摘要:

Abstract. Published data on the synthesis and properties of N-halohydroxylamine derivatives are described systematically and analysed. The bibliography includes 123 references. I. Introduction The development of the chemistry of N-halohydroxylamine derivatives started relatively recently, although the first represen- tative of this class, N-chloro-N-methoxy-p-toluenesulfonamine, was described as early as 1953.1 N-Halo-N,O-dialkylhydroxyl- amines are the nitrogen analogues of a-halo ethers, which are widely used in organic synthesis as highly reactive alkylating reagents.2 It is this formal analogy that attracted our attention about 20 years ago.This analogy has worked perfectly, and this permitted the synthesis of new classes of organic compounds by simple nucleophilic substitution of chlorine in N-chlorohydroxyl- amine derivatives.In particular, N-alkoxy-N-tert-alkyl-O-alkyl- hydroxylamines (nitrosoacetals),3, 4 N,N-dialkoxy-O-alkyl- hydroxylamines (trialkoxyamines, orthonitrites),5 and N-alkoxy- O-alkylhydroxylamines (NH-dialkoxyamines, orthoethers of nitrosyl hydride) 6 have been synthesised in this way, and oxida- tion of the latter compounds gave stable dialkoxyaminyl radicals 7 and their dimers (tetraalkoxyhydrazines).7, 8 The vast synthetic potential of N-halohydroxylamine derivatives is also due to the possibility of converting them into highly reactive intermediates such as N-alkoxynitrenes,9 N-substituted N-alkoxyaminyl radi- cals,7,10 and N-alkoxynitrenium ions.5, 11 Thus intramolecular cyclisation of N-chloro-O-alkylhydroxamates involving an aro- matic substituent present in the acyl or alkoxyl fragment of their molecules, which occurs via intermediate N-acyl-N-alkoxynitre- nium ions, is a general method for the synthesis of a large number of nitrogen-containing heterocycles.12 ± 18 N-Halohydroxylamine derivatives are also of interest from the stereochemical viewpoint, since the two s-electron-withdrawing substituents attached to the nitrogen atom markedly increase its configurational stability.4, 19 ± 26 This made it possible to prepare optically active derivatives of 2-chloro-1,2-oxazolidine 19, 20 and 2-chloroxaziridine 24, 25 with an asymmetric nitrogen atom.The non-symmetrically substituted N-alkoxy-N-tert-alkyl-O-alkylhy- droxylamines synthesised from N-chloro-N-tert-alkyl-O-alkylhy- droxylamines were partially resolved into optical antipodes, which were the first chiral compounds with asymmetric nitrogen atoms in an open chain.4 Some aspects of the chemical behaviour of O±N± Cl systems have been considered in a number of reviews.12, 27 ± 29 In the present review, we have attempted to survey all the currently available data on the synthesis, properties, and stereochemistry of N-halohydroxylamine derivatives.The stereochemistry and chemical properties of geminal oxy- gen ± nitrogen ± halogen systems are mostly determined by the stabilising interaction between the non-bonding (np) orbital of the oxygen atom and the antibonding s* orbital of the N± Hal bond, which is described in terms of the concept of valence bonds as hyperconjugation.According to the theory of molecular orbital perturbation, the energy of n ± s* stabilisation is approximately proportional to the overlap of the interacting orbitals and is inversely proportional to the difference between their energies.30, 31 The n ± s* interaction results in kinetic destabilisation of the N± Hal bond, i.e. in an increase in its tendency to dissociate (kinetic anomeric effect 32){ yielding a nitrenium ion stabilised due to the p-overlap between the vacant 2pz orbital of the N atom and the occupied 2pz orbital of the neighbouring O atom (Fig. 1).4, 13 ± 15, 33, 34 N-Alkoxynitrenium (nitrenium-oxonium ions) are ambident cations. They react with nucleophiles according to two competing pathways.5, 10, 35, 36 Hal7.R0 R0 Hal N RO N + RO V F Rudchenko, R G Kostyanovskii N N Semenov Institute of Chemical Physics, Russian Academy of Sciences, ul. Kosygina 4, 117977 Moscow, Russian Federation. Fax (7-095) 938 21 56 Received 24 February 1997 Uspekhi Khimii 67 (3) 203 ± 218 (1998); translated by Z P Bobkova UDC 541.127.4:542.91:547.435 Geminal oxygen ± nitrogen ± halogen systems. N-Halohydroxylamine derivatives V F Rudchenko, R G Kostyanovskii Contents I.Introduction 179 II. N-Halo-O-alkylhydroxylamines 180 III. N-Halo-N,O-dialkylhydroxylamines 180 IV. N-Halo-N-alkoxy-O-alkylhydroxylamines 185 V. Derivatives of N,N-dihalohydroxylamines 185 VI. N-Halo-N-alkoxyamides 186 VII. Structure and stereochemistry 189 VIII. Optically active cyclic N-chlorohydroxylamines 190 IX.Conclusion 191 { The structural consequences of the n ± s* interaction are considered in Section VII. Russian Chemical Reviews 67 (3) 179 ± 192 (1998) #1998 Russian Academy of Sciences and Turpion LtdThe addition of nucleophiles to the nitrenium centre [path- way (a)] is a reversible orbitally controlled reaction, which occurs in the case of `soft' nucleophiles with high-lying highest occupied molecular orbitals (HOMO).Conversely, dealkylation of the oxonium site [pathway (b)] is an irreversible charge-controlled reaction occurring in the case of `hard' nucleophiles having low- lying HOMO. The thermal stability and the reactivity of N-halohydroxyl- amine derivatives depend appreciably on the type of the halogen. The former decreases,13, 37 whereas the latter increases with an increase in the electron-withdrawing ability of the N± Hal bond.The above-mentioned properties of N-halohydroxylamine deriv- atives also depend substantially on the type of the third substitu- ent at the nitrogen atom. Electron-releasing substituents, which favour ionisation of the N± Hal bond to even a greater extent, diminish the thermal stability and increase the reactivity of these compounds.For instance, whereas N-chloro-N-tert-alkyl-O- alkylhydroxylamines 4 are stable distillable liquids, N-chloro-N- alkoxy-O-alkylhydroxylamines 5 cannot be detected by 1H NMR spectroscopy even at 730 8C. Conversely, electron-withdrawing substituents decrease the polarisation of the N± Hal bond, and in some cases, they even change the direction of polarisation; this, for example, occurs in the case of N-halo-N-alkoxy-substituted sulfo- namides 1, 38 and phosphamides.38 II.N-Halo-O-alkylhydroxylamines N-Halo-O-alkylhydroxylamines are unstable and have not been isolated in a pure state. Nevertheless, N-chloro-O-methylhydrox- ylamine, generated by treatment of O-methylhydroxylamine with N-chlorosuccinimide, has been involved in situ in the addition to tetramethylethylene.39 Two routes of transformation of N-chloro-O-tert-alkylhy- droxylamines have been discovered; in weakly alkaline solutions, they form N2O, while in strongly alkaline media (6 N or 8 N NaOH), they are converted into nitrosoalkanes.40 Dinitrogen monoxide was assumed to result from decomposition of nitrosyl hydride formed upon dealkylation of the intermediate nitrenium- oxonium ions.Nitroso-derivatives result from 1,2-rearrangement of the alkoxynitrenes,41, 42 which can be generated from N-chloro- O-alkylhydroxylamines via 1,1-elimination 40 or deprotonation of N-alkoxynitrenium ions.35 It has been assumed that the products of monohalogenation of O-diphenylmethylhydroxylamine 43 and 1,2-bis(aminooxy)- ethane 44 are mostly formed via decomposition of intermediate hyponitrites yielding N2 and alkoxy radicals, which, in turn, disproportionate to give an aldehyde (or ketone) and an alcohol.In some cases, hyponitrites are the final products of mono- halogenation of O-alkylhydroxylamines.45, 46 One of the possible pathways to hyponitrites in this reaction includes decomposition of N-halo-O-alkylhydroxylamines to alkoxynitrenes and their subsequent dimerisation.43 The inter- mediate formation of benzyloxynitrene has also been assumed 4 for the chlorination of O-benzylhydroxylamine.47 The bromination of O-methylhydroxylamine in the presence of C-nitroso-derivatives affords diazene oxides in 5%±20% yields.9 III.N-Halo-N,O-dialkylhydroxylamines This type of compound has been studied most comprehensively.Methods for synthesis of acyclic and cyclic N-chloro-, N-bromo-, and N-fluoro-derivatives have been developed; chemical proper- ties of these derivatives have been studied. Nu7 + N OR Nu7 N + OR N Hal OR N Nu OR O +RNu N a b 7Hal7 +Hal7 NCS is N-chlorosuccinimide. MeONH2 [MeONHCl] Me2C CMe2 Cl NHOMe Me2C CMe2 NClS 7Cl7 RONHCl OH7 OH7 7 RONCl RON RONH2 NaOCl + RO NH Cl7 [HNO] N2O, 7RCl 7H2O + RO NH Cl7 R=But, Me2EtC.RN O RON Ph2C NOCHPh2 +Ph2C O, NBS is N-bromosuccinimide; NH2OCH2CH2ONH2 HOCH2CH2OH+HOCH2CH NOH. ButOCl 778 8C 7N2 Ph2CHON NOCHPh2 ] Ph2CHONH2 NBS [ Ph2CHONHBr R=Et, But; XHal=Br2, NaOCl, chloramine T. RONH2 [RONHHal ] RON NOR 7HHal XHal PhCH2ONH2 PhCH2ON PhCH2ONHCl 7HCl PhCH PhCH2N O NOH PhCONH2 .(20%) 1. HOCl 2. EtONa XBr=Br2, NBS; R=Me2(NO2)C, MeO2CCMe2, Ph, 2,4-(NO2)2C6H3, MeONH2 [MeONHBr ] XBr RN O RN(O) NOMe NO2 , N N . Me O N , Me Me H N + O N p* p Figure 1. Interaction of the 2pz orbitals of nitrogen and oxygen in alkoxynitrenium ions. 180 V F Rudchenko, R G Kostyanovskii1. Synthesis The simplest representative of acyclic N-chloro-N,O-dialkyl- hydroxylamines, namely N-chloro-N,O-dimethylhydroxylamine 1, has been prepared by chlorination of N,O-dimethylhydroxyl- amine 48 and by cleavage of aminal 2 upon treatment with N-chlorosuccinimide.4 Compound 1 withstands vacuum distillation (b.p. 35 8C at 200 mmHg).It was characterised by spectroscopic meth- ods.4, 48, 49 However, the attempts to synthesise other N-chloro- N,O-dialkylhydroxylamines containing an a-hydrogen atom in the N-substituent failed.50, 51 These compounds are thermally unstable and are readily dehydrochlorinated to O-ethers of oximes.Conversely, N-chloro-O-alkyl-N-tert-alkylhydroxylamines 3a ± g for which this dehydrochlorination is impossible are ther- mally stable under ambient conditions. They have been prepared by chlorination of the corresponding hydroxylamines 4, 48, 52 ± 57 and by cleavage of N-tert-alkyl-N-methoxy-O-methylhydroxyl- amine 4 upon treatment with acyl chlorides or chlorotrimethylsi- lane.58, 59 The compound 3a in a pure state decomposes over a period of 12 h at 20 8C,4 whereas 3c can be distilled in vacuo and remains unchanged in solution (CCl4) for at least a month.4 The only known acyclic N-bromo-N,O-dialkylhydroxylamine 6 was synthesised by bromination of the oxime O-ether 5 in the presence of CsF.60 The attempt to prepare N-bromo-N-tert-alkyl-O-methylhy- droxylamine 7 was unsuccessful; this compound is unstable and undergoes further transformations under the reaction condi- tions.59 Acyclic fluorinated N-fluoro-N,O-dialkylhydroxylamines have been reported.These derivatives have been synthesised by fluorination of the corresponding NH-hydroxylamines,61 oxime O-ethers,60 and trifluoronitrosomethane 62 and by photochemical reaction of N,N-difluoroamines with trifluoromethyl peroxide.63 Apart from the acyclic N-halo-N,O-dialkylhydroxylamines described above, a number of cyclic analogues have also been described. 2-Haloxaziridines 8a ± e and 9a,d have been obtained in two ways: by halogenation of the corresponding NH-oxaziridines and by oxidation of N-chlorimine 10.24, 25, 64, 65 The chloroxaziridines 8a ± e can be distilled in vacuo without decomposition; in a pure state, they can be kept unchanged for 1 month at 0 8C, while as solutions in CDCl3, they are stable for 1 h at 100 8C in sealed tubes.The 2-bromoxaziridines 9a,d are stable for 1 weak at 0 8C in inert solvents; neat compounds can be stored at 778 8C but vigorously decompose to give the corresponding ketone at temperatures above 0 8C.65 Thermally stable 2-chloro- and 2-fluoro-perhalo-1,2-oxazeti- dines 11 have been prepared by halogenation of the corresponding 1,2-oxazetidines.23, 66 Dimethyl 2-chloro-1,2-oxazolidine-3,3-dicarboxylate 12 has been synthesised by the chlorination of 1,2-oxazolidine 13 20 and by the reaction of the methoxy-derivative 14 with SOCl2.20, 58 MeNHOMe MeN(Cl)OMe , ButOCl 710 8C 1 (5%) MeN(OMe)CH2N CO2Me H 1 . 2 NClS R1=Me, Et; R2=H, Et; R3=Et, Me. R=H, Me. ON CR2 ON CR2 R1R2CHNHOR3 . HCl [R1R2CHN(Cl)OR3] NaOCl 7HCl R1R2C NOR3 ONHCHR2 ONHCHR2 2ButOCl 2Et3N ON(Cl)CHR2 ON(Cl)CHR2 72HCl R1=MeO2CCMe2: R2=Me (a), Pri (b); R1=MeO2CCH2CMe2: R2=Me (c), Pri (d), PhCH2 (e); R1=MeO2CC(CF3)Me: R2=Me (f); R1=(EtO2C)2CHCMe2: R2=Me (g). R1NHOR2 R1N(Cl)OR2 3a7g ButOCl XCl=AcCl, CF3COCl, SOCl2, Me3SiCl.MeO2CCMe2N(OMe)2 4 XCl MeO2CCMe2N(Cl)OMe 3a CF2 5 NOCF2CF3 Br2, CsF CF3N(Br)OCF2CF3 . 6 (99%) MeO2CCMe2N(OMe)2 AcBr [MeO2CCMe2N(Br)OMe ] 7 MeO2CCMe2N(Ac)OMe +MeO2CNHOMe+ AcBr 7AcOMe +MeO2CCMe2N(O) NCMe2CO2Me.CF3NHOC2F5 F2 CF3N(F)OC2F5 ; (98%) CF3N(F)OCFXCF3 NOCFXCF3 CF2 F2 CF3N(F)OCF3+CF3ON(CF3)2+CF3ONF2 ; CF3N O F2 RFNF2+(CF3O)2 hn (5% ± 17%) RFN(F)OCF3+RFN(OCF3)2 (1% ± 5%) X=H (87%), Cl (62%) ; RF=CF3, C3F7. ButOCl MCPBA is m-chloroperbenzoic acid; MNPA is monoperphthalic acid. R=R0=Me (a); R=Me, R0=Et (b); R=R0=But (c); R,R0=(CH2)5 (d); R,R0 =CH2CH(Me)(CH2)3 (e); NCl 10 8d MCPBA or MNPA O NCl R R0 8a ± e O NH R R0 NBS 9a,d O NBr R R0 8d NClS O NCl Hal=Cl, F.Hal2 O NH F F F Hal O NHal F F F Hal 11 Geminal oxygen ± nitrogen ± halogen systems. N-Halohydroxylamine derivatives 1812. Reactions with nucleophiles The chemical properties of acyclic N-chloro-O-alkyl-N-tert-alkyl- hydroxylamines, 2-haloxaziridines, and 2-chloro-1,2-oxazolidine 12 have been studied.For these compounds, reactions with nucleophilic reagents are most typical. The transformations of N-chloro-O-alkyl-N-tert-alkyl- hydroxylamines under the action of nucleophilic reagents follow, as a rule, one of the four pathways shown in Scheme 1: (a) nucle- ophilic substitution of chlorine, (b) formation of C-nitroso- derivatives, (c) redox reactions, (d) 1,2-rearrangements.Scheme 1 a. Nucleophilic substitution of chlorine N-Chloro-O-alkyl-N-tert-alkylhydroxylamines 3a ± g react smo- othly with alcohols and glycols in the presence of bases to give N-alkoxy-N-tert-alkyl-O-alkylhydroxylamines 15 in 20% ±85% yields.4, 48, 53 ± 58,67 ± 69 In the case of secondary and tertiary alco- hols, the reaction also affords diazene oxides 16 resulting from a competing redox reaction [pathway (c)].In some cases, the compounds 16 are the only reaction products.4, 59 The intramolecular version of this reaction has been used to synthesise N-methoxy-1,2-oxazolidine 17; the product was obtained in 45% yield.4, 52 Methanolysis of 2-chloro-1,2-oxazolidine 12 also proceeds smoothly.20, 58 The products of nucleophilic substitution of chlorine were obtained in reactions of the compounds 3a,c with sodium acetate, thiocyanate, and cyanide and also with a Grignard reagent.10 When N-chloro-N-tert-alkyl-O-methylhydroxylamines 3a,c react with ammonia or amines, the corresponding N-methoxy- hydrazine derivatives formed initially upon nucleophilic substitu- tion of chlorine are unstable and decompose with elimination of MeOH.Thus the reaction of 3a with NH3 gave a-chloroisobutyr- amide and diazene oxide 16.70 This outcome can be explained by assuming that the intermediate N-methoxyhydrazine derivative decomposes to give NH-diazene, which undergoes homolytic cleavage yielding a tert-alkyl radical.The reaction of the latter with the initial compound 3a followed by ammonolysis gives the final products.In conformity with this scheme, the reactions of 3a with primary amines give rise to non-symmetrical diazenes 18a,b.70 Pyrazolidone 19 was obtained in 68% yield by the reaction of the compound 3c with dimethylamine.70 The formation of this product was explained in terms of a scheme including decom- position of the intermediate N-methoxyhydrazine derivative giv- ing a diazenium salt, which then undergoes a typical cleavage with abstraction of the a-proton and generation of a zwitter-ion.This ion adds dimethylamine giving rise to a non-symmetrical aminal, which disproportionates yielding bis(dimethylamino)methane and the corresponding hydrazine; cyclisation of the latter affords the final reaction product. When the compound 3a reacts with a less basic N,O-dime- thylhydroxylamine, the aminal formed by a similar scheme does not disproportionate; instead, it eliminates MeOH, which results in the formation of diazene 20 in 42% yield.70 O N CO2Me CO2Me Cl 12 O N CO2Me CO2Me H 13 O N CO2Me CO2Me OMe 14 ButOCl SOCl2 R1 R3 N R2 Nu OR4 R1 R3 R2 R1 R3 R2 Nu R3 N R2 R1 OR4 Nu7 b 7Cl7 NOR4 N O+R4Nu c 7Nu,7Cl7 d 7Cl7 7Cl7  R1 R3 N R2 Cl OR4 R3=Me, Et, Pri, But, PhCH2, CF3CH2, HO(CH2)2, HO(CH2)3; B=Et3N, R3ONa.R1N(Cl)OR2 3a ± g R1N(OR3)OR2+R1N(O) 15 16 NR1 R3OH, B HO(CH2)2CMe2N(Cl)OMe 17 Et3N Et2O O N Me Me OMe 12 MeOH Et3N 14 (86%) O N CO2Me CO2Me OMe R=MeO2C(CH2)nCMe2: n=0, 1; Nu=AcO, NCS, CN, Et. 3a,c RN(Cl)OMe RN(Nu)OMe Nu7 7Cl7 16 (12%) RN(O) NR R RN(OMe)NH2 H+ 7MeOH, 7H+ 3a NH3 7HCl RN NH 7N2,7H 3a NH3 7MeOH ClCMe2CONH2 (27%) [RNOMe] + RCl R=MeO2CCMe2. R=MeO2CCMe2; R0=Me [18a (99%)], Et [18b (93%)]. 7MeOH, 7H+ H+ RN NR0 RN(OMe)NHR0 3a R0NH2 7HCl R0NH2 7MeOH 18a,b R0NHC(O)CMe2N NR0 RN(OMe)NMe2 3c Me2NH 7HCl 7MeOH H+ + RN NMe2 7H+ R=MeO2CCH2CMe2. +N CH2 Me 7 RN RNHNMeCH2NMe2 Me2NH Me2NH 7(Me2N)2CH2 RNHNHMe 7MeOH N H N O Me Me 19 Me 182 V F Rudchenko, R G KostyanovskiiStable aziridine derivative 21 containing an N-methoxyhydr- azine moiety was synthesised in 70% yield as the product of nucleophilic substitution of chlorine by reaction of 3c with ethyl- ene imine.70 The instability of N-methoxyhydrazine derivatives 70 is due to the easy rupture of the N±O bond, caused by the vicinal nN(1) ± s*N±O orbital interaction.In the aziridine 21, this inter- action is weak, because the positive mesomeric effect of the ethylene imine nitrogen atom is low.This accounts for the stability of this compound. Nevertheless, the reaction of the compound 3c with 2,2- dimethylethylene imine under the same conditions affords the acyclic product 22.70 It was assumed that, due the fact that 2,2-dimethylethylene imine is more basic than ethylene imine, the aziridine ring in the chlorine substitution product 21a formed initially is cleaved under the action of HCl according to an SN2 mechanism. The reactions of 3a,c with pyridine also occur as nucleophilic substitution of chlorine.70 Based on kinetic studies, it was concluded that this reaction occurs by the SN2 mechanism.20 It was shown that methanolysis of optically active N-chloro- 1,2-oxazolidine (+)-12 (its synthesis is described in Section VIII) occurs with retention of the optical activity and affords N-methoxy-1,2-oxazolidine R-(7)-14 with an optical purity of about 3%.19, 20 However, since the optical purity and the absolute configuration of the starting (+)-12 are unknown, no reliable conclusions about the mechanism of this reaction can be drawn. b.Formation of C-nitroso-derivatives C-Nitroso-derivatives 23a,b have been prepared by hydrolysis of N-chloro-N-tert-alkyl-O-methylhydroxylamines 3a,c in the pres- ence of K2CO3 10, 71 or SbCl5.71 Hydrolysis of N-chloro-1,2-oxazolidine 12 also gives rise to a nitrosoalkane.However, this product is unstable and rearranges to oxime 24 as a result of migration of the methoxycarbonyl group from the C atom to the O atom.57 These reactions proceed presumably via intermediate nitre- nium-oxonium ions.Water is a hard nucleophile; therefore, in conformity with the general principles outlined in Section I, it reacts with these ions with dealkylation of the oxonium centre.10 c. Redox reactions When N-chloro-N-tert-alkyl-O-methylhydroxylamines 3a,c react with sterically hindered alcohols in the presence of Et3N,4 steri- cally hindered amines (Et3N, Et2NH),10 Grignard reagents,10 triphenylphosphine,10 or sodium ethanethiolate,10 nucleophilic substitution of chlorine is suppressed completely or partially by the competing redox reaction.This reaction affords N-substi- tuted-N-methoxyaminyl radicals, dimerisation of which results in the formation of unstable 1,2-dimethoxyhydrazine derivatives.Decomposition of these compounds yields the final products, diazene oxides 16a,c. d. 1,2-Rearrangements 1,2-Rearrangement with migration of the phenyl group to the electron-deficient nitrogen atom is the predominant route of transformation of N-chloro-O-alkyl-N-triphenylmethylhydroxyl- amines under conditions of alcoholysis. This rearrangement occurs spontaneously even in the absence of an external nucleo- phile.11 R=MeO2CCMe2. 3a [ RN(OMe)N(OMe)Me MeNHOMe 7HCl RNHN(OMe)CH2N(OMe)Me ] 7MeOH 20 RN NCH2N(OMe)Me 3c 7HCl NH 21 MeO2CCH2C Me Me N OMe N 3c+HN Me Me 7HCl HCl MeO2CCH2C N OMe 21a N Me Me Me Me 22 (84%) 7MeOH MeO2CCH2CN(OMe)NHCCH2Cl Me Me Me Me MeO2CCH2CMe2N NCMe2CH2Cl R=MeO2CCMe2, MeO2CCH2CMe2. RN(Cl)OMe + 3a,c N MeCN + RN OMe Cl7 N MeOH, KOH 778 8C (R)-(7)-14 O N CO2Me CO2Me OMe (+)-12 O N CO2Me CO2Me Cl R=MeO2CCMe2 [23a (88%)], MeO2CCH2CMe2 [23c (91%)]. 23a,c RN O RN(Cl)OMe 3a,c H2O H2O K2CO3 HO(CH2)2CN O CO2Me CO2Me MeO2CO(CH2)2C 24 (82%) CO2Me NOH 12 O N CO2Me CO2Me Cl RN(Cl)OR0 + [RN OR0 Cl7] 7HCl, 7R0OH H2O RN O R=MeO2CCMe2 (a), MeO2CCH2CMe2 (c). + RN N R OMe Nu 7Nu+Me 16a,c RN(O) NR 7MeO7 3a,c RNOMe Nu + 7Nu,7Cl7 N R MeO N R OMe Ph3CNHOR ButOCl + N Ph Ph OR Ph Cl7 Ph3CN(Cl)OR Geminal oxygen ± nitrogen ± halogen systems.N-Halohydroxylamine derivatives 183A 1,2-rearrangement with migration of the alkyl or methoxy- carbonyl group from the carbon atom to the nitrogen atom was found in the chlorination of the adducts of MeONH2 with cyclo- hexanone or dimethyl mesoxalate.The rearrangement products were isolated after treatment with Et3N.11 The mechanism of these rearrangements has not been studied; however, they are believed to involve the formation of intermedi- ate nitrenium ions. 2-Haloxaziridines 8 and 9, unlike N-chloro-N,O-dialkyl- hydroxylamines, possess relatively low reactivity with respect to nucleophiles. In addition, the reactions of these compounds with some nucleophiles, for example, MeO7, Me2NH, morpholine, dimethyl sulfide, and diisopropyl sulfide, occur as substitution of oxygen rather than chlorine, i.e.when the nucleophile attacks the nitrogen atom, an oxygen-containing anion becomes the leaving group.65 The intermediate A thus formed decomposes yielding the corresponding ketone and a substituted N-haloamine B.The latter reacts with amines giving rise to tetrazene 25 (apparently, via diazenium salt and N-aminonitrene) or reacts with dialkyl sulfides with formation of N-chlorosulfimines 26. The formation of sulfoxides 27 in the reactions of 8a,d with dialkyl sulfides was explained by hydrolysis of N-chlorosulfimines 26. 3. Other reactions N-chloro-N-tert-alkyl-O-methylhydroxylamine 3a 59 and oxazoli- dine 12 20 react with gaseous HCl to give the corresponding hydrochlorides 28 and 29.The reaction of the compound 3a with CF3COCl affords N-methoxytrifluoroacetamide 30, the hydrochloride 28, and methyl N-methoxycarbamate 31.59 Presumably, the trifluoroacetamide 30 is formed upon decom- position of the N-chloro-N-methoxy-N-trifluoroacetylammo- nium salt formed intermediately.N-Methoxcarbamate 31 results from 1,2-rearrangement of the compound 3a, accompanied by migration of the methoxycarbonyl group, and subsequent hydrol- ysis of the resulting intermediate C by traces of water. The HCl evolved during this process reacts with the initial compound 3a according to the scheme described above to give the hydrochloride 28. Treatment of 3a with reagents facilitating ionisation or heterolytic rupture of the N± Cl bond, for example, SO2 or BF3 .Et2O, also results in 1,2-rearrangement.59 This is why SO2 initiates the addition of N-chloro-N,O- dialkylhydroxylamines 3a,c to isobutene.55 The reactions of 3a and 3c with AgF .H2O in MeCN can follow different pathways depending on the nature of the alkyl substituent at the nitrogen atom.10 R=Me, MeO2CCMe2; R0=Me, Et.Ph2C(OR0)N Ph OR 7Et3N. HCl R0OH, Et3N R=Me 7MeCl (77%) N Ph Ph O Ph 1. MeONH2 2. ButOCl Et3N 7Et3N. HCl O N O Cl OMe H N OMe O (29%) (55%) MeO2CCN CO2Me OMe O 1. MeONH2 2. ButOCl (MeO2C)2C O Et3N 7Et3N. HCl MeO2C O H MeO2C N OMe Cl 8a ± d, 9a O NX R1 R2 25 R1R2C O+R32 NN NNR32 R1=R2=Me (a); R1=Me, R2=Et (b); R1=R2=But (c); R1, R2=(CH2)5 (d); R32 =Me2, (CH2)2O(CH2)2; R4=Me, Pri.R1R2C O +[R42 S NCl] H2O 26 R42 S O 27 7 + R1R2C O +[NuNX] R42 S R32 NH MeO7, CN7 X=Cl, Br X=Cl X=Cl O N Hal Nu R1 R2 Hal N O7 A R1 R2 + Nu B + 7 R1R2C O+[NuNHal ]. R=MeO2CCMe2. 12 HCl 29 (82%) + O N CO2Me CO2Me H H Cl7 HCl + R N Cl H OMe Cl7 HCl 7Cl2 RNHOMe . HCl 28 (73%) 3a R=MeO2CCMe2. 3a CF3COCl + R N Cl OMe COCF3 Cl7 30 , 7Cl2 31 .C H2O 7Me2C=O, 7HCl Cl7 Me Me +N CO2Me OMe 3a Me MeO2C Me N Cl OMe RN(OMe)COCF3+28+MeO2CNHOMe, 3a 30 (37%) 25% 31 (14%) CF3COCl R=MeO2CCMe2, MeO2CCH2CMe2. 3a,c + Me2C CH2 SO2 Me2C(Cl)CH2N(OMe)R 23a (20%) 32 (45%) MeO2CCMe2N O+MeO2CCMe2NO2, 3a AgF .H2O MeCN 3c AgF .H2O MeCN 33 (28%) 34 CHCO2Me. MeCONHCMe2CH2CO2Me+Me2C 184 V F Rudchenko, R G KostyanovskiiIn the case of 3c, this reaction was assumed to occur via elimination of methoxynitrene from the intermediate nitrenium ion to give a tert-alkyl cation, which is stabilised through the Ritter reaction with acetonitrile or deprotonation.10 The same transformation of 3a does not occur, because the corresponding tert-alkyl cation is less stable; therefore in this case, redox process predominates.10 N-Chloroxazolidine 12 is reduced by Na2SO3 to give 1,2-oxa- zolidine 13 in 67% yield.20 It was found that the sterically hindered 2-chloroxaziridine 8c, unlike non-hindered 8a,b,d, rearranges to a-chloronitrosoalkane 35.The rearrangement of pure 8c at 0 8Cproceeds slowly, whereas that in CDCl3 at 60 8C occurs over 15 ± 20 min.64, 65 It was assumed 65 that this rearrangement occurs as intramolecular attack by chlorine on the carbon atom of the oxaziridine ring.During photolysis of 2-chloroxaziridine 8c, the corresponding oxaziridinyl radical was detected by EPR spectroscopy.72 IV. N-Halo-N-alkoxy-O-alkylhydroxylamines The only fluorine-containing compound of this type was prepared in 3% yield by photochemical reaction of N,N-difluoro-O-tri- fluoromethylhydroxylamine with trifluoromethyl peroxide.63 N-Bromo- and N-iodo-derivatives of N-alkoxy-O-hydroxyl- amines are unknown.N-chloro-derivatives are thermally unsta- ble, due to the easy heterolysis of the N± Cl bond yielding dialkoxynitrenium ions stabilised by resonance; these ions undergo irreversible dealkylation through the action of the internal nucleophile, i.e. the chlorine atom.For instance, chlori- nation of N-benzyloxy-O-methylhydroxylamine by ButOCl gives benzyl chloride and methyl nitrite resulting from decomposition of the intermediate N-chloro-N-benzyloxy-O-methylhydroxyl- amine, which could not be detected by 1H NMR spectroscopy even at730 8C.5 Nevertheless, N-chloro-N-methoxy-O-methylhydroxylamine was in situ involved in several reactions. It was found that in reactions with Me3N and pyridine, this compound, unlike N-chloro-N,O-dialkylhydroxylamines (Section III), acts as a methylating rather than an aminating reagent.5 This difference between the behaviour of N-chloro-N,O- dialkylhydroxylamines and N-chloro-N-alkoxy-O-alkylhydroxyl- amines in the above reactions was explained 5 by the fact that the nitrogen electrophilic centre in the latter is `harder' and, hence, the orbitally controlled addition of nucleophiles to the nitrenium centre becomes possible only for very `soft' nucleophiles contain- ing high-lying HOMO. A similar difference in the properties has also been noted for monoalkoxy- and dialkoxycarbenium cati- ons.73 In accordance with the foregoing, nucleophilic substitution of chlorine in N-chloro-N-alkoxy-O-alkylhydroxylamines occurs under the action of sodium methoxide and yields trialkoxyamines 36a,b .5, 74 V. Derivatives of N,N-dihalohydroxylamines The only known compounds of this type are N,N-difluorohydr- oxylamine derivatives.The data on the synthesis and properties of compounds of the general formula XONF2, where X = CF3, C2F5, Cl(CF2)2, FOCH2, F2NOCH2, (CF3)2N, FCO2, FSO2, SF5, published before 1976 have been surveyed in reviews.27, 28 Later, a method for the synthesis of N,N-difluoro-O-perfluoroalkyl- hydroxylamines by reaction of perfluroalkyl hypofluorites with difluoroamine ± alkali metal fluoride complexes has been devel- oped.75 N,N-Difluorohydroxylamine derivatives have also been syn- thesised by reactions of trifluoroamine oxide with fluoroalkenes in the presence of Lewis acids (BF3, AsF5, SbF5).76, 77 7H+ 3c 7MeON AgF .H2O 7AgCl, 7F7 + [MeO2CCH2CMe2NOMe] 33 MeCN H2O + MeO2CCH2C Me Me 34 + MeC NCCH2CO2Me Me Me 7MeF 23a 3a O2 MeO2CCMe2NOMe 7AgF Ag + MeO2CCMe2NOMe F7 AgF .H2O 7AgCl MeO2CCMe2NOMe O 7Ag AgF MeO2CCMe2NOMe + O F7 7MeF 32. 12 O N CO2Me CO2Me H 13 Na2SO3 H2O Et2O Cl But But N O d+ d7 + Cl But But N O7 35 (87%) O N But But Cl 8c But 2C N O Cl CF3ONF2+(CF3O)2 (CF3O)2NF.hn R=PhCH2. RONHOMe RON(Cl)OMe ButOCl + RONOMe Cl7 RCl+MeON O (79%) RON + OMe Cl7 NOMe + RO Cl7 Nu=Me3N, Py. (MeO)2NH [(MeO)2NCl] Nu+Me Cl7 ButOCl 778 8C Nu 7MeONO R=Me [36a (64%)], Et [36b (50%)]; DME is dimethoxyethane. BuiONHOR [BuiON(Cl)OR] ButOCl 778 8C MeONa DME BuiO N OMe OR 36a,b RF=CF3, iso-C3F7, FOCF2; MF=KF, RbF, KF7NaF.(10%720%) RFOF+F2NH.MF RFONF2+MF.HF Geminal oxygen ± nitrogen ± halogen systems. N-Halohydroxylamine derivatives 185N,N-Difluorohydroxylamines 37 are cleaved by AsF5 to give FNO, which is formed as a nitrosonium salt.78 In reaction with perfluorocyclobutene, O-fluorosulfonyl- N,N-difluorohydroxylamine 38 acts as a pseudohalogen.79 The reactions of 38 with ethylnitramine, ethylenedinitramine, and salts of azoles (imidazoles, pyrazoles, triazoles) lead to N-fluorosulfonylation products.80 The reaction of 38 with the sodium salt of pyrazole was reported to be an example.80 The reaction of the sodium salt of 4-nitrotriazole with the compound 38 is accompanied by ring opening.Subsequent hydrolysis affords formylnitrodiazomethane in 36% yield.80 VI.N-Halo-N-alkoxyamides As noted above, a typical feature of systems containing the O±N± Hal triad is polarisation of the N± Hal bond towards the halogen, which accounts for its anionic mobility. When the nitro- gen atom carries an electron-withdrawing substituent, polarisa- tion of the N± Hal bond decreases or even changes its direction, which has a substantial influence on the properties of these systems. 1. N-Halo-N-alkoxyphosphamides N-Halo-N-alkoxyphosphamides 39,81 40,38 and 41 82 have been prepared by halogenation of the corresponding NH amides. The compounds 39a ± d are stable in a benzene solution (withstand refluxing for several hours); however, in a pure state they decompose over a period of 2 days at 0 ± 5 8C. They give the typical `positive chlorine' reaction.81 The addition of 39c to styrene occurs by a radical mechanism and is not regiospecific yielding adducts 42 and 43 and some other products.81 The attempts to carry out nucleophilic substitution of the chlorine atom in N-chloro-N-alkoxyphosphamides 39a and 40a failed.The reaction with MeONa in MeOH involves the phos- phoryl group and is accompanied by elimination of the pseudo- halogen fragment N(Cl)OMe.38 The compound 39a reacts with MeOH in the presence of Et3N giving rise to amide 44.38 Photolysis of a solution of N-bromo-N-tert-butoxyphosph- amide 41 in cyclopropane at7100 8C gave radical 45, which was detected by EPR spectroscopy.82 2.N-Halo-N-alkoxysulfonamides N-Halo-N-alkoxysulfonamides 46a ± e 1, 38, 83 and 47 84, 85 have been described.The attempt to prepare the N-Br analogue of the compound 46a resulted in the formation of 1,2-dimethoxyhydrazine deriva- tive 48.83 Halogenation of compound 49 yields hyponitrite 50, appa- rently via the intermediate thiadiaziridine dioxide.46, 86 N-Halo-N-alkoxysulfonamides 46a,b,d act as chlorinating reagents with respect to H2O and MeOH, being thus converted into the corresponding NH amides.38, 83 Thus compound 46c chlorinates aniline into the aromatic ring.1 Photolysis of solutions of N-bromo-N-alkoxysulfonamides 47 in CFCl3 or in a mixture of CFCl3 with CH2Cl2 at730 to760 8C is accompanied by the generation of the corresponding N-alkox- ysulfonamidyl radicals, which have been detected by EPR spec- troscopy.84, 85 R=F, Cl, Br, C2F5O, C3F7OCF(CF3)CF2O; R=CF3, C5F11, SF5, FCO.Cat. RCF2CF2ONF2 RCF CF2+F3NO RCF CF2+F3NO RFC(CF3)ONF2 Cat. R=CF3, SF5. RONF2 RF+NO+AsF¡6 37 AsF5 FSO2ONF2+ F F F F F NF2 F OSO2F (80%) 38 160 8C F F F F F F 38+ Na+ N N SO2F N N 7 38 + Na+ H2O N N 7 N O2N NSO2F O2N N2 N N N O2N SO2F CHO O2N N2 R=Me (a), PhCH2 (b), Bu (c), Et (d). (EtO)2PN(Cl)OR O (Me2N)2PN(Cl)OR O (EtO)2PN(Br)OBut O 39a7d 40a,b 41 +PhCH(Cl)CH2Cl+ClCH2CH(Ph)CH(Ph)CH2Cl. 39c 42 (EtO)2PN(OBu)CH2CH(Cl)Ph + O PhCH CH2 43 +(EtO)2PN(OBu)CHPhCH2Cl +(EtO)2POBu + O O (EtO)2PNHOMe. 44 (71%) MeOH, Et3N 39a O R=Me, R0=Me (a), MeSO2 (b); R=4-MeC6H4, R0=Me (c); R=Me2N, R0=PhCH2 (d); R=2,6-Me2-4-ButC6H2, R0=But (e). R=Me, Me2N, 4-O2NC6H4, R0=But; R=4-MeC6H4, R0=Me, Et, Pri, But, PhCH2; R=2,6-Me2-4-ButC6H2, R0=Me, But, PhCH2.RSO2N(Cl)OR0 46a7e RSO2N(Br)OR0 47 MeSO2NHOMe NBS [MeSO2N(Br)OMe] MeSO2(MeO)N N(OMe)SO2Me. 48 XHal ButONHSO2NHOBut 49 7SO2 S NOBut ButON O O XHal=NaOCl, NaOBr, chloramine T. NOBut ButON 50 EtOH AcOH 46c+PhNH2 4-MeC6H4SO2NHOMe +4-ClC6H4NH2. 186 V F Rudchenko, R G Kostyanovskii3. N-Halo-N-alkoxycarbamates The only known compound of this type, methyl N-chloro-N- methoxycarbamate 52, was synthesised by chlorination of the N-methoxycarbamate 51.It can be distilled in vacuo without decomposition; upon treatment with MeOH and Et3N it is converted into the initial N-methoxycarbamate 51 and hydrazine derivative 53.87, 88 4. N-Chloro-N-alkoxyureas N-Chloro-N-alkoxyureas 54,89, 90 55a ± h,38, 91, 92 56, 8, 93, 94 and 57a ± i 95 have been prepared by chlorination of the corresponding NH ureas with ButOCl.The compound 55a was also obtained by cleavage of N,N-di- methoxyurea 58 upon treatment with acetyl chloride.96 N-Chloro-N-alkoxyureas 54, 55a± h, 56, and 57a ± i are non- distillable liquids or crystalline solids stable under ambient con- ditions. At room temperature in a CCl4 solution, 55a completely decomposes over a period of 25 days to give methyl N,N- dimethylcarbamate 59 and N,N-dimethylcarbamoyl chloride 60 in 1 : 1 ratio.38 The carbamoyl chloride 60 results presumably from a con- certed rearrangement with migration of chlorine to the carbonyl group and ejection of methoxynitrene (HERON rearrange- ment).97 The driving force of this migration is the orbital no ± s*N± Cl interaction in the O±N± Cl structural fragment.The N,N-dimethylcarbamate 59 is formed in this process upon inter- action of N,N-dimethylcarbamoyl chloride with the MeOH pro- duced among the products of decomposition of the intermediate methoxynitrene;38 this process is shown below: Among N-chloro-N-methoxyureas 57a ± i, only those which contain electron-withdrawing substituents in the aryl group are thermally stable under ambient conditions.The compound 57a isomerises at room temperature giving N-(4-chlorophenyl)-N0- methoxyurea.95 N-Chloro-N-alkoxyureas readily undergo nucleophilic sub- stitution of chlorine atoms, because the carbamoyl group does not have any significant effect on the polarisation of the N± Cl bond. This also accounts for the similarity of chemical properties of N-chloro-N-alkoxyureas and N-chloro-N,O-dialkylhydroxyl- amines (see Section III).Thus N-chloro-N-alkoxyureas 54 ± 56 react with alcohols and glycols in the presence of bases affording N,N-dialkoxyureas.8, 38, 89 ± 94 For instance, these reactions have been described for 55a ± h.38 Intramolecular cyclisation of N-chloro-N-alkoxyurea 55h gives rise to 1,3,2-dioxazolidine 61.98 Products of nucleophilic substitution of chlorine in N-chloro- N-methoxyureas 55a and 57b were also obtained with other nucleophilic reagents.95, 99 On treatment with bases, N-chloro-N-methoxyureas 57a ± h cyclise to give 1-methoxy-2-benzimidazolinones 62.95 ButOCl MeO2CNHOMe MeO2CN(Cl)OMe 51 52 MeOH, Et3N + 7Et3N,7Cl7 53 (36%) (30%) 51+MeO2C(MeO)N N(OMe)CO2Me.[MeO2CNOMe] RNHCN(Cl)OR0 54 O Me2NCN(Cl)OR 55a7h O R=Me (a), Bui (b), CH2 CHCH2 (c), PhCH2 (d), MeO2CCH2 (e), MeO2CCH2CH2 (f), MeO2CCMe2 (g), HOCH2CMe2 (h).R=Me, R0=Me, PhCH2; R=Et, R0=Me. n=0, 2, 3. Me2NCN(Cl)OCH2(CH2OCH2)nCH2ON(Cl)CNMe2 56 O O ArNHCN(Cl)OMe 57a7i O Ar=Ph (a), 4-ClC6H4 (b), 4-O2NC6H4 (c), 4-MeC6H4 (d), 4-CNC6H4 (e), 4-MeSO2C6H4 (f), 3-F3CC6H4 (g), 3,4-Cl2C6H3 (h), 2,6-Me2C6H3 (i). Me2NCN(OMe)2 58 O AcCl + Me2NCN O OMe Me Ac Cl7 O 7AcOMe 55a.(86%) 60 59 Me2NCOMe+Me2NCCl. 55a Me2NCN(Cl)OMe O O O 2MeO 59. 55a MeON MeON NOMe 7N2 7Me2NCOCl 7CH2O Me2NCOCl MeOH 57a PhNHCN(Cl)OMe O 4-ClC6H4NHCNHOMe. O B=R0ONa, collidine, NaOAc; R0=Me, Et, Bui, CH2 CHCH2, CF3CH2, HO(CH2)2. Me2NCN(Cl)OR O 55a7h R0OH, B Me2NCN(OR)OR0 O (14%785%) B is collidine. Me2NCN(Cl)OCMe2CH2OH O 55h B CH2Cl2 61 (68%) Me2NCN O O O Me Me Nu=Me3N, Py; R=Me, Ph.O Me2NCN O Nu+ Cl7 OMe 55a TsN(Na)R MeCN Me2NCN O N(R)Ts OMe Et2O Nu 57b+ THF N Na Cl O N OMe (28%) N N Cl Cl MeONCNH O OMe 57a7h B R R0 N N O Cl MeO H Geminal oxygen ± nitrogen ± halogen systems. N-Halohydroxylamine derivatives 187The formation of non-aromatic intermediate D during the cyclisation of 57i was proved by isolation of its adduct with MeOH.95 The reaction of N-chloro-N-methoxyurea 55a with Me2NH affords bis(dimethylamino)methane 63 and the urea 64;99 these products are formed via a scheme similar to that considered in Section III for the reaction of N-chloro-N-tert-alkyl-O-methyl- hydroxylamine 3c with Me2NH.Dimethylurea 64 results appa- rently from carbamoylation of Me2NH by an intermediate product.The reaction of N-chloro-N-methoxyurea 55a with N,O-di- methylhydroxylamine affords diazene 65 in 38% yield.99 This reaction proceeds presumably according to a similar scheme except that the intermediate aminal does not disproportionate, but instead, it eliminates MeOH yielding the product 65. In the reaction of N-chloro-N-methoxyurea 55a with Et3N, nucleophilic substitution of chlorine is hampered for steric rea- sons.Therefore, a redox reaction occurs, the final products of which are formed upon transformations of the N-methoxycarb- amidyl radical and substituted 1,2-dimethoxyhydrazine formed as intermediates.99 5. N-Halo-O-alkylhydroxamates Among compounds of this type, only N-chloro-derivatives are known; they are synthesised by the chlorination of the corre- sponding NH hydroxamates with ButOCl.The attempts to iodinate and brominate N-methoxyamide of biphenyl-2-carbox- ylic acid resulted in the formation of N-methoxy-phenathridone 66, hydrazine derivative 67, and the product of its decomposition 68.13, 37 Conversely, N-chloro-N-methoxyamide 69 is stable under ordinary conditions; its photolysis is accompanied by rupture of the N± Cl bond and gives the hydrazine 67, resulting from dimerisation of the intermediate N-methoxyamidyl radical, and compound 68.13, 37 N-Alkoxyamidyl radicals have been detected by EPR spectro- scopy during the photolysis of N-chloro-O-alkylhydroxamates 70,100 72, and 73. 101 Similarly to N-chloro-N-alkoxyureas, N-chloro-O-alkyl- hydroxamates are able to undergo nucleophilic substitution of chlorine.Thus reaction of the compound 70 with sodium salt 74 gives hydrazine derivative 71 in a quantitative yield.100 B=ButOK, NaH. N N OMe R0 R H O N N OMe H R0 R 62 (56%783%) O 57i KOH, MeOH MeOH N NH (35%) D O Me OMe Me OMe N N O OMe Me Me 55a Me2NH 7HCl H+ 7MeOH Me2NCN O NMe2 OMe Me2NCN O + NMe2 7H+ CH2 + 7 Me2NCN O N Me Me2NH 63 (26%) 64 (9%) (Me2N)2CH2+(Me2N)2C O.O Me2NCNHNMeCH2NMe2 Me2NH 7MeOH Me2NCNHN(OMe)CH2N(Me)OMe O Me2NCN O NCH2N(Me)OMe. 65 MeONHMe 55a 7HCl Me2NCN O N(Me)OMe OMe (23%) (17%) (7%) Me2NCNHOMe +Me2NCNHNHCNMe2+64 . O O O CONHOMe ButOX CON(X)OMe X=Br, I. NOMe O + CON OMe 2 + CO2Me 66 67 68 69 CON(Cl)OMe hn 7Cl 67+68 . (33%) (35%) CONOMe R=H, Ph; X=Et2C, PhN, MeN. N ButO OBut N 71 C O C O CON(Cl)OBut 7Cl hn CONOBut 70 O R O 72 N Cl X O O O 73 N Cl CON(Na)OBut Et2O 70 + 71 . 74 55a Et3N + 7Et3N,7Cl7 Me2NCNOMe O N Me2NC MeO O N CNMe2 OMe O 188 V F Rudchenko, R G KostyanovskiiTwo other reactions of N-chloro-N-alkoxybenzamides 75 � alcoholysis102, 103 and transformation into N-acetoxy-N-alkoxy- benzamides 76 on treatment with silver acetate in ether � also proceed smoothly.102, 104 However, alcoholysis of N-chloro-N-alkoxyarylacetamides 77a ± h does not result in nucleophilic substitution of chlorine.105 Depending on the nature of the substituent R, these reactions give mostly compounds 78 ± 80.An impnt characteristic feature of N-chloro-O-alkylhy- droxamates is their ability to be converted on treatment with Lewis acids into highly reactive intermediates, N-acyl-N-alkoxy- nitrenium ions,12 ± 18, 33 which tend to undergo inter- and intra- molecular electrophilic aromatic substitution.As a rule, these reactions occur at room temperature in the presence of metal ions (Ag+, Zn2+, Pd2+, Fe2+, Hg2+). The best results were achieved by using AgBF4 ± THF, Ag2CO3 ±CF3COOH, and Zn(OAc)2 ± MeNO2 systems. For instance, cyclisation ofN-chloro-N-methox- yarylacetamides 81 is an efficient method for the synthesis of 2-hydroxy-1-methoxyindoles 82.12, 14, 16 ± 18 In principle, the electrophilic attack by the intermediate nitrenium ion is directed at one of three positions in the aromatic ring, C(1), C(2), or C(6).18 When the aromatic ring contains an ortho-substituent, the nitrenium ions attack C(2) and C(6).Cyclisation of N-chloro-O-alkylhydroxamates 83 can involve the aromatic ring of both arylacyl and arylalkoxyl substitu- ents.14, 15 In the former case, the reaction yields g, d, and e-N-alkoxy- lactams 84, while in the latter case, it gives 2,1-benzoxazepines and 2,1-benzoxazines but not benzoxazoles.It was shown experimen- tally 15 that benzoxazepines result from the ipso-attack of the intermediate nitrenium ion on the aromatic nucleus followed by 1,2-migration of the alkyl group, while benzoxazines are formed upon the ortho-attack.The cyclisation of N-chloro-O-alkylhydroxamates containing an ortho- or para-methoxy-substituted aryl group occurs through the ipso-attack of the intermediate nitrenium ion on the aromatic ring and affords spirodienones.14, 18 For example, compounds 86 and 87 have been prepared in this way.18 N-Chloro-O-alkylhydroxamates are able to accomplish electro- philic amination of aromatic compounds in the presence of silver salts to giveN-alkoxy-N-arylamides in 20%± 93%yields.12 ± 14, 16, 18 VII.Structure and stereochemistry The stereoisomerism of molecules with a tricoordinated nitrogen atom is due to its pyramidal configuration.However, usual R=Et, Bu, C8H17, PhCH2. PhCON(Cl)OR 75 PhCON(OR0)OR PhCON(OAc)OR 76 (69%798%) AgOAc R0OH, H2O RCH2CNOR0 O 7HCl 7Cl+ R=Ph, R0=Me (a), PhCH2 (b), But (c); R=4-MeOC6H4, R0=Me (d); R=2-naphthyl, R0=Me (e); R=4-O2NC6H4, R0=Me (f); R=PhCO, R0=PhCH2 (g); R=PhCH2, R0=Me (h). EtOH RCH2CN(Cl)OR0 O Et3N EtOH RCH(OEt)CNHOR0 O 78a7f 77a7h R CH NOR0 O7 RCH CNHOR0 O7 Cl+ RCH(Cl)CNHOR0 O 79f,g N CCH2R OR0 O 80h N RCH2C R0O O 7Cl + R1=R3=R4=H, R2=Cl, Br, Me, MeCONH, NO2; R1=R2=MeO, R3=R4=H; R1=R3=R4=H, R2=NO2; R1=R4=H, R2=MeO, R3=Me; R1=H, R2=MeO, R3=R4=Me.R1 R2 R4 R3 + NOMe O 7H+ R1 R2 N R3 R4 OMe 82 (32%796%) O R1 R2 N(Cl)OMe R4 R3 81 O Ag2CO3, CF3COOH 7AgCl R=(CH2)nPh. 83 Ph(CH2)nCN(Cl)O(CH2)nPh O (CH2)n N COR 85 (n=2, 3) O (CH2)n N OR 84 (n=1, 2, 3) O 7AgCl Ag+ n=3 (a); 2 (b).ortho-attack ipso-attack 7H+ 85b H + N COR (CH2)n O + H (CH2)n O N COR 85a 7H+ + Ph(CH2)nONCR O H + N COR (CH2)n O Yield of 86 (%): 71 (n=1), 83 (n=2), 83 (n=3); Yield of 87 (%): 82 (n=2), 39 (n=3). + (CH2)n NOMe O Me O OMe (CH2)nCN(Cl)OMe O (CH2)n NOMe O O 86 Ag2CO3 CF3COOH OMe (CH2)nON(Cl)CMe O (CH2)n NCOMe O O 87 Ag2CO3 CF3COOH ArH=C6H6, PhMe, p-MeC6H4, PhOMe, C10H8, PhCOOMe.Ag+ 7AgCl RCN(Cl)OR0 +ArH O ArN(OR0)CR O Geminal oxygen ± nitrogen ± halogen systems. N-Halohydroxylamine derivatives 189N-alkylamines possess low configuration stabilities; the barriers to their pyramidal inversion (DG=) amount to 20 ¡À 30 kJ mol71.106 ¡À 108 An electronegative substituent at the nitro- gen atom increases the inversion barriers by inducing rehybridisa- tion of the bonding orbitals of the N atom accompanied by an increase in the p-character of the N¡ÀX s-bond and, correspond- ingly, by an increase in the s-character of the nitrogen lone pair orbital.106 ¡À 108 In addition, the planar transition state of the inversion is destabilised by the four-electron interaction between the lone pairs of the two linked heteroatoms.Both effects act in the same direction increasing the pyramidal stability. Indeed, the barriers to inversion in N,N,O-trialkylhydroxylamines 109 and in N-chloro- 110, 111 and N-fluoroamines 111 are as high as 40 ¡À 50, 38, and 63 kJ mol71, respectively. The presence of two heteroatoms at the nitrogen atom in the O¡ÀN¡À Hal system accounts for the substantially increased configurational stability of the cor- responding derivatives.In this case, the orbital nO ¡À s*N¡À Hal interaction also contributes to the stabilisation of the pyramidal configuration of the N atom.29, 34, 112, 113 The inversion barriers in the N-chloro-N,O-dialkylhydroxylamines 3a,c,g are 79 ¡À 83 kJ mol71.4 The inversion barrier predicted 4 for N-fluoro- N,O-dialkylhydroxylamines is 113 kJ mol71.When the N atom is incorporated in a ring, its pyramidal stability further increases, because in this situation, the change of the nitrogen hybridisation to sp2 in the planar transition state to inversion increases ring strain. Naturally, this effect is more pronounced in rigid bicyclic systems and in small rings. For instance, the inversion barrier in 2-chloro-1,2-oxazolidine 12 (DG==87 kJ mol71) 19, 20 is, on the average, 7 kJ mol71 higher than that in its acyclic analogues 3.4 The nitrogen inversion in 2-chloro- and 2-fluoro-substituted perhalo-1,2-oxazetidines 11 is also hindered under normal conditions,22 and the corresponding 4-monochloro-derivatives have been obtained as stable inverto- mers.23 The record-holding height of the barrier to the pyramidal inversion of nitrogen via a planar transition state (DH== 184.8 kJ mol71) was predicted for non-substituted 2-chloroxazir- idine by ab initio calculations in the 3 ¡À 21G basis set. 26 However, the barrier for 2-chloroxaziridine found experimentally based on the kinetics of racemisation was almost three times lower (DH== 63.9 kJ mol71, DS= = 43.5 e.u., and DG= = 132 kJ mol71 at 100 8C in n-heptane), and, besides, the rate of inversion increased by an order of magnitude on going to a polar solvent (n-buta- nol).26 This was explained by assuming that in this case, inversion occurs by a dissociative mechanism, because inversion via a planar transition state is characterised by low DS= values and slows down in polar solvents due to the more efficient solvation of the ground state, which is more polar (the calculation for 2-chlorox- aziridine in the same basis set predicts the values m=2.7 and 3.6D for the transition and ground states, respectively).The transition state to the dissociative inversion is represented by a contact ion pair, because ionic mobility of chlorine is not manifested in reactions of 2-chloroxaziridines 8a ¡À d and 9a.65 The driving force of ionisation is the orbital nO ¡À s*N¡À Cl interaction, which results, according to the 2-chloroxaziridine structure optimised in the 6 ¡À 31G* basis set, in a substantially lengthenedN¡À Cl bond.25 The introduction of a p-electron-withdrawing substituent to the nitrogen atom sharply decreases the barrier to its inversion, because p-conjugation is most efficient in the planar transition state.106, 107 In fact, the calculated inversion barrier for N-chloro- N-formyl-O-methylhydroxylamine is only 10.5 kJ mol71.34 For this compound, two other hindered processes can be expected, namely, amide rotation and rotation around the N¡ÀO bond with the maximum barriers of 47 and 55 kJ mol71, respectively.34 The latter is*10 kJ mol71 higher than the barriers to rotation around theN¡ÀObond inN,N,O-trialkylhydroxylamines.109 This increase in the barrier to the rotation around the N¡ÀO bond in N-chloro- N-formyl-O-methylhydroxylamine is due to the orbital nO ¡À s*N¡À Cl interaction, which makes the N¡ÀO bond `more double'.34 The height of this barrier is in good agreement with the experimental DG6��N¡¦O value, e to 58 kJ mol71, which was determined for N-chloro-N-alkoxyurea 55g by dynamic NMR.114 In N-chloro-N-alkoxyureas 55a,e,g, hindered rotation around the Me2N¡ÀCamide bond is also observed, the corresponding barriers being 60 ¡À 67 kJ mol71.114 There are no experimental data about the structures of N-hal- ohydroxylamines.The results of semiempirical and non-empirical calculations for 2-chloroxaziridine,25, 65, 115 N-chlorohydr- oxylamine,33 N-chloro-N-methylhydroxylamine,65 N-chloro-N-for- mylhydroxylamine,33 N-chloro-N-formyl-O-methylhydroxyl- amine,34 N-fluorohydroxylamine,116 ¡À 121 N,N-difluorohydroxyl- amine,119, 121 N,O-difluorohydroxylamine,119 ¡À 121 trifluorohydrox- ylamine,119, 121 and N,N-difluoro-O-fluorosulfonylhydroxyl- amine 122 demonstrate that their geometry is affected by the anomeric nO ¡Às*N¡ÀHal interaction, which is manifested as elonga- tion of theN¡ÀHal bonds and shortening of theN¡ÀObonds in these compounds.In addition, it was shown that the energetically preferred conformation for N-chloro- 33 and N-fluorohydroxyl- amines 116 ¡À 121 as well as for N-chloro-N-formylhydroxylamine 33 andN-chloro-N-formyl-O-methylhydroxylamine 34 is that in which the H¡ÀO (or C¡À O) and N¡ÀHal bonds are nearly perpendicular to each other and, hence, the anomeric nO ¡Às*N¡ÀHal interaction is the maximum (Fig. 2, conformation A). In the case of di- and trifluorohydroxylamines, conformation B (Fig. 2) has been predicted to be more stable; this conformation is stabilised by the nN ¡À s*O¡À F interaction, which is the most efficient in O-fluorohydroxylamine.VIII. Optically active cyclic N-chlorohydroxylamines The first optically active compound of this type, (+)-2-chloro-1,2- oxazolidine (+)-12, in which only the nitrogen atom was chiral, was prepared in a quantitative yield by cleavage of aminal (S)-(7)-88 by chlorine at778 8C.19, 20 However, the time needed for its half-racemisation at 13 8C is only 8 min.The high configurational stability of 2-chloroxazir- idines, the half-racemisation time of which at 20 8C is 833 years, made it possible to synthesise them in optically active forms by asymmetric chlorination and kinetic resolution.24, 25 R=MeO2C. (S)-(7)-88 778 8C Cl2, Et2O O N R R Cl (+)-12 +N R H Cl7 +CH2 N R H O N R R CCl is N-chlorocamphorimide; (+)-CCl O NH (2S)-(7)-8d Cl O N O X Y F O X Y Hal A B Figure 2.Newman projections along the O¡ÀN bond for N-halohydr- oxylamine derivatives XON(Y)Hal. A: X=Y=H, Hal=Cl, F; X=H, Y=HCO, Hal=Cl; X=Me, Y=HCO, Hal=Cl; B: X=H, Y=F; X=F, Y=H; X=Y=F. 190 V F Rudchenko, R G KostyanovskiiThe absolute configurations of 8d and 8e were determined by NMR and CD methods.25 The compound (5S)-(+)-8e was prepared as a mixture of two diastereoisomers in a ratio of 1.5 : 1, the absolute configuration of the predominant isomer being (2R,3S,5S).The sign of the Cotton effect of the long-wave band in the CD spectrum of optically active 2-chloroxaziridines was shown to be mostly determined by the absolute configuration of theNatom and to depend only slightly on the configurations of the carbon chiral centres.25 IX.Conclusion Analysis of the published data demonstrates that during the four decades of studies on the chemistry of N-halohydroxylamine derivatives, substantial progress has been achieved in this field. All main types of these compounds have been synthesised, namely, acyclic and cyclic N-halo-N,O-dialkylhydroxylamines, N-fluoro-N-alkoxy-O-alkylhydroxylamines, derivatives of N,N- difluorohydroxylamine, and N-halo-N-alkoxyamides.It was found that N-halo-O-alkylhydroxylamines and N-chloro-N- alkoxy-O-alkylhydroxylamines are thermally unstable under ambient conditions; nevertheless, they can be involved in situ in various chemical reactions. The halogen atom in most of the systems containing the O±N± Hal triad can be easily removed as the anion, which is due to the hyperconjugation nO ± s*N± Hal.Hence, N-chloro-N- tert-alkyl- and N-chloro-N-alkoxy-O-alkyl-hydroxylamines are powerful alkoxyaminating reagents. A fundamentally important result obtained along this line is the discovered possibility of nucleophilic substitution of chlorine in `N-chloramides': N-chloro-N-alkoxyureas and N-chloro-O- alkylhydroxamates the chlorine atom of which, unlike that in their N-alkyl analogues, possesses anionic mobility.Under the action of Lewis acids, N-chloro-O-alkylhydroxamates form highly reactive intermediates, N-acyl-N-alkoxynitrenium ions, capable of reacting with aromatic compounds in intermolecular and intra- molecular processes of elecrophilic aromatic substitution. Compounds containing O±N± Hal fragments belong to one of the few types of derivatives characterised by high pyramidal stability of the nitrogen atom.As a result, 2-chloroxaziridines and 2-chloro-1,2-oxazolidine derivatives have been partially resolved into optical antipodes with an asymmetric N atom. The orbital nO ± s*N± Hal interaction in O±N± Hal systems accounts for the predominant existence of the conformation with nearly anti-periplanar p-orbital of the oxygen atom in relation to the N± Hal bond.N-Amino-substituted derivatives of N-halo-O-alkylhydroxyl- amines remained beyond the scope of this review, because, similarly to N-halohydrazines, they exist entirely as alkoxydi- azenium salts R2N+=NOR Hal7.123 These compounds act as aminoaminating reagents and undergo O-dealkylation upon treatment with nucleophiles [see Scheme 1, pathways (a) and (b)].The vicinal orbital interaction considered for the O±N± Hal system and related anomeric effects are typical properties of many other geminal systems, for example, N±E ± Hal and O± E ± Hal, where E=C, N, O, P, S. The review was supported by the Russian Foundation for Basic Research (Project No. 97±03±33021) and the INTAS (Grant No. 94±2839). References 1. M Murakami, T Nashima Mem. Inst. Sci. Ind. Res. Osaka Univ. 10 175 (1953); Chem. 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(Engl. Transl.) c�Chem. Heterocycl. Compd. (Engl. Transl.) 192 V F Rudchenko, R G Kostyan

