摘要:

L e t t e r Hydroxylation of aromatic drugs by the electro-Fenton method. Formation and identi–cation of the metabolites of Riluzole Mehmet A. Oturan,a* Jean Pinson,a Nihal Oturanb and Dominique Deprezc a L aboratoire dœElectrochimie (CNRS UMR 7591), Paris7-Denis Diderot, Moleç culaire Universiteç 75251 Paris Cedex 05, France. E-mail : oturan=paris7.jussieu.fr de Marne la Descartes, Bat. L avoisier, 77420 Champs sur Marne, b Universiteç V alleç e, Citeç France Rorer, Centre de Recherche de V itry-Alfortville, 13 quai Jules Guesdes, BP14, c Rho� ne-Poulenc 94403, V itry sur Seine, France Received (in Strasbourg, France) 22nd April 1999, Accepted 14th June 1999 The electro-Fenton method permits hydroxyl radicals to be produced by simultaneous electrochemical reduction of dioxygen and ferric ions.These hydroxyl radicals react with the neuroprotective drug Riluzole to give four identi–ed hydroxylated compounds. These hydroxyl compounds are identical to the natural metabolites, the electrochemical behaviour of which is investigated. The electrochemically assisted Fenton reaction could therefore provide a convenient method for obtaining metabolites of aromatic drugs.The electro-Fenton method permits hydroxyl radicals (OH~) to be generated by the simultaneous electrochemical reduction of dioxygen and catalytic amounts of ferric ion in an acidic aqueous medium, on a carbon electrode.1h3 An acidic aqueous medium prevents the precipitation of iron hydroxide and allows the electrolysis to be performed without additional supporting electrolyte. O2]2e~]2H`]H2O2 H2O2]Fe2`]Fe3`]OH~]OH~ Fe3`]e~]Fe2` Hydroxyl radicals are powerful hydroxylating agents and their reaction with aromatic compounds provides hydroxylated derivatives,2,4h6 as shown, for example, on benzoic and salicylic acids.Metabolites of aromatic drugs are often hydroxylated compounds7h9 and we want to show in this report that the electrochemically assisted Fenton reaction oÜers an easy and fast way of preparing such metabolites in small amounts, sufficient, however, for identi–cation.We will consider the example of Riluzole,10,11 a neuroprotective drug that has proved to be efficient against amyotrophic lateral sclerosis (ALS). In an acidic aqueous medium (pH 2, room temperature), saturated with dioxygen and containing Riluzole (1»5 mM) and ferric chloride (1 mM), the potential of a carbon working electrode was set at [0.5 V vs.SCE, a potential at which both dioxygen and ferric ions are reduced.12 Aliquots were withdrawn at diÜerent charge amounts and analysed by high performance liquid chromatography (HPLC) using a Shadon ODS Hypersil C-18 reversed phase column (250 mm]4.6 mm i.d. ; 5 lm mean particle diameter).The column was eluted with methanol»phosphate buÜer 0.01 M)»acetic (Na2HPO4, acid (60 : 40 : 0.2 v/v) with a —ow rate of 0.7 mL min~1. The detection was performed by UV absorption at 263 nm. The HPLC retention times (Fig. 1) were 24.80 min for Riluzole, 22.62 min for II, 16.70 min for III and 9.95 min for IV and V. To analytically separate IV and V, the column was eluted with a linear gradient composed of solvent A (acetonitrile»water» phosphoric acid, 20 : 80 : 0.2 v/v) and solvent B (acetonitrile» water»phosphoric acid, 90 : 10 : 0.2 v/v) at mL min~1.The percentage of B was 10 at initial time, 10 at 5 min, 90 at 45 min and 10 at 60 min. The retention times under these conditions were 25.60 min for V and 28.50 for IV. The reaction products were identi–ed by comparison with authentic samples using HPLC and HPLC-mass spectrometry (LCQ2 system, electrospray HPLC/MS/MS, chemical ionization ; [M]1]` at 235 for Riluzole and at 251 for the four hydroxylated metabolites).In this way, we could identify the four known metabolites of Riluzole (Scheme 1). The maximum yield of the metabolites was obtained after 300 C had been passed through the solution (125 mL of a 1 mMRiluzole solution, reaction time about one hour): II (3%), III (7%), IV (4%), V (3%). These monohydroxylated derivatives underwent further hydroxylation and ring opening reactions.It has been shown previously that and are the –nal oxidation CO2 H2O products of organic compounds by hydroxyl radicals.13,14 The polyhydroxylated and ring opened compounds are observed as a series of peaks at the begining of the chromatogram.The electrochemical behaviour of these four hydroxylated derivatives was examined by cyclic voltammetry on a glassy carbon electrode with a scan rate equal to 0.2 V s~1. Riluzole itself at pH 6.5 50 : 50 v/v) shows a two- (MeOH»H2O, Fig. 1 Chromatogram of the electrolysis solution of Riluzole.Separation of IV and V was achieved under gradient conditions (see text for conditions). New J. Chem., 1999, 23, 793»794 793Scheme 1 electron irreversible oxidation wave at ]1.07 V vs. SCE, leading to an azo dimer5. The ring hydroxylated compounds are irreversibly oxidized : III V vs. SCE), V (Ep\]0.55 (Ep\ ]0.65 V vs. SCE), and IV \]0.67 V vs. SCE); they also (Ep present a broad wave at approximately ]1.10 V vs.SCE. The –rst wave likely corresponds to the oxidation of the phenolic function while the second one located at the same potential as Riluzole itself should correspond to the oxidation of the amino function. The behaviour of II is diÜerent : it presents in the same medium a reversible wave located at E°\]0.44 V vs. SCE. The reversibility of this voltammetric wave can be con–rmed by spectroelectrochemistry : II presents an absorption maximum at nm, which disappears upon oxi- jmax\292 dation at ]0.5 V while a new one appears at 387 nm; if the potential of the grid is returned to [0.5 V the initial spectrum reappears, thus showing the reversibility of the system on a time scale of a few minutes.This reversible system can be con- –dently assigned to the oxidation of the hydroxylamine function into a nitroso group on the basis of the well established behaviour of hydroxylamines in aqueous medium:15 The hydroxylated derivatives of Riluzole are therefore more easily oxidized than the starting compound, in particular II whose behaviour is similar to that of an antioxidant.In conclusion, the electrochemically assisted Fenton reaction, despite the low yields, appears as an easy and fast method to obtain hydroxylated derivatives of aromatic drugs, which may be identical to the biological metabolites.There is indeed a present lack of good methods to selectively oxidize aromatic molecules into phenols. References 1 R. Tomat and A. Rigo, J. Appl. Electrochem., 1985, 15, 167 and references therein. 2 M. J. Clifton and A. Savall, J. Appl. Electrochem., 1986, 16, 812. 3 M. A. Oturan and J. Pinson, J. Phys. Chem., 1995, 99, 13948. 4 G. B. Buxton, C. L. Greenstock, V. P. Helman and A .B. Ross, J. Phys. Chem. Ref. Data, 1988, 17, 513. 5 C. Walling, Acc. Chem. Res., 1975, 8, 125. 6 M. A. Oturan, J. Pinson, D. Deprez and B. Terlain, New J. Chem., 1992, 16, 705. 7 Biotransformations : a Survey of the Biotransformations of Drugs and Chemicals in Animals, ed. D. R. Hawkins, Royal Society of Chemistry, London, 1988»1993, vol. I»V. 8 Oxygen Radicals in Biology and Medicine, eds. M. G. Smic, K. A Taylor, J. F. Ward and C. von Sonntag, Plenum Press, New York, 1988. 9 B. Halliwell and H. Kaur, Free Rad. Res., 1997, 27, 239. 10 G. Bensimon, L. Lacomblez, V. Meininger, and the ALS/Riluzole study group, New Eng. J. Med., 1994, 330, 585. 11 P. Couratier, P. Sindou, F. Esclaire, E. Louvel and J. Hugon, NeuroReport, 1994, 5, 1012. 12 M. A. Oturan, J. Pinson, M. Traikia and D. Deprez, J. Chem. Soc., Perkin T rans. 2, 1999, 619. 13 G. Merga, H.-P. Schuchmann, B. S. Madhava Rao and C. von Sonntag, J. Chem. Soc., Perkin T rans. 2, 1996, 1097. 14 Y. Sun and J. J. Pignatello, Environ. Sci. T echnol., 1993, 27, 304. 15 W. Kemula and T. M. Krygowski, in Encyclopedia of the Electrochemistry of the Elements, Organic Section, eds. A. J. Bard and H. Lund, M. Dekker, New York, 1979, vol. 13, p. 78. L etter 9/03791H 794 New J. Chem., 1999, 23, 793»7

ISSN:1144-0546    

DOI:10.1039/a903791h

出版商:RSC    

年代:1999

数据来源: RSC

摘要:

L e t t e r Direct electron transfer involving a large protein : glucose oxidase§ Catherine Godet, Mohamed Boujtita and Nabil El Murr* Groupe Electrochimie[L AIEM (UPRSA»CNRS: 6006), des Sciences et des Faculteç T echniques, de Nantes; 2 rue de la 92208, 44322 Nantes Cedex 3, Universiteç Houssinie` re[BP France. E-mail : nabil.elmurr=chimbio.univ-nantes.fr Received 10th March 1999, Accepted 3rd June 1999 Electrochemical behavior of glucose oxidase –xed in the bulk of a graphite paste electrode, with no additives available to play the role of mediator or redox activator, shows the oxido reduction of the prosthetic FAD group that is still attached to the apoenzyme with no loss of enzyme activity. Since Updike and Hicks1 described the –rst enzyme electrode in 1967, a great amount of work has been devoted to the study of the contacts and exchange reactions between enzymes and electrodes.The association between an electrode material and biological macromolecules, such as proteins, and the study of their interactions may lead to useful information. At –rst sight, this combination could be used as a model for the investigation of the intrinsic redox behaviors of proteins.Moreover, the ììprotein electrode complexœœ may worthily be used as an analogue of the protein»protein complexes involved in a large number of physiological electron transfer reactions. This implies that the communication between the protein and the electrode material should be as direct and fast as in physiological systems. On the other hand, an efficient exchange between the redox proteins and the environment which uses them is essential in order to make the best bene–t of their peculiar properties in biotechnological applications such as biosensors or biocatalysis.Until recently, it was believed to be difficult to achieve direct and reversible electron transfer between redox proteins and electrodes. Extensive research has been done over the last twenty years to study the electrochemical behaviour of small redox proteins.2,3 For several metalloproteins such as cytochrome c, —avodoxin, ferredoxin, rubredoxin or plastocyanin, direct electron transfer was observed.Electrochemical studies with large redox enzymes have proved to be less successful. A recent review4 on fundamentals and analytical applications of enzyme-catalysed direct electron transfer related that a number of enzymes were found to be capable of interacting directly with an electrode while catalysing the corresponding enzymatic reaction, whereas controversial results were reported on the direct electrocatalysis of glucose oxidase (GOD).Several studies indicated that no enzymatic activity associated with the observed electron transfer has been detected for GOD,5,6 and that its electrocatalytic activity decreases rapidly while catalysing glucose electrooxidation.7 This suggests that the enzyme has undergone important structural transformations during its –xing to the surface of the electrode and that the observed electron transfer does not concern the enzyme itself, or that the activity of the protein has been destroyed following the transfer of electrons.In the two cases the electrochemical signal observed cannot be directly linked to the enzymatic activity of the protein nor is it indicative of the oxidation state of the enzyme. § Supplementary data available : cyclovoltammetric data. Available from BLDSC (No. SUP 57580, 4 pp.). See Instructions for Authors, 1999, Issue 1 (http ://www.rsc.org.njc).Previous studies on the direct electrochemistry of enzymes, in the absence of mediators or activators, have been attempted using mercury or conventional solid electrodes on the surface of which the enzyme was adsorbed or covalently attached.8,9 Although the carbon paste electrode is now widely used, few attempts have been made to exploit the intimate association of proteins with the transducer in order to observe the direct electrochemical communication between them.Yabuki et al.10 reported that the cyclic voltammogram of the carbon paste electrode containing GOD modi–ed with polyethylene glycol (PEG-GOD) shows oxidation and reduction peaks respectively at [0.3 and [0.45 V vs. Ag»AgCl. They also reported that in contrast no redox current peak was detected around these potentials when unmodi–ed GOD was used in the enzyme electrode.In the present study, we report an example of direct electron transfer involving a large protein such as glucose oxidase with no loss of its apparent enzyme activity. This electron transfer is directly linked to the enzyme that is –xed in the bulk of a graphite paste electrode in the absence of additives or modi–ers.Cyclic voltammetry using a GOD modi–ed electrode [GOD-electrode] in deaereted 0.1 M phosphate buÜer solution, pH\7.2, without previous chemical or electrochemical pre-treatment of the electrode surface shows a well de–ned and reproducible electrochemical response [Fig. 1(a)]. A nearly reversible redox system is observed (Ered p \[0.46, V vs.SCE; *Ep\80 mV and for Eox p \[0.38 iox p /ired p \0.65 scan rate of 0.5 mV s~1). Cyclic voltammetry using graphite paste electrode with no GOD in its bulk shows no response in phosphate buÜer in the presence or in the absence of GOD in solution. In order to determine whether the electron transfer is provoked by the reduction of FAD linked to the protein or by the reduction of free FAD molecules released from the enzyme during the carbon paste electrode preparation, a FAD modi- –ed graphite paste electrode [FAD-electrode] was prepared in Fig. 1 Cyclic voltammogram of (a) the GOD and (b) the FADelectrode in 0.1 M phosphate buÜer, pH 7.2, (» » » ») in the absence and (»»») in the presence of oxygen. Electrode area, 0.07 cm2. Scan rate, 0.5 mV s~1. New J.Chem., 1999, 23, 795»797 795the same way as the GOD-electrode. The FAD-electrode shows, in deaereted phosphate buÜer solution (0.1 M, pH\7.2), a diÜerent behaviour to that observed with the GOD-electrode. Its cyclic voltammogram [Fig. 1(b)] displays a reproducible electrochemical signal showing a reversible system at [0.50 V vs. SCE for the cathodic peak and [0.44V vs. SCE for the anodic one as well as a cathodic prewave at around [0.4 V vs.SCE. When a mixture of GOD and free FAD are included in the carbon paste electrode, the cyclic voltammogram in the presence or in the absence of oxygen is the sum of the two independent voltammograms. Three diÜerent sources may be attributed to the electron transfer observed with the GOD-electrode: a direct transfer to the active enzyme, an electron transfer involving the detached FAD or the oxido reduction of the FAD that is still attached to a denatured enzyme.It has been shown11 that in the latter case, even when FAD is not divorced from the apoenzyme, the cyclic voltammogram is completely consistent with the one of free FAD. Fig. 1 clearly indicates that cyclic voltammograms of the GOD and FAD-electrode are diÜerent implying that in the GOD-electrode, FAD groups are still attached to the active enzyme.Investigations concerning the oxido reduction of FAD have been made using electrochemical techniques.12h14 Cyclic voltammetry indicates that FAD is strongly adsorbed on the surface of the electrode (mercury, graphite, gold, platinum) and shows a pair of cathodic and anodic peaks corresponding to a two-electron transfer process.A cathodic prepeak is also observed depending on the pH and the electrode material. The overall reduction of FAD implies two electrons and two protons are involved leading to the dihydro—avin compound FADH2 : FAD]2e~]2H`°FADH2 (1) The electrochemical behaviour of entrapped FAD in a carbon paste matrix is also consistent with this scheme at least at neutral pH.It has been reported15 that the prosthetic FAD group can be easily detached from the apoenzyme when GOD is reacted with a highly concentrated solution of urea. We immersed the GOD-electrode in a 6 M urea solution for 30 s and recorded the cyclic voltammogram in phosphate buÜer. Such a treatment leads to exactly the same voltammogram as the one obtained with the FAD-electrode.This indicates that the cathodic and anodic peaks observed with the GOD-electrode are likely due to the oxido reduction of the prosthetic FAD group which is still attached to the apoenzyme. In order to validate that the electron transfer involved in the GOD-electrode does not result from detached FAD the two modi–ed electrodes were submitted to the same reactant, namely and their respective behaviours were compared.O2 , Fig. 1 shows that the two electrodes are aÜected when is O2 present in the buÜer solution but they behave diÜerently. For the GOD-electrode the reduction peak increases while the oxidation peak decreases, particularly at slow potential scan rates. For the FAD-electrode only the cathodic prepeak is strongly aÜected in the presence of indicating that the two O2 species generated after the electron transfers involving the GOD and the FAD-electrode are diÜerent.By comparison with the mechanism recognised for the reduction of free FAD, the electrochemical behaviour of the GOD-electrode in the absence of oxygen is the following : GOD-FAD]2e~]2H`°GOD-FADH2 (2) In the presence of oxygen, the reduced enzyme is oxidised very quickly at the surface of the electrode.GOD-FADH2]O2 ]GOD-FAD]H2O2 (3) The catalytic regeneration of the enzyme in its oxidised form causes the loss of reversibility and the increase in size of the cathodic peak. This is still visible even for relatively high potential scan rates (1 V s~1) because of the very important reactivity of the enzyme in its reduced form towards the oxygen.Another important diÜerence between the GOD and the FAD-electrode is observed when glucose is added to the nondeaerated buÜer solution. Glucose does not aÜect the FADelectrode behaviour, while the reduction peak observed with the GOD-electrode decreases when the glucose concentration increases until it attains the height of the peak observed in the absence of (Fig. 2). This is concordant with a competitive O2 reaction occurring at the vicinity of the electrode surface : GOD uses the dissolved oxygen to enzymatically oxidise the glucose, this leads to the depletion of the oxygen from the surface of the electrode making its reaction with GOD- [reaction (3)] less favourable. A Mickaeé lis type curve FADH2 is obtained when current is plotted against glucose concentration showing for the enzyme an apparent of 4.4 mM.The KM maximum rate, V m, of the reaction is limited by the oxygen concentration. The plot is linear between 0 and 0.55 mM of glucose. This competitive reaction shows that GOD is still active at the surface of the electrode and does not suÜer during the electrode preparation. This activity is not modi–ed even when repetitive cyclic voltammetry of the GODelectrode is run for a long time before adding the glucose.Under anaerobic conditions, enhancement of the anodic peak of the GOD-electrode, on addition of glucose, was observed using cyclic voltammetry. This could result from the catalytic regeneration of the reduced form of GOD by glucose in the absence of oxygen.The enzymatic activity of the electrode surface was also con–rmed by the increase of the oxidation current after the addition of glucose to the deaerated buÜer solution when the potential of the GOD-electrode is set at 0.2 V vs. SCE. Fig. 3 shows the maximum increased current at each glucose concentration. The increased current is proportional to the glucose concentration and the apparent is KM equal to 5.5 mM.Again the GOD-electrode, set at 0.2 V vs. SCE, shows anodic signals when used in a —ow injection analysis mode (FIA). The heights of the signals are proportional to the glucose concentration. At this potential no signal is observed when hydrogen peroxide or fructose solutions are injected or when the FAD-electrode is used or glucose solutions are injected.The electrochemical behaviour of the GOD-electrode in the absence and in the presence of glucose may account for a direct electron transfer between the electrode and the enzyme. Additional experiments, including FIA assays, on the use of such a direct electron transfer for the design of new biosensors Fig. 2 Cyclic voltammogram of the GOD-electrode in the presence of oxygen and in increasing amounts of glucose. Electrode area 0.07 cm2.Scan rate 2 mV s~1. 796 New J. Chem., 1999, 23, 795»797Fig. 3 The response of the GOD-electrode to the addition of glucose solution. Insert : plot of the current vs. concentration of glucose/mmol dm~3. and for a better understanding of redox reactions of proteins will be the topic of future accounts from this laboratory.Experimental Glucose oxidase (GOD) from Aspergillus niger [EC.1.1.3.4 was purchased from Biozyme Ltd (154 U mg~1, M\160 000)]. FAD disodium salt (M\829.5) (Sigma, F-6625), glutaraldehyde (Sigma, G-6257) and bovine serum albumin (BSA, Sigma, A-2153) were obtained from Sigma Chemical Ltd. Castor oil (cat. No 83905) was obtained from Fluka Chemical Ltd. Graphite powder (cat.No 16858) was purchased from Le Carbone Lorraine. Cyclic and linear sweep potential voltammetry measurements were performed with an Amel potentiostat, model 472, connected to a BD90 XY recorder. A three-electrode system with a SCE reference electrode and a platinum wire as auxiliary electrode was used. All potential values are given versus SCE. The working electrodes are either GOD or FAD carbon paste modi–ed electrodes.The geometric area of the working carbon paste electrode is 7 mm2. All experiments were conducted at room temperature. The carbon paste electrodes used were prepared using GOD and BSA, glutaraldehyde being used to chemically cross-link the proteins via the amino groups present. The standard method is described here : the active enzyme powder (1 g quantity) was prepared by dissolving 50 mg of BSA in 0.8 ml of phosphate buÜer (0.01 M, pH\7.2), after which 160 mg of glucose oxidase were added.Following the dissolution of the enzyme, 0.29 g of a 0.25% w/w glutaraldehyde solution was added to form a cross-linked gel. The enzyme mixture was stirred for 10 min, after which 0.79 g of graphite powder was slowly added and further stirred to allow homogenisation.The mixture was dried either in a vacuum dessicator or lyophilised at [20 °C in a freeze drier. The dried powder was crushed in a mortar and sieved (80 lm) and then mixed with castor oil : 23.66% (m/m) of oil. The resulting paste was then packed into a plastic tube (cartridge) with an internal diameter of 0.3 cm forming the carbon paste working electrode.A graphite rod was inserted into one end of the cartridge to form an electrical contact with the sensing element and the measuring device. Before use, a small amount of carbon paste is expelled from the plastic cartridge and the surface of the working electrode is polished on a paper and dipped in the electrolytic solution. Variants of this method of preparation have been equally achieved.Thus BSA and the enzyme have been dissolved in distilled water instead of the buÜered solution, BSA has not been incorporated in the electrode or glutaraldehyde has not been used. In all cases the electrochemical characteristics of consequent electrodes were identical, only the stability of the surface of the electrode was best when the standard method described previously was used.In order to load a FAD modi–ed carbon paste electrode with an equivalent amount of protein as the GOD-electrode, 208.5 g of BSA were dissolved in 0.8 ml phosphate buÜer solution (0.01 M, pH\7.2), after which 1.5 mg of FAD were added (this approximately represents the ratio of FAD present in the 160 mg of GOD), followed by the addition of 0.29 g of a 0.25% w/w glutaraldehyde solution.The mixture was stirred for 10 min, after which 0.79 g of graphite powder was slowly added and stirred until homogenisation. The resulting mixture was dried, mixed with castor oil (23.66% m/m) and packed into the plastic cartridge to be used as the working electrode in the same way as for the GOD-electrode. References 1 S. J. Updike and G.P. Hicks, Nature (L ondon), 1967, 214, 986. 2 M. J. Eddowes and H. A. O. Hill, J. Chem. Soc., Chem. Commun., 1977, 771. 3 F. A. Armstrong, H. A. O. Hill and N. J. Walton, Acc. Chem. Res., 1988, 21, 407. 4 A. L. Ghindilis, P. Atanasov and E. Wilkins, Electroanalysis, 1997, 9, 661. 5 Z. Wen, B. Ye and X. Zhou, Electroanalysis, 1997, 9, 641. 6 R. M. Ianniello, T. J. Lindsay and A. M. Yacynych, Anal. Chem., 1982, 54, 1098. 7 A. I. Yaropolov and A. L. Ghindilis, Biophysics (Engl. T ransl.), 1990, 35, 711. 8 F. Scheller, G. Strand, B. Neumann, M. Kué hn and W. Ostrowski, Bioelectrochem. Bioenerg., 1979, 6, 117. 9 L. Jiang, C. J. McNeil and M. Cooper, J. Chem. Soc., Chem. Commun., 1995, 1293 10 S. Yabuki, F. Mizutani and T. Katsura, Biosens. Bioelectron., 1992, 7, 695. 11 Q. Chi, J. Zhang, S. Dong and E. Wang, Electrochim. Acta, 1994, 39, 2431. 12 B. Janik and P. J. Elving, Chem. Rev., 1968, 68, 295. 13 M. M. Kamal, H. Elzanowska, M. Gaur, D. Kim and V. I. Birss, J. Electroanal. Chem., 1991, 318, 349. 14 M. J. Honeychurch and M. J. Ridd, Electroanalysis, 1996, 8, 362. 15 Y. Degani and A. Heller, J. Phys. Chem., 1987, 91, 1285. L etter 9/01967G New J. Chem., 1999, 23, 795»797 797

ISSN:1144-0546    

DOI:10.1039/a901967g

出版商:RSC    

年代:1999

数据来源: RSC

摘要:

L e t t e r Heterogeneous N-oxidation of pyridines using a combined oxidant of hydrogen peroxide and nitriles catalysed by basic hydrotalcites Kazuya Yamaguchi, Tomoo Mizugaki, Kohki Ebitani and Kiyotomi Kaneda* Department of Chemical Science and Engineering, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama, T oyonaka, Osaka 560-8531, Japan. Phone/fax : ]81 6 6850 6260; e-mail : kaneda=cheng.es.osaka-u.ac.jp Received (in Montpellier, France) 2nd June 1999, Accepted 21st June 1999 The oxidation of pyridines using a combined oxidant of hydrogen peroxide and benzonitrile catalysed by a basic hydrotalcite, gave high yields of the corresponding Mg10Al2(OH)24CO3 , pyridine N-oxides.This solid hydrotalcite catalyst was easily separated from the reaction mixture and could be reused with retention of its high catalytic activity and selectivity.Recently, we have developed a series of hydrotalcite-catalysed oxidation reactions such as the Baeyer»Villiger oxidation of ketones,1 oxidative dehydrogenation of alcohols,2 oxygenation of aromatic compounds,3 and epoxidation of ole–ns,4 using hydrogen peroxide and molecular oxygen as oxidants. The basic hydrotalcite, in the presence Mg10Al2(OH)24CO3 , of a combined oxidant of hydrogen peroxide and nitriles, had especially high catalytic activity for the epoxidation of terminal ole–ns.4a Here, we report the efficient heterogeneous oxidation of various pyridines to yield pyridine N-oxides using this same mono-oxygenation catalyst system.Pyridine N-oxides are versatile synthetic intermediates and are usually prepared by oxidation of pyridines using peracids such as meta-chloroperbenzoic acid (m-CPBA) and peracetic acid.5 Catalytic methods for oxidation processes using aqueous hydrogen peroxide and molecular oxygen as oxidants in place of stoichiometric reagents are, however, now more desirable because of the need for the chemical industry to minimize waste production associated with stoichiometric reagents.6 Over the years, many researchers have studied the N-oxidation of pyridines using hydrogen peroxide in the presence of metal complex catalysts.7 However, such homogeneous catalysts are typically not easily recovered from the reaction mixture, are not reusable, and sometimes require halogenated solvents to obtain high yields of N-oxides. The heterogeneous N-oxidation system using hydrotalcite catalysts we describe here has the following advantages: (1) simple work-up procedures and reusable catalysts, (2) use of nonpolluting natural mineral catalysts, and (3) use of nonhalogenated solvents.Various hydrotalcite catalysts were obtained from NaOH, and (or Mg(NO3)2 … 6H2O, Al(NO3)3 … 9H2O, Na2CO3 by the coprecipitation method, according to liter- Na2SO4) ature procedures.8 A typical hydrotalcite-catalysed Noxidation of pyridines was accomplished as follows.Into a reaction vessel with a re—ux condenser were successively placed the hydrotalcite, (0.10 g), meth- Mg10Al2(OH)24CO3 anol (20 cm3), pyridine (8 mmol), benzonitrile (16 mmol), and 30% aqueous hydrogen peroxide (4.0 cm3).After the reaction mixture had been stirred at 60 °C for 24 h, the hydrotalcite was separated by –ltration. The recovered hydrotalcite could then be reused as a catalyst. The –ltrate was treated with to decompose the remaining hydrogen peroxide. The MnO2 solution was poured into brine (10 cm3) and extracted with ethyl acetate (4]10 cm3). The combined organic layers were dried over and the crude product was puri–ed by MgSO4 column chromatography on silica gel (Wako Gel C-200) with ethyl acetate»methanol (9 : 1) as eluent to give 0.61 g of pyridine N-oxide (80% yield). Nitriles, hydrogen peroxide, and hydrotalcites were indispensable components of this oxidation and methanol was the best solvent.Water-immiscible solvents like benzene, toluene, and cyclohexane were poor solvents.A survey of various base catalysts and nitriles for the N-oxidation of pyridine is shown in Table 1. Among the nitriles used (entries 1»3), benzonitrile was the most eÜective. The yields of pyridine N-oxide increased with increasing calorimetric heats of benzoic acid adsorption on the hydrotalcites. The calorimetric heat of benzoic acid adsorption is a measure of the basicity of the Table 1 N-Oxidation of pyridine using nitriles and various base catalystsa H2O2 , Yield of N-oxide Convn of pyridineb based on pyridineb Heat of adsorptionc/ Surface area/ Entry Catalyst Nitrile (%) (%) J g~1 m2 g~1 1 Mg10Al2(OH)24CO3 Benzonitrile 98 92 14.2 44.3 2 Mg10Al2(OH)24CO3 Acetonitrile 50 49 14.2 44.3 3 Mg10Al2(OH)24CO3 Propionitrile 47 42 14.2 44.3 4 Mg6 Al2(OH)16CO3 Benzonitrile 75 75 6.3 43.5 5 Mg6 Al2(OH)16SO4 Benzonitrile 38 38 5.1 57.0 6 MgOd Benzonitrile 35 32 8.4 119.3 7 Mg(OH)2 Benzonitrile 84 79 6.4 24.9 8 None Benzonitrile 0 0 » » a Reaction conditions : pyridine (4 mmol), nitrile (8 mmol), catalyst (0.05 g), MeOH (10 cm3), 30% aq.(2 cm3), 60 °C, 24 h. b Determined by H2O2 HPLC using an internal standard technique.c See ref. 1(d). d MgO calcined at 400 °C was used. New J. Chem., 1999, 23, 799»801 799solid surfaces.1d had much higher cata- Mg10Al2(OH)24CO3 lytic activity than other solid base catalysts such as MgO and (entries 6 and 7), which might be due to the high Mg(OH)2 basicity of its surface. It is likely that the basic hydroxyl functions of the hydrotalcites play an important role in this Noxidation.Table 2 shows the oxidation of various pyridines using hydrogen peroxide and benzonitrile in the presence of In the oxidation of pyridine, the yield of Mg10Al2(OH)24CO3 . pyridine N-oxide based on the consumed hydrogen peroxide reached 70%. In our oxidation system, the corresponding pyridine N-oxides were exclusively obtained without other oxidation 800 New J.Chem., 1999, 23, 799»801Scheme 1 products. For example, 4-pyridylcarbinol was oxidized to give 4-pyridylcarbinol N-oxide without the formation of other oxidation products, pyridinealdehyde and pyridinecarboxylic acid (entry 9), while the use of m-CPBA resulted in the formation of a mixture of 4-pyridylcarbinol N-oxide and 4- pyridinecarboxylic acid (89 : 11).Pyrazine aÜorded the mono-N-oxide exclusively. No di-N-oxide was detected (entry 15).9 The N-oxidation of 3- and 4-substituted pyridines proceeded smoothly and 2-substituted pyridines (except for those with electron withdrawing groups such as 2-chloropyridine and 2-phenylpyridine10) gave high yields of the corresponding N-oxides (entries 4»13). Recently, Sharpless et al.have reported the efficient N-oxidation of pyridines using hydrogen peroxide catalysed by methyltrioxorhenium (MTO).7d The Sharpless system is not eÜective for 2-substituted pyridines ; it requires a large amount of the expensive rhenium catalyst to attain high yields, even for 2-picoline and 2,6-lutidine. A polysubstituted pyridine like quinoline also could be smoothly oxidized with our hydrotalcite catalysts (entry 14).11 The spent hydrotalcite catalyst was easily separated from the reaction mixture by –ltration and could be reused without an appreciable loss of catalytic activity and selectivity for the oxidation of pyridine (entries 2 and 3, in Table 2).Furthermore, we found that adding an anionic surfactant, sodium dodecyl sulfate (SDS), to the above reaction system promoted the N-oxidation. For example, 4-picoline N-oxide was quantitatively obtained within 5 h in the presence of SDS but the reaction required 24 h in the absence of SDS.This Noxidation occurred in three phases: the aqueous, organic and solid phases. We speculate that the main roles of the surfactant are to increase the contact area of the interface between the aqueous and organic phases and to enhance the transfer of the lipophilic benzonitrile from the organic phase to the aqueous phase.A possible reaction mechanism for this reaction is shown in Scheme 1. Hydrogen peroxide reacts with a basic hydroxyl function on the surface of the hydrotalcite to form HOO~ species, which attacks the nitrile to generate a peroxycarboximidic acid as an active intermediate oxidant.Oxygen transfer from peroxycarboximidic acid to a pyridine then occurs.4a Interestingly, 2-cyanopyridine could react with hydrogen peroxide in the absence of nitriles. In this case, picolineamide N-oxide was formed in high yield without the formation of 2-cyanopyridine N-oxide : presumably via intramolecular oxygen transfer by the intermediate peroxycarboximidic acid.In conclusion, a heterogeneous system consisting of hydrogen peroxide, nitriles, and basic hydrotalcites efficiently oxidize various kinds of pyridines to the corresponding pyridine N-oxides. The oxidation proceeds smoothly under mild reaction conditions and the hydrotalcite catalysts are recyclable. 12 At present, we are continuing to study selective oxidations using functionalised hydrotalcites with the aim of developing environmentally benign chemical processes.References and notes 1 (a) K. Kaneda, S. Ueno and T. Imanaka, J. Chem. Soc., Chem. Commun., 1994, 797. (b) K. Kaneda, S. Ueno and T. Imanaka, J. Mol. Catal., 1995, 102, 135. (c) K. Kaneda and T. Yamashita, T etrahedron L ett., 1996, 37, 4555. (d) S. Ueno, K. Ebitani, A. Ookubo and K.Kaneda, Appl. Surf. Sci., 1997, 121/122, 366. 2 K. Kaneda, T. Yamashita, T. Matsushita and K. Ebitani, J. Org. Chem., 1998, 63, 1750. 3 T. Matsushita, K. Ebitani and K. Kaneda, Chem. Commun., 1999, 265. 4 (a) S. Ueno, K. Yamaguchi, K. Yoshida, K. Ebitani and K. Kaneda, Chem. Commun., 1998, 295. (b) K. Yamaguchi, K. Ebitani and K. Kaneda, J. Org. Chem., 1999, 64, 2966. 5 (a) G. B.Payne, P. H. Deming and P. H. Williams, J. Org. Chem., 1961, 26, 651. (b) G. B. Payne, J. Org. Chem., 1961, 26, 668. (c) D. C. Edwards, T etrahedron L ett., 1966, 4767. (d) E. Ochiai, Aromatic Amine Oxides, Elsevier, Amsterdam, 1967. (e) G. E. Chivers and H. Suschitzky, J. Chem. Soc., Chem. Commun., 1971, 28. 6 (a) R. A. Sheldon, CHEMT ECH, 1994, March, 38. (b) P. T. Anastas and J.C. Warner, Green Chemistry: T heory and Practice, Oxford University Press, Oxford, 1998. (c) J. H. Clark, Green Chem., 1999, 1, 1. 7 (a) K. Takabe, T. Yamada and T. Katagiri, Chem. L ett., 1982, 1987. (b) R. W. Murray and K. Iyanar, J. Org. Chem., 1996, 61, 8099. (c) A. Goti and L. Nannelli, T etrahedron L ett., 1996, 37, 6027. (d) C. Copeç ret, H. Adolfsson, T. V. Khuong, A.K. Yudin and K. B. Sharpless, J. Org. Chem., 1998, 63, 1740. (e) C. Copeç ret, H. Adolfsson, J. P. Chiang, A. K. Yudin and K. B. Sharpless, T etrahedron L ett., 1998, 39, 761. 8 (a) S. Miyata, Clays Clay Miner., 1980, 28, 50. (b) F. Cavani, F. Tri–ro` and A. Vaccari, Catal. T oday, 1991, 11, 173. 9 By contrast, use of the MTO catalyst resulted in 91% yield of di-Noxide. 10 2-Chloropyridine and 2-phenylpyridine aÜorded the corresponding N-oxides in 17 and 63% yields, respectively, under the same reaction conditions as in Table 2. 11 For this substrate, the resultant reaction mixture was poured into ethyl acetate, which was extracted with water. After removal of water, 0.57 g (87%) of quinoline N-oxide monohydrate was obtained. 12 In this system, the resultant benzamide could be further employed for the N-oxidation : benzamide was also formed as a co-product from benzonitrile, which was recovered quantitatively by column chromatography. By using dehydrating reagents such as and P2O5 benzamide could be easily regenerated to benzonitrile. SOCl2 , L etter 9/04446I New J. Chem., 1999, 23, 799»801 801

ISSN:1144-0546    

DOI:10.1039/a904446i

出版商:RSC    

年代:1999

数据来源: RSC

摘要:

L e t t e r Silver-catalyzed cyclization of acetylenic alcohols and acids : a remarkable accelerating eÜect of a propargylic CñO bond V. Dallaa and P. Pale*b a L aboratoire de Chimie Organique Physique (CNRS UMR 6519), de Universiteç Reims-Champagne-Ardenne, BP 347, 51100 Reims, France b L aboratoire de et Organique (CNRS UMR 7509), Institut L e Bel, Reç activiteç Synthe` se L . Pasteur, 3 rue Blaise Pascal, 67000 Strasbourg, France.Fax: ]33 3 88 41 60 42; Universiteç E-mail : ppale =chimie.u.strasbg.fr Received (in Montpellier, France) 4th May 1999, Accepted 8th June 1999 The heteroannulation catalyzed by silver salts of alkynols 1, 2 or alkynoic acids 3 is considerably enhanced by the presence of a propargylic CñO bond. This method allows for a rapid access to highly functionalized heterocycles, such as a-methylene oxolanes 4 or oxanes 5 and as c-methylene pentanolactones 6.Tetrahydrofurans and pentanolactones are frequently encountered structures in natural products.1,2 This has motivated numerous eÜorts to develop strategies for the synthesis of these compounds.1,2 Particularly, methods that utilize transition metal-catalyzed ring closure between a hydroxyl or an acid and an unsaturated compound, either ole–nic, acetylenic or allenic, have gained considerable preeminence in recent times.1h5 [Pd3, Hg4, and Ag5 have been the most currently used metals for this purpose].We recently reported that acetylenic alcohols and acids could be efficiently cyclized by a catalytic amount of silver carbonate in re—uxing benzene.5 The cyclization proved to be regiospeci–c and the exocyclic a-methylene heterocycles resulting from an exo-dig ring closure were always exclusively isolated.This cyclization is now becoming a useful tool for the synthesis of tetronic acid6 and tetrahydrofuran7 derivatives. In this paper, we report on further studies that demonstrate the strongly accelerating eÜect of the propargylic oxygenated substituent on the ring closure of both acetylenic alcohols and acids and thus expand the scope of this novel route towards oxygenated heterocycles.Our previous work has shown a marked diÜerence between the cyclization of 4-pentynoic or 5-hexynoic acids and the cyclization of the corresponding alcohols. Acetylenic alcohols cyclized faster but the process was less general.The results suggested that the cyclization seemed to be dependent on the structure of the substrate, requiring a predisposed orientation of the hydroxyl and the acetylenic unit as in cis 2,3-epoxy pent-4-yn-1-ols.5 Considering the mechanism of such transition metal-assisted electrophilic cyclizations8 (Scheme 1), we Scheme 1 * Present address : Laboratoire de Chimie Organique et Macromole ç culaire (CNRS UMR 6519), Universiteç des Sciences et des Technologies de lœUniversiteç de Lille, 59655 Villeneuve dœAscq, France.reasoned that any factor altering the p system would also aÜect its coordination to the electrophilic metal and eventually the cyclization. We thus studied the cyclization of several acetylenic alcohols and acids bearing various substituents at the propargylic position with diÜerent electronic properties.With respect to a coordination-cyclization mechanism, the size of the propargylic substituent would also be expected to play a role in the reaction by preventing access to the triple bond. We chose to study acetylenic compounds incorporating an oxygen atom at the propargylic position since both its electronic in—uence and its size could be easily modulated by the proper selection of substituent.Various pentynols 1a»f with an oxygenated group at the propargylic position were prepared according to conventional sequences from 1-tertbutyldiphenylsiloxypent- 4-yn-3-ol. This compound was obtained from 1,3-propanediol after monoprotection, oxidation and addition of lithium trimethylsilylacetylide (Scheme 2).Contrary to the parent unsubstituted 4-pentynols, which do not cyclize under our standard conditions (Table 1, run 1), all the substituted 4-pentynols 1a»e readily cyclized in high yields (runs 2»6) except the acetate 1f (run 7). Diol 1a underwent complete cyclization in only 1 h (run 2), while propargylic ethers 1b»d required longer but reasonable periods of time to be totally consumed (runs 3»5).However, the silyloxy deriv- Scheme 2 (i) tBDPSCl, 68%. (ii) PCC, 90%. (iii) TMSacetylide, 75%. (iv) MeOH, 95%. (v) 67»86%. (vi) NaH, K2CO3 , TBAF… 3H2O, HMPA and MeI, 78%. (vii) MOMCl, 79%. (viii) Isoprene, montmorillonite K10, 22%. (ix) 4-DMAP, 100%. (x) 2.2 equiv. TBAF… 3 Ac2O, 96%. (xi) a: TMSCl, b: tBDPSCl, c : citric acid, MeOH, 75%.H2O, New J. Chem., 1999, 23, 803»805 803Table 1 Silver-catalyzed cyclization of miscellaneous alkynols bearing a propargylic oxygenated substituent Acetylenic alcohol Runa n Z Time/h Product (yield/%)b 1 1 H 1 6 4 (0) 2 1 OH 1a 1 4a (99) 3 1 OMe 1b 2.5 4b (99) 4 1 OMOM 1c 3 4c (99) 5 1 OtBu 1d 4 4d (99) 6 1 OtBDPS 1e 24 4e (99) 7 1 OAc 1f 2.5 1f recovered 8c 1 OAc 1f 6 Decomposition 9 2 H 2 6 2 recovered 10 7.5 2a a Reactions were carried out in re—uxing benzene with 0.1 equiv.as the catalyst. b Yields were evaluated from the 1H NMR Ag2CO3 spectra of the crude products. c The reaction was performed in re—uxing toluene. ative 1e proved to be only slowly cyclized (run 6). The bene–- cial in—uence of a propargylic C»O bond is also clear in the homologous 5-hexynol series.Although 5-hexynol 2 does not cyclize in the presence of a catalytic amount of silver carbonate (run 9), derivative 2a methoxylated at the propargylic position does cyclize (run 10). All the a-methylene oxolanes 4 so obtained proved to be very air- and moisture-sensitive and decomposed rapidly under exposure to ambient atmosphere or to slightly acidic conditions, thus preventing puri–cation by silica gel chromatography.Nevertheless, due to the cleanliness of the reaction, they could be kept intact for several months by storing in a matrix of benzene or deuteriated benzene at [20 °C after simple –ltration of the heterogeneous reaction medium.4 NMR in deuteriated benzene clearly showed in each case the sole presence of the exocyclic enol ether function with the two methylene protons (4.01 and 4.48 ppm for 4a, 3.81 and 4.4 ppm for 4b, 4.17 and 4.6 ppm for 4c, 4.11 and 4.54 ppm for 4d, 4.16 and 4.57 ppm for 4e, 3.93 and 4.8 ppm for 5a) and the characteristic exocyclic carbons (81 and 166.2 ppm for 4a, 83.4 and 161.7 ppm for 4b, 83.4 and 162 ppm for 4c, 82.6 and 161.9 ppm for 4d, 81.5 and 163.4 ppm for 4e, 93.3 and 127.6 ppm for 5a).The failure observed in the case of 1f, in which an electronwithdrawing acetate is present (entry 7), supports the crucial role of electronic eÜects. These results are in agreement with one of our hypotheses. Hyperconjugation between the adjacent C»O bond and the triple bond would impoverish the electronic density and preclude complexation with silver ion and thus the cyclization.Since the size of the propargylic substituent would also in—uence the reaction by preventing access to the triple bond, one may reason that the reaction is slower for the more sterically demanding propargylic oxygenated group. This is what was indeed observed (Table 1, entries 2»6). The hindered tert-butyldiphenylsilyl derivative 1e underwent complete cyclization but required a reaction time 4 times Table 2 Silver-catalyzed cyclization if miscellaneous alkynoic acids bearing a propargylic oxygenated substituent Runa Acetylenic acid Product 1b 2 3 4 5 a Reactions were performed in re—uxing benzene with 0.1 equiv.of The reactions proceeded in 5 min., except as noted. Yields Ag2CO3 . were evaluated by 1H NMR spectroscopy on the crude product and were 99% in all cases.b The reaction proceeded in 10 h. longer than the cyclization of the analogous tert-butyl 1d (entry 5 vs. 6). The remarkable eÜect of a propargylic C»O bond reached its maximum in the cyclization of 4-pentynoic acids. Thus, syn or anti 2-hexadecyl-3-hydroxy-4-pentynoic acids as well 3asóa , as their 2-(z-tetradec-7-enyl) analogs readily cyclized 3bsóa , within a few minutes (Table 2, runs 2»5) when reacted under our standard conditions, while the unsubstituted parent 3 required 10 h for complete transformation (run 1).Surprisingly, no diÜerence was detected between the two syn and anti diastereoisomers in each series, although the cyclization forces the two substituents into a cis relationship in the case of the anti isomer.In conclusion, this work shows that the presence of oxygen at the propargylic position of acetylenic alcohols and acids greatly favored their cyclizations catalyzed by silver carbonate. The results reported here improve the scope of our reaction, which thus appears to be a method of choice for the synthesis of highly oxygenated 5- and 6-membered oxacycles. The authors thank the CNRS for –nancial support.One of us (V.D.) thanks the Ministe` re de la Recherche et de la Technologie for a doctoral fellowship. References 1 For reviews on oxacycles, see : U. Koert, Synthesis, 1995, 115; T. L. B. Boivin, T etrahedron, 1987, 43, 3309. 2 For a recent review on lactones, see : G. Rousseau, T etrahedron, 1996, 52, 2577. 804 New J. Chem., 1999, 23, 803»8053 G.Cardillo and M. Orena, T etrahedron, 1990, 46, 3321; M. Cavicchioli, S. Decortiat, D. Bouyssi, J. Gore and G. Balme, T etrahedron, 1996, 52, 11463. 4 M. Riediker and J. Schwartz, J. Am. Chem. Soc., 1982, 104, 5842; M. Suzuki, A. Yanagisawa and R. Noyori, T etrahedron L ett., 1983, 24, 1187. 5 P. Pale and J. Chuche, T etrahedron L ett., 1987, 28, 6447. 6 V. Dalla and P. Pale, T etrahedron L ett., 1994, 35, 3525; J. Nokami, H. Ohtsuki, Y. Sakamoto, M. Mitsuoka and N. Kunieda, Chem. L ett., 1992, 1647. 7 P. Pale, J. Chuche, J. Bouquant, P. Vogel and P. A. Carrupt, T etrahedron, 1994, 50, 8035; V. Dalla and P. Pale, T etrahedron L ett., 1996, 37, 2777; V. Dalla and P. Pale, T etrahedron L ett., 1996, 37, 2781; V. Dalla and P. Pale, T etrahedron L ett., 1992, 33, 7857. 8 In such transition metal-assisted electrophilic cyclizations, the accepted mechanism postulates a previous activation of the p system by coordination to the metal cation, rendering the soformed complex electrophilic enough for further reaction with a nucleophile. See, for example: K. Utimoto, Y. Fukuda and H. Nozaki, Heterocycles, 1987, 25, 297. L etter 9/03587G New J. Chem., 1999, 23, 803»805 805

ISSN:1144-0546    

DOI:10.1039/a903587g

出版商:RSC    

年代:1999

数据来源: RSC

摘要:

L e t t e r Symmetric bis-substituted and asymmetric mono-substituted nitridotechnetium complexes with heterofunctionalized phosphinothiolate ligands§ Cristina Bolzati,a Erica Malago` ,a Alessandra Boschi,a Aldo Cagnolini,b Marina Porchiab and Giuliano Bandoli*c a L aboratorio di Medicina Nucleare, Dipartimento di Medicina Clinica e Sperimentale, Universita` di Ferrara, 44100 Ferrara, Italy b ICT IMA, Consiglio Nazionale delle Ricerche, 35127 Padova, Italy c Dipartimento di Scienze Farmaceutiche, di Padova, 35131 Padova, Italy.Universita` E-mail : bandoli=pdfarm3.dsfarm.unipd.it Received (in Montpellier, France) 5th May 1999, Accepted 4th June 1999 The mixed bidentate ligand 2-(dicyclohexylphosphino)- ethanethiol (HL) reacts with labile nitrido-Tc precursors to aÜord a rare example of an asymmetric monosubstituted species 1, along with the symmetric bis- [TcN(L)Cl(PPh3) ] , substituted complex 2.The latter compound, as [TcN(L)2 ] , assessed by TLC and HPLC chromatography, was found to possess the same molecular structure as the agent produced at the ìnon carrier addedœ level utilizing the 99mTc nuclear isomer. Nitrido-Tc(V) chemistry is receiving growing attention in the last few years owing to the recent availability of a standard method for producing the [Tc3N]2` moiety at ìnon carrier addedœ level utilizing the 99mTc nuclear isomer.1 In this connection, the –rst 99mTc radiopharmaceutical carrying the nitrido group, namely (NOEt\N-ethyl- [99mTcN(NOEt)2] N-ethoxydithiocarbamate), has been proposed as a myocardial perfusion agent2 and is now under phase III clinical trials.The proposed co-ordination of the complex is [TcN(NOEt)2] square pyramidal with the terminal nitrogen group at the apex and the two substituted dithiocarbamate ligands at the base of the pyramid, as previously assessed for the related diethyldithiocarbamato (dedc) compound [TcN(dedc)2],3 which exhibits a trigonality index q of 0.02 (q\0 for ideal square-pyramidal geometry and q\1 for ideal trigonalbipyramidal geometry4). A geometry approaching trigonal bipyramidal (q\0.76) is instead exhibited by [Tc(L@)2(O2L@)], a Tc(III) complex containing the bidentate 2-(diphenylphosphino) ethanethiol (HL@) ligand, in which two phosphorus atoms are located at the apexes and the three thiolate sulfur atoms are equatorial, leaving the oxidized phosphorus atom of the third ligand dangling outside the co-ordination sphere.5 These phosphino-thiolato Tc(III) complexes are generated via reduction-substitution reactions starting from and [TcO4]~ an excess of the relevant phosphinothiol. The formation of intermediate oxo-Tc(V) species was not detected, even when using a stoichiometric amount of the ligand, indicating that if these Tc(V) adducts were formed, they rapidly underwent further reduction to the thermally stable Tc(III) species.However, similar reduction-substitution reactions, conducted at the ìnon carrier addedœ level starting from in [99mTcO4]~ the presence of a nitrido source such as hydrazine or substituted hydrazines and an excess of phosphinothiol, evidenced § Supplementary material available : HPLC traces for 2 and 2m.Available from BLDSC (No. SUP 57578, 2 pp.). See Instructions for Authors, 1999, Issue 1 (http ://www.rsc.org/njc). the existence of novel species having diÜerent retention times when compared to those exhibited by the related Tc(III) species. Moreover, these new derivatives have shown promising heart uptake when injected in rats and primates.6 We were consequently stimulated to elucidate the molecular structure of these agents at the macroscopic level by utilizing the 99gTc isotope.The understanding that the nitrido group is much more resistant toward reduction than the corresponding oxo group in Tc(V) complexes,7 directed our investigation on the reactivity of labile pre-reduced nitrido-Tc precursors with representative P,S ligands.Reaction of with a three-fold [TcVINCl4]~ molar excess of 2-(dicyclohexylphosphino)ethanethiol (HL) in (2 : 1 v/v) aÜorded a yellow»brown solution CH2Cl2»EtOH from which an orange»yellow powder of 2 was isolated upon removal of the solvent. The IR spectrum exhibits a medium intensity stretching vibration at l\1045 cm~1 indicating the retention of the terminal nitrido group, as well as vibrations characteristic of the cyclohexyl rings.Elemental analysis is in agreement with the presence of two phosphinothiolate ligands, and the singlet in the 31P NMR indicates the magnetic equivalence of the P nuclei. Recrystallization from CH2Cl2»MeOH gave pale yellow plates suitable for X-ray determination. In spite of some disorder (see Experimental), the co-ordination about Tc in 2 is well established (Fig. 1), and is best described as intermediate between square pyramidal and trigonal bipyramidal (q1\0.49 and q2\0.60 for the two types of molecules). In the trigonal-bipyramidal description the nitrido group and two thiolate sulfur atoms occupy the equatorial sites with the phosphine phosphorus at the apexes of the bipyramid.The twist-envelope –ve-membered chelate rings of both phosphinothiolato ligands are virtually perpendicular to the S(1)N(1)N(1A) plane and form an angle of 40.4° to each other. The cyclohexyl rings adopt a chair conformation and (D3d) make a dihedral angle of 56.5° to each other. The mono-substituted complex 1, was [TcN(L)Cl(PPh3)], produced by reaction of with a slight excess [TcNCl2(PPh3)2] of HL in (2 : 1 v/v) mixtures.The 31P NMR CH2Cl2»EtOH spectrum of 1 displays two broad signals at room temperature. On lowering the temperature the signals become narrower, and the appearance of two doublets at 250 K Hz) indicates the existence of two magnetically (2JPP\162 inequivalent trans-positioned P donors. The IR spectrum shows vibrations characteristic of both the cyclohexyl rings and of co-ordinated triphenylphosphine, along with the typical medium intensity band of the terminal nitrido group at l\1066 cm~1.The X-ray analysis con–rmed a –ve-co- New J. Chem., 1999, 23, 807»809 807Fig. 1 View of structure 2 showing the numbering scheme adopted. Thermal ellipsoids are drawn at the 40% probability level. Selected bond lengths and angles (°) [in square brackets for the molecule (Aé ) generated by a pseudo-mirror plane]: Tc»S(1) 2.362(6) [2.46(1)], Tc»P(1) 2.389(5) [2.38(1)], Tc»N(1) 1.64(3) [1.63(6)], S(1)»C(2) 1.82(2) [1.87(3)], P(1)»C(1) 1.85(2) [1.80(4)] ; S(1)»Tc»S(1A)* 129.2(3) [124.1(5)], S(1)»Tc»P(1) 82.2(2) [81.5(3)], P(1)»Tc»P(1A)* 158.5(3) [160.1(7)], S(1)»Tc»N(1) 115.4(2) [117.9(3)], P(1)»Tc»N(1) 100.8(2) [99.9(3)], Tc»S(1)»C(2) 110.5(8) [107(1)], Tc»P(1)»C(1) 106.1(7) [108(2)]. * At [x, y, 12 [z.ordiate structure (Fig. 2) approaching the square-pyramidal environment (q\0.28) with the nitrido group at the apex and the remaining PSPCl donors at the base of the pyramid. In the square-pyramidal description the Tc atom is displaced by 0.65 from the mean equatorial plane toward the nitrido Aé nitrogen atom and the four basal donors are displaced by ^0.16 as well.However, if we use the trigonal-bipyramidal Aé desription the Tc ion is virtually within the S(1)N(1)Cl plane (deviation of only 0.005 and the twist-envelope –ve- Aé ) membered chelate ring of the phosphinothiolate ligand is orthogonal to this plane. The cyclohexyl rings behave as in 2 and make a dihedral angle of 65.8° to each other.The three phenyl groups of the triphenylphosphine ligand are almost orthogonal to each other, making dihedral angles of 79.0, 86.5 and 110.1°. 2 and the corresponding agent prepared utilizing 99mTc, 2m, have shown identical chromatographic pro–les, available as supplementary material, con–rming the existence of nitrido- Tc(V) species containing phosphinothiolato ligands at the ìnon Fig. 2 View of structure 1 showing the numbering scheme adopted. Thermal ellipsoids are drawn at the 40% probability level. Selected bond lengths and angles (°) ; Tc»Cl 2.377(2), Tc»P(1) 2.387(2), (Aé ) Tc»P(2) 2.430(2), Tc»S(1) 2.303(2), Tc»N(1) 1.590(5), P(1)»C(1) 1.817(6), C(1)»C(2) 1.471(9), S(1)»C(2) 1.817(6) ; P(1)»Tc»P(2) 156.4(1), N(1)»Tc» S(1) 109.0(2), N(1)»Tc»Cl 111.4(2), S(1)»Tc»P(1) 82.9(1), Cl»Tc»S(1) 139.6(1), Tc»P(1)»C(1) 105.8(2), Tc»S(1)»C(2) 110.1(2).carrier addedœ level. As expected from the relative 99mTc/ ligand concentrations, no trace of the mono-substituted complexes has been detected at the ìnon carrier addedœ level. Experimental Syntheses Ligand 2-(dicyclohexylphosphino)ethanethiol (HL).This ligand was prepared by Argus Chemicals according to the method previously described by Chatt et al. for the synthesis of 2-(diphenylphosphino)ethanethiol.8 Dicyclohexylphosphine and 2 M n-BuLi were used instead of diphenylphosphine and methyl lithium. 1. Solid (0.085 g, [Tc(N)(L)Cl(PPh3) ] , [TcNCl2(PPh3)2] 0.129 mmol) was suspended in (5 cm3) and HL (0.064 CH2Cl2 g, 0.249 mmol) dissolved in EtOH (5 cm3) was added. The mixture was stirred at re—ux for 1 h until a clear orange» yellow solution appeared.After cooling the solvent was removed by a gentle stream of dinitrogen and the residue was treated with EtOH (5 cm3). A yellow precipitate was collected by –ltration, washed with (2]10 cm3) and dried under Et2O vacuum (yield 72%).Anal. found (calcd. for C, 56.97 (57.52) ; H, 6.36 (6.18) ; N, 2.31 C32H41P2SNClTc): (2.10) ; S, 5.31 (5.22)%. FT IR (KBr pellets, cm~1) : 1066 (l[Tc3N]). 1H NMR d: 7.95»7.30 15 H), 3.05» (CDCl3) (Ph3P, 3.08 26 H). 31P NMR 250 K) d: (Cy2P, SCH2CH2P, (CDCl3 , 86.3 (d, Hz), 39.3 (d, Hz). Cy2P, 2JPP\162 Ph3P, 2JPP\162 2. Solid (0.082 g, 0.129 [Tc(N)(L)2 ] , [AsPh4][TcNCl4] mmol) was suspended in (10 cm3) and HL (0.100 g, CH2Cl2 0.387 mmol) dissolved in EtOH (5 cm3) was added.The mixture was stirred at room temperature for 1 h until the solution became clear and brown coloured. The solvent was then removed by a gentle stream of dinitrogen and the residue was treated with EtOH (5 cm3). A pale yellow solid was collected by –ltration, washed with (2]10 cm3) and dried Et2O under vacuum (yield 91%).Anal. found (calcd. for C, 52.99 (53.56) ; H, 8.41 (8.35) ; N, 2.32 C28H52P2S2NTc): (2.24) ; S, 11.01 (10.21)%. FT IR (KBr pellets, cm~1) : 1045 (l[Tc3N]). 1H NMR d: 2.87 (m, 4 H), 2.75» (CDCl3) SCH2», 1.10 48 H). 31P NMR 298 K) d: 85.9 (Cy2P, PCH2», (CDCl3 , (s). 2m. In a 5 cm3 borosilicate vial containing [99mTc(N)(L)2 ] , 1 mg of N-methyl-S-methyldithiocarbazate and tin chloride (0.1 mg) dissolved in water (0.1 cm3), ethanol (0.75 cm3) and [99mTc]pertechnetate solution (0.5 cm3, 5 mCi) were added.The solution was left to react at room temperature for 30 min, after which the pH was adjusted to 8 by means of a 0.5 M carbonate buÜer (0.1 cm3). Then 0.25 cm3 of a 0.01 M HL ethanolic solution was added. The vial was quickly capped and placed into an oil bath at 100 °C for 30 min.The reaction mixture was left to cool at ambient temperature; the vial was then opened and aliquots of the reaction mixture were analyzed by HPLC, giving the desired product of radiochemical purity : 88»94%. Chromatography HPLC. column (Beckman) equipped with a ODS-C18 C18 guard column. Isocratic elution methanol»water 90 : 10 at a —ow rate of 1 mL min~1.TLC. Silica gel 60 plates (Merck). Elution with an F254 ethanol»chloroform»benzene 0.5 : 2.0 : 1.5 mixture gave rf values of 0.7 for both 2 and the corresponding 99mTc agent 2m. 808 New J. Chem., 1999, 23, 807»809Crystallography Crystal data for 1. M\667.1, mono- C32H41P2SNClTc clinic, a\12.437(4), b\13.441(4), c\19.060(6) P21/n, Aé , b\102.05(3)°, U\3116(2) Mo-Ka radiation : j\0.71073 Aé , Z\4, T \294 K, k(Mo-Ka)\7.40 cm~1. 4563 absorp- Aé , tion corrected re—ections (( scans,9 I\0.452»0.763), 4320 unique Structure solution and re–nement on F2 (Rint\0.038). using SHELXTL/PC and SHELXTL-93 program packages,10 with application of anisotropy only to the heavy atoms. At –nal convergence, S\1.027 for 339 R1\0.043, wR2\0.099, parameters and 3107 re—ections with I[2p(I) using the following weighting scheme: w\1/[p2(F0 2 )](0.0678P)2 DiÜerence electronic density ]0.0486P], P\(F02]2Fc2)/3.features lie within the range 0.70 to [0.66 e Aé ~3. Crystal data for 2. M\626.8, monoclinic, C28H52P2S2NTc C2/c, a\10.528(9), b\11.731(9), c\24.25(2) Aé , b\95.32(9)°, U\2982(4) Mo-Ka radiation : j\0.71073 Aé 3, Z\4, T \294 K, k(Mo-Ka)\7.48 cm~1. 2718 Absorp- Aé , tion corrected re—ections (w scans, I\0.563»0.813), 1409 unique Unfortunately, the diÜracting ability of (Rint\0.056). some samples mounted on the diÜractometer fell oÜ rapidly with increasing Bragg angle, and much of the higher angle data were —agged as weak and bore negative intensity. As a consequence, the data collection was restricted to 2H'\ In addition, the re–nement of the structure was relatively 40°.difficult due to the siting of the atoms; in fact, the Patterson map and subsequent observed Fourier synthesis showed a double image of the molecule: the atoms of one molecule at x, y, z (major component) together with the others at ca. x, y@, z (minor component). They were re–ned with occupancies of 2/3 and 1/3, and only Tc and Tc@ were assigned anisotropic thermal parameters.No attempt was made to include hydrogen atoms. Re–nement, performed with the same procedure used for 1, converged at and with 144 R1\0.078 wR2\0.203 parameters. In the diÜerence electron density map the largest peak and hole were 0.58 and [0.62 e respectively. Some Aé ~3, caution is required in considering the metrical data of 2 because of the pseudo-symmetry, which caused some difficulties in re–nement, leading to high estimated standard deviations.CCDC reference number 440/121. See http ://www.rsc.org/ suppdata/nj/1999/807/ for crystallographic –les in .cif format. References 1 R. Pasqualini, V. Comazzi, E. Bellande, A. Duatti and A. Marchi, Appl. Radiat. Isot., 1992, 43, 1329. 2 R. Pasqualini, A.Duatti, E. Bellande, V. Comazzi, V. Brucato, D. HoÜschir, D. Fagret and M. Comet, J. Nucl. Med., 1994, 35, 334. 3 J. Baldas, J. Bonnyman, P. M. Pojer and G. A. Williams, J. Chem. Soc., Dalton T rans., 1981, 1798. 4 A. W. Addison, T. N. Rao, J. Reedijk, J. Van Rijn and G. C. Verschoor, J. Chem. Soc., Dalton T rans., 1984, 1349. 5 F. Tisato, F. Refosco, G. Bandoli, C. Bolzati and A. Moresco, J. Chem. Soc., Dalton T rans., 1994, 1453. 6 C. Bolzati, A. Boschi, L. Uccelli, E. Malago` , A. Duatti, R. Pasqualini, M. Giganti and A. PiÜanelli, in T echnetium, Rhenium and Other Metals in Chemistry and Nuclear Medicine, no. 5, ed. M. Nicolini and U. Mazzi, SGEditoriali, Padova, Italy, 1999, p. 615. 7 K. Dehnicke and J. Strahle, Angew. Chem., Int. Ed. Engl., 1992, 31, 955. 8 J. Chatt, J. R. Dilworth, J. A. Schmutz and J. A. Zubieta, J. Chem. Soc., Dalton T rans., 1979, 1595. 9 A. C. T. North, D. C. Philips and F. Matheus, Acta Crystallogr., Sect. A, 1968, 24, 351. 10 (a) G. M. Sheldrick, SHEL XT L /PC, Version 5.03, Siemens Analytical X-ray Instruments Inc., Madison, WI, 1994. (b) G. M. Sheldrick, SHEL XL -93, University of Goé ttingen, Goé ttingen, 1993. L etter 9/03679B New J. Chem., 1999, 23, 807»809 809

ISSN:1144-0546    

DOI:10.1039/a903679b

出版商:RSC    

年代:1999

数据来源: RSC

摘要:

L e t t e r Synthesis of mono- and di-nuclear palladium diselenolenes from bis(cycloalkeno)-1,4-diselenins : X-ray crystal structure of [Pd(C7H10Se2)(PBu3)2 ] Susan Ford,a Christopher P. Morley*a and Massimo Di Vairab a Department of Chemistry, University of W ales Swansea, Singleton Park, Swansea, UK SA2 8PP. E-mail : c.p.morley=swan.ac.uk b Dipartimento di Chimica, degli studi di Firenze, V ia Maragliano 75/77, 50144 Universita` Firenze, Italy Received 17th June, 1999 The reaction between (dba = dibenzylidene- [Pd2(dba)3 ] acetone), tributylphosphine, and a bis(cycloalkeno)-1,4-diselenin leads to either a mononuclear [ Pd{SeC(R1)2 or a dinuclear C(R2)Se}(PBu3)2 ] , [ Pd2 {SeC(R1)2 diselenolene n= 4, 5, C(R2)Se} 2(PBu3)2 ] (R1ñR2= (CH2)n ; 6) depending on the stoichiometry employed; the structure of the monomeric product with n= 5 has been determined by X-ray crystallography.While organosulfur chemistry, and speci–cally that of dithiolene complexes, has been widely investigated, the –eld of organoselenium chemistry and diselenolenes had, until recent years remained relatively little studied. The current increase in research in this area is due to the greater accessibility and range of applications of these materials, i.e.the realisation that they may possess potentially useful properties for the electronics industry.1 Despite their ready preparation from 1,2,3-selenadiazoles2 (Scheme 1) little research into the chemistry of 1,4-diselenins has been documented.3h5 We previously reported that cycloalkeno-1,2,3-selenadiazoles react with the palladium(0) phosphine fragment ìPd(PR3)2œ (R\Et, Bun) generated in situ from the reaction of with to give the azo-complexes shown [Pd2(dba)3] PR3 , below (Scheme 2).6 When the 1,2,3-selenadiazole (n\5, 6) is replaced by the analogous 1,4-diselenin, a deep purple diselenolene is formed Scheme 1 Scheme 2 after heating the reaction mixture to re—ux in toluene for 1 h.To obtain the corresponding product when n\4 a higher temperature (re—ux in xylene) and a longer reaction time are required. By manipulation of the stoichiometry the product obtained after column chromatography can be either the mononuclear (1a»c) or dinuclear (2a»c) complex (Scheme 3).§ The detailed mechanism of the reaction is not yet clear. However, based on previous research showing cleavage of the C»Se bond in 1,4-diselenins,3 we postulate a –rst step of metal insertion into the heterocyclic ring system, followed by loss of the cycloalkyne (Scheme 4). It is interesting to note the forcing conditions required to obtain a product when bis(cyclohexeno)-1,4-diselenin (n\4) is employed.The dependence of product formation on ring size is not unusual for 1,4-diselenins.5 Indeed this observation is in accordance with the proposed mechanism, as expulsion of a cycloalkyne fragment would be favourable for n\6, cyclooctyne being an isolable species, but not for n\4, the ring strain in cyclohexyne being signi–cantly larger. Scheme 3 Scheme 4 New J.Chem., 1999, 23, 811»813 811We believe that this is the –rst example of a palladium diselenolene which has been isolated in both mononuclear and dinuclear forms.Monomeric palladium diselenolenes and even dithiolenes containing ancillary ligands such as phosphines appear to be unknown in the literature, although their platinum analogues are well established.7 It is interesting here that no reaction was observed under a variety of conditions, when the corresponding platinum chemistry was examined.The colour of the reaction mixture remained unchanged and the starting material was recovered quantitatively by column chromatography. Perhaps the greater lability of palladium complexes is partly responsible for this diÜerence in behaviour. Very little has been documented regarding dinuclear diselenolenes, although we recently prepared an analogous complex containing triphenylphosphine.8 A diÜerence between these two reactions is that no mononuclear product containing triphenylphosphine could be isolated, despite an excess of phosphine being present in solution.Also, these complexes are dark green unlike their deep purple n-butyl analogues, although this appears to be due to intensity changes in the absorption spectra, rather than any signi–cant diÜerences in electronic structure. The molecular structure of 1b has been determined by X-ray crystallographyî and is shown in Fig. 1. It consists of a distorted square-planar core, with only the outer PdSe2P2 atoms of the hydrocarbon ring and the butyl chains protruding signi–cantly from this plane. The Se»Pd»Se bond angle (86.66°) is smaller than those previously reported in the related complexes 3 (91.5°)9 and [NBun4]2[Pd(C3Se5)2] 4 (90.74, 91.18°).10 This is presum- [NMe4][Pd(C3S3Se2)2]2 ably due to steric repulsion between the large adjacent trialkylphosphine ligands (P»Pd»P\100.2°).The Pd»Se bond lengths in 1b (2.402 and 2.419 are similar to those in 3 and Aé ) 4 [2.409 (av.) and 2.383 respectively]. Aé Aé The molecular formulae of 1a»c and 2a»c have been established by elemental analysis and mass spectrometry.The mass spectrum of each complex shows an intense cluster corresponding to the molecular ion, with the expected isotope distribution. The NMR spectroscopic data are in accord with the proposed structures. For the mononuclear species analysis of the satellites in the 31P NMR spectra allows evaluation of the Fig. 1 Structure of 1b with H atoms omitted for clarity. Selected bond lengths and angles (°) : Pd»P(1) 2.318(3), Pd»P(2) 2.326(3), (Aé ) Pd»Se(1) 2.402(1), Pd»Se(2) 2.419(1), P(1)»Pd»Se(2) 84.3(1), Se(2)»Pd» Se(1) 86.66(4), Se(1)»Pd»P(2) 88.8(1), P(2)»Pd»P(1) 100.2(1). coupling of the equivalent selenium atoms to the corresponding cis- (J\16 Hz) and trans- (J\65 Hz) phosphorus atoms.There is also coupling between the two phosphorus nuclei (J\44 Hz) (cf. J\8 Hz; cis-[PdCl2(PMe3)2], cis- J\80 Hz).11 As expected in these sym- [PdCl2MP(OMe)3N2], metrical molecules there is only one 13C resonance for the ole–nic carbon atoms in the alicyclic ring, and only one signal in the 77Se NMR spectrum. For the dinuclear species the selenium NMR spectra contain two signals corresponding to the terminal and bridging selenium atoms.The resonance due to the bridging selenium atoms is a doublet of doublets, due to coupling to the cis- (J\9 Hz) and trans- (J\111 Hz) phosphorus atoms. These are similar coupling constants to those observed in the analogous triphenylphosphine complexes.8 The signal corresponding to the terminal selenium atoms is an apparent singlet, as the expected small coupling to the neighbouring cis-phosphorus atoms is not resolved.Owing to the delocalisation of the electrons present in the metal ligand bonding system, diselenolenes, like their sulfur analogues, are expected to display rich and varied chemical behaviour. The properties and reactivity of 1a»c and 2a»c are currently being investigated.thank EPSRC for the provision of a studentship (to We S. F.), Johnson Matthey for the loan of palladium salts, and the Ministero dellœUniversita e della Ricerca Scienti–ca e Tecnologica for –nancial support (to M. Di V.). Notes and references § Synthesis of palladium diselenolenes 1b and 2b: [0.16 g, 0.8 PBu3 mmol (1b) or 0.08 g, 0.4 mmol (2b)] was added to a toluene solution (100 cm3) of (0.23 g, 0.2 mmol).After stirring at [Pd2(dba)3] … dba room temperature for a short time, bis(cyclohepteno)-1,4-diselenin (0.14 g, 0.4 mmol) was added and the mixture heated to re—ux for 1 h. Removal of the solvent under reduced pressure left a dark coloured oil, which was puri–ed by column chromatography on an alumina column, with toluene as the eluent. Collection of the deep purple band and recrystallisation from hexane gave 1b or 2b as an analytically pure solid. 1b: yield : 0.042 g (14%); mp 119 °C. 1H NMR (400 MHz, CDCl3 , d 2.47 (m, 4H), 1.73 (m, 12H), 1.56 (m, 4H), 1.40 (m, 14H), 1.34 SiMe4), (m, 12H), 0.85 (t, 18H); 13C NMR (100 MHz, d 131.4 CDCl3 , SiMe4), [average J(13C»31P) 7 Hz], 39.9, 29.7, 27.3, 26.2 [average J(13C»31P) 12 Hz], 24.9 [average J(13C»31P) 7 Hz], 24.4, 13.7 ; 31P NMR (101 MHz, external 85% d 0.80 ; 77Se NMR (47.7 MHz, CDCl3, H3PO4), external d 533 [J(77Se»31P) 65, 16 Hz]; UV»VIS CDCl3, SeMe2 ), (hexane) : (e/dm3 mol~1 cm~1) 560 (140), 410 (420), 322sh (4600), jmax 285 (24000), 225 (29000) nm; MS (FAB): m/z (%) 764 (15) [M`], 203 (100) [PBu3 `]. 2b: yield : 0.12 g (54%); mp 112 °C. 1H NMR (400 MHz, CDCl3 , d 2.53 (m, 8H), 1.65 (m, 12H), 1.54 (m, 12H), 1.41 (m, 24H), SiMe4), 0.93 (t, 18H); 13C NMR (100 MHz, d 151.1, 120.7, CDCl3 , SiMe4), 43.6, 40.6, 33.0, 31.0, 26.4, 24.4, 24.2, 24.0, 13.8 ; 31P NMR (101 MHz, external 85% d 11.2 ; 77Se NMR (47.7 MHz, CDCl3, H3PO4), CDCl3 , external d 498, 434 [J(77Se»31P) 111, 9 Hz] ; UV»VIS SeMe2), (hexane) : (e/dm3 mol~1 cm~1) 565 (2100), 405 (8700), 325 (16000), jmax 285 (24000), 270 (25000), 240 (49000) nm; MS (FAB): m/z (%) 1122 (5) [M`], 203 (100) [PBu3 `]. î Crystal data for 1b: M\763.08 ; crystal size C31H64P2PdSe2; 0.30]0.40]0.60 mm, monoclinic, space group (no. 14) ; P21/c a\10.447(2), b\22.124(4), c\16.772(2) b\94.80(1)°, Aé , U\3863(1) (by least squares re–nement on setting angles of 24 Aé 3 re—ections, Z\4), F(000)\1576, g cm~3, k(Mo-Ka)\ Dc\1.312 2.46 mm~1. Data collection (Enraf-Nonius CAD4, graphitemonochromated Mo-Ka radiation, j\0.71069 T \295 K), x»2h Aé , scans, 2.5\h\25°. 5924 measured re—ections (^h, ]k, ]l), 5458 unique Structure solution by direct methods, with (Rint\0.047). SIR,12 and heavy-atom procedures with SHELXL-93.13 Empirical absorption correction (t scans ; min., max. correction factors 0.71, 1.00).Final re–nement cycles performed against F2 with all nonhydrogen atoms anisotropic and hydrogen atoms in calculated positions. Re–nement on 331 variables converged at (based on R1\0.054 2858 re—ections with (on all re—ections), Fo[4pFo), R1\0.154 GOF\1.021. Max., min. peaks in the –nal diÜerence wR2\0.150, map 0.47, [0.42 e Aé ~3. 812 New J. Chem., 1999, 23, 811»813CCDC reference number 440/126. 1 J. Arnold, Prog. Inorg. Chem., 1995, 43, 353; M. L. Steigerwald and C. R. Sprinkle, J. Am. Chem. Soc., 1987, 109, 7200; P. OœBrien, Chemtronics, 1991, 5, 61. 2 H. Meier and E. Voigt, T etrahedron, 1972, 28, 187. 3 C. M. Bates, P. K.Khanna, C. P. Morley and M. Di Vaira, Chem. Commun., 1997, 913. 4 M. R. J. Dorrity, A. Lavery, J. F. Malone, C. P.Morley and R. R. Vaughan, Heteroatom Chem., 1992, 3, 87; A. Chesney, M. R. Bryce, A. S. Batsanov and J. A. K. Howard, Chem. Commun., 1997, 2293. 5 A. J. Mayr, H.-S. Lien, K. H. Pannell and L. Parkanyi, Organometallics, 1985, 4, 1580. 6 S. Ford, C. P. Morley and M. Di Vaira, Chem. Commun., 1998, 1305. 7 C. E. Johnson, R. Eisenberg, T. R. Evans and M. S. Burbery, J. Am. Chem. Soc., 1983, 105, 1795; C.M. Bollinger and T. B. Rauchfuss, Inorg. Chem., 1982, 21, 3947; R. D. McCullough, J. A. Belot, J. Seth, A. L. Rheingold, G. P. A. Yap and D. O. Cowan, J. Mater. Chem., 1995, 5, 1581: D. M. Giolando, T. B. Rauchfuss and A. L.Rheingold, Inorg. Chem., 1987, 26, 1636. 8 S. Ford, P. K. Khanna, C. P. Morley and M. Di Vaira, J. Chem. Soc., Dalton T rans., 1999, 791. 9 C. Faulmann, J. P. Legros, P. Cassoux, J. Cornelissen, L. Brossard, M. Inokuchi, H. Tajima and M. Tokumoto J. Chem. Soc., Dalton T rans., 1994, 249. 10 G. Matsubayashi, S. Tanaka and A. Yokozawa, J. Chem. Soc., Dalton T rans., 1992, 1827. 11 S. Berger, S. Braun and H. O. Kalinowski, NMR Spectroscopy of the Non-metallic Elements, Wiley, London, p. 954. 12 A. Altomare, M. C. Burla, M. Camalli, G. Cascarano, C. Giacovazzo, A. Guagliardi and G. Polidori, J. Appl. Crystallogr., 1994, 27, 435. 13 G. M. Sheldrick, SHELXL-93, Program for Crystal Structure Re–nement, University of Goé ttingen, 1993. L etter 9/04853G New J. Chem., 1999, 23, 811»813 813

ISSN:1144-0546    

DOI:10.1039/a904853g

出版商:RSC    

年代:1999

数据来源: RSC

摘要:

L e t t e r SchiÜ-base chemistry of niobium: unexpected products of radical elimination and diastereoselective addition Paul R. Woodman,a Christopher J. Sanders,a Nathaniel W. Alcock,a Peter B. Hitchcockb and Peter Scott*a a Department of Chemistry, University of W arwick, Coventry, UK CV 4 7AL . E-mail : peter.scott=warwick.ac.uk b T he Chemistry L aboratory, University of Sussex, Falmer, Brighton, UK BN1 9QJ Received 8th June 1999 The two main products from the reaction of a chiral SchiÜ-base proligand with are the amidoimido species H2L [NbIV(NEt2)4 ] and the highly unusual [LNbV(NEt)(NEt2)] [LºNbV(NEt2)2 ] in which a 1-ethylaminoethyl radical has undergone highly diastereoselective addition to one of the coordinated imine groups.Our current work on the development of synthetic routes to chiral multidentate SchiÜ-base complexes of the transition metals is inspired by the success of this type of compound in the mediation of enantioselective catalytic processes.1 We note that while there is an extensive chemistry of achiral SchiÜ-base vanadium compounds,2 there is a paucity of such complexes of the heavier Group 5 congeners niobium and tantalum.3,4 This might be blamed on the apparent ease with which the latter elements may move between closely spaced oxidation states, but is no doubt also caused in part by the inherent reactivity of the imine ligand.5 Not unexpectedly then, attempts to extend our work on zirconium complexes of a biphenyl bridged quadridentate SchiÜ-base system6 to niobium were initially plagued by complex side-reactions, but we are now able to report a reliable route to such complexes of this element.The highly unexpected products formed cast light on the type of side reactions which have prevented the development of this chemistry in the past. We have prepared zirconium diamides in good yield via a protonolysis reaction between with metal amides H2L Accordingly, reaction of (Scheme 1) with [Zr(NR2)4].6 H2L was examined.Unexpectedly the target mol- [Nb(NEt2)4] ecule, green 1 (identi–ed by its mass spectrum [NbL(NEt2)2] and EPR signal) was produced in only trace quantities. The two main products formed in this reaction, the (diethylamido)(ethylimido) complex 2 [NbL(NEt)(NEt2)] (43%) and 3 (16%) contain pentavalent [NbL@(NEt2)2] niobium centres.The compounds were readily separated by crystallisation.§ We propose a simple mechanism for the formation of 2 and 3 (Scheme 1). The reaction of and gives H2L [NbIV(NEt2)4] 1 and two equivalents of diethylamine as [NbIVL(NEt2)2] expected. Loss of an ethyl radical from 1 to give 2 is driven by the concomitant oxidation NbIV to NbV and formation of the strong bond Nb2N. The ethyl radical is rapidly quenched by the two-fold excess of diethylamine present in the reaction mixture to produce the more stable 1-ethylaminoethyl radical which adds in an intermolecular fashion to one of the two equivalent imine groups of further 1 to give 3 directly.Pure yellow 2 is obtained by recrystallisation of the reaction mixture from pentane, and single crystals were obtained by allowing acetonitrile to diÜuse slowly into a concentrated solution of the compound in dichloromethane»pentane.The molecular structure as determined by X-ray diÜractionî (Fig. 1) is a rare example of the cis»trans structure (class II)6 N2O2 in which the two imine nitrogen atoms N(1) and N(2), one phenoxide oxygen atom O(1) and the amido nitrogen atom N(3) form the plane of a distorted octahedron.The second phenoxide oxygen atom O(2) and the imido ligand nitrogen atom N(4) occupy mutually trans sites with O(2)»Nb»N(4) of 168.4(2)°. The angle at niobium between the amido and imido ligands is 96.1(2)°. The three types of Nb»N bond are clearly distinguishable in this structure ; the Nb»N(imine) bond lengths are 2.388(5) and 2.240(6) the Nb»N(amido) length Aé Aé , is 2.012(5) and the Nb»N(imido) bond is 1.786(7) The Aé Aé .imido linkage C(49)»N(4)»Nb is almost linear at 172.2(5)°, as is common in early transition metal imido chemistry.7 The amido nitrogen atom N(3) is coplanar with Nb, C(46) and Scheme 1 Formation of complexes 1»3. New J. Chem., 1999, 23, 815»817 815Fig. 1 Thermal ellipsoid plot of the molecular structure of 2; hydrogen atoms omitted.C(47) (sum of angles\359.9°) indicating normal sp2 amido» metal bonding.8 The 1H NMR spectrum of 2 contains four resonances assigned to tert-butyl groups and two resonances assigned to aromatic methyl groups, as expected for an unsymmetric complex containing L. It is interesting to note that all four methylene protons of the diethylamido group are inequivalent, indicating that rotation about the bond is slow Nb»NEt2 compared to the 1H NMR chemical shift timescale.Although the formation of complex 2 under these mild conditions is quite unexpected, mixed amido»imido complexes of the Group 5 metals are known. For example [Ta(2NEt) is a product of the thermolysis of and (NEt2)3] [Ta(NEt2)5],9 (M\Nb, Ta) results from [MCl2(NBut)(NHBut)(NH2But)]2 the reaction of with However, these reac- MCl5 ButNH2 .10 tions proceed without change in oxidation state at the metal centre ; to our knowledge 2 is the –rst such complex produced by an oxidative process from an amido precursor. The third product of the reaction between and H2L i.e. 3 is obtained from the [Nb(NEt2)4], [NbL@(NEt2)2] pentane mother-liquor of 2 above after exposing the solution brie—y to air in order to decompose any NbIV species.Single crystals of 3 were grown from a saturated solution of the pure compound in a pentane»acetonitrile mixture and the molecular structure was determined by X-ray diÜraction.î The niobium atom is in a distorted octahedral environment (Fig. 2) with the diethylamido ligands occupying mutually cis coordination sites.The Nb»N(2) and Nb»N(1) distances for the chelate ligand nitrogen atoms of 2.336(3) and 2.032(3) are Aé Aé indicative of imino and amido functionality respectively. All three dialkylamido(niobium) fragments are essentially planar and have bond angles consistent with sp2 hybridisation. The structural parameters within the diethylamine fragment which has become bonded to C(7) do not show any peculiarities.Nevertheless, the pendant amine nitrogen N(5) makes a close approach (ca. 3.05 to the phenolic carbon C(5). Although Aé ) the hydrogen atom at C(5) was not located, its calculated position is ca. 2.34 from N(5), commensurate with the presence Aé of an intramolecular C»H]N hydrogen bond. This is highlighted in Scheme 1. The 1H and 13C NMR spectra of 3 have been fully assigned by standard two-dimensional methods and are consistent with the observed solid state structure.For example, the unusually low –eld 1H NMR resonance of one of the phenolic aromatic protons (d 8.45) is assigned to the hydrogen-bonded C»H group (see above). Also, the protons of the diastereotopic Fig. 2 Thermal ellipsoid plot of the molecular structure of 3; hydrogen atoms omitted. 816 New J. Chem., 1999, 23, 815»817methylene group C(46) adjacent to the amine group are separated by an unusually large chemical shift diÜerence (0.68 ppm), suggesting that the rotation of the diethylamine fragment is restricted by the hydrogen bond. The highly unusual trianionic quadridentate ligand L@ in 3 contains three elements of chirality : the two stereogenic centres marked * (Scheme 1) and the biaryl group.Despite this, close examination of the 1H and 13C NMR spectra of the crude reaction mixture provided no evidence for the presence of signi–cant amounts of more than one diastereomer (d.e. [95%). The con–gurations of the stereogenic centres in 3 are readily explained. Intermolecular attack of the radical at the imine carbon from the side opposite to the metal leads to the observed stereochemistry of C(7).That of C(45) is determined by the close proximity of the biaryl group below and a vacant space above the nascent C(7)»C(45) vector as drawn in Fig. 2. It is possible also that one of these interactions ultimately controls the stereochemistry at both carbon centres. The above mentioned hydrogen bond also may have had a part to play in the highly diastereoselective formation of 3.The chiral pentavalent complex 2 and [NbL(NEt)(NEt2)] the fascinating diastereomerically pure 3 are [NbL@(NEt2)2] thus shown to have been formed from the reaction of H2L and The Nb(IV/V) radical redox processes which [Nb(NEt2)4]. gave rise to these species are probably characteristic of the kinds of side reactions which have beleaguered earlier attempts to synthesise SchiÜ-base complexes of the heavier Group 5 metals.Notably, the complexes and [LNbCl2] are readily accessible, and we will report their [LNb(NMe2)2] chemistry in due course. Acknowledgements wishes to thank P–zer Ltd. and SmithKline Beecham for PS support. The SMART X-ray diÜraction facility was supported by EPSRC and Siemens Analytical Instruments.Notes and references § Experimental: 2. A solution of (1.00 g, 1.55 [NbL(NEt2)(NEt)] H2L mmol) in diethyl ether (30 ml) was added dropwise to a solution of (0.60 g, 1.57 mmol) also in diethyl ether (10 ml). The [Nb(NEt2)4] mixture was stirred for 12 h to give a green solution from which the volatile components were removed under reduced pressure.The solid remaining was extracted into pentane and cooled to [30 °C to give a dark green solid and yellow-brown supernatant liquor. The solid was collected and dried in vacuo, then dissolved in the minimum quantity of diethyl ether, brie—y exposed to air to turn the solution orange, and cooled to [30 °C to give yellow crystals (570 mg, 43%). Found (Calculated for C, 70.44 (70.57) ; H, 8.24 (8.17) ; N, C50H69N4O2Nb): 6.68 (6.58)%. 1H NMR d 8.08 (s, 1H, N2CH), 7.74 (s, 1H, (C6D6) : N2CH), 7.69 (d, 1H, phenolic), 7.57 (d, 1H, phenolic), 7.43 (d, 1H, biaryl), 7.18 (t, 1H, biaryl), 7.02 (d, 1H, phenolic), 6.85 (d, 1H, biaryl), 6.70 (d, 1H, phenolic), 6.65 (t, 1H, biaryl), 6.53 (d, 1H, biaryl), 6.47 (d, 1H, biaryl), 4.58 (sextet, 1H, 4.07 (sextet, 1H, Nb»NCH2), Nb»NCH2), 3.91 (sextet, 1H, 3.65 (sextet, 1H, 2.76 (sextet, Nb»NCH2), Nb»NCH2), 1H, 2.56 (sextet, 1H, 1.95 (s, 3H, Me), 1.79 (s, Nb2NCH2), Nb2NCH2), 9H, But), 1.71 (s, 9H, But), 1.70 (s, 3H, Me), 1.45 (t, 3H, 1.27 (s, 9H, But), 1.16 (s, 9H, But), 1.02 (t, 3H, Nb»NCH2CH3), 0.34 (t, 3H, 13C-M1HNNMR Nb»NCH2CH3), Nb2NCH2CH3).d 167.95 (N2C), 166.67, 166.25 (aromatic), 164.88 (N2C), (C6D6) : 154.49, 151.90, 140.98, 138.86, 138.66, 138.01, 136.93, 136.05, 131.94, 131.26, 131.12, 129.06, 128.30, 128.11, 127.87, 127.61, 127.01, 124.18, 123.75, 120.55, 118.87, 115.26 (aromatic), 59.48, 52.71, 48.02 35.89, 35.74, 34.14, 33.86 31.66, 31.37, 30.28, (NCH2CH3), (CMe3), 30.15 19.92 (Me), 18.09, 16.60, 16.52 IR (Nujol) (CMe3), (NCH2CH3).cm~1: 1612 (m), 1595 (m), 1551, 1530, 1321, 1302, 1259 (m), 1199, 1169, 1067, 1005, 970, 886, 841, 825, 789, 774, 722 (m), 610, 561, 547.MS (EI) : m/z 850 [M`], 813, 778 [M`[NEt2] 3. The yellow-brown supernatant liquor from the [NbL@(NEt2)2] above experiment was evaporated to dryness and the solid remaining was washed with pentane (3]2 cm3). These portions of pentane were combined and brie—y exposed to air. The solution became dark red.Acetonitrile was added until solids began to precipitate. The solution was then cooled to [30 °C to give red crystalline 3 (240 mg, 16%). Found (Calculated for C, 70.70 (70.64) ; H, 8.99 C56H84N5O2Nb): (8.89) ; N, 7.21 (7.35)%. 1H NMR d 8.45 (d, 1H, phenolic), 7.64 (C6D6) : (s, 1H, N2CH), 7.51 (d, 1H, phenolic), 7.26 (d, 1H, phenolic), 7.07 (d, 1H, biaryl), 7.04 (d, 1H, biaryl), 6.87 (t, 1H, biaryl), 6.84 (t, 1H, biaryl), 6.73 (d, 1H, phenolic), 6.63 (d, 1H, biaryl), 6.44 (d, 1H, biaryl), 5.56 (d, 1H, 4.50 (sextet, 2H, 4.19 (sextet, 2H, N»CHC6H2), Nb»NCH2), 3.86 (sextet, 2H, 3.80 (sextet, 2H, Nb»NCH2), Nb»NCH2), 3.50 (q of d, 1H, diethylamino 2.56 (m, 1H, Nb»NCH2), NCHCH3), 2.16 (s, 3H, Me), 1.88 (m, 1H, 1.73 (s, 18H, NCH2CH3), NCH2CH3) 2But), 1.68 (s, 3H, Me), 1.47 (s, 9H, But), 1.38 (d, 3H, 1.08 N»CHCH3), (s and t, 15H, But and 0.75 (t, 6H, Nb»NCH2CH3), Nb»NCH2CH3), 0.69 (t, 3H, 13C-M1HN NMR d 165.79 (N2C), NCH2CH3).(C6D6) : 161.50, 159.67, 152.Z, 148.17, 139.44, 139.31, 138.07, 137.11, 136.74, 134.73, 133.31, 129.74, 127.8, 127.67, 127.59, 127.36, 126.88, 126.18, 123.98, 123.51, 122.51, 119.11, 115.26, 112.64, 105.57 (aromatic), 68.58, 58.97, 45.97, 45.20, 43.20, 35.57, 35.43, 34.67, 33.93 32.32, (CMe3), 31.82, 31.29, 30.55 21.10, 20.07, 18.83, 15.61, 13.78, 13.31 (Me).(CMe3), IR (Nujol) cm~1: 3331, 1618, 1551, 1538, 1299, 1257, 1231, 1201, 1172, 1145, 1124, 1100, 1037, 1004, 949, 915, 875, 837, 824, 797, 772, 735, 722, 685, 668, 592, 547, 534.4, 502, 473. MS (EI) : m/z 879 [M` 807 735 [NEt2], [M`[2NEt2], [M`[3NEt2]. î Crystal data for 2: M\869.02, triclinic, C51.5H69N4NbO2 a\12.5569(10), b\13.0513(5), c\16.5008(5) a\80.481(5), Aé , b\92.26(3), c\82.328(5)°, U\2511.7(2) T \180(2) K, space Aé 3, group Z\2, k\0.279 mm~1, 14 770 re—ections collected, 10 931 P1 6 , independent Final R indices [I[2p(I)] were (Rint\0.1857).R1\ For 3: M\952.2, mono- 0.0939, wR2\0.2028.C56H84N5NbO2 clinic, a\13.670(2), b\20.289(14), c\19.765(11) b\92.26(3)°, Aé , U\5478(5) T \293(2) K, space group (no. 14), Z\4, Aé 3, P21/c k\0.26 mm~1, 10 018 re—ections collected, 9597 independent (Rint\ Final R indices [I[2p(I), 6753 re—ections] were 0.0456). R1\0.048, The structures were solved using full-matrix least wR2\0.117. squares on F2. Crystals of 2 desolvated rapidly and consequently diffracted weakly, leading to a high R value.A disordered solvent molecule (presumably pentane) was modelled as three 0.5 occupancy C atoms near to an inversion centre. CCDC reference number 440/122. See http ://www.rsc.org/suppdata/nj/1999/815/ for crystallographic –les in .cif format. 1 See M. Palucki, N. S. Finney, P. J. Pospisil, M. L. Guler, T.Ishida and E. N. Jacobsen, J. Am. Chem. Soc., 1998, 120, 948 and refs. therein. 2 H. Kneifel and E. Bayer, J. Am. Chem. Soc., 1986, 108, 3075; J. A. Bonadies, W. M. Butler, V. L. Pecoraro and C. J. Carrano, Inorg. Chem., 1987, 26, 1218; J. A. Bonadies and C. J. Carrano, J. Am. Chem. Soc., 1986, 108, 4088; R. L. Robson, R. R. Eady, T. H. Richardson, R. W. Miller, M. Hawkins and J.R. Postgate, Nature (L ondon), 1986, 322, 388; A. Hills, D. L. Hughes, G. J. Leigh and J. R. Sanders, J. Chem. Soc., Dalton T rans., 1991, 325; S. A. Fairhurst, D. L. Hughes, U. Kleinkes, G. J. Leigh, J. R. Sanders and J. Weisner, J. Chem. Soc., Dalton T rans., 1995, 321. 3 C. Floriani, M. Mazzanti, S. Ciurli, A. Chiesi-Villa and C. Guastini, J. Chem. Soc., Dalton T rans., 1988, 1361. 4 S. Chomal and G. C. Shivahae, Acta Chim. Hung., 1985, 118, 31; B. U. Khan, M. U. Rahman and N. Ahmad, T ransition Met. Chem., 1988, 13, 392. 5 E. Solari, C. Maltese, M. Latronico, C. Floriani, A. Chiesi-Villa and C. Rizzoli, J. Chem. Soc., Dalton T rans., 1998, 2395. 6 P. Woodman, P. B. Hitchcock and P. Scott, Chem. Commun., 1996, 2735. 7 D. E. Wigley, Prog. Inorg. Chem., 1994, 42, 239; V. C. Gibson, Angew. Chem., Int. Ed. Engl., 1994, 15, 1565; S. C. Dunn, P. Mountford and D. A. Robson, J. Chem. Soc., Dalton T rans., 1997, 293. 8 M. F. Lappert, P. P. Power, A. R. Sanger and R. C. Srivastava, Metal and Metalloid Amides, Ellis Horwood, Chichester, 1980. 9 D. Bradley and I. Thomas, Can. J. Chem., 1962, 40, 1355. 10 K. C. Jayaratne, G. P. A. Yap, B. S. Haggerty, A. L. Rheingold and C. H. Winter, Inorg. Chem., 1996, 35, 4910. L etter 9/04579A New J. Chem., 1999, 23, 815»817 817

ISSN:1144-0546    

DOI:10.1039/a904579a

出版商:RSC    

年代:1999

数据来源: RSC

摘要:

