CHLOROPHYLL and HEMOGLOBINT W O NATURAL PYRROLE PIGMENTS

EMMA M. DIETZ*
Harvard University, Cambridge, Massachusetts

HLOROPHYLL is the brilliant green pigment

found in certain special cells of all green plants.
I t absorbs energy from the sun and in some unknown way uses it for the manufacture of sugar,
starch, and proteins (1). This important process is
known as photosynthesis and ultimately provides food
for all plants and animals on the earth. Hemoglobin
is the red pigment coloring the red blood cells of animals.
It is necessary for the maintenance of l i e through respiration-the process which provides a constant supply of oxygen to the tissues. In the slow development
of the chemistry of these two pigments, it has been an
increasing source of wonder to chemists to find that
two substances of such widely different origin and function are yet so remarkably similar in structure.
The pure pigments as isolated in the laboratory are
very different in appearance. Chlorophyll, when separated from the leaf structure, is a dark green wax which
is really not one substance but two closely related ones,
called chlorophyll a and b by Willstatter. Hemoglobin
is a compound molecule consisting of a colorless protein (globin) attached to four molecules of a red-brown
crystalline pigment called heme. In the process of
isolation from hemoglobin, heme changes over to its
familiar oxidation product, bemin, which is a brownred crystalline compound of high melting point. When
chlorophyll is dissolved in ether or chloroform a brilliant
green solution results; hemin forms a red-brown solution. When observed through a spectroscope, solutions of either pigment show the remvkable property,
due to similar elements of structure, of absorbing sharp
bands of visible light. There results an unusual banded
spectrum in the visible region, which is characteristic
for each compound and serves as a valuable means of
identification. An important diierence between the
two pigments which is immediately apparent from a
chemical analysis, is that chlorophyll contains magnesium, whereas hemin contains iron incorporated in
the large organic molecule.
The widespread distribution and importance to life
of both substances early stimulated the interest of sdeutists. The chemistry of chlorophyll was confused for
some time by the use of drastic methods of isolation
which altered or destroyed the molecule and introduced
metallic impurities. I t is still a matter of concern to
the chemist to find means of isolating this complex

* Sarah Berliner Fellow. American Association of University

Women, 1934-1935, at present engaged in research at the University of Munich, Munich, Germany.

material without introducing subtle changes in structure.

Heme, the pigment now believed to be present in hemoglobin, 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 the
chemistry of natural products where faulty isolation
bas obscured the true structure for years.
RELATIONSHIP

BETWEEN

CHLOROPHYLL AND

HEMIN

The first suggestion of a relationship between hemin

and chlorophyll was made as early as 1851 by Verdeil
(Z),though on the basis of invalid evidence. He unwittingly introduced iron into his preparations of
chlorophyll by employing crude methods and reagents,
and as a result thought that it might be related t o
hemin, which was already known to contain iron. Actually, the similarity in the two pigments is not in the
metallic constituents but in the rest of the molecule.
However, Verdeil's hypothesis stimulated research on
the problem for years and culminated in the series of
brilliant investigations by Hans Fischer for which he
was awarded the Nobel Prize .in 1930 (3). He and
his co-workers fmally established the correct structure
of natural hemin and heme by synthesis, and showed
their true relationship to chlorophyll. Research on
the plant pigment benefited enormously by these results, but its structure is not yet absolutely clear and its
complete synthesis has not been accomplished. In the
present discussion of the subject it has been found convenient to follow the historical development, and t o
consider 6rst the structure of hemin and its relationship to chlorophyll, before presenting the detailed
structure of the latter.
The 6rst real evidence of the connection between
chlorophyll and hemin was theformation of very similar
red,.crystalline compounds called porphyrins, from both
blood and plant pigments. These were first obtained
by Hoppe-Seyler (4) in 1879, later by Schunck and
Marchlewski (5) by drastic chemical treatment of the
two pigments. These porphyrins formed bright red
solutions in ether with striking four-banded absorption
spectra in the visible region, and they were actually
close chemical relatives though not identical as a t first
supposed.
In 1901, Nencki (6) reduced both hemin and a crude
preparation of chlorophyll to mixtures of volatile bases
called pyrroles (seeI),thus showing for the f i s t time that
pyrrole nuclei were involved in the structure of both
pigments. Willstatter (7) later identified the same

-.

Methyl Ethyl Maleie Imlde

pyrrole fragments from both chlorophyll and hemoglobin, thus proving a very close relationship. Kiister
(8) studied the products of drastic oxidation (11) as
well as reduction and proposed in 1910 the correct
general formula for the nucleus common to the porphyrin, hemin, and chlorophyll molecules. It was not
until twenty years later, however, that the Kiister
formula was completely justified by the synthetic results of Fischer.
THE FORMULA OF HEMIN

X = Fe++) the iron is in the reduced form, without

the halogen atom. The interconversion between protoporphyrin and its two iron derivatives is readily accomplished in the laboratory. On drastic reduction of
hemin and heme the metal drops out and the porphin
ring breaks on either side of the bridge carbon atoms,
forming the pyrroles shown in I ; oxidation removes the
bridge carbon atoms completely, forming hematinic
acid (see 11) as stated above.
I t might be mentioned here that hemin and heme are
class names applied to Feel++ and Fe++ complexes of
all porphyrins, but have been adopted as the specific
names for the blood pigment derivatives, more correctly called protohemin and protoheme. Further, the
use of non-systematic names, wherever possible in this
field, is preferable to the very lengthy Geneva nomenclature, though somewhat confusing at first. These
incidental names are usually derived from Greek stems
denoting color, i. e., chlorophyll = green leaf, porphyrin
= purple.