ISSN:0036-021X    

出版商:RSC    

年代:1998

数据来源: RSC

摘要:

Abstract. Data on the methods of synthesis, structures, and reactivity of organic derivatives of monocoordinated tellurium, namely, compounds with C=Te bonds and phosphine tellurides R3P=Te, are described systematically and generalised. The use of the latter compounds as synthetic equivalents of elemental tellu rium for the preparation of metal tellurides is considered. The bibliography includes 139 references.I. Introduction Organic derivatives of monocoordinated tellurium, namely, com- pounds with C=Te bonds and phosphine tellurides R3P=Te, constitute a relatively little studied class of organotellurium compounds. In fact, the first phosphine telluride, tributylphos- phine telluride, was synthesised in 1963,1 while the first and still the only one known stable telluroketone, 1,1,3,3-tetramethylin- danetellone, which is stable only in solution, was synthesised only in 1993.2 The synthesis, structure, and chemical properties of tellurocarbonyl compounds and phosphine tellurides have been considered most comprehensively in a monograph devoted to the chemistry of organic derivatives of selenium and tellurium.3, 4 However, during more than a decade, which has passed after the publication of this monograph, the number of studies dealing with the derivatives of monocoordinated tellurium has approximately doubled. New fields of application of tellurocarbonyl compounds have been discovered, for example, as dienophiles in the syntheses of six- and seven-membered tellurium-containing heterocycles.Of special interest is the use of phosphine tellurides as synthetic analogues of elemental tellurium for the synthesis of some organo- tellurium compounds, which undergo thermal decomposition under relatively mild conditions to give metal tellurides 5 possess- ing semiconductor and other valuable properties.II. Synthesis and reactions of compounds containing C=Te bonds Up to now, four types of tellurocarbonyl compounds have been described in the literature: telluroketones (telluroaldehydes) R2C=Te (R=H, Alk, or Ar), tellurocarboxylic acids RC(=Te)OH, O-alkyl tellurocarboxylates RC(=Te)OR1, and telluroamides RC(=Te)NR1R2 and their analogues (tellurohy- drazides and telluroureas).These compounds sharply differ in stability. Only representatives of the two latter types have been isolated in a pure state.The formation of telluroketones (telluro- aldehydes) and tellurocarboxylic acids is judged either from the NMR spectra of reaction mixtures or from the products of their chemical transformations. However, the stability of tellurocar- bonyl compounds of all these types is substantially enhanced when the tellurocarbonyl carbon atom carries bulky organic substitu- ents or relatively strong p-electron-donating groups (OR, NR2, NR1NR22 ) or upon coordination to transition metal compounds.It should be noted that one of the most efficient methods used to increase the stability of other classes of organotellurium deriva- tives, such as tellurenyl halides, azides, acetates, and isothiocya- nates, diorganyl tritellurides, etc., has not been used so far in the chemistry of tellurocarbonyl compounds.This method consists in the introduction, into appropriate positions of aryl or alkenyl substituents of functional groups (CHO, COR, CO2R, NO2, N=N, CH=N, CH2NMe2, etc.) containing sp2 (or, more rarely, sp3) hybridised O or N atoms, which form strong intramolecular coordination bonds with Te atoms.6 1. Telluroaldehydes and telluoroketones Despite the apparent similarity of chalcogens (S, Se, Te),7 their properties are appreciably different; therefore, in some cases, methods that are used successfully for the synthesis of organic derivatives of sulfur and selenium cannot be used to prepare their tellurium analogues.For example, `dialkyl telluroketones' have been described in a study,10 which is cited in monographs dealing with the chemistry of organotellurium compounds.8, 9 They were synthesised by a method similar to a method used to prepare thioketones, namely, by the reaction of the corresponding ketones with H2Te in a strongly acidic medium.However, in reality, the reactions of H2Te, generated in situ from aluminium telluride and water, with aldehydes and ketones afford alcohols 11 or mixtures of dialkyl ditellurides with alcohols.12 Alcohols are formed in 50%± 100% yields when H2Te is passed through solutions of carbonyl compounds in THF.11 When solutions of aldehydes contain strong acids (CF3COOH, PhSO3H, H2SO4), dialkyl ditellurides 1 are formed as the main reaction products (yields 34%± 56%); in this case, only traces of primary aliphatic alcohols are formed.12 Ketones react in a similar way, but the yields of the corresponding dialkyl and di(cycloalkyl) ditellurides are lower.12 I D Sadekov, A A Maksimenko, V L Nivorozhkin Research Institute of Physical and Organic Chemistry of the Rostov State University, prosp.Stachki 194/2, 344090 Rostov-on-Don, Russian Federation. Fax (7-863) 228 56 67. Tel. (7-863) 228 08 94 (I D Sadekov) Received 3 June 1997 Uspekhi Khimii 67 (3) 219 ± 235 (1998); translated by Z P Bobkova UDC 283.283.4+547.286.4+547.1 118 Organic derivatives of monocoordinated tellurium I D Sadekov, A A Maksimenko, V L Nivorozhkin Contents I.Introduction 193 II. Synthesis and reactions of compounds containing C=Te bonds 193 III. Synthesis, reactions, structures, and spectroscopic characteristics of phosphine tellurides R3P=Te 200 IV.Conclusion 206 Russian Chemical Reviews 67 (3) 193 ± 208 (1998) #1998 Russian Academy of Sciences and Turpion LtdAccording to the scheme proposed by the researchers cited,12 the ditellurides 1 result from the reduction of telluroaldehydes 2 with hydrogen telluride. The influence of pH on the outcome of these reactions is due to the fact that in strongly acidic media the acid ± base equilibrium 3 4 is almost entirely shifted to the right: a.Stable telluoroketones As noted above, 1,1,3,3-tetramethylindanetellone 5 is the only stable, although only in solution, telluroketone that has been characterised by various spectroscopic methods.2 This compound was obtained in an almost quantitative yield, together with alkenes 6a and 6b, upon thermolysis of 1,3,4-tellurodiazoline 7; the compound 7 was synthesised in 26% yield by treating the hydrazone of 1,1,3,3-tetramethylindanone with tellurium dichlor- ide in the presence of Et3N.13 The telluroketone 5 is very sensitive to oxygen and light; on exposure to UV radiation, it is converted into a mixture of ditelluride 8 and 1,1,3,3-tetramethylindanone.2 The 13C NMR signal for the C=Te-group carbon atom (d 301 ppm) is substan- tially shifted downfield with respect to the signals for other C=X groups (C=O, d 226.0 ppm; C=S, d 282.1 ppm, C=Se, d 294.0 ppm).The same is true for the 125Te signal (d 2858.3 ppm in relation to Me2Te), which is the lowest-field signal in the series of organotellurium compounds known. The long-wave absorption band (l=825 nm) in the UV spectrum of the ketone 5 undergoes substantial bathochromic shift with respect to the bands of the corresponding selenium (l=667 nm) and sulfur (l=527 nm) derivatives.The compound 5 enters into 1,3-dipolar cycloaddition with mesitylnitrile oxide yielding a derivative of a new heterocyclic system, oxatellurazole 9. Like other tellurocarbonyl compounds (see below), the ketone 5 enters into the Diels ± Alder reaction with 2,3-dimethylbutadiene 2 to give compounds 11 and 8 in addition to 1-telluracyclohex-3-ene derivative 10.The complex 12 was synthesised in a yield of more than 70% by the reaction of the telluroketone 5 with the complex (CO)5W. THF.14 Earlier, complexes of this type had been obtained only by insertion of a Te atom into C=M bonds in transition metal complexes.The compound 12 was isolated as dark-violet crystals. Unlike the initial telluroketone, it is resistant to heating and exposure to light. The signals for the C=Te-group atoms in the 13C and 125Te NMRspectra are shifted upfield with respect to the corresponding signals in the spectra of 5; they are manifested at d 285 and 1783 ppm, respectively.The C=Te bond length in the complex 12 found by X-ray diffraction analysis 14 amounts to 1.987A, which is close to the length of the similar bond in S=C=Te (1.904A); 15 this indicates that the order of the C=Te bond in 12 is closer to two. The telluroketone 5 is a relatively weak donor; in fact, when the complex 12 is heated in MeCN at 60 8C, the tellurium- containing ligand is quantitatively replaced by MeCN. b.Methods for the generation of unstable telluroaldehydes and telluoroketones. Dimerisation and the Diels ± Alder reaction All the telluroaldehydes and telluoroketones, except for the ketone 5, are unstable. Conclusions about the formation of these compounds are based on the structures of products of their subsequent chemical transformations, first of all, dimerisation (or trimerisation in the case of telluroformaldehyde) and the Diels ± Alder reaction.Telluroformaldehyde was the first telluroaldehyde reported in the literature. It was obtained by treatment of tellurium mirrors with the carbene generated by thermal decomposition of diazo- methane 16 ± 18 or by photodissociation of ketene.17 On cooling, the gaseous monomeric telluroformaldehyde trimerises to yield 1,3,5-tritelluracyclohexane 13.RCHO H2Te, H+ H2Te (RCH2)2Te2+RCH2OH RCH2OH 1 7 3 RCHO HTe7 R CH TeH O7 R CH Te7 OH 7Te +H+ 7H+ R CHOH H+ RCH2OH R CH TeH OH 4 7H2O R CH Te 2 H2Te 7Te (RCH2)2Te2 1 CDCl3 D N N Te 7 5 Te N2 + 7N2 5 hn, O2 H 2Te2 O + 8 + CH2 6b 6a Te N Te O Ms 9 Te + 8+ 10 5 MsC O7 + Te H 11 N 5 Te +(CO)5W.THF 7THF W(CO)5 Te 12 MeCN 60 8C (CO)5W.MeCN +5. 194 I D Sadekov, A A Maksimenko, V L NivorozhkinThe structure of the heterocyclic compound 13 was confirmed by IR spectroscopy and mass spectrometry.18 The reactions of the compound 13 with bromine or iodine give the corresponding dihalomethanes.17 Apart from the trimer 13, telluroformaldehyde can also exist as a polymeric form, poly(methylene telluride) 14.Disodium methaneditellurolate resulting from reduction of poly(methylene ditelluride) 15 19 with sodium tetrahydridoborate in ethanol dis- proportionates into sodium telluride and telluroformaldehyde, which then polymerises to give the polytelluride 14.20 It has been suggested21 that the synthesis of poly(methylene telluride) occurs according to a scheme which includes reduction of the polyditelluride 15 with sodium tetrahydridoborate in methanol and alkylation of sodium methyleneditellurolate with dibromo(or diiodo)methane.In our opnion, this mechanism is erroneous. We believe that the polymer 14 results from dispro- portionation of the dianion as described above rather than from the alkylation. Although in recent years several methods for the generation of telluroaldehydes and telluroketones have been developed, it is fairly difficult to judge their synthetic potential, because their chemical properties have not been extensively studied.The most efficient method for the preparation of telluroaldehydes 2 and telluroketones 16 is the reaction of bis(dimethylaluminium) tel- luride with aldehydes or ketones in boiling dioxane.22, 23 The driving force of these reactions is the formation of thermodynami- cally favourable Al ±O bonds.The tellurocarbonyl compounds 2 and 16 formed initially can be trapped by 2,3-dimethylbutadiene or cyclopentadiene; this gives Diels ± Alder adducts 17 ± 19 in 44%± 62% yields.22 These reactions are the only methods known to date for the synthesis of 1-telluracyclohex-3-enes 24 and 2-tellurabicyclo[2.2.1]hept-5-enes.The formation of the telluroketone 16c is indicated by the blue colouration of the solution. Owing to the low stability of this compound, it was impossible to determine its spectral character- istics. Unlike the sulfur and selenium analogues which form complexes with pentacarbonylchromium and -tungsten, the reac- tion of the ketone 16c with (CO)5M.THF (M = Cr, Mo, W) occurs as detelluration and affords tetraferrocenylethylene in a quantitative yield.23 In the absence of dienes, the ketone 16a dimerises to give 1,3- ditelluretane derivative 20 (yield 28%);22 reactions of this type for other ketones have not been reported.It is noteworthy that the first ditelluretanes, cis- (21a) and trans- (21b) 2,4-dibenzylidene-1,3-ditelluretanes (the structure of the trans-isomer was confirmed by X-ray diffraction analysis 25), were obtained in low yields upon protonation of sodium phenyl- ethynyltellurolate with a solution of HCl in ether.25, 26 It has been reported 27 that ditelluretane 21a and cis-3,5-dibenzylidene-1,2,4- tritellurole 22 are the main products of this reaction; the structure of the latter was determined by X-ray diffraction analysis.27 The protonation of sodium phenylethynyltellurolate with CF3COOH affords a mixture consisting of 21a and cis- and trans-2,6- diphenyl-1,4-ditellurafulvalene, 23a and 23b, respectively.28 Apparently, these reactions involve intermediate formation of a telluroketone, which either dimerises yielding the compounds 21 or 23, or reacts with phenylethynylditellurolate anion being thus converted into the tritellurole 22.27 Tellurium-containing heterocycles have also been synthesised from tellurocarbonyl compounds generated by other methods.One of these methods, which has been used previously in the chemistry of selenoaldehydes,29 is based on the reaction of benzylidenetriphenylphosphorane with powdered tellurium in boiling toluene.30 The resulting tellurobenzaldehyde 2a is trapped by 2,3-dimethylbutadiene to give the heterocyclic derivative 17a in a yield of only 11%.This way of generation of telluroaldehydes is much less efficient than the method described above in which bis(dimethylaluminium) telluride acts as the tellurating reagent. CH2+Te H2C Te Te Te Te 13 Na2Te+ CH2 Te ( CH2 Te )n . 14 CH2 Te Te NaBH4 n H2C 15 TeNa TeNa R1=H; R2=Ph (2a), Pr (2b), But (2c);22 R1R2C O +(Me2Al)2Te 7(Me2Al)2O R1R2C Te 2a7c; 16a7c R1, R2= (16a), (16b);22 R1=R2=C5H5FeC5H4 (16c).23 R=Ph [17a, (49%)]; Pr [17b, (44%)]; But [17c, (62%)]. Te R H + Me Me Te Me Me R 2 17a7c Te Te 16a 18 (55%) + Te + Te 16b 19 (52%) Te Te Te 16a 20 PhC CTeNa H+ PhC CTeH C Te H Ph Te Te Ph H H Ph Te Te Ph H Ph H + 23a 23b 21b Te Te H Ph Ph H Te Te Ph H Ph H + 21a Te Te Te Ph H H Ph 22 PhC C Te Te7 Organic derivatives of monocoordinated tellurium 195In view of the fact that phosphine tellurides (see below) are efficient synthetic analogues of elemental Te, it would be of interest to check whether these compounds could be used in this reaction instead of elemental Te.The reaction of the phosphorane with an equimolar or catalytic amount of tellurium in the absence of a diene affords only trans-stilbene in a yield of more than 60%.30 Yet another way of generating telluroketones, which has also been borrowed from the chemistry of organoselenium deriva- tives,31 consists in the elimination of HCN from tellurocyanate 24.32 Hydrolysis of this compound under anaerobic conditions gives rise to a mixture of the anti- and syn-isomers of 1,3- ditelluretane 26a and 26b (yields 45% and 6%, respectively) resulting from dimerisation of the intermediate telluroketone 25.One more approach to the generation of tellurocarbonyl compounds reported in the literature, which is not associated with their formation from transition metal complexes, has been studied in relation to tellurocarbonyl difluoride 27.33, 34 This compound is formed in a fairly low yield upon treatment of bis(trifluoromethyltelluro)mercury with diethylaluminium iodide and rapidly dimerises to give 2,2,4,4-tetrafluoro-1,3-ditelluretane 28a.33, 34 The telluretane 28a was obtained in higher yields (50% ± 60%) by pyrolysis of trifluoromethyltellurotrimethylstannane.34 Co-condensation of the compound 27 with selenocarbonyl difluoride results in the formation of 2,2,4,4-tetrafluoro-1- selena-3-telluretane 29.34 The reaction of the compound 27 with 2,3-dimethylbutadiene gives 6,6-difluoro-3,4-dimethyl-1-tellura- cyclohex-3-ene 30.This heterocyclic derivative was prepared in 48% yield by heating trifluoromethyltellurotrimethylstannane with 2,3-dimethylbutadiene in a sealed tube.The synthesis of dichloro- and dibromotelluretanes 28b,c by treatment of the tetrafluoride 28a with boron halides also deserves attention.34 c. Telluration of transition metal complexes Yet another method for stabilisation of telluroaldehydes and telluroketones is the formation of complexes with transition metal compounds.These complexes, unlike the complex of telluroketone with pentacarbonyltungsten (12) described above,14 are formed upon interaction of metal complexes of various structures with tellurium-containing nucleophiles or ele- mental tellurium, TeCN7 and HTe7 being usually employed as the tellurium-containing nucleophiles. For example, complexes 32a 35 and 32b 36 have been obtained in 18% and 12% yields, respectively, by the reaction of complexes 31 with TeCN7.The complex 32a is thermally fairly unstable and is readily converted into binuclear complex 33 (the yield given above refers to 33). The complexes 32 enter into reactions typical of tellurocar- bonyl derivatives, in particular, into the Diels ± Alder reaction, i.e. they behave similarly to `free' tellurocarbonyl compounds.Their reaction with 2,3-dimethylbutadiene leads to pentacarbonyltungs- ten complexes of 1-tellura-3,4-dimethylcyclohex-3-enes 34a,b in 12%37 and 8%36 yields, respectively. It has been reported 37 that the reactivity of complexes of the (CO)5W±X=CPh2 type, where X=S, Se, and Te, sharply increases in the series S<Se<Te. In fact, whereas the reaction of 32b with 2,3-dimethylbutadiene at 725 8C is completed over a period of 45 min, the reaction of the selenium analogue with the same diene requires 10 hours at room tempersture.Finally, pentacarbonyl(diphenylthione)tungsten does not react with this diene at all under these conditions. The complex 34a exists in solution as an equilibrium mixture of two conformers in which the Ph group occupies an equatorial position.36 The reactions of the complexes 32 with cyclopentadiene occur faster than with 2,3-dimethylbutadiene and afford tungsten com- plexes of 2-tellurabicyclo[2.2.1]hept-5-enes 35a,b 36, 37 in 31% and 24% yields, respectively.The formation of the complex 35a is highly stereoselective: the ratio of the exo- and endo-isomers is 11 : 1.36 The structure of the major isomer, exo-35a, was estab- lished by X-ray diffraction analysis.36 Ph3P CHPh +Te 7Ph3P PhCH Te Me Me 17a 2a TeCN OSiMe3 H2O 7HCN Te O 24 25 O Te Te O O O Te Te + 26a 26b (CF3Te)2Hg+Et2AlI CF3TeSnMe3 7HgI2, Et2AlF 7Me3SnF D F2C Te 27 F2C=Se +28a+ Se Te F F F F 29 Se Se F F F F Te Te F F F F 28a BX3 Te Te X X X X 28b,c X =Cl (b), Br (c) 27 Te Me Me F F 27 30 Me Me R=H (a), Ph (b).W OC CO CO OC CO +TeCN7 7CN7 W OC CO CO OC CO R=H 31a,b 32a,b OC CO CO OC CO 33 C R Ph Te C Ph R Te C Ph H W(CO)5 W R=H (a), Ph (b). Ph Ph + Me Me Te Ph R Me Me (CO)5W 32a,b 34a,b (CO)5W Te C 196 I D Sadekov, A A Maksimenko, V L NivorozhkinReactions of the complex 32b with 1-diethylaminoprop-1-yne and bis(diethylamino)acetylene have also been studied.38 They occur as [2+2]-cycloaddition followed by electrocyclic ring open- ing and give rise to telluroacrylamide complexes 36a,b.38 The complex 36b decomposes on prolonged heating in an ethereal solution under a CO atmosphere to give W(CO)6, Te, and Et2NCH=C(NEt2)CHPh2.Metal complexes have also been used to stabilise chalcogeno- ketenes including telluroketenes.39 Complexes 38 as well as their sulfur and selenium analogues have been synthesised in relatively high yields (68% ± 87%) by the reaction of vinylidene rhodium complexes 37 with chalcogens in benzene.Methylation of 38 (R= H) with methyl triflate and subsequent treatment with NH4PF6 results in the formation of an ionic complex 39.39 Finally, we would like to mention the synthesis of tellurocar- bonyl complex 41a (X = Te) in which the C=Te group acts as a carbonyl ligand.40 The complex 41a as well as its sulfur (41b) and selenium (41c) analogues were obtained as crystals, stable in air, on treatment of osmium dichlorocarbene complex 40 with HX7 anions (X=S, Se, Te).The n(C=X) absorption frequencies in the IR spectra of the complexes 41a ± c regularly decrease in the order S (1315 cm71)> Se (1156 cm71)>Te (1056 cm71).40 The tellurium-containing groups in the complexes 32 and 38 are bound to metal atoms either through the Te atom or by the C=Te double bond, i.e.these compounds are Z1-complexes. At the same time, a number of heterobinuclear m-alkylidene com- plexes of the dimetallacyclopropane type containing C and M in three-membered rings have also been described.Complexes of this type, for example, 43 and 45, have been prepared as blue crystalline compounds by treatment of the manganese complexes 42 41 or 44 42 with diazoalkanes.41, 43 The reactions of osmium complexes 46 44 and 47 45 with hydrogentelluride anions 44 or elemental tellurium 45 resulted in the formation of telluroformaldehyde complexes 48a,b. The reaction of the complex 48a with HCl involves the Os ±CH2 bond and gives rise to complex 49.43 In the reaction with methyl iodide, the attack is directed at the tellurium atom; the cationic complex 50 thus formed is easily reduced by sodium tetrahydridoborate with opening of the three-membered ring to give complex 51.44 Rhodium complexes 53 containing rigid three-membered rings have been synthesised in relatively low yields by reaction of rhodium complexes 52 with sodium hydrogentelluride.46, 47 Te (CO)5W R Ph Te Ph R (CO)5W + endo-35a,b exo-35a,b R=H (a); Ph (b).(CO)5W Te C R Ph 32a,b + R=Me (36a), NEt2 (36b). 32a,b + RC CNEt2 36a,b (CO)5W Te C NEt2 C R CPh2 (CO)5W Te C Ph Ph R=H, Me, Ph. Rh C Pri 2P C H R Te Rh Pri 2P C C H R Te R =H 1. CF3SO3Me 2. NH4PF6 37 38 Rh Pri 2P C CH2 MeTe + PF6 7 39 X=Te (a), S (b), Se (c). Os PPh3 PPh3 Cl CO CCl2 Cl HX7 Os PPh3 PPh3 Cl CO CX Cl 40 41a7c R=H, Me; Cp* is C5Me5 .Cp*(CO)2Mn Te Mn(CO)2Cp*+R2C N2 7N2 42 Cp*(CO)2Mn Te Mn(CO)2Cp* CR2 43 [Cp(CO)2Mn]3Te+CH2N2 7Cp(CO)2Mn 7N2 44 Cp(CO)2Mn Te Mn(CO)2Cp CH2 45 Cp is C5H5. Os PPh3 PPh3 L2 L1 CH2 Te HTe7 Te 48a,b L1=L2=CO (46, 48a); L1=NO, L2=Cl (47, 48b).Os PPh3 PPh3 L2 L1 CH2 47 Os PPh3 PPh3 L2 L1 CH2I I 46 48a Os PPh3 PPh3 OC OC CH2 Te HCl MeI 50 Os PPh3 PPh3 OC OC CH2 TeMe + I7 Os PPh3 PPh3 OC OC Me TeMe 49 Os PPh3 PPh3 OC OC Cl TeMe NaBH4 51 L=Me3P,46, 47 Pri 3P,47 CO.47 Cp(L)Rh CH2I+NaTeH I 7NaI Cp(L)Rh Te CH2 52 53 Organic derivatives of monocoordinated tellurium 1972. Telluronic acids The synthesis and some reactions of tellurocarboxylic acids have been described only quite recently.These acids were prepared by acidolysis of the corresponding cesium tellurocarboxylates 54 with an ethereal solution of HCl.48 At low temperatures in a polar solvent like THF, they exist mostly as the telluoroxo form 55 (telluronic acids).48 The structure of the acids 55 has been confirmed by their spectral characteristics. The 1H and 13C NMR spectra of these compounds recorded at790 8C contain no signals corresponding to the TeH and C=O groups present in the isomeric tellurolic acids 56.The singlets observed at d 16.02, 16.48 and at d 222.9, 223.1 ppm were attributed to the OH and C=Te fragments of the acids 55a and 55b, respectively. When the temperature was raised to 770 8C, the signals mentioned above were markedly broad- ened; this was attributed 48 to a dynamic exchange process involv- ing the formation and rupture of hydrogen bonds (Te ±H_O,O± H_O) in the above equilibrium.The telluroxo structure of these compounds is also supported by the magnitudes of 125Te chemical shifts (d 952 ppm for 55a and d 1024 ppm for 55b). The acids of the aromatic series are coloured dark-green (lmax = 652 and 673 nm for 55a and 55b, respectively), whereas tellurotrimethylacetic acid 55c is blue-violet (lmax=594 nm).The isomeric tellurobenzoic Te-acid 56a was prepared by treatment of the salt 54a with liquid hydrogen chloride at 7195 8C followed by raising the temperature to 790 8C. At 7110 8C, the dark-brown salt 54a was converted into the solid yellow acid 56a, which is soluble in toluene at790 8C.The signal observed in the 125Te NMR spectrum of this compound at d 535 ppm was assigned to the TeH group. When THF was added to the resulting solution, the acid 56a was converted into the isomeric acid 55a.48 Tellurocarboxylic acids of both types are unstable at ambient temperature and are very sensitive to oxygen; on exposure to air even at 790 8C, they immediately decompose with liberation of elemental Te.The tellurolic acids 56 are appreciably less stable than telluronic acids 55.48 Tellurocarboxylic acids 55a,b react with aryl isocyanates in THForCH2Cl2 at770 8Cto give stable acyl carbamoyl tellurides 57a ± c, their yields exceeding 80%.48 The synthesis of free acids from their salts proved to be possible only for cesium salts.49 These salts, which are relatively stable (for R = Ar) under an inert atmosphere, as well as their rubidium analogues, have been prepared in 40% ±60% yields by the reactions of O-trimethylsilyl tellurocarboxylates 58 (see Sec- tion II.3) with cesium and rubidium fluorides.49 The presence of a carbonyl group in the compounds 54 was confirmed by an X-ray diffraction study of Te-cesium 2-methoxy- tellurobenzoate 54a.49 It should be noted that salts of type 54 containing lighter alkali metal cations, that is, lithium,50 sodium 51, 52 and potassium,53 have been synthesised earlier using one of the reactions presented below: However, it was impossible to carry out X-ray diffraction analyses of these salts, because they were sensitive to oxygen and thermally unstable and, hence, it was difficult to purify them.The same features precluded preparation of the corresponding acids from these salts. 3. O-Alkyl and O-trimethylsilyl tellurocarboxylates O-Alkyl tellurocarboxylates 59 were the first representatives of organotellurium compounds with the C=Te bond to be isolated in a pure state. These compounds were prepared by Barton et al.54, 55 by the reaction of (1-chloro-2,2-dimethylpropyl- idene)dimethylammonium chloride 60 with alcohols and sodium hydrogentelluride, synthesised from NaBH4 and Te in ethanol in the absence of air and light.The yields of the compounds 59 varied over wide limits, namely, from 6% in the case of 59b to 91% for 59c. Evidently, the higher stability of the compound 59a is due to the presence of bulky tert-butyl groups in their molecules.In fact, when the analogous phenyl derivative is used instead of the chloride 60, the reactions with alcohols and sodium hydrogentelluride afford the corresponding benzyl ethers as the final products. Telluro- benzoates 61 were postulated as intermediate compounds.56, 57 Since these reactions occur under relatively mild conditions, it was proposed 56, 57 to use them for protecting hydroxy groups in carbohydrates and other compounds, labile in alkaline media.R=4-MeOC6H4 (a), 4-MeC6H4 (b), Me3C (c). O TeCs R 54a7c Te OH R 55a7c 54a HCl 71958C HCl 790 8C O TeH 4-MeOC6H4 THF 55a 56a R=4-MeOC6H4: R1=Ph (57a), 4-MeC6H4 (57b); R=4-MeC6H4: R1=Ph (57c). Te OH R +R1 N C O 770 8C O Te R NHR1 O 55a,b 57a7c Te OSiMe3 R +MF 58 M=Cs, Rb; R=Me, But, Ph, 2-MeC6H4, 4-MeC6H4, 2-MeOC6H4, 4-MeOC6H4, 4-ClC6H4.MeCN, 20725 8C, 30 min 7Me3SiF O Te7 M+ R 54 M=Li, Na. M=Na, K. RCOCl+M2Te 7MCl O Te7M+ R O Te R R O +EtOM 7RCOOEt O Te7M+ R R=CH2But (a), (7)-menthyl (b), adamantylmethyl (c), 5a-cholestan-3b-yl (d), D9-tigogenyl-3 (e). 7NaCl, 7Me2NH. HCl Te OR But 59a7d + Me2N C But Cl Cl7+ROH+NaTeH 60 + Cl7 Me2N C Cl Ph 7NaCl, 7Me2NH.HCl +ROH+NaTeH 61 Te RO Ph HTe7 H+ RO C Ph Te7 RO CH Te Ph 7Te ROCHPh ROCH2Ph. 198 I D Sadekov, A A Maksimenko, V L NivorozhkinThe compounds 59 are coloured violet, which is due to absorption of the tellurocarbonyl chromophore with lmax= 584 ± 596 nm; the 13C NMR signals for the carbon nuclei of the C=Te group occur at d 229 ppm.54, 55 The esters 59 are quite stable to atmospheric oxygen in the dark.However, they are readily oxidised by phenylseleninic anhydride; in the case of 59d, the ester of the corresponding carboxylic acid is formed in an almost quantitative yield.54, 55 The reaction of the telluroesters 59 with excess sodium hydro- gentelluride initially yields a mixture of oligotellurides 62 and finally gives esters 63 and 64.The formation of these compounds was explained in terms of the scheme given below.55 The silyl esters 58 represent yet another type of telluroesters with C=Te bonds. They have been synthesised by reactions of bis(acyl) tellurides 65 with bis(trialkylsilyl) tellurides 66 [pathway (a)], of acyl chlorides with equimolar amounts of bis(trimethyl- silyl) tellurides [pathway (b)],58 ± 61 and of sodium tellurocarbox- ylates with trialkylchlorosilanes [pathway (c)].52 Compound R R03 Pathway a Me Me3 a, b b But Me3 a, b c Me PriMe2 a d 2-MeC6H4 Me3 c e 2-MeC6H4 ButMe2 c f Ph ButMe2 a The compounds 58 gradually decompose in solutions with liberation of tellurium giving mixtures of E- and Z-isomers of alkenes 68 58 ± 61 (when R = R0 = Me, the E: Z ratio amounts to 55 : 45 57).The stability of the esters 58 increases with increase in the size of the substituents R and R0. For example, in the reaction of bis(acetyl) telluride with bis(trimethylsilyl) telluride, the mix- ture of alkenes 68a is formed in almost quantitative yield over a period of 30 min, whereas the reaction of bis(acetyl) telluride with bis[(dimethyl)isopropylsilyl] telluride giving rise to a mixture of the alkenes 68c is completed only over a period of 12 h.58, 61 Te-Silylated derivatives 67 are the precursors of the esters 58; they either completely isomerise into O-silylated esters 58 (for R=Ar 52) or exist as equilibrium mixtures with them.Thus the reaction of pivaloyl chloride with silyl telluride 66 (R1 = Me) affords an equilibrium mixture of the compounds 67b (35%) and 58b (65%),60 as indicated by the spectral parameters of the blue liquid formed in this reaction (lmax=624 nm58, 60).The 13C NMR spectrum exhibits signals at d 207.5 (C=O) and d 251.2 ppm (C=Te); the 125Te NMR signal corresponding to the compound 58b is manifested at d 1418 ppm.60 Similarly to telluroaldehydes and telluroketones (see Section II.1), the esters 58 enter into [2+4]-cycloaddition to 2,3-dime- thylbutadiene and cyclopentadiene giving the corresponding tellurium-containing heterocycles.60 4.Telluroamides and their analogues The first telluroamides and tellurohydrazides 71 were synthesised at about the same time as telluroesters, in 1979.62 They have been prepared similarly to selenium analogues,63 namely, by treating the salts 70 with the hydrogentelluride anion 69 generated from Et3NandH2Te in CH2Cl2.62 The yields of the compounds 71 were low (5% ± 19%).Telluroamides have also been synthesised from bis(dimethy- laluminium) telluride, which had been used previously to prepare telluroaldehydes and telluroketones. 