Syntheses of isotopically labelled derivatives of protoporphyrin-IX for spectroscopic and biosynthetic studies

lnr. J. Btu‘hr.m. Voi 12. pp 689 to 694 0 Pergamon Press Ltd 1980. Pnnted III Great EMam

SYNTHESES OF ISOTOPICALLY LABELLED DERIVATIVES OF PROTOPORPHYRIN-IX FOR SPECTROSCOPIC AND BIOSYNTHETIC STUDIES KEVIN M. SMITH and KEVIN C. LANGRY

Department of Chemistry, University of Caiifomia at Davis. Davis, CA 95616, U.S.A. Abstract.--This paper describes: 1. Recent methods developed to accomplish regioseiective deuteriation of derivatives of protoporphyrin-IX. 2. New syntheses of coproporphyrin-III, harderoporphyrin, and isoharderoporphyrin, from deuteroporphyrin-IX using a mercuration and paIladium/olefin reaction sequence.

lNTRODUCTiON A large variety of spectroscopic methods have been employed (Smith, 1975) for investigations of the physical, chemical, and biological characteristics of the porphyrin system. One of the most successful of these techniques has been nuclear magnetic resonance (NMR) spectroscopy. If low spin iron(II1) complexes of porphyrins are used, then it is possible not only to study free ferrihemes, but also the same compounds embedded in a protein matrix. On account of the unpaired electron in low spin iron(II1) porphyrins. any NMR resonances associated with the iron atom (i.e. on the porphyrin or the coordinated imidazoles) suffer Iarge paramagnetic shifts which cause these resonances to be situated clear of the thousands of peaks associated with the diamagnetic envelope of the protein. Moreover, the chemical shifts of the porphyrin-associated resonances can be important for determination of the electronic structure in the heme, since the magnitude of the paramagnetic shift is related to the spin density at that position in the prosthetic heme (Shulman et al., 1971). In recent years, the amount of information which can be gleaned from heme protein NMR spectra has been limited mainly by the fact that compiete interpretation of the spectra, and the electronic effects which they indicate, cannot be made with certainty until the resonance assignments are made in a definitive way. In this paper we describe recent work involving deuteriation of the methyls, vinyls, and meso-positions in protoporphyrin-IX dimethyl ester (l), which can be used, by difference NMR, to make assignments in complex heme protein NMR spectra.

in protoporphyrin-IX itself to give partially deuteriated (1) was also discovered (Evans et al., 1977); this procedure was unsatisfactory for production of large quantities of heavily 1,3-deuteriated material (2) because of decomposition during the required prolonged treatment with strong base. We therefore reasoned that the methyl exchange in rings A and B would be facihtated if more strongly electronwithdrawing groups (than vinyl) were situated at the 2 and 4 positions in protoporphyrin-IX dimethyl ester (1). Treatment of 2,4-diacetyldeuteroporphyrin-IX dimethyl ester (6) with NaOMe/MeOD under reflux for only 5 hr caused almost complete exchange of the 1 and 3 methyls, as well as the acetyl methyls and the propionate methylenes adjacent to the ester carbonyls. Reduction with borohydride gave (7) which

DEUTERIATION OF METHYL GROUPS Methyl groups in the proton NMR spectrum (Fig. 1) of the low spin dicyanoferriheme from (1) can be assigned in a qualitative fashion merely by integration of peak areas. However, this approach does not give definitive assignments to show which of the four lowest field 3-proton singlets is the 1, 3, 5, or 8 methyl. We have previously described total syntheses of the methyl deuteriated derivatives (2H5) (Cavaleiro et al., 1974a; Smith et al., 1979a). An unexpected basecatalyzed method for exchange of the 1 and 3 methyls

