CHLOROPHYLL and HEMOGLOBINTWO NATURAL PYRROLE PIGMENTS

8/9/2019 CHLOROPHYLL and HEMOGLOBINTWO NATURAL PYRROLE PIGMENTS

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CHLOROPHYLL nd HEMOGLOBIN-T W O NATURAL PYRROLE PIGMENTS

EMM M. DIETZ*

Harvard University Cambridge Massachusetts

C LOROPHYLL is the brilliant green pigmentfound in certain special cells of all green plants.I t absorbs energy from the sun and in some un-known way uses it for the manufacture of sugar,starch, and proteins 1). This important process isknown as photosynthesis and ultimately provides foodfor all plants and animals on the earth. Hemoglobinis the red pigment coloring the red blood cells of animals.It is necessary for t he maintenance of l i e through res-piration-the process which provides a constant sup-ply of oxygen to the tissues. In the slow development

of th e chemistry of these two pigments, it has been anincreasing source of wonder to chemists to find thattwo substances of such widely different origin and func-tion are yet so remarkably similar in structure.

The pure pigments as isolated in the laboratory arevery differen t n appearance. Chlorophyll, when sepa-rated from the leaf s truc ture , is a dark green wax whichis really not one substance but two closely related ones,called chlorophyll a and by Willstat ter. Hemoglobinis a compound molecule consisting of a colorless pro-tein (globin) attached to four molecules of a red-browncrystal line pigment called heme. In t he process ofisolation from hemoglobin, heme changes over to itsfamiliar oxidation product, bemin, which is a brown-

red crystalline compound of high melting point. Whenchlorophyll is dissolved in ether or chloroform a brilliantgreen solution resu lts; hemin forms a red-brown solu-tion. When observed through a spectroscope, solu-tions of either pigment show the remvkabl e property,due to similar elements of s truc ture, of absorbing sharpbands of visible light. There results an unusual bandedspectrum in the visible region, which is characteristicfor each compound and serves as a valuable means ofidentification. An important diierence between thetwo pigments which is immediately apparent from achemical analysis, is that chlorophyll contains mag-nesium, whereas hemin contains iron incorporated inthe large organic molecule.

The widespread distribution and importance to lifeof both substances early stimulated the interest of sdeu-tists. The chemistry of chlorophyll was confused forsome time by the use of dras tic methods of isolationwhich altered or destroyed the molecule and introducedmetallic impurities. I t is still a matt er of concern tothe chemist to find means of isolat ing this complex

Sarah Berliner Fellow. American Association of UniversityWomen 1934-1935 at present engaged in research at the Uni-versity of Munich Munich Germany

material without introducing subtle changes in structure.Heme, the pigment now believed to be present in hemo-globin, was long mistaken for its close relative hemin,to which it is readily converted during isolation.There are many similar instances in the history of thechemistry of natural products where faulty isolationbas obscured the true structure f or years.

RELATIONSHIP BETWEEN CHLOROPHYLL ND HEMIN

The first suggestion of a relationship between heminand chlorophyll was made as early as 1851 by Verdeil

Z), hough on th e basis of invalid evidence. He un-wittingly introduced iron into his preparations ofchlorophyll by employing crude methods and reagents,and as a result thought that it might be related tohemin, which was already known to contain iron. Ac-tually, the similarity in the two pigments is not in themetallic constituents b ut in the rest of the molecule.However, Verdeil s hypothesis stimulated research onth e problem for years and culminated in the series ofbrilliant investigations by Hans Fischer for which hewas awarded the Nobel Prize in 1930 3). He andhis co-workers fmally established the correct structur eof natural hemin and heme by synthesis, and showedtheir true relationship to chlorophyll. Research onthe plant pigment benefited enormously by these re-sults, but its structure is not yet absolutely clear and itscomplete synthesis has not been accomplished. In t hepresent discussion of the subject i t has been found con-venient to follow th e historical development, and t oconsider 6rst the structure of hemin and its relation-ship to chlorophyll, before presenting the detailedstructure of the latter.

The 6rs t real evidence of the connection betweenchlorophyll and hemin was theformation of very similarred,.crystalline compounds called porphyrins, from bothblood and plan t pigments. These were first obtainedby Hoppe-Seyler (4) in 1879, later by Schunck and

Marchlewski (5) by drastic chemical treatment of thetwo pigments. These porphyrins formed bright redsolutions in ether wi th str iking four-banded absorptionspectra in the visible region, and they were actuallyclose chemical relatives though not identical as a t firstsupposed.

