ORGANIC CHEMISTRY.1. INTRODUCTION.A FURTHER step forward has been taken this year towards the objective ofmaking this section a true Annual Report. Some two-thirds of the spaceavailable is devoted to this purpose under the headings : Stereochemistry,General Methods, Aliphatic Compounds, Homocyclic Compounds, Hetero-cyclic Compounds, and Macromolecules ; the section on Theoretical OrganicChemistry deals with addition and elimination reactions and molecularrearrangements which are topics which have not been discussed fully in theseReports in recent years. It has beeh thought necessary to include specialisedarticles on Nucleic Acids and Porphins to cover obvious gaps. With the verylarge number of publications appearing in the space of one year, it is inevit-able that each aspect of organic chemistry cannot be dealt with every year asthere are still severe limitations on space.For this reason treatment of, forexample, sugars and many of the heterocyclic nitrogenous compounds hasbeen postponed for a later Report.We propose next year to devote the whole of our space to an account ofthe progress made during the year which will be organised under the generalheadings we have just mentioned; of these that devoted to Macromoleculesis a new departure which we think necessary in view of the ever increasinginterest of such substances from the organic chemical, ke., structural, pointof view. It seems inevitable, however, that occasional essay articles willbe required even under the new policy.A. W.J.H. N. R.2. THEORETICAL ORGANIC CHEMISTRY.A. Addition Rmctions.-Homolytic additions, particularly of halogenacids to olefinic systems, have recently been considered in these Reports,lbut heterolytic addition processes have received little attention. In 1931,Ingold and Ingold,2* cfs3 examining by a competition method the effects ofsubstituents on the rate of addition of bromine to ethylene derivatives, madeit clear that halogens are usually electrophilic in their attack on unsaturatedcompounds. These authors also adduced evidence that powerful electron-withdrawal from the olefinic centre might polarise the double link sufficientlyto make bromine a nucleophilic reagent. Knowledge of the kinetics of halogenaddition reactions has since been extended, partly by Anantakrishnan’sSmith, Ann.Reports, 1939,36, 219 ; Hey, ibid., 1948,45, 149.Ingold and Ingold, J., 1931, 2354.Anantakrishnan and Ingold, J . , 1935,984,1396DE LA MARE : THEORETICAL ORGANIC CHEMISTRY. 127school 4 and by Nozaki and Ogg 5 but most extensively by P. W. Robertsonand his co-workers,6 who have examined the reactivities of many ethyleniccompounds under various conditions of medium and of catalysis. Theresults obtained in acetic acid (in this solvent complications due to homolytic,heterogeneous, and photochemical reactions may be avoided) have recentlybeen surveyed,’ and they are summarised in the following section.Chromones, Flavones and Flavono1s.-Schmid and his colleagues 98 havecarried out an extensive study of the chromone constituents of carnations,Eugenia aromaticu and E .mryophyllata. Among the products isolated wereeugenin (XXXVIII ; R = R” = H, R’ = Me), eugenitin (XXXVIII ;R = R’ = Me, R” = H), isoeugenitin (XXXVIII; R = H, R’ = R” =Me), and isoeugenitol (XXXVIII; R = R’ = H, R” = Me). The simul-taneous occurrence of eugenitin and the iso-structures suggests the presenceof a monocyclic intermediate, e.g., (XXXIX), which can cyclise in two ways.Syntheses of eugenitin, isoeugenitin, and isoeugenitol were described.The Allan-Robinson synthesis of flavones, by heating of an o-hydroxy- oran o-aroyloxy-acetophenone with an aromatic anhydride and the salt of thecorresponding acid, suffers from the disadvantage that 3-acylflavones areobtained as by-products, often in appreciable amount, and the hydrolyticfission of the acyl group may result in considerable Baker andGlocking loo have discussed the mechanism of this formation of acylflavonesand have devised a method depending on a condensation with benzylamine93 Johnson, Robertson, and Whalley, J., 1950, 2971.94 Brown, Cartwright, Robertson, and Whalley, J., 1949, 859.95 Cram, J .Amer. Chem. SOC., 1948,70,440,4244; Frye, Wallis, and Dougherty, J .Org. Chem., 1949,14, 397.Cartwright, Robertson, and Whalley, J., 1949, 1563.@’ Cram, J . Amer. Chem. Soc., 1950,72, 1001.98 Schmid et al., Helv. Chim. Acta, 1048, 31, 1603; 1949, 32, 813, 1358; 1950, 33,917, 1770.Baker and Butt, J . , 1949, 2142. loo J., 1050, 2759230 ORGANIC CHEMISTRY.for distinguishing between the two possible isomeric acylflavones.If theo-substituted acetophenones are heated alone in glycerol a t 250°, the flavonesthemselves are obtained,lol a method which is also applicable to the synthesisof flavonols.Paper chromatography has been applied to the separation of mixtures offlavones and related compounds.lo2 Briggs and Locker lo3 have examinedthe flavonol content of the bark of certain Melicope spp. and have described avariety of new compounds, some of which are ethers of quercetin(3 : 5 : 7 : 3’ : 4’-pentahydroxyflavone) and others also have substituents inthe 6- or the 8-position. Methods of synthesis were evolved, e.g., of ternatin(XL) consisting of a combination of the Allan-Robinson flavonol method andSeshadri’s nuclear hydroxylation method. lo4 The structure of ginketin, thecolouring matter of the autumn leaves of the maidenhair tree, is still underconsideration but it is probably more complex than the simple flavoneformulation originally put forward.lo5 Moewus lo6 has reviewed the partplayed by flavonols in the sexual processes of certain flowering plants, e.g.,Forsythia.Lactones.