22, 23 (see Section II.1). Treat- ment ofN-methylformanilide with this tellurating reagent resulted in the synthesis of telluroformanilide 71d in a yield of about 90%.64 However, other telluroformanilides could not be isolated because they decomposed during purification. The compounds 71 are stable at room temperature in the absence of water.They are rapidly hydrolysed in moist air or in wet solvents to give the corresponding amides and elemental tellurium resulting from fast oxidation of H2Te.62 The hydrolysis is catalysed by alumina and silica gel; this apparently accounts for the fact that other telluroformanilides could not be isolated.The relatively high stability of telluroamides (hydrazides) 71a ± c is caused by the presence of strong electron-donating substituents Te OR But +[PhSe(O)]2O 7Ph2Se2, 7Te O OR But 59d RO CH But Te NaHTe (RO CHBut 2)Ten 62 (n=174) 7Te RO CH But ROCH2But 63 Te RO But 59 59+ROCHBut RO CH But C But RO Te 7Te RO CH But C But RO RO CH But CH RO But 64 OSiR03 R R03SiO R R OSiR03 R03SiO R + (E)-68a ë f (Z)-68a ë f (RC)2Te+(R03Si)2Te O 65 66 RCOTeNa +R03SiCl RCOCl+(Me3Si)2Te a b c RCOTeSiR03 67a7f RCOSiR03 Te 58a7f Te Me Me But Me3SiO ButC OSiMe3 Te 58 Me Me Te But OSiMe3 R1=Ph: R2=R3=Me (a); R2=Me, R3=NMe2 (b).R1C SMe NR2R3 + I7 +[Et3NH]+HTe7 Ar, 740 8C R1C NR2R3 Te 70a,b 71a,b 69 NMe NMe I7 4-ClC6H4 +69 + SMe 70c NMe NMe 4-ClC6H4 Te 71c HC N O Me Ph +(Me2Al)2Te 7(Me2Al)2O HC N Te Me Ph 71d Organic derivatives of monocoordinated tellurium 199(NR2, NR1NR22 ) at the tellurocarbonyl carbon atom. Evidently, the stability of telluroamides, like that of telluroesters 59, could be increased by introducing bulky substituents into their molecules.However, compounds of this type have not yet been synthesised. The long-wave absorption bands associated with the n? p* transition in the C=Te fragment in the UV spectra of the telluroamides 71a,d shift bathochromically with respect to those in the spectra of the analogous selenium derivatives by more than 100 nm and are located at 540 nm.62, 64 The 1H NMR data imply that the barriers (DG) to rotation of the NMe2 groups in the amides PhC(X)NMe2 vary in the follow- ing sequence: C=O(62.3 kJ mol71)<C=S(77.0 kJ mol71)< <C=Te(80.5 kJ mol71)<C=Se (80.8 kJ mol71).65 The deviation of the DG value for the telluroamide 71a from the general tendency was attributed 65 to the increase in the steric strain in the ground state caused by the repulsion between the bulky Te atom and the (Z)-N-methyl group.The stability of telluroureas 73 and 75 is also associated with the presence of electron-donating substituents at the tellurocar- bonyl group. These compounds have been synthesised by the oxidation of alkenes 72 66 or 1,3-dimethylbenzimidazoline with elemental Te.The compounds 73 were formed in 42% ±45% yields, and the tellurourea 75 was obtained in*9% yield. The properties of the tellurourea 75 have scarcely been studied. It has been reported 67 that the long-wave absorption bands in the UV spectra of 1,3-dimethylbenzimidazolinechalco- genones shift bathochromically in the series: thione (l=310 nm), selone (l=318 nm), tellurone (l=372 nm).The properties of the telluroureas of type 73 have been studied somewhat more extensively.66 These compounds are sensitive to air and decompose with the liberation of tellurium both in solutions and in the solid state. Complexes 74, which are formed in 50%± 75% yields in the reaction of the tellurourea 73b with Cr, Mo, and W carbonyls, are more stable to air.The length of the C± Te bond in the complex 74a corresponds to the length of a single C± Te bond and is equal to 2.12A; this is much longer than a similar bond in the complex 12 formed from the telluroketone 5 and tungstenpentacarbonyl (1.987A14). This was explained 66 by the assumption that the resonance-stabilised zwitter-ionic struc- tures 76 make the predominant contributions to the ground state of the complexes.III. Synthesis, reactions, structures, and spectroscopic characteristics of phosphine tellurides R3P=Te 1. Synthesis of phosphine tellurides The first compound with the P ± Te bond was synthesised by Foss about 50 years ago.68 The reaction of elemental Te with an ethanolic solution of potassium diethyl phosphite has resulted in the synthesis of (EtO)2P(O)TeK.Later, the corresponding sodium derivative was used as an efficient dehalogenating reagent for a-chloro- and a-bromoketones,69 and also for deoxygenation of epoxides.70 As mentioned above, the first phosphine telluride, tributyl phosphine telluride, was synthesised by Zingaro in 1963.1 This compound and similar compounds 77 have been prepared by the interaction of triorganylphosphines with elemental tellurium in anhydrous toluene.1, 71, 72 The yields of the compounds 77 varied from 5% to 45%.However, the reaction of tris(tert-butyl)- phosphine with tellurium carried out under the same conditions gave the corresponding phosphine telluride in an almost quanti- tative yield.73 Phosphine tellurides have been synthesised in substantially higher yields (75% ± 80%) by heating trialkyl(cy- cloalkyl)phosphines with tellurium without a solvent.74 It should be noted that there is no direct correlation between the solubility of tellurium in phosphines and the capability of the latter of being converted into phosphine tellurides. In fact, the solubility of tellurium in dimethyl(phenyl)phosphine (defined as the ratio of the number of moles of dissolved Te to the number of phosphine moles multiplied by 100) 71 is 16.7, and that for dibutyl(phenyl)phosphine is 9.1; however, only the latter com- pound yields phosphine telluride upon interaction with tellurium.No triorganylphosphine tellurides containing more than one phenyl group have been obtained so far. For example, all attempts to prepare triphenylphosphine telluride failed.However, this compound has been obtained as a complex with triphenylphos- phine (78) by detelluration of TeCN7 with triphenylphosphine in acetonitrile in the presence of lithium perchlorate.75 N N Te . M(CO)5 Et Et 74a ë c R=Me (a), Et (b);M=Cr (74a), Mo (74b),W(74c). N N Me Me +Te D N N Me Me Te 75 N N N N R R R R +Te PhMe, D N N Te R R 72a,b 73a,b M(CO)5 .MeCN R=Et N N Te Et Et (CO)5Cr + N N Te Et Et (CO)5Cr + 76a 76b 7 7 RCCH2X+(EtO)2PTe7 O O 7X7 RCCH2 Te P(OEt)2 O O HOEt RCCH2Te7 O 7Te RC CH2 O7 RCMe .O 7(EtO)3PO, 7H+ H+ (EtO)2P O O 7Te R (EtO)2P O Te 7 O R 7(EtO)2P(O)O7 Te R 7Te R (EtO)2PTe7 O O R (EtO)2PTe7 O O R + R3P Te R3P+Te 77a7i R=Me (a),72 Et (b),71 Pr (c),71,74 Bu (d),1,71,74 But (e),73 C5H11 (f),74 cyclo-C6H11 (g),74 C8H17 (h),74 R3=PhBu2 (i).71 Ph3P+NCTe7 7CN7 [Ph3P Te] Ph3P Ph3P Te . PPh3. 78 200 I D Sadekov, A A Maksimenko, V L NivorozhkinThe structure of the adduct 78 was determined by X-ray diffraction analysis.75 It contains a linear P ± Te ± P fragment with non-equivalent P ± Te bonds. The length of one of these bonds is 2.27 ± 2.42A; this falls into the range of typical P=Te bonds in phosphine tellurides (2.31 ± 2.37A76 ±80); the other bond, which is 3.41 ± 3.95A long, corresponds to a `secondary interac- tion' 81 between the Te atom and the phosphorus atom in triphenylphosphine.The reversible reaction between triorganylphosphines and NCTe7 anions was used to estimate quantitatively the `telluro- philicity' of phosphines;78 for this purpose, the equilibrium con- stants (K) for the reaction were measured.The following results were obtained: R Me2N Et2N Bu K 8.000 0.600 0.540 0.270 0.170 0.009 R Pri 3N EtO MeO BuO Ph K 0.048 *0.003 *0.001 *0.003 0 The K values are in fairly good agreement with other findings. Thus the fact that Ph3P does not form phosphine tellurides is consistent with the K value found (0.000).At the same time, various triaminophosphines characterised by relatively large K readily react with elemental tellurium giving tris(dialkylamino- phosphine) tellurides 79 in high yields.78, 82 ± 85 Amidophosphine tellurides of type 80 83, 86 ± 88 and 81 88 have been obtained in a similar way. Cyclic phosphine tellurides, which are relatively resistant against water and air compared to other phosphine tellurides, have been synthesised by the reactions of the corresponding diazadiphosphetidines with tellurium.89 ± 91 Depending on the ratio of the reactants, these reactions gave diazadiphosphetidine monotellurides 82 89 ± 91 and ditellurides 83.89 Dichloro(methyl)phosphine does not react with tellurium,92 although the chloro-substituted diazadiphosphetidine forms the corresponding telluride 82a in 17% yield.89 The fairly low tellurophilicity of trialkyl phosphites accounts for the fact that no trialkyl phosphotelluroates (RO)3P=Te have been synthesised so far.However, phosphonites RP(OR0)2 are able to form compounds 84 with a P=Te bond. The stability of these compounds is largely determined by the nature of the hydrocarbon substituents attached directly to the phosphorus atom.For example, 84a resulting from reaction of diethyl ethyl- phosphonite with Te decomposes during distillation,93 whereas phosphonotelluroate 84b formed from diethyl allylphosphonite is relatively stable under an inert atmosphere.94 Phosphonotelluroate 85 was obtained in 44% yield by the reaction of bis(trimethylsilyl) hydrogenphosphonite with Te in dioxane.95 Finally, it should be noted that N-diphenyltellurophosphi- noyl(triphenyl)phosphazene 86 is formed in a yield of 87% when the corresponding phosphazene is heated with elemental tellurium in benzene.96 Thus, synthesis of all types of compounds containing P=Te bonds is based on the reactions of P(III) derivatives with elemental tellurium.Other sources of tellurium do not afford phosphine tellurides, although their sulfur and selenium analogues are used to prepare the corresponding phosphine chalcogenides. For example, unlike diorganyl disulfides and diselenides, which react with triorganylphosphines to give triorganylphosphine sulfides or selenides,97 diorganyl ditellurides subjected to photolysis in the presence of tertiary phosphines yield only mixtures of organyl tellurides and elemental tellurium.98 2.Reactions of phosphine tellurides Triorganylphosphine tellurides R3P=Te are golden-yellow crys- talline compounds, which partially decompose in air or during recrystallisation. Therefore, they are normally recrystallised in the presence of the corresponding phosphines. In many cases, heating of solutions of phosphine tellurides in hydrocarbon solvents results in the formation of tellurium mirrors.71 Only tris(tert- butyl)phosphine telluride is thermally stable both in the solid state and in solutions.73 Tris(dialkylaminophosphine) tellurides are also quite sensitive to air and decompose with liberation of elemental Te.82 Phosphine tellurides containing residues of cyclic nitrogen-containing compounds at the phosphorus atom are more stable.78 Diazadiphosphetidine tellurides 82 and 83 are very stable both in the crystalline state and in solutions.88 ± 90 a.Exchange reactions The 1H, 31P and 125Te spectra of mixtures of phosphine tellurides with phosphines recorded at room temperatures indicate that the Te atom migrates rapidly, on the NMR time scale, between the R3P+NCTe7 R3P Te+CN7 N N N O (R2N)3P+Te (R2N)3P Te 79 N (c),78 R2N =Me2N (a),82, 84, 85 Et2N (b),83 N (d),78 N (e).78 O R1n P(NR2R3)37n+Te n=1: R1=R2=Me;83,87 R1=Me, R2=R3=Et;88 R1=Ph, Et; R2=R3=Me;82 n=2: R1=But; R2=H, R3=Pri, cyclo-C6H11.86 (Et2N)2P(CH2)4P(NEt2)2+2Te (Et2N)2P(CH2)4P(NEt2)2.Te Te 81 R1n P(NR2R3)37n 80 Te N R1P N PR3 R2 R2 +2 Te N P N PR3 R1 Te R2 R2 82a7c 82a: R1=Me, R2=But, R3=Cl;89 82b: R1=R3=NMe2, R2=But;90 82c: R1=R3=But, R2=Me.91 N P N P But But Me Me +Te N P N P But But Te Te Me Me 83 R=R0 =Et (a); R=CH2CH=CH2, R0 =Et (b). RP(OR0)2+Te RP(OR0)2 Te 84a,b (Me3SiO)2PH+Te (Me3SiO)2PH Te 85 Ph3P N PPh2 Ph3P N PPh2 Te 86 Organic derivatives of monocoordinated tellurium 201phosphine molecules.73, 98 ± 101 Apparently, this reaction occurs via a transition state like 87.99 However, in the But 3P=Te/But 2PSiMe3 system, the Te atom is rapidly and irreversibly transferred to the silylphosphine; the intermediate phosphine telluride 88 immediately rearranges into compound 89 containing a Te ± Si bond.101 However, this com- pound and also similar derivatives with Te ± Ge and Te ± Sn bonds have not been isolated in an analytically pure state due to disproportionation, which is described by the following scheme:101 Fast, on the NMR time scale, transfer of a tellurium atom between phosphorus atoms has been observed for cyclic phos- phine tellurides 82b,c.90, 91 The concentration dependence of the coalescence temperature indicates that this process can occur not only as intramolecular exchange, which is facilitated by the non-planarity of the diazadi- phosphetidine ring,76 but also intermolecularly.b. Protonation and alkylation The protonation of phosphine tellurides with strong acids affords cations [R3PTeH]+.102 However, no salts with these cations have been isolated in a pure state; their formation in solutions has been judged only by NMR spectra. Under mild conditions (benzene, room temperature), phos- phine tellurides are alkylated with methyl iodide to give methyl- tellurophosphonium salts 90, their yields being about 90%.103 The salts 90 are soluble in nitromethane but decompose soon after dissolution.It is of interest that not only phosphine tellurides but also their complexes with metal carbonyls can be methylated.104 Thus the reaction of the complex 91 with methyl iodide gave cationic complex 92.4 The P ± Te bonds in the salts 90 tend to be cleaved by strong nucleophiles. For instance, treatment of these salts with methyl- lithium affords dimethyl telluride and triorganylphosphines.103 c.Oxidation The phosphine tellurides 77c,d are oxidised with the ferrocenium salts Cp2Fe+X7 (X = BF4, PF6, SbF6) at low temperatures to give dications 93, the yields of which are about 30%, and ferrocene.105 The salt 93 (R = But, X = SbF6) was studied by X-ray diffraction analysis.105 Based on the bond lengths Te ± Te (2.71A) and P ± Te (2.50A, which corresponds to a single bond between Te and P atoms) and on the Te ± Te ± Te (109 8) and Te ± Te ± P (108.6 8) bond angles, this compound was described 105 as a Te2á 3 dication stabilised by complex formation with But 3P.In a more recent study,106 tributylphosphine telluride has been used to stabilise the tellurenyl cations RTe+. These species were generated by oxidation of diorganyl ditellurides with the nitro- sonium salts NO+X7 (X = BF4, ClO4) and isolated as stable complexes [Bu3P?TeP]+X7. d. Complex formation Phosphine tellurides form complexes with some metal salts.Like their sulfur and selenium analogues, phosphine tellurides act as donors in complex formation. Moreover, since in this case the bipolar character of the (P=Te P+± Te7) bond is more pronounced, phosphine tellurides are stronger donors than phos- phine sulfides or phosphine selenides. Thus tributylphosphine telluride reacts with mercury(II) bro- mide to give a complex of the composition (HgBr2)4(TePBu3)3, which is fairly sensitive to oxygen and heating.107 According to X- ray diffraction data, this complex consists of dimeric [(HgTePBu3)3Br5]2á 2 cations and binuclear [Hg2Br6]27 anions.The reactions of trimethylphosphine telluride with nickel and cobalt salts follow a different pathway, which includes decom- position of phosphine telluride and liberation of elemental tellu- rium even at730 8C.72 The first phosphine telluride complexes 91a ± c were prepared in almost quantitative yields on irradiation of mixtures of Cr, Mo, and W carbonyls with tris(tert-butyl)phosphine telluride in THF.104 They appear as dark-red crystals fairly stable in air and at elevated temperatures.The relatively high donor ability of the phosphine telluride ligands is indicated by the substantial upfield shift of the signals of the CO groups in the 13C NMR spectra of the complexes 91.This is also confirmed by the fact that the phosphine telluride ligands in the complexes 91, unlike those in similar complexes derived from phosphine sulfides (CO)5M±S=PR3,108 are not replaced by Ph3P or CO on heating in solutions at 80 8C.104 The length of the P ± Te bond in the complex 91c is 2.439A, which corresponds to a single P ± Te bond.104 The results ofNMRstudies of complexes 94 also indicate that phosphine tellurides are stronger donors than other phosphine R13 P Te+R23 P Te7 PR23 ] + 87 R13 P+Te PR23 R1=R2=But,73, 99, 100 Me2N,99 Bu;100 R1=But, R2=Me2N.73, 99 [R13 P But 2P TeSiMe3 89 (Me3Si)2Te+(But 2P)2Te.But 3P Te+But 2PSiMe3 7But 3P But 2PSiMe3 88 Te R1=Me2N, R2=But (b);90 R1=But, R2=Me (c).91 N P N P R1 R1 R2 R2 Te N P N P R1 R1 R2 R2 Te 82b,c R=Me, Pri, Bu, But, Me2N. R3P Te+MeI [R3PTeMe]+I7 90 (CO)5W Te PBut 3 +MeI (CO)5W Te PBut 3 Me + I7 92 91 [R3PTeMe]+I7+MeLi 7LiI R3P+Me2Te 90 R=Pri (c), But (d); X =BF4, PF6, SbF6. [R3P Te Te Te PR3]2+2X7 93 R3P Te+(C5H5)2Fe+X7 77c,d 7Cp2Fe But 3P Te Te Te PBut 3 2+ M=Cr (a), Mo (b),W(c).M(CO)6+Te PBut 3 hn 7CO (CO)5M TePBut 3 91a7c 202 I D Sadekov, A A Maksimenko, V L Nivorozhkinchalcogenides. These compounds have been synthesised 109 by a reaction similar to that used earlier 110 to prepare the correspond- ing sulfur and selenium derivatives. The dark-brown complexes 94 were formed in almost quantitative yields on the interaction of salts 95 with phosphine tellurides in dichloromethane at room temperature.The compounds 94 are stable in air in the solid state but slowly decompose in solutions with liberation of tellurium to give complexes 96.109 The complex 94e, which is the least stable in this series, decomposes yielding [CpFe(CO)3]+BF4 7 and the corresponding phosphine telluride.The attempts to replace one of the CO groups in the cation 94 by a second R3PTe molecule, which would have resulted in the formation of the cations [CpFe(CO)(TePR3)2]+, failed; heating or irradiation of mixtures of 94 with R3P=Te was accompanied by intense decomposition. Meanwhile, treatment of the cations 94 with iodine resulted in the rupture of the Fe ± Te bonds and in the formation of [CpFe(- CO)2I]2.109 It should be noted that the reaction of the uranium cyclo- pentadienyl complex 97 with tributylphosphine telluride, unlike the reactions of the salts 95, is accompanied by elimination of tributylphosphine giving rise to compound 98.111 Triphenylphosphine sulfide and selenide react with the com- plex 97 yielding compounds with similar structures.In the case of triphenylphosphine oxide, this gives the complex (MeC5H4)3U. OPPh3.111 e. Phosphine tellurides as synthetic equivalents of elemental tellurium. Preparation of metal tellurides The use of phosphine tellurides as synthetic equivalents of elemental tellurium to introduce tellurium into molecules of organic and organoelement compounds is based on the fact that phosphine tellurides readily dissociate on heating. In some cases, phosphine tellurides are preferred over elemental tellurium, since the reactions can be carried out at lower temperatures and the products can be obtained in higher yields.Moreover, some telluration reactions can occur only with phosphine tellurides. Low-temperature synthesis of metal tellurides is among the most significant synthetic applications of phosphine tellurides. Thus triethylphosphine telluride reacts with metallic mercury in toluene at room temperature to give mercury telluride in an almost quantitative yield.112 Phosphine tellurides eliminate metallic tellurium at lower temperatures than the majority of widely used sources of tellurium such as Me2Te and Et2Te, which generate tellurium at temper- atures above 200 8C.5, 113 The easy thermal dissociation of phosphine tellurides has served as the basis for the recently developed synthesis of semi- conductor nanosized crystals 114 by pyrolysis of a mixture of organometallic substrates upon their injection into a hot coordi- nating solvent.Thus cadmium telluride with a size of crystals ranging from 12 to 115A was obtained by the introduction of (C8H17)3P=Te andMe2Cd into (C8H17)3P=Oat 240 8Cfollowed by keeping the reaction mixture at 190 ± 220 8C to grow the crystals.114 The reactions of phosphine tellurides with some organoele- ment compounds result in insertion of tellurium into C± element bonds.For example, the formation of mercury telluride in the reaction of Et3P=Te with diorganylmercury derivatives occurs according to the following scheme:112 It should be noted that various Ar2Hg react with elemental tellurium only under fairly drastic conditions (>200 8C) to give diaryl tellurides.8,9 Insertion of Te atoms into Sc ± C(sp3) or Sc ± C(sp2) bonds occurs relatively easily when permethylscandoceno-alkyls or -aryls are made to react with tributylphosphine telluride or elemental tellurium.115 ± 118 The reactions of scandocenes with phosphine telluride occur faster than the reactions with elemental tellurium, and compounds 99 are formed in higher yields (31% ± 71%).The dimeric scandium m-telluride 100 has been prepared in a similar way;115, 117 its structure was determined by X-ray diffrac- tion analysis.The advantages of Bu3P=Te over elemental tellurium are illustrated by the synthesis of the scandocene derivative 101.118 When elemental tellurium is used, the reaction is completed over 4 ± 5 h, whereas in the case of Bu3P=Te, this reaction occurs immediately after mixing the reactants. Yet another example of the insertion of a Te atom into a C± element bond is the reaction of complexes 102 with tri- alkylphosphine tellurides yielding alkyltelluride manganese com- plexes 103.119, 120 Pyrolysis of one of these products (103b) gives rise to manganese telluride.A series of metal tellurides including manganese telluride have been prepared using complexes of metals in low oxidation states. Depending on the reaction conditions, the reactions involving these compounds either directly afford metal tellurides or initially [CpFe(CO)2 .THF]+BF4 7+Te PR3 7THF 95 [CpFe(CO)2TePR3]+BF4 7 7Te 94a7e [CpFe(CO)2PR3]+BF4 7 96a7e R=Me (a), Pri (b), But (c), Me2N (d), O (e). N (MeC5H4)3U.THF+Bu3P Te 7THF, 7Bu3P [(MeC5H4)3U]2Te . 97 98 Et3P Te+Hg 7Et3P HgTe. R=Et, Ph. RHgR +Et3P Te PhMe, D 7Et3P RTe Hg TeR 7R2Te HgTe RTe HgR Et3P Te 7Et3P (C5Me5)2Sc R Bu3P Te or Te (C5Me5)2Sc TeR 99 R=Me3SiCH2, PhCH2, erythro-CHDCHDBut, CH2CHDCH2CH2CH=CMe2, Ph, cyclo-C5H9CH2.(C5Me5)2Sc D Bu3P Te or Te 7D2 [(C5Me5)2Sc]2Te. 100 [Me2Si(ButC5H3)2]Sc CH2SiMe3 Bu3P Te 7Bu3P [Me2Si(ButC5H3)2]Sc TeCH2SiMe3 . 101 R =CH2Ph: R0 =Me (103a), Et (103b); R03P Te PhMe, D RTeMn(CO)3(PR03)2 103a,b 300 8C, 45 min R0 =Et MnTe RMn(CO)5 102 Et3P Te PhMe, D MeTeMn(CO)3(PEt3)2 + MeMn(CO)3(PEt3)2. 103c MeMn(CO)5 Organic derivatives of monocoordinated tellurium 203afford clusters the pyrolysis of which under relatively mild conditions leads to tellurides. Manganese 121 and cobalt 122, 123 tellurides have been obtained using carbonyls of the correspond- ing metals 104 the reactions of which with triethylphosphine telluride in toluene give rise to binuclear complexes 105; these products result from the insertion of two tellurium atoms into metal ± metal bonds in the initial complexes. The dimeric molecules of 105 are built like diorganyl ditellur- idesR± Te ± Te ± R, but contain (Et3P)2(CO)n72Mgroups instead of the organic radicals.The Te ± Te bond lengths (2.763A in 105a 121 and 2.765A in 105b 122) are somewhat larger than that in diphenyl ditelluride (2.712A124).The pyrolysis of the complex 105a giving crystalline manganese telluride occurs under substan- tially milder conditions (300 8C, 16 h) than does the synthesis of this telluride from the elements (heating to 800 8C followed by keeping the reaction mixture at 500 8C121). When the Co2(CO)8 : (Et3P=Te) : Et3P ratio was varied, clus- ters 106 122 and 107a 122, 123 were isolated in addition to the compound 105b. The former of these clusters can also be obtained from 105b, Co2(CO)8, and Et3P;122 treatment of the complex 105b with Et3P=Te and Et3P affords the cluster 107a.122 The chromium-containing cluster 107b, similar to the cobalt derivative 107a, was prepared by treatment of bis(triethylphos- phinediallyl)chromium or bis(2,4-dimethylpentadienyl)chrom- ium with triethylphosphine telluride.125 According to X-ray diffraction data, the clusters 107a,b are built of octahedra consisting of six cobalt (the Co ± Co bond length is 3.23A122) or chromium (the Cr ± Cr bond length is 2.94A125) atoms; the octahedra are incorporated into a cube combining 8 tellurium atoms; the triethylphosphine groups are bound only to metal atoms.Pyrolysis of the clusters 107a (300 8C, 45 min 122, 123) and 107b (315 8C, 19 h 125) gives rise to polycrystalline b-CoTe and Cr3Te4, respectively. In a relatively recent study,126 the cluster (Ph2PPr)6Co6Te8 the composition of which is close to that of 97a was synthesised by the reaction of CoCl2 with Ph2PPr and trimethyl(phenyltelluro)silane.The complexes bis(cyclooctadiene)nickel 108a 127 and (cyclo- octatetraene)iron 108b 128 served as the starting compounds for the preparation of the tellurides of these metals. Thus heating of bis(cyclooctadiene)nickel 108a in boiling toluene, which normally results in its dissociation to Ni and cyclooctadiene, affords polycrystalline nickel telluride when carried out in the presence of Et3P=Te.127 The synthesis of this telluride from the elements is possible only upon prolonged heating at 400 ± 600 8C.Nickel telluride 127 as well as a mixture of b- and g-FeTe 128 have been synthesised by pyrolysis of clusters 109 ± 111 formed initially. These clusters, like those considered above, are inter- mediate compounds on the way from organic substrates to inorganic solid materials.The nickel clusters 109 and 110 contain Et3P ± Ni ± Te fragments with tetracoordinated nickel and tellu- rium atoms;127 in the cluster 111, the tellurium atoms are tricoordinated, and the iron atoms are tetracoordinated.128 Like nickel telluride, palladium telluride was synthesised using two procedures: by heating of an equimolar mixture of Pd(PPh3)4 and Et3P=Te in toluene 129 and via the clusters 112 and 113; however, the clusters were isolated in fairly low yields (10% and 2%, respectively).To complete the discussion of the methods for synthesis of metal tellurides, we shall mention a new approach developed recently,130 which is based on the use of phosphine tellurides other than R3P=Te. Complexes 114, which have been obtained in almost quantitative yields by the reaction of phosphine tellurides 80 with zinc and cadmium bis[N-di(trimethylsilyl)amides], pro- vide an example.130, 131 The complexes 114 are crystalline compounds readily soluble in toluene.Based on the results of X-ray diffraction analysis of the selenium analogue of 114a,131 a distorted tetrahedral structure has been ascribed to these complexes.Pyrolysis of the complexes 114a,b at 300 8C and 114d at 320 8C gives rise to cubic zinc telluride with an admixture of elemental tellurium and to cubic cadmium telluride, respectively.130 M=Mn, n=5 (a);M=Co, n=4 (b). [(Et3P)2(CO)n72MTe]2 D MnTe 105a,b (CO)nM M(CO)n +Et3P Te 7Et3P 104 105b Co2(CO)8, Et3P Et3P Te, Et3P (Et3P)6Co6Te8 107a D CoTe (Et3P)4(CO)6Co4Te2 106 n=3, m=5; n=7, m=11.(Et3P)6Cr6Te8 107b 315 8C, 19 h Cr3Te4 (Et3P)2Cr(CnHm)2+ Et3P Te L=cyclooctadiene. NiL2+R3P Te PhMe, D 7L,7R3P NiTe 108a ML2+Et3P Te 108a,b Et3P, C6H14, 20725 8C Ni9Te6(PEt3)8 109 7L Et3P, PhMe, 20725 8C Ni20Te18(PEt3)12 110 7L PhMe, 20725 8C 7L (Et3P)4Fe4Te4 111 280 8C 18 h FeTe M=Ni, L=cyclooctadiene (108a); M=Fe, L2=cyclooctatetraene (108b). Pd(PPh3)4+Et3P Te PhMe, D PdTe C7H167PhMe 20725 8C (Et3P)4Pd2Te2+(Et3P)8Pd6Te6 112 113 250 8C 14 h PdTe M[N(SiMe3)2]2+2 But 2P Te NHR 80 7HN(SiMe3)2 But 2P N M Te R Te PBut 2 N R 114a7d M=Zn: R=Pri (114a), cyclo-C6H11 (114b); M=Cd: R=Pri (114c), cyclo-C6H11 (114d). 204 I D Sadekov, A A Maksimenko, V L NivorozhkinPhosphine tellurides have also been used to synthesise com- pounds containing M± Te ±M fragments. For example, the reac- tion of tributyltinhydride with tributylphosphine telluride in the presence of catalytic amounts of (C5Me5)2TiH resulted in the formation of bis(tributylstannyl) telluride 115.132 We shall not discuss the mechanism of the catalytic action of (C5Me5)2TiH;132 however, it should be noted that without this compound, the formation of the telluride 115 proceeds very slowly.Compounds 116 containing theM± Te ±M0 fragment (M and M0 are atoms of different elements) were prepared by the reactions of dimetal derivatives 117 with tributylphosphine telluride.132 However, unlike 116a, which rapidly disproportionates to give symmetrical tellurides 118a and 119a, its phenyl analogue dis- proportionates quite slowly. The reaction of the telluride 118 with tributylphosphine telluride giving rise to compound 120 is the only example of transformation of a telluride into a ditelluride known at present.132, 133 It is of interest to verify whether diorganyl tellur- ides R2Te would be converted into diorganyl ditellurides R2Te2 upon treatment with tributylphosphine telluride. The compound 120 can be regarded as a synthetic equivalent of Te2¡ 2 , because its interaction with benzyl chloride affords dibenzyl ditelluride.133 3. X-Ray diffraction analysis and spectroscopic characteristics of phosphine tellurides. a.X-Ray diffraction analysis of phosphine tellurides The data considered in this Section are mostly concerned with the lengths of P ± Te bonds. Detailed information about the fine structure of phosphoryl groups R3P can be found in original publications.The molecular and crystal structures of six com- pounds with P=Te bonds have been studied by X-ray diffraction analysis. The corresponding bond lengths are listed in Table 1. It can be seen from Table 1 that the P=Te bond lengths vary over a narrow range and are intermediate between the theoret- ically predicted lengths of single (2.48A134) and double (2.27A134) P=Te bonds.These lengths have been reported 80 to correspond to a bond order of 1.5; this implies a substantial contribution of the bipolar P+± Te7 structure to the ground state of phosphine telluride molecules. Comparison of the P ± chalc- ogen bond lengths in tris(morpholinophosphine) telluride 79e and its selenium analogue 78 with the sums of the radii of double bonds formed by the corresponding elements indicates that the contri- bution of the bipolar structure to the resonance hybrid in compounds with the P=Te bonds is larger than that in com- pounds with the P=Se bonds.This is indirectly supported by the conclusion, based on measurements of the dipole moments of phosphine chalogenides, that the polarity of compounds increases in the sequence oxide < sulfide < selenide.135 However, the values of the bond dipole moments of P=X bonds (X=O, S, Se, Te) calculated from the dipole moments of (C8H17)3P=X136 do not confirm this conclusion.The diazadiphosphetidine rings in the compounds 82c 76 and cis-83 77 are markedly non-planar, whereas trans-83 77 is planar. Although tellurium atoms in other types of organotellurium compounds tend to form both intramolecular 6 and intermolecu- lar bonds with atoms of other elements,137 none of the phosphine tellurides has been found to contain any intermolecular contacts between tellurium and phosphorus atoms that would be shorter than the sum of their van der Waals radii.b. Spectroscopic characteristics of phosphine tellurides.Ultraviolet spectra of phosphine tellurides have been relatively little studied, which is apparently due to the fact that these compounds readily decompose in solutions. Therefore, the UV spectra have been reported only for the most stable compounds 79 in which the phosphorus atom is bound to nitrogen atoms incorporated into heterocycles. The absorption maxima of these compounds are observed at 280 nm.78 Although the nature of the electronic transition responsible for this absorption is not known exactly, presumably,78 it is the n?p* transition.Particular interest is aroused by comparison of theUVspectra of compounds of the Ph3P=X type containing O, S, Se, and Te atoms. The data for these derivatives presented in Table 2 indicate that the absorption bands shift bathochromically in the series O, S, Se, and Te.Bu3SnH+Bu3P Te (C5Me5)2TiH 7Bu3P, 7H2 (Bu3Sn)2Te. 115 R=Bu (a), Ph (b). R3+Bu3P 117a,b (C5Me5)2Ti Sn Te (C5Me5)2Ti TeSnR3 116a,b 7Bu3P 119a,b [(C5Me5)2Ti]2Te+(Bu3Sn)2Te 118a,b [(C5Me5)2Ti]2Te+Bu3P Te 7Bu3P 118 (C5Me5)2Ti Te Te PhCH2Cl 7(C5Me5)2TiCl2 (PhCH2)2Te2. 120 Table 1. Lengths of the P=Te bonds in phosphine tellurides. Compound Bond Ref.Compound Bond Ref. length length /A /A But 3P=Te 2.368 80 2.354 76 2.355 79 2.305 77 2.357 78 2.327 77 N ButP N P But Te Me Me 82c 3 P Te 79c N Me Me N P N P But Te But Te cis-83 79e P Te 3 N O trans-83 N P N P But Te Te But Me Me Table 2. Data of the UV spectra for compounds R3P=X.78 R X lmax/ nm lg e O <205 S *210 *3.70 Se *225 *3.56 Te 286 2.79 Te 288 2.85 Te 281 2.80 N O N O N O N O N N Organic derivatives of monocoordinated tellurium 205According to several publications,73, 74, 78, 84, 96 the P=Te stretching bands lie in the range of 445 ± 526 cm71.However, Watari,138 who studied the IR and Raman spectra of Me3P=Te and (CD3)3P=Te, did not find any absorption bands between 400 and 450 cm71; the bands for the P=Te stretching vibrations were observed at 376 cm71.It should be noted that the order of variation of the force constants of the P=X bonds in the series of phoshphine chalcogenidesR3P=Xdiffers depending on R. For R = Me2N, the force constants change in the order O > Te > Se> S,84 whereas in the case of R = Me, this order is O > S > Se>Te.138 Data on the 31P and 125Te NMR spectra of phosphine chalcogenides of various types are listed in Table 3.The 125Te NMR spectra (provided that the solution contains no free phosphine) are well resolved doublets arising due to 31P ± 125Te spin-spin coupling.139 In the case of triphenylphos- phine telluride which exists, as shown above, as a complex with triphenyl phosphine, the 125Te signal is exhibited as a broad singlet at d 7491.8 ppm, owing to rapid migration of the Te atom between the Ph3P groups.139 The 125Te chemical shifts range from d 7513 to 71000 ppm (in relation toMe2Te) indicating that the Te nuclei are appreciably shielded and that the bipolar structures R3P+± Te7 make sub- stantial contributions to the ground states of phosphine telluride molecules.The 31P ± 125Te spin-spin coupling constants are 1660 ± 2150 Hz, the values for tris(dialkylaminophosphine) tellurides being approximately 300 ± 400 Hz greater than those for phos- phine tellurides R3P=Te.IV. Conclusion The data presented in this review demonstrate certain progress in the chemistry of organic derivatives of monocoordinated tellu- rium achieved over the past decade. Efficient methods for the generation of telluroaldehydes and telluroketones have been developed and the first relatively stable telluroketone was syn- thesised.Dimerisation and Diels ± Alder reactions of these com- pounds have led to the synthesis of ditelluretanes, 1-telluracyclohex-3-enes and 2-tellurabicyclo[2.2.1]hept-5-enes. New complexes of tellurocarbonyl derivatives with transition metal compounds have been prepared and their chemical trans- formations have been studied.It has been shown that at low temperatures in polar solvents like THF, tellurocarboxylic acids exist in the telluroxo-form. The chemistry of telluroesters and telluroamides has been developed to a lesser extent. Development of new methods for the synthesis of compounds with C=Te bonds, study of the possibility of using these derivatives in the preparation of tellurium-containing heterocycles, and attempts to synthesise stable telluroaldehydes and telluroketones by introduc- ing substituents, able to form intramolecular coordination bonds with the C=Te groups, into their molecules appear to be promis- ing lines of research in this field.As regards phosphine tellurides, the main progress has been achieved in the investigation of their reactivity towards alkylation, complex formation, oxidation, and other types of transformation. However, the discovery and study of reactions in which phosphine tellurides act as synthetic substitutes of elemental tellurium is the most important achievement.This property of phosphine tellur- ides forms the basis for using them in low-temperature syntheses of metal tellurides and new types of tellurium-containing clusters.In addition to further development of these studies, it appears of interest to attempt using phosphine tellurides as tellurating reagents in the synthesis of some organotellurium derivatives (the transformation of diorganyl tellurides into diorganyl ditel- lurides, reactions with halogen-containing substrates, etc.) The review was supported by the Russian Foundation for Basic Research (Project No. 96 ± 03 ± 32502a) and the INTAS (Grant No. 94 ± 4675). References 1. R A Zingaro J. Organomet. Chem. 1 200 (1963) 2. M Minoura, T Kawashima, R Okazaki J. Am. Chem. Soc. 115 7019 (1993) 3. 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ISSN:0036-021X    