In 1901, Nencki (6) reduced both hemin and a crudepreparation of chlorophyll to mixtures of volatile basescalled pyrroles (seeI),thus showing for the f i s t ime th atpyrrole nuclei were involved in the structure of bothpigments. Willstatter (7) later identified the same


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pyrrole fragments from both chlorophyll and hemo-globin, thus proving a very close relationship. Kiister(8) studied the products of drastic oxidation (11) aswell as reduction and proposed in 1910 the correctgeneral formula for the nucleus common to the por-phyrin, hemin, and chlorophyll molecules. It was notuntil twenty years later, however, that the Kiisterformula was completely justified by the synthetic re-sults of Fischer.

THE FORMULA OF HEMIN

A detailed examination of th e hemin formula willbring out several features, shared by chlorophyll aswell. It contains four pyrrole, or modified pyrrole nucleibound into a large symmetrical ring structure by fourcarbon atoms. For purposes of nomenclature, th eunsubsti tuted molecule is called a porphin ring. Inhemin there is an unbroken alternation of single anddouble bonds such as is assumed for all porphyrins,and is found on a smaller scale in benzene. As in ben-zene, th e exact location of each bond is not determinedand various arrangements are possible. The evidenceof years indicates tha t most compounds exist in onlyone of the possible electromeric forms, and it remainsfor physico-chemical methods, such as electrometrictitration [as applied by Conant t o some chlorophyllderivatives 9 ) ] to determine which form is present.

Heme Protoheme X F e + +Hcmin Protohemin X l eCltt

Only one possible electromeric structure of each com-pound is shown here for convenience.

In hemin (Formula 111; X FeCl++) two hydrogenatoms of the parent porphyrin, protoporphyrin (For-mula IV; X -CH=CHe), have been replaced by anFeCl++ grouping which is doubtless coordinately boundto all four nitrogen atoms. In heme (Formula 111:

.

H

Methyl Ethyl Maleie Imlde

X Fe++) the iron is in the reduced form, withoutthe halogen atom. The interconversion between proto-porphyrin and its two iron derivatives is readily ac-complished in the laboratory. On dras tic reduction ofhemin and heme the metal drops out and the porphinring breaks on either side of the bridge carbon atoms,forming the pyrroles shown in I ; oxidation removes thebridge carbon atoms completely, forming hematinicacid (see 11) as stated above.

I t might be mentioned here tha t hemin and heme areclass names applied to Fe el ++ and Fe ++ complexes ofall porphyrins, but have been adopted as the specificnames for the blood pigment derivatives, more cor-rectly called protohemin and protoheme. Further, theuse of non-systematic names, wherever possible in thisfield, is preferable to the very lengthy Geneva nomen-clature, though somewhat confusing a t first. Theseincidental names are usually derived from Greek stemsdenoting color, i e. chlorophyll green leaf, porphyrin

purple.:

BLOOD ND CELOROPHY2.L WRPHYFSNS

The correctness of these, structures for protopor-phyrin and its iron derivatives was proved throughthe isolation and identification of their degradationproducts and finally through t he synthesis of the whole

series of blood porphyrins by Fischer. In protopor-phyrin, as in hemin, there a refourmethylgroups (-


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Hemoglobin Globin 4 Hemexidation

HeminJ -F l

Protoporphyrin

+4H

Synthetic Mesoporphyrin IX Mesoporphyrin

Synthetic Btioporphyrin I11 -

groups forming ethyl groups (-GHd, (Formula VII,X CHtCH2COOH). Bi op orphyr in (more accu-rately called meso-etioporphyrin) is th e oxygen-freeand completely alkylated compound in which even thetwo propionic acid groups of proto- and mesoporphy-rins have been converted to ethyl groups by loss of

carbon dioxide (Formula VI; X C2H Meso-etioporphyrin consists therefore of a porphin ring sub-stituted by four ethyl and four methyl groups and isa very stable compound of high melting point. Theserelationships are summarized above (V). I t is evidentfrom the formula that four isomers of this aetiopor-phyrin are possible, depending on the order of the alkylgroups around the porphin ring. The etioporphyrinsare of great interest since they represent th e final stagein th e drastic a W m e degradation of chlorophyll aswell as of hemin.

HrVI

Meso-=tioporphyrin X - CIHZ Blwd Derivative)Pyryrm-etioporphyrin X - H (Chlmophyll Derivative)

Chlorophyll is not truly a porphyrin derivative likehemin but it is nevertheless capable of yielding a series

of porphyrins when treated with suitable reagents.Thus on drastic alkali treatment one obtains a dibasicacid, rhodoporphyrin (Formula VII ; X COOH), andfrom this by loss of carbon dioxide a monobasic acid,pyrroporphyrin (Formula VII; X H). I t will benoted that mesoporphyrin is really pyrroporphyrinwith a propionic acid group replacing the hydrogenatom a t position 6. Further removal of carbon dioxidefrom pyrroporphyrin forms the oxygen-free, completelyalkylated porphyrin derivative, pyrro-zetioporphyrin-th e aetioporphyrin of the chlorophyll series. (For-mula VI; X H.)