-The occurrence of the unsaturated lactone structure 1°7 inmany natural products had led to a great deal of work on the synthesis andproperties of these compounds, Methods of synthesis in the heart-poison logand toad-venom 109 fields have been reviewed. An important syntheticroute for the +unsaturated lactones is that depending on acetylenic inter-mediates,l1° e.g.:The addition of methanol to the triple bond yields the unsaturated methoxy-lactones, as exemplified by Raphael’s synthesis of penicillic acid ;ll1 similarOMe /\, r‘C:CH*CO,H 1 \ ,,CH*CH,-CO,Hi / IPh*CH:CH(O,CO co-o co-0 -(XLI.) (XLII.) (XLIII.)additions to the Py-acetylenic alcohols 112 lead to six-membered unsaturatedlactones, as in the synthesis of (&)-kawain (XLI).l13101 Dunne, Gowan, Keane, O’Kelly, O’Sullivan, Roche, Ryan, and Wheeler, J.,1950, 1252.102 Bate-Smith et al., Biochim. Biophys. Actu, 1950, 4, 427, 441 ; Lindstedt, ActuChem. Scand., 1950,4,448, 1042.lo3 J., 1949, 2157, 2162; 1950,864,2376,2379.104 Proc. Indian Acad. Sci., 1949,3Q, A , 333.106 Baker, Flemons, and Winter, J . , 1949, 1560.106 Angew. Chem., 1950, 62, 496.108 Turner, Chem.Reviews, 1948, 43, 2 3 ; Heusser, “ Fortschritte der Chemie109 Deulofeu, ibid., 1948, 5, 241.11s Fowler and Henbest, i b d . , 1950, 3642.Haynes, Quart. Reviews, 1948,2, 46.Organischer Naturstoffe,” Vienna, 1950, Vol. VII, p. 87.110 Jones, J . , 1950, 756.112 Henbest, Jones, and Walls, ibid., 1949, 2696. J., 1948, 1508JOHNSON AND RYDON : HETEROCYCLIC COMPOUNDS. 23 1Similar principles have been used by Jones et ul.ll* to synthesise analoguesof the plant growth hormone, auxin b, as formulated by Kogl :116[CH,],>XH R*CH( OH)*CH,*CiC*CO,Me ____ -+ R*CH( OH)*CH2*COCH,*C0,MeR-yH-CH,*C( OH) :CH (R = A / ) o------co ! I! --+In contrast to the reported properties of auxin b,l15 the product could not behydrolysed to the corresponding acid and attempts to prepare the acidinvariably led to the formation of the lactone.Further work on the isolationand properties of the natural compound is necessary to remove theseanomalies. Other analogues containing the same side chain were alsodescribed (R = Me, Ph, propenyl, spirocyclohexyl) and some progress hasbeen made towards building of the auxin-a side-chain .l15a Meanwhile Kogland de Bruin 116 have also synthesised the cyclopentenyl analogue of auxin bby means of a Reformatsky reaction of cyclopentenealdehyde with y-bromo-p-ethoxycrotonic ester. No comparisons of the product with auxin b itselfwere made other than the ultra-violet absorption spectrum.The bearing of Bredt's rule on the enol lactonisation of y- and 8-keto-acidshas been discussed by Fawcett,l17 and Linstead and his colleagues havestudied the formation of lactonic acids by cyclodehydration of keto-dicarboxylic acids.Thus treatment of p-ketoadipic acid with acetylchloride-hydrogen chloride gave the lactone (XLII), the properties of whichresembled those of the &-unsaturated lactones (see below). Richter ll9prepared the ester of (XLII) by cyclisation of the monoester of p-ketoadipicacid, but his claim to have prepared the free acid is considered doubtfulby the English workers. Lactonisation of cis-cis-muconic acid gave the+unsaturated lactone (XLIII) ,121 the corresponding ester of which, ontreatment with sodium methoxide,122 gave the cis-truns-isomer of the mono-methyl ester of muconic acid, from which the corresponding acid, not formerlydescribed, was obtained.Johnson et ~ 1 . l ~ ~ have studied the decarboxylationof y-substituted paraconic acids.with N-bromosuccinimide and then treated with silver acetate, to give adiacetate with an extra acetoxy-group in the 2-position of the pyrone ring.Removal of acetic acid as before gave patulin itself although in small yield.Hydrogenation and hydrolysis of the above diacetate gave deoxypatulinicacid (LXII), which was an intermediate in the synthesis of allopatulin(LVIII), as well as a second patulin synthesis via 5-chlorodeoxypatulinicacid.147 The new patulin formula (LVII) was used 144 to interpret theextensive degradative work described by earlier workers.lP1 J., 1944, 415.l a 3 Woodward and Singh, J .Amer. Chem. SOC., 1950,72, 5351.144 Idem, Experientia, 1950, 6, 238.la6 Woodward and Singh, ibid., 1950, 72, 1428.147 Idem, Nature, 1950, 165, 928.la8 Naute, Oosterhuis, van der Linden, van Duyn, and Dienske, Rec. Trau. chim.,142* 148Ira Helv. Chim. Acta, 1949,32, 1166, 1752.Dauben and Weisenborn, J . Amer. Chem. SOC., 1949, 71, 3853,1946,65, 865; Cohen, Chem. and Ind., 1949, 640230 ORGANIC UHEMISTRY.Coumarins.-The fluorescent behaviour of coumarins and its variationwith pH is recommended as a means of identification in this series.149 Therecognition of the blood-anticoagulant and rodenticide properties of a number(LXII.) (LXIII.) (LXIV.)of 3-substituted 4-hydroxycoumarins 150 has intensified studies on thereactions of the 4-hydroxycoumarins, e.g., acylation,151 Michael addition,152and aldol ~0ndensation.l~~ Koelsch and Sundet lM have described Michaeladditions with 4-acetylcoumarin.The coumarin photodimers probablyhave a cycbbutane structure as 3-phenylcoumarin gives a product analogousto that obtained from coumarin i t ~ e 1 f . l ~ ~ Parker and Robertson 156 havesynthesised a chromono(2’ : 3’-3 : 4)coumarin having the nucleus ofrotenonone (LXIII).Methods for the synthesis of isocoumarins (LXIV) have been surveyed.15’Dioxans.-1 : 3-Dioxans, formed by the Prins reaction 15* with substitutedethylenes, may be hydrogenolysed to 1 : 3-diols lSQ or 3-substituted primaryalcohols. 160Sulphur ring systems.Ethylene Sulphides.