A detailed examination of the hemin formula will

bring out several features, shared by chlorophyll as
well. It contains four pyrrole, or modifiedpyrrole nuclei
bound into a large symmetrical ring structure by four
carbon atoms. For purposes of nomenclature, the
unsubstituted molecule is called a porphin ring. In
hemin there is an unbroken alternation of single and
double bonds such as is assumed for all porphyrins,
and is found on a smaller scale in benzene. As in benzene, the exact location of each bond is not determined
and various arrangements are possible. The evidence
BLOOD AND CELOROPHY2.L WRPHYFSNS
of years indicates that most compounds exist in only
The correctness of these, structures for protoporone of the possible electromeric forms, and it remains phyrin and its iron derivatives was proved through
for physico-chemical methods, such as electrometric the isolation and identification of their degradation
titration [as applied by Conant to some chlorophyll products and finally through the synthesis of the whole
derivatives ( 9 ) ] to determine which form is present.

:.

Heme = Protoheme
Hcmin
Protohemin

X
Fe++
X = l'eCltt

Only one possible electromeric structure of each compound is shown here for convenience.
In hemin (Formula 111; X = FeCl++) two hydrogen
atoms of the parent porphyrin, protoporphyrin (Formula IV; X = -CH=CHe), have been replaced by an
FeCl++ grouping which is doubtless coordinately bound
to all four nitrogen atoms. In heme (Formula 111:

series of blood porphyrins by Fischer. In protoporphyrin, as in hemin, there arefourmethylgroups (-<Hz)

at positions 1, 3, 5, and 8; two vinyl groups (-CH=CHI) at 2 and 4; and two propionic acid groups (-CHr
CHCOOH) at 6 and 7, all substituting beta positions of
the pyrrole rings. Mesoporphyrin results from the addition of two hydrogen atoms to each of the two vinyl

Hemoglobin = 1 Globin

4 Heme
oxidation
Hemin
-F&l
Protoporphyrin
+4H
Synthetic Mesoporphyrin IX = Mesoporphyrin

Synthetic Btioporphyrin I11

groups forming ethyl groups (-GHd, (Formula VII,

X = CHtCH2COOH). Bioporphyrin (more accurately called meso-etioporphyrin) is the oxygen-free
and completely alkylated compound in which even the
two propionic acid groups of proto- and mesoporphyrins have been converted to ethyl groups by loss of
carbon dioxide (Formula VI; X = C2H& Mesoetioporphyrin consists therefore of a porphin ring substituted by four ethyl and four methyl groups and is
a very stable compound of high melting point. These
relationships are summarized above (V). I t is evident
from the formula that four isomers of this aetioporphyrin are possible, depending on the order of the alkyl
groups around the porphin ring. The etioporphyrins
are of great interest since they represent the final stage
in the drastic a W m e degradation of chlorophyll as
well as of hemin.

Willstatter prepared the etioporphyrins from both

hemin and chlorophyll (10) and believed them to be
identical. Actually, meso-etioporphyrin, the sptioporphyrin of blood, contains an ethyl group a t position 6
in place of a free hydrogen atom of the chlorophyll
derivative, but the rest of the groups are identical and
in the same order. Therefore meso-etioporphyrin is
really 6-ethyl-pyrroetioporphyrin,and both chlorophyll and hemin may thus be regarded as related to
a common porphyrin.
PORPHYRIN SYNTHESIS

The complete demonstration of this relationship and

the elucidation of the structures of the individual blood
and chlorophyll porphyrins did not occur until after
Fischer's enormous development of synthetic pyrrole
and porphyrin chemistry extending over fifteen years.

Hr

VI
Meso-=tioporphyrin
Pyryrm-etioporphyrin

--

CIHZ(Blwd Derivative)
H (Chlmophyll Derivative)

Chlorophyll is not truly a porphyrin derivative like

hemin but it is nevertheless capable of yielding a series
of porphyrins when treated with suitable reagents.
Thus on drastic alkali treatment one obtains a dibasic
acid, rhodoporphyrin (Formula VII; X = COOH), and
from this by loss of carbon dioxide a monobasic acid,
pyrroporphyrin (Formula VII; X = H). I t will be
noted that mesoporphyrin is really pyrroporphyrin
with a propionic acid group replacing the hydrogen
atom at position 6. Further removal of carbon dioxide
from pyrroporphyrin forms the oxygen-free, completely
alkylated porphyrin derivative, pyrro-zetioporphyrinthe aetioporphyrin of the chlorophyll series. (Formula VI; X = H.)