Selective solvent extraction of tetrahedrally-coordinating transition metal ions from acidic aqueous media using benzimidazoleñphosphinate ligands : speci–city for zinc(II) over copper(II) Christopher D. Edlin,a David Parker,*a Justin J. B. Perry,a Christine Chartrouxb and Karsten Gloeb a Department of Chemistry, University of Durham, South Road, Durham, UK DH1 3L E b Institut Anorganische Chemie, T echnische Dresden, Mommsenstrasse 13, f ué r Universitaé t Dresden, D-01062, Germany Received 26th April 1999, Accepted 29th May 1999 The synthesis, solution complexation behaviour and solvent extraction ability of related chelating mono-and bis-(benzimidazole)phosphinate ligands 8 and 9 has been assessed. 31P NMR, —uorescence and absorption spectroscopy and electrospray mass spectrometry revealed preferential formation of 1 : 1 complexes with the divalent ions Zn, Co and Ni, and complex formation with the bidentate ligand.Extraction studies showed ML2 that 9 extracted with no extraction of copper(II), but avid extraction of copper(I), generated in situ, Zn[CoANi consistent with the ligandœs preference for the binding of tetrahedrally-coordinating metal ions. The selective solvent extraction of transition metal ions from 0.01 M acidic aqueous solutions, requires the use of a lipophilic ligand with sufficient avidity for the target metal ion that the derived complex partitions selectively into the organic phase.Such hydrometallurgical processes are therefore of commercial use for the recovery of metal ions,1 as they may avoid some of the high costs associated with pyrometallurgical methods, involving elevated temperatures.2 For the recovery of zinc, a mixture of hydrochloric acid and ferric chloride is often used as the leach solution, and during solvent extraction the pH is held below 2.5 to inhibit precipitation of ferric hydroxide.The organic phase may then be extracted with 0.1 M sulfuric acid to generate a zinc sulfate solution which is subsequently electrolysed to give the pure metal.The electrolyte solution must be free of other metal ion impurities to prevent formation of metals which possess a lower reduction potential than ZnII. It is particularly important to achieve high selectivity over FeIII and CuII, as they are common contaminants which are readily reduced.The properties of the ligand that will allow the selective extraction of zinc are well-de–ned: it must bind zinc rapidly at pH 2 and release it quickly below pH 1; it must be readily synthesised and be sufficiently robust to withstand many cycles of complexation and decomplexation in a continuous recovery process ; it must be sufficiently lipophilic to avoid ligand loss to the aqueous phase and the metal complex must be soluble in the organic solvent ; the zinc complex must be charge-neutral to prevent co-transport of chloride ions to the pure zinc sulfate stream, otherwise chlorine evolution may occur at the anode.The solvent extraction of zinc is now used in a limited number of operating plants, using extractants such as di(2-ethylhexyl)phosphoric acid 1,3 di(n-octyl)phosphinic acid 2,4 and ìCyanex 302œ 35 which is a thiophosphinic acid proposed to enhance extraction at lower pH.A particular problem with these three ligands is co-extraction of FeIII, and to a lesser extent Cu(II). Some of these problems may be addressed by the thiophosphoramide ligands e.g. 4,6 being developed by Zeneca (UK), although 4 is sensitive to oxidation, especially in the presence of one-electron redox couples.The issue of the selectivity of a ligand for zinc(II) over Fe(III) and Cu(II) may be discussed in terms of respective donor atom, coordination number and geometric preferences. Thus zinc is the only one which may prefer a tetrahedral geometry with hard donors and diÜerent types of tetrahedrallycoordinating ligands have been examined recently.7h9 The phosphinic acid group is strongly acidic and sufficiently favours binding of zinc(II) over copper(II) that the Irving» Williams stability series may be inverted.10 N-Substituted benzimidazole ligands have been studied for the purpose of zinc extraction for some time11h14 but selectivity for zinc only occurs in aqueous media containing P3 M Cl~, consistent with the formation of overall neutral or L2M2Cl4 LMCl2 complexes observed by crystallography with 5 and 6, respectively. 11,15 Following earlier work with 78 which forms a charge neutral complex, we set out to examine the Zn2L2 behaviour of the benzimidazole»phosphinates 8, 9 and 10.16 It was envisaged that these may form neutral ML and ML2 , complexes17 respectively with ì tetrahedralœ ions.Solu- M2L2 bility problems limited the study of complex formation with 10,18 so that details are presented here only for the complexation behaviour of 8 and 9. New J. Chem., 1999, 23, 819»826 819Results and discussion Ligand synthesis Practicable syntheses of 8 and 9 were devised starting from 1,2-diamino-3-bromobenzene, 11, using a palladium-catalysed activation of the aryl halide to promote carbon»phosphorus bond formation, (Scheme 1).The precursor amine 11 was prepared by a modi–cation of the literature route19 wherein nitration of N-acetyl-2-bromoaniline used a 4 : 3 v/v mixture of fuming nitric acid and tri—uoroacetic with 1% added water; the reaction took place over 10 h to generate a roughly 1 : 1 mixture of ortho and para-nitration products which were separated by crystallisation from at 0 °C.Formation of CHCl3 the benzimidazole 12a involved a modi–ed Phillips procedure20 and regioselective N-alkylation occurred at the least-hindered nitrogen to give 12b, con–rmed by a 1H NOE enhancement (3%) between the benzylic and H-7 of the CH2 benzimidazole ring. Cross-coupling of the N-alkylbromobenzimidazole with ethyl phenylphosphinate, in the presence of in boiling toluene, followed a Pd(PPh3)4»Et3N procedure reported by Xu and Huang21 and yielded the desired phosphinate ester in 62% yield, following —ash chromatography.Condensation of the diamine 11 with dimethyl glutarate in polyphosphoric acid22 at 180 °C for 18 h aÜorded the bis(benzimidazole) 13a. Alkylation at N-1 with tertbutylbenzyl bromide in DMF followed by a palladiumcatalysed coupling with ethyl phenylphosphinate yielded the diester 13c from which ligand 9 was obtained by acid hydrolysis, in 42% overall yield from 11.Scheme 1 (a) 110 °C; (b) DMF, Ac2O, H3O`; 4-tBuC6H4CH2Br, 25 °C; (c) PhPH(O)(OEt), 110 °C, PhMe; Cs2CO3 , Pd(PPh3)4, Et3N, (d) 6 M HCl, 110 °C, 16 h. Solution complexation behaviour The behaviour of the ligands 8 and 9, in the presence of Zn2` and related metal ions, was studied by 31P NMR, —uorescence, absorption spectroscopy and electrospray mass spectrometry, prior to an examination of their extraction ability.Anhydrous metal tri—ates were used as the salts and the solvent used was methanol or methanol»chloroform mixtures. Incremental addition of zinc tri—uoromethanesulfonate to a solution of 8 and 9 was monitored by 31P NMR (293 K, 75% and the variation of the 31P shift CDCl3»25% CD3OD), plotted as a function of M : L ratio.With 9, the phosphorus signal shifted to higher frequency with increasing M : L ratios, and a single peak was observed indicating that free and bound ligands were in fast-exchange on the NMR timescale (293 K, 101 MHz). The variation of with Zn : L ratio (Fig. 1), is *dp consistent with formation of a 1 : 1 complex with KMLP104. With the monobenzimidazole, 8, a similar overall binding curve was obtained but with a positive deviation from linearity over the M : L range 0 to 1 : 1. Such behaviour suggested that an complex may be formed at lower zinc concentra- ML2 tions. Accordingly a Job plot23 was obtained to allow the determination of the predominant complex stoichiometry in solution.The turning point was observed at a ligand percentage of ca. 66% (Fig. 2), and is consistent with formation of an complex. ML2 While 31P NMR is very useful for monitoring phosphinate binding to the metal ion, coordination of the benzimidazole Fig. 1 31P NMR titration of 9 with (293 K; 75% Zn(CF3SO3)2 showing behaviour consistent with formation CDCl3»25% CD3OD), of an ML complex. 820 New J. Chem., 1999, 23, 819»826Fig. 2 Variation of complex concentration with the percentage of ligand 8 (Job plot ; 293 K; 75% CDCl3»25% CD3OD). nitrogen may be probed by changes in the ligand —uorescence emission. Variations in the —uorescence emission spectrum of 8 were recorded as a function of pH over the range 1.2 to 8 (293 K; 10% MeOH).Under acidic conditions H2O»90% (pH\5), the emission spectrum gave a peak with maximum intensity around 326 nm, which increased in intensity and shifted by ca. 15 cm~1 to shorter wavelength at pH[6. NProtonation changes the relative energies of the HOMO and LUMO§ and the energy gap is less for the protonated species. Protonation will also aÜect the internal charge transfer (ICT) excited state that is present in such benzimidazoles.Evidence for the occurrence of an ICT state was provided by an examination of the —uorescence emission spectrum of 8 in solvents of increasing polarity. In the series THF, CH2Cl2, CH3 CN, MeOH and (with polarities of 0.21, 0.31, 0.46, 0.76 and H2O 1.0 on the scale),25 the emission maximum shifted to ET[30] the red in direct proportion to solvent polarity.The variation of emission intensity from 8 as a function of pH (293 K, 10% MeOH) revealed a pattern of H2O»90% behaviour that may be interpreted in terms of the changing proportions in solution of the appropriate diÜerently protonated species, (Scheme 2 and Fig. 3). Over the pH range 1 to 3, the emission intensity at 326 and 308 nm increased, reached an approximately stable value over the range 3 to 5, before rising again beyond pH 5. Given that diphenylphosphinic § Gas phase energies of the HOMO for the parent benzimidazole and benzimidazolium cation ([8.87 and [13.51 eV) and LUMO energies of [0.09 and [5.20 eV were calculated using semi-empirical orbital calculations using MOPAC 6.024 and the AM force-–eld operating in commercial software packages (CaChe, 1996, Oxford Molecular).Fig. 3 Variation in emission intensity for ligand 8 as a function of pH 290 nm, 308 or 326 nm, 90% MeOH»10% 293 K). (jexc jem H2O, acids typically have a of ca. 2.9 26,10 and that the ground- pKa state in the polar medium used is not likely to be very pKa diÜerent from the of the –rst singlet (p,p*) excited state, pKa the in—ection observed at a pH of between 2.6 and 3.0 may be associated with the maximal concentration of the zwitterionic structure, formed at this pH value.This species may be stabilised by an intramolecular hydrogen bond providing extra rigidity which may be associated with the emission intensity enhancement. At higher pH, deprotonation of the benzimidazole nitrogen occurs, and in the anion the phosphorus oxygen double bond is less likely to be conjugated with the benzimidazole ring, as lone-pair conjugation requires that the moiety at the tetrahedral P centre is coplanar.The PO2~ in—ection in emission intensity around pH 5 is consistent with the ground-state values of ca. 5.5 observed for related pKa benzimidazoles.27 A similar pattern of behaviour with varying pH was shown by 9.Incremental addition of to a methanolic solu- Zn(CF3SO3)2 tion of 8 (10 lM, 293 K, 90% MeOH»10% eÜective pH H2O, 4.4), was monitored by —uorescence (Fig. 4). An increase in the intensity of emission at 309 nm was observed, with the emission maximum at 326 nm shifting to this lower value. Complexation of zinc by 8 under these conditions leads to a displacement of the bound proton on the benzimidazole nitrogen and the Zn»N bond length is likely to be about 1 longer Aé than the N»H distance, so that there is much less perturbation of the p system in the zinc chelate structure.By assuming a 1 : 1 limiting binding stoichiometry (most likely via an intermediate complex at higher L : M ratios), non-linear ML2 least-squares analysis of this binding isotherm indicated that M~1, in agreement with the estimate KZnL\1.3(^0.15)]104 Scheme 2 New J.Chem., 1999, 23, 819»826 821Table 1 Major species observed by electrospray mass spectrometrya in the complexation of 8 and 9 by metal perchlorate salts Complex Observed species Observed mass Calculated mass Relative intensity (%) Zn/9 [LZn]~ 911.14 911.28 100 [LZnCl]~ 945.09 945.36 77 [LZnClO4]~ 1012.63 1012.84 33 12 [LZn]~ 456.26 456.15 56 Cu/9 [LCuCl]~ 943.95 943.24 73 [LCuClO4]~ 1008.60 1008.77 41 Ni/9 [LNiCl]~ 938.76 938.74 5 [LNiClO4]~ 1004.63 1004.32 3 Zn/8 [LH]` 419.65 419.88 73 [L2ZnH]` 899.19 899.28 100 [L2ZnNa]` 922.83 922.27 89 Cu/8 [LH]` 419.65 419.88 100 [L2CuH]` 898.10 898.43 33 [L2CuNa]` 920.28 920.63 67 Ni/8 [LH]` 419.65 419.88 100 [L2NiH]` 893.24 893.37 9 [L2NiNa]` 915.23 915.65 7 a 30 V cone voltage, 60 °C source temperature, 5]10~5 M solutions of metal salt and ligand in MeOH.Only the most intense peak in the isotope cluster is shown: in all cases good agreement was obtained between observed and calculated isotope distribution patterns. Fig. 4 Variation in emission intensity for 8 (293 K, 10 lM, 90% MeOH»10% 270 nm, nm) as a function of added H2O; jexc jem\309 zinc tri—uoromethanesulfonate, showing the –t (line) to the observed data for M~1.KML\1.3(^0.15)]104 provided by 31P NMR analysis.î That relatively little change occurs to the energy of the frontier (p) orbitals in the benzimidazole ring system on binding to zinc(II) was con–rmed by examination of the absorption spectrum. In the presence of a –ve-fold excess of (M\Zn, Co, Cu and Ni) in 90% M(ClO4)2 MeOH, the absorption spectrum in the region 250»290 nm underwent no signi–cant shifts in absorption maxima, compared to the free ligand spectrum.A 70]90% enhancement in the intensity of the higher energy transition at 210 nm was observed in each case, with a reduction in intensity in each band, in the region 250]280 nm.No time-dependence was observed over a period of 24 h and similar behaviour was shown by ligand 9. Electrospray mass spectrometry Speciation in methanolic solution was studied using ESMS,28 recording spectra at 1 : 1 M : L ratios over a range of sample concentrations from 10~4 to 10~5 M using metal perchlorate salts.With 9, more consistent results were obtained by examiî Under identical conditions the variation of emission intensity of the tetradentate bis(benzimidazole) 9 with added zinc showed a rather different dependence on added metal concentration. The —uorescence intensity reached a maximum at an M : L ratio of 0.5 : 1 and then remained constant. In this case, intermediate formation of an ML2 complex may also occur in which the zinc must be bound preferentially by 4 nitrogen donors: at higher added metal concentrations the ML complex will form but with no observable change in the —uorescence as the N-5 nitrogen remains bound.Such an interpretation is consistent with the 31P NMR study and the result of extraction experiments (see below). nation of the negative ion spectrum, and ML complexes were observed as perchlorate or chloride adducts (Cl~ arose from the hydrochloride salt of 9 used in these studies), with peak intensity falling in the series Zn[Cu[Ni (Table 1).No peaks were observed for complexes of Fe(III), under these conditions. For the monobenzimidazole ligand, 8, positive ion mass spectra were most informative and complexes were ML2 identi–ed as their proton or sodium adducts.The observed intensity of the species followed the order [ML2H]` Zn[Cu[Ni and again no complex species were observed in the presence of ferric ions. Liquidñliquid extraction studies In order to assess the ability of ligands 8 and 9 to transport metal ions across an aqueous»chloroform interface, radiolabelled metal salts were used and the distribution of metal ions in each phase measured at equilibrium by counting the solution activity (Table 2).Studies were carried out in microreaction vials with equal volumes of and a buÜered CHCl3 aqueous medium at pH 2 (298 K, NaOAc»HCl buÜer 0.1 M). The initial metal salt concentration in the aqueous phase was 10~4 M and the ligand concentration was varied from 0.25 to 5.0 mM. Equilibrium values were obtained within 30 min for and whereas a 2 h 64CuCl2, 65ZnCl2, 60Co(NO3)2 203HgCl2, incubation was used for experiments with and 59FeCl3 Even under these conditions, equilibrium was 63Ni(NO3)2 .not deemed to have been obtained for 8 with and in 59FeCl3 the extraction of by 9. Experiments with 63Ni(NO3)2 64CuCl2 were also carried out in the presence of a –fty-fold excess of the reducing agent hydroxylammonium sulfate, in order to generate the copper(I) complex in situ.Table 2 Estimated extraction constants using ligand 9 (298 K, pH 2, [Mn`]\0.1 mM)a CHCl3 , Metal ion log KML ex log KML2 ex Zn 4.1 6.3 Hg 3.0 6.1 Co 3.4 4.8 Fe(III) 2.7 6.9 Cu(I)b n.d. 11 a Ligand 8 was insufficiently soluble at higher ligand concentrations to allow reliable measurements of extraction constants, but with copper(I) and copper(II) complex formation occurred readily at sufficiently low ligand concentrations to allow estimates of extraction constants : 8/Cu(I) : log 8/Cu(II) : log log KML2 ex \8.7 ; KML ex \3.9, KML2 ex \ b For Cu(II), no extraction was observed i.e.log 8.6. KML\0.1. 822 New J. Chem., 1999, 23, 819»826Fig. 5 Percentage extractability of metal ions from an aqueous acidic phase (pH 2) to chloroform [298 K, 10~3 M ligand, 10~4 metal salt : front 8; rear 9. N.b. Cu(II) extraction by 9 is \0.1%]. The relative efficiency of ligands 8 and 9 to extract the different metal ion shows some striking diÜerences in behaviour (Fig. 5). Thus, the mono(benzimidazole), 8, extracted copper-(I) and -(II) and zinc(II) in preference to Hg(II) and virtually no extraction of nickel(II) and cobalt(II) occurred. With 9 , zinc(II) and copper(I) [and to a lesser extent Hg(II) and Co(II)] were extracted efficiently, but no measurable (\ 0.1%) extraction of copper(II) was found.Evidently the preference for a tetrahedral geometry in the 1 : 1 complex of 9 (when it acts as a tetradentate ligand) is being dramatically demonstrated by the failure to extract the copper(II) ion, which although adopting a range of coordination geometries in coordination numbers 4 to 6, tends to favour square planar or square pyramidal coordination geometries.On the other hand, copper(I), zinc(II) and cobalt(II) are known to form tetrahedral complexes in aqueous media, and even the kinetically sluggish Ni(II) ion is extracted to some extent (11%).Time dependent extraction was also noted for with 8, although 9 extracted it efficiently 59FeCl3 (99.5%). Information regarding the overall stoichiometry of complexation was obtained by measuring the ratio of cation concentrations in the organic»aqueous phase, as a function (DM) of total ligand concentration. From the variation of log DM with the logarithm of the ligand concentration, information on the stoichiometry (from the slope) and extraction affinity may be obtained (Fig. 6). In all cases at higher concentrations of 9 there was some evidence for the formation of com- ML2 plexes. In the case of copper(I), only an complex was ML2 observed with 9. The sequence of extraction constants for the 1 : 1 complexes of 9 follows the order Zn(II)[Co(II)[ consistent with the ligandœs preference to act as Ni(II)ACu(II), a tetradentate ligand, rigidly imposing a tetrahedral N2O2 coordination geometry at the metal. Competitive forma- ML2 tion with copper(I) and the observed affinity for the ferric ion, suggest that the ligand can also act as a bidentate donor, (N2) presumably via coordination of the N-7 nitrogen.Further work is therefore warranted using either acid-stable, lipophilic carbamate, arylsulfonyl, or amide derivatives (protecting the N-7 nitrogen site), or exploring the behaviour of the related Fig. 6 Variation of log with log[9] for the extraction of Hg2`, DM Zn2`, Co2` and Cu` (298 K; pH 2, [metal salt]\0.1 mM). CHCl3 ; series of indole ligands, which lack this alternative binding site.Conclusions The study of the solution complexation behaviour of ligands 8 and 9 has been aided by the complementarity of information deduced from 31P NMR, —uorescence and absorption spectroscopy, electrospray mass spectrometry and liquid»liquid extraction studies. The bidentate, monobasic ligand 8 forms complexes (31P NMR, ESMS, extraction data) with an ML2 affinity that follows the sequence Cu[Zn[HgACo[Ni.For the tetradentate ligand 9, 1 : 1 complex formation was consistent with all data obtained, and the complexes with Zn, Co and Ni probably involve ligation. No extraction of N2O2 copper(II) was observed with ligand 9. With copper(I), binding also involved an species but in this case the ligand may ML2 act as a bidentate donor, possibly via N-7 coordination.N2 Such a binding mode may also occur in the presence of excess ligand with other metal ions (e.g. FeIII) and in seeking to eliminate this competitive coordination, future work should perhaps target the behaviour of derivatives of 9 in which N-7 coordination may not occur. Experimental All reactions were carried out under argon in apparatus which had been oven dried.Basic alumina refers to Merck Alumina activity II-III, alumina refers to Merck alumina pre-soaked in ethyl acetate for at least 24 h prior to use and silica refers to Merck Kieselgel 230»400 mesh. 1H, 13C and 31P NMR spectra were obtained with a Bruker AC 250 operating at 250.13, 62.9 and 101.26 MHz respectively, a Varian Mercury 200 operating at 200, 63 and 81 MHz respectively ; proton and carbon spectra were also obtained using a Varian Unity 300 operating at 300 and 75 MHz or a Varian VXR 400s operating at 399.96 and 100.58 MHz respectively.Spectra are described in ppm to higher frequency of Sime4 with coupling constants, J in Hz. Infrared spectra were recorded on a Perkin-Elmer 1600 FT-IR spectrometer as KBr discs, neat –lms or using a Golden Gate accessory.Ultraviolet spectra were recorded using a Unicam 2 spectrometer and —uorescence emission spectra were recorded with a Perkin-Elmer LS50B spectro—uorimeter using FLwinlab as the data collection program. Mass spectra were recorded on a VG 7070E, operating in FAB, EI` or DCI ionisation modes as stated. Electrospray ionisation mass spectra were obtained on a VG platform II (Fisons Instruments), where major fragments are quoted as a percentage of the base peak intensity.Accurate mass spectroscopy was performed by the EPSRC Mass Spectroscopy service using FAB, EI` or DCI ionisation modes. Combustion analysis was performed using an Exeter Analytical Inc CE440 elemental analyser and metal concentration was