-The preparation and properties of the ethylenesulphide ring system have been studied by Davies and his co-workers inMelbourne.lG1 They have reviewed the methods of preparation and havereported favourably on the reaction of epoxides with thiourea or relatedcompounds, except in those cases where a strongly polar group was adjacentto the oxide ring.In many respects, although not all, the ring-openingreactions are similar to those of the epoxides, but there is a much greaterGoodwin and Kavanagh, Arch. Biochem., 1950,27, 152.l 5 0 E.g., FuEik, Prochbzka, LQbler, and Strof, Nature, 1950, 166, 830.161 Ukita, Nojima, and Matsurnoto, J . Amer. Chem. Soc., 1950, 72, 5143; Badcock,Dean, Robertson, and Whalley, J . , 1950,903 ; Muller, Syrovatka, and Wlasak, Monatsh.,1950,81, 174.154 Seidman, Robertson, and Link, J.Amer. Chem. SOC., 1950, 72, 5193.153 Ikaws and Link, ibid., p. 4373.154 Ibid., pp. 1681, 1844.166 Schiinberg, Lrttif, Moubaaher, and Awad, J., 1950, 374.1 5 8 Ibid., p. 1121.1 5 7 Kamal, Robertson, and Tittensor, ibid., 1950, 3376 ; Johnston, Kadow, Langs-160 Cf. Johnson, Ann. Reports, 1949,46,151.159 Price and Krishnamurti, J . Amer. Chem. SOC., 1950,72,5335.1‘0 Emerson, Heider, Longley, and Shafer, ibid., p. 5314.161 J., 1949, 278, 282; 1950, 317, 892.See also J . , 1949, 2049.joen, and Shriner, J . Org. Chem., 1948,18,477JOHNSON AND RYDON : HETEROCYCLIC COMPOUNDS. 237tendency of the cyclic sulphides to polymerise. Unlike that in thiophen,the sulphur in a three-membered ring cannot exist in a higher valency state.mophen.-Several papers have been devoted to the substitution of thethiophen ring for aromatic or other heterocyclic rings in natural productsand their prototypes, as well as in the chemotherapeutic field, especiallyantihistamines,162 but this work generally follows the standard reactions ofthiophen chemistry and its main interest is biological.to occur to the extent of 20--30%in the residual " tar " from the preparation of thiophen by reaction of buta-diene with s u 1 p h ~ r .l ~ ~ Liquid thiophen polymers of low molecular weighthave been described, containing predominantly the trimer (LXV).165Osmium tetroxide oxidation of protoporphyrin can be made to yieldspirographis porphyrin (oxidation of only one vinyl group to formyl) or2 : 4-diformyldeuteroporphyrin according to the conditions.Improvementsin the yield of the latter substance have recently been achieved.*7 Fischer and Deilmann, ibid., 1944, 280, 186.2. physiol. Chm., 1936, 242, 133.Lemberg and Falk, Biochem. J . , in the press.Ibid., 1941, 272, 1FALK AND RIMINQTON : PORPHYRINS. 273The glycol which is an intermediate stage in this oxidation, -CH:CH, --+-CH(OH)CH,*OH -+ -CHO --+ -CO,H, can also be ~btained.~The elegant synthesis (I1 + I11 --+ IV) of aetioporphyrin I in excel-lent yield under physiological conditions due to Siedel and Winkler lo has notpreviously been reported. Andrews, Corwin, and Sharp l1 have also reporteda new porphyrin synthesis which proceeds smoothly at room temperature,leading to a porphyrin with four carbethoxy-groups, although such groupshad previously been held to inhibit porphyrin formation.The methoddepends on the fact that the l-methylpyrrole-%aldehyde (V) can supply thecarbon atom for condensation of 2 molecules of (VI) to give a tripyrryl-methane (VII), which is specifically split to the dipyrromethene (VIII).When, instead of (VI), 2 molecules of the dipyrrylmethane (IX) are used, thedihydroporphyrin (X) is formed and is autoxidised to the porphyrin (XI)which crystallises from the reaction mixture in 40 yo yield.Me-C02Et Me -CO,Et Et0,C -Me II II Et0,C ----Me \g’ II IIMe II II CH2-\N/ \I$ Me11 [ICHOMe H\N/(V.1 (VI -1 (IX.)Et0,C -Me Me=CO,EtMel(N!J-CH=(N/MeH (VIII.)-1-I1 IIMe‘N/Me-C0,EtMeEt0,C H, C0,EtMe,(\()OMe/-NH N-\/ \ \--N HN //MJ I T M eEt0,C C0,Et\/\/\/Aluminium isopropoxide under mild conditions reduces the formylside-chain without reducing the isocyclic ring carbonyl group of mesopyro-ph%ophorbide-b.l2 The vinyl group, when present, is not affected by thisreduction.Fischer’s school clung long to the idea of “ Kekulb ” isomers, dependingon the exact position of the double bonds in both the porphyrin and thechlorophyll series.Not a little work has centred around the related question* Fischer and Pfeiffer, Annalen, 1944,556, 131.lo Ibid., 1943, 554, 162.l2 Fischer, Mittenzwei, and Hevkr, Annalen, 1940, 545, 154; “ Organic Reactions,”l1 J . Amer. Chem. SOC., 1950, 72,491.Vol.11, p. 184274 ORGANIC CHEMISTRY.of hydrogen bonding between pyrrole nitrogens.13 The evidence now pointsstrongly to complete resonance in these molecules, and has been well sum-marised by Lemberg and Legge.3aExtra ” Hydrogen Atoms.-The isolationby Fischer and Wendroth in 1940 l4 of the optically active acid (XII) insteadof haematinic acid (XIII) on degradative oxidation of chlorins confirmedthe previous amignment of the ( ( extra ” hydrogen atoms to positions 7 and8, affording also, for the first time, a degradation product which, like theoriginal chlorophyll, was optically active. Since it was already known thatchlorophyll-b differed from chlorophyll-a only in having CHO instead ofCH, a t position 3 (conversion of a phaeophorbide-b derivative, by reduction,into a phaophorbide-a derivative had been achieved), this fixed the ‘( extra ”hydrogens of chlorophyll-b also a t positions 7 and 8.CH,*CH2*C0,H HO,C*CH, CH,*CH,*CO,HH-TY1-H (iNiCH2*C02H O=’ ‘ 1-0The Chlorophyll Series : The“/HO = w = O(XII.) O = w = O H (XIII.) (XIV.)HBromination of chlorins leads to substitution of one of these hydrogensby bromine.15 Chlorination of chlorins leads to substitution of hydrogen atpositions 7 and 8 by hydroxyl groups,16 and of porphyrins to substitution ofmethene-bridge hydrogen by chlorine,17 though in porphyrins like phzeopor-phyrin-a, or phylloerythrin substitution by chlorine occurs first a t position 10.Removal of Iron from Haems.