出版商:RSC    

年代:1998

数据来源: RSC

摘要:

Abstract. The methods of synthesis, properties, and reactivities of azo-compounds based on rhodanine and thiopropiorhodanine are examined. The possibilities of the wide scale practical employment of this series of reagents, including their modified and immobilised forms, in the analytical chemistry of platinum and heavy metals are discussed. The bibliography includes 115 references. I. Introduction Azo-compounds based on rhodanine (2-thioxo-4-thiazolidinone), thiorhodanine (2,4-dithioxothiazolidine), and aromatic amines containing salt-forming groups were first synthesised in 1971; these azo-compounds were obtained at the same time as analytical reagents.1, 2Adistinctive feature of these reagents is their ability to enter into highly sensitive, selective, and contrasting colour reactions with noble (NM) and heavy (HM) metals in solutions and in the solid phase.3±13 Systematic studies on azo-compounds synthesised from 3-substituted derivatives of rhodanine, thiohy- dantoin (2-thiazolidine), selenoisorhodanine [4-thioxo-2-(1,3- selenazolidinone)], the six-membered analogue thiopropiorhoda- nine (2,4-dithioxo-1,3-thiazane), as well as bisazo-compounds based on rhodanine resulted in the creation of a whole series of promising analytical reagents.14 ± 24 By virtue of their exception- ally valuable properties, azorhodanines and azothiopropiorhoda- nines have found extensive applications in analytical chemistry.For example, various methods for the determination of 17 elements (photometric, differential photometric, extraction-pho- tometric, sorption-photometric, including those using sensitive photometric cells, etc.) have been developed using these com- pounds.The method may be applied in the analysis of objects of different composition and with different contents of the compo- nents. It may be that azorhodanines and azothiopropiorhoda- nines are some of the most promising analytical reagents proposed during the last 25 years.Rhodanine is known as the analytical reagent for the precip- itation and gravimetric determination of silver, mercury, and other platinum metals.25 ± 30 However, the low sensitivity of rhodanine (as well as the products of its interaction) and the low sensitivity of the reactions precluded the wide scale employment of this reagent for the requirements of analytical chemistry, especially photometry.Because of the enhanced reactivity of the 5-methylene group, rhodanine interacts with compounds of different classes � aldehydes, ketones, and diazo-compounds.31 ± 42 This leads to the possibility of obtaining from it new reagents containing the chromophoric groups =C=CH7 and 7N=N7. The sensitiv- ity and contrasting properties of the reactions of rhodanine condensation products with metal ions are greatly superior to those of rhodanine.Thus p-dimethylaminobenzylidenerhodanine has found fairly extensive applications in the photometric deter- mination of silver and gold.29, 30, 43 ± 49 In contrast to the 5-condensed derivatives, the azo- compounds based on rhodanine and aromatic amines containing salt-forming groups (OH, SO3H, COOH AsO3H2) 1 ± 3, 13, 14, 16, 21, 24 are of interest for the determination of not only gold and silver but also of platinum, palladium, rhodium, iridium, ruthenium, and heavy metals. Such compounds are more stable in solution, dissolve relatively easily, and their reactions with the elements are characterised by an unusually high sensi- tivity [the molar absorption coefficient (e) reaches (3 ± 9)6105 in certain cases], good contrasting properties, and high selectivity.The selectivity is based on the sharp differences between the conditions and kinetics of the colour reactions with the elements and the spectroscopic characteristics and stability of the com- plexes formed.50 ± 58 II. Synthesis of the reagents and methods for their identification About forty 5-azo-compounds based on rhodanine (1a ±m), thiorhodanine (2a ± c), thiohydantoin (3a, b), pseudothiohydan- toin (4a ± d), 3-aminorhodanine (5a ± f), N-substituted 3-amino- rhodanine (6a, b), bisazo-compounds based on rhodanine (7a ± c), selenoisorhodanine (8a ± d), a six-membered structural analogue NH O C S S H2C 1 2 3 4 5 R F Gur'eva, S B Savvin V I Vernadskii Institute of Geochemistry and Analytical Chemistry, Russian Academy of Sciences, ul.Kosygina 47a, 117975 Moscow, Russian Federation. Fax (7-095) 938 20 54. Tel. (7-095) 137 28 78 (S B Savvin) Received 24 March 1997 Uspekhi Khimii 67 (3) 236 ± 251 (1998); translated by A K Grzybowski UDC 543.43 Azorhodanines, azothiopropiorhodanines, and their analytical application R F Gur'eva, S B Savvin Contents I.Introduction 209 II. Synthesis of the reagents and methods for their identification 209 III. Properties of the reagents 210 IV. Reactions with metal ions and the properties of the complexes formed 215 V. Characteristic features of the interaction with acido-complexes of platinum metals 218 VI. Applications 219 VII. Conclusion 222 Russian Chemical Reviews 67 (3) 209 ± 224 (1998) #1998 Russian Academy of Sciences and Turpion Ltdof rhodanine�thiopropiorhodanine (9a ± d), as well as aromatic amines containing predominantly salt-forming groups have been synthesised as possible analytical reagents.1 ± 3, 13, 14, 16, 21, 24 The structural formulae and names of reagents of each group are presented in Table 1.The azo-compounds are synthesised in accordance with a general scheme: by treating the heterocyclic azo-component with the corresponding diazotised amine under conditions suitable for each reaction (nature of the solvent, pH, time during which the reaction mass is allowed to stand, and the temperature regime).This ensures the preparation of reagents in high yields and reduces to the maximum extent the possibility of the formation of side products.The heterocyclic azo-components are obtained as follows: rhodanine � by the condensation of monochloroacetic acid with ammonium thiocyanate on heating;25 thiorhodanine� from rhodanine by replacing the carbonyl group by the thiocar- bonyl group on treatment with P2S5;1 thiohydantoin � by the reaction of potassium thiocyanate with the hydrochloride of the ethyl ester of glycine;59 pseudothiohydantoin � by condensing urea with monochloroacetic acid on heating;25 3-aminorhoda- nine �by condensing sodium monochloroacetate with hydrazine dithiocarbazinate;60, 61 3-aminobenzylidenerhodanine � by con- densing 3-aminorhodanine with aldehydes in acetic acid; thiopro- piorhodanine � by treating propiorhodanine with P2S5 in dry dioxane;62 propiorhodanine � by condensing chromotropic acid with freshly prepared ammonium dithiocarbamate and subse- quent heating of thiocarbamoyl-b-thiolactic acid with acetic anhydride;63 selenoisorhodanine�by the interaction of 1,3-sele- nazolidine-2,4-dione with P2S5 in dry dioxane;64 1,3-selenazoli- dine-2,4-dione � by condensing monochloroacetic acid with selenourea on heating.In order to obtain purer products and higher yields of the main substances, the synthesis is carried out in the presence of metal (calcium, lead) salts, but the solubility of the reagents is then reduced. The azo-compounds based on aminophenols and sele- noisorhodanine could not be isolated. The azo-compounds are identified by paper chromatography, column chromatography, electrophoresis using various buffer systems and also from the characteristic absorption spectra of the reagents themselves (concentrated H2SO4, neutral and alka- line solutions) and their complexes. The purity of the preparations is established by the potentiometric titration of the reagents with alkali and by spectrophotometric titration with solutions of metal salts.3, 7, 16, 65 The study of 5-azo-compounds based on propiorhodanine [2-thioxo-4-(1,3-thiazanone)] showed that, in contrast to rhoda- nine, propiorhodanine does not react with diazonium salts even in the presence of activators of the azo-coupling reactions (pyridine, alkoxides).The reduced reactivity of the methylene group in the 5-position is due to the fact that the activating effp in the 4-position extends to the 5-methylene group, while the thioxo-group activates the 6-methylene group.On the other hand, in rhodanine the activating effect of the two groups is directed to the 5-methylene group of propiorhodanine. At the same time, the azo-coupling of thiopropiorhodanine with diazonium salts becomes possible as a result of the increase in electron density on the carbon atom in the 5-position as a consequence of enolisation and the formation of a thioenolate anion.62, 63 III. Properties of the reagents The reagents are powdered substances the colour of which varies from yellow to dark-red depending on the nature of the diazo- and azo-components; they dissolve in dioxane, dimethylformamide, ethanol, acetic acid, solutions of alkalis, and concentrated acids.The reagents containing sulfo-groups are readily soluble in water. In a dry form, they retain their analytical properties for more than three years. The state of the reagents in solution is one of the principal factors determining the reactivity, the mechanism of the complex formation reaction, and the nature of the colour reactions with the elements.Solutions of the reagents undergo a sharp colour change as the pH is altered, which is due to the dissociation of the acid groups and the protonation of the basic groups as well as tautomeric transformations. For the azo-substituted rhodanines 1a ±m, the diazo-component of which contains a OH group, it is possible to identify several coloured forms: red (conc. H2SO4), yellow (pH 1 ± 4), orange (pH 4 ± 8), red (pH 8 ± 10), and violet (pH>11).For reagents without OH groups, a colour change from lemon-yellow (acid and neutral media) to yellow-orange (pH 9 ± 10) and red (pH 11 ± 13) is observed. Only three coloured forms have been identified for the azothiorhodanines 2a ± c, which contain an OH group: dark-red (acid and neutral media), violet (weakly alkaline media), and blue (strongly alkaline media).The azothiorhodanines are unstable in strongly acid media. The above pH values and the sequence in which the acid groups of the reagents ionise have been established on the basis of the results of potentiometric and high-frequency titrations as well as the analysis of the absorption spectra at the pH corresponding to the potential jumps.3, 7, 16, 22, 65 The nature of the heteroatom (O, S) influences appreciably the pK for the dissociation of theNH group of the heterocycle.In the azothiorhodanines 2a ± c, the NH group is titrated at lower pH; on the other hand, the nature of the substituents in the diazo-component influences the pK of the OH group in the benzene ring. For the majority of azorhodanines, pKNH<pKOH.However, in the case of disulfophenolazorhodanine 1b, the NH and OH groups are titrated simultaneously, whilst in picramineazorhoda- nine 1e, containing two strong electron-accepting substituents, the dissociation of the OH group in the benzene ring precedes that of the NH group. In concentratedH2SO4, a shift of the long-wavelength absorp- tion band (Dl=70 nm) is observed for all 5-azo-substituted rhodanines relative to their absorption band in neutral solutions.According to calculated data,65 the bathochromic shift in strongly acid media is due to the protonation of the reagents: the heter- oatoms of the rhodanine ring are protonated but not the hydroxy- group in the benzene ring or the nitrogen atom of the azo- or hydrazo-group, because the highest negative charge is present on the heteroatoms of the rhodanine ring.Several types of tautomerism are possible for azo-substituted rhodanines: If the reagent contains aOHgroup in the benzene ring, there is yet another form of tautomerism: hydroxyazoquinone ± hydrazone. thione ± thiol N N S NH O S N N S N O SH keto ± enol N N S NH HO S N N S NH O S N N S N HO S HN N S NH O S N N S NH O S azo ± hydrazone 210 R F Gur'eva, S B SavvinTable 1.Structural formulae and names of the reagents. Azo-compound Number Name according to IUPAC nomenclature Trivial name 1a 5-chloro-2-hydroxy-3-[(4-oxo-2-thioxo- sulfochlorophenolazo- 5-thiazolidinyl)azo]benzenesulfonic acid rhodanine (SCPAR), rhodazol KhS 1b 3,5-disulfo-2-[(4-oxo-2-thioxo-5-thiazol- disulfophenolazo- idinyl)azo]phenol rhodanine 1c 2-hydroxy-5-nitro-3-[(4-oxo-2-thioxo- sulfonitrophenolazo- 5-thiazolidinyl)azo]benzenesulfonic acid rhodanine 1d 3-carboxy-2-hydroxy-5-[(4-oxo-2-thioxo- carboxysulfophenol- 5-thiazolidinyl)azo]benzenesulfonic acid azorhodanine 1e 3,5-dinitro-2-[(4-oxo-2-thioxo-5-thiazol- picramineazorhodanine idinyl)azo]phenol 1f 4-chloro-2-[(4-oxo-2-thioxo-5-thiazol- rhodazol Kh idinyl)azo]phenol 1g 2-[(4-oxo-2-thioxo-5-thiazolidinyl)- o-phenolazorhodanine azo]phenol 1h 3-[(4-oxo-2-thioxo-5-thiazolidin- m-phenolazorhodanine yl)azo]phenol 1i 4-[(4-oxo-2-thioxo-5-thiazolidin- p-phenolazorhodanine yl)azo]phenol 1j 2-carboxy-[(4-oxo-2-thioxo-5-thi- carboxybenzene- azolidinyl)azo]benzene azorhodanine 1k 2-[(4-oxo-2-thioxo-5-thiazolidin- o-sulfobenzeneazo- yl)azo]benzenesulfonic acid rhodanine N N S NH OH HO3S Cl O S N N S NH OH HO3S HO3S O S N N S NH OH HO3S O2N O S N N S NH OH HOOC HO3S O S N N S NH OH O2N O2N O S N N S NH OH O S Cl N N S NH OH O S N N S NH HO O S N N S NH O S HO N N S NH COOH O S N N S NH SO3H O S Azorhodanines, azothiopropiorhodanines, and their analytical application 211Table 1 (continued).Azo-compound Number Name according to IUPAC nomenclature Trivial name 1l 3-[(4-oxo-2-thioxo-5-thiazolidinyl)azo]- m-sulfobenzeneazo- benzenesulfonic acid rhodanine 1m 4-[(4-oxo-2-thioxo-5-thiazolidinyl)- p-sulfobenzeneazo- azo]benzenesulfonic acid rhodanine 2a 5-chloro-2-hydroxy-3-[2,4-dithioxo- sulfochlorophenol- 5-thiazolidinyl)azo]benzenesulfonic acid azothiorhodanine 2b 2-carboxy-[(2,4-dithioxo-5-thiazolidinyl)- carboxybenzeneazo- azo]benzene thiorhodanine 2c 2-arsono-[(2,4-dithioxo- arsonobenzeneazo- 5-thiazolidinyl)azo]benzene thiorhodanine 3a 2-hydroxy-5-nitro-3-[(4-oxo-2-thioxo- sulfonitrophenolazo- 5-imidazolidinyl)azo]benzenesulfonic thiohydantoin acid 3b 2-[4-oxo-2-thioxo-5-imidazolidinyl)azo]- o-phenolazothio- phenol hydantoin 4a 5-chloro-2-hydroxy-3-[(2-imino-4-oxo- sulfochlorophenolazo- 5-imidazolidinyl)azo]benzenesulfonic pseudothiohydantoin acid 4b 2-hydroxy-5-nitro-3-[(2-imino-4-oxo- sulfonitrophenolazo- 5-imidazolidinyl)azo]benzenesulfonic pseudothiohydantoin acid 4c 3,5-disulfo-2-[(2-imino-4-oxo-5-imidazol- disulfophenolazo- idinyl)azo]phenol pseudothiohydantoin N N S NH HO3S O S N N S NH O S HO3S N N S NH OH HO3S Cl S S N N S NH COOH S S N N S NH AsO3H2 S S N N N NH OH O S HO3S O2N H N N N NH OH O S H N N S NH OH O NH HO3S Cl N N S NH NH OH O HO3S O2N N N S NH NH OH O HO3S HO3S 212 R F Gur'eva, S B SavvinTable 1 (continued).Azo-compound Number Name according to IUPAC nomenclature Trivial name 4d 3-carboxy-5-sulfo-2-[(imino-4-oxo- carboxysulfophenol- 5-thiazolidinyl)azophenol azopseudothiohydantoin 5a 5-chloro-2-hydroxy-3-[(3-amino-4-oxo- sulfochlorophenolazo- 2-thioxo-5-thiazolidinyl)azo]benzenesulfonic 3-aminorhodanine acid 5b 2-hydroxy-5-nitro-3-[(3-amino-4-oxo- sulfonitrophenolazo- 2-thioxo-5-thiazolidinyl)-azo]benzene- 3-aminorhodanine sulfonic acid 5c 3-[(3-amino-4-oxo-2-thioxo-5-thiazolidin- sulfophenolazo-3-amino- yl)azo]-2-hydroxybenzenesulfonic rhodanine acid 5d 4-[(3-amino-4-oxo-2-thioxo-5-thiazol- p-phenolazo-3-amino- idinyl)azo]phenol rhodanine (PATAR) 5e [(3-amino-4-oxo-2-thioxo-5-thiazolidinyl)- carboxybenzeneazo- azo]-2-carboxybenzene 3-aminorhodanine 5f 2-[(3-amino-4-oxo-2-thioxo-5-thiazolidin- o-sulfonbenzeneazo- yl)azo]benzenesulfonic acid 3-aminorhodanine 6a 5-chloro-2-hydroxy-3-({2-thioxo-3-[(20-hydr- 7 oxy-30-methoxyphenyl)methyleneamino]-4- oxo-5-thiazolidinyl}azo)benzenesulfonic acid 6b 5-chloro-2-hydroxy-3-({2-thioxo-3-[(20-hydr- 7 oxyphenyl)methyleneaminol-4-oxo-5-thiazol- idinyl)azo]benzenesulfonic acid 7a 4-[(4-oxo-2-thioxo-5-thiazolidinyl)azo]- benzeneazobenzeneazo- benzene rhodanine (BABAR) N N S NH NH OH O HOOC HO3S N N S N OH O HO3S Cl S NH2 N N S N OH O HO3S O2N S NH2 N N S N OH O HO3S S NH2 N N S N O S NH2 HO N N S N O S NH2 COOH N N S N O S NH2 SO3H N N S N O S N OH HO3S Cl CH OH OCH3 N N S N O S N OH HO3S Cl OH CH N N N N S NH O S Azorhodanines, azothiopropiorhodanines, and their analytical application 213Table 1 (continued).Azo-compound Number Name according to IUPAC nomenclature Trivial name 7b 3-hydroxy-4-[(4-oxo-2-thioxo-5-thiazol- sulfophenolazoben- idinyl)azo]benzenesulfonic acid zeneazorhodanine (SPABAR) 7c 20-hydroxy-50-[4-oxo-2-thioxo-5-thiazol- sulfobenzeneazo- idinyl)azo]benzeneazobenzenesulfonic phenolazorhodanine acid 8a 5-chloro-2-hydroxy-3-[(2-oxo-4-thioxo- sulfochlorophenol- 5-selenazolidinyl)azo]benzenesulfonic selenoisorhodanine acid 8b 2-hydroxy-5-nitro-3-[(2-oxo-4-thioxo- sulfonitrophenolazo- 5-selenazolidinyl)azo]benzenesulfonic selenoisorhodanine acid 8c 2-carboxy-[(2-oxo-4-thioxo-5-selen- carboxybenzeneazo- azolidinyl)azo]benzene selenoisorhodanine 8d 4-nitro-[(2-oxo-4-thioxo-5-selenazol- nitrobenzeneazo- idinyl)azo]benzene selenoisorhodanine 9a 5-chloro-2-hydroxy-3-[(tetrahydro- sulfochlorophenol- 2,4-dithioxo-1,3-thiazin-5-yl)azo]benzene- azothiopropiorhodanine, sulfonic acid tyrodine 9b 2-hydroxy-5-nitro-3-[(tetrahydro- sulfonitrophenolazo- 2,4-dithioxo-1,3-thiazin-5-yl)azo]benzene- thiopropiorhodanine sulfonic acid 9c 2-carboxy-2-hydroxy-5-[(tetrahydro- carboxysulfophenol- 2,4-dithioxo-1,3-thiazin-5-yl)azo]benzene- azothiopropiorhodanine sulfonic acid 9d 2-carboxy-[(tetrahydro-2,4-dithioxo- carboxybenzene- 1,3-thiazin-5-yl)azo]benzene azothiopropiorhodanine N N N N S NH O S HO HO3S S NH O S N N N N HO HO3S N N Se NH S O OH HO3S Cl N N Se NH S O OH HO3S O2N N N Se NH S O COOH N N Se NH S O O2N N N S NH S S OH Cl HO3S N N S NH S S OH O2N HO3S N N S NH S S OH HO3C HOOC N N S NH S S COOH 214 R F Gur'eva, S B SavvinThe molecules of azo-substituted rhodanines exist in aqueous solutions predominantly in the azodiketo-form.On complex formation, the equilibrium shifts towards the enolic form.This is confirmed by data concerning the number of protons liberated on formation of the complex between copper ions and SCPAR 1a. The formation of a 1 : 1 complex is accompanied by the liberation of two protons.3 The concentration of the azothiol tautomer is low. Picramineazorhodanine 1e, for which the contents of the diketo- and thiol forms are approximately the same, is an exception. For the reagents containing a OH group, the first bathochromic shift (pH 6 ± 10) is due to the transition from the azo-form of the reagent to the quinone hydrazone form.In strongly alkaline solutions, a shift of the thione ± thiol equilibrium towards the thiol azo-tautomer is observed, this being accompa- nied by a bathochromic shift of the long-wavelength absorption band.The presence of electron-accepting substituents in the benzene ring, for example in compounds such as sulfonitrophe- nolazorhodanine 1c and picramineazorhodanine 1e, `shifts' the pH of the thione ± thiol transition towards lower values.65 A series of azorhodanines have been investigated by 13C NMR.It was concluded that the reagents containing OH groups in the benzene ring exist in the form of two isomers in organic solvents (OS) such as CD3OD, DMSO, and CD3COOCD3.66, 67 Analysis of IR spectroscopic data for azo-derivatives of rhodanine, 3-aminorhodanine, and thiopropiorhodanine showed that the transition from rhodanine to thiopropiorhodanine entails significant changes in the nature of the interaction of the individ- ual fragments of the heterocycle and a displacement of the equilibrium towards the quinone hydrazone tautomer.68 5-Azo-substituted thiohydantoins 3a, b and pseudothiohy- dantoins 4a ± d represent the least investigated series of reagents.Their stability is inferior to that of the azorhodanines 1a ±m and azo-3-aminorhodanines 5a ± f.The ionisation sequence of the acid groups is retained. In the case of 5-azo-compounds based on 3-aminorhodanine and N-substituted rhodanines 6a, b, the same features of the influence of substituents in the diazo-component on the spectro- scopic characteristics and acid ± base properties of the reagents are observed as for azorhodanines. For the same sequence of the dissociation of the acid groups, a strengthening of the acid properties of the OH groups in the benzene ring is observed.For example, pKOH=8.5 for sulfochlorophenolazorhodanine 1a, while pKOH=7 for sulfochlorophenolazo-3-aminorhodanine 5a. The bathochromic effect in concentrated H2SO4 is weaker than in the case of azo-substituted rhodanines.16 The introduction of a second areneazo-group into the reagent molecule affects the state of the reagent in acid media.Whereas the protonation of azorhodanines, accompanied by a deepening of the colour { (from 420 to 500 nm), is observed in 10 ± 15 MH2SO4, the bisazo-compounds based on rhodanines 7a ± c are protonated already in 1 ± 2 M H2SO4 whereupon the colour becomes even deeper (from 420 to 580 nm). The influence of thermochrom- ism � the change in colour on heating in acid media � has been observed for the bisazo-compounds.A sharp change in the colour of benzeneazobenzeneazorhodanines as a function of pH is observed in the presence of surfactants (for example, cetylpyridi- nium chloride) and certain organic solvents (ethanol, propanol, acetone), provided that the content of the latter exceeds 85%.At least two new coloured forms of the reagents, undetected in aqueous solutions in the absence of organic additives, appear under these conditions. It has also been noted that, in the presence of surfactants, the reagents are protonated in more acid media. The deepening of the colour in alkaline and acid solutions is accompanied by an increase in optical density at the absorption maximum.21 ± 23 5-Azo-substituted selenoisorhodanines 8a ± d are unstable in solutions.When the pH changes, the colour of solutions of the reagents is altered. The nature of the heteroatom (Se) intensifies the acid properties of azoselenoisorhodanines. The protonation of reagents takes place in more dilute acid solutions and is accom- panied by a more pronounced deepening of the colour compared with azorhodanines (lmax'530 and 490 nm respectively). 5-Azo-substituted derivatives of thiopropiorhodanine have a deeper colour in neutral and alkaline solutions than the corre- sponding rhodanines (lmax'540 and 430 nm respectively). A change in the dissociation sequence of the NH and OH groups has been noted for these reagents. The OH group in the benzene ring is the first to dissociate (at lower pH).This can be due both to the difference between the acid properties of the azo-components themselves (pKNH=5.8 for rhodanine and 8.1 for thiopropio- rhodanine) and to the difference between the mechanisms of the tautomeric transformations. A deepening of the colour of the reagents, as in the case of azorhodanines and azo-3-aminorhoda- nines, is not observed for azothiopropiorhodanines in strongly acid media.The yellow colour of the reagents in weakly acid media, characteristic of the neutral form, is retained even in concentrated H2SO4. A strong influence of surfactants and organic solvents on the colour of the reagents over a wide pH range is observed both for azothiopropiorhodanines and for azorhodanines and rhodanine-based bisazo-compounds.22, 23 IV.Reactions with metal ions and the properties of the complexes formed The reagents based on aminophenols give rise to the most sensitive and contrasting colour reactions. In the case of 5-azorhodanines, it is possible to identify three complex-formation pH ranges.1, 5, 7, 9, 65 Pd, Ag, Cu, Fe, Co, Ni, Zn, Cd, Pb, and Be react in the pH range 6 ± 10.The colour then changes from orange (480 nm) to violet (530 ± 580 nm); e=26104. Cu, Pd, Ag, and Hg interact at pH 1 ± 6, the colour changing from yellow (430 nm) to red-orange (430 ± 500 nm); e=(1.5 ± 3)6104. In acid and strongly acid media, noble metals (Pt, Pd, Rh, Ir, Ru, Au, Ag) and Hg interact with the reagent; the reactions are contrasting (430?500 ± 530 nm) and highly sensitive; e=(2 ± 12)6104.The reactions in strongly acid media proceed as a rule over a period of time and in the pres of a 30 ± 50-fold excess of the reagent. The sensitivity and rate of the reaction as well as the solubility of the reaction products depend on the nature and concentration of the acids (mixture of acids) in which the reaction is performed. Heating increases the rate of the reactions of noble metals with azorhodanines, but the yield of the complexes of certain metals diminishes under these conditions.For example, the chloride complexes between platinum(IV) and SCPAR give rise to a colour reaction in the presence of reductants both in HCl and in a mixture of HCl with H2SO4 or H3PO4; heating decreases the yield of the complex. The colour development time is 20 and 4 h, while e=3.36104 and 1.06105 in HCl and in the HCl ±H3PO4 mixture respectively (Table 2). Rhodium and iridium give rise to a colour reaction with azorhodanines in H3PO4 and H2SO4; in the presence of CH3COOH, the sensitivity of the reactions increases; heating to 70 and 90 8C respectively increases the rate of the reaction. OH N N O N NH N N H O C O H NH S S O H N N S NH O H S { The bathochromic shift of the main absorption band.Azorhodanines, azothiopropiorhodanines, and their analytical application 215Gold interacts with azorhodanines in HCl solutions at room temperature, the reaction being completed after 5 ± 10 min. The presence of H2SO4 and H3PO4 does not affect the sensitivity and rate of the reaction, but the solubility of the complexes increases and the permissible acidity range expands in H3PO4.Thus the optical density of solutions of the gold complexes of SCPAR is constant in 2 ± 10 M H3PO4 and 0.5 ± 1.3 M HCl, whereas in the absence of H3PO4 the maximum development of colour is observed in 1 M HCl. The silver complexes, like the palladium complexes, have different molar absorption coefficients at the absorption max- imum (lmax), compositions (reactant : metal ratio in the complex), solubilities, and other characteristics depending on the reaction conditions.In neutral and weakly acid solutions, palladium and silver form readily 1 : 1 complexes with azorhodanines. Saturation is then attained in the presence of a 2 ± 3-fold excess of the reagent. Among the reagents of the azorhodanine series, those which exist predominantly in the form of the diketo-tautomers in acid and neutral media are best, for example SCPAR, carboxysulfopheno- lazorhodanine 1d, and o-phenolazorhodanine 1g.Reagents which contain comparable amounts of diketo- and thiol forms in weakly acid media, for example sulfonitrophenolazorhodanine 1c and picramineazorhodanine 1e, are less sensitive.According to quan- tum-chemical studies, the reactions of silver with benzeneazorho- danines containing electron-donating substituents in the benzene ring should be characterised by a high sensitivity. The presence of a hydroxy-group enhances the contrasting properties of the reaction as a result of the hydroxyazoquinone ± hydrazone tauto- meric transition. In strongly acid media, the reactions of Ag and Pd as well as those of Rh, Ir, Ru, and Pd proceed over a period of time and in the presence of a large excess of the reagent.The sensitivity of the reactions in strongly acid media is higher than in Table 2. Analytical characteristics of the colour reactions of azorhodanines and tyrodine. Initial form of Reaction conditions lmax /nm 1073 e Ref. the elements Sulfochlorophenolazorhodanine Pt(IV), chloride 3 M HCl, 4 h 500 32 1, 5, 50 2 M HCl +2 M H3PO4, 4 h b 500 100 1, 5, 50 Pt(III), sulfate 2 M H2SO4 +11 M H3PO4, 30 min, 50 8C 500 100 18, 51, 54, 74 Pt(III), phosphate 12 ± 15 M H3PO4, 50 min, 50 8C 500 100 18, 54, 58 Pd(II), chloride a 2 M H2SO4 +8 M H3PO4 520 120 1, 5, 75 1 M HCl 520 50 Rh(III), chloride 7 M H2SO4(H3PO4) +6 M CH3COOH, 60 min, 70 8C 510 65 4, 5, 51 Rh(II), phosphate 14 M CH3COOH, 60 min, 70 8C 510 8 8, 9 Rh(II), sulfate 7 M H2SO4+6 M CH3COOH, 10 min, 70 8C 510 17 76 Rh(III), acetate 7 M H2SO4 +6 M CH3COOH, 15 min 510 11 76 Ir(IV), chloride 10 M H2SO4 +6 M CH3COOH, 1 h, 90 8C 510 45 5, 8, 9 Ir(IV), sulfate, phosphate weak reaction <5 5, 8, 9 Ru(III), Ru(IV), 2 M HCl +10 M CH3COOH, 30 min, 80 8C 500 16 18, 53, 54, 56, 74 sulfate, phosphate, chloride Au(III), chloride 1 M HCl, 10 min 530 53 1, 51 Ag, nitrate pH 2 ± 6 480 16 1, 7, 77 pH 6 ± 10 560 15 1, 7, 77 1 ± 6 M H2SO4, 30 min 500 40 1, 7, 77 Tyrodine Pt(IV), chloride 7.6 M H3PO4, 3 h b 510 65 14, 16 Pt(III), phosphate, sulfate 3 M H2SO4 +5 M CH3COOH, 30 min b 510 65 18, 74 Ag, nitrate 1 M H3PO4 +10 M CH3COOH 535 53 16, 17 Extraction, pH 5 c 550 ± 580 450 ± 900 57, 78 Pb(II), nitrate pH 2 ± 6 590 42 20, 69 Cd(II), sulfate pH 4 ± 5.5, 10 min 520 30 72 p-Phenolazo-3-aminorhodanine Hg(I), Hg(II), nitrate 15 M H3PO4 or 7 ± 10 M H2SO4 510 20 19, 73 Cu(III), chloride 1 M HCl, pH 1 b 520 40 12 Benzeneazorhodanine Hg(I), Hg(II), nitrate pH 7 ± 9, surfactant 570 100 21 ± 23 Cu(II), chloride pH 7 ± 9, surfactant 570 20 21 ± 23 Au(III), chloride pH 7 ± 9, surfactant 570 100 21 ± 23 Re(IV) 2 M NaOH+0.1 M SnCl2 570 cmin=0.052 ± 79 ± 81 ± 0.005 mg ml71 Sulfobenzeneazophenolazorhodanine Pd(II), chloride pH 1 ± 3, surfactant 540 300 21 ± 23 Au(III), chloride pH 1 ± 3, surfactant 540 600 21 ± 23 Rh(III), chloride pH 1 ± 3, surfactant 540 100 21 ± 23 Ru(III), Ru(IV), chloride pH 1 ± 3, surfactant 540 40 21 ± 23 a For the same sensitivities of the reactions of Pd(II) in the form of chlorides and in the form of sulfates, phosphates, and nitrates, the rates of the last three reactions are higher.b The reaction is carried out in the presence of reductants. c The two reagents `tyrodine ± macrocyclic compound' method. 216 R F Gur'eva, S B Savvinneutral and weakly alkaline solutions (pH 7 ± 9).In terms of selectivity, the colour reactions of silver and palladium supple- ment one another. In strongly acid media, the selectivity of the determination in relation to nonferrous metals is higher and in relation to platinum metals is lower than in weakly acid and neutral solutions (pH 1 ± 6). 5-Azothiorhodanines enter into colour reactions with gold, platinum, and palladium in 1 ± 6 MHCl.The colour changes from red to blue-violet; e=(2 ± 5)6104. Silver interacts with 5-azothio- rhodanines at pH 1.5, the sensitivity of the reactions being low. 5-Azothiorhodanines and 5-azothiopropiorhodanines do not react with rhodium and iridium, whereas thiopropiorhodanine- based azo-compounds enter into highly sensitive colour reactions with Pt, Pd, Au, and Ag in acid and strongly acid media; the colour changes from yellow to red.The presence of HCl has a negative influence on the reactions of gold and platinum. For platinum and gold, the optimum acid mixture comprises H3PO4 (H2SO4) and CH3COOH. For platinum, the rate of reaction and the yield of the complexes increase in the presence of reductants.16, 17 For silver, it is possible to identify two regions of interaction with azothiopropiorhodanines: neutral (pH 5 ± 9, Dl=30 nm, e=2.06104) and acid (CH3COOH±H3PO4 mixture, Dl= 75 nm, e=5.36104) media.The formation of a coloured silver compound in strongly acid media was first noted for azorhoda- nines. The interaction of silver with azothiopropiorhodanines has a series of characteristic features compared with azorhodanines.In the case of azothio propiorhodanines, the most sensitive colour reaction with silver takes place in a CH3COOH±H3PO4 acid mixture, while in the case of azorhodanines the addition of even insignificant amounts of CH3COOH masks the reaction with silver, which can be accounted for by the greater stability of the silver complexes of azothiopropiorhodanines.The reaction of silver with azothiopropiorhodanines takes place in the presence of a 2 ± 5-fold excess of the reagent, while that with azorhodanines requires a 20 ± 50-fold excess of the reagent for saturation. Among the characteristic features of the reactions of silver with azothio- propiorhodanines, one should include also their high selectivity in relation to copper and other nonferrous metals, which is of great practical interest. Tyrodine 9a proved to be a promising reagent for lead.20, 69 ± 71 Lead(II) interacts with tyrodine, forming a violet complex.The colour reaction develops over a period of 20 ± 30 min. An increase in the temperature of the reaction mixture and changes in pH and in the reagent concentration do not lead to an increase in the rate of reaction.The curve for the time dependence of the optical density A=f(t) is characterised by an induction period of 15 ± 20 min, after which the reaction goes to completion in 1 ± 2 min. It has been noted that the presence of up to 2% of the lead complex of tyrodine accelerates the reaction so much that the induction period is eliminated.An autocatalytic mechanism of the colour reaction between lead and tyrodine has been proposed and can be described as follows in general terms.70 The first stage � the formation of a simple lead salt PbR involving the sulfo-group which is ionised under these condi- tions � is not accompanied by a colour change. This reaction is rapid (pH 3 ± 4), the salt formed is sparingly soluble under the reaction conditions, and a weak opalescence of the solutions is observed.The first stage involves the formation of a 1 : 1 complex (PbR) with participation of the hydroxyazothioketone group of the reagent as a result of either an intramolecular rearrangement or interaction with a second molecule of the reagent (which is present in solution in an excess relative to lead).This stage is slow; 2 ± 3 min after its onset, a change in the colour of the solution is observed and the opalescence vanishes. The third stage � the appearance of the final reaction product PbR2 � is also slow, since both interacting species, PbR and R, are relatively unreac- tive: lead is partially hydrolysed and the hydroxy-group in the benzene ring of the reagent is undissociated;16 both species are to a considerable extent hydrated.It has been suggested that the final product PbR2 actually possesses catalytic activity: in the fourth stage, it dissociates, as a result of which compounds or radicals (PbR . andR . ) appear and are in the most active form at the instant of their formation (for example, as a result of minimal solvation). In the subsequent rapid reactions PbR+R .PbR2 . and PbR . +R PbR2 the number of PbR2 molecules increases, which is in fact the basis of the catalytic effect. Other mechanisms for the reaction of lead with tyrodine have also been proposed.70 From the practical point of view, it is important that the development of the colour reaction between lead and tyrodine is not affected by large amounts of Ni, Co, Fe, Zn, Au, Pt, Rh, Be, Mn, Sn, and Ca; Ag, Hg,and Cu interfere.The high selectivity of the reaction permits the direct determination of lead in a series of systems (for example, in waste waters of the lead industries, anodic and cathodic copper, sulfuric acid) without preliminary separa- tion from the accompanying elements.20 A series of quantitative characteristics of the colour reaction of cadmium with tyrodine have been obtained.72 The colour reaction of cadmium is unaffected by 500 ± 10 000-fold excess amounts of alkali and alkaline earth elements and 10 ± 100-fold excess amounts of heavy metals; Pb, Hg, Ag, and Cu interfere.The influence of copper and mercury may be eliminated with the aid of thiourea. The colour reaction between cadmium and tyrodine has served as the basis of methods for the determination of cadmium in various types of water.Like lead, cadmium forms complexes with Cd :R=1 : 2 under analytical conditions.72 The colour reactions of azo-compounds based on 3-amino- rhodanine 5a ± f and substituted 6-aminorhodanine 6a,b with Pt, Pd, Au, and Ag are close to the corresponding reactions of azorhodanines as regards sensitivity, contrasting properties, and rate of development of the colour.On the other hand, like azothiorhodanines and azothiopropiorhodanine, these com- pounds do not react with rhodium and iridium, which is appa- rently associated with their instability in strongly acid media on heating, i.e. under conditions where the kinetically inert rhodium and iridium complexes react. The azo-compounds based on 3- aminorhodanine, for example p-phenolazo-3-aminorhodanine 5d, are of considerable interest for the determination of monovalent and divalent mercury primarily by virtue of their high selectivity.Mercury interacts with the reagent in strongly acid media at room temperature with formation of a stable raspberry-red complex. Large amounts of Pb, Zn, Bi, Be, Ba, Ca, Mo, Ir, Ru, and Rh do not interfere with the determination; Ag, Au, and Pd do interfere.The reagent has been used in the analysis of biological objects and industrial waters without preliminary separation of mercury from the accompanying elements (in contrast to the widely familiar method for the determination of mercury with the aid of pyridy- lazoresorcinol).19 Derivatives of benzeneazobenzeneazorhodanine 7a ± c enter into colour reactions with noble and heavy metals, but the sensitivity of the reactions is low.In terms of sensitivity, these reactions are inferior to the corresponding reactions with rhoda- nine-based monoazo-compounds. The colour reactions of azo- benzeneazorhodanines proceed in less acid media. This can apparently be accounted for by the characteristic features of the structure of the reagents, namely by the presence of an extended p-electron system.This decreases the acidity required for the protonation of the reagents, which is accompanied by a pro- nounced deepening of the colour (lmax for the neutral forms of benzeneazobenzeneazorhodanines is 440 ± 480 nm, while that for the protonated forms is 580 ± 620 nm).One of the important features of these reagents is that they enter into highly sensitive and contrasting colour reactions with noble and heavy metals in the presence of surfactants [csurf<CMC (CMC is critical micelle concentration)] and certain organic solvents when the content of the latter is more than 80%± 85%.21 ± 23 Two types of colour