I 20

I

IO

I

O

ppm

Fig. 1. 100 MHz proton NMR spectra in CD,OD of the dicyanoferrihemes from A, protoporphyrin-IX dimethyl ester (If; B, 1,3-di-(trideuteriomethyl)protoporphyrin-IX dimethyl ester (8). Assignments Iabelled 1, 3, 5, and 8 refer to the methyl groups at the corresponding ring position. Other assignments in A are: a, vinyl a-CH; b, propionic methylenes adjacent to the porphyrin ring; c. vinyl /KH,; s, solvent. 689

KEWN M. SMITH and KEVIN C. LANERY

6) R’= R%W$ (91 R’= R2= W (13) R’- &

0

(16) R’= R2”CH=CW~Me

R’

I14)M=Zn,

Me

R’=@=H

(15) M = Zn, R’= R’= Hgtl (20lM=Cu,

R’=R’=H

(21) M = Cu, R’= COMe, ?f- H (22) M = Cu, R’= H, @= CCtvle (23) M = Cu, R’= COMe ;R’= &&I WJM=Cu,

R’=H@;

fi”~COMe

125fNI=C;u;R1-COhrle,fJECX4CJJC0,Nte iE6J M = Gil, R’- CH-CHCa, 127) M = Cu; R!- Cme;

Me, f?%Ol&

R_2-&H,CH,Co*Me

ES> M = Cu; R’= CHsCH,CO$ie;

R%X%le

691

Labelled derivatives of protoporphyrin-IX was dehydrated using toluene psulfonic acid, to give (8). Figure 1 shows the NMR spectrum of the dicyanoferriheme from (I), and from (8), and ~90% exchange of the 1 and 3 methyl groups is obvious. We have employed our regioselectively methyl labelled hemes in a variety of studies with heme proteins. The most recent example (La Mar et al., 1978) is in characterization of the phenomenon of “heme disorder” in reconstituted heme proteins. We have shown that minor peaks in the NMR spectrum of deuterohemin-reconstituted myoglobin can be attnbuted to the presence, in the invariant heme pocket, of small amounts of disordered material which differs from the natural material by a 180” rotation of the heme about the ay axis. It was also possible to vary the proportions of the un-natural and natural species by careful choice of pH. Given the availability of labelled hemes from (2)-Q) it was a simple matter to demonstrate this unique effect. DEUTERIATION

OF VINYL GROUPS

Simple synthetic schemes (Scheme 1) (Budd et al., 1979) can be employed, using 2,4_diacetyldeuteroporphyrin-IX dimethyl ester (6), for regioselective

C_H-CH,

deuteriation of the vinyl a-H and /?-H in (1). However, these protons can also be exchanged by merely treating protoporphyrin-IX dimethyl ester (1) with deuteriated toluene p-suifonic acid. Figure 2 shows the proton NMR spectrum of a sample treated in this way. The /?-vinyl resonances are clearly diminished by cu. 90%, but to our surprise, the a-vinyl protons were also about 45% exchanged. The results imply intermediacy, not only of the expected secondary vinyl-derived carbonium ion (Par-&HCH,), in acid, but also the primary one (PorCH,&H,) or the corresponding cyclopropyl analog

Por:j1

.CH* j ‘CH, I .

Work in hand, using carbon-13 labelled materials, will test the feasibility of the last of these three possibilities, but there exists already good evidence for such unusual carbonium ions from NMR spectroscopy (Smith & Unsworth, 1975), and in a novel vinyl cyclization reaction of the bacteriochlorophylls-c from Chloropseudomonas ethylicum (Kenner et al., 1978).