-Fischer and his school devised severaldifferent methods for the removal of iron from haemin.These all dependedon the action of a reducing agent in an acid medium and yielded either proto-porphyrin or mesoporphyrin in which the two vinyl groups of the formerhave also suffered hydrogenation. Side reactions made the purification ofthe porphyrins difficult.A very convenient technique, leading directly to protoporphyrin dimethylester in good yield and applicable to haemoproteins such as haemoglobin, isdue to Grinstein;l9 for work on a micro-scale, the use of pyruvic acid forremoval of iron is excellent.Z0 For splitting the thio-ether linkage whichunites the porphyrin moiety to the protein in cytochrome-c, Paul 21 employssalts of Ag, Hg, Pb, Cu, or Cd, preferably the first mentioned. The porphyrinrelease is a first-order reaction.Improvements have been suggested.18l3 Vestling and Downing, J.Amer. Chem. SOC., 1939, 61, 3511 ; McEwen, iCid., 1936,l4 Annalen, 1940, 545, 140.l5 Fischer and BalQz, ibid., 1943, 555, 81 ; Fischer, Kellermann, and Balaz, Ber.,l7 Fischer and Klendauer, ibid., p. 123.lo Ibid., 1947, 167, 515.21 Acta Chem. Scand., 1950, 4, 239.58, 1124; Ellingson and Corwin, ibid., 1946, 68, 1112.1942,75, 1778. l6 Fischer and Diet], Annalen, 1941, 547, 234.Grinstein and Watson, J. Biol. Chem., 1943,147, 667.20 Paul, personal communication, 1950FALK AND RIMINBTON : PORPHYBINS. 275The relatively inefficient Fischer-Kdgl method for transformation ofprotoporphyrin into mesoporphyrin, modified 22 with improvement of yield,has been further studied by Grinstein and Watson23 who have raised theyield to 60%.The necessity for obtaining maximum yield in isotope erxperi-ments involving step-wise degradation of the porphyrin ring has encouragedfurther study of this transformation. Catalytic hydrogenation, with methylmethacrylate as the supporting colloid, affords yields ofThe biological conversion of one porphyrin into another has never beensatisfactorily demonstrated either in the whole animal or by uae of survivingtissues (excepting the bacterial degradation of haemin into meso- and deutero-porphyrin in the gut), although Fischer and others assumed that this necess-arily took place. Van den Bergh, Grotepass, and Reever's 25 claim that theliver transformed protoporphyrin into coproporphyrin has not beensubstantiated.2sThe administration of protoporphyrin intramuscularly or by mouth failsto raise the blood protoporphyrin, but a small increase in this level is said tofollow intramuscular injection of haernat~porphyrin.~~ Further evidencewill be needed before a biological conversion of haematoporphyrin to proto-porphyrin can be accepted.All modern theories of haem biosynthesis (see below), based on theinterpretation of experiments with isotopically-labelled substances, postulatethe formation first of a highly carboxylated pyrrole derivative which is thenprogressively decarboxylated either before or after cyclisation to the porphyrinring.The occurrence in Nature, under normal or pathological conditions, ofporphyrins representing intermediate stages betweeen the fully carboxylateduroporphyrins (8 C0,H groups) and coproporphyrins (4 C0,H groups) orprotoporphyrin (2 C0,H groups) might thus be expected.Nicholas andRimingtonF8 using paper chromatography, have indeed obtained evidenceof the existence of such porphyrins with seven, six, five, and three carboxylgroups in various materials ; a pentacarboxylic porphyrin has been isolatedin nearly pure condition from a porphyria patient.29The " conchoporphyrin " from pearl mussel shells, described by Fischerand Jordan30 as a pentacarboxylic porphyrin, has on the other hand beenshown by paper chromatography to 'be a mixture of uroporphyrin andc~proporphyrin.~~Chromic acid oxidation of porphyrins causes rupture of the macrocyclicring with production of maleinimide derivatives.The synthesis of ethyl-a2 Schultze, J . Biol. Chem., 1942,142, 89 ; Rimington, Biochem. J., 1938, 32, 460.24 Muir and Neuberger, Biochem. J . , 1949,45, 163. 25 Klin. Woch., 1932, 11, 1534.2o Watson, Pass, and Schwartz, J . Biol. Chem., 1941,139,583 ; Salzburg and Watson,27 Schumm and Beckermann, Arch. exp. Path. Phczrm., 1948,205,98.2s McSwiney, Nicholas, and Prunty, Biochem. J . , 1950, 46, 147.so 2. physiol. Chem., 1930, 190, 75.31 Nicholas and Comfort, Biochern. J . , 1949, 45, 208,J . Biol. Chem., 1943, 147, 671.ibid., p. 593.Scand. J . Clin. Lab. Invest., 1949,1, 12, and personal communication, 1950276 ORGANIC CHEMISTRY.methylmaleimide and of haematinic acid have been improved and theconditions laid down, from a study of their physical characteristics, for theoptimal separation of these substances.24Anmcal.-The distribution of the chromoprofeins, haemoglobin,myoglobin, and cytochrome-c in the tissues of different animals has beenreported by Drabkin 32 who also describes the preparation of crystallinehaemoglobin on a large scale.Directions for the large-scale preparation ofhaemin have been given.33 Determination of porphyrins in body fluids isreviewed by With and by B r ~ g s c h , ~ ~ while Salamanca, Mesorana, et ~ 1 . have studied the determination and characterisation of copro- and uro-porphyrins. Photoelectric and fluorimetric methods are reported for thedetermination of protoporphyrin in blood.36* 36 Attention has been drawnto the necessity for certain preliminary treatments in the determination ofurinary c~proporphyrin,~' and a micro-determination of porphyrins bytitration with a copper salt has been described.38 The use of phosphoricacid in place of hydrochloric acid in the fluorimetric determination of por-phyrins has been re~ommended.~QThe most notable advance in the analysis of mixtures of porphyrins is theapplication to these substances of filter-paper chromatography employinglutidine-water as the solvent system.28* 29 Under the prescribed conditions(requiring 5-10 vg.of porphyrin mixture) individual porphyrins areseparated so that their R, values are linearly related to the number of carboxylgroups in the molecule.The method is applicable to porphyrin-metalcomplexes. A partition-chromatographic method, using columns of silicagel, has also been reported.40 Nicholas 41 has undertaken a thorough studyof adsorption chromatography of porphyrins under carefully standardisedconditions.The molecular extinction coefficients of pure coproporphyrins I andI11 42 have been measured, also those of the uroporphyrin~.