A partial synthesis of the diastereomeric mixture of methyl pheophorbides 2 from the chlorophyll degradation product chlorin e6 trimethyl ester (4) is described. Compound 2 is related to band 6 of the bacteriochlorophylls c (Chlorobium chlorophylls, "660" series) and also to bacteriochlorophyll c" recently isolated from Chloroflexus aurantiacus. Separation of the R,S diastereomeric mixture [at the 2-(l-hydroxyethyl)group] is readily accomplished by using reverse-phase high-performance liquid chromatography (high-performance LC). Markownikoff hydration of methyl pyropheophorbide a (17) similarly gives the R,S diastereomeric mixture [16(R),16(S)] which can in turn be separated by high-performance LC. On account of Woodward's earlier total synthesis of optically pure chlorin e6 trimethyl ester (4), this work constitutes a formal total synthesis of the optically pure methyl pheophorbides of band 6 of the bacteriochlorophylls c (equivalent to methyl bacteriopheophorbide cs) and of band

Incorporation experiments with labeled sirohydrochlorin and trimethylisobacteriochlorin demonstrate that ring contraction in vivo to the corrin macrocycle of vitamin B12 liberates acetic acid. The C-20 atom of the precursors becomes the acetate carboxyl carbon.The trimethylisobacteriochlorin isolated (1, 2) from the vitamin B12 producer Propionibacterium shermanii was proved (3,4) to have structure 11 in which, surprisingly, the third methyl group to be inserted is located at C-20 (Fig. 1). The surprise stems from the fact that the carbon atom at C-20 of structure 11 has to be extruded at some stage during the demonstrated conversion of the trimethyl system (structure 11)* into cobyrinic acid (structure 13) by using cell-free enzyme preparations from P. shermanii (ref. 7 and refs. therein) or Clostridium tetanomorphum (1). Double-labeling experiments further proved (4, 7) that the C-20 methyl group is also lost during the conversion of 11 to 13 and, hence, study of the nature of the extruded fragment is of considerable interest; the necessary experiments are outlined here.There are, in principle, several approaches to this problem based on different known precursors of cobyrinic acid (structure 13). [20-14C, 2,7-methyl-3H]Sirohydrochlorin (structure 8), [20-14C, 2,7-methyl-'4C]sirohydrochlorin (structure 9), and [2,7,20-methyl-14C]trimethylisobacteriochlorin (structure 12) were selected to avoid formation of formaldehyde, which can arise chemically from uroporphyrinogen-III (structure 2). Incubation of the former precursor (structure 8) with a cobaltcontaining cell-free enzyme system (8) from P. shermanii (Table 1, Exps. 1 and 2) yielded first the trimethylisobacteriochlorin (structure 10) in dihydro form,* followed by further biological transformation into cobyrinic acid (structure 13). From the incubation mixtures were isolated cobyrinic acid (structure 13) as its ester (structure 14), acetic and formic acids as their p-bromophenacyl esters, and acetaldehyde and formaldehyde as their dimedone derivatives. In each case, radioinactive carrier material was added to assist the isolation. The results in Table 1 (Exps. 1 and 2) demonstrate reproducible incorporation of sirohydrochlorin (structure 8)* into cobyrinic acid (structure 13) with formation of labeled acetic acid. The isolated formic acid (Exp. 2) and formaldehyde and acetaldehyde (Exps. 1 and 2) carried negligible activity (<2 X 10-3 equiv.), and this trace also appeared in the corresponding materials from the blank run (Exp. 3). This parallel blank also showed clearly that the production of acetic acid in Exps. 1 and 2 is an enzymic process.These results were confirmed with triply 14C-labeled sirohydrochlorin (structure 9; Exp. 4), in which a remarkably high conversion of sirohydrochlorin (structure 9)* into cobyrinic acid (structure 13) was achieved. The acetic acid isolated from Exps.

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