Willstatter prepared the etioporphyrins from bothhemin and chlorophyll (10) and believed them to beidentical. Actually, meso-etioporphyrin, the sptio-porphyrin of blood, contains an ethyl group a t positionin place of a free hydrogen atom of the chlorophyllderivative, but th e rest of the groups are identical and

in the same order. Therefore meso-etioporphyrin isreally 6-ethyl-pyrroetioporphyrin nd both chloro-phyll and hemin may thus be regarded as related toa common porphyrin.

PORPHYRIN SYNTH SIS

The complete demonstration of thi s relationship andth e elucidation of the structures of th e individual bloodand chlorophyll porphyrins did not occur until afterFischer's enormous development of synthetic pyrroleand porphyrin chemistry extending over fifteen years.

Meroporphyrin X CH,CH,COOHPyrroporphytin X - HRhodoporphyrio X COOH

.He not only found methods for synthesizing the naturalpyrrole degradation products of the porphyrins bu t also

learned how t o join these in to so-called dipyrryl meth-enes of known structures and finally to couple suitablepairs of these methenes to form porphyrins, whosestruc tures would therefore also be known. VIII showshow two differen t pairs of methenes were combiied toform the same porphyrin (11). The f a d tha t bothsyntheses yielded a porphyrin with the same spectrumand melting point showed that no rearrangement hadtaken place and made the assigned structure morecredible.

This last step in t he synthesis of porphyrins is apyrolysis carried out in an organic acid melt, in which


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carbon dioxide, halogen acid, hydrogen, and freehalogenmay be lost as in this instance. The method is ratherlaborious as the yields are usually low and isomericporphyrins almost always result. It may be claimedthat such syntheses are too complicated in mechanismto afford an absolute proof of structure, bu t the consis-

tency of t he cumulated da ta lends weight to the methodand certainly makes it valid as confirmatory to theevidence from degradation reactions.

H z C = = C ~ H II

group, obtaining a product identical with mesopor-phyrin from hemin. The reverse transformation ofmeso- to pyrroporphyrin has also been accomplished

16), though in a more complicated series of steps.Such interconversions afford final proof th at the

nucleus and order of substituent groups in chlorophyll

and hernin are the same and perhaps make a transitionbetween the two pigments in the animal body moreprobable. As will appear later, however, the pigments

H a C 7 C H rI

IS L C I H ~ H ; L C Z sVIII Two SYNTEESE~ 8 P o a m w o ~ m e m s

By similar synthetic methods Fischer has success-fully prepared several blood and chlorophyll porphyrins,thus establishing beyond a doubt t he Kiister ring for-mula for the nuclei of both pigmentsand thefundamentalrelationship between them. For example, he synthe-sized twelve of th e fifteen possible isomers of meso-porphyrin and proved th at one of these, called meso.porphyrin IX ll ) , is identical with the natural prod-uct as judged by mixed melting-points and absorp-tion spectra. He also prepared the four possible meso-

aetioporphyrins Formula VI; X CsHr) and identi-fied that obtained from hemin as ztioporpbyrin I12). In the chlorophyll series, furthermore, he suc-

ceeded in preparing natural pyrro- and rhodoporphy-rins Formula VII) by the use of similarsyntheticmeth-ods 13).

The synthesis of hemin itself was accqmplisbed in anumber of steps starting with deuteroporphyrin 14)

Formula IV, X H) which was prepared from meth-enes by a method similar to that shown in VIII, butwhich also occurs in nature as a putrefaction product ofblood. Two acetyl groups were introduced into deu-teroporphyrin, these were reduced to -CHOHCHsgroups and then dehydrated, the product being proto-

porphyrin Formula IV, X -CH=CHs), whichwas readily converted into i ts FeCl++ complex, bemin.Up to th is point the proof th at both chlorophyll and

blood porphyrins are built on the same structural plan,with the same order of substituents on the porphinring, rested on independent syntheses in each series.The final step consisted in attempting a transition be-tween the two series without possibility of rearrange-ment. This was also achieved by Fiscber in the inter-conversion of pyrro- and mesoporphyrins. Thu sFischer and Riedl 15) succeeded in replacing the freebeta position in pyrroporphyrin by a propionic acid

themselves are far less closely related than these twoporphyrins and thus far the nature of their degrada-tion products in the animal body and in the laboratorydoes not support such a possibility.