~3>~~ An im-proved method for the determination of uroporphyrin in urine has beendescribed which employs a spectrophotometric correction for absorbingimpurities and thus makes it possible to carry out the determination directlyupon diluted porphyria ~rines.~4Separation and quantitative determination of the coproporphyrin32 J . Biol. Chem., 1950,182, 317 ; Arch.Biochem., 1949, 21, 224.33 Org. Synth., 1941, 21, 54.s4 Scand. J . Clin. Lab. Invest., 1949, 1, 164 ; 2. Qes. inn. Med., 1949, 4, 253.35 Arch. Med. Expt. (Madrid), 1949,12, 25, 39.3~ Grinstein and Watson, J. Biol. Chem., 1943, 147, 675; Grinstein and Wintrobe,37 Mmchling, J. Lab. Clin. Med., 1940-41,26, 1676; Raine, Biochem. J . , 1950,47, xiv.38 Oliver and Rawlinson, Biochem. J., in the press.39 Kliewe, 2. Qes. inn. Med., 1948, 3, 543.40 Lucas and Orten, Ped. Proc., 1950,9, 197.Jope and O'Brien, ibid., 1945, 39, 239.43 Nakamiya, Bull. Inst. Phys. Chem. Res., Tokyo, 1942, 21, 252.Sveinsson, Rimington, and Barnes, Scand. J. Clin. Lab. Inveat., 1949,1,2 ; Riming-ibid., 1948, 172, 459.41 Biochem. J., in the press.ton amd Sveinsson, ibid., 1950,2,209FALK AND RIMINGTON : PORPHYRINS. 277isomers, which so often occur together in biological materials, have alwaysbeen a matter of great difficulty.A study of the melting points of mixturesof known composition showed the unreliability of this criterion.42 Separationof the esters by chromatography on alumina, with elution of the type-111isomer by 35% aqueous acetone, has been claimed 45 but not confirmed.42* 46More recently Schwartz et aL4' have employed the quenching a t low temper-ature of fluorescence of coproporphyrin I in 30% aqueous acetone solutionas a means of determining the quantities of the isomers present in a mixture.Figures are published, based upon this method, for the daily coproporphyrinI and I11 excretion in normal urine.4sThe pigment of the malaria parasite has been extracted by a methodavoiding the use of alkali at all stages and has been identified as haematin.49Haem a, The Prosthetic Group of Cytochome-&-Earlier work on thisporphyrin derivative is summarised by Warburg 50 in his book on metallo-porphyrins and enzyme action.Rawlinson and Hale 5 l described a methodfor its isolation from cells of Corynebacterium diphtheriae and from heartmuscle, and studied its spectral and chemical properties, which indicated thepresence of at least one aldehyde group. Simultaneously Lemberg and Falk 52studied the same subject by comparing the absorption spectra of haem andporphyrin-a with the spectra of a series of synthetic haems and porphyrins.Short reports from the two Schools were made to the 1st InternationalCongress of Bio~hemistry.5~ Porphyrin-a resembles in some respects spiro-graphis (ch1orocruoro)porphyrin.Kiese claimed 54 that the latter waspresent among the products of the action of nitrous acid on haemoglobin;the mixture, however, is very complex. Cystalline pigments, which may benitroso-derivatives, are formed, according to Sapir0,~6 by the action of nitrousacid on chlorophyll (mixture of a and b ) .Prodigiosh-The tripyrrylmethene pigment prodigiosin produced byB. prodigiosus (Serratia rnarcescem) is of interest on account of the possibleintervention of a tripyrrylmethene stage in the biosynthesis of porphyrins.Hubbard and Rimington56 have shown by the isotope technique that thenitrogen and the methylene-carbon atom of glycine are specifically utilisedin the bacterial synthesis of prodigiosin, but not the carboxyl-carbon atom.Both carbon atoms of acetic acid are specifically utilised.There is thusconsiderable resemblance between the biosynthesis of this pigment and thatof haem.45 Watson and Schwartz, PTOC. SOC. exp. Biol., 1940, 44, 7.46 Helwig, 2. Ges. inn. Med., 1949, 4, 415.4 7 Schwartz, Hawkinson, Cohen and Watson, Science, 1946,103,338 ; J . Biol. Chem.,** Wat.son, Hawkinson, Schwartz, and Sutherland, J . Clin. Incest., 1949,28,447.50 " Heavy metal prosthetic groups and enzyme action " (trans. Lawson), Clarendon5a Ibid., in the press.53 Rimington, Hale, Rawlinson, Lemberg, and Falk, 1949, Abstracts 1st Inter-1947,168, 133.Rimington, Fulton, and Sheinman, Biochem.J . , 1947, 41, 619.Press, Oxford, 1949.national Congress of Biochemistry, pp. 351, 378, 379.51 Biochem. J . , 1949,45, 247.54 NdtuTWiS8., 1946, 53, 123.5 6 Onderstepoort. J . Vet. Lcci. Animal Ind., 1950, $34,105. 5 6 Biochem. J . , 1950,40,220278 ORGANIC CHEMISTRY.A prodigiosin-like pigment, which may be a higher homologue of prodi-giosin itself, has been des~ribed.~' I n this case the organism was a mould(Actinmycetes), not a bacterium.Bacterial Porphyrins.-The porphyrin, produced by Corynebacteriumdiphdheriae grown in an iron-deficient medium, has been reinvestigated bychromatographic techniques and shown to consist of coproporphyrin IU:together with smaller quantities of uroporphyrin I and porphyrins with fiveand six carboxyl groups 68 Coproporphyrin 111 has alsobeen identified as a cellular constituent of several mycobacteria. 69Uroporphyrins.