determined by atomic absorption spectroscopy using a Perkin Elmer 5000 atomic absorption spectrophotometer.Melting points were determined on a Koé —er block melting point apparatus and are uncorrected. All solvents were dried by distillation from the appropriate drying agent and water was puri–ed by the PURITE system. NMR titrations NMR titrations were carried out in a 5 mm oven dried tube. A solution of the ligand was prepared (typically 0.04 M) in the deuteriated solvent (25% and 0.75 ml CD3OD»75% CDCl3) of the solution transferred to the tube by Gilson pipette.A solution of the metal tri—uoromethanesulfonate salt was also New J. Chem., 1999, 23, 819»826 823prepared in the deuteriated solvent (typically 0.4 M). The metal solution was then added in increments of known volume to the ligand solution and the shift of a given proton or phosphorus resonance monitored as a function of the M : L ratio.Typically the M : L ratios examined were 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0 : 1. The change in shift of the given resonance was then plotted against the M/L ratio and the binding isotherm analysed using standard least-squares –tting procedures to give an approximate value for the 1 : 1 formation constant, K.Fluorescence titrations Fluorescence titrations were carried out in a quartz cell ; stock solutions of the ligand (typically 1 mM) and metal perchlorate (10 mM) were prepared. For the pH titrations, 25 ml of a 0.01 mmol solution of the ligand was prepared in a 9 : 1 mixture of methanol and water.The pH was adjusted to approximately 1 with tri—uoracetic acid and sodium hydroxide solution (50 mM) was added to raise the pH. The pH of the solution was measured with a standard pH electrode. For the zinc titrations a 25 ml solution of the ligand (0.01 mM) was prepared in methanol. To this was added incremental amounts of zinc perchlorate solution (1 mM) so that the total volume change was less than 5%; typically M : L ratios of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0 : 1 were examined.Data were analysed by a least-squares –tting procedure operating in Microsoft Excel. Speciation analysis by electrospray mass spectrometry Stock solutions of the ligand and metal salts (tri—ate or perchlorate) were prepared (typically 1 mmol) in freshly distilled methanol (Aristar, MeOH). To a sample of the ligand solution (1 ml) and methanol (1 ml) was added the appropriate volume of metal tri—ate solution to make a 1 : 1 or 1 : 2 metal to ligand ratio.The resultant sample was diluted to give a –nal concentration of 1 to 10 lM. Mass spectra were obtained on a VG Platform (II) electrospray mass spectrometer, in positive or negative ionisation mode using a typical source temperature of 60 °C , a capillary voltage of 4 kV and a cone voltage of 30 V.A 10 ml sample was introduced into the —owing solvent using an injection valve with a 10 ml steel loop and transported to the electrospray capillary through a silica tube. LiquidñLiquid extraction Liquid»liquid extraction studies were carried out at the Technical University of Dresden.The extraction studies were performed at 25^1 °C in 2 ml micro-centrifuge tubes with mechanical shaking. Unless stated the shaking time was 30 min and the phase ratio was 1 : 1 (0.5 ml each). All V(org) : V(aq) samples were centrifuged after extraction. The determination of metal concentrations in both phases was carried out radiometrically using c-radiation measurement of 65ZnCl2 , and in a NaI(Tl) 64CuCl2 , 59FeCl3 , 60Co(NO3)2 203HgCl2 scintillation counter (Cobra II ; Canberra-Packard), and bradiation of in a liquid scintillation counter (Tricarb 63NiCl2 2500, Canberra-Packard).Aqueous 0.1 mM solutions of metal salts were prepared and the pH adjusted to 2.0 (^0.1) with NaOAc»HCl buÜer.Chloroform solutions of the ligands were prepared with a concentration of between 5 mM and 0.25 mM. For the extraction of copper(I), the procedure was carried out in the presence of a 50-fold excess of hydroxylammonium sulfate (0.05 M) Ligand synthesis N-Acetyl-2-bromo-6-nitroaniline. This was prepared by a modi–cation of the literature method:28 to a mixture of fuming nitric acid (6 ml), tri—uoroacetic acid (4.5 ml) and water (2 drops) was added N-acetyl-2-bromoaniline (4 g, 18.69 mmol) with gentle heating over a period of 30 min.The mixture was stirred at room temperature for 10 h and the reaction followed by TLC (silica, 1% methanol»99% dichloromethane, product\0.2). The mixture was poured onto Rf crushed ice (10 ml) and the brown solid collected by –ltration and washed with water (60 ml). Recrystallisation from chloroform gave the required isomer as white needle-shaped crystals (2.17 g, 45%), mp 193»194 °C (lit.,29 193 °C). 2.24 dH(CDCl3) (3H, s, 7.26 (1H, dd, H4), CH3), 3JH4H3\8.7, 3JH4H5\7.6, 7.71 (1H, br s, NH), 7.86 (1H, dd, 3JH3H4\8.7, 4JH3H5\1.4, H3), 7.91 (1H, dd, H5) 3JH5H4\7.6, 4JH5H3\1.4, 2-Bromo-6-nitroaniline. A 33% ethanol»hydrochloric acid mixture (50 ml; 6 M HCl) was added to N-acetyl-2-bromo-6- nitroaniline (4 g, 15.4 mmol) and the mixture heated under re—ux for 3.5 h.The mixture was poured onto a basic ice solution (10 ml) and the pH adjusted to 14 by addition of NaOH pellets. The resultant solid was collected by –ltration, washed with water and dried under vacuum to give a bright yellow crystalline solid which was used without further puri–cation (3.35 g, 100%), mp 74»75 °C (lit.,30 73»74 °C). 5.72 dH (CDCl3) (1H, br s, NH), 6.62 (1H, br s, NH), 6.61 (1H, dd, 3JH4H3\8.8, H4), 7.69 (1H, dd, 3JH4H5\12.1, 3JH4H5\12.1, 4JH5H3\1.1, H5), 8.14 (1H, dd, H3) 3JH3H4\8.8, 4JH3H5\1.1, 1,2-Diamino-3-bromobenzene 11. To a solution of tin(II) chloride dihydrate (8.36 g) in concentrated hydrochloric acid (43 ml) was added 2-bromo-6-nitroaniline (2.19 g, 8.45 mmol) and the mixture stirred at room temperature for 5 min.The mixture was heated under re—ux for 30 min, poured onto crushed ice and the solution made basic (pH 14, NaOH pellets) and the aqueous layer extracted exhaustively with diethyl ether (5]100 ml). The organic fractions were combined, dried and the solvent removed under reduced (K2CO3) pressure to give a pale yellow crystalline product (1.85 g, 96%), mp 52»53 °C (lit.,19 52»54 °C). 6.38 (1H, dH (CD3OD) dd, H5), 6.57 (1H, d, 3JH5H4\9.2, 3JH5H6\7.9, 3JH6H5\7.9, H6), 6.76 (1H, d, H4). 3JH4H5\9.2, 4-Bromo-2-methylbenzimidazole31 12a. To 1,2-diamino-3- bromobenzene (2.5 g, 13.37 mmol) was added acetic anhydride (15 ml) and the solution heated at 110 °C for 2 h.The mixture was left to cool to room temperature and water (20 ml) was added and the solution heated at 60 °C for 1 h. Hydrochloric acid (3M, 20ml) was added and the mixture heated at 100 °C for a further 2 h. Activated carbon (500 mg) was added, and the solution –ltered through a Celite plug. The aqueous –ltrate was made basic (ammonia 0.880 solution) and extracted with dichloromethane (4]100 ml), dried and the (K2CO3) volatile organics removed under reduced pressure to give an oÜ-white solid (2.82 g, 89%), mp 136»138 °C (lit.,31 137 °C).dH 2.65 (3H, s, 7.08 (1H, dd, (CDCl3) CH3), 3JH6H5\7.6, H6), 7.38 (1H, d, H7), 7.46 (1H, d, 3JH6H7\7.9, 3JH7H6\7.6, H5). 3JH5H6\7.9, 4-Bromo-1-(4-tert-butylbenzyl)-2-methylbenzimidazole 12b. Dimethylformamide (15 ml) was added to a mixture of 4- bromo-2-methylbenzimidazole (2.5 g, 11.84 mmol) and caesium carbonate (4.24 g, 13.03 mmol) and the mixture stirred under argon for 2 h. 4-(tert-Butyl)benzyl bromide (2.93 ml, d\1.236, 13.03 mmol) was added and the mixture heated at 40 °C for 12 h. After cooling, the DMF was removed by vacuum distillation, and the residue taken up in dichloromethane (15 ml), washed with water (3]20 ml), dried and the dichloromethane reduced to minimal (K2CO3) volume (1 ml) followed by precipitation of the product with 824 New J.Chem., 1999, 23, 819»826diethyl ether (10 ml). This precipitate was –ltered oÜ and dried to yield an oÜ-white solid (4.01 g, 95%), mp 134»135 °C. dH 1.25 (9H, s, tBu), 2.57 (3H, s, 6.91 (2H, d, (CDCl3) CH3 ), H10), 7.01 (1H, dd, 3JH10H11\2.6, 3JH6H7\8.1, 3JH6H5\7.6, H6), 7.14 (1H, d, H7), 7.28 (2H, d, 3JH7H6\8.1, 3JH11H10\ H11, 7.38 (2H, d, H5). 16.2 2.6, 3JH5H6\7.6, dC (CDCl3) 33.3 (tBu), 36.56 (C2), 49.3 110.8, 114.5, 125.2, (CH3), (CH2), 127.0, 127.9, 128.0, 134.3, 137.9, 153.2, 154.8 ; m/z (EI), 356 (51%, M`), 358 (49%, M`) ; Found: M` 356.0888. requires M` 356.0888. Found: C, 63.62 : H, C19H21N2Br 5.98 : N, 7.58.requires C, 63.87 : H, 5.92 : N, C19H21N2Br 7.58%. Ethyl 1-(4-tert-butylbenzyl)-2-methylbenzimidazol-4-yl- (phenyl)phosphinate. Using a procedure adapted from Huang,21 4-bromo-1-(4-tert-butylbenzyl)-2-methylbenzimidazole (300 mg, 0.84 mmol), ethyl phenylphosphinate (0.14 ml, 0.924 mmol) and triethylamine (0.39 ml, 2.77 mmol) were mixed in dry degassed toluene (5 ml).Tetrakis(triphenylphosphine) palladium(0) (46 mg, 0.04 mmol) was added and the mixture degassed three times (freeze»thaw cycle), then heated at 100 °C for 96 h. The solution was diluted with dichloromethane (10 ml), washed with 5% aqueous hydrochloric acid (2]10 ml) and water (3]20 ml), dried and the (K2CO3) solvent removed under reduced pressure to give an oÜ-white solid.Puri–cation by column chromatography [alumina, eluant 2% MeOH»98% dichloromethane increasing to 8% MeOH»92% dichloromethane, product\0.38 (8% Rf MeOH»92% dichloromethane)] gave a white solid (233 mg, 62%), mp 56»57 °C. 1.19 [9H, s, 1.32 dH (CDCl3) C(CH3)3], (3H, t, 3J\7.2, 2.52 (3H, s, 4.09 (2H, qd, OCH2CH3), 2-CH3), 3J\7.2, 5.18 (2H, s, 6.91 2JHP\2.2, OCH2CH3), NCH2Ar), (2H, d, H11), 7.01 (2H, ddd, 3JH11H10\8.4, 3JHmPhHoPh\7.4, HmPh), 7.12 (2H, d, 3JHmPhHpPh\6.9, 4JHmPhP\3.6, H10), 7.25 (1H, dd, 3JH10H11\8.4, 3JHpPhHmPh\6.9, HpPh), 7.31 (1H, ddd, 5JHpPhP\7.4, 3JH6H7\8.2, 3JH6H5\ H6), 7.47 (1H, d, H7), 7.79 (2H, 7.4, 4JH6P\2, 3JH7H6\8.2, dd, HoPh), 7.99 (1H, dd, 3JHoPhHmPh\7.4, 3JHoPhP\13.1, H5), 14.1 3JH5H6\7.4, 3JH5P\12.9, dC (CDCl3) [C(CH3)3], 16.5 (d, 31.1 34.4 3JCP\7.6, OCH2CH3), [C(CH3)3], 46.7 (2- 61.3 (d, (NCH2Ar), CH3), 2JCP\7.1, OCH2CH3), 113.7 (d, C6), 120.6 (d, CiPh), 121.4 (d, 3JCP\3.5, 1JCP\160, C5), 125.8 (C11), 125.9 (C12), 127. 4 (d, 2JCP\15.5, 4JCP\9.7, C7), 127.9 (d, CmPh), 131.6 (d, C8), 3JCP\16, 3JCP\3.4, 131.86 (d, CoPh), 132.2 (d, C4), 135.6 (d, 2JCP\13, 3JCP\168, CpPh), 143.2 (d, C9), 150.9, 153.1 (C2). 4JCP\15, 2JCP\12, dp 31.3. m/z (ES) 447.15 (100%, LH`). (KBr ) 3439, (CDCl3) tmax 2961, 1602, 1438, 1419, 1209, 1035 cm~1. Found: M` 446.2123. requires M` 446.2123. Found: C, C27H31N2PO2 72.83 : H, 7.16 : N, 6.35. requires C, 72.62 : H, C27H31N2PO2 6.99 : N, 6.27%. 1-(4-tert-Butylbenzyl)-2-methylbenzimidazol-4-yl(phenyl)- phosphinic acid hydrochloride 8. To ethyl 1-(4-tert-butylbenzyl)- 2-methylbenzimidazole-4-yl(phenyl)phosphinate (200 mg, 0.45 mmol) was added hydrochloric acid (6 M, 10 ml) and the mixture heated at 110 °C for 16 h.After cooling, the acid was removed under reduced pressure to give a white solid (202 mg, 99%), mp 172»174 °C. 1.25 (9H, s, tBu), 3.10 (3H, dH (CD3OD) s, 5.67 (2H, s, 7.02 (1H, dd, 2-CH3), NCH2Ar), 3JH6H7\8.4, H6), 7.09 (2H, d, H10), 7.30 (1H, 3JH6H5\8.4, 3JH10H11\8.3, dd, HpPh), 7.32 (2H, d, 3JHmPhHpPh\7.9, 4JHpPhP\7.8, H11), 7.50 (2H, ddd, 3JH11H10\8.3, 3JHmPhHoPh\7.2, HmPh), 7.59 (1H, d, 3JHmPhHpPh\7.9, 4JHmPhP\3.2, 3JH5H6\ H5), 7.17 (1H, d, H7), 8.07 (2H, dd, 8.4, 3JH7H6\8.4, HpPh). 22.6 3JHpPhHmPh\7.2, 5JHpPhP\12.4, dC (CD3OD) 31.2 34.6 (2- 48.5 [C(CH3)3], [C(CH3)3], CH3), (NCH2Ar), 114.6 (C2), 124.1 (C8@), 125.3 (d, CoPh), 126.3 2JCP\12.6, (C10), 126.6 (C11), 128.5 (d, CmPh), 129.1, 129.8.JCP\13.3, 129.8 (d, CiPh), 132.2, 132.3, 132.3 (d, 1JCP\143, 1JCP\132, C4), 152.1, 152.5. 28.0. m/z (ES) 419.29 (100%, dp (CD3OD) LH`). (KBr) 3418, 2958, 2864, 1678, 1546, 1544, 1424, tmax 1218, 1132 cm~1. Found: MH` 419.1888, C25H28N2PO2 requires MH` 419.1888. 4,4º-Dibromo-2,2º-propane-1,3-diyldi(1H-benzimidazole) 13a.To a mixture of 1,2-diamino-3-bromobenzene (730 mg, 3.90 mmol) and dimethyl glutarate (313 mg, 1.95 mmol) was added polyphosphoric acid (10 ml) and the mixture heated at 180 °C for 18 h. The black solution was allowed to cool to 100 °C and poured in a thin stream into well stirred water (100 ml), and stirring continued for 1 h. The mixture was –ltered, the pH adjusted to 8 by addition of NaOH solution (3 M) and the resultant dark solid was collected by –ltration and washed with water (2]10 ml).The solid was dissolved in methanol (50 ml) and treated with activated charcoal at 80 °C for 30 min, –ltered through Celite and solvent removed under reduced pressure to give an orange solid (500 mg, 59%), mp 85»87 °C. 2.34 (2H, t, 3J\6.7, 3-propyl dH (CDCl3) CH2), 3.05 (4H, t, 3J\6.7, 2-propyl 7.13 (2H, dd, CH2), 3JH6H7\ H6), 7.44 (2H, d, H7), 7.53 (2H, 8.0, 3JH6H5\7.9, 3JH7H6\8.0, d, H5). 18.4 36.2 107.8 3JH5H6\7.9, dC (CDCl3) (CH2), (CH2), (C-Br), 115.0, 131.1, 133.5, 134.5, 141.6 (C2). m/z (ES) 434.32 (45%, LH`), 435.54 (100%, LH`), 436.71 (51%, LH`). tmax (KBr) 3134, 2928, 2760, 1652, 1536, 1422, 1215, 1190, 1048, 932 cm~1.Found: C, 47.36 ; H, 3.62 ; N, 12.45. C17H14N4Br2 requires C, 47.03 ; H, 3.25 ; N, 12.90%. 4,4º-Dibromo-1,1º-di(tert-butylbenzyl)-2,2º-propane-1,3- diyldi(1H-benzimidazole) 13b. Dimethylformamide (15 ml) was added to a mixture of 4,4@-dibromo-2,2@-propane-1,3-diyldi( 1H-benzimidazole) (920 mg, 2.12 mmol) and caesium carbonate (1520 mg, 4.66 mmol, 2.2 equiv.) and the mixture stirred under argon for 2 h. 4-(tert-Butyl)benzyl bromide (0.86 ml, d\1.236, 4.66 mmol, 2.2 equiv.) was added and the mixture heated at 80 °C for 16 h. The DMF was removed by vacuum distillation, and the residue taken up into dichloromethane (40 ml), washed with water (3 40 ml), dried (K2CO3) and the dichloromethane reduced to a minimal volume (1 ml) followed by precipitation of the product with diethyl ether (5 ml).This yielded a pale orange solid (1477 mg, 96%), mp 79» 80 °C. 1.23 (18H, s, tBu), 2.44 (2H, t, 3J\6.6, 2- dH (CDCl3) propyl 2.95 (4H, t, 3J\6.6, 1- and 3-propyl 5.38 CH2), CH2), (4H, s, 7.21 (4H, d, H10), 7.33 (2H, dd, CH2), 3JH10H11\2.9, H6), 7.45 (2H, d, 3JH6H7\8.1, 3JH6H5\7.6, 3JH7H6\8.1, H7), 7.61 (2H, d, H11), 7.69 (4H, d, 3JH11H10\2.9, 3JH5H6\ H5). 16.1 18.0, 33.2 (tBu), 36.5, 49.2 7.6, dC (CDCl3) (CH3), 110.8, 114.5, 125.1, 126.9, 127.9, 127.9, 134.3, 137.9, (CH2), 153.1, 154.8. m/z (ES) 724.51 (55%, LH`), 726.33 (100%, LH`), 727.27 (60%, LH`). (KBr) 3388, 2954, 1894, 1732, tmax 1628, 1532, 1426, 1274, 1182, 1064, 924, 738 cm~1. Found: C, 64.2 ; H, 5.95 ; N, 7.79. requires C, 64.5 ; H, 5.79 ; C39H42N4Br2 N, 7.71%. 1,1º-Di(tert-butylbenzyl)-4,4º-di [ ethoxy(phenyl)phosphonyl ] - 2,2º-propane-1,3-diyldi(1H-benzimidazole) 13c.Using a procedure adapted from Huang,21 4,4@-dibromo-1,1@-di(tert-butylbenzyl)- 2,2@-propane-1,3-diyldi(1H-benzimidazole) (300 mg, 0.41 mmol), ethyl phenylphosphinate (0.14 ml, 0.87 mmol, 2.1 equiv.) and triethylamine (1 ml) were mixed in dry degassed toluene (3 ml). Tetrakis(triphenylphosphine)palladium(0) (10 mg) was added and the mixture degassed three times (freeze» thaw cycle), then heated at 125 °C for 48 h.The solution was diluted with dichloromethane (20 ml), washed with hydrochloric acid (1 M, 2]20 ml) and water (3]20 ml), dried and the solvent removed under reduced pressure to (K2CO3) give an oÜ-white solid. Puri–cation by column chromatography [silica gel, eluant 100% dichloromethane increasing to New J.Chem., 1999, 23, 819»826 8258% MeOH»92% dichloromethane, product\0.2 (8% Rf MeOH»92% dichloromethane)] gave a light orange solid (230 mg, 62%), mp 64»66 °C. 1.13 [18H, s, dH (CDCl3) C(CH3)3], 1.29 (6H, t, 3J\7.2, 2.41 (2H, t, 3J\6.6, 2- OCH2CH3), propyl 2.94 (4H, t, 3J\6.6, 3-propyl 4.09 (4H, CH2), CH2), qd, 3J\7.2, 4.97 (4H, s, 2JHP\2.0, OCH2CH3), NCH2Ar), 6.61 (4H, d, 3J\8.0, H10), 6.90 (4H, d, 3J\8.0, H11), 7.22 (2H, ddd, H6), 7.46 (4H, 3JH6H7\7.8, 3JH6H5\7.9, 4JH6P\2, ddd, HmPh), 3JHmPhHoPh\8.0, 3JHmPhHpPh\7.5, 4JHmPhP\3.6, 7.62 (2H, dd, HpPh), 7.56 (2H, 3JHpPhHmPh\7.5, 5JHpPhP\8.0, d, H7), 7.83 (4H, dd, 3JH7H6\7.8, 3JHoPhHmPh\8.0, 3JHoPhP\ HoPh), 8.16 (2H, dd, H5). 13.5, 3JH5H6\7.9, 3JH5P\13.6, dC 14.3 16.7 (d, 29.0, (CDCl3) [C(CH3)3], 3JCP\7.6, OCH2CH3), 31.2 34.6 46.7, 61.2 (d, [C(CH3)3], (NCH2Ar), 2JCP\7.0, 114.0 (d, C6), 120.9 (d, OCH2CH3), 3JCP\3.1, 1JCP\162.5, CiPh), 121.7 (d, C5), 126.1 (C11), 126.1 (C12), 2JCP\16.0, 127. 7 (d, C7), 128.2 (d, CmPh), 131.9 4JCP\9.8, 3JCP\16.5, (d, C8), 132.3 (d, CoPh), 132.4 (d, 3JCP\3.8, 2JCP\13.3, C4), 135.8 (d, CpPh), 143.5 (d, 3JCP\169, 4JCP\15.5, 2JCP\ C9), 151.2, 153.3 (C2). 29.2. m/z (ES) 905.31 12.8, dP (CDCl3) (100%, LH`). (KBr) 3420, 2962, 2868, 1725, 1709, 1690, tmax 1483, 1270, 1112, 1044, 1012, 830, 746, 666 cm~1. Found: MH` 905.4325. requires MH` 905.4324. C55H63N4P2O4 1,1º-Di(tert-butylbenzyl)-4,4º-di [ hydroxy(phenyl)phosphoryl ] -2,2º-propane-1,3-diyldi(1H-benzimidazole) dihydrochloride 9. To a solution of 1,1@-di(tert-butylbenzyl)-4,4@-di[ethoxy- (phenyl)phosphonyl]-2,2@-propane-1,3-diyldi(1H-benzimidazole) (100 mg, 0.11 mmol) in 1,4-dioxane (6 ml) was added concentrated hydrochloric acid (10 M, 4 ml).The solution was heated at 110 °C and the reaction followed by 31P NMR [dP (starting material)\29.22, (product)\16.44] until the dP reaction was complete (ca. 20 h). The solvent was removed under reduced pressure to give a pale orange solid (92 mg, 99%), mp 173»175 °C. 1.23 (18H, s, tBu), 2.01 dH (CD3OD) (2H, t, 3J\6.8, 2-propyl 2.65 (4H, t, 3J\6.8, 1- and CH2), 3-propyl 5.52 (4H, s, 7.35 (1H, dd, CH2), NCH2), 3JH6H7\8.5, H6), 7.09 (2H, d, H10), 7.30 (1H, 3JH6H5\8.3, 3JH10H11\8.3, dd, HpPh), 7.32 (2H, d, 3JHpPhHmPh\7.7, 5JHpPhP\7.2, H11), 7.62 (2H, ddd, 3JH11H10\8.3, 3JHmPhHoPh\7.1, HmPh), 7.72 (1H, d, 3JHmPhHpPh\7.7, 4JHmPhP\3.2, 3JH5H6\ H5), 7.89 (1H, d, H7), 8.23 (2H, dd, 8.3, 3JH7H6\8.5, HpPh). 22.6 3JHpPhHmPh\7.7, 5JHpPhP\12.0, dC (CD3OD) 28.0, 31.1 34.6, 48.8 114.9 [C(CH3)3], [C(CH3)3], (NCH2Ar), (C2), 124.3 (C8@), 125.5 (d, C3@), 126.5 (C10), 126.9 2JCP\12.2, (C11), 128.7 (d, 129.3, 130.0. 130.1 (d, JCP\13.8), 1JCP\ 132.5, 132.6, (d, 152.3, 152.8 ; 153.6), 1JCP\130.8), dP 16.4.m/z (ES) 847.23 (100%, M~). (KBr) 3316, (CD3OD) tmax 2966, 2924, 2860, 1852, 1725, 1709, 1690, 1648, 1512, 1258, 1140, 1090, 830, 794 cm~1. Found: MH` 848.3621. requires MH` 848.3620. Found: C, 71.8 ; H, C51H55N4P2O4 6.54 ; N, 5.68. requires C, 72.1 ; C51H55N4P2O4 … 2HCl … 2H2O H, 6.37 ; N, 5.85%. Acknowledgements thank EPSRC for support under the CASE scheme, the We Royal Society for a Leverhulme Trust Senior Research Fellowship (DP), and Professor Peter Tasker, Dr Domenico Cupertino and Dr Troy Leese (Zeneca Specialties, Blackley, UK) for support under the CASE scheme.References 1 C. K. Gupta and T. K. Mukherjee, Hydrometallurgy in Extraction Processes, CRC Press, Boca Raton, FL 1990, vol. 1, pp 1, 2, 35, 56. 2 C. B. Alcock, Principles of Pyrometallurgy, Academic Press, London, 1970. 3 E. D. Nogueira, J. M. RegiÜe and P. M. Blythe, Chem. Ind., 1980, 63; C. I. Sainzdiaz, H. Klocker, R. Marr and H. J. Bart, Hydrometallurgy, 1996, 42, 1. 4 N. Miralles, A. M. Sastre, M. Aguillar and M. Cox, Solv. Extr. Ion Exch., 1992, 10, 51. 5 N. B. Devi, K. C. Nathsarma and V. Chakravortty, Hydrometallurgy, 1997, 45, 169; S.Amer and R. Luis, Rev. Metal. (Madrid), 1995, 31, 351. 6 A. Davison and E. S. Switkes, Inorg. Chem., 1971, 10, 837; M. R. Churchill, J. Cooke, J. P. Fennessey and J. Wormald, Inorg. Chem., 1971, 10, 1031; H. Groeger and A. Schmidtpeter, Chem. Ber., 1967, 100, 3216. 7 G. B. Bates and D. Parker, J. Chem. Soc., Perkin T rans. 2, 1996, 1109. 8 G. B. Bates, D. Parker and P.A. Tasker, J. Chem. Soc., Perkin T rans. 2, 1996, 1117. 9 G. B. Bates and D. Parker, T etrahedron L ett., 1996, 37, 267. 10 G. B. Bates, E. Cole, D. Parker and R. Kataky, J. Chem. Soc., Dalton T rans., 1996, 2693. 11 S. Wigetors and J. Rydberg, Acta Chem. Scand, Ser. A, 1980, 34, 313. 12 D. P. Devonald, A. J. Nelson, P. M. Quan and D. Steward, Eur. Pat., 0196153B1 1986. 13 R.F. Dalton, A. Burgess and P. M. Quan, Hydrometallurgy, 1992, 30, 385. 14 C. J. Matthews, T. A. Leese, D. Thorp and J. C. Lockhart, J. Chem. Soc., Dalton T rans., 1998, 79 and refs. therein. 15 C. J. Matthews, W. Clegg, S. L. Heath, N. C. Martin, M. N. S. Hill and J. C. Lockhart, Inorg. Chem., 1998, 37, 199. 16 C. D. Edlin and D. Parker, T etrahedron L ett., 1998, 39, 2797. 17 Molecular modelling of the and ML zinc complexes of 8 and ML2 9 was performed using the Sybyl 6.3 programme (Tripos UK Ltd.) at Zeneca Specialties, Blackley.Charge distributions were calculated using MNDO routines and the structures minimised with the AM-1 force –eld. With 8, a methyl substituent at C-2 sterically inhibited the formation of square-planar or octahedral complexes when the ligand was constrained to act as an N, O bidentate chelate : attempts to place the ligand in a square planar or trigonal bipyramidal coordination geometry resulted in an energyminimised tetrahedral complex. With the ML complex of 9, the C3 chain only adopted an ideal gauche conformation when the metal was tetrahedral with all four donors binding cooperatively. (N2O2) 18 C. D. Edlin, Ph.D. Thesis, University of Durham, 1998. 19 S. Sanders and N. P. Peet, J. Heterocycl. Chem., 1979, 16, 33; T. Neilson, H. C. S. Wood and A. G. Wylie, J. Chem. Soc., 1962, 371. 20 M. A. Phillips, J. Chem. Soc., 1953, 1143. 21 Y. Xu, Z. Li, J. Xia, H. Guo and Y. Huang, Synthesis, 1983, 377. 22 B. A. Koshits-Porai, O. F. Ginsburg and L. F. Efros, Zh. Obshch. Khim., 1949, 1545. 23 A. Job, Ann. Chim. (Paris), 1928, 9, 113; K. A. Connors, Binding Constants, T he Measurement of Molecular Complex Stability, Wiley, New York, 1987. 24 QCPE, Indiana University, Bloomington, IN, USA. 25 C. Reichardt, Solvents and Solvent EÜects in Organic Chemistry, VCH, Weinheim, 1988, p. 365. 26 D. E. C. Corbridge, Phosphorus: An Outline of its Chemistry, Biochemistry and T echnology, Elsevier, Amsterdam, 4th edn., 1990, ch. 4 and 7. 27 R. M. Smith and A. E. Martell, Critical Stability Constants, Plenum, New York, 1974, vol. 1»6, 1986. 28 Recent studies using ESMS support the useful qualitative appreciation of solution complexation that may be obtained: M. Goodall, P. M. Kelly, D. Parker, K. Gloe and H. Stephan, J. Chem. Soc., Perkin. T rans. 2, 1997, 59; E. Leize, A. JaÜrezic and A. Van Dorsselaer, J. Mass Spectrom., 1996, 31, 537. 29 J. Gibson and B. Johnson, J. Chem. Soc., 1928, 3093. 30 F. H. Jackson and A. T. Peters, J. Chem. Soc. C, 1929, 268. 31 S. H. Dandegaonker and I. Recakar, J. Karnatak Univ. Sci., 1961, 6, 25. Paper 9/03534F 826 New J. Chem., 1999, 23, 819»826