WILLSTATTER S INVESTIGATIONS ON C~LOROPAYU

Our knowledge of t he exact chemistry of chlorophyllis due largely to t he remarkable researches of Will-s e t t e r and his co-workers 17). notably Stoll, from 1906to 1914. They developed methods of isolation and

purification and studied a number of degradation prod-ucts, but the formulas which they suggested weresubsequently proved to be somewhat incorrect. Re-search on the subject was taken up from this point inabout 1927 by Fischer and by Conant, and has con-cerned itself mainly with the proof of *he Kiister struc-ture for th e nucleus, with the study of the many deg-radation products, and with the identification of cer-tain very labile groupings in chlorophyll and its im-mediate derivatives.

To Willstatter we are indebted for a mild isolationprocedure permitting extractioq of tbe unaltered pig-ment from the plant, and for the invaluable methodof sepkration and purification of reaction products by

acid fractionation. This process makes use of thevarying basicity of chlorophyll derivatives which en-ables them to be extracted separately from a mixturein ether solution, by agitation with hydrochloric acidof suitable coucentratious. W i s e t t e r further showedth at there are two chlorophylls in nature, which he calleda and b, with the nearly constant ratio of 2.9 to n thehigher plant forms, but with chlorophyll b apparentlyabsent in certain alga?. He determined the correctanalytical formula of chlorophyll a Ca6H~sNaOsMg)and noted that the two chlorophylls differed by a singleoxygen atom. He proved the presence of magnesium


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and correctly characterized the two ester groups asbeing formed from methyl alcohol and phytol GoHa-OH). The latte r was a new alcohol with a long straightcarbon chain whose structure was established in 1929through a brilliant synthesis by Gottw alt Fischer 18).

Chlorophyll is readily obtained in a u d e form by

extraction of green leaves with aqueous acetone. Will-statter accomplished the separation into a and b com-ponents by distribution between methyl alcohol andpetroleum ether. A bette r method is th at of chromato-graphic adsorption on powdered sugar according to thedirections of Winterstein and Stein 19) who thus ob-tained very pure samples. This adsorption methodhas recently found considerable application in theseparation of closely related natural products. Freshlyisolated chlorophyll a and b were found by Stoll to beslightly optically active 20). Conant and Dietz 21)and Zschiele 22) have reported indications of a th irdform of chlorophyll. Wi te rs te in and Schon 19b)failed to find a chlorophyll in carefully controlled

adsorption experiments.In alcohol solution and in th e presence of the plant

enzyme, chlorophyllase, the phytyl group of chloro-phyll is replaced by a methyl or ethyl group to formcrystalline derivatives called chlorophyllides. M i dacid treatment of chlorophyll acts only to remove themagnesium atom, forming phaeophytin, a dark, waxysolid which finds commercial use in the fo m.of itsstable and brilliantly colored copper salt, a non-poison-ous green coloring material. More drastic acid treat-ment of chlorophyll a with concentrated hydrochloricacid) hydrolyzes the phytyl group and removes themetal as well, forming crystalline phaeophorbide a amonomethyl ester. This is easily est erz ed to a di-

methyl ester, methyl phgophorbide a The accom-panying diagram IX) shows these transformationswith t he accepted compositions of the various products.

the hasis of color and characteristic absorption spectra.More drastic alkali treatme nt of chlorophyll or anyof the above-mentioned degradation products, removesvarious groups leaving the simple dibasic and mono-basic acids, rhodo-, pyrro-, and phyllo- y-methylpymo-) porphyrins, all of which have been synthesized

by Fischer as described above.THE STRUCTURE OF CHL0ROPJiYL.L

We are now ready t o examine the formula of chloro-phyll in some detail. During the last few years threesomewhat dierent formulas have been proposed byFischer 23), Conant @I) , and Stoll 24) on the basisof results obtained in their respective laboratories.These are shown in formulas X, XI, and XII . TheFischer structure has undergone numerous revisions,the latest being shown here.) At the present time theFischer formula seems to he the best representation ofthe existing data, although some points are not yetclear. ll three st ructures are more fully discussed inth e excellent reviews of Fischer 25) and Stoll 26),and in a very complete account by Armstrong 27),which summarize the vast body of accumulated fac tsup to July, 1933.

EtOH and conc. acidchlorophyllase H1C CHO H

Ethyl Chlorophyllide a dil, acid Phmphorbide aCOOCH,COOH HC- H

lalkali 2 CH 2H2COOGoHloChlorin N Mg N

XHC-

Concentrated alkali brings about a more fundamental HsG-~ H ~

change in chlorophyll and in the phaeophorbides, de- XI. STOLL 933

veloping a third acid group, the product being the tribasic acid chlorin e from the a component and rhodin g The three formulas are, of course, similar inqgrossfrom the b component; both acids form trimethyl esters. structure since they involve a common interpretationHere, as in the case of the blood pigment, t he nomen- of the dat a given above. Thus all contain five oxygenclature is non-systematic and the class names such as atoms, in accordance with th e recent analyses of verychlorin, phzeophorbide, and porphyrin are assigned on pure samples of chlorophyll a and phzeophorbide a by


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XII. CONANT 933

Stoll (28). All contam esterified carboxyl and pro-pionic acid groups as described by Willstatter, thus ac-counting for four of the oxygen atoms. The nature ofthe group containing the fifth oxygen atom has beenmuch harder to ascertain. The position of the phytylgroup was fi s t correctly placed by Conant (29) andlate r substantiated by Fischer on the basis of indepen-dent evidence.