Meroporphyrin
Pyrroporphytin
Rhodoporphyrio

X = CH,CH,COOH
X
H
X = COOH

.He not only found methods for synthesizing the natural

pyrrole degradation products of the porphyrins but also
learned how to join these into so-called dipyrryl methenes of known structures and finally to couple suitable
pairs of these methenes to form porphyrins, whose
structures would therefore also be known. VIII shows
how two different pairs of methenes were combiied to
form the same porphyrin (11). The f a d that both
syntheses yielded a porphyrin with the same spectrum
and melting point showed that no rearrangement had
taken place and made the assigned structure more
credible.
This last step in the synthesis of porphyrins is a
pyrolysis carried out in an organic acid melt, in which

carbon dioxide, halogen acid, hydrogen, and freehalogen

may be lost as in this instance. The method is rather
laborious as the yields are usually low and isomeric
porphyrins almost always result. It may be claimed
that such syntheses are too complicated in mechanism
to afford an absolute proof of structure, but the consistency of the cumulated data lends weight to the method
and certainly makes it valid as confirmatory to the
evidence from degradation reactions.

group, obtaining a product identical with mesoporphyrin from hemin. The reverse transformation of
meso- to pyrroporphyrin has also been accomplished
(16), though in a more complicated series of steps.
Such interconversions afford final proof that the
nucleus and order of substituent groups in chlorophyll
and hernin are the same and perhaps make a transition
between the two pigments in the animal body more
probable. As will appear later, however, the pigments

HzC==-C~HI

H S L C I H ~

VIII. Two SYNTEESE~

08 A

By similar synthetic methods Fischer has successfully prepared several blood and chlorophyll porphyrins,
thus establishing beyond a doubt the Kiister ring formula for the nuclei of both pigmentsand thefundamental
relationship between them. For example, he synthesized twelve of the fifteen possible isomers of mesoporphyrin and proved that one of these, called meso.
porphyrin IX (ll), is identical with the natural product as judged by mixed melting-points and absorption spectra. He also prepared the four possible mesoaetioporphyrins (Formula VI; X = CsHr) and identified that obtained from hemin as ztioporpbyrin I11
(12). In the chlorophyll series, furthermore, he succeeded in preparing natural pyrro- and rhodoporphyrins (Formula VII) by the use of similarsyntheticmethods (13).
The synthesis of hemin itself was accqmplisbed in a
number of steps starting with deuteroporphyrin (14)
(Formula IV, X = H) which was prepared from methenes by a method similar to that shown in VIII, but
which also occurs in nature as a putrefaction product of
blood. Two acetyl groups were introduced into deuteroporphyrin, these were reduced to -CHOHCHs
groups and then dehydrated, the product being protoporphyrin (Formula IV, X = -CH=CHs),
which
was readily converted into its FeCl++ complex, bemin.
Up to this point the proof that both chlorophyll and
blood porphyrins are built on the same structural plan,
with the same order of substituents on the porphin
ring, rested on independent syntheses in each series.
The final step consisted in attempting a transition between the two series without possibility of rearrangement. This was also achieved by Fiscber in the interconversion of pyrro- and mesoporphyrins. Thus
Fischer and Riedl (15) succeeded in replacing the free
beta position in pyrroporphyrin by a propionic acid

HaC-7CHr

1I

I 1

H ; L C Z s
P o a mw oM
~m e m s

themselves are far less closely related than these two

porphyrins and thus far the nature of their degradation products in the animal body and in the laboratory
does not support such a possibility.
WILLSTATTER'S

INVESTIGATIONS ON C~LOROPAYU

Our knowledge of the exact chemistry of chlorophyll

is due largely to the remarkable researches of Wills e t t e r and his co-workers (17). notably Stoll, from 1906
to 1914. They developed methods of isolation and
purification and studied a number of degradation products, but the formulas which they suggested were
subsequently proved to be somewhat incorrect. Research on the subject was taken up from this point in
about 1927 by Fischer and by Conant, and has concerned itself mainly with the proof of *he Kiister structure for the nucleus, with the study of the many degradation products, and with the identification of certain very labile groupings in chlorophyll and its immediate derivatives.
To Willstatter we are indebted for a mild isolation
procedure permitting extractioq of tbe unaltered pigment from the plant, and for the invaluable method
of sepkration and purification of reaction products by
acid fractionation. This process makes use of the
varying basicity of chlorophyll derivatives which enables them to be extracted separately from a mixture
in ether solution, by agitation with hydrochloric acid
of suitable coucentratious. W i s e t t e r further showed
that there are two chlorophylls in nature, which he called
a and b, with the nearly constant ratio of 2.9 to 1in the
higher plant forms, but with chlorophyll b apparently
absent in certain alga?. He determined the correct
analytical formula of chlorophyll a (Ca6H~sNaOsMg)
and noted that the two chlorophylls differed by a single
oxygen atom. He proved the presence of magnesium