Azorhodanines, azothiopropiorhodanines, and their analytical application 217reactions are characteristic of modified derivatives of benzene- azobenzeneazorhodanine.The first type of the interactions of these reagents includes those with Cu, Hg, Ag, Pd, and certain other elements in neutral and alkaline solutions, the reactions being characterised by effective contrasting properties (Dl= 250 nm) and a higher sensitivity, the absorption coefficients e increasing by a factor of 5 ± 10.The interactions of the second type are those with Pt, Pd, Rh,and Ru (heavy metals and silver do not enter into reactions of this kind). The reactions proceed at pH 1 ± 3 and are characterised by an even more pronounced increase in sensitivity with virtually unchanged contrasting prop- erties.The reactions of Au and Pd with modified SBAPAR 7c are the most sensitive, the absorption coefficients e being 36105 and 66105 respectively. The sensitivity of reactions of the second type is greatly superior to that of the most sensitive reactions of noble metals with SCPAR and tyrodine. A high rate of development of the colour in these reactions should also be noted.The ratio of the reagent to the metal in the complex (molar ratios method) is 2 : 1 in the presence of cetylpyridinium chloride in the case of reactions of the first type. Compounds even more `saturated' with respect to the reagent are formed by reactions of the second type. The cause of such an increase may be associated both with the increase in the coordination capacity of the metal (for example, by virtue of dehydration on treatment with a surfactant or organic solvent) and with a change in the state of the reagent itself (for example, its aggregation, preceding the formation of the complexes) Table 2 presents the characteristics of reactions of metal ions with the reagents (including their modified forms).The com- pounds examined apparently belong to the class of the mosing reagents of the azorhodanine and azo-thiopropiorho- danine series for the determination of noble and heavy metals both in solutions and in the solid phase.13, 73 V.Characteristic features of the interaction with acido-complexes of platinum metals The reactions of sulfate (Pt, Pd, Rh), phosphate (Pt, Pd, Rh, Ir), acetate (Rh), and hydroxonitrate (Pd) complexes have been investigated together with the traditional chloride complexes using sulfochlorophenolazorhodanine and tyrodine as the reagents 1, 4, 8, 9, 53 ± 56, 82 (see {).The vast majority of methods for the determination of platinum elements are known to be applicable only to chloride systems. Analysis of other systems involves as a rule an additional stage in which the initial complexes are converted into chloride complexes.On the other hand, from the practical standpoint it is important to investigate the reactivities of acido-complexes of platinum metals based on various oxygen-containing acids (H2SO4, HClO4, H3PO4, CH3COOH). These acids are in fact largely employed at various stages in the processing of natural and industrial materials and in refining.Studies of the reactivities of dimeric complexes of platinum elements with a metal ± metal bond have acquired special importance. Such compounds have unusual thermodynamic and kinetic characteristics. They are readily aquated and enter into substitution reactions more vigorously than the chlorides. This leads to new possibilities in the analytical chemistry of the platinum elements.In the dimeric complexes,the platinum elements can manifest unusual chemical properties. Many of the difficulties in the development of methods for the photometric determination of platinum metals, for example in the sulfate or phosphate form, are known to be associated with hydrolysis and the formation of unreactive polymeric forms in dilute acid solutions.A characteristic feature of azorhodanines and their analogues consists in their ability to enter into highly sensitive colour reactions with noble metals in strongly acid media, in which readily hydrolysable forms of the platinum elements may retain their reactivity. Simultaneously with the investigation of the colour reactions of acido-complexes of platinum metals, the behaviour of the initial forms of the complexes under the conditions of their colour reactions has been investigated spectrophotometrically. Taking into account the results of such studies, a hypothesis has been put forward concerning the nature of the active forms of the platinum elements and the factors determining their yield.Procedures have also been proposed for the stabilisation (standardisation) of the active forms and in certain cases procedures for the conversion of the initial forms into active ones.53, 56 Dimeric platinum(III) phosphate and sulfate complexes as well as their chloride forms give rise to brightly coloured com- pounds with sulfochlorophenolazorhodanine and tyro- dine.8, 53 ± 56 Depending on the nature of the ligand and the oxidation state of the central atom in the initial complex, the conditions, kinetics, sensitivity, and selectivity of the colour reactions are different. Platinum phosphate and sulfate complexes interact with organic reagents in more acid media and a higher kinetic reactivity, compared with platinum(IV) chloride com- plexes, is characteristic of them.Markedly different effects of reductants on the colour reactions of acido-complexes of platinum have also been observed.Thus the reactions of platinum(IV) chloride complexes with organic reagents are strongly accelerated in the presence of reductants, whereas the influence of the latter on the colour reactions of the phosphates and sulfates is insignificant. The absorption spectra of platinum complexes with organic reagents are independent of the initial form of platinum.The characteristics (reaction conditions, solubility of the products, behaviour in relation to reductants) of the colour reactions of dimeric platinum sulfate and phosphate complexes are similar. The coloured compounds of platinum sulfate and phosphate with organic reagents are more soluble and more stable on heating than the chloride forms of platinum.In the reactions of the chloride complexes, heating of the solutions even to 40 ± 50 8C reduces the yield of the coloured compound, although the rate of reaction increases. Heating of the sulfate and phosphate com- plexes to 50 ± 70 8C shortens the reaction time from 4 h to 30 ± 60 min. Regardless of the nature of the ligand in the initial complex, the reaction with the organic reagent is apparently preceded by the stage involving the reduction of platinum(IV) to platinum(II).In the case of platinum(III) sulfate and phosphate complexes, the Pt7Pt bond is initially dissociated with formation of platinum(II) and platinum(IV), after which platinum(IV) is reduced to platinum(II). The reduction of platinum in the sulfate and phosphate complexes is faster than in the platinum(IV) chloride complexes and takes place at lower concentrations of the reductants in solution.One of the causes of the appreciable difference between the reactivities of the acido-complexes of platinum investigated in relation to organic reagents probably consists in the difference between the rates and conditions in the aquation and reduction of the initial forms. Evidently, the similarity of the behaviour of platinum sulfate and phosphate complexes and the characteristics of the colour reactions can be accounted for by the similar structures of such complexes.The reactions of dimeric platinum sulfate and phosphate complexes are highly sensitive and selective. The complexes exhibit a high kinetic reactivity. All these features make it possible to classify the reactions considered as the most promising for the determination of platinum.Whilst showing equal resistances to the interfering effects of alkaline earth and alkali metals, the { The sulfate complexes of platinum(III) {K5H3[Pt4SO4(HSO4)8O5]}, tri- and tetra-valent ruthenium {K2H[Ru2(O .H2O)2(SO4)3l}, and rhodium(II) [Cs4Rh2(SO4)4(H2O) .2H2O), the phosphate complexes of platinu- m(II, IV) {K2(H3O)3[Pt2(PO4)3(HPO4)H2O]}, palladium(II) {K3(H3O)3[Pd(P2O7)2] . 2H2O}, rhodium(III) [Na3(H3O)6Rh(P2O7)3], iri- dium(IV) {K6H2[Ir4O3(H2PO4)10(OH)8]}, and tri- and tetra-valent ruthe- nium [K3Ru2(H2P2O7)5 . 7H2O], the acetate complexes of rhodium(II) [Rh2(CH3COOH)4 . 2H2O] and rhodium(III) [Rh(CH3COO)3], and the hydroxonitrate complex of palladium(II) [Pd(NO3)2(OH)2] have been obtained by members of the Institute of General and Inorganic Chemistry of the Russian Academy of Sciences.83 ± 91 218 R F Gur'eva, S B Savvinselectivity of the sulfate and phosphate complexes with respect to Cu, Fe, and other transition metals as well as Rh and Ir is markedly higher compared with the chloride forms.However, the selectivity with respect to silver and mercury is higher for the chloride forms.On the other hand, from the practical point of view, it is important that conditions have been found under which platinum sulfates and phosphates are stable and retain their reactivity for a long time. The possibility of converting kinetically inert platinum chloride complexes into more reactive ones, for example the phosphate complexes, has also been demonstrated; in this case, the reaction takes place in concentratedH3PO4 solutions at 200 8C in the presence of perchloric acid, which decomposes the chlorides, oxidising Cl7 to Cl2, the aquohydroxo-complexes formed react directly with H3PO4.Palladium(II) sulfate, phosphate, hydroxonitrate, and chlor- ide complexes interact with SCPAR under similar conditions.The spectroscopic characteristics of these complexes are close, but the kinetic reactivity of the sulfates, phosphates, and hydroxonitrates is higher than that of the chlorides. The solubilities of the products of the interaction of the sulfates, phosphates, and hydroxonitrates are higher than those of the corresponding compounds derived from the chlorides.8, 9, 54, 82 Iridium sulfate and phosphate complexes are less active in reactions with organic reagents than the chloride complexes. On prolonged heating, iridium in the form of phosphates and sulfates reacts with sulfochlorophenolazorhodanine in strongly acid media.However, these reactions are less sensitive than those involving the chloride complexes.8, 9 Tri- and tetra-valent ruthenium sulfate, phosphate, and chloride complexes react with sulfochlorophenolazorhodanine to form coloured compounds with similar spectrophotometric char- acteristics.The reactions of ruthenium sulfate and phosphate with the reagent are preceded by a stage involving the conversion of the initial forms of ruthenium into the chloride form, which has been identified as [Ru2OCl10]47 on the basis of absorption spectra.Regardless of the nature of the ligand and the oxidation state of ruthenium in the initial complex, the aquochlororuthenium(III) complex apparently reacts with the organic reagent. The condi- tions of the colour reaction (a mixture of HCl with CH3COOH) and the reducing properties of the reagent ensure the conversion of the initial forms of ruthenium into the active form.53, 56 In the presence of organic solvents, for example CH3COOH, the formation of the chloride form from the initial phosphate and sulfate forms is possible in more dilute HCl solutions, for example in 0.5 ± 2 M solutions instead of 5 ± 7 M solutions.In the presence of organic solvents, the degree of hydrolysis of the chloride form apparently diminishes; under these conditions, the organic solvent is a stabilising additive.Furthermore, in the presence of organic solvents, the formation of the active form � the aquochloro- ruthenium(III) complex � and the substitution for the organic ligand take place more readily than in the case of large amounts of HCl, which entails an increase in the sensitivity of the reaction.Acido-complexes of ruthenium do not enter into colour reactions with sulfochlorophenolazorhodanine in sulfuric and phosphoric acid solutions, which is due to the formation of unreactive hydro- lysed forms under these conditions. The interaction of ruthenium with sulfochlorophenolazorhodanine results in the formation of 1 : 2 complexes. Under the conditions of its interaction with ruthenium, the reagent is in a neutral form 16 and apparently behaves as a monodentate ligand, ruthenium being coordinated to the reagent via the thioxo-group of rhodanine.Regardless of the nature of the ligand and the oxidation state of the central atom in the initial rhodium complexes � rhodiu- m(II) and rhodium(III) sulfates, phosphates, and acetates and rhodium(III) chlorides � coloured compounds with lmax=510 nm are formed in the reactions with SCPAR.Similar dependences of the yields of the reaction products on the nature and concentration of the acids and the reagent are observed for the above rhodium complexes. It has been suggested that the nature of the reactive form of rhodium in the reactions with SCPAR is the same for all the initial forms of di- and tri-valent rhodium.There are significant differences in the kinetics and sensitivities of the reactions. The dimeric rhodium(II) acetate and sulfate complexes are more reactive in the ligand substitution reaction than, for example, rhodium(III) chlorides and acetates; the highest reac- tivity has been noted for the dimeric acetate complex. The rate of formation of the coloured rhodium(II) acetate complex of SCPAR is unusually high.The reaction develops at room temperature over a period of 10 ± 20 min and is character- ised by a fairly high sensitivity. The high kinetic reactivity of the dimeric rhodium(II) complexes compared with the corresponding rhodium(III) complexes is due to their ability to form `more readily' the labile aquo-complexes, with participation of which many processes, including ligand substitution reactions, take place faster.In the case of the rhodium(III) sulfate, phosphate, and chloride complexes, one may postulate that one of the intermedi- ate stages of the overall complex formation reaction between rhodium and SCPAR is the conversion of the initial forms into the more reactive (dimeric acetate or acetate-sulfate) rhodium(II) complexes.This is confirmed by the fact that the conditions of the colour reactions of acido-complexes of rhodium with SCPAR (mixture of 7 M H2SO4 and 6 M CH3COOH, heating to 60 ± 70 8C, excess of the reducing agent) are virtually identical with those in the preparative synthesis of these complexes.4, 8, 9 The study of the state of solutions of rhodium acetate and sulfate complexes has shown that under certain conditions they retain their reactivity in relation to SCPAR for a fairly long time. The high kinetic reactivity of the dimeric rhodium complexes [in the first place, rhodium(II) acetate] as well as the high sensitivity of their reactions with SCPAR are properties which may serve as the basis for the creation of new high-speed methods for the determi- nation of rhodium via reactions of its dimeric complexes.VI. Applications 1. Photometry One of the main fields in which azorhodanines and azothiopro- piorhodanines are used is photometry. The distinctive features and advantages of azorhodanines as photometric reagents are as follows. Firstly, the high sensitivity of the determination of the elements, primarily the platinum metals, and, secondly, the interaction with metal ions over a wide pH range.These properties are due to the presence of several functional-analytical groups in the reagent molecules. For example, there are three regions corresponding to the interaction of silver with SCPAR � pH 2 ± 6 and 7 ± 10 and 1 ± 6 M H2SO4, which makes it possible to employ in practice the `optimum' region of the interaction, depending on the composition and the content of the object analysed.Thus, in the determination of silver with the aid of SCPAR, for example at pH 5 ± 7, the presence of the remaining platinum group elements (except palladium) is permissible, whereas the determination of silver in strongly acid media (for example, in 6 M H2SO4) permits the presence of a much greater excess of nonferrous and heavy metals.Thirdly, the reactivity of the reagents in relation to surfactants and certain organic solvents. Under certain conditions, this leads to the formation of modified forms of the reagents, the colour reactions of which with platinum and other metals are charac- terised by an unusually high sensitivity [e=(100 ± 600)6103].Fourthly, the interaction with platinum metals in the form of various acido-complexes (chlorides, phosphates, sulfates, and acetates). Such interaction is due to the fact that azorhodanines and azothiopropiorhodanines react with platinum metals in strongly acid media, where the presence of stabilising additives (CH3COOH, HCOOH, metal salts) is permissible. All these features hinder the hydrolysis of the platinum metals, i.e.hinder the formation of their unreactive forms. Azorhodanines, azothiopropiorhodanines, and their analytical application 219Fifthly, the possibility of analysing solutions containing both individual forms of platinum metals and their sum. For example, ruthenium is determined in chloride and sulfate solutions, which are used for its preparation in the course of the complex processing of copper ± nickel ores.The conditions in the colour reaction of ruthenium with SCPAR (mixture of HCl with CH3COOH) (Table 2) and the reducing properties of the reagent ensure a fairly rapid conversion of the initial forms of ruthenium into the reactive form�ruthenium(III) chloride. Sixthly, the high selectivity of the determination.Despite the fact that azorhodanines and azothiopropiorhodanines are group reagents, a high selectivity of the determination is attained by virtue of different spectroscopic characteristics of the colour reactions with the elements (sensitivity and contrasting properties of the reactions), reaction conditions (nature and concentration of the acids, pH), reaction kinetics, the stability of the complexes formed, and, in the case of noble metals, also the different reactivities of their initial forms.Highly sensitive methods have been developed for the deter- mination of Pt, Pd, Rh, Ir, orm of various acido- complexes), Au, and Ag with sulfochlorophenolazorhodanine, Pt, Pd, Au, Ag, Pb, Cd, and Cu with tyrodine, Pd, Pt, Ru, Rh,and Cu with sulfobenzeneazorhodanine derivatives, and Hg and Cu with p-phenolazo-3-aminorhodanine, which have found applications in the analysis of various natural and industrial objects.1, 2, 4 ± 11, 13 ± 23, 50 ± 56, 58, 69, 72, 74, 76, 77, 82, 92 ± 100 2.Differential photometry The problem of creating direct differential-photometric (DP) methods for the determination of noble metals which are no less accurate than gravimetric or titrimetric methods (or approach them in accuracy) but are greatly superior to the latter in the speed of the determination has been successfully solved by virtue of the application of azorhodanines and tyrodine.The success has been due to the ready solubility of both the reagents themselves and of their complexes with noble metals, the possibility of the direct determination of the noble metals and a series of accompanying elements in natural and industrial objects, the possibility of using various acido-complexes as the initial form in reactions with the reagent, the fairly high kinetic reactivity of some of these com- plexes (phosphates, sulfates, acetates), and, in the case of Pt, Rh, and Ir chloride complexes, the possibility of activation, for example with ascorbic acid or copper salts.In the differential measuring procedure, the fundamental light absorption law holds over a wide range of concentrations of the noble metal. The DF methods developed are universal and, when combined with the photometric version, make it possible to determine 0.2 ± 250 mg ml71 of the noble metal.High-speed DF methods are used in the analysis of alloys and composite materials for the direct determination of silver (Ag ± Cu, 10% ±90% Ag), platinum (Pt ± Rh, Pt ± Ir, 10%± 20% Pt), and ruthenium [lead ruthenite, bismuth ruthenite, 30% Ru; residue remaining after the combus- tion of the waste from the manufacture of materials for the electronic industry containing Pb and Bi (ash), 50% Ru] and also in the analysis of other objects.24, 58, 74, 77 3.Sorption-photometric methods The methods in which azorhodanines are used both for determi- nation and detection, on the one hand, and for the separation and concentration of reagents, on the other, merit special attention. Such are the electrophoretic and chromatographic (on paper and in a thin layer of sorbent) methods as well as methods based on the use of immobilised reagents.Their principal advantages are simplicity, clarity, and high speed of execution (primarily at the level of qualitative and semiquantitative visual determinations � test methods) and the possibility of combining separation (includ- ing the procedure based on the specific effect of the reagent) and concentration from large volumes under static or dynamic con- ditions.a. Chromatographic and electrophoretic method Conditions have been found and limits have been established for the detection of toxic heavy metals such as Cu, Pb, Zn, Fe, Ni, Cd, and Hg (the organic forms are methyl-, propyl-, and phenyl- mercury, while the inorganic forms are Hg+ and Hg2+) on a thin layer of sorbent (sorbfil, silufol) with the aid of azorhodanine derivatives�SCPAR and PATAR.The sensitivity of the visual determination is 0.1 ± 0.5 mg, and for Cu 0.01 mg, of the element in the zone. The possibility of the semiquantitative estimation of the content of both individual toxic elements and their sum has been demonstrated. The selectivity of the detection is achieved as a result of the specificity of the reagents, variation of the colour reaction conditions in a thin layer (nature of the acid, pH), and selection of the separation conditions on the chromatograph.Conditions have been developed for the separation of various forms of mercury � organic (phenyl-, methyl-, and propyl- mercury) and inorganic (Hg+, Hg2+). At the same time, when SCPAR is used in an ammoniacal medium as the mobile phase, it is possible to separate methylmer- cury (Rf=0.9) and propylmercury (Rf=0.45) from phenylmer- cury (Rf=0.0) and inorganic mercury (Rf=0.0) in the form of their coloured complexes with the reagent.The separation of organic and inorganic forms of mercury and their separation from other heavy metals with subsequent detection by means of SCPAR have also been achieved using other chromatographic systems. When chromatograms are sprayed with azorhodanines, red zones appear against the yellow background of the sorbent which is due to the formation of complexes of different forms of mercury with the reagent.The colour in the zones develops over a period of 1 ± 5 min and is maintained for between 2 h and several days. The methods for the determination of toxic elements in a thin layer of sorbents have been tested on food products.12 Procedures have been devised for the development of chro- matographic and electrophoretic zones of both individual noble metals (except osmium) and their sum in the form of various acido-complexes. The sensitivity of the visual detection is 0.5 ± 0.01 mg of the element in the zone.Together with high sensitivity and stability (as a function of time) of the colour, the advantage of SCPAR as the developer over other reagents consists in the fact that SCPAR develops zones corresponding to different acido-forms of the noble metal.Methods have been proposed for the electrophoretic and paper and thin-layer chromatographic separation with subsequent detection of the noble metal (in the form of chloride complexes) by means of SCPAR in the Pd ± Ir, Pd ± Rh, Ir ± Rh, Pt ± Pd ± Au, Pd ± Pt ± Ir, Pd ± Pt ± Rh, Pd ± Au, and Pt ±Rh systems.These methods have found applications in the analysis of alloys.101 ± 102 The electrophoretic mobilities of noble metals in the form of coloured compounds with azorhodanines in buffer solutions based on formic, acetic, and oxalic acids (pH 1.5 ± 1.7), acetic and boric acids (pH 2), and pyridine and oxalic acid (pH 5.9) have been investigated.The electrophoretic characteristics have been determined. A clear-cut separation of the zones is observed in the case of Pd ± Ag, Pt ± Ag, and Pd ± Au in the form of their complexes with SCPAR and other reagents.103 b. Sorption-photometric methods based on the use of immobilised reagents The prospects for the analytical application of the colour reactions of azorhodanines in the solid phase have been demonstrated.13, 73 Azorhodanines immobilised on a polymer matrix (for example, on a polyamide caprone membrane, a polyacrylonitrile fibre filled with finely disperse KU-2 cation exchanger, and a fabric with phosphate groups) retain their reactivity in relation to noble and heavy metals over a wide pH range (1 ± 10), the reactions being fairly rapid.The similarity of the spectroscopic characteristics of the reactions of azorhodanines in solution and in the sorbent phase has been noted. 220 R F Gur'eva, S B SavvinHigh-speed and sensitive methods for the determination of Pt, Pd, Au, Ag, Cu, and Hg with a 1 ± 3 mg ml71 detection limit have been developed with the aid of azorhodanines and azothiopropio- rhodanines immobilised on polymer supports.22, 23 A micropo- rous polyamide material (a caprone membrane) or a polyacrylonitrile fibre filled with a finely-disperse cation exchanger (KU-2) have been used as supports.The supports are employed in the form of discs 0.8, 1, and 2 cm in diameter and with a mass of 2 ± 10 mg.The reagents are immobilised by immersing the support disc in 1075 ± 1076 M solutions of the reagents in a weakly acid medium (pH 2 ± 5) for 15 ± 30 min. Two versions of the determination have been proposed � static and dynamic. In the first case, the coloured discs (photometric elements) are lowered into a flask containing the sample (the preliminary preparation of the sample is carried out � acidification and introduction of additives activating the colour reaction and givo a high rate of response) and are kept there for 5 ± 60 min at 20 ± 50 8C.In the second case, the coloured discs are placed in the cell of an optical sensor analyser or a vacuum pump is employed. The colour change on the discs is estimated visually (test methods) or from the change in the diffuse reflection coefficients.A high sensitivity of the determination is attained both as a result of the employment of qualitative reagents and the possibility of accu- mulating the element analysed from dilute solutions on the coloured support disc. The selectivity is ensured by the specificity of the immobilised forms of the reagents, the sorption conditions, and the kinetic differences between the colour reactions on the polymer matrix. A sorption-photometric method for the determination of silver in natural waters with a detection limit of 5 mg litre71 in accordance with the `sorbent ± metal ± reagent' scheme has been developed.It involves the sorption of silver on discs made of polyacrylonitrile fibre filled with KU-2 cation exchanger and subsequent detection of silver in the sorbent phase with the aid of rhodazol Kh (compound 1f) at 490 nm.23 4.Extraction-photometric methods The extraction-photometric method for the determination of silver (the `two reagents' method) is based on the ability of silver to form mixed coloured compounds with azorhodanines or azothiopropiorhodanines and certain macrocyclic compounds (MCC) of the type 12,13-dibenzo-1,15-diaza-5,11-dioxacyclononadeca-1,14-diene 12,13-dibenzo-1,15-diaza-5,8,11-trioxacyclonondeca-1,14-diene TheMCCare used initially for the selective extraction of silver in the form of ionic associated species [Ag-MCC] Pi (Pi=picrate).Then tyrodine, for example, is added to the organic layer.It interacts with the associated species to form mixed coloured compounds (reagent ± Ag-MCC) characterised by an unusually high sensitivity; e=(4 ± 9)6105. With the aid of the `two reagents' method, it has been possible to reduce by more than an order of magnitude the detection limit for silver and also to increase the selectivity of the determination relative to Au, Pd, Pb, and Hg (Table 3).57, 58 The method has been used in the determination of silver in drinking water.A somewhat greater complexity of this method compared with the usual photometric version must be included among its disadvantages. In order to increase the selectivity of the determination of silver with SCPAR in an inorganic raw material, silver was extracted (in the form of its complex with the reagent) with cyclohexanone (in the presence of tributyl phosphate) and photo- metric measurements on the coloured organic layer was subse- quently carried out.58 5.Automatic analytical schemes A version of the flow-injection analysis (FIA) has been used with the aid of SCPAR for the spectrophotometric determination of noble metals.104, 105 The difficulty of devising such methods arises from the kinetic inertness of noble metal complexes in reactions with the organic reagent.In order to investigate the conditions under which the colour reactions of Au and Ag take place, a two-channel flow system was employed. Best results in the determination of silver were obtained with the following parameters of the FIA system: microvolume of solution containing the metal to be determined V0=200 ml; length of thermostated reaction coil l=30 cm; flow rate of the support (dilute acid solution) with the metal to be determined u1=2.8 ml min71; flow rate of reagent solution u2=1.2 ml min71.The range of silver contents which can be determined is 0.3 ± 4.5 mg litre71. The speed of the analysis is 180 determinations per hour. In the case of the slower reaction involving gold chloride complexes, the peak height is a maximum at V0=200 ± 300 ml, u1=1.2 ml min71, u2=0.8 ml min71, and l=60 cm.The range of gold contents which can be determined is 2 ± 19 mg litre71. The speed of the analysis is 90 determinations per hour. Comparison of the conditions in the colour reactions of Au and Ag with SCPAR in the usual photometric version and in the FIA version demonstrates an appreciably lower optimum acid concentration in the latter case, which is necessary to ensure a higher rate of reaction and this is in its turn important for obtaining the narrowest and highest peaks and also attaining a higher number of determinations per unit time.The flow-injection method for the determination of palladium with SCPAR at pH 5 is characterised by a speed of analysis of 120 determinations per hour.105 The high speed of analysis in this case N O N O 10 N O N O O 11 Table 3.Photometric determination of silver with tyrodine by the direct method and by the `two reagents' (MCC± tyrodine) method. Characteristics Direct deter- `Two reagents' of method mination with method, determination tyrodine a in extracts see b see c Range of concentra- 0.2 ± 2.4 0.01 ± 0.10 0.02 ± 0.40 tions of compounds determined /mg ml71 Detection limit 0.04 0.015 0.008 /mg ml71 e (lmax/nm) 56104 (540) 4.56105 (550) 96105 (580) Selectivity factor Zn 56103 16104 16104 Co 16103 16104 76104 Pb 50 7 16104 Hg 1 200 200 Pd 1 16103 100 Au 1 500 7 Cu 56103 500 36103 Cl 1 25 25 a In 1 M H3PO4+10 M CH3COOH solution.17 b Tyrodine ±MCS (com- pound 10) in dichloroethane.78 c Tyrodine ±MCC (compound 11) in a chloroform ± triphenylphosphine mixture.57 Azorhodanines, azothiopropiorhodanines, and their analytical application 221is due primarily to the high rate of interaction of palladium with SCPAR in weakly acid media.The necessary selectivity of the method is achieved with the aid of masking substances.The range of concentration which can be determined is 0.045 ± 30 mg litre71. Thus a high number of determinations of noble metals per unit time is attained in FIA systems without loss of sensitivity and without narrowing the range of contents which can be determined compared with the static version employing the same reagent � SCPAR. Continuous flow analysis (CFA) also belongs to the class of high-speed automatic analytical methods.106 A photometric method has been proposed for the determination of silver with SCPAR in the presence of noniogenic surfactants (Triton- 100 X).107 The introduction of the surfactant into the system stabilises the hydrodynamic flow parameters (ensures the stability of the rate and uniformity of the flow) and improves the solubility of the reagent, which has a positive effect on the reproducibility of the results.The selectivity of the determination of silver relative to Pb, Co, Ni, Cu, and Zn also increases under these conditions. The principal characteristics of the method are as follows: range of silver contents which can be determined (0.4 ± 0.5)61075 M; relative standard deviation 0.05; Triton-100X content 0.3% ± 0.03%.The determination is carried out in 0.1 M HNO3. 6. Other methods The kinetic determination of rhenium is based on the catalytic effect of tetravalent rhenium ions on the reduction of benzene- azobenzeneazorhodanine 7a by tin(II) in an alkaline medium (1.5 ± 2.5 M NaOH) and on the time variation of the optical density of the reagent solution.The calibration plot is linear in the range 0.01 ± 0.1 mg of rhenium in 1 ml. The determination of 0.02 mg of rhenium in 1 ml is not interfered with by Mo, W, Ca, and Cu. The method has been used for the high-speed determi- nation of the rhenium content in specimens of cupriferous sand- stone.79 ± 81 In the concentration and separation of noble metals with chelating sorbents based on rhodanine, azorhodanine, and azo- thiopropiorhodanine, one uses, for example, cellulose, styrene ± - divinylbenzene copolymers, and sephadexes as the matrix.Polymeric compounds interact under approximately the same conditions as the monomers and the selectivity is also similar. The concentration and separation methods based on chelating sorbents have found extensive application in the analysis of ores, industrial solutions, and natural waters.108 ± 115 VII.Conclusion Using azorhodanines and azothioppiorhodanines as the reagents, it has been possible for the first time to approach the problem of the direct and high-speed determination of noble metals in a form other than the chlorides and to achieve a sensitivity of the determination which is unusually high for analytical reagents (e'106).There are diverse applications of azorhodanines and azothiopropiorhodanines, including their modified and immobilised forms, in inorganic analysis � in photometry, differential photometry, sorption photometry (pri- marily in methods where the reagent is used simultaneously for the concentration, extraction, separation, testing, and determination, including procedures employing sensitive photometric cells), in extraction photometry (including the `two reagents' method), and in chromatographic and electrophoretic analytical methods.The likely usefulness of azorhodanines and azothiopropiorhodanines in automatic analytical schemes (FIA, CFA) has been demon- strated. 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F I Danilova, V A Orobinskaya, V G Parfenova, R F Propistsova, S B Savvin Zh. Anal. Khim. 29 2150 (1974) a 93. G V Myasoedova, I I Antokol'skaya, L I Bol'shakova, O P Shvoeva, S B Savvin Zh. Anal. Khim. 29 2104 (1974) a 94. O P Shvoeva, N I Shcherbinina, G V Myasoedova Zh. Anal. Khim. 38 221 (1983) a 95. Yu G Rozovskii, Candidate Thesis in Chemical Sciences, Institute of the Geochemistry of Minerals, Academy of Sciences of the USSR, Moscow, 1974 96. N N Roeva, Candidate Thesis in Chemical Sciences, Institute of Geochemistry, Academy of Sciences of the USSR, Moscow, 1985 97. N N Basargin, Yu G Rozovskii, V A Sychkova, P I Slyunyaev Zavod. Lab. 39 3 (1973) 98. N N Basargin, Yu G Rozovskii, V A Sychkova, N N Nikol'skaya, Z A Ezhkova, in Metody Khimicheskogo Analiza Gornykh Porod i Mineralov (Methods of the Chemical Analysis of Rocks and Minerals) (Moscow: Nauka, 1973) p. 10 99. N N Basargin, Yu G Rozovskii, V A Sychkova Izv. Akad. Nauk SSSR, Ser. Khim. 2360 (1971) g 100. N N Basargin, Yu G Rozovskii, I V Malinina, in Teoreticheskie i Prakticheskie Voprosy Primeneniya Organicheskikh Reagentov v Analize Mineral'nykh Ob'ektov (Theoretical and Practical Problems in the Application of Organic Reagents in the Analysis of Mineral Objects) (Moscow: Nauka, 1976) p. 125 101. T G Akimova, R F Propistsova, S B Savvin Zh. Anal. Khim. 29 2365 (1974) a 102. M P Volynets, A N Ermakov, S I Ginzburg, R F Gur'eva, T V Dubrova, M I Yuz'ko, T A Fomina Zh. Anal. Khim. 32 914 (1977) a 103. T G Akimova, R F Propistsova, S B Savvin Zh. Anal. Khim. 28 2005 (1973) a 104. L K Shpigun, R F Gur'eva Zh. Anal. Khim. 46 2187 (1991) a 105. P M Shiundu, P D Wentsell, A P Wade Talanta 37 329 (1990) 106. L K Shpigun Zh. Anal. Khim. 45 1045 (1990) a 107. I M Maksimova, D N Korolev, E I Morosanova, Yu A Zolotov Zh. Anal. Khim. 50 919 (1995) a 108. G V Myasoedova, O P Eliseeva, S B Savvin, N I Uryanskaya Zh. Anal. Khim. 27 2004 (1972) a 109. G V Myasoedova, L I Bol'shakova, O P Shvoeva, S B Savvin Zh. Anal. Khim. 28 1550 (1973) a 110. N N Basargin, Yu G Rozovskii V MZharova, in Organicheskie Reagenty i Khelatnye Sorbenty v Analize Ob'ektov (Organic Reagents and Chelating Sorbents in the Analysis of Objects) (Moscow: Nauka, 1980) p. 82 111. G V Myasoedova, S B Savvin, in Khelatoobrazuyushchie Sorbenty (Chelating Sorbents) (Moscow: Nauka, 1984) 112. G V Myasoedova,M P Volynets, T A Koveshkina, Yu A Belyaev Zh. Anal. Khim. 29 2253 (1974) a 113. S B Savvin, I I Antokolskaja, G V Myasoedova, L I Bolshakova J. Cromatogr. 102 287 (1974) 114. G V Myasoedova, P N Komozin Zh. Neorg. Khim. 39 280 (1994) f 115. N N Basargin, Yu G Rozovskii, V A Volchenkova, Yu F Zibarov Zavod. Lab. 62 5 (1996) Azorhodanines, azothiopropiorhodanines, and their analytical application 223a�J. Anal. Chem. (Engl. Transl.) b�Pharm. Chem. J. (Engl. Transl.) c�Russ. J. Gen. Chem. (Engl. Transl.) d�Chem. Geterocycl. Compd. (Engl. Transl.) e�Dokl. Chem. Technol., Dokl. Chem (Engl. Transl.) f�Russ. J. Inorg. Chem. (Engl. Transl.) g�Russ. Chem. Bull. (Engl. Transl.) 224 R F Gur'e

ISSN:0036-021X    

出版商:RSC    

年代:1998

数据来源: RSC

摘要:

Abstract. The role of metal ions as enzyme effectors is considered. Data on inhibitory and activating effects of metal ions are summarised. The dual character of action of the effectors depend ing on their concentration and the nature of the enzyme is highlighted. The analytical applications of these effects are discussed. The bibliography includes 66 references. I. Introduction The role of metal ions in the mechanism of catalytic effects of enzymes is diverse.1 One group of enzymes is characterised by the presence of one or several tightly bound metal ions involved in catalytic processes (metalloenzymes).Other enzymes do not contain directly bound metal ions but manifest catalytic activity only when the ions are present (metal-dependent or metal- activated enzymes). For enzymes of the third group, metals are not obligatory participants of the catalytic reaction; however, their presence in the system influences the enzyme activity (metal- independent enzymes).Three types of metal ± enzyme complexes are generally formed in enzymic catalysis: (1) Complexes through the ligand L (substrate) as a bridge. Here, the metal ion binds only to the ligand and takes part in the catalysis not being directly bound to the enzyme (Fig. 1, path- way I ). These complexes are typical of many enzymes, e.g., creatine kinase,2, 3 and some other kinases and synthetases.4± 6 (2) Complexes in which the metal ion binds the ligand completely (Fig. 1, pathway IIa) or partly (Fig. 1, pathway IIb). The existence of enzyme ± metal ± ligand complexes has been demonstrated by NMR (pyruvate kinase),7 X-ray analysis (car- boxypeptidase),8 and infrared spectroscopy (carbonic anhy- drase).9 There exist at least two routes 1 for the formation of the enzyme ± metal ± ligand complexes with a bridge metal:1 where L is the ligand.A third route of enzyme ± metal ± ligand complex formation is also possible, where the metal ion non-bound to the enzyme manifests strong affinity for the ligand.(3) Complexes with the enzyme as a bridge. Here, the metal ion and the ligand interact with different sites of the enzyme (Fig. 1, pathway III ). This type of complex is characteristic of glutamine synthetase 10 and some other synthetases.11, 12 The catalytic activity of enzymes, which is their most distinc- tive feature, can vary widely depending on the mode of interaction of metal ions with enzymes.Therefore, the study of effects of metal ions on enzyme activity has both theoretical and practical signifi- cance. E7Mn+7L, (1) L E+Mn+ E7Mn+ E+L E7L E7Mn+7L, (2) Mn+ Mn++L Mn+7L E7Mn+7L. (3) E E P Medyantseva, MG Vertlib, G K Budnikov Kazan State University, ul. Lenina 18, 420008 Kazan, Russian Federation.Fax (7-843) 238 09 94. Tel. (7-843) 231 85 46 (E P Medyantseva), (7-843) 231 84 09 (G K Budnikov) Received 4 August 1997 Uspekhi Khimii 67 (3) 252 ± 260 (1998); translated by R L Birnova UDC 577.15.049 Metal ions as enzyme effectors E P Medyantseva,MG Vertlib, G K Budnikov Contents I. Introduction 225 II. The effect of metal ions on the catalytic activity of enzymes 226 III.Changes in the catalytic activity of enzymes in the presence of metal ions: an analytical aspect 230 IV. Conclusion 231 E I Mn+ E L E L E IIa E Mn+ L E Mn+ E III E L Mn+ L L Mn+ Mn+ E E L Mn+ IIb E L Mn+ E L Mn+ Mn+ L E L Mn+ E L Mn+ Mn+ L Figure 1. The pathways of interaction of the enzyme (E) with the metal ion (Mn+) and the ligand (L). For I ± III, see text. Russian Chemical Reviews 67 (3) 225 ± 232 (1998) #1998 Russian Academy of Sciences and Turpion LtdThis review will not consider metalloenzymes and metal- dependent enzymes.The role of metals in catalytic processes involving these enzymes is the subject of special studies and presents particular interest for biochemistry and medicine (see Refs 13 ¡¾ 15). The review summarises the papers devoted mainly to the effect of metal ions on catalytic properties of metal- independent enzymes.II. The effect of metal ions on the catalytic activity of enzymes During the last 10 ¡¾ 15 years interest in biochemical methods of determination of inorganic toxic agents based on the analysis of properties of native and immobilised enzymes in the presence of metal ions has increased. And the main contribution to the development of this field has been made by Russian scientists.The same conclusion can be made from the analysis of the Proceedings of the 5th International Symposium `Kinetics in Analytical Chemistry' held in Moscow in 1995, which shows that the chemico-analytical aspect of the effect of metal ions on the catalytic activity of enzymes was mostly covered by reports of Russian investigators.Most commonly, the catalytic activity of enzymes in the presence of metals is evaluated as changes in the rate of the enzymic reaction. This can be done by measuring the changes in the concentration of the substrate or reaction products with time. A physicochemical parameter (most often, optical density or an ionisation potential) related to the substance concentration is used as the analytical signal. 1. The inhibitory effect of metal ions Far greater attention has been given to the study of the inhibitory effect of metal ions on the catalytic activity of enzymes than to the activating effect. In most studies, the effect of doubly charged metal cations was examined. In particular, doubly charged ions, like Hg2+, Cu2+, Co2+, Cd2+, Zn2+, Pb2+, Ni2+, and some others were shown to inhibit bovine ribonuclease A,16 ure- ase,17 ¡¾ 19 cholinesterases,20 ¡¾ 22 and some others.The I50 value (i.e., the inhibitor concentration causing a 50% decrease in the enzyme catalytic activity) and the value of the inhibition constant, Ki, are commonly used for the quantitative estimation of the inhibitory effect. Thus in order to estimate the inhibitory effect of metal ions (Ag+, Hg2+, Cu2+, Zn2+, Pb2+, Ni2+, Cd2+, Co2+) on St.sap- rophyticus urease, the I50 values were determined for each of them.17 The smaller the I50 value the stronger the metal inhibits the enzyme. Ag+ions (I50=861076 mol litre71) appeared to be the most effective inhibitor of St. saprophyticus urease. According to the strength of their inhibitory effect, these ions can be arranged in the following series: Ag+ >Ni2+ >Cd2+ >Co2+ >Hg2+ > Cu2+ > Zn2+ > Pb2+.The inhibitory action of these ions begins to manifest itself at concentrations from 161077 to 2.561076 mol litre71. The maximum inhibitory effect (100%) is observed at the concentrations 561074 mol litre71 (Ag+) and 161073 mol litre71 (Hg2+, Cu2+, Zn2+, Pb2+).In the presence of Ni2+, Cd2+, and Co2+ ions (1073 mol litre71) in the reaction medium, complete suppression of the enzyme activity does not occur: urease retains up to 10% ¡¾20% of its original activity. The inhibition of urease by the ions assayed is independent of the enzyme concentration and the time of incubation with the solution of a metal salt.The inhibitory effect of all these ions is reversible and is easily eliminated by dilution or desalting of the enzyme on a Sephadex G-25 column. However, when the concentration of Pb2+, Hg2+, Zn2+, and Ag+ ions in the reaction medium exceeds 1073 mol litre71, irreversible denaturation of the enzyme takes place. The inhibition, which precluded reactivation of the enzyme, was not observed for Cu2+, Ni2+, Cd2+, and Co2+ ions in the whole range of the concentrations studied (161077 ¡¾ 161073 mol litre71).Although the authors do not draw such a conclusion, it may be assumed that in this particular case reversible non-specific inhibition takes place. The values of apparent inhibition constants, Ki, for Mg2+, Co2+, and Mn2+ ions, which competitively inhibit the activity of Ca2+-ATPase of skeletal muscle plasma membranes, have been determined.23 The smaller the Ki value the stronger the effect produced by the inhibitor.It was found that according to their affinity for Ca2+-ATPase, these ions can be arranged in the following order: Mg2+>Co2+>Mn2+. The inhibitory effect is a maximum at a Mg2+ concentration of 161074 mol litre71.A linear dependence of the degree of inhibition of the enzymic activity on the magnesium concentration in the range of 161075 ¡¾ 561075 mol litre71 was established. Further increase in the magnesium concentration influenced the inhibition to a lesser degree, i.e., it disturbed the linear dependence. Mn2+ and Co2+ ions used at concentrations 161075 ¡¾ 161074 mol litre71 have no effect on Ca2+-ATPase.Their inhibitory effect becomes apparent only at a concentration of ca. 2.561074 mol litre71; a linear dependence of the decrease in the enzyme activity on the cation concentration is observed at concentrations from 2.561075 to (2 ¡¾ 3)61073 mol litre71. The study 16 deserves special mention among other studies concerned with the inhibition of the catalytic activity of enzymes by metal ions.This not only quantitated the effect observed (i.e., the experimental values of inhibition coefficients defined by the author as the ratio of initial rates of substrate cleavage in the presence and in the absence of M2+), but also considered the influence of some other parameters (ionisation potentials, hydra- tion energy, ionic radii of cations) on the magnitude of the inhibitory effect.In the work cited, the effect of Ca2+, Mg2+, Ba2+, Sr2+,Mn2+, Fe2+, Co2+, Ni2+, Pb2+, Cd2+, Zn2+, Cu2+, and Hg2+ on the catalytic activity of bovine RNase A was studied. The inhibitory effect of these cations was found to increase in the series: Ba2+&Sr2+&Ca2+<Mg2+< <Mn2+< Fe2+< Co2+< Ni2+< Pb2+<Cd2+<Zn2+ Cu2+< Hg2+.It is seen that Hg2+, Cu2+, and Zn2+ cations manifest the strongest inhibitory activity; the cations of the iron subgroup (Mn2+, Fe2+, Co2+, and Ni2+) are weak inhibitors, whereas the cations Ba2+, Sr2+, and Ca2+ do not produce any inhibitory effect at all. In the author's opinion,16 the fact that the metal cations do not possess strict selectivity, i.e., that they can interact with functional groups in the enzyme active centre differing in electronic effect, a complex dependence of the mode of enzyme inhibition by the metal cations on their concentration, and their ability to form complexes both with enzymes and the substrates preclude the estimation of the inhibitory activity of metal cations from the inhibition constants.In order to evaluate the inhibitory strength of cations the author introduces a parameterW, which is equal to the sum of the electrostatic (Z2/r) and the reduced covalent (E7DH) character- istics: W a Z2 r a OE ¢§ DHU , where Z is the ionic charge, r is the ionic radius, E is the sum of the ionisation potentials for the first and second electrons, and DH is the energy of cation hydration.The higher inhibitory activity of Zn2+, Cu2+, and Hg2+ is largely determined by the reduced covalent characteristics.In the cations of the iron subgroup (Mn2+, Fe2+, Co2+, and Ni2+), the electrostatic characteristics are comparable with those of Zn2+, Cu2+, and Hg2+, while the values of the reduced covalent characteristics are 2 ¡¾ 3 times as low. These cations manifest only weak inhibitory activity.For Ba2+, Ca2+, Mg2+, and Sr2+ cations, the values of electrostatic and, particularly, reduced covalent characteristics are smaller than those for Zn2+, Cu2+, and Hg2+. It has been shown experimentally that these cations do not inhibit RNase at the concentrations from 161074 to 161072 mol litre71. 226 E P Medyantseva, MG Vertlib, G K BudnikovThe effect of rare-earth elements (REE) (Pr, Nb, Sm, Eu, Tb, Er, Tu, Yb) on the catalytic activity of inorganic pyrophos- phatases from bakers yeast and E.coli has been studied.24 Analysis of the dependence of the rate of the indicator reaction on the concentration of REE over the range of 1075 ± 1071 mg ml71 revealed that all these ions inhibited both enzymes; the inhibitory effect of different REE was manifested in different concentration ranges.Praseodymium (Pr3+) appeared to be the most effective inhibitor of pyrophosphatases. The inhibitory effect decreased on going from praseodymium to ytterbium, i.e., with a decrease in the ionic radius of REE. The mechanism of inorganic phosphatase inhibition by praseody- mium is consistent with complete competitive inhibition. The value of the inhibition constant, which reflects the stability of the enzyme ± inhibitor complex, demonstrates that Pr3+ ions form a catalytically inactive complex with pyrophosphatase, which is more stable than that with Eu3+ ions.In other studies,24 ± 32 only qualitative aspects of the effect of metal ions on the catalytic activity of enzymes have been consid- ered with a mere statement of the existence of the corresponding effect and indicating the range of concentrations where it is manifested.Cu2+ ions decrease the catalytic activity of glutamate dehy- drogenase from bovine liver 25 (I50=261076 mol litre71). At concentrations of Cu2+ equal to 161075 mol litre71 and higher, the residual activity makes up 8%± 10% of the original. This effect is a result of the direct interaction of the copper cations with glutamate dehydrogenase (the difference absorption spectrum of glutamate dehydrogenase in the presence of Cu2+ suggests the formation of an enzyme ± metal complex).This inhibition is allosteric. Zn2+ ions also inhibit this enzyme. Spectral data point to identical conformational changes in the substrate-binding site induced by Cu2+ and Zn2+ cations.25 Zn2+ cations also inhibit poly(ADP-ribose)polymerase from rat brain.At a Zn2+ concentration equal to 1.2561074 mol - litre71, the enzyme is fully suppressed. The changes in the enzyme activity are due to the binding of Zn2+ ions with the sulfhydryl groups of polymerase. The inhibitory effect is reversible, since dithiothreitol neutralises the effect of this cation on the polymer- ase activity.26 Pb2+ and Cd2+ions (1073 ± 1072 mg ml71) inhibit alkaline phosphatase from chicken intestine in the hydrolysis of p-nitro- phenyl phosphate.23, 27 The decrease in the activity is a maximum at Cd2+ concentrations of 1 ± 10 mg ml71 (Ref. 23). Cd2+ ions (161074 mg ml71) also inhibit horseradish peroxidase.27 The catalytic activity of peroxidase is also inhibited by Hg2+ cations (5610713 ± 561078 mol litre71).28 ± 30 A drop in peroxidase activity is also observed in the presence of Bi3+ [(2 ± 10)61074 mg ml71].At higher Bi3+ concentrations, the rate of this reaction remains virtually unchanged.31 Protein disulfide reductase from bovine liver catalyses the thiol ± disulfide exchange between glutathione and insulin and reactivates randomly oxidised ribonuclease.The activity of this enzyme is inhibited by Cu2+ and Hg2+ ions.29 The effects of Zn2+, Cu2+,Mn2+, Fe2+, Co2+, and Ni2+ ions on the activity of acetylcholinesterase (AChE) of human eryth- rocytes have been studied over a broad range of salt concentra- tions.20, 33 It was found that all the salts in question (with the exception of MnCl2, which has no effect on the AChE activity) are reversible competitive inhibitors of AChE. To a first approxima- tion, the inhibitory effect is directly proportional to the concen- tration of the corresponding metal.From the pH dependence of the AChE inhibition by the salts it was concluded that the AChE activity is inhibited by hydroxo complexes of the MOH+ type. Cu2+ and Cd2+ ions suppress the catalytic activity of chol- inesterase (ChE) isolated from pea sprouts.22 Co2+ and Al3+ ions also manifest inhibitory activity.According to another report,21 Ca2+ and Mn2+ used at concentrations of 161073 ± 161072 mol litre71 inhibit ChE from kidney bean roots, whereas Mg2+ has no effect on the enzyme. Calcium occupies a special position among metals, since it manifests an activating rather than inhibitory effect.34 However, it can also inhibit certain enzymes, e.g., yeast hexokinase 30 and alkaline phosphatase.27 It has been established that calcium ions taken at a 10 ± 100-fold excess over praseodymium enhance the inhibitory effect of the latter.The inhibitory action of calcium 24 is ascribed to the fact that its ionic radius is similar to those of REE and thus it can exert a similar effect on the enzyme.Ca2+ cations markedly decrease the activity of hexokinase.35 The catalytic activity of alkaline phosphatase from chicken intestine is also decreased in the presence of calcium ions (1073 ± 1 mg ml71). It should be noted that the inhibitory effect of calcium on the enzyme decreases with an increase in the ion concentration.27 Recently, some papers have been published, which deal with the study of the catalytic activity and properties of immobilised enzymes.18, 19, 36 ± 38 Naturally, metal cations also change the catalytic activity of immobilised enzymes.Urease immobilised on a solid carrier (a microporous styr- ene ± divinylbenzene copolymer) is inhibited by ions of toxic metals. The inhibitory effect of Fe2+ manifests itself even at a concentration of 10710 ± 1079 mol litre71, whereas that of most ions, at 1078 mol litre71.With respect to the strength of their inhibitory action, the metal ions can be arranged in the following series:18, 19 Ag+>Hg2+>Fe2+>Cu2+>Ni2+>Co2+> Al3+>Mg2+>Cd2+>Pb2+. As noted above, Ag+ is the strongest and Pb2+ is the weakest inhibitor of native urease.17 At the same time, the series of other metals for the native urease (see above) differs from that for the immobilised enzyme due to the changes caused by enzyme immobilisation. A number of papers have been devoted to the effects of Zn2+, Fe2+, Ni2+, Hg2+, Pb2+, Tl+, Bi3+, Cd2+, Cu2+, Cr3+, Hf 4+, Ti4+, and Zr4+ ions on the catalytic activity of cholinesterase immobilised by incorporation into cellulose nitrate films.36 ± 38 All these metals inhibit the immobilised enzyme. The greatest inacti- vating effect is produced by Cu2+.The minimum Cu2+ concen- tration (cmin), which causes a statistically significant decrease in the catalytic activity of immobilised ChE, is 4610710 mol litre71. A similar change in the catalytic activity in the presence of other ions is observed at the following concentrations:36 ± 38 Ion Zr (IV) Ti (IV) Nb (V) Hf (V) Ta (V) c /mol litre71 5610710 8610710 261079 361079 561079 Ion Cr (III) Pb (II) Fe (III) Cd (II) c /mol litre71 561078 561077 161076 161075 The inhibitory effect also depends on the catalytic activity of the enzyme itself.Thus, for ChE immobilised in a cellulose nitrate matrix with a specific activity of 1.120.04 mmol min71 cm72, cmin for Cu2+ is 4610710 mol litre71, whereas for the samples with a threefold lower specific activity (0.330.02 mmol min71 cm72), it is equal to 161079 mol litre71 (Ref. 38). Solutions of cysteine and EDTA restore completely the catalytic activity of immobilised ChE after the action of heavy metal ions. However, only 70% of totally inhibited ChE is observed in the case of Pb2+. This is probably due to the interaction of Pb2+ with the histidine residue of the active centre of ChE, especially in the presence of high (*1074 mol litre71) concentrations of Pb2+ (Ref. 36). The mechanism of the inhibitory effect of metal ions on the catalytic activity of enzymes is rather difficult to establish. The metal can compete with the substrate for the binding sites in the active centre, it can also interact with different groups in the protein molecule outside the active centre but influencing the catalytic functions of the enzyme,39 i.e., it can bind to the allosteric centre of the enzyme.It is believed that the effect of metal ions on the enzyme molecule can be due to the formation of thiolates with both Metal ions as enzyme effectors 227cysteine, which is a constituent of the enzyme, and products of cleavage of disulfide bridges.40 If the cysteine residue is incorpo- rated into the active centre of the enzyme or is responsible for the maintenance of the appropriate conformation of the protein molecule, the modification of its thiol group inactivates the enzyme.In addition, heavy metal ions can catalyse the oxidation of thiols to disulfides:41 RSH+O2+R0SH R7S7S7R0 +H2O2 . Most commonly, the inhibitory effect of metal ions is ascribed to their ability to form coordinate bonds.41 As is known,42 the nucleophilic residues of some amino acids incorporated into the active centres of enzymes, can form coordinate bonds with heavy metal ions. For example, histidine, which is a constituent of the esterase region of the active centre of ChE, can interact with metal ions.36 Some authors explain the difference in the inhibitory action of various metal ions by the difference in the ionic radii of cations.16, 29, 36 2.The activating effect of metal cations Metals can not only inhibit but also enhance the catalytic activity of enzymes. Most often, the activating effect is produced by ions of rare-earth metals. It should be noted that the processes related to the activation of enzymes with metal ions have been documented in far fewer publications than those on the inhibition processes.Unfortunately, in the case of studies of the inhibitory effect, most authors give only a qualitative outline of the activation pro- cess.43 ± 56 without quantitative estimates, which makes it difficult to compare the published data.The activating effect of Ca2+ cations on mitochondrial glyce- rol-3-phosphate dehydrogenase has been studied.43 Ca2+ cations are not selective activators. They interact with both electron- donor amino acid residues and those nitrogen atoms of the peptide bonds that are not necessarily incorporated into the enzyme active centre.It thus follows that not all Ca2+ cations that interact with the enzyme play the same role in the maintenance of its activity. Some of them that interact with the amino acid residues remote from the enzyme active centre not only are devoid of the activation effect, but, conversely, produce an inhibitory effect.Analysis of literature data reveals that the simultaneous presence of several metal cations, even of the same nature (Ca, Mg), has an ambiguous effect on the activity of the enzyme under study: one of the ions can either enhance the effect of the other ion (synergism) or attenuate it. Most often, the synergistic effect is produced by Ca2+ and Mg2+ ions.34 For example, even a short-term preincubation of phosphorylase kinase with these ions caused a sevenfold increase in the catalytic activity.It is believed that the simultaneous presence of Ca2+ and Mg2+ favours the interaction of phosphor- ylase kinase with glycogen.34 Ca2+ andMg2+ ions also activate myofibrillar creatine kinase from rat skeletal and cardiac muscles. Calcium activates the enzyme to a lesser degree than magnesium.In the presence of Mg2+ ions, calcium inhibits creatine kinase. Presumably, creatine kinase has different binding sites for Ca2+ and Mg2+ ions, since the latter does not eliminate the inhibitory effect of calcium even when used in a 100-fold excess.44 Ca2+ and Mg2+ ions activate myometrium actomyosin ATPase. As the Ca2+ concentration is increased (0.01 ± 5)61073 mol litre71 in a Mg2+-free medium or after addition of Mg2+ (161073 mol litre71), the ATPase activity is noticeably increased and then reaches a plateau.39 A change in the Mg2+ concentration from 161075 to 561073 mol litre71 in a Ca2+-free medium increases the activity of ATPase in the region of low concentrations after which it reaches a plateau.Significant induction of the ATP-hydrolase reaction is observed after addition of the physiological concen- tration of Ca2+ (161073 mol litre71) to the incubation medium. Ca2+ ions are more effective activators of the reaction of ATP hydrolysis.46 The effect of cations of alkaline and alkaline-earth metals on the activity of native ChE 21, 46 and immobilised butyrylcholin- esterase 36 has been studied. Na+, K+, and Mg2+ ions stimulate the cholinesterase activity in pea sprouts by 30%± 60%.21, 22 The catalytic activity of immobilised ChE increases in the presence of Na+, Mg2+, and Mn2+ ions in the concentration range of 861074 ± 261072 mol litre71.36 Presumably, this is due to the presence of an anionic group near the active centre of ChE, which prevents the interaction of the enzyme with the substrate.Intro- duction of these metal salts results in binding of this group by ions of ChE-activator metals, which creates the most favourable conditions for the formation of the Michaelis complex. It was found that the larger the radius and the charge on the activator metal the lower the concentration of this metal ion needed to block the anionic group of ChE. Ca2+ cations activate butyrylcholinesterase.47 ± 49 The max- imum rate of the reaction is increased in the presence of Ca2+, whereas the Michaelis constant remains practically unchanged.Apparently, Ca2+ ions do not compete with the substrate for the binding sites, since the activation cannot be eliminated by an excess of acetylcholine. It is believed that Ca2+ exerts an allosteric effect as it binds with specific sites.50, 51 Co2+, Ni2+, and Mn2+ ions do not influence the acetylcholine hydrolysis.47 Immobilised ChE is activated by microquantities of heavy metals, such as Pb, Hg, Cd, and Tl used generally at concen- trations below 1077 ± 1076 mol litre71.52, 53 Some platinum met- als (Pt2+, Rh3+) also activate the immobilised ChE.54, 55 It should be noted that the inhibitory effect correlates linearly with the concentration of the effector.27 ± 31, 37, 38 As to the activation, this process demonstrates a more complex dependence on the concentration of metal ions.35, 36 First of all, an increase in the catalytic activity is observed over a small range of concen- trations that differ usually by not more than a factor of 10 ± 15 (Fig. 2). The maximum catalytic activity is manifested sometimes only at a definite concentration of the effector (Fig. 2b). In some cases, a small plateau is observed (Fig. 2a). A deviation from the optimum concentration results in a decrease of the enzyme catalytic activity towards the control level. Among studies concerned with enzyme activation, that deal- ing with the mechanism of the activating effects of Mg2+ and Co2+ ions on glucose isomerase of Actinomyces olivocinereus 154 stands separately.57 In this study, the main kinetic constants have been calculated and quantitative estimates of the activating capacity of these cations (the activation constants) have been given.It was found that the activating effect of Mg2+ ions is much stronger than that of Co2+. The maximum activating effect of Mg2+ ions is observed at a concentration (2.0 ± 2.5)61072 mol litre71, that of Co2+ ions, at (2.5 ± 5)61074 mol litre71.Further increase in the concentrations of Mg2+ and Co2+ ions leads to the inhibition of glucose isomerase; for Co2+ ions, this phenomenon is more strongly pronounced and manifests itself even at a concentration of 1.2561073 mol litre71. Studies of the activating effect of Mg2+ and Co2+ ions added to the reaction mixture simultaneously revealed a complex dependence of the enzyme activation on both the absolute concentrations of these metal ions and their ratio.The maximum activity was observed at a Mg2+ ion concentration of 261075 mol litre71 and that of Co2+ at 561073 mol litre71. It should be noted that for Mg2+ the optimum concentrations of this activator ion used in a mixture were slightly lower than when it was added separately, whereas for Co2+ ions, they were tenfold higher.Mg2+ and Co2+ ions bind to different sites on glucose isomerase and influence its activity differently.57 Therefore, the mechanism of activation of the glucose isomerisation reaction by these ions is also different. The activation with Mg2+ ions is non- competitive, while that with Co2+ ions is synergistic; when Mg2+ and Co2+ ions are used simultaneously, the activation is of a mixed type.In the non-competitive activation, glucose and Mg2+ ions bind to the enzyme independently of each other to form an 228 E P Medyantseva, MG Vertlib, G K Budnikovactive bridge-like ternary complex (Fig. 1, pathway II).The value of the activation constant is very low (0.002), which points to a high stability of an active ternary complex (Mg2+ ± E ± S) and the activated enzyme (Mg2+ ± E) and to a small rate constant for their dissociation into the original enzyme and the enzyme ± substrate complex (ES). Thus, in the activation of glucose isomerase with Mg2+, the major part of the enzyme is bound into the ternary complex Mg2+ ± E ± S.As has been mentioned above, the activa- tion with Co2+ ions is synergistic. Co2+ ions bind to the glucose isomerase molecule and increase the enzyme affinity for glucose. In addition, the ES complex interacts with Co2+ ions much faster than the free enzyme. The authors suggest that the main role of Co2+ ions consists in conferring a conformation that is resistant to thermal inactivation on the enzyme quaternary structure rather than in the activation of the enzyme. Moreover, this conformation facilitates the attachment of glucose even in the absence of Mg2+ ions and accelerates the formation of a bridge-like ternary com- plex in their presence, as a result of which the rate of isomerisation increases. This is corroborated by experiments with simultaneous addition of Mg2+ and Co2+ ions to the reaction mixture.57 Attempts have been undertaken56, 58 to characterise quant- itatively the processes of activation of immobilised ChE in the presence of alkaline-earth metal ions on the basis of voltammetric measurements. The results of these studies indicate that the activation is controlled by many factors, such as substrate concentration, pH, activity of the immobilised enzyme (i.e., the type of immobilisation), activator concentration, incubation time, and the order in which the substrate and the effector act on immobilised ChE.The maximum activating effect was observed at pH 8.95 ± 9.05. The lower the catalytic activity of immobilised ChE the higher the concentration of metal ions needed for the manifestation of their activating effect.Thus immobilisation according to Nikolskaya 33 in which incorporation of the enzyme into a cellulose nitrate matrix was performed together with treat- ment with a bifunctional reagent (glutaraldehyde) gave immobi- lised ChE with a catalytic activity about 3 ± 4 times lower than that obtained by immobilisation of the enzyme followed by treatment with a bifunctional reagent.In order to increase the catalytic activity of these samples, it is necessary to use high concentrations of the activators (661073 ± 661074 mol litre71 Ca2+).37 To verify these effects, the authors carried out a radiochemical analysis with 85 Sr. The results of this study suggest that under optimum conditions (pH 9.05, 561076 mol litre71 Sr2+, sub- strate concentration, 261073 mol litre71) alkaline-earth metal ions change the catalytic activity of immobilised ChE in the order: Ca2+ > Sr2+ > Ba2+, which is consistent with their ionic radii.Similar correlation between the ion radii and their activating effect was observed earlier for alkali metal ions.36 As the substrate concentration is increased from 261073 to 461073 mol litre71, the type of activation changed from associative to pseudo- and then to two-parametrically disbalanced activation.The activation constants for Ca2+ ions are (3.70.3)61076 mol litre71 at low [(1.5 ± 3)61073 mol litre71)] and (1.80.2)61078 mol litre71 at higher (461073 mol litre71) substrate concentrations; that for Ba2+ ions is (8.400.3)61076 mol litre71.The experimental values of the activation constants correlate with a stronger activating effect of Ca2+ ions (the catalytic activity of immobilised ChE is increased more strongly in the presence of Ca2+). 3. The dependence of the mode of action of metal ions on their concentration It is well known that the same metals can increase or decrease the catalytic activity of enzymes depending on their concentration.Thus Cu2+ ions used in low concentrations stimulate the activity of purified poly(ADP-ribose)polymerase. At a Cu2+ concentration of 561076 mol litre71, the polymerase activity is increased 1.4-fold. Further increase in the Cu2+ concentration inhibits the enzyme activity and with 561075 mol litre71 Cu2+ the polymerase activity drops down to zero.Poly(ADP-ribose)- polymerase contains sites with different affinity for Cu2+ cations. At low Cu2+ concentrations, the binding of these ions to the high- affinity sites of the enzyme increases the polymerase activity, whereas at high Cu2+ concentrations, the binding may also occur to sites with a lower affinity for the metal, which results in the inhibition of the enzyme activity.26 Mg2+ ions (1073 ± 1 mg ml71) inhibit alkaline phosphatase from chicken intestine.They strongly activate the enzyme over the concentration range 1 ± 100 mg ml71. Aluminium ions slightly inhibit the enzyme when used at the concentrations of 1073 ± 1 mg ml71 but strongly activate it at the concentrations of 1 ± 10 mg ml71 (Ref. 27). Low concentrations of Mn2+, Ni2+, and Co2+ ions activate muscle pyruvate kinase, whereas higher concentrations inhibit the enzyme.Ca2+ ions (461074 ± 261074 mol litre71) inhibit liver glutamate dehydrogenase and activate it at concentrations above 461074 mol litre71. At the same time, Mg2+ ions inhibit gluta- mate dehydrogenase only in the concentration range of (2 ± 4)61074 mol litre71. Higher and lower concentrations of Mg2+ activate this enzyme.Some phosphatases are activated by low concentrations of Mg2+ (up to 561073 mol litre71), whereas higher concentrations of this ion cause their inhibition.34 The changes in the properties of enzymes in the presence of some metal ions are summarised in Table 1. Almost all investigators emphasise that the effect of metal ions on the activity of ChE is of dual character: activation at small concentrations and inhibition at high concentrations.47 Even such cations as Na+, Mg2+, Ca2+, Sr2+, and Ba2+ do not increase the catalytic activity of ChE when used at high concentrations.56 a Ip /mA 0 1 2 3 7lg cMn+ 3 2 1 4 6 2 b Ip /mA 0 2.5 5 106 cMn+ /mol litre71 1 2 3 4 5 6 7 8 Figure 2.Dependence of the analytical signal (electric current at a potential of 70.55 V) on the concentration of metal ions (pH 9.05; the concentration of butyrylthiocholine iodide, 0.6 mg ml71).Specific activity of immobilised ChE is 0.35 mmol min71 cm72 (a) and 1.17 mmol min71 cm72 (b). (a): (1)�Mn (II), (2)�Mg, (3)�Na; (b): (1)�Ca, (2)�Sr, (3)�Ba. Metal ions as enzyme effectors 229Pb2+, Hg2+, Cd2+, and Tl+ ions activate ChE at concentrations below 1077 ± 1076 mol litre71 and inhibit the enzyme at higher concentrations.36, 37, 52, 53 Most authors attribute the activating effect of cations to the possible formation of the ternary complex E ±M± S (Fig. 1, pathway II),1, 58 which provides conformation- ally more favourable conditions for the enzymic reaction. The inhibitory effect of high concentrations of metal ions is assigned to the competitive reaction between metal and substrate cations (if the substrate bears a positive charge) for the active centre of the enzyme.At high salt concentrations, the active centre of the molecule can be occupied by metal ions, thus being inaccessible to the substrate cations. As a result, the catalytic activity of both native and immobilised ChE may decrease.36, 59 According to other authors,60 the effect of metal ions on the enzyme activity is due to changes in the substrate solubility at high salt concen- trations.Obviously, the effect of metal ions on enzymes depends on many factors. It is therefore difficult and usually impossible to identify the determining factor. In particular, the effect of metals on the catalytic acivity of enzymes depends on the nature of the metal, its size, charge, ionisation potential, the nature of the enzyme, its state (native or immobilised), conditions of the enzymic reaction, etc.Most commonly, the effect of metal ions on the enzyme activity is associated with their ability to form enzyme ± substrate complexes, which may participate in the cata- lytic process by facilitating the transfer of electrons, to stabilise the active conformation of the enzyme or association of its subunits, and to act as an `anchor' group without being directly involved in the catalytic act.These and other effects may also occur in combination. Unfortunately, the majority of authors give only qualitative characteristics of changes in the enzymic activity, which naturally hampers comparative analysis of the experimen- tal data, and only very few try to estimate these effects quantita- tively and get an insight into the mechanism of action of metal ions.These studies have received the greatest attention in the present review. III. Changes in the catalytic activity of enzymes in the presence of metal ions: an analytical aspect Over a long period of time, investigators have merely noted the effects of metal ions on the catalytic activity of enzymes, but these effects did not find practical application.As the bulk of exper- imental data have been accumulated, the analytical application of these effects became possible. The studies on this subject covering the period of 1949 ± 1979 have been summarised in a review.61 One of the first studies by Russian authors on the application of the effects of metals on enzymes for analytical purposes was that by Dolmanova et al.28 in which the authors offered a highly selective procedure for the quantitation of mercury, which was based on the inhibitory effect of mercury on horseradish perox- idase [the detection limit is (1.00.5)61075 mg ml71].Further investigations 29 in this field were directed to the use of various substrates and the optimisation of conditions for enzymic reac- tions, which made it possible to lower the detection limit to 3.061077 mg ml71.Bismuth and cadmium ions interfere with the determination. A procedure for quantitation of mercury was proposed 30 (in which the lower limit of concentrations is 161075 mg ml71), which differed from the standard ones by being time-saving (the analysis lasted for 30 minutes instead of 4 hours) and which permitted determination of mercury in the presence of relatively high (51 mg ml71) concentrations of Fe3+.Iron is masked by tartaric acid, which has no effect on the catalytic activity of peroxidase under the given experimental conditions and at selected concentrations.Quantitation of mercury in natural water is carried out using the method of additives, which makes it possible to estimate the effect of other components of the matrix (particularly that of Fe3+) without masking. The inhibitory effect of Bi3+ on the catalytic activity of horseradish peroxidase was used as the basis for the kinetic method used for its quantitation.The lower limit of concen- trations that can be determined is 261074 mg ml71. This assay is not hampered by a 1000-fold excess of Ca, Mg, Zn, Ni, and Al, by Table 1. The effect of some metal ions on the catalytic activity of enzymes. Enzyme Metal Effect ceff a Ref. ion /mol litre71 Ca2+-ATPase Mg2+, Co2+ inhibitor 161075 ± 161073 23 EC 3.6.1.3. Mn2+ " 161075 ± 161073 23 ATPase Ca2+, activator 161075 ± 561073 45 EC 3.6.1.8.Mg2+ " 161075 ± 561073 45 Hexokinase Ca2+ inhibitor 161073 ± 561072 35 EC 2.7.1.1. Glycerol-3- Ca2+ activator 161076 ± 161075 43 phosphate dehydrogenase EC 1.1.1.8 Glutamate Cu2+, inhibitor 161076 ± 161074 25 dehydrogenase Zn2+ " 161076 ± 161074 25 EC 1.4.1.4. Glucose Mg2+, activator 161073 ± 961071 57 isomerase Co2+ " 161073 ± 961071 57 EC 5.3.1.18.Creatine kinase Ca2+, " 161073 ± 161072 44 EC 2.7.3.2. Mg2+ " 161073 ± 161072 44 Inorganic Pr, Nb, Sm inhibitor 161075 ± 161071 24 pyrophosphatase Eu, Tb, Er " 161075 ± 161071 24 EC 3.6.1.1. Tu, Yb " 161075 ± 161071 24 Peroxidase Hg2+ " 5610713 ± 561078 28 EC 1.11.1.7. Bi3+ " 161075 ± 261074 31 Poly(ADP- Zn2+ " 261075 ± 261074 26 ribose)poly- Cu2+ " 161075 ± 561075 26 merase Cu2+ activator 161076 ± 161075 26 EC 2.4.2.30 Ribonuclease Ba2+, Sr2+ no influence 161074 ± 161072 16 EC 3.1.4.8.Ca2+ " 161074 ± 161072 16 Hg2+, Cu2+ inhibitor 161074 ± 161072 16 Zn2+, Mn2+ " 161074 ± 161072 16 Fe2+, Co2+ " 161074 ± 161072 16 Ni2+ " 161074 ± 161072 16 Urease Ag+, Hg2+ " 161077 ± 161073 17 EC 3.5.1.5. Cu2+, Zn2+ " 161077 ± 161073 17 Pb2+, 7 ± 161073 17 Cd2+, Co2+ " 161077 ± 161073 17 Cholinesterase Pb2+ " 561077 ± 161073 36 EC 3.1.1.8. Pb2+ activator 161079 ± 161077 53 Cd2+ inhibitor 161076 ± 161073 37 Cd2+ activator 561079 ± 861077 53 Hg2+ inhibitor 561075 ± 161073 36 Hg2+ activator 1610712 ± 161076 53 Tl+ inhibitor 561075 ± 161073 37 Tl+ activator 1610711 ± 161075 53 Ca2+, Sr2+ " 161076 ± 161075 56 Ba2+ " 161076 ± 161075 56 Alkaline Ca2+ inhibitor 261078 ± 361075 27 phosphatase Ba2+ " 761079 ± 761077 27 EC 3.1.3.1. Cd2+ " 961079 ± 961074 27 Pb2+ " 561079 ± 561074 27 Mg2+ " 461078 ± 461075 27 Mg2+ activator 461075 ± 461073 27 a ceff is the concentration range where the effect is observed. 230 E P Medyantseva, MG Vertlib, G K Budnikov100-fold excess of Pb and Ag, and by a 10-fold excess of Cd.The presence of comparable amounts of Hg2+ interferes with the analysis.31, 62 A highly sensitive (cmin = 861076 ± 461074 mg ml71), selective enzymic method24 for the quantitation of REE is based on their inhibitory effect on the catalytic activity of bakers yeast and E. coli pyrophosphatases. The interference of Co2+, Ni2+, and Mn2+ ions was neutralised by introducing an excess of magnesium into the indicator reaction.The addition of 561073 mol litre71 of magnesium in the presence of 561075 mg ml71 of praseodymium and of 561075 ± 561073 mg ml71 of manganese, cobalt, and nickel to the system eliminates their interference; this magnesium concentration has no effect on the inhibitory effect of praseodymium. The interference of cal- cium ions was eliminated by masking it with tartrate.The high sensitivity of enzymic methods makes it possible to determine many components of various waters in small samples and without preliminary concentration at the maximum permis- sible concentration (MPC) level. For example, metal ions like Cu, Co, Ni, Fe, Mn, Cr, Hg, Cd, Bi, Pb, and Zn can be detected at concentrations of 1 ± 100 pg ml71 on the basis of their inhibitory effect on horseradish peroxidase and alkaline phosphatase.63 It was proposed to use enzymic methods in the study of formation of hydroxo complexes.The stability constants of hydroxo complexes, KMOH +, have been calculated for zinc, copper, iron, cobalt, and nickel.20 Analytical methods based on the use of enzymes also permit measurement of the equilibrium concentrations of hydroxide ions and of ions of some d-block elements in complex heterogenous systems and the study of salt hydrolysis with a high degree of sensitivity and selectivity unat- tainable by the majority of other methods.34 The use of native enzymes for some specific assays has a number of disadvantages, such as the instability of enzymes on storage and under the influence of various factors (e.g., heat treatment) and the impossibility to separate enzymes from the reagents and the reaction products.A contemporary approach to the solution of this task consists in the wide use of immobilised enzymes. Immobilisation favours the stabilisation of enzymes on long-term storage and increases their resistance to external factors. The possibility of multiple use of enzymes reduces considerably the cost of analysis. The use of immobilised enzymes in metal ion assays has been reviewed.64 Immobilisation of enzymes for determination of metal ions entails their incorporation into various membranes or bind- ing to glass beads.Polymeric films with immobilised enzymes incorporated in them are now often used as membranes for biochemical sensors.Thus an amperometric biochemical sensor for determination of metal ions has been constructed on the basis of ChE immobi- lised by incorporation into cellulose nitrate films.36 ± 38 A method for assaying Cr3+, Ti4+, Zr4+, and Hf 4+ (1610710 ± 161075 mol litre71) has been developed, which is based on the inhibitory effect of heavy metal ions on the catalytic activity of immobilised cholinesterase.38 It was suggested 65 that Cu2+ be determined using an enzymic electrode containing glucose oxidase; it is based on the inhibitory effect of these cations on the enzyme in the concentration range of 2.561074 ± 561073 mol litre71.Recently, much attention has been paid to semiquantitative test-methods, which allow rapid, simple, and reliable determina- tion of concentrations of toxic substances at the MPC level.The use of immobilised enzymes for the elaboration of these methods is also very promising. A test-method for the determination of Hg2+ using perox- idase immobilised as a solid solution in chitosan has been described.66 The assay is based on the inhibitory effect of mercury on the oxidation of o-dianisine, o-phenylenediamine, and 3,30,5,50- tetramethylbenzidine with hydrogen peroxide in the presence of thiourea catalysed by immobilised peroxidase.The change in the colour of the reaction solution due to the formation of red or orange products of enzymic oxidation allows visual monitoring of the reaction rate. The lower limits of mercury concentrations that can be determined in these reactions are 161075, 561075, and 161077 mg ml71, respectively.This method is noted for selectiv- ity (the assays are hampered only by a 105-fold excess of cadmium and bismuth), rapidity (the analysis is completed within 15 minutes), simplicity, and low cost. Enzymic methods for assaying metal ions are characterised by high sensitivity and in some cases, high selectivity.27 However, most of these assays are not sufficiently selective.A combination of high sensitivity of biochemical sensors based on immobilised enzymes with traditional methods of masking makes it possible to solve complex analytical problems. Thus, enzymes act as a new generation of analytical reagents. The use of biosensors for quantitation of metal ions is also a promising approach because it exploits the conditions modelling individual functions of a living organism.The elaboration of novel measuring tools including biosensors, especially those based on highly sensitive methods of registration, considerably extends the possibilities of evaluation of the effects of metal ions on the catalytic activity of enzymes. It may be assumed that in many cases the concentration range of inhibition or activation for a definite `metal ± enzyme' pair could not be determined because of the limited potentialities of the recording systems.In addition, in the case of specific assays, allowance should be made for the ionic composition of the medium and the composition of the buffer solutions used for each assay. Buffer solutions must be selected in such a way that their ionic composition ensures the maximum catalytic activity of the enzyme.This provides the conditions for the generation of a maximum analytical signal, which in turn extends the analytical capabilities of enzymic assays of various effectors including metal ions.37, 57 IV. Conclusion The above analysis of literature data shows that the effect of metal ions on the catalytic activity of enzymes is still a timely problem.At the same time, special mention should be made of some recent specific features of these studies. Thus in the 1960 ± 70's interest in these studies was mostly biological or medical, while in the last decade interest has been shifted towards the chemical aspects of the problem. This is primarily due to the pollution of the biosphere with various metals above the permissible level and secondly, due to the possibility of their estimation with the help of enzyme preparations possessing higher sensitivity and, in some cases, selectivity.Two main trends in these studies can be isolated: quantitative assay of inhibitors or activators from the magnitude of the induced effect and the use of the activating effect of metal ions for increasing the sensitivity of assays for a wide range of compounds of various natures.An overview of current literature demonstrates that it is difficult to predict a priori how one or another metal will influence the enzyme. This depends not only on the nature of the metal and the enzyme, but also on other specific conditions. The most important of those are the ionic radius and the concentration of the metal.One should not consider the problem of interaction of metal ions with the enzyme separately from the pH of the solution and the presence or the absence of the substrate. The activating or inhibitory effect of metal ions must be considered only with allowance for these factors. Unfortunately, the majority of authors restrict themselves currently to the qualitative description of effects of metal ions on the catalytic activity of enzymes, while very few papers deal with quantitative estimates and analysis of the mechanism of interac- tion between metals and enzymes.It may be supposed, however, that the number of such publications will be increasing with the development of highly sensitive methods of registration. Metal ions as enzyme effectors 231References 1.G Eichhorn (Ed.) Inorganic Biochemistry (Amsterdam: Elsevier, 1978) 2. M Cohn, J S Leigh Nature (London) 193 1037 (1962) 3. 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Chem. 16 429 (1988) 60. L P Kuznetsova, E B Nikol'skaya Ukr. Biokhim. Zh. 60 35 (1988) 61. I F Dolmanova, N N Ugarova Zh. Anal. Khim. 35 1597 (1980) c 62. T N Shekhovtsova, S V Chernetskaya, in Proceedings of the 5th International Symposium on Kinetics in Analytical Chemistry (Abstracts of Reports) Moscow, 1995 p. 48 63. I F Dolmanova, G A Zolotova, T N Shekhovtsova, in Tez. Dokl. XVMendeleevskogo S'ezda po Obshchei i Prikladnoi Khimii (Abstracts of Reports at the XVth Mendeleev Congress on General and Applied Chemistry) (Minsk: Navuka i Tekhnika, 1993) p. 332 64. T N Shekhovtsova, S V Chernetskaya, N V Belkova, I F Dolmanova Zh. Anal. Khim. 49 789 (1994) c 65. A M Donlan,G J Moody, J D R Thomas Anal. Lett. 22 1837 (1989) 66. T N Shekhovtsova S V Chernetskaya, E B Nikol'skaya, I F Dolmanova Zh. Anal. Khim. 49 862 (1994) c a�Biochemistry (Engl. Transl.) b�Dokl. Chem. Technol., Dokl. Chem. (Engl. Transl.) c�J. Anal. Chem. (Engl. Transl.) 232 E P Medyantseva, MG Vertlib, G K Bu

