Stereochemistry of a Methyl-Group Rearrangement during the Biosynthesis of Lanosterol

Abstract

2. Each mevalonate was converted into cholesterol by a rat liver preparation. 3. Each cholesterol specimen was converted into androsta-l,4-diene-3,17-dione by incubation with Mycobacteriumphlei in the presencd of 2,2'-dipyridyl. Each specimen of androsta-l,4-diene-3,17-dione was converted into androsta-l,4-dien-3-one-l7-ethylene ketal.4. The samples of androsta-1,4-dien-3-one-17-ethylene ketal were each converted chemically into oestrones in which the methyl group at C-18 is the only carbon atom that originated from C-6 in mevalonolactone.5. The oestrone from (3RS)-[6-3H1 ,6-'4C]mevalonolactone was oxidised chemically to acetic acid which was converted into p-bromophenacyl acetate and the 3H/14C ratio was measured.6. There was no overall loss of tritium from the methyl group of acetic acid, as measured by determining the 3H/14C ratios of the p-bromophenacyl esters, when the synthetic and degradative procedures 1 -5 were tested with [3H,,2-'4C]acetic acid.7. The oestrones derived from the 6R and 6S-mevalonolactones were oxidised. The chiralities of the resulting acetates were determined by an established procedure whereby the acetates were converted into 2s-malates which were examined for loss of tritium on equilibration with fumarate hydratase.8. The oestrone from (3RS,6R)-[6-2Hl,6-3Hl,6-'4C]mevalonate gave acetic acid which was converted into 2s-malate that retained 68.6 % of its tritium after treatment with fumarate hydratase ; the configuration of this acetic acid was R. 9. The oestrone from (3RS,6S)-[6-2Hl,6-3Hl,6-'4C]mevalonate was oxidised to acetic acid which was converted into 2s-malate that retained 31.9 % of its tritium after treatment with fumarate hydratase; the configuration of this acetic acid was S. 30. There was no overall change in the configuration of a chiral methyl group between C-6 of mevalonate and C-18 of oestrone. It is concluded that the intramolecular migration of a chiral methyl group from C-15 in 2,3-oxidosqualene to C-13 in lanosterol is stereospecific and occurs with overall retention of configuration.The stereochemical synthesis and configurational assay of the chiral methyl group [1,2] have led to the elucidation of the substrate stereochemistry of many enzymic reactions. The chiral methyl has been used to study the stereochemistry of two types of biological [4,11]. The work described in this paper represents a novel use of the chiral methyl group to elucidate the stereochemistry

Overall Retention of Methyl Stereochemistry during B12-Dependent Radical SAM Methyl Transfer in Fosfomycin Biosynthesis

“…Previous feeding experiments of fosfomycin-producing organisms with methionine that was labeled with 1 H, 2 H, and 3 H on its methyl group with defined stereochemistry showed retention of configuration between methionine and the final product fosfomycin . Such chiral methyl groups have been used to investigate the mechanisms of a variety of enzymatic reactions since their first reported characterization by Cornforth and Arigoni in 1969. , Investigations using chiral methyl groups have made critical contributions to our understanding of transformations involved in key biological processes ranging from the TCA cycle , to steroid biosynthesis, , natural product biosynthesis, and methanogenesis. , Some of the most striking results have come from feeding microorganisms with chiral methyl-labeled molecules to examine a pathway without knowing the specific target enzymes or reactions a priori , such as Arigoni’s demonstration that B 12 biosynthesis in Propionibacterium shermanii occurs with inversion of configuration at all seven methyl groups appended to the corrin ring . However, such feeding experiments do not directly report on the methyl group during each step of the biosynthesis, and thus, the fosfomycin feeding studies mentioned above can be used only to infer molecular-level insight into the mechanism of the unusual C -methylation reaction that converts 2-HEP-CMP to (2 S )-2-HPP-CMP.…”

“…41 Such chiral methyl groups have been used to investigate the mechanisms of a variety of enzymatic reactions since their first reported characterization by Cornforth and Arigoni in 1969. 42,43 Investigations using chiral methyl groups have made critical contributions to our understanding of transformations involved in key biological processes ranging from the TCA cycle 44,45 to steroid biosynthesis, 46,47 natural product biosynthesis, and methanogenesis. 48,49 Some of the most striking results have come from feeding microorganisms with chiral methyl-labeled molecules to examine a pathway without knowing the specific target enzymes or reactions a priori, such as Arigoni's demonstration that B 12 biosynthesis in Propionibacterium shermanii occurs with inversion of configuration at all seven methyl groups appended to the corrin ring.…”

Chiral Methyl Groups

“…Examples of class a are relatively recent; they include a study of the stereochemical fate of one of the migrating methyl groups in the conversion of squalene into lanosterol (61,66) and several examples of the transfer of the S-methyl group of S-adenosylmethionine catalyzed by various methyltransferases (43,64,67). In each case the ultimate chirality analysis involved conversion of the chiral methyl group into acetate followed by assay with malate synthase/fumarase.…”

Triterpenoid biosynthesis in Euphorbia lathyris latex

“…Very few of these were aimed at labelling the methyl group. There are two reports of the synthesis of 3'-0-or 3'-T-mevalonic acid [27], a synthesis of mevalonic acid with a chiral methyl group [28], and a communication reporting a very good synthesis of 3'-13C-mevalonic acid [29). The previous syntheses of the hydrogen-labelled compounds are not regiospecific, do not produce 1 00 atom %, or are very inefficient.…”

“…There have been many syntheses of carbon-and hydrogen-labelled mevalonic acids [5,[26][27][28][29]. Very few of these were aimed at labelling the methyl group.…”