and correctly characterized the two ester groups as

being formed from methyl alcohol and phytol (GoHaOH). The latter was a new alcohol with a long straight
carbon chain whose structure was established in 1929
through a brilliant synthesis by Gottwalt Fischer (18).
Chlorophyll is readily obtained in a u d e form by
extraction of green leaves with aqueous acetone. Willstatter accomplished the separation into a and b components by distribution between methyl alcohol and
petroleum ether. A better method is that of chromatographic adsorption on powdered sugar according to the
directions of Winterstein and Stein (19) who thus obtained very pure samples. This adsorption method
has recently found considerable application in the
separation of closely related natural products. Freshly
isolated chlorophyll a and b were found by Stoll to be
slightly optically active (20). Conant and Dietz (21)
and Zschiele (22) have reported indications of a third
form of chlorophyll. Witerstein and Schon (19b)
failed to find a chlorophyll c in carefully controlled
adsorption experiments.
In alcohol solution and in the presence of the plant
enzyme, chlorophyllase, the phytyl group of chlorophyll is replaced by a methyl or ethyl group to form
crystalline derivatives called chlorophyllides. M i d
acid treatment of chlorophyll acts only to remove the
magnesium atom, forming phaeophytin, a dark, waxy
solid which finds commercial use in the fom.of its
stable and brilliantly colored copper salt, a non-poisonous green coloring material. More drastic acid treatment of chlorophyll a (with concentrated hydrochloric
acid) hydrolyzes the phytyl group and removes the
metal as well, forming crystalline phaeophorbide a, a
monomethyl ester. This is easily esterzed to a dimethyl ester, methyl phgophorbide a. The accompanying diagram (IX) shows these transformations
with the accepted compositions of the various products.

EtOH and
chlorophyllase
Ethyl Chlorophyllide a

the hasis of color and characteristic absorption spectra.

More drastic alkali treatment of chlorophyll or any
of the above-mentioned degradation products, removes
various groups leaving the simple dibasic and monobasic acids, rhodo-, pyrro-, and phyllo- (y-methyl
pymo-) porphyrins, all of which have been synthesized
by Fischer as described above.
THE STRUCTURE OF CHL0ROPJiYL.L

We are now ready to examine the formula of chlorophyll in some detail. During the last few years three
somewhat dierent formulas have been proposed by
Fischer (23), Conant @I), and Stoll (24) on the basis
of results obtained in their respective laboratories.
These are shown in formulas X, XI, and XII. (The
Fischer structure has undergone numerous revisions,
the latest being shown here.) At the present time the
Fischer formula seems to he the best representation of
the existing data, although some points are not yet
clear. All three structures are more fully discussed in
the excellent reviews of Fischer (25) and Stoll (26),
and in a very complete account by Armstrong (27),
which summarize the vast body of accumulated facts
up to July, 1933.

conc. acid

H1C

CHOH

dil, acid Phmphorbide a

COOCH,
COOH

HC-

lalkali

Chlorin e

IX

Concentrated alkali brings about a more fundamental

change in chlorophyll and in the phaeophorbides, developing a third acid group, the product being the tribasic acid chlorin e from the a component and rhodin g
from the b component; both acids form trimethyl esters.
Here, as in the case of the blood pigment, the nomenclature is non-systematic and the class names such as
chlorin, phzeophorbide, and porphyrin are assigned on

N-Mg

2-

CH#2H2COOGoHlo

HCHsG-~

C
H
XI. STOLL
1933

The three formulas are, of course, similar inqgross

structure since they involve a common interpretation
of the data given above. Thus all contain five oxygen
atoms, in accordance with the recent analyses of very
pure samples of chlorophyll a and phzeophorbide a by

such a structure a hydrogen atom would be capable of

migrating to form an en01 modification (R-C=
I

C-COCH3
I

1
I

HO
and characteristic changes would take

R 0

XII. CONANT
1933

Stoll (28). All contam esterified carboxyl and propionic acid groups as described by Willstatter, thus accounting for four of the oxygen atoms. The nature of
the group containing the fifth oxygen atom has been
much harder to ascertain. The position of the phytyl
group was f i s t correctly placed by Conant (29) and
later substantiated by Fischer on the basis of independent evidence.
The controversial groups in chlorophyll are those on
the gamma bridge carbon atom and position 6 of the
pyrrole ring adjoining. The Fischer and Stoll formulas have a carbon bridge attached a t these positions.
In the Conant formula the distinguishing feature is a
lactam bridze
position 6 and a pyrrole
nitro- between .
-.
gen atom.
The carbocyclic ring postulated by kischer is really a
beta-ketonic acid monoine such as is ores&t in substitnted acetoacetic ester (R-C-CH-COCH~.
In

