N-Methylprotoporphyrin IX: Chemical Synthesis and Identification

Proc. Nati. Acad. Sci. USA Vol. 78, No. 3, pp. 1490-1494, March 1981 Biochemistry

N-Methylprotoporphyrin IX: Chemical synthesis and identification as the green pigment produced by 3,5-diethoxycarbonyl-1,4- dihydrocollidine treatment (N-alkyl porphyrins/porphyrin synthesis/ferrochelatase inhibitor/NMR) PAUL R. ORTIZ DE MONTELLANO, HAL S. BEILAN, AND KENT L. KUNZE Department of Pharmaceutical Chemistry, School of Pharmacy, and Liver Center, University of California, San Francisco, California 94143 Communicated by Rudi Schmid, November 24, 1980 ABSTRACT The hepatic pigment accumulated in 3,5-di- mitochondrial ferrochelatase (13, 14). The differential physio- ethoxycarbonyl-1,4-dihydrocollidine-treated rats, which has been logical effects of AIA and DDC are thus related, at least in part, reported to inhibit ferrochelatase, has been isolated and purified. to differences in the structures of the accompanying The pigment has been resolved into one major, one minor, and hepatic two trace components, all of which appear to be isomeric por- pigments. In concert with our efforts to define the structure of phyrins. The major fraction has been unambiguously identified the abnormal porphyrins due to AIA and other unsaturated byspectroscopic methods as the isomer of N-methyprotopor- agents (9, 10, 15, 16), we have undertaken a structural inves- phyrin IX (isolated as the dimethyl ester) in which vinyl-substituted tigation of the DDC-induced pigment. We report here un- pyrrole ring A is methylated. The minor product appears to be an ambiguous spectroscopic identification of the DDC pigment isomer of the same porphyrin with the N-methyl group on pro- as N-methylprotoporphyrin IX (dimethyl ester), confirmation pionic acid-substituted ring C, and the trace components have the same high-pressure liquid chromatography retention times as the of this structural assignment by chemical synthesis of the four other two possible isomers of the porphyrin. The four isomers of possible isomers of the structure, and use of the synthetic iso- N-methylprotoporphyrin LX have been chemically synthesized, mers to define the ratio of isomers in the biological product. independently characterized, and used to confirm the structures of the biological products. MATERIALS AND METHODS The heuristic perturbation of hepatic heme synthesis by chem- Isolation of Abnormal Porphyrins. Sprague-Dawley male ical agents has played a pivotal role in elucidation of the heme rats weighing approximately 250 g received aqueous sodium biosynthetic pathway and of the mechanism through which it phenobarbital (80 mg/kg of body weight daily) intraperitoneally is regulated. Two agents, allylisopropylacetamide (AIA) and for 4 days prior to injection by the same route of DDC (400 mg/ 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), have achieved kg) in dimethyl sulfoxide (100 mg/ml). The rats were decapi- particular prominence in this regard because the biosynthetic tated 4 hr after DDC injection and their livers were perfused derangements that they engender mimic in many respects the in situ and subsequently homogenized in ice-cold 0.9% saline inherited metabolic defects characteristic of certain human por- (1 ml of saline per liver). The resulting homogenate was added phyrias (1). Administration of DDC to animals results in inhi- to 5% (vol/vol) H2SO4methanol (100 ml per liver) and the bition of the hepatic mitochondrial ferrochelatase responsible mixture was stored at 40C in the dark for 17-20 hr. Filtration, for the insertion of iron into the porphyrin ligand and conse- addition of 0.5 vol of water, and extraction twice with meth- quently leads to the accumulation of protoporphyrin IX (2-4). ylene chloride yielded an organic extract, which was washed Direct inhibition of ferrochelatase by DDC, however, appears four times with equal volumes of water before 0.5 ml of meth- not to be involved because the enzyme is not inhibited in vitro anol saturated with zinc acetate was added. After drying over by this agent (2, 3). The mechanism of the inhibitory interaction anhydrous sodium sulfate and removal of the solvent on a rotary has therefore remained obscure. evaporator, a crude residue was obtained which was purified More than a decade ago, two independent groups observed by thin-layer chromatography