CH-CJ,

I

Fig. 2. 100 MHz proton NMR spectrum in CDCI, of protoporphyrin-IX dimethyl ester (1) after treatment with deuteriated toluene p-sulfonic acid in refluxing o-dichlorobenzene for 4 days. The inset shows an expansion of the meso proton region; Greek letters refer to the individual meso assignments. BC 12/5-6-c

KEVIN M. SMITH and KEVIN C. LANGRV

692 DEUTERIATION

AT THE MESO POSITIONS

Several convenient methods exist for deuteriation at the meso positions in porphyrins (Kenner er al.. 1973 and references therein), but most of these have been used only on symmetrically substituted porphyrins such as octaethyiporphyrin. Some earlier work (Woodward & Skaric, 1961) using rhodoporphyrin-XV concluded that the electronwithdrawing group at the &position deactivated the whole system toward exchange. However, examination of the inset in Fig. 2 shows that the four meso protons in protoporphyrin-IX dimethyl ester (1) have been differentially exchanged during treatment with deuteriated toluene p-sulfonic acid. These meso proton assignments are available (Abraham rt al., 1979; Janson & Katz, 1972) and they are given in the inset. The conclusion is that those protons furthest away from the vinyl groups react fastest in electrophilic deuteriation (i.e. r < p < 6 < y) and this very sample has enabled us to make definitive meso assignments in the NMR spectrum of the low spin dicyanoferri-

heme (Smith er al.. 1979b). Previously we had been attempting to do this by lengthy total syntheses (Kenner & Smith, 1973). We have also examined electrophilic meso deuteriation of other unsymmetrically substituted porphyrins, and the results are shown in Fig. 3. From these spectra it becomes obvious that the protoporphyrin-IX dimethyl ester(l) result is anomalous. Both deuteroporphyrin-IX dimethy ester (9) and 2,4-diacetyldeutero~rphyrin-IX dimethy ester (6) undergo meso-exchange with the order 6 < r < 1’ < p. Bearing in mind the electronic similarities between the protonated intermediates (lo), (11). and (12) [related to (1) (9), and (6), respectively], one would expect the exchange order at the meso positions to be paralleled in all three cases, Considering, for all NH tautomers, the number of occasions for (lo), (1 l), and (12) (full structures) that the positive charge can be conjugated most often with the a and 6 positions (18 times) and with the a and y positions (10 times), it would be concluded that the exchange order would be a-6
039

~

~__ 98

96 S twm

with deuteriated toluene Fig 3. 360 MHz proton NMR spectra in CDCI,. after 4 days treatment p-sulfonic acid in refluxing o-dichlorobenzene. of the meso proton region of A. Zinc(H) protoporphyrin-IX dimethyl ester [plus 2 mol-equiv of pyrrolidine to disaggregate spectrum (Abraham ef al., 1979)]; B. zinc(H) 2.4-diacetyldeuteroporphyrin-IX dimethyl ester (plus 2 mol-equiv of pyrrolidine); C, palladium(I1) deuteroporphyrin-IX dimethyl ester. The zinc(I1) complexes were used in A and B, after addition of pyrrohdine, to remove upfield shifts due to aggregation (Abraham ef al.. 1979). In the case of spectrum C, the paIladium(I1) complex (which also does not aggregate) was used because the zinc/pyrroiidine spectrum showed troublesome overlapping of the jI and 6 resonances. Numbers above each peak are the proportions of a proton present in each case. the data being obtained by integration relative to an internal standard. Greek letters below the peaks refer to the individual meso proton assignments.

693

Labelled derivatives OFprotoporphyr~n-IX this can be explained by once again postulating

the transient existence af the vinyl-derived primary carbonium ion (Par-CH&H& such an ion would be expected to inhibit electrophlic attack at the meso positions adjacent to it, i.e. CIand 8. NEW SYNTHESES OF CQPROPORPHYRIN-HI, HARDEROPORPHYRIN, I~~AR~EROPORPHYRlN