The controversial groups in chlorophyll are those onthe gamma bridge carbon atom and position of thepyrrole ring adjoining. The Fischer and Stoll formu-las have a carbon bridge attached a t these positions.In the Conant formula the distinguishing feature is alactam bridze between position 6 and a pyrrole nitro-.gen atom.

The carbocyclic ring postulated by kischer is really abeta-ketonic acid monoine such as is ores t in substi-tnted acetoacetic ester (R-C-CH-COCH~. In

I I

H ~ c H c - ~ ~ ~

XIII. RLORIN

such a structure a hydrogen atom would be capable ofmigrating to form an en01 modification (R-C=

HOC-COCH3 and characteristic changes would takeI I

R

place in th e presence of acid and alkali. All of thesequalifications are fulfilled by chlorophyll or the pheo-phorbides, which contain th e same grouping. Thu sStoll prepared a benzoyl derivative of methyl phipo-phorbide (30), showing th e presence of an hydroxylgroup. Late r Fischer discovered the conditions neces-sary for oxime formation (31) after fruitless attemptsby several investigators. This evidence indicates thatthe f ifth oxygen atom can function as part of either anhydroxyl or a carbonyl group. The star ting materialwas recovered unchanged from both derivatives byhydrolysis. I t was this evidence for a carbonyl groupwhich, in 1934 seemed to rule ou t th e Conant and Stollformulas, since neither as such contains a group capableof forming an oxime.

Alkali hydrolyzes the ester groups and splits thecarbocyclic ring of chlorophyll forming the tribasic acidchlorin (Formula XIII ). Strong acid retains thering structure, although hydrolyzing the ester groupsand removing carbon dioxide from the free bridge car-boxyl group, forming phylloerytbrin (Formula XIV).These reactions of chlorophyll in acid and alkali arealso characteristic of beta-keto esters and thereforesupport the Fischer formula.

Pbylloerythrin was first isolated by Marchlewski(32) and has since been found frequently as a degrada-tion product of chlorophyll in th e ani mal body (33).Fischer early suspected a close relationship between th etwo, and after proving th at phylloerythrii contains acarbocyclic ring was led to postulate a similar ring inchlorophyll. The structure of phylloerythrin wasthoroughly established by the synthesis from meth-enes of i ts reduction product, desoxyphylloerythrin

34), and by i ts alkaline degradation to phyllo-, pyrro-,and rhodoporphyrins. The same carbon ring is alsode6nitely present in pheoporphyrin s 35), a porpby-rin isomeric w i t h ph~ophorbide a and produced byvery mild hydrogen iodide trea tment of chlorophyllor phipophorbide.

Fischer X = >C-CHnCOOHConant, X = >C=CHCOOH >CHCHOHCOOH


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I t will be noted tha t there is an ethylidene grouping(=CHCH8) in Fischer s formula for chlorophyll awhich does not appear in any of the others. This wasrecently postulated as the result of newly discoveredevidence and i s thought to be present in ll derivativesof chlorophyll except porphyrins, which have an ethyl

gronp in its place. This newly detected group appearsto ta ke up two atoms of hydrogen, and with hydriodicacid to be oxidized to an acetyl group, which can besplit o ff, leaving a free hydrogen atom. Two com-pounds resulting from such degradation reactions havebeen synthesized and t he position of this group may beregarded as established although its exact nature is notquite so certain.

One very important reaction of chlorophyll whichalso centers in the disputed carbocyclic ring is calledallomerization and was discovered by Willstatter andUtzinger (36). This is a subtle change takimg placein alcohol solution, whereby the a bi it y of the pigmentto show a certain Molisch color reaction is lost, but

no change in color or spectrum is observed. Conantmade the valuable discovery that allomerization is adehydrogenation reaction 37) involving two chemicalequivalents, in which either oxygen or potassium molyb-dicyanide can serve as agents. He also found that al-lomerized chlorophyll ditrers in behavior on alkali treat-ment from the unallomerized material. Thus he dem-onstrated th e formation of a ketonic add (-CO-COOH), pheopurpurin 7 from allomerized chlorophyll(38), while unaltered chlorophyll yields an hydroxyacid (-CHOHCOOH), chlorin hydrate, which de-hydra tes on isolation (39). Conant therefore postu-lat ed an easily formed hydrate of chlorophyll (seeFormula XI I), with a secondary alcohol gronp capable

of being dehydrogenated to a carbonyl group, as theseat of allomerization. The proof of a similar dehydro-genation of a --CHOH group in chlorin hydra teseemed to lend weight to this hypothesis. Stoll late rincorporated Conant s interpretation of allomeriza-tion into his formula, but dropped the idea when Fischerdemonstrated th e presence of a carb8nyl group in un-altered chlorophyll.