on Analtech (1000 ,um) silica gel a rapid decrease in hepatic cytochrome P-450 levels in mice G plates using 3:1 (vol/vol) chloroform/acetone. The red-flu- treated with either AIA or DDC (5, 6). Subsequent studies es- orescing fraction (RF 0.4-0.6) was extracted with methanol and tablished that AIA-mediated loss of cytochrome P-450 reflected was rechromatographed under the same conditions. The puri- conversion of the enzyme prosthetic heme into an abnormal fied sample was then subjected to high-pressure liquid chro- green pigment (7, 8). We have established that the prosthetic matography (HPLC) on a 4.6 X 250 mm Partisil 10-PAC (What- heme is alkylated by an activated form of AIA produced during man) column that was eluted with a 20 min 0-100% linear catalytic processing of this substrate, andwe have demonstrated gradient of methanol into 1:1 (vol/vol) hexane/tetrahydro- that the AIA green pigment is a covalent adduct of AIA with furan. The zinc-complexed pigment, which migrated as a single protoporphyrin IX (9-11). The interaction of DDC with cyto- peak, was demetalated by passage through 5% (vol/vol) H2SOd chrome P-450, in contrast to the interaction of AIA with this methanol (10). HPLC of the metal-free sample on a 9.4 x 250 enzyme, has not been intensively investigated. Tephly, Gibbs, mm Partisil 10-PAC semipreparative column eluted isocrati- and De Matteis, however, have recently reported that a green cally with 97:97:6 (vol/vol) hexane/tetrahydrofuran/methanol hepatic pigment is also formed in DDC-treated mice (12, 13) resolved the porphyrin isomers present. These were collected and have made the further important discovery that this pig- separately and the principal isomer, used subsequently for ment, unlike that obtained with AIA, is a potent inhibitor of NMR studies, was further purified by reconversion to the zinc

The publication costs of this article were defrayed in part by page charge Abbreviations: AIA, allylisopropylacetamide (2-isopropyl-4-penten- payment. This article must therefore be hereby marked "advertise- amide); DDC, 3,5-diethoxycarbonyl-1,4-dihydrocollidine; HPLC, high- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. pressure liquid chromatography. 1490 Downloaded by guest on September 26, 2021 Biochemistry: Ortiz de Montellano et aL Proc. Nat. Acad. Sci. USA 78 (1981) 1491 were obtained on a modified Kratos MS-9 instrument. Electron II impact mass spectra were obtained on a Kratos MS-25 instrument at 70 eV. NMR spectra were taken at the University of ', California (Davis) NMR facility on a 360 MHz Nicolet NT-360 I Fourier transform NMR spectrometer. Generally, a 700 pulse was employed with a repetition time of 1.26 sec and an acqui- II sition time of 1.16 sec. Solutions of the porphyrins in deuterated chloroform were used for NMR studies. The zinc com- III A plexes were washed with sodium chloride solution prior to ramtu IV preparation of the solutions for NMR to ensure that chloride was the zinc counterion. All peaks are assigned relative to the chloroform signal at 7.21 ppm. Circular dichroism spectra were Cu3 w obtained in methylene chloride on a Jovan Dichrograph II. ascc co0 0 RESULTS The hepatic pigment formed in rats treated with DDC (400 mg/ kg), after carboxyl group methylation, extraction, and com- plexation with divalent zinc, was purified by sequential thin- /,f'\, layer chromatography and HPLC. A single band or peak was observed for the zinc complex throughout this purification se- quence. Removal of the zinc ion from the purified zinc complex, i however, followed by HPLC, led to resolution of the free base 2 6 10 14 18 into four relevant peaks (Fig. 1). The second and third of these Retentioin time, min peaks (in order of elution from the column) represent most of the isolated material. Of these two peaks, the former is quan- FIG. 1. HPLC analysis of the p)urified DDC pigment after demet- titatively the most important, whereas the first and fourth peaks alation but before fractionation (----) and the mixture of the four syn- represent relatively minor fractions. The ratio of the first two thetic isomers of the dimethyl estter of N-methylprotoporphyrin IX peaks to the latter two, all four being due to isomeric porphyrins (-). The synthetic isomers are numbered, in order of elution, as I-IV. Peaks with retention times of 2-7 min are due to impurities.