I,

1 ~
H~cHc-~~
XIII. CRLORINc

>C-CHnCOOH
>C=CHCOOH
>CHCHOHCOOH

Fischer, X =

Conant, X

place in the presence of acid and alkali. All of these

qualifications are fulfilled by chlorophyll or the pheophorbides, which contain the same grouping. Thus
Stoll prepared a benzoyl derivative of methyl phipophorbide a (30), showing the presence of an hydroxyl
group. Later Fischer discovered the conditions necessary for oxime formation (31) after fruitless attempts
by several investigators. This evidence indicates that
the fifth oxygen atom can function as part of either an
hydroxyl or a carbonyl group. The starting material
was recovered unchanged from both derivatives by
hydrolysis. I t was this evidence for a carbonyl group
which, in 1934, seemed to rule out the Conant and Stoll
formulas, since neither as such contains a group capable
of forming an oxime.
Alkali hydrolyzes the ester groups and splits the
carbocyclic ring of chlorophyll forming the tribasic acid
chlorin c (Formula XIII). Strong acid retains the
ring structure, although hydrolyzing the ester groups
and removing carbon dioxide from the free bridge carboxyl group, forming phylloerytbrin (Formula XIV).
These reactions of chlorophyll in acid and alkali are
also characteristic of beta-keto esters and therefore
support the Fischer formula.
Pbylloerythrin was first isolated by Marchlewski
(32) and has since been found frequently as a degradation product of chlorophyll in the animal body (33).
Fischer early suspected a close relationship between the
two, and after proving that phylloerythrii contains a
carbocyclic ring was led to postulate a similar ring in
chlorophyll. The structure of phylloerythrin was
thoroughly established by the synthesis from methenes of its reduction product, desoxyphylloerythrin
(34),and by its alkaline degradation to phyllo-, pyrro-,
and rhodoporphyrins. The same carbon ring is also
de6nitely present in pheoporphyrin as (35), a porpbyrin isomeric with ph~ophorbidea and produced by
very mild hydrogen iodide treatment of chlorophyll
or phipophorbide.

I t will be noted that there is an ethylidene grouping

(=CHCH8) in Fischer's formula for chlorophyll a
which does not appear in any of the others. This was
recently postulated as the result of newly discovered
evidence and is thought to be present in all derivatives
of chlorophyll except porphyrins, which have an ethyl
gronp in its place. This newly detected group appears
to take up two atoms of hydrogen, and with hydriodic
acid to be oxidized to an acetyl group, which can be
split off, leaving a free hydrogen atom. Two compounds resulting from such degradation reactions have
been synthesized and the position of this group may be
regarded as established although its exact nature is not
quite so certain.
One very important reaction of chlorophyll which
also centers in the disputed carbocyclic ring is called
allomerization and was discovered by Willstatter and
Utzinger (36). This is a subtle change takimg place
in alcohol solution, whereby the abiity of the pigment
to show a certain Molisch color reaction is lost, but
no change in color or spectrum is observed. Conant
made the valuable discovery that allomerization is a
dehydrogenation reaction (37) involving two chemical
equivalents, in which either oxygen or potassium molybdicyanide can serve as agents. He also found that allomerized chlorophyll ditrers in behavior on alkali treatment from the unallomerized material. Thus he demonstrated the formation of a ketonic add (-COCOOH), pheopurpurin 7, from allomerized chlorophyll
(38), while unaltered chlorophyll yields an hydroxy
acid (-CHOHCOOH),
chlorin e hydrate, which dehydrates on isolation (39). Conant therefore postulated an easily formed hydrate of chlorophyll (see
Formula XII), with a secondary alcohol gronp capable
of being dehydrogenated to a carbonyl group, as the
seat of allomerization. The proof of a similar dehydrogenation of a --CHOH group in chlorin e hydrate
seemed to lend weight to this hypothesis. Stoll later
incorporated Conant's interpretation of allomerization into his formula, but dropped the idea when Fischer
demonstrated the presence of a carb8nyl group in unaltered chlorophyll.
Fischer, on the basis of allomerization experiments
assumes that the dehydrowith qninone in alcohol (M),
genation takes place between the gamma and 10 positions, forming a double bond, and that the molecule
may then add water or alcohol to the carbon atoms at
positions 6 and 10 (1,4addition) as is shown below.

XV.

Thus according to Conant the addition of water must

precede dehydrogenation, while according to Fischer
water cannot add until dehydrogenation has taken
place. Careful analysis of purer samples of allomerized
products and a further study of the properties of Fischer's 10-hydroxy methyl ph~ophorbidea might help
to clear up this difficulty. This compound on Conant's
formulation would be a hydrate of the unallomerized
material. Against this view is the fact that the compound is always prepared in allomerizing media, and
also that it no longer forms chlorin e on alkali treatment, a criterion for unallomerized material.
Conant and Fischer have postulated d i e r e n t chlorin e
formulas consistent with the formation of chlorin e
with alkali, or its ester with diazomethane, from their
respective structures for ph~ophorbidea. The behavior of chlorin e hydrate as au alpha-hydroxy acid
in the presence of suitable oxidizing agents h d s no
explanation in Fischer's formula, which is satnrated
and cannot form a hydrate. This casts some doubt
not only on his formula for chlorin e but also for phaeophorbide from which it is derived.
Aside from the determination of the nature and location of substituent groups on the chlorophyll skeleton,
there remain problems in connection with the h e
structure of the nucleus which will doubtless require
new methods of attack. Conant and Kamerlimg compared the absorption spectra of chlorins and porphyrins
a t liquid air temperatures (41), and found the difference
similar to that between benzene and dihydrobenzene
under the same conditions. They suggested, therefore,
that there is a completely conjugated system of double
bonds in the porphyrins which-is broken in the chlorins
by partial hydrogenation. Cofiant has shown further
that chlorophyll can be converted into a simply constituted chlorin, chlorin f, by transformations which do
not affect the nucleus; and that chlorin f is a dihydro
derivative of a porphyrin, isorhodoporphyrin (4Z),
an isomer of rhodoporphyrin (43). This evidence indicates that chlorophyll has the nucleus of a chlorin
(or dihydro-porphyrin). Fischer accepts the assignment of additional hydrogen atoms to the inner porphin ring of the chlorins and chlorophyll but, since he
postulates a free ethylidene group on one pyrrole ring,
the total hydrogen content of both chlorins and porphyrins is kept the same. This assumption is based on
catalytic hydrogenation data indicating that they are
isomeric (44). Thus conflicting chemical evidence has