Fischer, on th e basis of allomerization experimentswith qninone in alcohol M), ssumes that the dehydro-genation takes place between the gamma and 10 posi-tions, forming a double bond, and that the moleculemay then add water or alcohol to the carbon atoms atpositions 6 and 10 1,4 ddition) as is shown below.

Thus according to Conant th e addition of water mustprecede dehydrogenation, while according to Fischerwater cannot add until dehydrogenation has takenplace. Careful analysis of purer samples of allomerizedproducts and a further study of the properties of Fisch-er s 10-hydroxy methyl ph~ oph orb ide a might help

to clear up this difficulty. This compound on Conant sformulation would be a hydrate of the unallomerizedmaterial. Against this view is the fact that the com-pound is always prepared in allomerizing media, andalso that it no longer forms chlorin e on alkali treat-ment, a criterion for unallomerized material.

Conant and Fischer have postulated d i e r e n t chlorin eformulas consistent with th e formation of chlorin ewith alkali, or its ester with diazomethane, from theirrespective structures for ph~ophorbide a. The be-havior of chlorin e hydrate as au alpha-hydroxy acidin the presence of suitable oxidizing agents h d s noexplanation in Fischer s formula, which is satnratedand cannot form a hydrate. This casts some doubt

not only on his formula for chlorin e but also for phaeo-phorbide from which it is derived.Aside from the determination of the nature and loca-

tion of substi tuen t groups on the chlorophyll skeleton,there remain problems in connection with the h estruct ure of the nucleus which will doubtless requirenew methods of attack. Conant and Kamerlimg com-pared th e absorption spectra of chlorins and porphyrinsa t liquid air temperatures (41), and found the differencesimilar to that between benzene and dihydrobenzeneunder the same conditions. They suggested, therefore,th at there is a completely conjugated system of doublebonds in the porphyrins which-is broken in the chlorinsby partial hydrogenation. Cofiant has shown further

th at chlorophyll can be converted into a simply consti-tuted chlorin, chlorin f, by transformations which donot affect the nucleus; and th at chlorin is a dihydroderivative of a porphyrin, isorhodoporphyrin (4Z),an isomer of rhodoporphyrin (43). This evidence indi-cates tha t chlorophyll has the nucleus of a chlorin(or dihydro-porphyrin). Fischer accepts the assign-ment of addit ional hydrogen atoms to th e inner por-phin ring of the chlorins and chlorophyll but , since hepostulates a free ethylidene group on one pyrrole ring,the total hydrogen content of both chlorins and por-phyrins is kept the same. This assumption is based oncatalytic hydrogenation data indicating that they areisomeric (44). Thus conflicting chemical evidence has

XV. Frscmds INTERPRETATION ALLOMERIZATION


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led to a diierence of opinion as to the sta te of oxidationof the chlorophyll nucleus. Conant believes th e chloro-phyll skeleton to be th at of a dihydro-porphyrin,Fischer thinks i t is th at of an isomerized porphyrin.

A further application of physico-chemical methodsto the determination of nuclear structure is th e electro-

metric titration by Conant of the individual basicpyrrole groups in various chlorophyll derivatives inglacial acetic acid solution (9).

CHLOROPHYLL B

Chlorophyll b, as Willstiitter s analytical resultsshowed, d 8e rs from the a component by only one oxy-gen atom. Conant (45) [and later also Warburg( )I obtained carbonyl derivatives of compounds ofthe b series and so placed this oxygen atom in a carbonylgroup (capable of enolization) on one of the bridgecarbon atoms of his structure for chlorophyll a. Heassigned it to a position on the nucleus in order to ac-

count for its enormous effect on the absorption spec-trum. Fischer f i s t placed this group in the beta posi-tion of the propionic acid residue (47), but has recentlymoved i t to a formyl group in position 3 of pyrrole ringI1 in his formula for chlorophyll a (48).

