[DIVISION OF
FRUITPRODUCTS, UNIVERSITY
OF CALIFORNIA]
SPECTRA OF PORPHYRINS AND THEIR ACID SALTS S. ARONOFF
AND
C. A. WEAST
Received February 17, 1941 INTRODUCTION
H. Fischer's work confirmed without doubt, more than a decade ago, Kuster's conception of the porphyrin nucleus as that of a tetrapyrrole ring, the pyrroles being joined by methine carbons. The precise nature of the inner ring is, however, in doubt and has been the subject of considerable speculation. The present investigation was undertaken in an attempt to determine spectroscopically the existence of various acid porphyrin forms. The Fischer-Kuster formula could make possible a molecule with four basic dissociation constants, Le., the nitrogens being secondary or tertiary and adding H+ according to: NH+ Hf = NH2+, and h' H+ = KH+
+
FIG.1
With few exceptions, however, porphyrin salts have been of the composition P.2HC1 as shown by both potentiometric methods (1) and chemical analyses (2, 9). The usual method of obtaining porphyrin salts does not involve high acidity ( i e . , greater than concentrated HC1). Some of the nitrogens involved in salt formation may be of extremely weak basicity and their behavior may be shown in concentrated acid spectroscopically (3). It has generally been presumed that each acid form will display its own characteristic spectrum. Thus a compound capable of tetrasalt formation should display five spectral forms, one for each of the salts, and one for the free base. Similarly, a compound capable of disalt formation should show three forms, etc. Disalt formation as well as color and equivalence of the pyrrole rings, 550
SPECTRA OF PORPHYRINS
551
Desmophylloerythrin
PhySloe~hriu
FIG.3
are readily explained by Fischer's formula. The free base is assumed to resonate among eight homopolar structures (Fig. 1) etc., and forty-eight ionic structures, e.g., Fig. 2. It is, of course, possible that the tautomerism
552
S. ARONOFF AND C. A. WEAST
indicated (conceivably a resonance because of the small distance to be traversed) does not exist (6, 4), and the number of structures would be halved. Both the x-ray analyses of phthalocyanines ( 5 ) and infra-red studies of porphyrins (lo), however, indicate N-H--N bonding. Forms containing a double charge-separation could also be written, but their contribution is of much smaller magnitude than those with single chargeseparation. The different homopolar formulas readily show the equivalence of the @-positionsof the pyrroles, while the charge-separated structures promote color formation. We believe that in acid solution the charge-separated forms predominate, that is, approximate more closely the true state of the resonating structure, showing the interaction of the nitrogens with the rest of the system and explaining the acid spectra.
660
600
650
m !
FIG.4. ETIOPORPHYRIN I11 1. 96% H~SOI. 2. Pyridine-H&O, mixture. 3. Pyridine
Four porphyrins were studied, three of which displayed two spectral curves and one, three spectral curves. The compounds used were: etioporphyrin 111, mesoporphyrin IX, desoxophylloerythrin, and phylloerythrin; their skeletal structural relationships are shown in Fig. 3
[Pr =
- (CH2)&OOH].
EXPERIMENTAL
Etioporphyrin I11 was obtained as a sublimation product by the decarboxylation of mesoporphyrin IX. This was accomplished by heating mesoporphyrin in a potassium nitrate bath at 350" in a high vacuum (10-6mm. Hg).' Mesoporphyrin itself was obtained by hemin reduction by the method of Fischer (Z),except for the 1 Courtesy of Dr. H. J. Almquist, Division of Poultry Husbandry, University of California.
553
SPECTRA OF PORPHYRINS
use here of 57% hydriodic acid (Merck's reagent sp. g. 1.7). Hemin waa prepared by the usual method of Schalfejeff from defibrinated cow blood. Desoxophylloerythrin
FIG. 5. DESOXOPHYLLOERYTHRIN 1. 96% &Sod. 2. 48% HtSO4. 3. Pyridine
....
I
I
650
* 800
I
560 .cy
FIG. 6. PHYLLOERYTHRIN 1. 96% Hasod. 2. 22.8% HCI. 3. Pyridine. 4. 11.4% HCl was prepared from a chlorophyll mixture with hydrobromic-acetic acid, and phylloerythrin by hydriodic-acetic acid reduction.
554
5. ARONOFF AND C. A. WEAST
Absorption measurements were made visually with a Bausch and Lomb Universal Spectrophotometer. As the acid solvent, 96% sulfuric acid was utilized, in which all the compounds were stable. Acidity was decreased by addition of pyridine down to a 1:l ratio of pyridine-sulfuric acid, (with further dilution, insoluble pyridine-HZSOd is formed). Pyridine-hydrochloric acid mixtures were therefore substituted for the lower acidities. The results are shown in figures 4 , 5 , and 6. To avoid confusion, the curves for lower acidities are not included. They were in all cases practically identical v i t h the lowest acid spectra shown. It is seen that except for phylloerythrin there is no significant form change in concentrated acid solution apart from solvent effects (band maxima shift and volume contraction), cf. figure 7.