complex (saturated methanolic zinc acetate) followed by HPLC purification as described above for the zinc complexes. Synthesis of the N-Methylprotoporphyrin IX (Dimethyl Ester) Isomers. Methylfluorosulfonate (1.0 ml, 12 mmol) and 500 mg of the dimethyl ester of protoporphyrin IX (0.84 mmol) in dry methylene chloride (50 ml) were stirred in the dark at room temperature for 3 days. After washing with water, drying over anhydrous sodium sulfate, and solvent removal, the product was chromatographed on a 4 x 37 cm column of 10% (wt/ 01) wt) water-deactivated Merck silica gel 60, using 20:1 (vol/vol) w chloroform/methanol as the eluting solvent. Unreacted starting cucc porphyrin (75 mg), the dimethyl ester of N-methylprotopor- a0 phyrin IX (250 mg), and the dimethyl ester of N,N-dimethyl- -o protoporphyrin IX (50 mg) were eluted, in that order, from the column. The N-monomethylated fraction was then resolved into the four possible isomers of N-methylprotoporphyrin IX by HPLC on a 9.4 X 250 mm Partisil 10-PAC column, using 97:97:6 (vol/vol) hexane/tetrahvdrofuran/methanol as eluting solvent. The isomers were individually characterized. Synthesis of 3,5-Diethoxycarbonyl-1,4-Dihydro[15N]collidine. A modification of the procedure reported by De Matteis and Prior (17) was used to prepare ['5N]DDC. To a solution of 95% labeled [15N] ammonium chloride (Stohler Isotope Chem- icals, Waltham, MA) (0.50 g, 9.2 mmol) and sodium carbonate (0.49 g, 4.6 mmol) in 5 ml of water at 40C were added 2.4 g Wavelength, nm (18.4 mmol) of ethyl acetoacetate and 0.40 g (9.2 mmol) of acetaldehyde. After 6 days at 40C the precipitate was collected FIG. 2. Electronic absorption spectra of the zinc complexes of the by filtration and recrystallized from ethanol/water. After drying major isomer present in the DDC pigment (A), isomerII of the dimethyl under reduced pressure, 1.1 g (45% yield) of 3,5-diethoxycar- ester of syntheticN-methylprotoporphyrin IX (B), and isomer IV of the mp 125-1270C (uncorrected) dimethyl ester ofN-methylprotoporphyrin IX (C). The spectrum of the bonyl-1,4-dihydro['5N]collidine, zinc complex of synthetic isomer I is similar to spectrum B, and that [literature mp 131'C (17)] was obtained. of isomer III is similar to spectrum C. The Soret bands are recorded Spectroscopic Studies. Electronic absorption spectra were at a 10-fold higher attenuation: the molar absorbance values for the recorded in dichloromethane or chloroform on a Varian-Carv synthetic isomers are 124,000 at 431 nm, 7600 at 545 nm, 12,900 at model 118 spectrophotometer. Field desorption mass spectra 594 nm, and 2600 at 632 nm. Downloaded by guest on September 26, 2021 1492 Biochemistry: Ortiz de Montellano et. aL Proc. Nad Acad. Sci. USA 78 (1981) (vide infra), is approximately 6.5. The second and third peaks The principal DDC-derived abnormal porphyrin was recom- were collected and. their electronic absorption spectra, after plexed with zinc and was purified once more by HPLC prior recomplexation with. zinc, were determined. The spectrum of to determination of its NMR spectrum (Fig. 3). All the protons the zinc-complexed second fraction is given in Fig. 2 (spectrum expected from incorporation of protoporphyrin IX into the ab- A); the corresponding spectrum of the third peak (not shown) normal porphyrin can be discerned in the spectrum: four meso is superimposable on that reproduced in Fig. 2 (spectrum C). protons (10.2-10.4 ppm), two one-proton multiplets due to the The predominant (second) fraction has been subjected to fur- internal protons on the vinyl substituents (8.0 and 8.2 ppm), a ther spectroscopic study. Field desorption mass spectrometric four-proton multiplet due to the terminal protons on the vinyl analysis of the free base gave a very clean spectrum with a well- groups (6.2-6.4 ppm), eight protons due to the four methylene defined molecular ion peak at mass-to-charge ratio (m/e) 605. units in the propionic acid sidechains (4.3 and 3.3 ppm), and The electron impact mass spectrum, also obtainable in this in- six singlets due to the four methyl groups on the ring plus the stance, exhibited a molecular ion at m/e 604 with a smaller peak two methyl ester moieties (3.5-3.7 ppm). Only one other rel- at m/e 606. The latter peak reflects reduction. of the porphyrin evant signal appears in the spectrum, a three-proton