Besides the carbonyl group characteristic of thisseries, chlorophyll b seems to contain the same carbo-cyclic ring as the a compound. Thu s Stoll isolated adioxime of methyl phaeophorbide b (49) and Fischerhas prepared a number of porphyrins paralleling theph m - and chloroporphyrins of the a series (50). Thegreater scarcity of chlorophyll b and the greater diffi-culty of purification of its derivatives have delayedprogress in this series and its formula is less certain

than tha t of the a derivative. There is no evidence ofth e interconversion of the two series in the p lant noris there any explanation of their very constan t ratioas observed by W illstitte r.

We are now in a better positiqn to compare the de-

tailed st ructures of chlorophyll and of hemin from thepoint of view of their interconversion in the animalbody. As explained above, chlorophyll has either adihydro- or an iso-porphyrin ring rather than a por-phyrin ring, and its subs tituent groups correspond moreclosely to mesoporphyrin than they do to hemin.

Therefore, no simple theoretical transformation canbe postulated. This, however, does not exclude thebiological possibility, although the degradation prod-ucts of chlorophyll thus far found in the human andanimal body are not suggestive of a direct transforma-tion. Nevertheless, many investigators have claimedto 6nd a positive reaction of chlorophyll in anemia pre-vention and therapy in animals. The common typeof pernicious anemia is believed to be due not to lackof red pigment but to other factors necessary for theorigination of red blood cells in the bone marrow.Hence chlorophyll feeding might not be expected tohelp. Perhaps a more significant experiment is thatof Patek and Mmot (51) who investigated human pa-

tients with a rarer type of anemia caused by pigmentscarcity and observed a small positive increase inhemoglobin concentration on intravenous injection ofa chlorophyll derivative (chlorin e . The very smalleffect makes it probable that under the conditions ofthe experiment, chlorophyll and its derivatives merelybreak into simple pyrrole fragments in the body whichare then available for recombination to hemiu, buttha t the conversion is not very e5c ient .

The tetrapyrryl ring structure not only plays animportant r61e in the photosynthesis of plants and inthe respiration of animals, bu t has recently been de-tected in such powerful body.,catalysts as catalase(52) and peroxidase (53) and in the cytochrome c of

yeast (54). Traces of porphyrins are found very wide-spread in nature but usually without any obvious bio-logical function. Future research will undoubtedlyuncover further examples of the intervention of thistype of molecule in important natural processes.

C LITERATURE CITED

1) Recent developments on the nature of photosynthesis and 10) WXLLSTATTER JND F ~ s c m n , Die Stammsubstanzen derthe rdle played by chlorophyll are summarized in a review Phyll ine und Porphyrine, Ann. , 400, 182 1913).by H. A. Spoehr: The chemical aspects of photosyn- 11) Flscnsn UND STANGLER, Synthese des Mesoporphyrins,thesis, Stanford Universi ty Annual R m of B w c h - Mesoh5,mins. und iiber d ~ e onstitution de Himins,istry. 2, 453 1933). ibid., 459, 53 1927).

2) V E ~ E ~ ,ompt. rend.. 33, 689 1851). 12) FISCHER rn TREIBS, Jh= ~tiopa rphyri ne aus Blatt-(3) FISCHER. obelvortrag Stockholm 1931; Z. o na m . C h i c , und Blutfarbstoffporphyrine, ibid 466, 188 1928).

44, 617 1931).4) HOPPE-SEYLER, Uber das Chlorophyll der Hamen, 2

physiol. Cham.. 3, 339 1879); 4, 193 1880).5) SWIUNCK, I O C . oy. Soc. 50, 302 1891); SCRUNCK rnMARGUEWSKI, inid., 57, 314 1895); Zur Chemie desChlorophylls, Ann.. 284, 81 1894).

6) NE NW u rn ZALESKI, Uber die Reduktionsprodukte desHimins und iiber die Konstitution des Hiimins und seinerDerivate, Bcr.. 34, 997 1901).

7) WILLSTKTTER UND ASCHINA. 0nydatian.der ChlorophyllDerivate, Ann., 373, 227 1910); Uber die Reduk-tion des Chlorophylls. I. ibid. 385, 188 1911).

(8) Kiismn, Beitrige zur Kenntnis des Bilirubins und Hiim-ins, 2 hysiol. Cham.,82, 463 1912).

9) CONANT HOW. ND DIETZ, Chlorophyll series XIV. Po-tentiometric titration in acetic acid solution of the basicgroups in chlorophyll derivatives, J . A m . C h . oc.,56,2185 1934).

133) FISCHER. E R D VND SWIORYOLLER,Synthesen der Chloro-phyllporphyrine Rhodo- und Pyrroporphyrm, s o ~ l e esI'vrro~trooomhvrin5. bid 480. 109 (1930)

und Deuteroporphyrjns, ibid., 466,178 1928).15) FIscHER UND RIEDL, Uberfiihrung von Chlorophyll-pyrro-

porphyrin in Mesaporphyrin aus Himin, inid., 486,1 7 9 , l o l l ,

Y , ZY.,.