ISSN:0036-021X    

出版商:RSC    

年代:1998

数据来源: RSC

ISSN:0036-021X    

出版商:RSC    

年代:1998

数据来源: RSC

摘要:

Abstract. Data on the methods employed for cross-linking of DNA strands and for the synthesis of oligonucleotide duplexes with cross-links between strands are summarised. Existing meth- ods are systematised; their advantages and drawbacks are dis- cussed. The examples of applications of DNA duplexes with covalently cross-linked chains for the study of protein ± nucleic acid recognition and mechanisms of action of nucleic acid-binding proteins for gaining information about the spatial structure of nucleic acids, and for the solution of other problems of molecular biology are given.The bibliography includes 131 references. I. Introduction Studies of fine details of cellular processes related to the expres- sion and processing of genetic information require that a new generation of modified oligonucleotide derivatives be created that can be used as tools in these studies.Therefore, in recent years the chemical approach employing modified oligonucleotides for the study of mechanisms of inter- action of proteins with nucleic acids has been widely used in combination with physicochemical methods, such as X-ray struc- tural analysis andNMRspectroscopy.The direct modifications of the nucleic acid chemical structure in protein ± nucleic acid com- plexes and the analysis of properties of such synthetic systems has made it possible to gain information about the spatial structure of these complexes and the nature of protein-nucleic acid contacts. Directed modification allows useful properties to be conferred on oligonucleotides, such as resistance against nuclease degradation, enhanced thermodynamic stability of the duplexes formed by them, low toxicity, and the ability to penetrate cellular mem- branes.It also favours selective recognition and site-specific (including covalent) binding of DNA with proteins.1±9 Oligonucleotides and their reactive derivatives are important tools for the study of protein ± nucleic acid interactions in tran- scription, translation, replication, repair, recombination, and modification of nucleic acids.Until fairly recently, it was mainly derivatives of single-stranded oligonucleotides that were used to this end. Meanwhile, a great variety of processes, such as repair, recombination, initiation of transcription by the corresponding factors, etc., occur with the involvement of double-stranded nucleic acids.Consequently, it is the double-stranded structures that should be used as models and affinity reagents in studies of these processes. Modified nucleic acid duplexes have found use in elucidating the mechanisms of action of enzymes that interact with nucleic acids and in establishing the features of protein ± nucleic acid recognition.The method based on the use of cross-linked nucleic acid duplexes is one of the most promising and popular proce- dures for investigation of protein ± nucleic acid interactions.DNA duplexes with covalently cross-linked strands can be used as models for the study of the majority of the aforementioned cell- mediated processes and for the construction of affinity reagents and `decoys' for proteins binding double-stranded DNA.10 ± 16 For example, if the mechanism of action of an enzyme includes the unwinding of the double helix of DNA, covalent cross-links between the strands will prevent this process and thus strongly suppress the action of the enzyme.Duplexes stabilised by cross- linking can be potential inhibitors of nucleic acid-binding pro- teins.10, 13 Duplexes with covalently cross-linked chains are prom- ising for the isolation of proteins by affinity chromatography. Covalent cross-links formed in specific positions can stabilise thermodynamically unstable DNA structures.15, 17 Two main methods exist for the synthesis of this type of compound.Historically, the first of them consists in non-specific treatment of sufficiently long DNA molecules with bifunctional alkylating agents, such as derivatives of psoralen, pyrroles, and some other compounds.1±5, 18 The second method, which leads to the formation of DNA duplexes with covalently cross-linked chains, is based on the use of derivatives of modified oligonucleo- tides containing chemically reactive groups.One of the first to begin the study of reactions between the strands of DNA duplexes more than 30 years ago was a group of Russian investigators from the Siberian Branch of the Russian Academy of Sciences directed by Academician D G Knorre.1 ± 3, 19 ± 23 These studies have ushered in the method S I Antsypovich, T S Oretskaya Department of Chemistry, MV Lomonosov Moscow State University, Leninskie Gory, 119899 Moscow, Russian Federation.Fax (7-095) 939 31 81. Tel. (7-095) 939 31 48 Received 11 February 1997 Uspekhi Khimii 67 (3) 274 ± 293 (1998); translated by R L Birnova UDC5 47.963.32; 577.113.4 Double-helical nucleic acids with cross-linked strands: synthesis and applications in molecular biology S I Antsypovich, T S Oretskaya Contents I. Introduction 245 II. The main methods for the synthesis of cross-linked DNA duplexes 246 III.Methods for the synthesis of modified oligonucleotides 246 IV. The use of psoralens for the synthesis of covalently linked DNA duplexes 247 V. Cross-linked nucleic acid duplexes synthesised using pyrrole derivatives 250 VI. The main methods for the synthesis of nucleic acid duplexes with cross-links in predetermined positions of the double helix 252 VII.The use of sulfhydryl derivatives for the synthesis of cross-linked nucleic acid duplexes 256 VIII. Synthesis of cross-linked nucleic acid duplexes using sugar moieties of nucleosides 259 IX. Conclusion 261 Russian Chemical Reviews 67 (3) 245 ± 262 (1998) #1998 Russian Academy of Sciences and Turpion Ltdof `targeted chemical modification' which has been used in the structural analysis of nucleic acids.Conceptually, this method is related to the construction of synthetic analogues of nucleases specific for a definite type of DNA base. Later, this method gave way to the method of `complementarily targeted modifica- tion'.1 ± 3, 24 ± 31 The latter consists in the introduction of a reactive alkylating group into an oligonucleotide.After hybridisation, this group interacts site-specifically with the target DNA, i.e., it alkylates the target at a predetermined position. Thus, the majority of studies connected with the use of alkylating reagents aimed at the modification of nucleic acids dealt with the synthesis of highly reactive derivatives, which reacted with the target nucleic acid resulting in labilisation of the glycosidic bond in one of the nucleosides and, as a consequence, in the cleavage of the phosphodiester bonds of the oligonucleo- tide.1 ± 3 Irrespective of the nature of the alkylating reagent, double-stranded complexes with covalently cross-linked chains formed were unstable and underwent spontaneous hydrolysis in aqueous solutions. These investigations gave information about the reactions between the chains of double-stranded nucleic acids; however, the synthesis of DNA duplexes with covalently cross- linked chains was not the aim of these studies, and for this reason they are not considered in detail in the present review.This review encompasses studies devoted predominantly to the preparation of synthetic oligonucleotide duplexes with cova- lently cross-linked chains and to the methods of nonspecific cross- linking of DNA chains.It does not consider covalently closed structures of nucleic acids, such as cyclic oligonucleotides and dumbbell-like structures, since this is an independent trend of research, which is also being developed in recent years. II. The main methods for the synthesis of cross- linked DNA duplexes It is known that bifunctional alkylating reagents that can form cross-links between the chains (such as 2-chloroethylnitrosourea, etc.) can also inhibit nucleic acid biosynthesis.In many cases, such reagents are efficient antitumour drugs.5, 6, 32 ± 34 This is due to the fact that nonspecific cross-linking of DNA chains is highly toxic for the cell,5, 34 since it prevents expression of the genetic material.The ability to form cross-links in double-stranded DNA is the reason for the cytotoxicity of many compounds employed in clinical practice. A great number of compounds that can cross-link nucleic acid chains have been described. Psoralen 7, 11, 12, 18, 35 ± 64 and pyr- role 34, 65 ± 71 derivatives are the most popular to this end. Other compounds used for the formation of interchain cross-links in double-stranded nucleic acids include aziridinolbenzoquinones (2,5-bisaziridino-1,4-benzoquinone 72, 73 or alkylaziridino-1,4- benzoquinones),74 aziridinonitrobenzamides (5-aziridino-2- nitro-4-nitrosobenzamide and 5-aziridino-4-hydroxylamino-2- nitrobenzamide 75), anthracyclines (daunomycin,76 adriamycin,77 anthramycin,78, 79 mitomycin C80 ± 83 and its analogues 84), mela- mine derivatives, e.g., trishydroxymethyl(trimethyl)melamine,85 acridine derivatives, e.g., 1-nitro-9-(3-N,N-dimethylaminopropy- l)aminoacridine,86 chloroambucyl,87, 88 4-hydroperoxycyclophos- phamide,89 tris(2-chloroethyl)amine,90, 91 1,3-bis(2-chloro-ethyl)- 1-nitrosourea,92 2-aminomethylpyrrolidine(cyclo-butane-1,1- dicarboxy)platinum(II),93 cisplatin,34, 80, 92, 94 and cis-diamminedi- chloroplatinum(II).95 Synthetic cross-linked oligonucleotide duplexes are the most convenient tools for the study of gene structure and functions.Their application has a number of salient advantages over non- specific alkylation of nucleic acids. The presently known bifunc- tional electrophilic reagents usually recognise those sequences in nucleic acids the length of which does not exceed two nucleotide residues. As a rule, the reactions with the participation of these reagents yield mixtures of monoadducts and products of intra- strand cross-links.Heterogeneity of the reaction mixture resulting from the alkylation of DNA with non-specific bifunctional reagents 18 is often an obstacle in the studies of effects of individual interstrand cross-links on DNA processing.At present, the synthesis of oligonucleotide duplexes cross- linked at the predetermined positions of the double helix is complicated because the cross-links between the chains are either unstable 96 or endow significant disturbances in the secondary structure of DNA.35, 36 New approaches to the synthesis of DNA duplexes with interstrand cross-links have recently appeared, which permit cross-links to be created at any predetermined site of the DNA duplex and to introduce several cross-links into the same duplex.The main requirements for the methods of synthesis of modified oligonucleotides that are used in the preparation of duplexes with covalently cross-linked chains can be formulated as follows: (1) the possibility of targeted incorporation of modified units into any predetermined position of the oligonucleotide chain; (2) the applicability of standard procedures of oligonucleo- tide synthesis, and (3) the possibility of incorporation of several modified units into the same oligonucleotide for subsequent synthesis of an oligonucleotide duplex with several cross-links between the chains.III. Methods for the synthesis of modified oligonucleotides Generally, an oligonucleotide can be modified at heterocyclic bases, phosphate groups (internucleotide or terminal), or carbo- hydrate residues. The potentialities of modern chemical synthesis of nucleic acid fragments allow targeted incorporation of one or several modified units (identical or different) into the oligonucleo- tide.Compounds carrying primary aliphatic amino groups as well as aldehyde, carboxy, sulfhydryl, and phosphate groups are used for the cross-linking of the duplex chains. The general requirement for the formation of a cross-link is the localisation of the reactive group at the end of a spacer (a cross-linking arm). The length of the spacer can vary widely depending on the mutual arrangement of the reactive groups within the duplex.Thus in the case of psoralens, a relatively short, two-carbon-atoms-long spacer is sufficient for the interaction with the opposite chain.37, 38 A functional group can be incorporated into the oligonucleo- tide by the following methods:8 (a) using a modified nucleotide component carrying the corresponding functional group in the chemical oligonucleotide synthesis; (b) incorporating a precursor compound into the oligomeric chain followed by treatment with the corresponding reagents that give the target-modified unit either in the course of the oligonu- cleotide synthesis or after its completion; (c) post-synthetic modification of an oligonucleotide.The potentialities of the latter approach are limited.It can be used only for preparing oligonucleotide analogues carrying numerous statistically modified units (e.g., units with identical heterocyclic bases undergoing modification). In the former two methods, modification is envisaged in the very pattern of the chemical synthesis of oligonucleotides. The first method is the most versatile. It consists in prelimi- nary formation of modified units and their subsequent incorpo- ration into the oligonucleotide in the process of synthesis.This method does not constrain either the number or the chemical nature of the units to be introduced into the oligonucleotide chain. Moreover, it significantly simplifies isolation of the target prod- uct. Side reactions in the process of oligonucleotide synthesis are minimised because all chemically reactive functional groups of the oligonucleotide have been preliminarily protected.The initial modification of the nucleoside to be inserted is usually carried out in non-aqueous media, which permits the use of a broad spectrum of modifying reagents, whereas post-synthetic modifi- cations are usually performed in aqueous solutions. 246 S I Antsypovich, T S OretskayaIV.The use of psoralens for the synthesis of covalently linked DNA duplexes Psoralens {7H-furo[3,2-g] [1]benzopyran-7-ones} { are polycyclic aromatic compounds of the general formula 1; they have a planar structure and are capable of intercalating between the base pairs of the double helix.36, 39 ± 41 These compounds are specific to the thymidine and uridine residues.On UV irradiation of DNA duplexes at l 320 ± 360 nm, psoralen incorporates into the double helix and binds covalently with the pyrimidine bases of DNA, preferentially with thymine, at positions 5 and 6 to form four-membered rings (Scheme 1).37, 42, 43 Scheme 1 Psoralens can enter into photoaddition reactions with cyti- dine. However, this reaction occurs 15 times more slowly than with thymidine.44 In DNA, psoralens predominantly bind with the 50-d(TpA) sequence.40, 43, 45, 46 The efficiency of psoralen photoaddition to this sequence is at least one order of magnitude higher than that to thymidine in any another environment.The same regularities hold for the synthesis of cross-linked RNA duplexes.11, 12, 47, 48 Since psoralen is a bifunctional reagent, the photo-induced reaction can lead either to the monoadduct of the type 2, when the psoralen molecule is added to only one chain of nucleic acid, or to the bisadduct of the general formula 3.In the latter case, a duplex with covalently cross-linked chains is formed. The formation of the bisadduct 3 requires the absorption of two photons 49 and occurs in two steps (Scheme 1).7, 11, 45 If the pyrone ring of psoralen enters into the reaction at the first stage, the bisadduct is not formed, since the furan ring of the monoadduct becomes incapable of absorbing UV light.11 Intercalation of psoralen into the double helix, which usually increases the thermodynamic stability of the duplex, is a prereq- uisite for the reaction between the chains.The formation of adducts can be accompanied by photoinactivation of psoralen, which consists in photochemical degradation of the pyrone ring.38 The adducts of nucleic acids with psoralen are decomposed into the original components on irradiation with UV light with a wavelength that depends on the structure of the psoralen deriva- tive;39, 40, 42, 45 as a rule, it is equal to 254 nm.Thus, the cross- linking of DNA chains with psoralen is reversible. In the absence ofUV irradiation, psoralens are non-toxic. The ability of these compounds to form cross-links between DNA chains makes possible their application in in vitro and in vivo studies of theDNA structure. Psoralens are used both in medicine for photochemotherapy of some diseases 39, 40, 50, 51 and in inves- tigations of some molecular-biological processes.Psoralen has been used for the analysis of the secondary structure of RNA35, 47, 48, 50 ± 52 and the structure of the DNA± RNA-poly- merase transcription complex.12, 53 ± 55 Currently, psoralen deriv- atives are used for the study of the secondary and tertiary structure of nucleic acids as well as for the analysis of mechanisms of DNA repair.56 ± 58 Methods of targeted incorporation of psoralen into oligonu- cleotides (directly in oligonucleotide synthesis or after its comple- tion and removal of protective groups) are of prime interest.The monoadduct of psoralen with the oligonucleotide can be produced post-synthetically using an activating reagent.12, 59, 60 Such a monoadduct was obtained in the reaction of a 50-phosphorylated self-complementary oligonucleotide with 40-aminoalkyl-4,50,8-tri- methylpsoralen in the presence of 1-ethyl-3-(3-dimethyl- amino)propylcarbodiimide hydrochloride, EtN=C=N(CH2)3 ± NMe2 .HCl (water-soluble carbodiimide, CDI). Hybridisation and irradiation yield a DNA duplex, which is cross-linked at the both ends. The second method consists normally in the addition of psoralen to the 50-end of the oligonucleotide in the oligonucleotide synthesis.Phosphoramidite derivatives of psoralen are used at the last stage of the synthesis.37, 38 A typical synthetic scheme for the incorporation of psoralen into oligonucleotides is described in Ref. 38: 4,50,8-trimethylpsoralen (4) was converted into the phos- phoramidite 6 (Scheme 2) and the latter was used for the oligo- nucleotide synthesis. 4,50,8-Trimethylpsoralen 4 was added to the 50-end of an 18- membered oligonucleotide; the resulting oligonucleotide 7 was introduced into the cross-linking reaction of DNA duplex chains (Scheme 3). 4,50,8-Trimethylpsoralen 4 was shown 38 to be stable both under conditions of synthesis and post-synthetic treatments of the oligonucleotide.It is noteworthy that psoralen-containing oligonucleotides of the type 7 are characterised by higher hydro- phobicity, which is manifested in the increased retention time (in comparison with non-modified analogues) on HPLC.38 In this case, the nucleotide sequence of the oligonucleotides constituting the duplex was selected so that psoralen 4 intercalated into a double helix and reacted with thymidine in the 50-d(TpA)/ 30-d(ApT) sequence.61 It was found that the maximum yield of the DNA duplex 8 could be obtained when the CH27O7CH2CH2 group was used as the spacer for the addition of psoralen to the oligonucleotide (Scheme 3).As a rule, the yield of the product 8 increases within the first 10 ± 30 min after the beginning of irradiation (360 nm, 20 8C) and reaches 70% ±80% after 60 min.Aquantitative yield could not be attained even with a 100-fold excess of the modified oligonucleo- tide 7 with respect to theDNAmatrix 38 due to partial inactivation of psoralen in the irradiation.The 50-d(TpA)/30-d(ApT) motif is present in signal sequences of many genes. They represent potential targets for psoralen- R1, R2, R3=H, Alk.O O R2 O R3 R1 3 4 5 0 4 0 1 O N R1O HN O R2O Me O O O O hn O N R1O HN O R2O Me O O O O R3 T R4 2 hn R1 ±R4 are fragments of oligonucleotide chains. O N R1O HN O R2O Me O O O O HN N Me O O O R3O OR4 3 { Henceforth, the nomenclature accepted in the original publication is used. Double-helical nucleic acids with cross-linked strands: synthesis and applications in molecular biology 247modified oligonucleotides of the type 7.Thus oligonucleotides of this type can serve as convenient probes for the study of gene expression. A method for the synthesis of psoralen-containing oligonu- cleotides using a modified deoxyadenosine { phosphoramidite 9 has been proposed.37 4,50,8-Trimethylpsoralen 4 was attached to the C(8) atom of deoxyadenosine through the sulfur atom and the pentamethylene spacer. Subsequent phosphitylation afforded compound 9.To achieve the maximum yield (90%) in the coupling of the phos- phoramidite 9 with an oligonucleotide, 5-(4-nitrophenyl)-1H- tetrazole was used instead of tetrazide; the duration of condensa- tion was 8 min. The optimum length of the spacer was established by molec- ular modelling. In this particular case, the optimum length of the spacer needed for the psoralen intercalation into the 50-d(TA)/30- d(AT) site is five carbon atoms.An analogous derivative of deoxyadenosine of the type 9 but with a two-carbon-atoms-long spacer manifested a very weak cross-linking capacity. The effi- ciency of photoaddition to the complementary matrix chain exceeded 90% after 1-hr UV irradiation at l 345 nm.This resulted in the formation of several products, which correspond to different possibilities of psoralen incorporation into the double helix. It was noted that psoralen was effectively incorporated both into the 50-d(TA)/30-d(AT) and the 50-d(AT)/30-d(TA) sequences, where it reacted both with the thymidine unit in the opposite chain and the adjacent one. Owing to the fact that the ends of DNA duplexes of the type 8 with covalently cross-linked chains remain free, various labels (radioactive, fluorescent, or any other) can be easily attached to these ends.In addition, such duplexes can be incorporated into more extended DNA structures.37 The reaction of oligonucleotide methylphosphonates (OMP) bearing the psoralen derivative, 40-[N-(2-aminoethyl)amino- methyl]-4,50,8-trimethylpsoralen 10 (PS-OMP) attached to their 50-ends with single- and double-stranded DNA, has been studied. Compound 10 was coupled with the 50-end of OMP carrying a phosphate group to give compound 11 (Scheme 4).Water-soluble CDI was used for the activation of the phosphate group. It was shown that the P ±N bond is resistant to nucleases; however, it is cleaved in an acidic medium to give the original 50-phosphorylated OMP.On irradiation, PS-OMP specifically reacted with the tem- plate. The presence in the duplex of less stable nucleotide pairs (e.g., G. T or G. A) reduced the yields of the duplex with covalently cross-linked chains.12 This is due to the decrease in thermal stability of the original duplex. The formation of by- products that result from the interaction between the chains in O O CH2 O (CH2)5 S NHCOPh N N N N Pri 2N O(CH2)2CN P DMTr O OCH2 O Me Me O Me 9 DMTr=(4-MeOC6H4)2PhC.O O O Me Me Me CH2 NH(CH2)2NH2 10 5 O O Me Me Me ClCH2 HO(CH2)2OH O O O Me Me Me HO(CH2)2OCH2 Pri 2NP(Cl)O(CH2)2CN O 6 O O Me Me Me CH2 O (CH2)2 O O P NPri 2 O(CH2)2CN O O Me Me Me MeOCH2Cl 4 O Scheme 2 O 30 GCC AT CGGCGATAGCCAATCA 50 50 TA GCCGCTATCGGTTAGT 30 7 O O O Me Me Me CH2O(CH2)2O P O O7 hn 8 N HN O7ACCG 30 O O O O Me 50-ACTAACCGATAGCGGC 50 CH2O(CH2)2O P O O7 O TAGCCGCTATCGGTTAGT 30 O Me Me O O Me Scheme 3 { Hereinafter, the term `deoxynucleosides' will refer to 20-deoxyribonu- cleosides. 248 S I Antsypovich, T S Oretskayanon-canonical duplexes depends on temperature: by-products are formed at 20 8C and are absent at 40 8C.This can be explained by dissociation of less stable non-canonical duplexes with an increase in temperature. In the case of a canonical duplex, the temperature dependence of the yield of the covalently cross-linked duplex is not observed below its melting point. At higher temperatures, the yield of the target product drops drastically.The efficiency of the reaction between the chains of aDNAduplex is also influenced by the ratio of the rate of this reaction to the rate of psoralen photoinactivation. The yield of the cross-linked DNA duplex increases with increasing PS-OMP concentration. However, if the PS-OMP concentration is twice as high as that of the matrix, the latter becomes saturated and the yield of the target product does not further increase.12 The efficiency of the interaction of various oligonucleotides with the target under identical conditions differs markedly,12 although the differences in the nucleotide sequences should not be manifested at 4 8C.Molecular simulation has shown 12 that psoralen can be inserted between the nucleotides of the target in two modes: either between the terminal nucleosides N(n+1) and N(n) or between the nucleosides N(n) and N(n71), where N(n+1) is the 30-terminal nucleoside of the target and N(n) is the nucleoside that is complementary to the 50-terminal nucleoside of PS-OMP (Scheme 4).In the former case, the spatial structure of the reaction centre is the most favourable for the photoaddition of psoralen. The reaction between the chains is the most effective, when thymidine occupies position (n+1) of the template and position (n71) in OMP, which precedes the psoralen-modified unit, and adenosine is in the positions n.In this case, the reaction occurs with two possible sites and the yield of the duplex with covalently cross-linked chains reaches 95%. Oligonucleotides of the type 11 were used for the analysis of the secondary structure of nucleic acids.12 It was shown, in particular, that the cross-linking with the single-stranded region of DNA occurs 10 ± 30 times more efficiently than with the double-stranded one.Since OMP are characterised by enhanced resistance in the cell, these oligonucleotide derivatives can be used in in vivo studies.12 A series of PS-OMP that are complementary to the template and non-template strands of a 57-membered DNA duplex con- taining a 17-membered T7 promotor of RNA-polymerase were synthesised.12 The effects of PS-OMP on in vitro transcription were investigated.It was shown that PS-OMP, which is covalently cross-linked with the encoding region of DNA, effectively sup- pressed transcription, whereas the oligonucleotide cross-linked with the non-coding region had little inhibitory effect.Thus, the oligonucleotide that is covalently cross-linked to the template in the promotor region prevents binding of polymerase with the double helix of DNA either directly or by changing its conforma- tion in the promotor region. Being an agent that can directly react with DNA, psoralen can induce various mutations.11 Study of mutagenesis is necessary for the understanding of regularities of the origin of cancer, heredi- tary diseases, and evolutionary processes.11, 56, 57 It remained unclear which of the psoralen-induced mutations were generated by the monoadduct and which resulted from the presence of cross- links between the chains.The use of a plasmid containing a single, definite damaged site has made it possible to relate the initial damage of DNA induced by psoralen to the origin of subsequent mutations.11 The use of statistically modified DNA did not allow a correlation between the nature of mutations and the type of the adduct.The cyclic closed double-stranded plasmid containing the site-specifically incorporated psoralen as a mono- or bis-adduct (in the latter case, the cross-link is formed between the strands of the plasmid), has been synthesised in vitro and incorporated into the cells of E.coli. The recombinant DNA contained only one site with thymine in the form of a monoadduct with psoralen. Synthesis of the psoralen-containing plasmid was carried out using T4-DNA- polymerase and an oligonucleotide with psoralen at the 30-end as the primer.In this case psoralen did not influence either the elongation of the chain or its subsequent ligation. The mono- adduct was converted into the bisadduct by UV irradiation; the plasmid with covalently cross-linked chains was isolated taking advantage of its acquired ability to undergo reversible thermal denaturing. In contrast to the plasmid with cross-linked chains, denaturing of an ordinary plasmid is irreversible.The type and frequency of mutations was followed using two different DNA molecules. It was found that both types of psoralen-induced damages (the formation of the both monoadduct and the cross- links between the chains) can lead to severe genotoxic effects.11 This finding is consistent with the results obtained by other authors.58, 62, 63 The mutation induced by the formation of the monoadduct was eliminated by the repair mechanism of the cell.In the case of the bisadduct, a great number of secondary mutations (deletions, substitutions, inversions) evolved. The activity of DNA with cross-linked chains was 1.6% of the native one.11 Thus, it was shown that it is by bisadducts that the toxicity of psoralen is determined, and their formation results in severe damage to DNA.Treatment of nucleic acids with psoralen was shown to prevent replication and transcription processes.11 It was proposed 64 to couple psoralen with distamycin-like imidazole 12 and pyrrole 13 derivatives in order to promote the delivery of psoralen to specific sequences of DNA. Like the oligopeptide distamycin, these compounds bind with DNA in the region of the small groove.H H N N Me N O Me O O O O N O N N H 12 O N N Me Me N denotes deoxynucleoside residues, m56, k50. 6 11 O O O Me Me Me 50 CH2 NH(CH2)2NH P O O O7 N O P O O O7 N O P O O Me N OH 30 30 HO N O P O O O7 Nn+1 O P O O O7 Nn O P O O O7 Nn71 O P O O O7 N OH 50 k m DNA target Scheme 4 Double-helical nucleic acids with cross-linked strands: synthesis and applications in molecular biology 249The use of oligonucleotides for the delivery of psoralen to specific sequences ofDNAhas posed a number of problems.They are connected with the low ability of the oligonucleotides to penetrate through hydrophobic cellular membranes and their susceptibility to nuclease hydrolysis. This circumstance restricts the application of such compounds. The derivatives 12 and 13 are devoid of these drawbacks and can deliver psoralen to both GC-rich (imidazole analogues of distamycin, 12) and AT-rich (pyrrole analogues, 13) regions of DNA.It was found that the derivatives 12 and 13 modifyDNA10 and 1000 times more efficiently than psoralen. Compound 13 is 100 times more efficient than compound 12.Hence, psoralen, being attached to reagents that possess affinity with respect to DNA, manifests enhanced efficiency in the formation of multiple cross-links between the chains of extended DNA. V. Cross-linked nucleic acid duplexes synthesised using pyrrole derivatives Bifunctional pyrrole derivatives 14, together with psoralen deriv- atives, are widely used for cross-linking of chains of double- stranded nucleic acids.34, 65 ± 71 Only bifunctional pyrrole deriva- tives can cross-link the nucleic acid chains.Cross-linking with pyrrole derivatives 14 involves exocyclic amino groups of heterocyclic bases of DNA (usually, deoxygua- nosine) and those functional groups of pyrrole derivatives that can undergo nucleophilic attack. Hence, pyrrole derivatives are alky- lating agents.A hypothetical mechanism of this reaction has been proposed (Scheme 5).65 Scheme 5 Pyrrole derivatives as such are not specific reagents and can react with many reaction centres of DNA. Therefore, treatment of DNA with bifunctional pyrrole derivatives of the type 14 cannot be used to introduce cross-links at predetermined positions of DNA. The main drawback of pyrrole derivatives as antitumour agents is their low selectivity (i.e., the affinity for sequences with the length of no more than two nucleotides).Such limited selectivity of pyrrole derivatives is due to the high probability of occurrence of certain short repeated nucleotide sequences in the genome. Enhancement of selectivity of bifunctional pyrrole derivatives is an important problem, particularly as regards their application as therapeutic agents.This can be achieved through a more targeted delivery of pyrrole derivatives to a definite genome region, i.e., to a more rare nucleotide sequence. However, the solution of this task demands that the affinity of such agents for nucleic acids be increased.2, 34, 65 ± 71 As a rule, it is sufficient to achieve selective interaction of the cross-linking reagent with sequences more extended than four nucleotide pairs.The main route for attacking the selectivity problem is the addition of a bifunctional alkylating agent (which has little or no affinity for nucleic acids) to compounds manifesting high affinity for definite sequences of DNA, e.g., to oligopeptides.34, 65 ± 70 Synthesis of a new irreversible reagent 15 has been described.66 Compound 15 belongs to the group of pyrrolobenzodiazepine dimers (PBD dimers).It binds with DNA through the small groove and reacts with the amino group of guanine (Scheme 6). The preferential sequence for the binding of this compound is the Pu-G-Pu triplet, where Pu is the purine nucleotide (A or G). The yield of the interstrand reaction product is about 50%.H H N Me N O Me O O O O N O N 13 O N N Me Me N R3 R4 R2 R5 R1 14 R1 ±R5=H, Alk, CH2OH, CH2OC(O)Alk. Nu is the exocyclic amino group of guanine. N AcO OAc Me Nu 7AcOH + N AcO Me N AcO Nu Me Nu 7AcOH + N Me Nu N Nu Me Nu R1, R2 are fragments of oligonucleotide chains. O R1O R2O N N O H2N HN N N N H O O (CH2)3 O OMe O H N N MeO 15 +2 N N H O O (CH2)3 O OMe O H N N MeO N O NH NH N N R2O R1O O H H OR2 R1O O HN O N N HN N Scheme 6 250 S I Antsypovich, T S OretskayaThe synthesis of C(8)-linked PBD dimers of the type 16, which are homologous to compound 15, has been described by the same authors,67 and their relatively high affinity for DNA has been demonstrated.The highest efficiency of the interchain reaction was achieved with compounds 15 and 16b (three- and five-carbon- atoms-linking groups, respectively).Treatment of double-stranded DNA with any PBD dimers increases the melting point of the duplexes.67 The presence of cross-links in the linear double-stranded DNA prepared from the ring plasmid accounts for its rapid renaturation into a double- stranded form as the temperature decreases following thermal denaturing.67 The ability of extended DNA to undergo rapid renaturation is a sign of cross-linking between the chains.Thus, PBD dimers can stabilise double-stranded DNA. The cytotoxicity data 67 correlate with the yields of duplexes with covalently cross-linked chains. The PBD dimer (n=3), i.e., compound 15, possesses the highest toxicity. On the other hand, the monomer of the type 17 hardly exerts any noticeable effect on the stability of DNA duplexes and is non-toxic.Thus a possibility exists for the application of these com- pounds for the treatment of tumours. Their main shortcoming is relatively low affinity for DNA. The mechanism of cross-linking of theDNAchains by bifunc- tional pyrrole derivatives was investigated.65 It was found that all the pyrrole derivatives including natural (e.g., the antitumour antibiotic mitomycin C 18), and synthetic pyrroles 19 and 20 react withDNAby the same mechanism, they are directed to a common binding site and an alkylation site.Mitomycin C is converted into a bifunctional alkylating agent following reduction with specific enzymes. A binding site in DNA molecules, which is common for all the pyrrole derivatives in question, has been identified.The exocyclic amino group of deoxyguanosine in the 50-d(CG)/30-d(GC) sequence is the common target site in DNA for all the alkylating pyrrole derivatives.65 The formation of a cross-linked duplex requires the reaction of exocyclic amino groups of both deoxyguanosines with pyrrole derivatives. The structure of the adduct 21, the reaction product of two molecules of deoxyguanosine and mitomycin C, is given below.DNA duplexes containing only adenosine and thymidine residues have been studied by Wou et al.65 These duplexes differed in the nature of nucleosides in several nucleotide pairs. To obtain evidence for the involvement of the exocyclic amino group of guanine in the reaction between the chains, deoxyguanosine was substituted by inosine.This gave the duplexes containing the 50-d(ACGT)/30-d(TICA) and 50-d(ACIT)/30-d(TICA) fragments. When even one deoxyguanosine was substituted by inosine, the interchain cross-linking product disappeared completely. It was also shown 65 that the reaction in the 50-d(CG)/30-d(GC) sequence occurs at least ten times more efficiently than in any other sequence.The structure of compounds thus obtained was established by an analysis of the products of total enzymic hydrolysis of the cross-linked duplexes and by mass spectroscopy. It is by this method that the structure of compound 21 has been established.65 The same method has been used for structural elucidation of the adducts of the synthetic pyrrole derivatives 19 and 20.It should be emphasised that computer simulation of a cross-linked DNA duplex revealed a relatively significant distortion of the native B-form of the DNA helix irrespective of the nature of the pyrrole derivatives, which is associated with the torsional rearrangement of the DNA structure. Unfortunately, the attempts to rationalise the reason for such specificity failed. The reaction between the chains is a multistage process, which includes the formation of a mono- and then of a bis-adduct.Each of these stages can be responsible for the specificity. Since both the synthetic pyrrole derivatives 19 and 20 and natural compounds having a more complicated structure manifest identical specificity, it was concluded 65 that the specific- ity of pyrrole derivatives is inherent in the common elements of their structure.On the other hand, the significant differences in the efficiency of pyrrole derivatives as the reagents that cross-link DNA chains point to the important role of individual structural features of pyrrole derivatives. Recent developments in this area 68, 69 have been mainly focussed on the enhancement of the selectivity of pyrrole deriva- tives.In order to increase the efficiency of affinity binding of the alkylating reagent with the nucleic acid target, it was pro- posed 68, 69 to prepare its conjugate with an oligopeptide that can specifically recognise the small groove of the double helix. The affinity reagent 22 containing the oligopeptide distamycin, which recognises the 50-d(AATT) sequence (through the small groove), and pyrrole derivative, which recognises the 50-d(CG) sequence, was obtained.68, 69 n=4(a), 5(b).(CH2)n O OMe O H N N 16a,b N N H O O MeO OH OMe O H N N 17 N OAc AcO Me 20 N OAc AcO Me 19 N O O Me H2N O NH C O H2N 18 O OH N N O HN HN N OH NH2 Me H2N O N 21 O O OH N NH N O N N HO H n=2 (22), 3 (23), 4 (24). (CH2)n O 22724 N OH OH H Me N N O 3 HN NMe2 (CH2)3 Double-helical nucleic acids with cross-linked strands: synthesis and applications in molecular biology 251Thus, the affinity reagent 22 selectively recognises the nucleo- tide sequence 50-d(CGAATT)/30-d(GCTTAA) and produces a cross-link between the chains.The activity of this reagent was tested both with a synthetic duplex and a linearised plasmid.It was found that the derivative 22 is three orders of magnitude more efficient as a cross-linking reagent than a pyrrole derivative non- bound with the peptide. After thermal denaturation and cooling, the plasmid treated with this reagent underwent rapid renatura- tion into a double-stranded form, which can be explained by the presence of cross-links between the chains. The effect of compound 22 on DNA samples with different nucleotide sequences was studied.It was found that only theDNA containing the 50-d(CGAATTCG)/30-d(GCTTAAGC) sequence efficiently reacted with compound 22 (the yield of the cross-linked product is about 70% with respect to DNA). This sequence contains both 50-d(AATT), the binding site for distamycin, and 50-d(CG), the consensus binding site for pyrrole derivative.By- products were formed in all the cases studied as a result of the reaction of compound 22 with two deoxyguanosine residues of the same DNA chain. The synthesis of a series of bifunctional reagents that are homologous to compound 22 has been described.69 In these reagents (23, 24), 2,3-bis(hydroxymethyl)pyrrole is also linked to the oligopeptide distamycin.They differ from compound 22 only in the length of the linker connecting pyrrole derivative with the oligopeptide. Compounds 22 ± 24 are three orders of magnitude more efficient and more selective than ordinary pyrrole deriva- tives. They can form both intrachain and interchain cross-links in DNA depending on the nucleotide sequence in the cross-linking site of pyrrole derivatives.The interchain cross-link is formed in the duplex containing the 50-d(CGAATT)/30-d(GCTTAA) sequence, while the intrachain cross-link is formed in the case of the 50-d(GGAATT)/30-d(CCTTAA) sequence. The structures of nucleosides linked through pyrrole deriva- tives and isolated following total enzymic hydrolysis of internu- cleotide bonds are identical for both the interchain and intrachain reaction products.Comparison of binding efficiency of various substrates with each of compounds 22 ± 24 revealed that both binding sites [50-d(CG)/30-d(GC) (pyrrole derivative) and 50-d(AATT)/30-d(TTAA) (distamycin)] play an important role. Nucleotide substitution in any of these sites (CG for GC or A. T for G. C) sharply decreased the efficiency of the reaction.Thus, the 50-d(CGAATTCG)/30-d(GCTTAAGC) sequence was found to be the consensus sequence.69 Compounds 22 ± 24 were brought into reaction with a line- arised plasmid containing twenty seven 50-d(AATT)/30-d(TTAA) sites conjugated with 50-d(GC)/30-d(CG).69 The efficiency of the interchain reaction was estimated according to Hartley.5, 70, 71 This method is based on the ability of DNA with covalently cross-linked chains to undergo rapid renaturation into a double- stranded form after thermal denaturation.The efficiency of this reaction increased 69 with an increase in the reagent :DNA ratio up to 5 : 1 (these conditions favour saturation of the peptide- binding sites). The highest yield (80%) was observed on treatment of the oligonucleotide duplexes containing the 50-d(CGAATTCG)/30-d(GCTTAAGC) sequence with the com- pound 22.For the 50-d(CGAAAAACG)/30-d(GCTTTTTGC) sequence, the yield of the cross-linked product was about 50%, whereas that for the 50-d(GC...)/30-d(CG...) sequences was no more than 10%. In the case of compound 23, the yield for the 50-d(CGAATTCG)/30-d(GCTTAAGC) sequence was 40%. Sigurdsson and Hopkins 69 could not rationalise the decreased efficiency of this reaction with an increase in the linker length.Probably, this is due to the geometrical disorientation of pyrrole derivative in the reaction centre. The structures of compounds thus obtained were analysed by studying the composition of products of total enzymic hydrolysis of duplexes with covalently cross-linked chains. Even prolonged hydrolysis of DNA with covalently cross-linked chains by a mixture of snake venom phosphodiesterase and alkaline phos- phatase did not result in its complete disintegration into nucleo- sides. Increased amounts of snake venom phosphodiesterase in combination with endonucleases (DNAase I and DNAase II) were used for complete hydrolysis of the phosphodiester bonds of the duplex.The cytotoxicity of the compounds in question was relatively low and comparable with that of natural pyrrole derivatives.The use of compounds of the type 22 ± 24 for cross-linking of DNA chains has a number of advantages over psoralen deriva- tives. Psoralen recognises the large groove of DNA and binds thymidine residues in 50-d(TA)/30-d(AT) sequences through inter- calation into the double helix.In contrast, pyrrole derivatives coupled with peptides (compounds 22 ± 24) recognise the small groove. It is noteworthy that a more extended (six nucleotides) sequence is recognised in this case, and the reagent itself remains outside the double helix of DNA without causing its destabilisa- tion. However, the use of such targeted-action agents is now limited due to the lack of the general concept of the design of oligopeptides that bind to a predetermined nucleic acid sequence.34 Thus, a series of compounds capable of cross-linking nucleic acid chains has been described.11, 12, 18, 34 ± 95 Each of them binds with the duplex at a definite site and produces covalent cross-links with two nucleotides from the opposite chains.However, the majority of compounds described in the literature have a number of disadvantages. They are devoid of high binding specificity with a unique site in nucleic acids.This results, as a rule, in the formation of multiple interchain and intrachain cross-links. These reagents are not versatile, because they cannot form cross- links at predetermined positions between nucleic acid chains, but interact only with definite heterocyclic bases.In some cases, they destabilise the double helix at the cross-linking region, which results in disturbances in the geometry of the canonical spatial structure of nucleic acids. Psoralens, pyrrole derivatives, and other compounds mentioned above have found use both in clinical practice (as antitumour agents) and in the study of mechanisms of functioning of some molecular-biological systems.Nevertheless, it is a new generation of chemically designed synthetic systems devoid of the above drawbacks that will be helpful for the study of peculiarities of structural organisation and mechanisms of functioning of intracellular structures at the molecular level. VI. The main methods for the synthesis of nucleic acid duplexes with cross-links in predetermined positions of the double helix The use of nucleic acid duplexes with cross-links at predetermined positions of the double helix is the most promising for the study of protein ± nucleic acid recognition and mechanisms of action of enzymes.As a rule, synthesis of such duplexes requires the use of reactive oligonucleotide derivatives containing modified nucleo- sides.The use of these derivatives has a number of advantages over the above-described methods. In particular, they provide a full control over the localisation of cross-links to be incorporated between the chains and the completion of the synthetic process. Various types of reactive derivatives have been proposed. R is an oligopeptide. 