Frscmds INTERPRETATION
OF ALLOMERIZATION

led to a diierence of opinion as to the state of oxidation tailed structures of chlorophyll and of hemin from the
of the chlorophyll nucleus. Conant believes the chloro- point of view of their interconversion in the animal
phyll skeleton to be that of a dihydro-porphyrin, body. As explained above, chlorophyll has either a
dihydro- or an iso-porphyrin ring rather than a porFischer thinks it is that of an isomerized porphyrin.
A further application of physico-chemical methods phyrin ring, and its substituent groups correspond more
to the determination of nuclear structure is the electro- closely to mesoporphyrin than they do to hemin.
metric titration by Conant of the individual basic Therefore, no simple theoretical transformation can
pyrrole groups in various chlorophyll derivatives in be postulated. This, however, does not exclude the
biological possibility, although the degradation prodglacial acetic acid solution (9).
ucts of chlorophyll thus far found in the human and
animal body are not suggestive of a direct transformaCHLOROPHYLL B
tion. Nevertheless, many investigators have claimed
Chlorophyll b, as Willstiitter's analytical results to 6nd a positive reaction of chlorophyll in anemia preshowed, d8ers from the a component by only one oxy- vention and therapy in animals. The common type
gen atom. Conant (45) [and later also Warburg of pernicious anemia is believed to be due not to lack
(&)I obtained carbonyl derivatives of compounds of of red pigment but to other factors necessary for the
the b series and so placed this oxygen atom in a carbonyl origination of red blood cells in the bone marrow.
group (capable of enolization) on one of the bridge Hence chlorophyll feeding might not be expected to
carbon atoms of his structure for chlorophyll a. He help. Perhaps a more significant experiment is that
assigned it to a position on the nucleus in order to ac- of Patek and Mmot (51) who investigated human pacount for its enormous effect on the absorption spec- tients with a rarer type of anemia caused by pigment
trum. Fischer fist placed this group in the beta posi- scarcity and observed a small positive increase in
tion of the propionic acid residue (47), but has recently hemoglobin concentration on intravenous injection of
moved it to a formyl group in position 3 of pyrrole ring a chlorophyll derivative (chlorin e). The very small
I1 in his formula for chlorophyll a (48).
effect makes it probable that under the conditions of
Besides the carbonyl group characteristic of this the experiment, chlorophyll and its derivatives merely
series, chlorophyll b seems to contain the same carbo- break into simple pyrrole fragments in the body which
cyclic ring as the a compound. Thus Stoll isolated a are then available for recombination to hemiu, but
dioxime of methyl phaeophorbide b (49) and Fischer that the conversion is not very e5cient.
The tetrapyrryl ring structure not only plays an
has prepared a number of porphyrins paralleling the
phm- and chloroporphyrins of the a series (50). The important r61e in the photosynthesis of plants and in
greater scarcity of chlorophyll b and the greater diffi- the respiration of animals, but has recently been deculty of purification of its derivatives have delayed tected in such powerful body.,catalysts as catalase
progress in this series and its formula is less certain (52) and peroxidase (53) and in the cytochrome c of
than that of the a derivative. There is no evidence of yeast (54). Traces of porphyrins are found very widethe interconversion of the two series in the plant nor spread in nature but usually without any obvious biois there any explanation of their very constant ratio logical function. Future research will undoubtedly
as observed by Willstitter.
uncover further examples of the intervention of this
We are now in a better positiqn to compare the de- type of molecule in important natural processes.
C

LITERATURE CITED

(1) Recent developments on the nature of photosynthesis and

the rdle played by chlorophyll are summarized in a review
by H. A. Spoehr: "The chemical aspects of photosyn-

thesis," Stanford University Annual R&m of B w c h istry. 2, 453 (1933).