singlet at within the electron impact source (18). To determine if the -4.5 ppm which is not lost by proton exchange on addition of nitrogen atom from DDC was incorporated into the abnormal deuterated water. The extreme upfield position of this signal porphyrin, a possibility suggested by the fact that the m/e 605 uniquely identifies it as an N-methyl substituent centered in molecular ion corresponded to the sum of protoporphyrin IX the ring current of the porphyrin. The additional (extraneous) (dimethyl ester) plus an -NH moiety, '5N-labeled DDC was peaks in the spectrum are due to chloroform (7.21 ppm), an im- synthesized. The labeled agent was administered to rats and the purity (1.5 ppm), and water (1.2 ppm). Identification of all the principal abnormal porphyrin formed was isolated as before. protons from the dimethyl ester of protoporphyrin IX and of The porphyrin gave the same m/e 604 and 606 molecular ion three N-methyl group protons, two structural components doublet as the porphyrin obtained with unlabeled agent. These whose molecular weights add up to the observed molecular ion results establish that the true molecular ion is at m/e 604, that at m/e 604, unambiguously defines the structure of the abnor- the field desorption peak at m/e 605 was a monoprotonated mal porphyrin as N-methylprotoporphyrin IX (dimethyl ester) molecular ion (M+ + H), and that the DDC nitrogen was not (Fig. 4). incorporated into the abnormal porphyrin. We have usually Chemical reaction of the dimethyl ester of protoporphyrin observed monoprotonated molecular ions in the field desorp- IX with methyl fluorosulfonate yielded, after purification and tion mass spectra of biological adducts (10, 15, 16). separation by HPLC of the isomers (Fig. 1), authentic samples

r I I I, , I , 12 11 10 9 8 7 6 5 4 3 -4 -5 ppm FIG. 3. NMR spectrum of the zinc.complex of the principal porphyrin isomer present in the DDC pigment. The peak at 7.21 ppm is due to CHC13. Not shown are peaks at approximately 1.5 ppm due to an impurity and at approximately 1.2 ppm due to water. Essentially all of the other signals are due to the abnormal porphyrin. The sample was not prewashed with NaCi, so the zinc counterion may not be chloride. Downloaded by guest on September 26, 2021 Biochemistry: Ortiz de Montellano et aL Proc. Natl. Acad. Sci. USA 78 (1981) 1493 whereas this shoulder is not present in the spectra of isomers III and IV (Fig. 2, spectrum C). The synthetic isomers exhibited a molecular ion at m/e 604 in their field desorption mass spectra even when the spectra were obtained on the same day (and with the same emitter) as the spectrum of the biological adduct. The consistent observation of a monoprotonated (m/e 605) molecular ion with the biological sample must therefore reflect an altered ionization mode due to the presence of trace impurities in the sample. The electron impact spectra of the synthetic and biological samples, however, were identical. The NMR spectra of the four synthetic isomers (Fig. 5) can be divided into two sets. Isomers I and II are differentiated from isomers III and IV by the presence in the first pair of two distinct internal vinyl proton signals (8.0 and 8.2 ppm) but of only FIG. 4. Isomer II of N-methylprotoporphyrin IX (dimethyl ester). one signal for each of the two types of methylene groups in the Pyrrole ring A is alkylated in the structure. propionic acid side chains (3.3 and 4.3 ppm). Isomers III and IV, conversely, exhibit only one internal vinyl proton multiplet but four distinct signals due to the propionic acid side chain of the four possible N-methylprotoporphyrin IX dimethyl ester methylene groups (4.3, 4.2, 3.3, and 2.9 ppm). These differ- isomers. In order of elution from the HPLC column, these ences clearly establish that isomers I and II are N-methylated isomers are designated as I-IV. The electronic absorption spec- on vinyl-substituted pyrrole rings, whereas isomers III and IV tra of the zinc complexes of these isomers differ slightly, the are those methylated on the propionic acid-substituted rings. major difference being that the Soret band in isomers I and II This follows from the fact that tilting of the N-methylated ring (Fig. 2, spectrum B ) has a shoulder at longer wavelengths, will alter the position of its substituents relative to the ring cur-

IIn W~~~~~~~~~~~II.