-The presence of a porphyrin in the urine of Petry, apatient with congenital porphyria, had been noted by Salkowski, Giinther,Schumm, and other early workers but all had confused it with haemato-porphyrin produced chemically from haemin by Nencki.In 1916 Fischer 6oisolated the main porphyrin from Petry's urine and obtained its methyl esterin hair-like crystals, m. p. 293", in quantities of 200-300 mg. per day. Hea t first described it as a heptacarboxylic acid, but later analytical data anddetermination of the carbon dioxide yielded on decarboxylation showed thatthere were eight carboxyl groups in the The most convenientmethod for partial decarboxylation is heating with 1% HCI a t 180-190",624 mols.of carbon dioxide being lost and a coproporphyrin produced. Identi-fication of the isomer type of the latter indicates that of the parent uropor-phyrin also and is thus a valuable aid to structural characterisation. Theuroporphyrin from Petry's urine belonged to the stioporphyrin series I.Of the three possible structures for uroporphyrin I , 'uiz., 1 : 3 : 5 : 7-tetramethylporphin-2 : 4 : 6 : 8-tetrakismethylmalonic acid, 1 : 3 : 5 : 7-tetramethylporphin-2 : 4 : 6 : 8-tetrasuccinic acid, and 1 : 3 : 5 : 7-tetrakis-carboxymethylporphin-2 : 4 : 6 : $-tetrapropionic acid, the last was eventuallyaccepted by Fischer and Hofmann 63 because the carboxylated haematinicacid produced by chromic oxidation of natural uroporphyrin was found to beidentical with a synthetic preparation of the expected material (XIV) .and was stated to bederivable from turacin, the copper-containing pigment of turaco feathers.65Preparations from urines of patients with porphyria exhibited m.p.s (ofthe octamethyl esters) ranging from 255" to about 290". In 1936 Walden-strom 66 and Mertens 67 independently reported the isolation, from urines ofacute porphyria patients, of a new uroporphyrin with ester m. p. 255-260".Since on decarboxylation it yielded coproporphyrin I11 it was claimed thatUroporphyrin I was also found in marine shells6 7 Dietzel, Naturwise., 1948, 35, 345 ; 2.physiol. Chem., 1949, 284, 262.6 8 Gray and Holt, Biochem. J., 1948,43, 191.6 1 Fischer and Hilger, ibid., 1925, 149, 65.62 Fischer and Zerweck, ibid., 1924.137, 242. 6s Ibid., 1937, 246, 15.84 Fischer and Haarer, ibid., 1932, 204, 101; Nicholas and Comfort, Biochem. J . ,1949, 45, 208 ; Comfort, Nature, 1948, 162, 851 ; Science, 1950, 112, 279 ; Tixier, Bull.SOC. Chim. biol., 1946, 28, 394.6 5 Fischer and Hilger, 2. physiol. Chem., 1923,128, 167 ; 1924,138,49.6 8 Deut. Arch. klin. Med., 1935, 178, 38; 2. physiol. Chem., 1936, 239, iii; Walden-etrom, Fink, and Hoerburger, ibid., 1935, 233, 1. 67 Ibid., 1936, 238, i ; 1937, 250, 57.69 Todd, ibid., 1949, 45, 386.2. physiol. Chem., 1915, 95, 34FAT;# AND RIMMQTON : PORPHYRINS. 279this pigment was uroporphyrin 111.In 1945, however, Watson et U Z . ~ ~disputed the homogeneity and nature of such " Waldenstrom esters,"claiming that by chromatography on calcium carbonate they were often (butnot always) separable into a zone regarded as uroporphyrin I (ester m. p.284") since it yielded coproporphyrin I on decarboxylation, and a secondzone (ester m. p. 208') believed from analytical data to be a heptacarboxylicporphyrin (of the isomeric series I11 since it yielded coproporphyrin I11 ondecarboxylation) . Even chromatographically homogeneous Waldenstromesters were stated to yield mixtures of the I and the I11 series coproporphyrinson decarboxylation. Support for these claims has been offered by P r ~ n t y . ~ ~ ~Watson's view is that the I and I11 series porphyrins in the Waldenstromesters form molecular associations which crystallise as an individual material,but the contradictory decarboxylation results obtained by Waldenstrom andMertens on the one hand, and Watson and Prunty on the other, are difficult toreconcile.The identity of the uroporphyrin in turacin has also been called in question.Fischer and Hilger 65 carried out no decarboxylation of their supposeduroporphyrin I ; Rimington 70 found that turacins from eleven differentspecies of Turacos yielded only coproporphyrin 111 when decarboxylated.This has recently been confirmed by Nicholas and Rimington 71 who haveprepared unequivocal uroporphyrin 111 in pure state from turacin.It hasm. p. 264", similar to that of the Waldenstrom ester.Porphobilinogen.-In acute porphyria urines, Waldenstrom 72 detected asubstance, porphobilinogen, a colourless precursor of uroporphyrin.Furtherstudies 73 suggested that porphobilinogen had a molecular weight (by diffusion)of about 350 and thus contained only two pyrrole rings; when this materialwas heated in acid solution, union of two molecules took place. The mainproducts of this reaction were an amorphous pigment, porphobilin (probablyrelated to urobilin) and uroporphyrin 111, together with some of the I isomer.Porphobilinogen can be detected by the formation, on treatment withEhrlich's aldehyde reagent, of a red pigment not extractable by chlor~form.~~Its excretion appears to be characteristic of acute porphyria, Hammond andWelcker 75 having found no false positive reactions in urine of 1000 casesexamined,The Ehrlich reaction affords a method for quantitative determination.76The conversion into urDporphyrin has been the subject of special studies.77* 76That the porphyrin obtained from porphobilinogen-containing urines by6 8 Grinstein, Schwartz, and Watson, J . Biol. Chem., 1945, 157, 323; Watson,Schwartz, and Hawkinson, ibid., p. 345.6B Arch. intern. Med., 1946, 77, 623.7 1 Personal communication, 1950.73 Waldenstrom and Vahlquist, 2. physiol. Chern., 1939, 260, 189.7 5 J . Lab. Clin. Med., 1948,33, 1254.76 Jorgensen and With, Nord. Med., 1945, 27, 1341; Prunty, Biochem. J . , 1945,'I7 Grieg, Askevold, and Sveinsson, Scand. J . Clin. Lab. Invest., 1950,2, 1.70 PTOC.Roy. SOC., 1939, B , 127, 106.72 Acta Med. Scand., 1934, 83, 281.Watson and Schwartz, Proc. SOC. exp. Biol., 1941,47, 393.39, 446280 ORGANIC CHEMISTRY.boiling is not the same as that present in the fresh urine is suggested byGibson and Harrison.78 .Biosynthesis of Porphyrins.