(2) V E ~ ECompt.
~ , rend.. 33, 689 (1851).
(3). FISCHER.
Nobelvortrag Stockholm 1931; Z. onam.
. Chic,

44, 617 (1931). ,,

(4) HOPPE-SEYLER,
"Uber das Chlorophyll der Hamen," 2.
physiol. Cham.. 3, 339 (1879); 4, 193 (1880).
PIOC.Roy. Soc., 50, 302 (1891); SCRUNCK
urn
(5) SWIUNCK,
MARGUEWSKI, inid., 57, 314 (1895); "Zur Chemie des
Chlorophylls," Ann.. 284, 81 (1894).
"Uber die Reduktionsprodukte des
(6) NENW urn ZALESKI,

Himins und iiber die Konstitution des Hiimins und seiner

Derivate," Bcr.. 34, 997 (1901).
(7) WILLSTKTTER UND ASCHINA."0nydatian.der Chlorophyll
Derivate," Ann., 373, 227 (1910); "Uber die Reduktion des Chlorophylls. I." ibid., 385, 188 (1911).
(8) Kiismn, "Beitrige zur Kenntnis des Bilirubins und Hiimins," 2.ghysiol. Cham., 82, 463 (1912).
(9) CONANT,
CHOW.
AND DIETZ,"Chlorophyll series XIV. Potentiometric titration in acetic acid solution of the basic
groups in chlorophyll derivatives," J. Am. C h . Soc., 56,
2185 (1934).

(10)

WXLLSTATTER
IJND F ~ s c m n ,"Die Stammsubstanzen der

Phylline und Porphyrine," Ann., 400, 182 (1913).

UND STANGLER,
"Synthese des Mesoporphyrins,
Mesoh5,mins. und iiber d ~ eKonstitution de Himins,"
ibid., 459, 53 (1927). ,,
(12) FISCHERurn TREIBS,
YJh=2 ~tioparphyrine aus Blattund Blutfarbstoffporphyrine," ibid.. 466, 188 (1928).
HERD, VND SWIORYOLLER,
"Synthesen der Chloro(133) FISCHER.
phyllporphyrine Rhodo- und Pyrroporphyrm, s o ~ l edes
I'vrro~trooomhvrin5."rbid 480. 109 (1930)

(11) Flscnsn

. .

und Deuteroporphyrjns," ibid., 466,178 (1928).

(15) FIscHER UND RIEDL,"Uberfiihrung von Chlorophyll-pyrroporphyrin in Mesaporphyrin aus Himin," inid., 486,
179
"Y

,,'ZY.,.
loll,

(16) FwcnEn urn E B E R ~ G E F ~"iiber

E R , Mesorhodin und seinen
Cbergang m Chloraphyll-parphyrinen. sowic Oxydation
dss Phylloerythrinr." ibid.. 509, 19 (19334).
(17), WILLSTAI-TUR
USD STOLL.
"Unwr~uchunzen"ber Chloro-

phyll." Julius springer, Berlin, 1913; Translated by

Scherzand Mertz. Science Publishing Co., 1928. Theoriginal papers occur in issues of the Annolen from 1906 to 1914.
F. G., "Die Konstitution des Phytols," Ann., 464,
(18) FISCHER,
69 (1928); FISIXER,F. G. IJND L~WENBERG,
"Die Synthese
des Phytols." ibid.. 475, 183 (1929).