4. 11 10 9 8 7 6 5 4 3 -4.6

ppm

FIG. 5. NMR spectra of the four synthetic isomers of N-methylprotoporphyrin IX (dimethyl ester). The methyl group signals at approximately 3.6 ppm are shown truncated. Isomers II and IV are slightly contaminated with isomers I and III, respectively. Chloride was the counterion in all these samples. Downloaded by guest on September 26, 2021 1494 Biochemistry: Ortiz de Montellano et alPProc. Nad Acad. Sci. USA 78 (1981)' rent and.cause them to shift upfield in the NMR spectrum rel- tive that they must have been formed in a stereoselective ative to similar substituents on the unmethylated pyrrole rings process. (18). Definitive characterization of the DDC-engendered por- Preliminary circular dichroism studies of biologically formed phyrin fills a critical void in our understanding of the mecha- isomers II and III show that these. abnormal porphyrins are nism of action of this porphyrinogenic agent. In conjunction optically active. No circular dichroism curves are obtained, of with our studies of the abnormal porphyrins induced by other course, with the corresponding synthetic structures because agents (10, 15, 16), this investigation also lays the foundation they are racemic mixtures. for elucidation of the mechanisms by which the abnormal porphyrins are formed. The origin of the N-methyl group. in the DISCUSSION present case and the chain of events that results in, its intro- duction are intensely interesting questions for two reasons. The The esterified pigment from DDC-treated rats has been re- first is that, unlike other abnormal porphyrins in which the ad- solved into four porphyrin components (Fig. 1), the principal 15, 16), it is one of which has been unambiguously identified by electronic ministered agent becomes the N-alkyl group (10, absorption (Fig. 2, spectrum A), NMR (Fig. 3), and mass spec- not evident that the N-methyl group is contributed by the orig- trometric studies as the dimethyl ester of N-methylprotopor- inal DDC framework. The second is that a hepatic pigment phyrin IX. Chemical synthesis of the four possible isomers of identical to that induced by DDC has been claimed to be pres- this N-methylated porphyrin has allowed independent confir- ent in untreated mice (12). If confirmed, it may mean that the mation of the structural assignment: the retention time on formation of N-methylprotoporphyrin IX is a physiological HPLC (Fig 1), the electronic absorption spectrum (Fig. 2, spec- rather than aberrant process. The synthetic availability of the trum B), and the NMR spectrum (Fig. 5) of synthetic isomer isomers of this porphyrin, described here, should expedite the II are identical to those of the major biologically formed prod- effective resolution of these problems. uct. In addition, the minor but still significant.component of This work was supported by National Institutes of Health Grants GM the biological sample (the third peak) exhibits the same reten- 25515 and P50 AM-18520 and by an Alfred P. Sloan Fellowship awarded tion time (Fig. 1) and electronic absorption spectrum (Fig. 2, to P.R.O. M. Field desorption studies were carried out at the Berkeley spectrum C) as synthetic isomer III. The two trace components Biomedical Mass Spectrometry Resource supported by National Insti- in the biological sample appear, on the basis of their. retention tutes of Health Grant RR00719. Generous access to the mass spec- time, to be isomers I and IV of the N-methylated porphyrin, trometer provided by Prof. A. Burlingame, the facility director, is grate- although their detailed-study has not been possible. fully acknowledged. The synthesis and individual characterization of the four N- 1. De Matteis, F. (1967) Pharmacol. Rev. 19, 523-557. methylprotoporphyrin IX isomers have enabled us to establish 2. Onisawa, J. & Labbe, R. F. (1963)J. Biol Chem. 238, 724-727. that isomers I and II have the N-methyl group on the vinyl-sub- 3. Tephly, T. R., Hasegawa, E. & Baron, J. (1971) Metab. Clin. Exp. stituted pyrrole rings. Isomers III and IV, on the other-hand, 20, 200-214. on the acid-substituted pyrrole rings. 