-The last ten years have seen a remarkableincrease in knowledge concerning the materials and methods utilised byliving cells in synthesising substances containing the porphin ring. Isotope-labelling techniques have been almost entirely responsible. The N atom ofglycine is specifically utilised for haem production in man 79 and in avian andimmature mammalian erythrocytes in vitro. The rate of disappearanceof labelled haem from the blood stream in the first case leads to the conclusionthat haemoglobin is outside the general metabolic interchange, and alsopermits an estimate of normal red-cell longevity.81 Since rings (I and 11)and (111 and IV) of haem are labelled to an equal extent, it is probable thatglycine supplies the nitrogen of all four pyrrole rings.82 Carbon labellingshowed that the methylene- 83* but not the carboxyl-carbon atom 83* 85 ofglycine is incorporated in haem.Both carbon atoms of acetic acid are alsospecifically utilised,86 making a contribution, most probably, to the pyrroleP-side chains. The methene carbon atoms of the porphin ring are derivedfrom gly~ine.~' By means of a careful, step-wise degradation of the haeminsynthesised by avian erythrocytes in the presence of N- and a-C-labelledglycine, Wittenberg and Shemin 88 have localised the C atoms derived fromglycine as in (XV).The major steps in the degradation were as follows :fro, Haemin -+ Protoporphyrin --+ MesoporphyrinEthylmeth ylmaleinimidefrom rings I & I1E t h ylme th ylmaleinimide --C%Haematinic acidNaClO, E thylmethylmaleinimides (separately) oso,> EthylmethyltartarimideEt*CO,H =MnO4 x-Ketobutyric Pyruvicacid acid78 Biochem. J . , 1950, 46, 154.70 Shemin and Rittenberg, J. BioE. Chem., 1946, 166, 621. 'Shemin, London, and Rittenberg, ibid., 1950, 183, 749, 757.Shemin and Rittenberg, ibid., 1946,166, 627.8a Muir and Neuberger, Biochem. J., 1949,45, 163; Wittenberg and Shemin, J. BioE.83 Radin, Rittenberg, and Shemin, &id., 1950,184, 745.Chem., 1949,178,47.Altman, Casarett, Masters, Noonan, and Salomon, ibid., 1950, 176, 319; Altman,Salomon, and Noonan, ibid., 1949,177,489 ; Altman and Salomon, Science, 1950,111,117.8 5 Grinstein, Kamen, and Moore, J .Biol. Chem., 1948, 174, 767; 1948,179, 359.86 Bloch and Rittenberg, ibid., 1945,159, 45 ; Radin, Rittenberg, and Shemin, ibid.,1950, 184, 755 ; Pontecorvo, Rittenberg, and Bloch, ibid., 1949,179, 839.87 Muir and Neuberger, Biochem. J., 1949, 46, xxxiv; 1950, 47, 97; Wittenbergand Shemin, Fed. Proc., 1950, 9, 247. J . Biol. Chem., 1950,185, 103FALK AND RIMINGTON : PORPHYRINS. 281Radioactivity * was confined to the carboxyl-carbon atom of a-ketobutyricacid and the carbon dioxide arising from the four methene carbon atoms of theCH,:CH MeMe/\\Y+\\/%H:CH, ~LNH NA (0 indicates C derived from h (XV.) methylene of glycine.)HO,C*CH,*CH, CH,*CH,*CO,Hring in the initial chromic acid oxidation.The fact that eight glycinea-carbon atoms are utilised for every four nitrogen atoms 89 indicatesthe occurrence of deamination reactions to provide a C, residue. Neitherformate nor carbon dioxide is utilised for haem synthesis by thissys ternLemberg and Legge 3a and others 91 have reviewed various theories of thebiosynthetic mechanism. Neuberger, Muir, and Gray 92 recently suggestedthat the initial stage is the union of 2 molecules of a-ketoglutaric acid and1 molecule of glycine to give a highly carboxylated pyrrole derivative (XVI)from which the two a-carboxyl groups are then lost, to give (XVII).Addi-tion at one or other a-position of a two carbon fragment (e.g., glyoxylic acid)is then thought to occur, to give, after decarboxylation, either A (XVIII)or B (XIX). Porphyrin formation is postulated by condensation of foursuch units (cf. Siedel and Winkler lo).HO,C*CH, CH,*CH,*C02H I H02C-CH, CH,*CH,*CO,H(XVII .)H02C1Q1C0,H H (XVI.1 \g'If no restriction is placed on this final stage, all four porphyrin isomers couldbe expected. Since only types I and I11 are found in nature, it is postulatedthat in the condensation, the M (potential methene) groups of B but not ofA can be activated enzymically, while both units can act as acceptors of thepotential methene carbon atoms. Such a condition would preclude A-Alinkage and give rise to porphyrin molecules of only the I or the I11 series, thelatter predominating.Isotopic studies have been made of the biosynthesis of porphyrins andRadin, Rittenberg, and Shemin, Fed.Proc., 1949, 8, 240.Bufton, Bentley, and Rimingtoh, Biochem. J . , 1949,44, xlix.01 Shemin, Cold Spring Harbor Symp., 1948, 13, 185; Maitland, Quart. Reuiews,1950,4,45; Rimington, ref. 3b. 9a Nature, 1950, 165, 948282 ORGANIC CHEMISTRY.bile pigments in porphyria and other diseasesQ3 and of bile pigments innormal man.94 The conversion of injected haematin into bile pigment 95has also been demonstrated by this means. The biosynthesis of chlorophyllhas been studied; both glycine and acetate are specifically utilisedjS6 andprotoporphyrin Mg complex, Mg vinylphaeoporphyrin-a5, and protochloro-phyll have been identified as intermediate The relation betweenthe biosynthesis of porphyrins and haems by C .diptheriae has been investi-gated.gs An important role of lactoflavin in regulating the porphyrinproduction of yeast is described by S t i ~ h . ~ ~ Pyridoxine 99a, and folic acid andvitamin B,, s9b may also be important in porphyrin biosynthesis.Physicochemical Aspects.-The infra-red spectra of coproporphyrins I andI11 and stercobilin,lm and of zetioporphyrin I lol have been measured.Vestling and DowninglO1 found a band at 3320 cm.-l in aetioporphyrinin carbon tetrachloride solution which was considered lo1a to be due to a NHvibration lowered by H bonding. This has been confirmed by Willis andFalk,lolb using protoporphyrin; it was found that there is only a smallfrequency shift (from 3320 to 3280-3200 cm.