AND ESTERS

In order to obtain 2,4~ideuteriodeuteroporphyrin-IX dimethyl ester (13) for use in connection with NMR assignments in deuterohemin-r~onst~tut~ myoglobin, we decided to investigate mercuration and acidic demercuration of deuteroporphyrin. We have already shown (Hudson & Smith, 1975, 1976) that treatment of metal-free porphyrins with mercury(H) acetate gives “double sandwich” mercury complexes in which the metal atoms are associated with the central porphyrin nitrogen atoms. We supposed that something else might happen if we masked the porphyrin nitrogens by pre-forming a metal complex. Treatment of zinc(H) deutero~rphyrin-IX dimethyl ester (14) with mercury(H) acetate gave a > lOO*? yield of the ~ripherally dimercurat~d complex (1.5) after addition of chloride. (The “high” yield is presumably due to inclusion of extra mercury atoms, possibly by formation of some “double sandwich” complex). Treatment of (15) with DC1 gave a good yield of the required 2.4”dideuteriodeuteroporphyrin (13), which had also suffered some regioseiective meso deuteriation during the DC1 treatment. This successful mercuration experiment encouraged us to attempt the palladium/ole~n reaction (Heck, 1974) on this substance. Treatment of (15) with the methyl acrylate and LiPdCl, in acetonitrile gave a good yield of the bisacrylate (16) after demetallation. Catalytic hydrogenation of (16) in formic acid gave an excellent yield of copro~rphyrin-III tetramethyl ester (I 7), which was shown to be identical with an authentic sample To further illustrate the use of this chemistry, we chose to synthesize harderoporphyrin trimethyl ester (18) and its 2,4-isomer (19). Harderoporphyrin has been identified (Cavaleiro et ul., 1974b; Jackson et al., 1976) as the tripropionic acid porphyrin present in the Harderian gland of certain rodents; its ~~hyrinogen (hexahydro derivative) has also been shown (Cavaleiro er al., 1974s; Games et al,, 1976) to be a key intermediate in the biosynthesis of protoporphyrin-IX from coproporphyrinogen-III. Mono-acetylation of copper(H) de~teroporphyrin-IX dimethyl ester (20) with acetic anhydride and tin(IV) chioride gave a mixture of the two isomers (21) and (22). These were mercurated with mercuryfIIf acetate, followed by treatment with sodium chloride, to give (23) and (24), stitl as a mixture. With methyl acrylate and LiPdCl, in acetonitrile. these compounds gave (25) and (26) respectively, which were separated using preparative thick iayer chromatography. The fastest running band correlated with hardero~rphyrin (18). The individual copper compfexes were hydrogenated in tetrahydrofuran over patladized charcoal to give the tripropionate porphyrins (27) and (28), which were demetallated (using sulfuric acid in trifluoroacetic acid) to give (29) and (30). Reduction of the acetyl groups with sodium borohydride, and dehydration with toluene

p-sulfonic acid in o-dichlorobenzene gave harderoporphyrin trimethyl ester (18) and isoharderoporphyrin trimethyl ester (19) in good yield. The identity of each compound was established by melting point, mixed melting point with an authentic sample. as well as proton NMR and high performance liquid chromatography comparison (Cavaleiro et al., 1974b). Compounds (18) and (19) could also be obtained by use of the previously separated and identified monoacetyIdeutero~rphyrin-IX isomers. The 2-acetyi. isomer, of course, was correlated with harderoporphyrin trimethyl ester (18), using the same sequence of reactions as outlined above. SUMMARY f . Methyl groups at the I and 3 positions in proto~rphyrin-IX can be deuteriated by a simple base-catalyzed scheme using 2,4-dia~etyldeuteroporphyrin-IX. 2. Vinyl groups in protoporphyrin-IX and meso positions in it and its derivatives can be efficiently deuteriated using acid, and in a regioselective manner. 3. Coproporphyrin-III, harderoporphyrin, and isohardero~rphyrin methyl esters can be readily synthesized from deutero~rphyrin-IX using a synthetic sequence invofving mercuration, foflowed by a palladium/olefin reaction.

~ck~owl@dge~enrs-We are pleased to acknowledge financial support of this research by the National Institutes of Health (HL 22252). and a grant from the Research Corporation which enabled purchase of the HPLC equipment.

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