30 T T A A G C 50 A A T T C G NN2 NH2 N R OH HO 252 S I Antsypovich, T S OretskayaThe synthesis of a series of oligonucleotides 25 containing N4,N4-ethano-5-methyldeoxycytidine dCe has been de- scribed.97 ± 99 These oligonucleotides interact with the comple- mentary chain of DNA, and N4,N4-ethano-5-methyldeoxycyti- dine reacts with the oppositely located nucleotide immediately after hybridisation with the complementary target oligonucleotide under mild conditions and without treatment with activating reagents.97 This method was named `hybridisation-triggered alky- lation'.A phosphoramidite derivative of 5-methyldeoxycytidine car- rying a triazole residue at position 4 was used in the synthesis of oligonucleotides of the type 25. Oligonucleotides 26 containing this residue are stable under the conditions of the phosphorami- dite procedure of oligonucleotide synthesis.Treatment of the oligonucleotide 26 with ethyleneimine con- verts it into the oligonucleotide 25 in quantitative yield.97 This makes it possible to incorporate a reactive acid-labile group into the oligonucleotide following all acidic treatments needed for the removal of the dimethoxytrityl group in the process of oligonu- cleotide synthesis.N4,N4-Ethano-5-methyldeoxycytidine is unstable under con- ditions of standard treatment of oligonucleotides with ammonia (5 h, 55 8C, concentrated NH4OH). Therefore, the 9-fluorenyl- methoxycarbonyl group 97 (Fmoc) was used to protect the exocy- clic amino groups of the nucleosides; this protective group is removed faster and under milder conditions of b-elimination than the benzoyl group routinely used for this purpose.Fmoc-pro- tected deoxyadenosine and deoxycytidine phosphoramidites were synthesised. Attempts to obtain a Fmoc-derivative of deoxygua- nosine in satisfactory yield failed. Conditions have been selected for removal of the protective groups with retention of the ethyl- eneimine ring of compound 25.The reaction with the oligonucleotide template is carried out for several days (the semi-conversion period is about 30 h at 24 8C).98 The efficiency of the reaction between the chains substantially depends on the nature of the nucleotide that reacts with the dCe unit of compound 25. The following order of the efficiency of this reaction was established: A&T C&G.The reaction occurs with each of the four bases to a certain degree. In some cases, only one by-product is formed in the interchain reaction. It is assumed 98 that under these conditions the reaction involves not only the opposite base in the other chain, but also one of the neighbouring bases of the duplex. The melting point of duplexes with cross-linked chains is increased, on the average, by 15 8C (from 22 8C to 37 8C for a 21-membered AT-enriched duplex consisting of fifteen A.T pairs and six G.C pairs). The main disadvantage of this method is the necessity of substitution of standard nucleoside phosphoramidites on Fmoc- containing ones. The Fmoc-group is extremely labile, therefore the yields of Fmoc-protected nucleosides are low and deoxyino- sine must be used instead of deoxyguanosine.Moreover, the rate of the interchain reaction is too low (semiconversion period is 30 h) and therefore these oligonucleotides cannot be used as inhibitors of mRNA expression in vitro. On the other hand, the proposed method of interchain cross-linking 97 is devoid of such shortcomings as the necessity of activation of the alkylating function and low level of specificity.The latter can be due to the excessive reactivity of the alkylating group. The majority of the known methods for the preparation of duplexes with covalently cross-linked chains are based on the interaction of a single-stranded DNA with oligodeoxy- ribonucleotide containing a reactive group. Recent reports sug- gest an alternative variant, which consists in the cross-linking of an oligonucleotide to double-stranded DNA within a triple helix (triplex).7, 88, 100 ± 109 Thus, a method has been proposed for specific cross-linking of dCe oligonucleotides with units of the type 25 described above to double-stranded DNA within the triplex.100 In this case, the oligonucleotide is recognised by Hugsteen's pairs and intercalates into the large groove of the target DNA duplex.In the dCe+ .G.C triplex, the electrophilic methylene group of N4,N4-ethano-5-methyldeoxycytidine comes close to the N(7)- and O(6)-nucleophilic centres of deoxyguano- sine. The ethyleneimine ring interacts with one of the guanine bases of the double helix of the target DNA. The reaction between the chains occurs under physiological conditions in more than 95% yield and is completed within 16 h.This method can be promising for specific in vivo inhibition ofDNAtranscription and replication due to the formation of an untwistable triplex.100, 101 As has been mentioned above, one of the main problems connected with the introduction of new functional groups into the oligonucleotide is that under conditions of automated synthesis the DNA chain that is elongated is exposed to the action of acids, bases, and electrophilic reagents. The functional group to be introduced must withstand these treatments either as such or after protection.Since N4,N4-ethano-5-methyldeoxycytidine is sensitive to nucleophiles,101 it can be incorporated only into oligothymidylates but not into an oligonucleotide containing nucleosides of all the four types, because only the former do not require post-synthesis ammonia treatment to remove protective R1 ±R3 are fragments of oligonucleotide chains.O N R1O N O R2O Me N O N RO N O R2O Me HN (CH2)2NHR3 H2NR3 25 R1 and R2 are fragments of oligonucleotide chains. N N N R1O Me O N N O OR2 26 O N N O Me N R1O OR2 25 NH R1 ±R3 are oligonucleotides.N N N N R2 O H N H H H H H N R1 O N H H N R3 O N N + N N N N R2 O H N H H H N R1 O N H H N R3 O N N Me + N N Double-helical nucleic acids with cross-linked strands: synthesis and applications in molecular biology 253groups. This restricts the possible application of such oligonucleo- tides as substrates that can covalently bind to the DNA target. An attempt to apply this synthetic scheme to deoxyadenosine was even less successful.110 It was found that N6,N6-ethanodeoxyade- nosine was a weaker electrophile than N4,N4-ethano-5-methyl- deoxycytidine.In a recent study, devoted to the synthesis of oligonucleotide duplexes with covalently cross-linked chains Shabarova et al.13 also described modification of the heterocyclic bases of deoxy- cytidin. Two approaches to the design of DNA duplexes with cross-links between oligonucleotides in the predetermined posi- tions of the double helix were proposed. The first one consisted in the formation of an amide bond between the amino and carboxy groups of modified bases localised in the opposite chains, while the second one entails the formation of a covalent `bridge' as the Schiff's base between the two amino groups using dialdehydes of the aliphatic series. To this end, deoxycytidine in the self-comple- mentary oligonucleotide 50-d(TAATGCATTA) was replaced by a 5-methyldeoxycytidine derivative containing an aliphatic amino group (oligonucleotide 10n, 27) or a carboxy group (oligonucleo- tide 10c, 28).These functional groups occupied terminal positions at the spacer linked to the exocyclic amino group of 5-methyl- deoxycytidine.In the formation of the DNA duplex 29, the carboxy and amino groups turned to be close to each other, which enabled the reaction resulting in the formation of an amide bond between the chains to occur. Synthesis of a self-complementary oligonucleotide precursor, which is common for compounds 27 and 28, viz., 50-d(TAATG- C*ATTA) 29, where C* is the cytidine residue containing triazole at position 4, was carried out by the phosphoramidite method, and the triazolyl derivative of thymidine 30 was introduced in the reaction.After completion of the synthesis, the polymer with the immobilised oligonucleotide was divided into two equal parts. One of them was treated with ethylenediamine to give compound 27, while the other one was treated with b-aminopropionic acid to give compound 28. The reaction between the chains was carried out with water-soluble CDI as the condensation reagent at 0 8C, which facilitated the formation of a double-stranded complex.It must be noted that this system favours the formation of three types of oligonucleotide duplexes: (10n) : (10c), (10n) : (10n), and (10c) : (10c), since the original oligonucleotide is self-comple- mentary.It was found that the (10n) : (10n) complex was the most stable, presumably owing to the electrostatic contacts between the amino and phosphate groups of the opposite chains. The predom- inance of the (10n) : (10n) complex in solution is responsible for the low yield of the target product, which can be formed from the (10n) : (10c) complex.The optimum pH value for this reaction lies in the range of 5.5 ± 6.0. Any deviation from this interval increases the amount of by-products as a result of modification of hetero- cyclic bases with carbodiimide. In addition, the formation of an adduct of the oligonucleotide (10c) with carbodiimide involving the carboxy group took place as a side process.In the alternative version, the reaction between the chains of the 50-d(TAATGC*ATTA)/30-d(ATTAC*GTAAT), (10n) : (10n), duplex was carried out with glutaraldehyde. The Schiff's base formed initially was reduced with sodium borohydride to give a stable secondary amine. The yield of the target, cross-coupled duplex was brought to 20%. The low yield can be explained by several reasons.First, besides the aliphatic amino groups that have been introduced, oligonucleotides contain other reaction centres capable of react- ing with glutaraldehyde (e.g., exocyclic amino groups of hetero- cyclic bases). Second, it is difficult to select conditions where two aliphatic amino groups of the modified duplex will react with the same molecule of glutaraldehyde, since the reaction is carried out with an excess of the aldehyde.Third, most probably, the optimum mutual arrangement of reactive amino groups was not realised, since they were localised in adjacent base pairs. Prelimi- nary molecular modelling of the optimum mutual arrangement of reactive groups in the reaction centre has now become a standard procedure. At the same time, it was demonstrated that oligonucleotides containing an aliphatic amino and a carboxy group can be used for the synthesis of DNA duplexes with an amide bond between the chains.13 It must be noted that the system was designed in such a way as to enable polymerisation of the duplex with covalently O N R1O N O R2O Me NH O N R1O N O R2O Me NH + CDI 27 29 28 O N R1O N O R2O Me NH (CH2)2 O NH C (CH2)2 NH Me R2O O N R1O N O (CH2)2NH2 (CH2)2 C HO O R1 and R2 are fragments of oligonucleotide chains.CDI is EtN=C=N(CH2)3NMe2 . HCl. N N N P Me O O N DMTrO N O Pri 2N O(CH2)2CN 30 O N R1O N O R2O Me NH (CH2)2 NH2 + 27 27 O N R1O N O R2O Me NH (CH2)2 H2N OHC(CH2)3CHO NaBH4 O N R1O N O R2O Me NH (CH2)2 NH (CH2)5 NH O N R1O N O R2O Me NH (CH2)2 R1 and R2 are fragments of oligonucleotide chains. 254 S I Antsypovich, T S Oretskayacross-linked chains under the action of DNA-ligase. This gives a DNA-like polymer containing a repeating consensus Pribnow sequence, which is the main binding site for RNA-polymerase in the initiation of transcription. This requires unwinding of the DNA double helix with the formation of an open complex. As the presence of cross-links between DNA chains prevents unwinding, these compounds can be used for the study of initiation of transcription and, apparently, for influencing this process.Gottikh et al.111 have synthesised an unusual oligonucleotide derivative 31 with covalently cross-linked chains. This compound has a `snail-like' structure and represents a chimeric oligonucleo- tide comprising an a-nucleotide cluster.Unlike ordinary DNA, the a- and b-oligonucleotides form a parallel duplex, which allows the chimeric oligonucleotide to occur as an intramolecular duplex. The cross-link is formed between the amino group attached through a spacer to a thymidine analogue in a single-stranded region and the 30-terminal phosphate of the a-fragment. As expected, no reaction occurred directly between these groups because of steric hindrances.Therefore, an amino acid NH27(CH2)n7COOH (n=1, 2, 7) was coupled with the termi- nal phosphate of the a-fragment activated in the form of N-hydroxybenzotriazole ester. Then the reaction was carried out between the incorporated carboxy and amino groups using water- soluble CDI as the coupling reagent. Owing to the presence of an interchain cross-link in compound 31, this is highly resistant to nuclease degradation.It has been shown that RNAase H cleaves an oligoribonucleotide in the chimeric duplex involving compound 31. The study 111 illustrates the possibility of conferring useful properties to a compound by cross-linking of oligonucleotide chains. Presumably, the oligonu- cleotide derivative 31 can be effectively used for the regulation of RNA expression.111 A method for the synthesis of DNA duplexes in which the chains are cross-linked through a substituted pyrophosphate group was developed (Scheme 7).96 This involves the reaction between the activated terminal phosphate group linked to an oligonucleotide through a spacer and the spatially close internu- cleotide phosphate group of the complementary oligonucleotide.The activation of the terminal phosphate group was carried out with water-soluble CDI. TheDNAduplexes containing a trisubstituted pyrophosphate internucleotide group in one of the chains are stable in aqueous buffer solutions (pH 6.0 ± 8.75) in the absence of strong nucleo- philes. At the same time, they are readily and selectively cleaved at this group with nucleophilic reagents, such as N-methylimidazole, amines, and some amino acids. In this case, the nucleophilic substitution reaction involves both phosphate groups of the trisubstituted pyrophosphate group and is accompanied by a cleavage of the interchain bond and transfer of one of the duplex residues to the nucleophile.96 In contrast with DNA duplexes containing a disubstituted pyrophosphate internucleotide group in one of the chains, the reaction of nucleophilic substitution at the phosphorus atom in theDNAduplexes containing a trisubstituted pyrophosphate interchain internucleotide group can occur with Scheme 7 the involvement of both di- and tri-substituted phosphate groups to yield a complex mixture of oligonucleotides.Obviously, such DNA duplexes will be unstable in cell cultures.The method considered allows the synthesis of DNA duplexes with a reactive group between the chains. The interest in the synthesis of such compounds is connected with the possibility of their application for binding with proteins. A method of preparation of cross-linked duplexes involving deoxyadenosine residues was proposed.112 N6-(2-Aminoethyl)- deoxyadenosine (32) is converted into the phosphoramidite deriv- ative 33, which can be incorporated at any predetermined position of an oligonucleotide.N6-(2-Aminoethyl)deoxyadeno-sine 32 was obtained by treatment of 6-iododeoxyadenosine with ethylenedi- amine, and the amino group was protected by subsequent treat- ment of the reaction product with S-ethyltrifluorothioacetate.The trifluoroacetyl protection is ideally suited for the incorporated aliphatic amino group. A dinucleoside monophosphate containing the N6-modified deoxyadenosine 32 with an aliphatic amino group was synthes- ised.112 The synthesis was carried out by the phosphoramidite method in solution; the yield was 95%. Higher oligonucleotides were not synthesised; however, this method is a model study aimed at the incorporation into oligonucleotides of compound 33 as a potential tool for interchain cross-linking.The oligonucleotide synthesis involving the phosphoramidite derivative 33 was free of side reactions.112 However, the effective- ness of this modification for the synthesis of duplexes with covalently cross-linked chains is unclear.It was proposed 112 to perform the reaction between the chains in the following way:112 after hybridisation with the template, the aliphatic amino group has to be localised in the vicinity of the pyrimidine base of the complementary chain. The reaction of this base with the bisulfite ion at positions 5 and 6 facilitates the subsequent nucleophilic attack at position 4. The aliphatic amino group of N6-(2-amino- ethyl)deoxyadenosine 32 will act as the nucleophile.This method of interchain cross-linking must not bring significant disturbance to the structure of the double helix.Adisadvantage of this method is its inapplicability to cross-linking of oligonucleotides to extended DNA, since in the presence of the bisulfite anion T T T T CTTAGCTGTACCGCACAAGTTGCG TCTT TTTCCCCCCTTACTT TCT X T T T 50 AGAAAAAGGGGGGAATGAAAGA NH (CH2)n 24b 22b P O O7 O NH C O 22a 31 R is the deoxyribose residue.X= N N N O (CH2)6 O R O H ; n=1, 2 7; H 30 O (CH2)n O O O7 P 50 R1 R5 R4 NH N C O7 O O P + OR3 50 O7 P 30 R2 O 7R4HNCNHR5 O R1 ±R3 are fragments of oligonucleotide chains; n=3, 4, 7, 12. (CH2)nR 30 O O O7 P 50 R1 O O7 O7 P O O P O 30 R2 R3 50 O OH N N NH N N HO (CH2)2 H2N O O N N NH N N DMTrO (CH2)2 HN C F3C O 32 33 P O(CH2)2CN NPri 2 Double-helical nucleic acids with cross-linked strands: synthesis and applications in molecular biology 255deoxycytidine in single-stranded regions can undergo side con- versions into 20-deoxyuridine, which is undesirable.VII. The use of sulfhydryl derivatives for the synthesis of cross-linked nucleic acid duplexes Oligonucleotides containing SH-groups have received wide acceptance in recent years.They not only allow the synthesis of various types of oligonucleotide derivatives, but also of cross- linkedDNAduplexes in the predetermined positions of the double helix. The so-called `convertible nucleoside approach' of oligonu- cleotide synthesis has become widespread.113 It consists in the incorporation of a precursor of the modified nucleoside carrying an SH group into the oligonucleotide that is synthesised.This precursor carries a different group, which is usually more resistant to synthetic and post-synthetic treatments. Later (sometimes even after hybridisation with the template), the precursor is converted into the target nucleoside with a thio group, which becomes involved in the reaction between the chains.This strategy has considerable promise for it permits an access to various types of modified oligonucleotides from the same precursor because the incorporation of a functionalised linker is performed after completion of the oligonucleotide synthesis. This approach does not envisage the synthesis of a new starting monomer or a new modified oligonucleotide in order to change the length of the linker or the nature of the functional group.Another advantage of this method is that it allows a post-synthetic addition to oligonucleotide derivatives of ligands (oligopeptides, etc.) unable to withstand the treatments used in the oligonucleo- tide synthesis. A characteristic feature of this method is that the monomer used in the synthesis contains a leaving group, which undergoes nucleophilic substitution.When a bifunctional nucleophile is used, reactive oligonucleotide derivatives are formed. At the same time, the bifunctional nucleophile fulfils the function of a linker. In order to attain the maximum efficiency, a number of conditions must be observed.113 The nucleoside in question must be stable under conditions of automated synthesis ofDNAand be rapidly and completely converted into a reactive derivative by a nucleophile.Incorporation of a linker must cause minimum disturbance to the structure of the DNA double helix. As a rule, this process should not involve the terminal oligonucleotide units so as to retain the possibility of further enzymic reactions, such as radioactive labelling, elongation of the oligonucleotide, or its incorporation into a plasmid.Ferentz and Verdine 113 have `converted' deoxyinosine into modified deoxyadenosine. Earlier, these authors had shown 114 that O6-phenyldeoxyinosine 34 was quantitatively converted into N6-alkyldeoxyadenosine 35 on aminolysis with alkylamines. O6-Phenyldeoxyinosine 30-phosphoramidite was synthesised and introduced into automated synthesis to give the oligonucleo- tide 50-d(GCGAI*TTCGC) 36, where I* is the residue of the nucleoside 34.After completion of the synthesis, the oligonucleo- tide 36 was treated with bis(2-aminoethyl) disulfide or bis(3- aminopropyl) disulfide, resulting in compound 37. The oligonucleotide 37 obtained was self-complementary and formed a duplex in which the modified bases are localised in neighbouring units.Treatment of the duplex with dithiothreitol led to the conversion of the disulfide into thiol and then the disulfide bond between the chains was closed by oxidation with the atmospheric oxygen. This binding is reversible. Repeated treatment of the duplex with dithiothreitol results in the cleavage of the interchain cross-link.It was shown 113 that the products of enzymic hydrolysis of the DNA duplex with covalently cross- linked chains contain the dinucleoside 38; the shape of the helix of the DNA duplex corresponds to the B-form of DNA, while the modified bases form ordinary Watson ± Crick pairs. Computer simulation has revealed that the linker is localised in the large groove of DNA and does not cause any significant distortions in the spatial structure of the duplex.The original duplex and the duplex with covalently cross-linked chains have similar melting curve profiles. This indicates that the cross-linking between the chains does not disturb the duplex structure itself. Under these conditions, the melting point of the duplex with covalently cross-linked chains (75 8C) was by 18 8C higher than that for the original duplex in 1 MNaCl, whereas in 0.1 MNaCl it was by 30 8C higher.Thus, the formation of a disulfide bond between the chains has a number of advantages. First, it increases significantly the stability of the DNA duplex. Second, it does not result in noticeable distortions in the DNA structure.DNA duplexes with chains linked by disulfide bonds can be used for the study of processes involving the unwinding of DNA by enzymes, e.g., transcription, replication, and recombination. Yet another approach to the synthesis of a DNA duplex with covalently cross-linked chains by the `convertible nucleoside approach' has been described.14 O6-[2-(p-Nitrophenyl)ethyl]-2- fluorodeoxyinosine 115, 116 was introduced into a self-complemen- tary 18-membered oligonucleotide in the process of oligonucleo- tide synthesis.Treatment of this oligonucleotide with bis(aminoalkyl) disulfides (Scheme 8) or N-(2-methylthioethyl)- amine 39 led to the substitution of the êuorine atom for N-(o- aminodithioalkyl)amino group or the N-(2-methylthioethyl)a- mino group.14 Following treatment with dithiothreitol, the reaction between the chains was carried out in the presence of atmospheric oxygen.The presence of a cross-link was proved by an analysis of a correlation between the melting point of the duplex with cova- lently cross-linked chains and the ability of these cross-links to induce entropic stabilisation of DNA.117 dR is the 20-deoxyribose residue.N N N N dR OPh Alk NH2 N N N N dR NH Alk 34 35 R1 and R2 are fragments of the oligonucleotide chain; n=2, 3. O OR2 N N OPh N N R1O O OR2 N N S(CH2)nNH N N R1O H2N(CH2)nS H2N(CH2)nS 36 37 H2N(CH2)nS7 n=2, 3. O OH N N HN N N HO (CH2)n S S (CH2)n HO N N NH N N OH O 38 256 S I Antsypovich, T S OretskayaScheme 8 The melting point of the duplex cross-linked by a bis(2- aminoethyl) disulfide linker (n=2) was lower than that of the duplex cross-linked with a bis(3-aminopropyl) disulfide linker (n=3).Presumably, a shorter linker causes greater torsional tension in the DNA structure. Comparison with an oligonucleo- tide duplex containing the (2-methylthioethyl)amino groups revealed that this modification as such has little effect on the melting point of the duplex.14 The DNA duplexes thus synthesised with covalently cross- linked chains were used for the study of DNA methylation.This process plays an important role in the development of mammals and in the resistance of bacteria to viral infections.14 DNA-5- cytosine-methyltransferase (CMTase) catalyses transfer of the methyl group from S-adenosyl-L-methionine to cytosine. The enzyme recognises the symmetrical site of DNA, viz. 50-d(GGCC)/30-d(CCGG), and transfers the methyl group to the internal deoxycytidine residue of each chain. It was assumed 14 that if the methylation site contains an interchain cross-link, the unwinding of the DNA double helix as an element of the enzyme action mechanism will be hampered considerably during the transfer of the methyl group.It turned out that the enzyme actually induces a distortion of the double helix. In order to establish a correlation between the effect of interchain cross-linking and the recognition of the methylation site by the enzyme, the equilibrium binding has been studied under non-catalytic conditions. It was found that the duplex with a dithiaoctamethylene bridge between the chains binds to the enzyme with the same efficiency as does the non- modified duplex, whereas the duplex with a dithiahexamethylene bridge binds 43 times more weakly.Thus, the presence of a cross- link between the chains has no effect on the enzyme binding with DNA (at least, for a longer linker). Moreover, in the latter case cross-linking between the chains results in allosteric activation of the protein binding.Most probably, this is due to the fact that the presence of an interchain cross-link generates torsional tension, which decreases the thermodynamic barrier to DNA unwinding. However, in contrast to cross-linking, methylation occurs much more slowly in comparison with the natural duplex. Nevertheless, under conditions of substrate saturation both duplexes with covalently cross-linked chains serve as substrates for CMTase and the degree of methylation is 10% relative to that of the non- modified duplex.This study illustrates the applicability of DNA duplexes with covalently cross-linked chains for gaining insight into the mechanisms of action of DNA-recognising enzymes. Erlanson et al.14 have carried out computer simulation of the spatial structure of DNA duplexes with covalently cross-linked chains.The data obtained were compared with the results of X-ray analysis.118 The molecular dynamics methods were used to study the effect of the interchain cross-linking on the secondary struc- ture of DNA. This made it possible to estimate the extent of distortions induced by cross-linking in the duplex structure and to evaluate its effect on the dynamic behaviour of DNA.Spatial structures of the self-complementary decanucleotide 50-d(GCGAATTCGC) in the form of a duplex and of two modified duplexes with the same primary structure and a cross- link between the amino groups of deoxyadenosine residues in the central A. T pair were studied. The lengths of the linkers were six (dithiahexamethylene) or eight dithiaoctamethylene) atoms (the nitrogen atoms were not taken into account).It was shown 118 that the canonical B-form of DNA and the Watson ± Crick type of base pairing are preserved by all DNA duplexes in question. The parameters of the duplex helices with covalently cross-linked chains, of the large and small grooves, and of the base pairs, as well as the torsional angles of the sugar ± - phosphate backbone are also mostly similar to the corresponding parameters for the crystalline structure of the non-modified duplex.Yet, the presence of cross-links between the chains endows certain disorder in the geometry of the double helix. The devia- tions observed are insignificant: no more than 1.2 A from the canonical B-form of DNA and no more than 1.3 A from the experimentally determined parameters for the crystal of a non- modified duplex.These changes are more substantial with the shorter interchain linker. The dynamic properties of the duplexes with covalently cross-linked chains are also similar to those of the non-modified duplex. Certain changes are observed in a region adjacent to the cross-linking site and are especially pronounced in the case of a shorter linker.Structural distortions of general character are manifested in decreased mobility at the cross-link site irrespective of the linker length. Thus, the changes in structural parameters originate mostly from the modification of the amino groups of deoxyadenosines rather than from the presence of a cross-link between the chain. On the whole, the presence of a cross-link between the chains induces insignificant changes in the duplex structure;118 therefore, these duplexes may be used for the study of proteins that interact with nucleic acids. Yet another original procedure for obtaining DNA duplexes with covalently cross-linked chains on the basis of thiols has been proposed.119 Two residues of N3-(2-mercaptoethyl)thymidine 40 were incorporated into an oligodeoxyribonucleotide and two residues of N3-(2-mercaptoethyl)uridine 41, into an oligoribonu- cleotide.The phosphoramidite derivative of compound 40 was attached to the 50- and 30-ends of an oligodeoxyribonucleotide in the automated synthesis. The disulfide bond between the ends of a hairpin was closed by oxidation of the thio groups with atmos- pheric oxygen.The structure of the interchain cross-link at the end of the hairpin formed by two residues of compound 40 is shown below: R1 and R2 are fragments of the oligonucleotide chain; n=2, 3. O OR2 N N N N R1O NH O (CH2)2 O2N (CH2)n SH O OR2 N N N N R1O F O (CH2)2 O2N 1. 2. H2N(CH2)nS H2N(CH2)nS HSCH7CHSH OH OH 39 40: R1=Me, R2=H; 41: R1=H, R2=OH. O N HO N O HO R1 O R2 HS(CH2)2 40, 41 N N (CH2)2 O Me O S S O Me O (CH2)2 O N HO N O O OH O G C A A T C C C A T T T G G A T T G C Double-helical nucleic acids with cross-linked strands: synthesis and applications in molecular biology 257The structures of a covalently closed hairpin and its natural analogue are shown in Scheme 9.Their conformational structure was studied by NMR spectroscopy. It was found that the 8-membered double-stranded region in both hairpins has a canonical B-form; structurally both hairpins are completely iso- morphic. Scheme 9 In this case, the presence of an interchain cross-link neither influences the spatial structure of the hairpin nor interferes with the native B-conformation of the double helix.It should be noted that although in this study the cross-link was formed at the end of the DNA duplex, the modified nucleoside could be incorporated into any predetermined position of an oligonucleotide. This method can thus be applied for obtaining a set of various cross- linked oligonucleotide duplexes.Thus, the synthesis of duplexes with covalently cross-linked chains based on sulfhydryl derivatives has a number of advantages over the use of alkylating agents, which do not ensure sequence- specific cross-linking of chains.The latter circumstance restricts their application in the engineering of interchain cross-links. The disulfide method was used to cross-link fragments of the ribozyme RNA chain.17 The reaction was carried out between spatially close (but not complementary) regions of the RNA chain.Hence, here we do not deal with the formation of cross- links between the RNA chains within the duplex; however, this method is so elegant that it deserves special mention. Eckstein et al.17 took advantage of modification of the 20-positions of certain sugar residues. Oligoribonucleotides with incorporated 20-amino- 20-deoxyribonucleotides were synthesised for this purpose.Syn- thesis of 20-amino-20-deoxyribonucleosides has been described earlier.120 ± 126 The sulfhydryl groups were introduced as follows. First, the oligoribonucleotides were treated with the bifunctional reagent 42 (Scheme 10) containing the isothiocyanate and a protected sulf- hydryl group. It was shown that compound 42 selectively reacts with the 20-amino groups of the modified ribonucleotides.17 The reaction between the fragments of the ribozyme chain was initiated by dithiothreitol, which deprotected the sulfhydryl groups.This enabled the reaction between the ribozyme chains under the action of atmospheric oxygen (Scheme 10). The pres- ence of a cross-link between the chains was confirmed by limited alkaline hydrolysis. One cleavage of the ribozyme chain at the site between the modified residues gave a single product with the same molecular weight as that of the original ribozyme.Ribozymes with covalently cross-linked chains obtained by the above-described procedure were used to study the tertiary structure of the ribozyme in relation to the catalytic mechanism of RNA cleavage. The parameters of the ribozyme tertiary structure obtained earlier by X-ray analysis (Scheme 11, A) and fluores- cence resonance energy transfer (FRET) solution measurements (Scheme 11, B) were different.[Ribbon representation of the tertiary structures of the hammerhead ribozyme based on (A) X-ray crystallography and (B) FRET solution measurements. Ribozymes and substrates are coloured black and light gray, respectively.Residues that are close in space according to the X-ray structure and the FRET model (dark gray) are connected by solid lines and the broken lines represent the corresponding long distances in the other model.] At the same time, both putative structures had identical general geometry. The difference between them lay in the orientation of the two helical regions of RNA relative to each other (helices I and II, Scheme 11).In order to compare these two spatial models of the ribozyme and to choose the model corresponding to the genuine biologically active struc- ture, synthetic ribozymes with covalently cross-linked chains were used. The authors 17 have succeeded in stabilising the spatial struc- ture of the ribozyme according to each of the putative models.After hybridisation of the modified ribozymes with a non-hydro- lysable substrate analogue containing a deoxynucleoside in the reaction centre, the reaction mixtures were treated with oxygen for 30 h. This permitted a highly efficient reaction between the chains irrespective of the localisation of the modified units in the ribozymes (Scheme 12). Roman numerals indicate the number of the helices.An arrow indicates the site of cleavage. Residues contained a 20-amino function and utilised for cross-linking are filled by dark gray colour. The yield of the ribozymes with covalently cross-linked chains after all purification procedures was 15%. The cross-links between the chains stabilised the spatial structure of the ribozyme in accordance with the predetermined one, which made it possible to measure the catalytic activity of two synthetic ribozymes with different spatial structures.As expected, the catalytic activity of the ribozyme depends on the localisation of the cross-link between the chains. The cross-link between the adjacent regions of the helices corresponding to the X-ray model (Schemes 11, 12, A) had practically no effect on the catalytic A C T T T G C G C A T T A T A G C C G T T S S A C T T T G C G C A T T A T A G C C G T A + NCS S S N 42 NH2 20 NH2 20 NH C NH S SH NH C NH S SH O2 NH C NH S S S S NH C NH NH C NH S S S N NH C NH S S S N HSCH7CHSH OH OH Scheme 10 II I III 33A 11A A 13A 32A B Scheme 11 258 S I Antsypovich, T S Oretskayaactivity of the ribozyme.On the other hand, the cross-link between the adjacent nucleotides according to the other model (Schemes 11, 12, B) decreased the rate of the ribozyme-induced cleavage of the corresponding substrate by three orders of magnitude.It was thus concluded that the X-ray model corre- sponds to the real structure and that the previous methods that have previously been used in the study of the tertiary structure of RNA are inadequate.The results obtained by Eckstein et al.17 have provided the basis for further studies on ribozymes.127 VIII. Synthesis of cross-linked nucleic acid duplexes using sugar moieties of nucleosides In contrast to studies devoted to the modification of heterocyclic bases and phosphate groups of oligonucleotides, reports on the reactions between chains involving sugar residues are scarce.A method for the synthesis of a duplex was proposed,15 which entails the formation of a cross-link between the amino group attached through a spacer to position 2 of the sugar residue of adenosine, on the one hand, and the aldehyde group in a non- nucleoside insert of the complementary chain, on the other hand. Synthesis of this type of compound has been patented.16 In this case, the aldehyde group results from enzymic elimination of uracyl by uracyl-DNA-glycosylase from 20-deoxyuridine incorpo- rated into the oligonucleotide.The elimination of the heterocyclic base is accompanied by the sugar ring opening and the formation of the aldehyde group. Treatment of the latter with the primary amine gives a Schiff's base, which is reduced to give a stable secondary amine.However, enzymic treatment does not yet allow the preparative synthesis of these compounds, which restricts their application as therapeutic agents. As mentioned above, the main drawback of many procedures employed in the synthesis of cross-linked DNA duplexes is their low specificity. It is the specific cross-linking of DNA chains that allows the development of the most convenient tools for the study of protein-nucleic acid interactions.Therefore, in recent years most studies have been aimed at the elaboration of methods of site-specific cross-linking of chains in synthetic DNA duplexes. A series of publications 128 ± 131 are devoted to elaboration of a new effective method for the synthesis of cross-linked DNA duplexes that involves sugar residues of nucleosides.This method required the use of oligonucleotides modified at the carbohydrate fragment and containing a primary aliphatic amino group or a carboxy group. The existing procedures for incorporation of modified units into oligonucleotides permit introducing them into any predetermined position of the oligonucleotide chain or incorporate several modified units into the same oligonucleotide by a standard protocol of solid-phase phosphoramidite oligonu- cleotide synthesis.The reaction between the primary amino group and the carboxy groups selected for the cross-linking of the duplex chains is activated by water-soluble CDI and results in the formation of an amide bond between the chains. In the early 90's, several methods for the synthesis of 20-amino- 20-deoxynucleotides were developed.120 ± 126 High reactivity of the 20-amino groups of 20-amino-20-deoxynucleosides in acylation reactions120 ± 122 has led to an idea of formation of a cross-link between the 20-amino group in one chain and the carboxy group derived upon acylation of the 20-amino-20-deoxynucleoside of the complementary chain by a dicarboxylic acid anhydride.However, preparation of purine 20-amino-20-deoxynucleosides by transgly- cosylation (substitution of a heterocyclic base) of preformed 20-amino-20-deoxyuridine 123, 124 inevitably resulted in the forma- tion of a complex mixture of products and extremely low yield of the target product. Thus, a non-nucleoside insert was used instead of purine 20-amino-20-deoxynucleosides. On the one hand, this disturbs the hydrogen bonds in the pair in which one heterocylic base is missing.On the other hand, the use of this nucleoside substitution has a number of benefits. Compound 43 can be synthesised by a combination of simple high-yielding chemical reactions.128, 129 The distance of three carbon atoms between the phosphate groups, which is characteristic of natural oligonucleotides, is preserved in the structure of the non-nucleoside insert.This compound is resistant to synthetic and post-synthetic treatments of the oligonucleotide. Synthesis of specifically protected pyrimidine 20-amino-20- deoxynucleosides 44 and 45 has been described.120, 121 The reac- tion between the chains of a modified duplex is performed in the following steps.First, a carboxy group is introduced into one chain. This is achieved by acylating the amino group incorporated R1 ±R4 are the fragments of oligonucleotide chains. OR4 O O7 O O P OR3 G O O7 O O P HO O O NH O R2O G O O A O P O O O7 O7 O O P O R1O C O C Het=U (44); CBz (45). O Het O HO NHCCF3 O DMTr 44, 45 DMTrOCH2 CH CH2 OH NH C O CH2 CH2 NH 43 CH2OC O A G G C C A C G G C G A A A A U G U C G A U U A G U A A G U C G G U C G G C 30 C C A G C C G 50 C 50 30 B G U A A G G C C A C G G G A A A A U G U C G A U U A G U A A G U C G G U C G G C 30 C C A G C C G 50 C 50 30 II I III C G U Scheme 12 Double-helical nucleic acids with cross-linked strands: synthesis and applications in molecular biology 259into the oligonucleotide with a dicarboxylic anhydride.Molecular simulation allowed one to select succinic anhydride as the acylat- ing reagent. Then the reaction between the chains was conducted in a morpholinoethyl sulfonate buffer (pH 5.0 ± 6.7) for 1 ± 3 days at 0 ± 20 8C using water-soluble CDI as a condensation reagent (Scheme 13). Previously, it had been demonstrated 128, 129 that the reaction between the strands of modified DNA duplexes with a primary aliphatic amino group in one of them and a carboxy group in the other proceeds much more efficiently with the free amino group of 20-amino-20-deoxycytidine. This is explained by the fact that the pKa value for this amino group (6.2) 121 is much lower than that for the amino group of aliphatic amines, so this group exists mainly in a deprotonated form under the reaction conditions.The effects of some other factors, such as the mutual arrange- ment of modified units, their number, nucleotide environment, thermodynamic stability of the precursor duplex, etc., on the efficiency of the reaction between the chains, were studied.130, 131 Under optimum conditions, the yield of the corresponding DNA duplexes with covalently cross-linked chains reached 85% for certain systems.The yields of the target products differ considerably for a 20- and a 25-membered systems (26% and 85%, respectively). This is accounted for by the fact that the efficiency of the reaction increased with an increase in the melting point of the original modified duplex. Depending on the nucleotide environment of the modified units, the yield of the target product varied from 50% to 85%.130, 131 Substitution of 20-amino-20-deoxyuridine for 20-amino-20-deoxy-arabino-adenosine does not affect significantly the yield of the DNA duplex with covalently cross-linked chains.The thermodynamic stability of DNA duplexes with incorpo- rated modified units was in all cases lower than that of non- modified duplexes of the same primary structure.The decrease in the melting point depends on the length and the composition of the DNA duplex. In contrast, for duplexes with covalently cross- linked chains the melting point was higher than that for the corresponding modified and non-modified duplexes. The increase in the melting point was 4 ± 8 8C relative to natural analogues, while that for the corresponding modified duplexes was 15 ± 33 8C.128, 129 Duplexes with covalently cross-linked chains are much more resistant to the action of a mixture of snake venom phosphodies- terase and alkaline phosphatase than those devoid of interchain cross-links.Under conditions where natural and modified duplexes are completely hydrolysed to give nucleosides, duplexes with covalently cross-linked chains maintain their structure by no less than 70%.Such a low level of hydrolysis under these conditions is due to the presence of a cross-link between the chains. Comparative kinetics of enzymic hydrolysis of a 25-membered duplex with covalently cross-linked chains and of a non-modified duplex of the same primary structure by snake venom phospho- diesterase was carried out.132 Under selected conditions, enzymic hydrolysis of single-stranded oligonucleotides and a non-modified duplex occurred at a high rate.Single-stranded oligonucleotides were completely hydrolysed to give nucleotides in 5 min, whereas the duplex with covalently cross-linked chains was not completely hydrolysed under identical conditions even in 3 h. Relatively easy splitting of two or three units was observed together with accumulation of products of partial hydrolysis with chains short- ened by no more than 3 ± 8 units.After scission of eight units, the reaction is appreciably retarded. Thus, the presence of a cross-link between the chains causes strong inhibition of the effect of snake venom phosphodiesterase. In order to prove the ability of compounds obtained to serve as substrates of enzymes binding with unique nucleotide sequen- ces of DNA and to confirm their structure, DNA duplexes with covalently cross-linked chains were site-specifically cleaved with restriction endonuclease Alu I.130, 131 The primary structure of covalently cross-linked DNA duplexes corresponded to the 25- membered non-modified DNA duplex; its nucleotide sequence is shown in Scheme 14.The recognition site of restriction endonu- clease Alu I is given in italics; the sites of incorporation of the corresponding modified units 43 and 44 are underlined. The position of cross-links between the chains relative to the recognition site of restriction endonuclease Alu I in DNA duplexes determines the composition of the hydrolysate.The formation of oligonucleotides of different lengths resulting from hydrolysis of DNA duplexes with covalently cross-linked chains and the non-modified duplex is illustrated by Scheme 14. Scheme 14 The composition of reaction mixtures for all DNA duplexes is in agreement with the theoretical Scheme 14. Thus, it has been shown that the presence of cross-links between the chains in synthetic DNA duplexes does not prevent their cleavage with restriction endonuclease Alu I.The formation of a series of products of a definite length confirms the presence and position of cross-links between the chains in the synthetic DNA duplexes. The experimental results 128 ± 131 suggest that DNA duplexes with covalently cross-linked chains can be used as efficient `traps' for proteins binding with nucleic acids. 50 CGGTAGAGCTCACTT TCCGAGTGGC 30 30 GCCATCTCGAGTGAAAGGCTCACCG 50 p* is 32P-phosphate. +Alu I Alu I Alu I 50 p* p* +Alu I Alu I p* 50 p* Alu I +Alu I p* 25 25 p* +Alu I Alu I 50 p* p* + + + + + + + + 17 34 34 17 p* p* p* p* p* p* 8 p* 8 16 16 p* Alu I Het=U, CBz; R1 ±R4 are fragments of oligonucleotide chains. O pH 6.0 CDI (CH2)2 HN (CH2)2 HO O + NH2 O Het O P OR2 O O7 O7 O O P R1O O O HN O O7 P OR4 O P R3O O O7 (CH2)2 O HN O (CH2)2 R1O P O O O7 O7 O OR2 P O Het O NH O O HN O O7 P OR4 O P R3O O O7 Scheme 13 260 S I Antsypovich, T S OretskayaIX.Conclusion The data presented in this review testify to the existence of efficient methods for preparing DNA duplexes with covalently cross- linked chains. Cross-linking ofDNAchains can be achieved either nonspecifically (statistically) or at predetermined positions of the double helix.Owing to existing methods, the synthesis of these compounds is no longer a very complicated task. DNA duplexes with covalently cross-linked chains have become a convenient tool for the study of protein ± nucleic acid interactions. Convincing evi- dence for the significant potential of this approach can be derived from the investigations cited in the review.References 1. 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ISSN:0036-021X    

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年代:1998

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