ISSN:1144-0546    

DOI:10.1039/a903534f

出版商:RSC    

年代:1999

数据来源: RSC

摘要:

Asymmetric oxidations at sulfur catalyzed by engineered strains that overexpress cyclohexanone monooxygenase§ Gang Chen,a Margaret M. Kayser,*a Marko D. Mihovilovic,îa Megan E. Mrstik,b Carlos A. Martinezb and Jon D. Stewart*b a Department of Physical Sciences, University of New Brunswick, P.O. Box 5050, Saint John, NB E2L 4L 5, Canada b Department of Chemistry, University of Florida, Gainesville, FL 32611, USA.Fax: ]1 (352) 846-2095; e-mail jds2=chem.u—.edu Received (in Gainesville, FL) 15th March 1999, Accepted 2nd June 1999 Recombinant strains of bakerœs yeast (Saccharomyces cerevisiae) and Escherichia coli expressing cyclohexanone monooxygenase from Acinetobacter sp. NCIB 9871 have been used as whole-cell biocatalysts for oxidations of several sul–des, dithianes and dithiolanes to the corresponding sulfoxides.The enantio- and diastereoselectivities of these reactions compare favorably with oxidations catalyzed by the puri–ed monooxygenase or the parent microorganism (a class II pathogen). The facility of handling yeast reactions makes these biotransformations an attractive alternative route to optically pure sulfoxides. Methods that exploit the ability of chiral sulfoxides to control the stereochemical outcome of reactions at nearby centers have become important additions to the repertoire of organic synthesis.1h7 All of these strategies depend on access to homochiral sulfoxides and a variety of methods for their synthesis have been described.8 Several useful sulfoxides are accessible in high optical purities by chemical oxidations followed by diastereomeric separation.9 The direct asymmetric oxidations of prochiral thioethers provide a more direct route, but these reactions often display a variable degree of enantiomeric enrichment that is highly dependent on the nature of the substrate. 10h16 These limitations, coupled with a desire to develop ecologically more benign processes, have inspired a search for bioorganic oxidation methods.A large number of microorganisms17h19 including bakerœs yeast20 catalyze oxidations at sulfur. These biooxidations, however, have their own problems. Substrate acceptability is often limited and enantioselectivity can vary dramatically with substrate structure. Moreover, isolated redox enzymes require cofactors that must be regenerated for preparative-scale reactions and this adds to both the cost and complexity of these processes.Thus, there remains a need for simple, general oxidants that provide homochiral sulfoxides in high chemical and optical yields with minimal environmental impact. Since the pioneering work by Walsh and co-workers,21 Acinetobacter sp. NCIB 9871 cyclohexanone monooxygenase has been shown to accept a wide variety of thioethers and provide good yields of the corresponding sulfoxides.22h31 Unfortunately, a variety of practical difficulties have limited the appeal of this enzyme to those with the expertise to handle the pathogenic Acinetobacter strain grown in the presence of organic inducers, purify the protein and set up a regeneration system for the essential NADPH cofactor.32 § Supplementary material available : experimental details of the construction of an E.coli overexpression plasmid for cyclohexanone monooxygenase. For direct electronic access see http ://www.rsc.org/ suppdata/nj/1999/827/, otherwise available from BLDSC (No. SUP 57581, 5 pp.) or the RSC Library. See Instructions for Authors, 1999, Issue 1 (http ://www.rsc.org/njc). î Present address : Vienna University of Technology, Institute for Organic Chemistry, Getreidemarkt 9, A-1060,Vienna, Austria. We have recently described a recombinant strain of bakerœs yeast (Saccharomyces cerevisiae) that overexpresses Acinetobacter sp.cyclohexanone monooxygenase.33 These cells are non-pathogenic, simple to handle and monooxygenase production is induced by adding galactose to the growth medium.Whole cells of this ììdesigner yeast œœ strain have been used in place of the puri–ed enzyme to carry out Baeyer»Villiger oxidations and provide a variety of chiral d- and e-lactones, usually in optical purities [95%.34h36 The growing yeast cells provide a constant supply of the enzyme and the NADPH cofactor, which dramatically simpli–es the process.37 Moreover, these oxidations do not require specialized equipment or training.In the case of Baeyer»Villiger oxidations where comparisons were possible, the yields and optical purities of the products from yeast-mediated reactions were virtually identical to those obtained from reactions utilizing the puri–ed enzyme.38 Given the success of our ììdesigner yeastœœ in catalyzing asymmetric Baeyer»Villiger oxidations and the demonstrated ability of cyclohexanone monooxygenase to oxidize prochiral thioethers, we have applied the same yeast strain to synthesize chiral sulfoxides and these eÜorts are described here.Our results have shown that the engineered yeast strain can carry out asymmetric thioether oxidations ; however, better results are sometimes obtained by using a strain of Escherichia coli that overexpresses the same enzyme.This latter reagent avoids side-reactions catalyzed by yeast enzymes and aÜords higher yields in some cases. Results and discussion Oxidations of acyclic thioethers by a bakerœs yeast strain expressing cyclohexanone monooxygenase Phenyl methyl sul–de 1a was oxidized by our engineered yeast strain to aÜord the optically pure sulfoxide 2a in 95% yield with [99% ee (Scheme 1).This result is comparable to that obtained from the oxidation using puri–ed cyclohexanone monooxygenase and an NADPH regeneration system22 although no enzyme isolation or cofactor regeneration is required for reactions involving the engineered yeast strain. The optical purity of 2a was determined by chiral-phase HPLC and its absolute con–guration was assigned from its New J.Chem., 1999, 23, 827»832 827Scheme 1 optical rotation and comparison to literature values.15 Likewise, tert-butyl methyl sul–de 1b was oxidized to the corresponding (R)-sulfoxide 2b in 99% ee as determined by chiral-phase GC and optical rotation values. The initially low isolated yield from this reaction was improved signi–cantly (from 11% to 47%) by the use of a sealed —ask to minimize substrate loss by evaporation.Using these conditions, n-butyl methyl sul–de 1c was converted to 2c in 53% isolated yield and 74% ee. In other cases, however, attempts to use our engineered yeast strain to eÜect thioether oxidations were less successful. 39 For this reason and because a recombinant E. coli strain expressing a bacterial dioxygenase has been used successfully in arene40 and sul–de oxidations,41 we elected to create an efficient E.coli expression system for cyclohexanone monooxygenase and evaluate its potential for sulfoxide synthesis. An efficient E. coli overexpression system for cyclohexanone monooxygenase We constructed an E. coli overexpression plasmid for cyclohexanone monooxygenase using standard techniques (Fig. 1).42 The polymerase chain reaction was –rst used to correct a three base pair deletion present in the 5@ region of our originally cloned gene.33 Fortunately, this alteration in the Nterminal amino acid sequence did not appear to aÜect the catalytic activity or enantioselectivity of the protein expressed from the mutant gene.38 A subsequent polymerase chain reaction incorporated a restriction site (NdeI) required to insert the gene encoding cyclohexanone monooxygenase into E.coli expression plasmid pET 22b(]). In this –nal construct, designated pMM4, protein expression is driven by the strong T7 promoter that can be controlled by adding isopropylthio-b-Dgalactoside to the culture medium. This plasmid was used to transform E.coli BL21(DE3) to create the –nal strain [BL21(DE3)(pMM4)] that overexpresses cyclohexanone monooxygenase at a level of ca. 20% of total protein. This quantity is approximately –ve-fold higher than the previous E. coli overexpression strain43 and ten- to twenty-fold higher than typical expression levels in S. cerevisiae.44 The recombinant E. coli strain is a very efficient catalyst for asymmetric Baeyer»Villiger oxidations and these results will be described in detail elsewhere. Fig. 1 Partial restriction map of pMM4. This plasmid directs the efficient synthesis of Acinetobacter sp. cyclohexanone monooxygenase when present in E. coli BL21(DE3). The CHMO gene is transcribed by T7 RNA polymerase and the protein accumulates in soluble form in the cytoplasm.The bla gene encodes b-lactamase to confer resistance to ampicillin and the lacI gene product is involved in regulating monooxygenase expression. Oxidations of dithianes using engineered bakerœs yeast and E. coli strains Colonna and co-workers have used puri–ed cyclohexanone monooxygenase along with an NADPH regeneration system to oxidize dithiane 3a to (R)-sulfoxide 5a in 81% yield and [98% ee along with a 19% yield of sulfone 7a (Scheme 2; Table 1).25 The high optical purity of 5a was due to both the enzymeœs preference in the oxidation of 3a and subsequent enantioselective oxidation of (S)-5a, also catalyzed by cyclohexanone monooxygenase.45 Similar results were obtained when Furstoss and co-workers used whole Acinetobacter sp.NCIB 9871 cells in place of puri–ed cyclohexanone monooxygenase (Table 1).28 On the other hand, when our engineered yeast cells expressing cyclohexanone monooxygenase were grown in the presence of 3a, sulfoxide 5a was isolated in only 18% yield (Table 1).A control experiment in which the unmodi–ed yeast cells were grown in the presence of 3a revealed that native yeast enzymes were also capable of forming both sulfoxide and sulfone products.To avoid this competition, the E. coli strain that overexpresses cyclohexanone monooxygenase was used to oxidize 3a to the (R)- sulfoxide in 73% isolated yield. Loss of the volatile substrate and/or product by evaporation, either during the oxidation or during workup, likely accounts for the slightly depressed yield of 5a relative to the amount expected from GC analysis.This oxidation was complete within 20 h and only a small amount of starting material and sulfone were present at the end of the reaction (Fig. 2). The absolute con–guration of 5a was assigned by comparing the sign of its optical rotation to literature values. Given the results of Colonna et al.,25 it is likely Scheme 2 828 New J. Chem., 1999, 23, 827»832Table 1 Biocatalytic oxidations of dithianes Sulfoxide 5 Sulfone 7 Substrate Catalyst 3 : 5 : 7a trans : cis Yieldb (%) % ee Yieldb (%) % ee Reference 3a Isolated enzyme 0 : 81 : 19 »c 81 [98 19 » 25 Acinetobacter N.R.d » 76 98 20 » 28 S.cerevisiae 15C 82 : 1 : 17 » N.D.e N.D. N.D. » This paper 15C(pKR001) 1 : 42 : 57 » 18 90 19 » This paper E. coli BL21(DE3) 100 : 0 : 0 » » » » » This paper BL21(DE3)(pMM4) 3 : 91 : 6 » 73 84f N.D.» This paper 3b Isolated enzyme 0 : 100 : 0 50 : 1 N.R. 28 » » 29 S. cerevisiae 15C 92 : 8 : 0 N.D. N.D. N.D. » » This paper 15C(pKR001) 66 : 34 : 0 9 : 1 30 30f,g » » This paper E. coli BL21(DE3) 100 : 0 : 0 » » » » » This paper BL21(DE3)(pMM4) 34 : 66 : 0 19 : 1 N.D. 12f,g » » This paper a Determined by gas chromatography. b Isolated yield after chromatographic puri–cation.c Not applicable. d Not reported. e Not determined. f Optical purities were determined by chiral-phase GC. g Optical purity of the major diastereomer. that extending the reaction time in the presence of the engineered E. coli cells would result in an even greater optical purity of the sulfoxide by preferential oxidation of (S)-5a to the sulfone.A control experiment established that native E. coli enzymes are not capable of oxidizing 3a (Table 1). Moreover, incubating racemic sulfoxide 5a with the unmodi–ed E. coli host strain for extended times failed to yield any sulfone product (Scheme 3). The engineered E. coli strain thus acts as the whole-cell equivalent of puri–ed cyclohexanone mono- Fig. 2 Oxidation of 1,3-dithiane 3a by E. coli BL21(DE3)(pMM4). This oxidation was performed with 8.3 mM substrate and monitored periodically by chiral-phase GC. Symbols: dithiane 3a; (R)- (K) ()) sulfoxide 5a; sulfone 7a. (L) Scheme 3 oxygenase that does not require enzyme puri–cation or cofactor regeneration. Similar results were observed for 2-phenyl-substituted compound 3b (Table 1).While the engineered yeast strain aÜorded the expected (R)-sulfoxide, its efficiency was compromised by competing oxidations carried out by native yeast enzymes. This competition was avoided by substituting the engineered E. coli strain. Unfortunately, cyclohexanone monooxygenase itself displays relatively low enantioselectivity for 3b, which limits the utility of all routes based on this catalyst.Oxidations of dithiolanes using engineered bakerœs yeast and E. coli strains Chiral sulfoxides derived from 1,3-dithiolanes are also valuable synthetic intermediates and several groups have investigated the use of cyclohexanone monooxygenase as a chiral oxidant for their synthesis. Puri–ed cyclohexanone monooxygenase converts unsubstituted 4a to the (R)-sulfoxide in 94% yield and [98% ee, although this is accompanied by 6% of sulfone 8a (Scheme 2; Table 2).25 Unfortunately, in this case, both the yeast and E.coli strains engineered to overexpress cyclohexanone monooxygenase aÜorded mainly the sulfone product. Control reactions in which the unmodi–ed yeast and E. coli strains were grown in the presence of dithiolane 4a or racemic sulfoxide 6a showed no reaction (Table 2 and Scheme 3, respectively), demonstrating that the overoxidation observed was due to cyclohexanone monooxygenase.Since puri–ed cyclohexanone monooxygenase oxidizes 6a to sulfone 8a more slowly than it converts 4a to 6a, we suspect that the rate of diÜusion of sulfoxide 6a across the cell membrane is relatively slow, which would allow 6a to accumulate in the presence of the monooxygenase.A similar situation was found for methyl-substituted dithiolane 4b (Table 2). By contrast, both the engineered yeast and E. coli strains oxidized phenyl-substituted 4c to the corresponding (R)-sulfoxide with high chemo- and diastereoselectivity (Table 2). Because native yeast enzymes also oxidized 4c (Table 2), the engineered E. coli strain was preferred for this reaction.Native E. coli enzymes did not oxidize sulfoxide 6c (Scheme 3). Here, however, as with phenyl-substituted dithiane 3b, cyclohexanone monooxygenase displayed relatively low enantioselectivity for this substrate. Using either the engineered yeast or E. coli strains, dimethyl-substituted dithiolane 4d aÜorded the (S)-sulfoxide, in agreement with the results obtained by Pasta and coworkers using puri–ed enzyme (Scheme 2; Table 2).29 Reversals of stereoselectivity that accompany changes in substrate structure are well-precedented for cyclohexanone monooxygenase. 27 The enantioselectivities of oxidations using New J.Chem., 1999, 23, 827»832 829Table 2 Biocatalytic oxidations of dithiolanes Sulfoxide 6 Sulfone 8 Substrate Catalyst 4 : 6 : 8a trans : cis Yieldb (%) % ee Yieldb (%) % ee Reference 4a Isolated enzyme 0 : 94 : 6 »c 94 [98 6 » 25 Acinetobacter N.R.d » 71 95 16 » 30 S.cerevisiae 15C 100 : 0 : 0 » » » » » This paper 15(pKR001) 5 : 25 : 70 » 20 75e 45 » This paper E coli BL21(DE3) 100 : 0 : 0 » » » » » This paper BL21(DE3)(pMM4) 0 : 15 : 85 » 13 86 47 » This paper 4b Isolated enzyme 0 : 94 : 6 P50 : 10 N.R. 50f N.R. N.R. 29 S. cerevisiae 15C 100 : 0 : 0 » » » » » This paper 15C(pKR001) 1 : 22 : 77 10 : 1 16 20e,f 15 76e This paper E. coli BL21 (DE3) 100 : 0 : 0 » » » » » This paper BL21(DE3)(pMM4) 34 : 31 : 35 5 : 1 35 40e,f 16 73e This paper 4c S. cerevisiae 15C 81 : 19 : 0 1 : 2 N.D.g 70f » » This paper 15C(pKR001) 6 : 94 : 0 32 : 1 74 20e,f » » This paper E. coli BL21(DE3) 100 : 0 : 0 » » » » » This paper BL21(DE3)(pMM4) 13 : 87 : 0 40 : 1 60 20e,f » » » 4d Isolated enzyme 0 : 100 : 0 »c N.R. 65 » » 29 S. cerevisiae 15C 100 : 0 : 0 » » » » » This paper 15(pKR001) 0 : 86 : 14 » 84 48e 10 » This paper E. coli (BL21(DE3) 100 : 0 : 0 » » » » » This paper BL21(DE3)pMM4) 9 : 67 : 24 » 46 69e 6 » This paper a Determined by gas chromatography. b Isolated yield after chromatographic puri–cation.c Not applicable. d Not reported. e Optical purities were determined by chiral-phase GC. f Optical puritiy of the major diastereomer. g Not determined either the engineered yeast or E. coli strains were virtually identical to that observed for the puri–ed monooxygenase. Over-oxidation to sulfone 6d was a relatively minor sidereaction when the engineered yeast strain was employed.Conclusion In summary, our results demonstrate that microbial strains genetically engineered to express cyclohexanone monooxygenase can be useful substitutes for the puri–ed enzyme in thioether oxidations. These reactions are simple to perform and provide chiral sulfoxides with chemical and optical yields comparable to those obtained by traditional methods. The major difficulty associated with this approach is the interference of native enzymes that compete for the thioether substrate.However, by judicious choice of host cell (S. cerevisiae or E. coli), such problems can be minimized. Experimental NMR spectra were obtained on Varian Unity 400, Bruker AMX 400 or Varian XL-200 instruments. All spectra were recorded in solutions unless otherwise indicated and CDCl3 referenced to solvent (d\7.24 for 1H and 77.0 for 13C spectra) or TMS.IR spectra were recorded from thin –lms on a Nicolet 520 FT-IR spectrophotometer. Optical rotations were measured on a Perkin Elmer 241 polarimeter operating at ambient temperature. All measurements were performed in chloroform solutions unless otherwise indicated.Packed column gas chromatography was performed on a Shimadzu GC-9A instrument equipped with a —ame ionization detector and a custom-packed column (1/8A]1 m, 5% OV-101 on 100/120 Supelcoport, Supelco, Inc.) with helium as carrier gas. Capillary gas chromatography was performed on a Hewlett- Packard 5890 chromatograph employing a 0.54 mm]15 m DB-1301 column (J&W, Inc.) or a Shimadzu GC-9A employing a 0.32 mm]30 m 225 column (Supelco).The injec- ê-Dex tor and detector temperatures were maintained at 225 °C. HPLC analyses were performed on a Beckman System Gold personal chromatograph using an Econosphere silica column (4.6]250 mm, Alltech) coupled with a Chiralcel OD-H column (4.6]150 mm, Daicel Chemical Industries, Ltd.) Mixtures of hexanes and 2-propanol were used as the mobile phase.Distillations were carried out using a Kugelrohr apparatus. Thin layer chromatography was performed on precoated silica gel 60 plates (Whatman). Flash chromatography was performed on silica gel (200»425 mesh, Fisher). Potassium carbonate was oven-dried at 80 °C and cooled to room temperature prior to use. Methylene chloride was dried over anhydrous distilled and stored over 3 molecular K2CO3, Aé sieves.Other solvents were puri–ed by fractional distillation. All other reagents were obtained from commercial suppliers and used as received. General procedure for synthesizing dithiolanes and dithianes The aldehyde or ketone (20 mmol) and 1,2-ethanedithiol (20 mmol, 1.7 mL) was dissolved in 40 mL of The solu- CH2Cl2 . tion was cooled in an ice-water bath and (7.9 BF3»OEt2 mmol, 1.0 mL) was added dropwise.The cold bath was then removed and the reaction mixture was vigorously stirred for 1 h at room temperature. The mixture was poured into saturated and ice. The organic layer was washed thor- NaHCO3 oughly with saturated and the combined aqueous NaHCO3 phases were extracted with The combined organic CH2Cl2 .phases were dried over anhydrous and concentrated MgSO4 by rotary evaporation. The residue was puri–ed when necessary by vacuum distillation or —ash chromatography on silica gel using a hexane»ethyl acetate mixture. Dithanes were synthesized in a similar manner except that 1,3-propanedithiol was used in place of 1,2-ethanedithiol. General procedure for yeast-mediated oxidations To a 250-mL baffled Erlenmeyer —ask was added 100 mL of YP-Gal medium (1% bacto-yeast extract, 2% bacto-peptone, 2% galactose), 100 lL of substrate (0.51»0.94 mmol, depending on the substrate), 3 lL of cyclohexanone and 0.2 g of frozen yeast cells.37 If b-cyclodextrin was required, it was also added at this time.46 The reaction mixture was shaken at 30 °C, 250 rpm until GC or HPLC analysis showed complete consumption of the starting material or an unchanged ratio of starting material and product over a 4 h time period.At this 830 New J. Chem., 1999, 23, 827»832time, the reaction mixture was centrifuged to remove yeast cells (5,000]g, 10 min). The supernatant was saturated with NaCl and extracted with EtOAc (5]50 mL). The cell pellet was resuspended in ca. 20 mL of water and extracted twice with 50 mL of EtOAc. The combined organic extracts were washed once with brine, dried over anhydrous and Na2SO4 concentrated by rotary evaporator. The residue was puri–ed by —ash chromatography on silica gel using 3 : 1 hexanes» acetone as the eluant. General procedure for E. coli-mediated oxidations Recombinant E. coli strain BL21(DE3)(pMM4) from a frozen stock was streaked on LB plates (1% bacto-tryptone, 0.5% bacto-yeast extract, 1% NaCl, 1.5% bacto-agar) containing 200 lg mL~1 ampicillin, then the plate was incubated at 30 °C until colonies were 1»2 mm in size.A single colony from this plate was used to inoculate 10 mL of liquid LB medium (1% bacto-tryptone, 0.5% bacto-yeast extract, 1% NaCl) containing 200 lg mL~1 ampicillin in a sterile 50-mL Erlenmeyer —ask.After shaking at 37 °C, 250 rpm overnight, 1 mL of this saturated culture was used to inoculate 100 mL of liquid LB medium containing 200 lg mL~1 ampicillin supplemented with 10 mL of 20% glucose in a 250-mL baffled Erlenmeyer —ask. The culture was shaken at 37 °C, 250 rpm until the was between 0.8 and 1.0. At this point, 10 lL of OD600 isopropyl-thio-b-D-galactoside (IPTG) stock solution (200 mg mL~1) was added followed by 100 lL of substrate (0.51»0.94 mmol, depending on the substrate).If b-cyclodextrin was needed for the reaction, it was added at this point. The –nal reaction mixture was shaken at room temperature, 250 rpm until GC showed complete consumption of the starting material or when the ratio of product and starting material remained unchanged over a 4 h time period.At this time, the reaction mixture was saturated with NaCl and extracted with ethyl acetate (5]50 mL). The combined extracts were washed once with brine, dried over anhydrous and concen- Na2SO4 trated by rotary evaporator. The residue was puri–ed by —ash chromatography on silica gel using 3 : 1 hexanes»acetone as the eluant.General procedures for monitoring biotransformations For GC analysis, 100 lL aliquots of the reaction mixture were removed periodically and mixed with 100 lL of EtOAc, vortexed vigorously for 30 s and centrifuged at 7,500 rpm in a microcentrifuge for 1 min. The EtOAc also contained 100 ppm methyl benzoate as an internal standard. The organic phase was removed and analyzed by GC.For HPLC analysis, 2 mL aliquots of the reaction mixture were removed and extracted with an equal volume of EtOAc. The organic layer was then concentrated to one half to one third of its original volume by a stream of nitrogen, then an aliquot of the residue was analyzed by HPLC. General procedure for chemical oxidations The sulfur compound (10 mmol) was dissolved in 50 mL of methanol and cooled to approximately 15 °C.To this was added (11 mmol, 2.35 g) dissolved in 10 mL of warm NaIO4 water at a rate that kept the reaction temperature below 20 °C. After the addition was complete, the reaction was stirred for 30»90 min at room temperature until TLC analysis showed complete consumption of the starting material. The mixture was evaporated to near dryness, then the residue was diluted with distilled water and extracted with EtOAc or The combined organics were dried over and CH2Cl2.Na2 SO4 concentrated by rotary evaporator. Pure products were obtained by —ash chromatography on silica gel using 2 : 1 petroleum ether»acetone as the eluant. Acknowledgements support by the Natural Sciences and Engineering Financial Research Council and the University of New Brunswick (M.M.K.), the National Science Foundation (J.D.S.; CHE- 9816318) and the FWF for a Schroé dinger Fellowship (M.D.M.; J1471-CHE) is gratefully acknowledged. We also thank Cerestar, Inc. for supplying the cyclodextrins used in this work. J.D.S. is a New Faculty Awardee of the Camille and Henry Dreyfus Foundation (1994»1999). This paper was taken from the Ph.D.thesis of G.C. References 1 G. Solladieç , Synthesis, 1981, 185. 2 M. Mikolajczyk and J. Drabowicz, T op. Stereochem., 1982, 13, 333. 3 M. R. Barbachyn and C. R. Johnson, in Asymmetric Synthesis, ed. J. D. Morrison and J. W. Scott, Academic Press, New York, 1984. pp. 227»261. 4 G. H. Posner, Acc. Chem. Res., 1987, 20, 72. 5 H. L. Holland, Chem. Rev., 1988, 88, 473. 6 J. Drabowicz, P. Kielbasinski, and M. Mikolajczyk, in T he Chemistry of Sulfones and Sulfoxides, ed. S. Patai, Z. Rappoport and C. J. M. Stirling, John Wiley and Sons, New York, 1988. pp. 233»278. 7 A. J. Walker, T etrahedron : Asymmetry, 1992, 3, 961. 8 E. G. Mata, Phosphorus, Sulfur, 1996, 117, 231. 9 R. F. Bryan, F. A. Carey, O. D. Dailey, R. J. Maher and R. W. Miller, J.Org. Chem., 1978, 43, 90. 10 P. Pitchen, P. P. E. Dun8 ach, M. N. Deshmukh and H. B. Kagan, J. Am Chem. Soc., 1984, 106, 8188. 11 H. B. Kagan, E. Dun8 ach, C. Memecek, D. Pitcher, O. Samuel and S.-H. Zhao, Pure Appl. Chem., 1985, 57, 1911. 12 S.-H. Zhao, O. Samuel and H. B. Kagan, T etrahedron, 1987, 43, 5135. 13 F. A. Davis, R. ThimmaReddy and M. C. Weismiller, J. Am. Chem. Soc., 1989, 111, 5964. 14 V. K. Aggarwal, G. Evans, E. Moya and J. Dowden, J. Org. Chem., 1992, 57, 6390. 15 J.-M. Brunel, P. Diter, M. Duetsch and H. B. Kagan, J. Org. Chem., 1995, 60, 8086. 16 P. B. C. Page, R. D. Wilkes, E. S. Namiwindwa and M. J. Witty, T etrahedron, 1996, 52, 2125. 17 B. J. Auret, D. R. Boyd and H. B. Henbest, J. Chem. Soc. C, 1968, 2371. 18 H. L. Holland, H. Popperl and R.W. Ninniss, Can. J. Chem., 1985, 63, 1118. 19 H. Ohta, Y. Okamoto and G. Tsuchihashi, Agric. Biol. Chem., 1985, 49, 671. 20 J. Beecher, P. Richardson, S. Roberts and A. Willets, Biotech. L ett., 1995, 17, 1069. 21 D. R. Light, D. J. Waxman and C. Walsh, Biochemistry, 1982, 21, 2490. 22 G. Carrea, B. Redigolo, S. Riva, S. Colonna, N. Gaggero, E. Battistel and D. Bianchi, T etrahedron : Asymmetry, 1992, 3, 1063. 23 F. Secundo, G. Carrea, S. Dallavalle and G. Franzosi, T etrahedron: Asymmetry, 1993, 4, 1981. 24 G. Ottolina, P. Pasta, G. Carrea, S. Colonna, S. Dallavalle and H. L. Holland, T etrahedron : Asymmetry, 1995, 6, 1375. 25 S. Colonna, N. Gaggero, A. Bertinotti, G. Carrea, P. Pasta and A. Bernardi, J. Chem. Soc. Chem. Commun., 1995, 1123. 26 G. Ottolina, P. Pasta, D.Varley and H. L. Holland, T etrahedron : Asymmetry, 1996, 7, 3427. 27 S. Colonna, N. Gaggero, P. Pasta and G. Ottolina, Chem. Commun., 1996, 2303. 28 V. Alphand, N. Gaggero, S. Colonna and R. Furstoss, T etrahedron L ett., 1996, 6117. 29 S. Colonna, N. Gaggero, G. Carrea and P. Pasta, T etrahedron : Asymmetry, 1996, 7, 565. 30 V Alphand, N. Gaggero, S. Colonna P. Pasta, and R. Furstoss, T etrahedron, 1997, 53, 9695. 31 S. Colonna, N. Gaggero, G. Carrea and P. Pasta, Chem. Commun., 1997, 439. 32 For recent advances in NADPH regeneration technology applied to cyclohexanone monooxygenase, see F. Secundo, G. Carrea, S. Riva, E. Battistel and D. Bianchi, Biotechnol. L ett., 1993, 15, 865 and S. Rissom, U. Schwarlinek, M. Vogel, V. I. Tishkov and U. Kragl, T etrahedron : Asymmetry, 1997, 8, 2523. 33 J. D. Stewart, K. W. Reed and M. M. Kayser, J. Chem. Soc., Perkin T rans. 1, 1996, 755. New J. Chem., 1999, 23, 827»832 83134 J. D. Stewart, K. W. Reed, J. Zhu, G. Chen and M. M. Kayser, J. Org. Chem., 1996, 61, 7652. 35 J. D. Stewart, K. W. Reed, C. A. Martinez, J. Zhu, G. Chen and M. M. Kayser, J. Am. Chem. Soc., 1998, 120, 3541. 36 M. M. Kayser, G. Chen and J. D. Stewart, J. Org. Chem., 1998, 63, 7103. 37 M. M. Kayser, G. Chen and J. D. Stewart, Synlett, 1999, 153. 38 J. D. Stewart, Curr. Org. Chem., 1998, 2, 211. 39 In the case of phenyl n-propyl sul–de, extended reactions in the presence of the engineered yeast strain gave only a low yield of the corresponding (R)-sulfoxide in 25% ee. Neither ethyl thiophenylacetate nor methyl b-(thiophenyl)ethyl ether aÜorded any sulfoxide products, even after extended reaction times. Moreover, when the racemic sulfoxides derived from these thioethers were incubated with the yeast strain, all traces of the sulfoxides disappeared within 78 h. We did not investigate the fates of these materials further. 40 G. J. Zylstra and D. T. Gibson, Genetic Eng., 1991, 13, 183. 41 C. C. R. Allen, D. R. Boyd, H. Dalton, N. D. Sharma, S. A. Haughey, R. A. S. McMordie, B. T. McMurray, G. N. Sheldrake and K. Sproule, J. Chem. Soc., Chem. Commun., 1995, 119. 42 Details of this construction are provided as Supplementary Material. 43 Y.-C. J. Chen, O. P. Peoples and C. T. Walsh, J. Bacteriol., 1988, 170, 781. 44 S. D. Emr, Methods Enzymol., 1990, 185, 231. 45 Using racemic sulfoxide 5a and puri–ed cyclohexanone monooxygenase, Colonna and co-workers estimated an enantioselectivity value of 12 for the oxidation of 5a to 7a. 46 R. Bar, T rends Biotechnol., 1989, 7, 2. Paper 9/02283J 832 New J. Chem., 1999, 23, 827»832