4. De Matteis, F., Abbritti, G. & Gibbs, A. H. (1973) Biochem. J. are methylated propionic 134, 717-727. Isotopic studies combined with NMR relaxation and nuclear 5. Wada, O., Yano, Y., Urata, G. & Nakao, K. (1968) Biochem. Phar- Overhauser measurements (details to be reported separately) macol 17, 595-603. have established that isomers I, II, III, and IV are N-methylated 6. Waterfield, M. D., Del Favero, A. & Gray, C. H. (1969) Biochim. on rings B, A, C, and D, respectively. The identity of the prin- Biophys. Acta 184, 470-473. cipal biological product with isomer II thus demonstrates that 7. Unseld, A. & De Matteis, F. (1976) in Porphyrins in Human Dis- pyrrole ring A in the heme from which the. adduct is derived ease, ed. Doss, M. (Karger, Basel, Switzerland), pp. 71-75. 8. De Matteis, F. (1978) in Heme and Hemoproteins, Handbook of is the most vulnerable to N-methylation. However, all four rings Experimental Pharmacology, eds. De Matteis, F. & Aldridge, W. appear to be biologically methylated, propionic acid-substituted N. (Springer, New York), Vol. 44, pp. 95-127. ring C (that which results in isomer III) being the second most 9. Ortiz de Montellano, P. R., Mico, B. A. & Yost, G. S. (1978) susceptible to N-alkylation. Biochem. Biophys. Res. Commun. 83, 132-137. Correlation of the electronic absorption spectra of isomers 10. Ortiz de Montellano, P. R., Yost, G. S., Mico, B. A., Dinizo, S. I-IV with the type of ring alkylated in each reveals a potentially E., Correia, M. A. & Kambara, H. (1979) Arch. Biochem. Biophys. 197, 524-533. very useful difference between N-alkylation of vinyl- and pro- 11. Ortiz de Montellano, P. R. & Mico, B. A. (1981) Arch. Biochem. pionic acid-substituted rings: a shoulder on the Soret band (Fig. Biophys. 206, 43-50. 2, spectrum B) is found in the former but not in the latter (Fig. 12. Tephly, T. R., Gibbs, A. H. & De Matteis, F. (1979) Biochem.J. 2, spectrum C). If general, this difference provides a simple 180, 241-244. means for the assignment of the type of ring alkylated in other 13. De Matteis, F., Gibbs, A. H. & Tephly, T. R., (1980) Biochem. biological adducts. The findings that alkylation by ethylene of J. 188, 145-152. 14. De Matteis, F. & Gibbs, A. H. (1980) Biochem. J. 187, 285-288. the prosthetic heme of cytochrome P450 (16, 19) yields N-(2- 15. Ortiz de Montellano, P. R. & Kunze, K. L. (1980)J. Biol. Chem. hydroxyethyl) protoporphyrin IX in which the N-alkyl substi- 255, 5578-5585. tutent is on a propionic acid-bearing ring (20) and that the Soret 16. Ortiz de Montellano, P. R., Kunze, K. L. & Mico, B. A. (1980) band. of the dimethyl ester of this porphyrin does not have a MoL Pharmacol. 18, 602-605. shoulder support the validity of the proposed spectroscopic cor- 17. De Matteis, F. & Prior, B. E. (1962) Biochem.J. 83, 1-8. relation. It is also apparent from the difference in the pyrrole 18. Jackson, A. H. & Dearden, G. R. (1973) Ann. N.Y. Acad. Sci. 206, 151-176. ring principally (or exclusively) alkylated in.these two different 19. Ortiz de Montellano, P. R. & Mico, B. A. (1980) Mol. Pharmacol. adducts that there is no universal preference for biological N- 18. 128-135. alkylation of one type of ring. It is also clear from the obser- 20. Ortiz de Montellano, P. R., Beilan, H. S., Kunze, K. L. & Mico, vation that the biologically isolated structures are optically ac- B. A. (1981) J. Biol. Chem. 256, in press. Downloaded by guest on September 26, 2021