16) FwcnEn urn EBER ~GE F~ ER, iiber Mesorhodin und seinenCbergang m Chloraphyll-parphyrinen. sowic Oxydationdss Phylloerythrinr. ibid.. 509, 19 19334).

17) WILLSTAI-TUR SD STOLL. Unwr~uchunzen ber Chloro-phyll . Julius springer, Berlin, 1913; Translated byScherzand Mertz. Science Publishing Co. 1928. Theorigi-nal papers occur in issues of the Annolen from 1906 to 1914.

18) FISCHER, . G., Die Konstitution des Phytols, Ann. , 464,69 1928); FISIXER, . G. IJND L~WENBERG, Die Synthesedes Phytols. ibid.. 475, 183 1929).


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(19) WINTERSTEIN urn STEIN. Fraktionieruna und Reindar- (36) WILLSTATTER W D UrzrNGen, ober die ersten Umwand-stellung organischer Snbstanzen nach dim Prinzip der . . lungen des Chlarophylls, Ann. , 382, 129 (1911).chromatographischen Adsorptionsaualyse. (a) 11. Mit- (37) CONANT, KAKERLINO, AND STEELE, The allomerirationteilung: Chlorophyll. Z. physiol: Chem.,220, 263 (1933); of chlorophyll, J. Am. Chem. Soc., 53, 3171 (1931);

6) WINTERSTEIN ND SCHON, lnd., 230,139 (1934). also reference (27); STEELE. Chlor ~phy ll series VI.(20) STOLL ND WIEDEMANN. Die optische Akti vita t des Chloro- Themechanis m of the phase test, i M . , 53, 3171 (1931).

phylls, Helu. Chim. Ad a, 16, 307 (1933). (38) CONANT, HYDE, MOYER, ND DIETZ, Chlorophyll series.(21) CONANT AND DIETZ, Chlorophyll studies. XI. The IV. ibid.. 53, 359 (1931); DIETZ AND ROSS, Chloro-

position of the methoxyl group,J.

A m . C h . oc.,55,

phyll series. XII. The phgopurpurins, ibid., 56, 159,P O 1 ( 111 > 11 0 ? d >- \* ,.ZSCHIELE, An improved method of purification of chloro-

phyll a and b, quant itat ive measurement of their absorp-tion spectr a; prwf for th e presence of a third chloro-phyll component. Bot. Gas 95, 529 (1934).

FLSCIIER first published a carbucycli~ ring srruc turc forchlorophyll in 1931. Zur Srrul;rur d r i Chloroplrsll 0,A n n 486. 130 (1931). The revised formula shown is~ u h l k h e d n AS;.. 513, 107 (1934). Neue Erkenntnissein dder Feinstruktni des Chlordphylii a.

STOLL ND WIEDEMANN. Der Reaktionwerlauf der Phasen-probe und die Konstitutiane von Chlorophyll a und b,Natum'ssrmschaffen, 20, 706 (1933).

FISCHER, Uher Chlorophyll a, Ann. , 502, 175 (1933);Pedler Lecture, J. Chem. Soc.. February, 1933. See alsoTREIBS, Z UngW Chem., 47,294 (1934).

STOLL ND WIEDEMANN, Die Zusammensetzung desChlorophylls, Helv. Chim. Acta, 16 183 (1933).

ARMSTRONG, The const ~tuti on f chlorophyll, CkrmistryIndustry, 52, 809 (1932).

STOLL ND WIEDEMANN, Uber Chlorophyll a eine phase-positiven Derivate nnd seine Allomerisation, Helv.Chim. Acfa, 16, 739 (1933); also reference (24) above.

CONANT, DIETZ, BAILEY. AND KAMERLING, 'Chlorophyllseries V. The s truc ture of chlarophyU a, J . Am. Chem.Soc.. 53. 2384 (19311.

STOLLUND IEUEMANN, Die Benzoylverbindungen undOxime Ton Methyl Pheophorhide a; Helo. Chim. Acte.17, 163 (1934).

FISFEER, RIEDMAIR, ND HASENKAMP, Uber Oxyporphy-nne. Ein Beitrag zur Kenntnis der Feinstruktnr vanChlorophyll a, Ann., 508,22 4 (1934).

MARCHLEWSKI. . physiol. Chem., 42, 464 (1904); herden Ursprung des Cholehamitins. i M . , 45, 466 (1905);

Chemie der Chloro~hvll. Braunschweie. Viewea. 1909.

~ l s&IEn UND H E S ~ Vorkommen von ~h

CHLOROPHYLL and HEMOGLOBINTWO NATURAL PYRROLE PIGMENTS

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