ISSN:1144-0546    

DOI:10.1039/a902283j

出版商:RSC    

年代:1999

数据来源: RSC

摘要:

Peptide nucleic acids (PNA) derived from N-(N-methylaminoethyl)glycine. Synthesis, hybridization and structural properties° Gerald Haaima,§a Hanne Rasmussen,b Gué nther Schmidt,a Dorte K. Jensen,a Jette Sandholm Kastrup,b Pernilla Wittung Stafshede,îc Bengt Nordeç n,c (the late) Ole Buchardta and Peter E. Nielsen*d a Center for Biomolecular Recognition, T he H. C. Institute, Universtitetsparken 5, –rsted DK-2100 Copenhagen, Denmark –, b Department of Medicinal Chemistry, Royal Danish School of Pharmacy, Universtitetsparken 2, DK-2100 Copenhagen, Denmark –, c Department of Physical Chemistry, Chalmers University of T echnology, S-41296, Gothenburg, Sweden d Center for Biomolecular Recognition, Department of Biochemistry and Genetics, L aboratory B, T he Panum Institute, Blegdamsvej 3c, DK-2200 N, Copenhagen, Denmark.E-mail : pen=imbg.ku.dk Received (in Cambridge, UK) 15th March 1999, Accepted 24th May 1999 Backbone N-methylated peptide nucleic acids (PNAs) containing the four nucleobases adenine, cytosine, guanine and thymine were synthesized via solid phase peptide oligomerization. The oligomers bind to their complementary target with a thermal stability that is 1.5»4.5 °C lower per ììN-methyl nucleobase unitœœ [dependent on the number and position(s) of the N-methyl] than that of unmodi–ed PNA.However, even fully N-methyl modi–ed PNAs bind as efficiently to DNA or RNA targets as DNA itself. Furthermore, the hybridization efficiency per N-methyl unit in a PNA decreased with increasing N-methyl content, and the eÜect was more pronounced when the N-methyl backbone units are present in the Hoogsteen versus the Watson»Crick strand in triplexes.(PNA)2-DNA Interestingly, CD spectral analyses indicate that 30% (3 out of ten) substitution with N-methyl nucleobases did not alter the structure of PNA-DNA (or RNA) duplexes or triplexes, and likewise CD spectroscopy and (PNA)2-DNA X-ray crystallography showed no major structural diÜerences between N-methylated (30%) and unmodi–ed PNA-PNA duplexes.However, PNA-DNA duplexes as well as triplexes adopted a diÜerent conformation when formed with all-N-methyl PNAs. Introduction Reagents that interact with DNA or RNA in a sequence speci –c manner are of great interest for the development of gene therapeutic drugs and diagnostic and molecular biology tools.1h3 Peptide nucleic acid (PNA) is a DNA mimic in which the sugar-phosphate backbone is replaced by a backbone consisting of N-(2-aminoethyl)glycine units.4h6 The chemical, biophysical and biological properties of PNA, not least the –nding that PNA oligomers hybridize strongly and sequence speci–cally to complementary DNA, RNA or PNA oligomers, have attracted attention towards this and analogous peptide nucleic acids.7h12 A signi–cant number of PNA derivatives with modi–cations in the backbone have been prepared and are currently being studied in order to understand the chemi- § Present address : Center for Drug Design and Development, University of Queensland, Brisbane QLD 4072, Australia.î Present address : Beckman Institute, California Institute of Technology, Pasadena, 91125 California, USA.° Abbreviations used: DCC dicyclohexylcarbodiimide ; DCU dicyclohexyl urea; DhbtOH 3,4-dihydo-3-hydroxy-4-oxo-1,2,3-benzotriazine ; HATU O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexa- —uorophosphate. cal and structural features which are important for nucleic acid binding and recognition as well as biological activity. 11h24 In order to further investigate the structure»activity relationships of PNA, a modi–ed PNA backbone, which contains an N-(2-methylaminoethyl)glycine was considered. This substitution eliminates the hydrogen bonding donor capacity of the backbone as well as changes the hydrophilicity and hydration properties. It has been proposed on the basis of molecular modeling that the carbonyl of the linker to the nucleobase constitutes a potential hydrogen bonding acceptor for this amide proton, either in the direction of the amide NH of the same or to the preceeding nucleobase unit.25,26 However, neither NMR27,28 nor X-ray crystallographic29 three-dimensional structure determinations of PNA-DNA/ RNA/PNA duplexes or a triplex have sup- (PNA)2-DNA ported such a hydrogen bonding pattern.In fact, the crystal structures show that the backbone amide protons of the Hoogsteen PNA strand in the triplex are (PNA)2-DNA involved in speci–c hydrogen bonds to the phosphates of the DNA backbone,29 whereas the backbone amide protons in the PNA-PNA duplex are hydrogen bonded to localized water molecules which bridge the nucleobase and the backbone. 30 In this paper we report the synthesis and hybridization New J. Chem., 1999, 23, 833»840 833analysis of PNA oligomers containing N-methylated glycine units at one or more positions along the backbone. Furthermore, the eÜect of backbone N-methylation on the structure of PNA complexes has been investigated by CD spectroscopy and X-ray crystallography. Experimental General All reagents were obtained from commercial suppliers and used without further puri–cation.Melting points are uncorrected. Flash chromatography was performed using Merck silica gel 60 (230»400 mesh ASTM). 1H and 13C NMR spectra were obtained in using a Varian 400 MHz Unity or DMSO-d6 a Bruker 250 MHz AMX spectrometer. Fast atom bombardment mass spectra were recorded on a JEOL Hx110/110 operating in positive ion mode.MALDI-TOF mass spectra of PNA oligomers were recorded on a Kratos, Kompact Maldi II operating in positive ion mode and using 3,5-dimethoxy-4- hydroxycinnamic acid as the matrix. Analytical HPLC was carried out on a 3.9]150 mm Delta Pak 5 lm C-18 100 column (Waters). BuÜer A: 99.9% Aé TFA; BuÜer B: 10% H2O»0.1% H2O»89.9% CH3CN»0.1% tri—uoroacetic acid (TFA).The solvents were heated to 50 °C while the —ow rates were 1.0 for analytical and 2.0 ml min~1 for preparative HPLC. RNA oligomers were purchased from DNA Technology, Aarhus, Denmark and were used as received, and DNA oligomers were synthesized by standard methods. Thermal denaturation studies (Tm) were run on a Gilford Response II spectrophotometer scanning from 5 to 90 °C at a rate of 0.5 °C per step (approx. 0.7 °C min~1). Prior to recording the Tm, the complexes were heated to 95 °C for 5 min and allowed to slowly cool to 4 °C. The oligomers were hybridized in the following buÜer: 100 mM NaCl»10 mM sodium phosphate»0.1 mM pH 7.0. The concentrations of PNA and oli- H4EDTA, gonucleotides were determined optically at 60 °C using the molar absorptivities of the four nucleosides.CD spectra were recorded on a Jasco model 720 spectropolarimeter equipped with a thermoelectrically controlled cell holder. Each spectrum is the average of at least eight scans, recorded at 20 °C using 1 cm optical path length. The samples were kept in a 5 mMsodium phosphate buÜer at pH 7.0. Monomer synthesis N-Methyl-1-aminopropane-2,3-diol 1. To a pre-cooled solution (0 °C) of methylamine (172 ml of a 40% solution in water, 2 mol) was added 2,3-epoxypropan-1-ol (25 g, 0.34 mol) at such a rate that the reaction temperature did not exceed 10 °C.After 3 h at 0°C excess methylamine and water were evaporated and the residue Kugelrohr distilled (103»105 °C, 0.5 mmHg) resulting in 25.6 g (76%) of colorless oil. 1H NMR (400 MHz, d 3.74 (m, 1H, CH), 3.56 (m, 2H, CDCl3) : CH2), 3.22 (m, 2H, 2.94 (s, 3H, FAB-MS: m/z 106 CH2), NCH3) ; (M`]H). 1- [N-(tert-Butyloxycarbonyl)-N-methyl ]aminopropane-2,3- diol 2. Di-tert-butyl dicarbonate (52.3 g, 0.24 mol) was added to a pre-cooled (0 °C) solution of 1 (21 g, 0.20 mol) in water (340 ml) and the mixture was allowed to warm to room temperature. The pH was maintained at 10.5 by addition of 4 M aqueous NaOH.After addition of 2 equivalents of NaOH the reaction was left to stir for 15 h. Upon cooling to 0 °C and adjusting the pH to 2.5 using 4 M aqueous HCl the reaction mixture was extracted with EtOAc (6]100 ml). The combined organic fractions were washed with half-saturated aqueous (3]150 ml) followed by brine (1]150 ml). KHSO4 The organic fraction was then dried and concen- (MgSO4) trated to an oil.Kugelrohr distillation (110»112 °C, 0.5 mmHg) furnished 31.3 g (81%) of the desired product as a colorless oil. 1H NMR (400 MHz, d 3.72 (m, 1H, CDCl3) : CH), 3.55 (m, 2H, 3.22 (m, 2H, 2.90 (s, 3H, CH2), CH2), 1.43 (s, 9H, t-Bu) ; FAB-MS: m/z 206 (M`]H). NCH3), 2-{ [N-(tert-Butyloxycarbonyl)-N-methyl ]amino}acetaldehyde 3. To a solution of 2 (20 g, 97 mmol) in water (100 ml) stirring under was added (24.68 g, 108 mmol).After N2 KIO4 2.5 h the reaction mixture was –ltered and the –ltrate extracted with (5]50 ml). The layers were CHCl3 CHCl3 dried and concentrated to an oil. Kugelrohr distilla- (MgSO4) tion (76»80 °C, 0.5 mmHg) provided 13.26 g (79%) of a colorless oil. 1H NMR (400 MHz, d 8.54 (s, 1H, major, CDCl3) : CHO), 8.51 (s, 1H, minor, CHO), 4.01 (s, 2H, major, CH2), 3.93 (s, 2H, minor, 2.95 (s, 3H, major, 2.93 (s, CH2), NCH3), 3H, minor, 1.47 (s, 9H, major, t-Bu) ; 1.42 (s, 9H, NCH3), minor, t-Bu) ; FAB-MS: m/z 174 (M`]H).Ethyl N-{2- [ (N-tert-butyloxycarbonyl)-N-methyl ]aminoethyl} glycinate 4. Ethyl glycinate hydrochloride (8.10 g, 58 mmol) dissolved in absolute EtOH (100 ml) was added dropwise to a cooled (0 °C) solution of 3 (10.0 g, 58 mmol) and NaOAc (9.32 g, 114 mmol) in absolute EtOH (130 ml). After addition of 10% Pd on carbon (1.65 g) the reaction was hydrogenated (1 atm) until one equivalent of was H2 absorbed.The reaction mixture was –ltered and to the –ltrate was added water (80 ml). After adjusting the pH to 8 with 2 M aqueous NaOH the mixture was extracted with CH2Cl2 (5]60 ml).The organic layers were dried and con- (MgSO4) centrated to an oil. Kugelrohr distillation (100»105 °C, 0.5 mmHg) furnished 10.6 g (74.3%) of a colorless oil. 1H NMR (250 MHz, d 4.18 (q, 2H, 3.42 (s, 2H, CDCl3) : CH2), CH2), 3.34 (t, 2H, 2.88 (s, 3H, 2.77 (t, 2H, 1.45 (s, CH2), CH3), CH2), 9H, t-Bu), 1.28 (t, 3H, FAB-MS: m/z 261 (M`]H).CH3) ; General procedure for the synthesis of the monomer esters 5añd To a pre-cooled (0 °C) solution of 4 (7.0 mmol), DhbtOH (7.7 mmol) and the appropriate carboxymethyl derivative of the nucleobase (7.7 mmol) in a 1 : 1 mixture of dry DMF»CH2Cl2 (60 ml) was added DCC (8.4 mmol). After stirring at 0 °C for 1 h followed by a further 3 h at room temperature the precipitated DCU was removed by –ltration and the reaction work-up was as follows for each derivative.Ethyl N-{2- [ (N-tert-butyloxycarbonyl)-N-methyl ]aminoethyl}- N- [ (thymin-1-yl)acetyl ] glycinate 5a. The reaction was diluted with (60 ml) and washed sequentially with CH2Cl2 half-saturated aqueous (3]30 ml), half-saturated NaHCO3 aqueous (2]30 ml) and brine (1]30 ml). The KHSO4 organic phase was dried and the solvent removed in (MgSO4) vacuo.The resulting foam was redissolved in cooled CH2Cl2 , to 0 °C and precipitated by slow addition of light petroleum (bp 40»60 °C) under vigorous stirring. The product was isolated as a white solid ; mp 93»95 °C; 1H NMR (400 MHz , d 11.20 (s, 1H, major, T-imid NH), 11.18 (s, 1H, CDCl3) : minor, T-imid NH), 7.52 (s, 1H, major, T-H6), 7.50 (s, 1H, minor, T-H6), 4.60 (s, 2H, major, 4.42 (s, 2H, CH2CON), minor, 4.32 (q, 2H, 4.25 (s, 2H, minor, CH2CON), OCH2CH3), 4.07 (s, 2H, major, 3.60»3.10 (m, CH2COOH), CH2COOH), 2.90 (s, 3H, major, 2.83 (s, 3H, minor, CH2CH2), BocNCH3), 1.79 (s, 3H, 1.39 (s, 9H, t-Bu), 1.34 (t, 3H, BocNCH3), CH3), FAB-MS: m/z 427 (M`]H). OCH2CH3) ; Ethyl N-{2- [ (N-tert-butyloxycarbonyl)-N-methyl ]aminoethyl}- N-{ [4-N-(benzyloxycarbonyl)cytosin-1-yl ] acetyl}- glycinate 5b.The reaction mixture was evaporated to dryness and to the residue was added diethyl ether (50 ml), this was stirred for 2 h and the solid –ltered oÜ. The solid was again stirred with 50 ml ether for 2 h and –ltered oÜ, washing the solid with half-saturated aqueous The solid was NaHCO3 . then dissolved in hot dioxane (60 ml) and precipitated by slow addition of water (60 ml).The product was further puri–ed by 834 New J. Chem., 1999, 23, 833»840column chromatography 5 : 95) ; mp (SiO2, MeOH»CH2Cl2 , 152»156 °C; 1H NMR (400 MHz, d 7.98 (s, 1H, CDCl3) : C-H5), 7.42»7.34 (m, 5H, Ph), 7.09 (s, 1H, C-H6), 5.19 (s, 2H, 4.85 (s, 2H, major, 4.62 (s, 2H, minor, PhCH2), CH2CON), 4.28 (q, 2H, 4.17 (s, 2H, minor, CH2CON), OCH2CH3), 3.99 (s, 2H, major, 3.42»3.03 (m, CH2COOH), CH2COOH), 2.97 (s, 3H, major, 2.89 (s, 3H, minor, CH2CH2), BocNCH3), 1.39 (s, 9H, t-Bu), 1.24 (t, 3H, BocNCH3), OCH2CH3) ; FAB-MS: m/z 546 (M`]H).Ethyl N-{2- [ (N-tert-butyloxycarbonyl)-N-methyl ]aminoethyl}- N-{ [6-N-(benzyloxycarbonyl)adenin-9-yl ] acetyl}- glycinate 5c. The reaction mixture was evaporated to dryness, redissolved in (300 ml) and washed successively with CH2Cl2 half-saturated aqueous (3]100 ml), half-saturated NaHCO3 aqueous (2]100 ml) and brine (1]100 ml). The KHSO4 organic phase was dried and the solvent removed in (MgSO4) vacuo. To the resulting foam in absolute EtOH (60 ml) was slowly added water (30 ml). The mixture was left stirring overnight and the product was isolated by –ltration ; mp 140» 143 °C; 1H NMR (400 MHz, CDCl3): d 9.19 (br s, 1H, ZNH), 8.74 (s, 1H, major, H-8), 8.06 (s, 1H, minor, H-8), 7.47»7.32 (m, 5H, Ph), 5.29 (s, 2H, major, 5.14 (s, 2H, minor, CH2CON), 5.24 (s, 2H, 4.27 (s, 2H, minor, CH2CON), PhCH2), 4.17 (q, 2H, 4.02 (s, 2H, major, CH2COOH), OCH2CH3), ; 3.54»3.03 (m, 2.93 (s, 3H, major, CH2COOH) CH2CH2), 2.86 (s, 3H, minor, 1.40 (s, 9H, t-Bu), BocNCH3), BocNCH3), 1.26 (t, 3H, FAB-MS: m/z 570 (M`]H).OCH2CH3) ; Ethyl N-{2- [ (N-tert-butyloxycarbonyl)-N-methyl ]aminoethyl}- N-{ [6-N-(benzyloxycarbonyl)guanin-9-yl ] acetyl}- glycinate 5d. The reaction mixture was evaporated to dryness, redissolved in (80 ml) and washed with half-saturated CH2Cl2 aqueous (3]40 ml). After drying and KHSO4 (MgSO4) removal of the solvent in vacuo the desired product was isolated by crystallization from EtOAc; mp 157»159 °C; 1H NMR (400 MHz, d 7.95 (s, 1H, major, G-H8), 7.88 (s, CDCl3) : 1H, minor, G-H8), 7.55»738 (m, 5H, Ph), 5.37 (s, 2H, PhCH2), 5.18 (s, 2H, major, 5.00 (s, 2H, minor, CH2CON), CH2CON), 4.36 (s, 2H, minor, 4.20 (q, 2H, 4.06 CH2COOH), OCH2CH3), (s, 2H, major, 3.62»3.32 (m, 2.94 (s, CH2COOH), CH2CH2), 3H, major, 2.80 (s, 3H, minor, 1.38 (s, BocNCH3), BocNCH3), 9H, t-Bu), 1.27 (t, 3H, FAB-MS: m/z 586 OCH2CH3) ; (M`]H).N-{2- [ (N-tert-Butyloxycarbonyl)-N-methyl ]aminoethyl}-N- [ (thymin-1-yl)acetyl ] glycine 6a. To a solution of 5a (2.41 g, 5.6 mmol) in MeOH (45 ml) at 0 °C was added 2 M NaOH (45 ml) and stirring was continued for 2 h.The pH was adjusted to 2 with 2 M HCl and extracted with ethyl acetate. The organic phase was dried and evaporated to dryness (MgSO4) yielding 1.45 g (52%) of a white solid ; mp 119»122 °C; 1H NMR (400 MHz , d 12.83 (s, 1H, COOH), 11.36 DMSO-d6) : (s, 1H, major, T-imid NH), 11.34 (s, 1H, minor, T-imid NH), 7.38 (s, 1H, major, T-H6), 7.34 (s, 1H, minor, T-H6), 4.72 (s, 2H, major, 4.54 (s, 2H, minor, 4.26 (s, CH2CON), CH2CON), 2H, minor, 4.04 (s, 2H, major, CH2COOH), CH2COOH), 3.70»3.10 (m, 2.92 (s, 3H, major, 2.84 (s, CH2CH2), BocNCH3), 3H, minor, 1.75 (s, 3H, 1.38 (s, 9H, t-Bu) ; BocNCH3), CH3), FAB-MS: m/z 399 (M`]H); Calc.for C, C17H26N4O7 : 51.25 ; H, 6.58 ; N, 14.07. Found: C, 50.98 ; H, 6.55 ; N, 13.90%. N-{2- [ (N-tert-Butyloxycarbonyl)-N-methyl ]aminoethyl}-N- { [4-N-(benzyloxycarbonyl)cytosin-1-yl ] acetyl}glycine 6b.To a solution of 5b (1.17 g, 2.3 mmol) in THF (40 ml) was added 1 M aqueous LiOH (60 ml). After 30 min the reaction was cooled to 0 °C and the pH was adjusted to 2 with 2 N aqueous HCl. The mixture was extracted with and CH2Cl2 the organic phase was dried and evaporated yielding (MgSO4) 0.72 g (27%) of white foam; mp 181»184 °C; 1H NMR (400 MHz, d 10.78 (s, 1H, COOH), 7.88 (s, 1H C-H5), DMSO-d6) : 7.41»7.32 (m, 5H, Ph), 7.01 (s, 1H, C-H6), 5.19 (s, 2H, PhCH2), 4.81 (s, 2H, major, 4.62 (s, 2H, minor, CH2CON), CH2CON), 4.17 (s, 2H, minor, 3.98 (s, 2H, major, CH2COOH), 3.42»3.03 (m, 2.94 (s, 3H, major, CH2COOH), CH2CH2), 2.87 (s, 3H, minor, 1.38 (s, 9H, t-Bu) ; BocNCH3), BocNCH3), FAB-MS: m/z 518 (M`]H); Calc.for C, C24H31N5O8 : 55.69 ; H, 6.04 ; N, 13.53. Found: C, 55.35 ; H, 5.99 ; N, 13.28%. N-{2- [ (N-tert-Butyloxycarbonyl)-N-methyl ]aminoethyl}-N- { [6-N-(benzyloxycarbonyl)adenin-9-yl ] acetyl}glycine 6c. To a solution of 5c (3.14 g, 5.5 mmol) in THF at 0 °C was added 1 N aqueous LiOH (25 ml). After 30 min the pH was adjusted to 1 with 2 N aqueous HCl.The precipitate was –ltered and dried yielding 1.62 g (57%) of a white solid ; mp 171»173 °C; 1H NMR (400 MHz, d 12.77 (broad, 1H, COOH), DMSO-d6) : 10.68 (broad, 1H, ZNH), 8.59 (s, 1H, major, A-H8), 8.35 (s, 1H, minor, A-H8), 7.48»7.32 (m, 5H, Ph), 5.37 (s, 2H, major, 5.18 (s, 2H, minor, 5.23 (s, 2H, CH2CON), CH2CON), 4.35 (s, 2H, minor, 3.99 (s, 2H, major, PhCH2), CH2COOH), 3.54»3.02 (m, 2.93 (s, 3H, major, CH2COOH), CH2CH2), 2.84 (s, 3H, minor, 1.39 (s, 9H, t-Bu) ; BocNCH3), BocNCH3), FAB-MS: m/z 542 (M`]H); Calc.for C, C25H31N7O7 : 55.44 ; H, 5.77 ; N, 18.10. Found: C, 55.10 ; H, 5.71 ; N, 17.81%. N-{2- [ (N-tert-Butyloxycarbonyl)-N-methyl ]aminoethyl}-N- { [6-N-(benzyloxycarbonyl)guanin-9-yl ] acetyl}glycine 6d. To a solution of 5d (2.33 g, 4.0 mmol) in MeOH (40 ml) was added 2 M aqueous NaOH (40 ml).After 1 h at room temperature the reaction mixture was cooled to 0 °C and the pH was adjusted to 2 using 2 M aqueous HCl. The precipitate was –ltered, dried and recrystallised from absolute EtOH yielding 1.27 g (43%) of a white solid ; mp 189»192 °C; 1H NMR (400 MHz, d 11.42 (broad, 1H, COOH), 7.92 (s, 1H, DMSO-d6) : major, G-H8), 7.85 (s, 1H, minor, G-H8), 7.55»738 (m, 5H, Ph), 5.33 (s, 2H, 5.18 (s, 2H, major, 5.00 PhCH2), CH2CON), (s, 2H, minor, 4.32 (s, 2H, minor, CH2CON), CH2COOH), 4.05 (s, 2H, major, 3.62»3.30 (m, 2.95 CH2COOH), CH2CH2), (s, 3H, major, 2.82 (s, 3H, minor, 1.38 BocNCH3), BocNCH3), (s, 9H, t-Bu) ; FAB-MS: m/z 558 (M`]H); Calc.for C, 53.86 ; H, 5.60 ; N, 17.59. Found: C, 53.54 ; C25H31N7O8 : H, 5.54 ; N, 17.30%.Synthesis of PNA oligomers Coupling of the monomers was carried out according to published protocols31 using a 4-(methylbenzhydryl)amine resin down-loaded to 0.12 mmol g~1 with Boc-L-lysine(2-Cl- Z)OH. The PNAs were cleaved from the resin using a tri- —uoromethanesulfonic acid (TFMSA)»TFA procedure.31 Syntheses typically gave a crude product of greater than 90% purity as judged by reversed-phase HPLC.The oligomers were puri–ed by HPLC and characterised by MALDI-TOF mass spectrometry. Crystallization of H-CMeGTMeACMeG-(L-Lys)-NH2 A 5 mg ml~1 solution of self-complementary N-methylated PNA [H- was screened for CMeGTMeACMeG-(L-Lys)-NH2] crystallization conditions using the sparse-matrix screen of Jancarik and Kim32 from Hampton Research.Optimization of initial crystallization conditions resulted in crystals of size 0.600]0.250]0.075 mm. The hanging-drop vapor-diÜusion method was used at room temperature with drop sizes of 5 ll (3 ll N-methylated PNA solution and 2 ll well solution) and 0.5 ml of well solution. The well solution contained 0.4 M 5% ethanol, 0.075 M TRIS … HCl pH 7.2, and 3.0 M MgCl2 , hexane-1,6-diol.N-Methylated PNA crystallizes in space group with cell dimensions a\48.94, b\31.00, P21 c\50.74 b\111.78°. Aé , New J. Chem., 1999, 23, 833»840 835Table 1 Diffraction data and refinement statistics DiÜraction data Resolution/Aé 2 25.0»2.2 (2.24»2.20) Unique re—ections 6794 Completeness (%) 92.0 (93.0) Multiplicity 1.6 Rmerge(I)a (%) 7.5 (30.0) I/pI 6.6 (2.2) Re–nement Resolution/Aé 6.0»2.2 Number of re—ections 4049 p cut-oÜ 3 R-value/Rfree-valueb (%) 21.4/29.4 Total number of non-hydrogen atoms 1343 Water molecules 207 Average B-values of PNA units/Aé 2 19 Average B-values of L-Lys units/Aé 2 51 Average B-values of water molecules/Aé 2 41 R.m.s.deviation of bond lengths/Aé 2 0.015 R.m.s. deviation of bond angles/° 3.7 between symmetry related re—ections. a Rmerge\agreement b Rfree\ R-value for test set of re—ections (10%) omitted cross-validation during the re–nements.Crystallography Data collection. A diÜraction data set of N-methylated PNA was collected to 2.2 resolution using one crystal on a Aé Rigaku RU-200 rotating-anode generator equipped with an R-AXIS II imaging plate detector (j\1.542 50 kV, 180 Aé , mA, normal focus). The data collection was performed at room temperature with a crystal»detector distance of 100 mm and with 180° rotation in steps of 3° oscillations.Autoindexing and data processing were performed with DENZO33 and the CCP4 suite of programs.34 The statistics of the data set are tabulated in Table 1. Structure determination and re–nement. The structure of Nmethylated PNA was solved by molecular replacement using the program AMoRe35 from CCP4.Two right-handed and two left-handed double helices have been located per asymmetric unit. The two right-handed and the two left-handed helices, respectively, are related by a translation of 0.50, 0.50, 0.15, as indicated in a native Patterson map. Initially, a righthanded double helix, coaxially stacked with a left-handed helix, of an unmodi–ed PNA-PNA hexamer was used as search model30 in the resolution range 10.0»3.0 One clear Aé .solution to the translation function appeared with the best solution from the rotation search. A second translation search was conducted, –xing the –rst solution to the translation function. After each translational search, ten cycles of rigid-body re–nement were performed using AMoRe, resulting in R factors of 56.3% and 52.7%, respectively, and correlation coef- –cients of 64.4 and 72.3.The four double helices were subjected to a positional re–nement protocol in X-PLOR36 using data in the resolution range 6.0»2.2 and a 3p cut-oÜ, alternating with graphical Aé sessions using the program O.37 Non-crysytallographic symmetry restrains were applied during re–nement. B-Factor re–nement and water molecules were included in the early cycles of re–nement. The –nal R-value was 21.4% and the –nal re–nement statistics and average B-values are tabulated in Table 1.The atomic coordinates of N-methylated PNA have been deposited in the Brookhaven Protein Data Bank, accession code RCSB009127. Results and discussion Synthesis of N-methyl monomers and oligomerization Monomers suitable for incorporation using Boc-based peptide synthesis were prepared according to Scheme 1.Essentially the chemistry was adapted from existing chemistry used to Scheme 1 B\a thymine, b N4-benzyloxycarbonylcytosine, c N6- benzyloxycarbonyladenine, d N2-benzyloxycarbonylguanine. construct the regular PNA monomers.31,38,39 Reaction of aqueous methylamine with 2,3-epoxypropanol produced 1 which after Boc protection of the amine was oxidatively cleaved with to produce 3.The backbone 4 was KIO4 obtained from reductive alkylation of ethyl glycinate with 3. The nucleobase acetic acids were synthesised as described in the literature.38 Coupling of the four nucleobase acetic acids using DCC»DhbtOH followed by alkaline hydrolysis yielded the four monomers 6a»d. Duplex formation and stability Decamer PNA oligomers of mixed pyrimidine»purine content were used for studies of duplex formation with complementary DNA, RNA and PNA oligomers.Two PNAs were synthesised ; PNA I containing three N-methyl backbone thymines and PNA II in which all monomers derived from the Nmethyl backbone.Thermal stability studies of the duplexes formed between these PNAs and DNA, RNA or PNA are presented in Table 2. The data clearly demonstrate that inclusion of N-methyl backbone units results in a decrease in thermal stability of the PNA-nucleic acid duplexes of 1»3 °C per Nmethyl unit. It is also observed that the eÜect is less pronounced for RNA (and PNA) binding and that the eÜect of having more N-methyl units is not ììadditiveœœ, since the cost per N-methyl is higher for PNA I than for PNA II.Furthermore, we note that even the fully methylated PNA II exhibits PNA-nucleic acid duplex stabilities that are equal to (DNA) or signi–cantly higher (RNA) than that of the corresponding DNA oligonucleotide. Finally, it is interesting that the relative 836 New J.Chem., 1999, 23, 833»840Table 2 Thermal stabilities (Tm/°C) of PNA-DNA, PNA-RNA, PNA-PNA duplexes Anti-parallel DNA Parallel DNA PNA-DNA duplexa duplexb Mismatchc RNAd RNA mismatche PNAf H-GTMeAGATMeCACTMe-NH2 (I) 41.5 (2.8)g 37.5 D37h 48.5 (1.8) 39 63.0 (1.3) H-(GTAGATCACT)Me-LysNH2 (II) 33.0 (1.8) 34.5 » 44.0 (1.2) » 61.5 (0.6) H-GTAGATCACT-NH2 (IIIa) 50 38.5 D40h 54 44 67 H-GTAGATCACT-LysNH2 (IIIb) 51 38 D37h 56 46 67 5@-GTAGATCACT-3@ 33.5 26.5 34 32 49 a 5@d(AGTGATCTAC).b 5@d(CATCTAGTGA). c 5@d(AGTGGTCTAC). d 5@(AGUGAUCUAC). e 5@(AGUGGUCUAC). f H-AGTGATCTACg *Tm per N-methyl unit given in parentheses. h Due to a thermal transition of the single stranded PNA itself around 40 °C, this value LysNH2 . is difficult to determine accurately.Table 3 Thermal stabilities (Tm/°C) of triplexes (PNA2)-DNA PNA DNA (ap)a DNA (p)b RNAc PNAc H-T10-LysNH2 (IV) 71.5 (0)d 81 70 H-TTTTTTMeTTTT-LysNH2 (V) 66.0 (2.7) 56 H-TTTTTMeTTMeTTT-LysNH2 (VI) 61.5 (2.5) 63.5 H-TTTTMeTTMeTTMeTT-LysNH2 (VII) 57.0 (1.5) 58 56.5 H-Gly-(TTTTTTTTTT)Me-LysNH2 (VIII) 43.0 (1.4) 42 48 H-TJTJTTT(eg)3-TTTCTCT-LysNH2 (IX) 64.0 (0) 49 H-TMeJTMeJTMeTMeTMe(eg)3TTTCTCT-LysNH2 (X) 42.0 (4.4) B39 H-TJTJTTT(eg)3TMeTMeTMeCMeTMeCMeTMe-LysNH2 (XI) 40.5 (3.3) 31 TJTJTTT(eg)3TMeTMeTMeCTMeCTMe-LysNH2 (XII) 48.5 (2.2) 32.5 TJTJTTT(eg)3TTTCMeTCMeT-LysNH2 (XIII) 55.5 (4.3) 32 a Sequences: or ap: anti-parallel, p: parallel.b Sequence c Sequence: 5@d(CGCA10CGC) 5@d(CGCAGAGAAACGC), 5@d(CGCAAAGAGACGC). d *Tm per N-methyl unit given in parentheses. A10 .preference of PNA for binding to DNA in the antiparallel orientation is diminished (PNA I) or completely abolished (PNA II) for the N-methylated PNAs. Triplex formation and stability Two formats were chosen for the study of tri- (PNA)2-DNA plexes. The –rst consisted of a with an increasing number T10 (1, 2, 3 or 10) of N-methyl substitutions. The second format was a bis(PNA) in which the N-methyl substitutions were made in the Watson»Crick or the Hoogsteen strand, respectively.The results are presented in Table 3. Analogously to what was observed for duplex formation, inclusion of the Nmethylated PNA backbone decreases the thermal stability of the triplexes by an average 2 °C per N-methyl (PNA)2-DNA unit, and the eÜect is diminished with an increasing number of N-methyl units (compare PNAs V»VIII). [Using PNA VII as a representative case, we performed a titration (Job-plot) to ascertain that indeed triplexes are formed by (PNA)2-DNA these N-methyl substituted PNAs (Fig. 1)]. In order to address whether the eÜect of the methylation was con–ned to Fig. 1 Titration (Job-plot) of the binding of PNA VII to a complementary oligonucleotide (dA10).the Watson»Crick interaction or was also aÜecting the Hoogsteen strand, we synthesized a series of linked bis(PNAs) in which the preferred binding mode (Watson»Crick versus Hoogsteen) can be controlled by the orientation of the PNA strands (antiparallel orientation is preferred by the Watson» Crick PNA strand whereas parallel orientation is preferred by the Hoogsteen PNA strand), as well as the position of pseudoisocytosine (J) nucleobases, which do not require low pH for optimal J»G Hoogsteen type interactions.40 Thus for the triplexes of PNAs IX»XIII presented in Table 3, Hoogsteen binding should be preferred by the ììJ-strandœœ, while Watson» Crick binding should take place with the ììC-strandœœ.This assignment is supported by the fact that all of these PNAs bind more strongly to the DNA complement that is antiparallel to the PNA ììC-strandœœ than to the DNA complement that is parallel to this strand (Table 3).Therefore by comparing the result obtained with PNA X having –ve (thymine) N-methyl units in the Hoogsteen strand (*Tm\4.4 °C) with that of PNA XII having –ve (thymine) N-methyl units in the Watson»Crick strand (*Tm\2.2 °C) we conclude that N-methylation aÜects the stability of both strands, and that the interference seems greater in the Hoogsteen strand. CD spectroscopy We applied circular dichroism spectroscopy to monitor any eÜects of the backbone N-methylation on the structure of the PNA-nucleic acid complexes. The results presented in Fig. 2 and 3 clearly show that no major structural change occurs in the PNA-DNA or PNA-RNA duplexes or tri- (PNA)2-DNA plexes upon introduction of 30% (three out of ten) N-methyl units in the PNA strand(s). However, most interestingly, the complexes (duplex or triplex) formed by the fully methylated PNAs II and VIII show distinctly altered CD spectra, strongly suggesting that these complexes also have structures that distinguish them from those formed by the normal PNAs.On the other hand no major diÜerence between the PNA-PNA duplexes formed by the fully methylated PNA II as compared to that of the normal PNA I was apparent from CD spectroscopy (Fig. 4). New J. Chem., 1999, 23, 833»840 837Fig. 2 Circular dichroism (CD) spectra of the duplexes between PNAs I (»»»), II (» » » ») or the control PNA IIIa (»… … …») and the antiparallel DNA (a) or RNA (b) oligonucleotide (5@-AGTGATCTAC) . CD spectra of the free DNA or RNA are shown by (»…»).This is also consistent with the observation that N-methyl backbone substitutions are more easily accommodated in PNA-PNA duplexes (*TmB0.6»1.3 °C per N-methyl substitution) than in PNA-DNA (RNA) duplexes (*Tm 1.2» 2.8 °C per substitution). It has been shown that a PNA-PNA duplex adopts a novel helical conformation, the P-form30 that shows a much larger helical pitch (B18 bases per turn) and diameter (B28 than the natural B- or A-form conforma- Aé ) tions which are closer to the structures adopted by PNA-DNA and PNA-RNA duplexes.27,28 Thus it appears that the N-methyl PNA backbone can be quite easily accommodated in the P-type helix, whereas it is more difficult in the B- or A-type helices.The triplexes also adopt a P-type conforma- (PNA)2-DNA tion,29 but nonetheless do not seem to accommodate the NFig. 3 CD spectra of the triplexes between and PNAs IV dA10 T10 (control) (»…»), V (one N-methyl) (» » » »), VII (three N-methyl) (- - - - - - -), and VIII (full N-methyl) (»»»). Fig. 4 CD spectra of the duplexes formed between PNA II (»»») or the control PNA I (- - - - - - -) and a complementary PNA (having a C-terminal L-lysine).methyl backbone any better than PNA-DNA (RNA) duplexes. Furthermore, the largest eÜect is seen when the N-methyl backbone is placed in the Hoogsteen strand (compare PNAs X and XIII). This is most likely due to disruption of N» H… … …phosphate hydrogen bonding interactions between the backbone of the DNA and the Hoogsteen PNA strand as observed in the crystal structure and suggested to provide extra stabilization of the triplex.29 (PNA)2-DNA Crystal structure determination The structure of a 50% N-methylated PNA hexamer [Hwas determined in order to CMeGTMeACMeG-(L-Lys)-NH2] establish how the N-methyl backbone is accommodated in the P-type helix of PNA-PNA duplexes. The asymmetric unit of the crystal contains two pairs of coaxially stacked righthanded and left-handed double helices (Fig. 5). As previously observed,30 this results, by crystallographic symmetry, in the formation of a continuous pseudo-helix, alternating between right- and left-handed forms. The two right-handed and the two left-handed helices are very similar.The N-methylated PNA hexamer adopts the P-form helix (Table 4) with helical parameters close to those of the unmodi–ed PNA-PNA duplex.30 Fig. 5 The N-methylated PNA hexamer crystallizes with two righthanded (cyan) and two left-handed (green) double helices in the asymmetric unit. The right-handed and left-handed helices are coaxially stacked. The two right-handed and the two left-handed helices, respectively, are related by non-crystallographic translational symmetry.The unit cell with axes is shown in stereo. The –gure was generated using the program O.37 838 New J. Chem., 1999, 23, 833»840Table 4 Helical parameters (average)a of the N-methylated PNA-PNA duplex Twist (%) Rise/Aé Base tilt (%) Displacement/Aé Bases per turn N-Me PNAb Right1 18.9 3.8 0.2 4.8 18 Right2 19 3.8 0.1 4.9 18 Left1 [20 3.5 0 7.2 18 Left2 [20 3.5 0.3 7.2 18 Unmodi–ed PNAc 19.8 3.2 1 8.3 18 a The helical parameters were determined using the program CURVES.41 b N-Me PNA: backbone N-methylated PNA-PNA duplex [Hc Unmodi–ed PNA: unmodi–ed PNA-PNA duplex (H-CGTACG- CMeGTMeACMeG-(L-Lys)-NH2].NH2).30 The major structural diÜerence between N-methylated and unmodi–ed PNA is an alteration in the conformation of the N-methyl substituted amide moiety.In the N-methylated PNA, the backbone carbonyl oxygens of the aÜected amide moieties are turned inward towards the nucleobases (Fig. 6) whereas the carbonyl oxygens are directed into solution in the structure of unmodi–ed PNA.30 In the structure of the unmodi–ed PNA hexamer speci–c water molecules that form a bridge between the backbone amide NH and the nucleobase were located for each base step.This hydrogen bonding donor has been eliminated in the modi–ed PNA backbone. However, the water bridge is retained by coordination of the carbonyl oxygen and the water molecule, thereby leaving the N-methyl moiety exposed to the solvent. The water bridge between the backbone amide NH and the nucleobase is retained in the unmodi–ed amide moieties of the N-methylated PNA, either through backbone amide NH or through the carbonyl oxygen, as two of these non-methylated amide moieties in each strand adopt two diÜerent conformations.The backbone»nucleobase interactions through water molecules seem important for stabilizing the P-form helix of PNA. These interactions are still possible within the modi–ed PNA backbone, and probably explain why no major overall structural diÜerence is apparent between the PNA-PNA duplexes formed by N-methylated PNA as compared to normal PNA. Conclusion The present results demonstrate that PNA duplex (with DNA, RNA or PNA) and to a lesser extent triplex (PNA)2-DNA Fig. 6 Backbone (amide NH or O) forms hydrogen bonds to water molecules, bridging the nucleobase and backbone in the double helical structure of the methylated PNA-PNA hexamer.Three representative base pairs are shown. PNA is colored by atom-types, and water molecules are depicted as red spheres. The –gure was generated using the program O.37 structures can without major loss in stability accommodate a limited number (at least 30%) of N-methyl substitutions in the backbone.Most interestingly, duplexes and triplexes with fully methylated PNAs adopt helix conformations which judged by CD are distinctly diÜerent from the regular PNA structures. The crystal structure of the 50% modi–ed PNA-PNA duplex shows that the N-methyl group is accommodated in the double helical structure by an alteration in the conformation of the aÜected amide moiety.This clearly indicates that NH hydrogen bonding is not crucial for stabilization of the structure as long as the possibility for formation of a nucleobase» backbone bridge through a water molecule is present. In conclusion these results indicate that N-alkylation can with only a moderate loss of hybridization potency be exploited as a means to modulate, for example the pharmacological properties of PNA.Acknowledgements technical assistance of Annette and Brian The J‘rgensen Rosenberg is gratefully acknowledged. The work was supported by grants from The Danish National Research Foundation, PharmaBiotec and The Lundbeck Foundation. References 1 E. Uhlmann and A. Peyman, Chem. Rev., 1990, 90, 544. 2 A. De Mesmaeker, K.H. Altmann, A. Waldner and S. Wendeborn, Curr. Opin. Struct. 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ISSN:1144-0546    

DOI:10.1039/a902091h

出版商:RSC    

年代:1999

数据来源: RSC