.2
.1
1. 96% HZSO,.
FIG.7. PRYLLOERYTRRIN 2. 86.4%. 3. 76.7%. 4. 67.3%. 5. 48% HzSOd DISCUSSION
The change of the acid spectrum of phylloerythrin in concentrated sulfuric acid may be correlated with the oxygen on the isocyclic ring. This oxygen is capable of oxonium formation, viz.,
H N H’
\
+ H+ cII
0
-
H N
”\
C
II
H-O+
SPECTRA OF PORPHYRINS
555
The latter structure can then resonate with one of the ring nitrogens, e.g.,
I
HO resulting in a spectral shift toward the red. The keto group of the free base involves the resonance :
H
I
0-
resulting in significant effect on the free base spectrum, but this chargeseparation involves more work than that occasioned by the oxonium resonance. In the enolic free base, a resonance similar to the ketonic is not possible, and thus the ketonic form probably predominates. Treibs (9) has cited evidence (7) that various degrees of porphyrin-acid compounds exist, the ratio of porphyrin:acid varying from 1:l to 1:5, types greater than 1:2 being addition compounds. He concludes that there are two acid spectral types, the highest and normally observed type being a disalt, The existence of a higher type, however, has been demonstrated here in phylloerythrin, and may be expected in similar compounds such as pheoporphyrin ab, etc. In an attempt to find the intermediate type (monosalt) of a typical porphyrin (mesoporphyrin), pyridine-acetic acid spectra have been measured. It is seen (Fig. 8) that the intermediate forms may be calculated by the assumption of a salt/free base ratio, and the addition of the proportionate amount of the corresponding acid and free base curves. (To avoid confusion with the calculated values, the ordinates of the observed mixture have been displaced vertically.) Spectroscopic evidence of an intermediate type has, therefore, not been found. The monosalt must then be formed in such a manner as to have a spectrum identical with that of khe free base or the disalt, or its existence must be limited within an extremely short range of acidity. We believe the latter to be more probable for
556
S. ARONOFF AND C. A. WEAST
three reasons. Representing the porphyrin, its mono- and di-salts by
*e
-.5
I
I
6M)
t
I
I
I
650
600
I
I
I
soow
FIG.8. MESOPORPHYRIN IX 1. Glacial acetic acid. 2. Mixture, calculated, Pyridine-acetic acid (6:4). 2' Mixture, observed. (To avoid confusion with the calculated values, the ordinates of the observed mixture have been displaced vertically.) 3. Pyridine.
1
2
3
FIG.9
(a) the spectrum of the monosalt cannot be identical with that of the free base because the addition of the first hydrogen (structure 2) should result in an increase in color (shift to the red) owing to the ease of chargemigration (without charge-separation),
SPECTRA OF PORPHYRINS
557
(b) nor can the spectrum of the monosalt be identical with that of the disalt (structure 3) because nitrogen-carbon charge-separated resonance structures and H-tautomers cannot exist in the latter, though they may in the former, (c) because of the unsymmetrical nature of the monosalt, its existence should be limited to a short range of acidity, on the basis of the work of Schwarzenbach and co-workers (8). The monosalt spectrum may possibly be determined by appropriate calculations in the narrow region where, by virtue of its presence, the salt/free base curve deviates from calculated values. SUMMARY
Four porphyrins, etioporphyrin 111, mesoporphyrin IX, phylloerythrin and desoxophylloerythrin were studied. 1. Spectroscopic investigation reveals no change in acid form with increasing acidity up to 96% sulfuric acid, except where additional structures may be formed and the resonances increased, as oxonium formation on the ketonic oxygen of phylloerythrin. 2. “Intermediate” types of porphyrin spectra are shown to be mathematically deducible by the addition of the acid and free base curves, assuming a salt/free base ratio. 3. The existence of porphyrin monosalts must be limited within a narrow range of acidity. The authors wish to acknowledge the criticism and numerous helpful suggestions of Drs. M. Calvin and G. Mackinney. BERKELEY, CALIF. REFERENCES (1) COXANT, CHOW,AND DIETZ,J . Am. Chent. Soc., 66, 2185 (1934). (2) FISCHER, ORTH,AND STERN,“Die Chemie des Pyrrols,” Vol. II., parts 1 and 2. Akademische Verlagsgesellschaft M. B. H., Leipzig, 1937, 1940. (3) FLEXSER, HAMMETT, AND DINGWELL, J . Am. Chem. Soc., 67, 2103 (1935). (4) KNORR AND ALBERS, J . Chem. Phys., 9, 197 (1941). (5) ROBERTSON, J . Chem. SOC.,1936, 1195. (6) ROTHEMUND, J . Am. Chem. SOC.,61, 2912 (1939). (7)SCHUMM, Z. physiol. Chem., 181, 141 (1929). (8) SCHWARZENBACH, OTT AND HAGGER, Helv. Chim. Acta, 20, 490 (1937). (9) TREIBS,Ann., 476, I (1929). (10) VESTLING AND DOWNING, J . Am. Chem. Soc., 61, 3511 (1939).