(19)
"Fraktionieruna und Reindar- (36) WILLSTATTER WD UrzrNGen, "ober die ersten Umwand. . WINTERSTEINurn STEIN.
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 allomeriration
teilung: Chlorophyll." Z. physiol: Chem., 220, 263 (1933);
of chlorophyll," J. Am. Chem. Soc., 53, 3171 (1931);
(6)WINTERSTEIN
UND SCHON,
zlnd., 230,139 (1934).
also reference (27); STEELE. "Chlor~phyll series VI.
(20) STOLLAND WIEDEMANN.
"Die optische Aktivitat des ChloroThemechanism of the phase test," i M . , 53, 3171 (1931).
phylls," Helu. Chim. Ada, 16, 307 (1933).
(38) CONANT,HYDE,MOYER,AND DIETZ, "Chlorophyll series.
(21) CONANTAND DIETZ, "Chlorophyll studies. XI. The
IV." ibid.. 53, 359 (1931); DIETZAND ROSS, "Chloroposition of the methoxyl group," J. Am. C h . Soc., 55,
phyll series. XII. The phgopurpurins," ibid., 56, 159,
PO0
/
1
(
1
1
1
>
110?d>
-"" \*""",.
I ZSCHIELE,"An improved method of purification of chlorolhlorophyll series. X. The
phyll a and b, quantitative measurement of their absorption spectra; prwf for the presence of a third chlorophyll component." Bot. Gas.. 95, 529 (1934).
FLSCIIER first published a carbucycli~'ring srructurc for
chlorophyll in 1931. "Zur Srrul;rur d r i Chloroplrsll 0,"
Derivate." Ann., 495 (1532).
Ann.. 486. 130 (1931). The revised formula shown is 141)
ll
VII. Evi. . CONANTAND KAMERLING," C h l o r ~ ~ h v series
~ u h l k h e din AS;.. 513, 107 (1934). "Neue Erkenntnisse
dence as to structure from mea&&ment of absorption
in dder Feinstruktni des Chlordphylii a."
spectrum," J. Am. Chem. Soc., 53,3522(1931).
STOLLUND WIEDEMANN.
"Der Reaktionwerlauf der Phasen- (42) CONANTAND BAILEY, "ChlorophyU series. IX. Transprobe und die Konstitutiane von Chlorophyll a und b,"
formations establishing the nature of the nucleus,"
Natum'ssrmschaffen,20, 706 (1933).
ibid.. 55, 795 (1933).
FISCHER,"Uher Chlorophyll a," Ann., 502, 175 (1933);
Pedler Lecture, J. Chem. Soc.. February, 1933. See also (43) DIETZAND WERNER,"Chlorophyll series. XIII. Nuclear
isomerism of the porphyrins," ibid.. 56, 2180 (1934).
TREIBS, Z. UngW. Chem., 47,294 (1934).
STOLLUND WIEDEMANN,"Die Zusammensetzung des (44) F ~ s c m nWNn LAKATOS,"Katalytische Hydrierung in der
Chlorophyll Reihe," Ann., 506, 123 (1933); FISCAER,
Chlorophylls," Helv. Chim. Acta, 16, 183 (1933).
LAKATOS,
UND SCHNELL,
ibid., 509, 201 (iQ34); FISHER
ARMSTRONG,
"The const~tutionof chlorophyll," Ckrmistry
DND SPIELBEROER.
"Teilsynthese von Athyl Chloro&Industry, 52, 809 (1932).
phyllide b, sowie iiber 10-Athoxy Methyl Phzophorbide
STOLL
UND WIEDEMANN,
"Uber Chlorophyll a, seine phaseb." ibid.. 515, 130 (1935).
positiven Derivate nnd seine Allomerisation," Helv.
Chim. Acfa, 16, 739 (1933); also reference (24) above.
(45) CONANT,DIBTZ,AND WERNER,''Chlorophyll studies. VIII.
''Chlorophyll
CONANT,DIETZ, BAILEY. AND KAMERLING,
The structure of chlorophyll b," J. Am. C h . Soc., 53,
series V. The structure of chlarophyU a," J. Am. Chem.
4436 (1931).
Soc.. 53. 2384 (19311.
(46) W n a u n o , "lfber Phmphytin b," Biochmn. Z.. 235, 240,
STOLLUND
WIEUEMANN,
"Die Benzoylverbindungen und
244, 9 (1932).
(1931); *bid.,
Oxime Ton Methyl Pheophorhide a;' Helo. Chim. Acte. (47) FISCHER,BROICH,BUEITNER.DND NUSSLER, her Chloro17, 163 (1934).
phyll b (I)," Ann. 498,228 (1932); FISCHER,B m I m n .
FISFEER, RIEDMAIR,
UND HASENKAMP, "Uber OxyporphyUND NUSSLER, %her Chlorophyll b (11),
HENDSCHEL,
ixd., 503, 1 (1933).
nne. Ein Beitrag zur Kenntnis der Feinstruktnr van
Chlorophyll a," Ann., 508,224 (1934).
(48) FISCHERUND BREITNER, "lfber Chlorophyll 4" ibid., 511,
MARCHLEWSKI.
Z. physiol. Chem., 42, 464 (1904); her
183 (1934): ibid., 516,61 (1935).
den Ursprung des Cholehamitins." i M . , 45, 466 (1905); (49) STOLL
UND WIEDEMANN,"Die Oxime der Phaophorbide
"Chemie der Chloro~hvll." Braunschweie. Viewea. 1909.
b." HA.Chim. Ada, 17,456 (1934).
(50) h s c n s n , HENDSCAEL,
UND:NOSSLER, "Nachweis des isacyclisches Ring in Chlorophyll b," Ann., 506,83 (1933).
(51) PATEKAND MINOT, "Bile pigment and hemoglobin regeneration. The effect of bile pigment in cases of chronic
~ls&IEn UND H E S ~"Vorkommen von ~h<lloerytkrinih
hyperchromic anemia,'' Am. J. Med. Sci., 188, 206-15
Rindergallenstein," Z. physiol. Chem.,,j87, 133 (1930):
(Aug.. 1934).
FISCHER,MOLDENRAUER,
DND Sus, "Uher Phyllo- und
Z s n E mrr, HELLSTROM,
"ober die aktive Grnppe der Leber
(52)
Ann..
485.
1
11931):
FISCHER
Pseudo~hvlloervthrin."
- ~ ~ - ~
Katalase," Z. physiol. Chem,..l92, 171 (1930).
;& G E ~ M A I R"synth&
,
d e s ~esoy-phylloerythrins
,
UND FLORKIN,
''Vber die Natur der Peroxyder Grnndsubstanz des ChlorophyUs, ibid., 490, 91 (53) K ~ NHAND,
dase," ibid., 201, 255 (1931).
(1~.)31),
L N O B A ~ I L B '.cher
R.
Ph:&. und Phylloeryrhro- (54) HILL AND KEILIN, PIOC.ROY. So<. (London), B107, 286,
FISCHCR
(1930); ZEILE UND REUTER,"Uher Cytochrome c," Z..
powllyrin~." ibid.. 474, 65 (19?\1); "Cber I'hmporphyphysiol. Ckem., 221, 101 (1933).
riue," iM.,
480, I K ( IXjIl).

~.

. -,.~ ~