-l) for the NH vibration ongoing from dilute carbon tetrachloride solution to the solid state.Infra-redspectra can distinguish porphyrin isomers of the I and I11 series,100. l o l b andtentative assignments have been made for the frequencies of different typesof carbonyl groups in porphyrin side chains.lolb Rabinovitch lo2 attempteda theoretical interpretation of the absorption spectra of porphyrins andchlorophyll. Kuhn lo3 has calculated porphyrin spectra on the basis of hisuniform-potential, free-electron-gas model ; this has been criticised byDewar.lm Simpson lo5 made a quantum-mechanical interpretation somewhatsimilar to Kuhn's.The molecular-orbital method has been applied byLonguet-Higgins, Rector, and Platt lo6 to porphin and tetrahydroporphin.83 London, West, Shemin, and Rittenberg, J. Biol. Chem., 1950,184,365; Neuberger,Muir, and Gray, Nature, 1950, 165, 948 ; Grinstein, Aldrich, Hawkinson, and Watson,, J . Biol. Chem., 1949, 179, 983 ; Gray and Neuberger, Biochem. J., 1950, 47, 81 ; Gray,Neuberger, and Sneath, ibid., p. 87; Grinstein, Wikoff, de Mello, and Watson, J. Biol.Chew., 1950, 182, 723; London, Shemin, West, and Rittenberg, ibid., 1949, 179, 463;London and West, ibid., 1950,184, 359.84 London, West, Shemin, and Rittenberg, ibid., p. 351.9 5 London, ibid., p. 373.Q6 Salomon, Altman, and Della Rosa, Fed. Proc., 1950,9, 222.Gilder and Granick, J.Gen. Physiol., 1947, 31, 103; Granick, J. Biol. Chem.,Q* Hale, Rawlinson, Gray, Holt, Rimington, and Wilson Smith, Brit. J. Exp. Path.,OD Naturwisa., 1950, 9, 212; Deut. med. Woch., 1950, 37, 1217; Stich and Eisgruber,1948,172, 717; 1948,175,333; 1950,183, 713.1950, 31, 96.Klin. Woch., 1950, 28, 133.Cartwright and Wintrobe, J. Biol. Chem., 1948, 172, 557.BOnard, Gajdos, and Gajdos-Torok, Compt. rend. SOC. Biol., 1950, 144, 38, 350.loo Gray, Neuberger, and Sneath, Biochem. J., 1950, 47, 87.lor Vestling and Downing, J. Amer. Chem. SOC., 1939, 61, 3511.lola Ruswell, Downing, and Rodebush, ibid., p. 3252.lol* Willis and Falk, personal communication, 1950.lo2 Rev. Mod. Physics, 1944,16, 226.lo' J., 1950, 2329.lo9 J .Chem. Physics, 1949,17, 1198.lo6 J . Chem. Physics, 1949, 17, 1218. lo6 Ibdd., 1950, 18, 1174BALK AND RIMINQTON : PORPHYRINS. 283Preliminary X-ray crystallographic analysis lo’ shows that tetramethyl-haematoporphyrin, like the phthalocyanines, is probably planar. A verybrief X-ray examination has also been made of aetioporphyrin I crystals.lo8From X-ray crystallographic studies of haemoglobin, the molecule appearsto be composed of superposed layers of folded polypeptide chains, the haemresidues being attached tangentially ; Perutz’s log* 3* data indicated fourpolypeptide layers in the structural unit, but Dornberger-SchS’s 110calculations of Patterson and Fourier projections suggest a unit of 7layers. Myoglobin 1 1 1 s 3 b appears to be closely analogous in structure tohaemoglobin.In studies 112 of the photo-oxidation of the zinc complex of tetraphenyl-chlorin evidence has been found for an intermediafe triplet state of the chlorinmolecule when p-naphthaquinone is the oxidising agent.This chlorin isoxidised by o- or p-quinones to the corresponding porphyrin, the zinccomplex eight times as fast as the magnesium complex; oxygen also acts asoxidising agent in a similar manner, a secondary reaction with hydrogenperoxide leading, however, to further oxidation. In a study of substanceswhich do and do not quench the fluorescence of chlorophyll-a, it was found 113that those which quench most efficiently are oxidising agents. The complexstructure of the fluorescence spectrum of the magnesium complexes ofphthalocyanine and chlorophyll has been studied.l14Clark and his collaborators have continued their systematic studies of thedissociation constants of ferriporphyrins and of other metalloporphyrins.Values of pK have been determined for the dissociation of ferriproto-porphyrin hydroxide (hydroxyhaemin) 116* 117 and of nicotine ferro-porphyrins (haemochromogens) .l18 Older values for the haemochromogensformed by ferroprotoporphyrin with a number of different bases have beensummarised.llQ A value has been given for the dissociation of hydroxylfrom ferricoproporphyrin hydroxide, and for the dissociation of hydroxylfrom some ferriporphyrin base hydroxides (hydroxymethaemochromo-lZo Lemberg 1-21 has given a pK value for pyridine protomethaemo-chromogen. Because the equilibrium involved is not well understood, pKvalues are not useful as an index of the affinity of bases for ferriporphyrins;it had been found, however, that the linkage of bases to ferroprotoporphyrinlo7 O’Daniel and Damaschke, 2.K r i ~ t . , 1942, 104, 114.lo8 Robertson, Amer. Min., 1942, 27, 219.loo Proc. Roy. SOC., 1949, A , 195, 474.ll1 Kendrew, Proc. Roy. SOC., 1950, A , 201, 62.112 Calvin and Dorough, J . Amer. Chem. SOC., 1948,70,699; Huennekens and Calvin,113 Livingston and Chun-Lin Ke, ibid., 1950, 72, 909.11‘ Gachkovskii, Doklady Akad. Nauk. S.S.S.R., 1950, 71, 509.116 Clark, Cold Spring Harbor Symp., 1939,7, 1.116 Clark and Perkins, J. Biol. Chem., 1940,135, 643.118 Davies, ibid., 1940, 135, 597.120 Vestling, J . Biol. Chem., 1940, 135, 623.121 Lemberg and Foulkes, quoted in ref. 3a, p. 178.1 1 O Acta Cryetall., 1950, 3, 143.ibid., 1949, 71, 4024,4031.Shack and Clark, ibid., 1947, 171, 143.119 Ref. 3a, p. 175284 ORGANIC CHEMISTRY.(haem) is stronger than to ferriprotoporphyrin hydroxide,122 and it has beenpossible 123 to calculate the approximate extent to which the affinity of ferro-porphyrins for bases is greater than that of ferriporphyrins.The (ferrous-ferric) oxidation-reduction potentials of iron-porphyrincompounds have been studied very intensively, the haem-haematin systemand the haemochromogen-methaemochromogen system by Barron lZ4 andby Clark’s school.l18* l2Ov 122,125 Lemberg and Legge have discussed Clark’stheoretical treatment in detai1.126 Systems where the base is protein innature have also been studied, e.g., haem~globin,~~~ myoglobin,12* and cyto-~hr0me-c.l~~The combination of cyanide and alkyl cyanides, and of carboalkylamineswith ferro- and ferri-porphyrins has been further studied.130