A Tandem Enzymatic sp²‐C‐Methylation Process: Coupling in Situ S‐Adenosyl‐l‐Methionine Formation with Methyl Transfer

Abstract

A one-pot, two-step biocatalytic platform for the regiospecfic C-methylation and C-ethylation of aromatic substrates is described. The tandem process utilises SalL (Salinospora tropica) for in situ synthesis of S-adenosyl-l-methionine (SAM), followed by alkylation of aromatic substrates by the C-methyltransferase NovO (Streptomyces spheroides). The application of this methodology is demonstrated for the regiospecific labelling of aromatic substrates by the transfer of methyl, ethyl and isotopically labelled CH CD and CD groups from their corresponding SAM analogues formed in situ.

S‐Adenosyl Methionine Cofactor Modifications Enhance the Biocatalytic Repertoire of Small Molecule C‐Alkylation

“…As identified in an earlier study,M TANw as added to the reaction mixture in order to degrade the SAH analogue,which inhibits NovO. [34] In almost all examples,t he 2amino-and 2-chloro-modified cofactors outperformed SAM in methylating 5-13.O ne exception was triazole 11,i nw hich no C-(m)ethylation was observed using any of the cofactors tested. %conversions determined by RP-HPLC using aratio of the peak area (254 nm) of the ClDA analogue to SAM/SAE analogue and 5'-methylthioadenosine (or analogue).…”

“…As identified in an earlier study,M TANw as added to the reaction mixture in order to degrade the SAH analogue,which inhibits NovO. [34] The2 -modified alkyne cofactor 4i displayed comparable conversion (5a,5 0%)t oS AM, whereas the formation of the 2-amino-6-chloro analogue (4g)i nsitu did not form 5a. ClDA/ClDAanalogue (400 mm), l-Met/l-Ethionine (2.00 mm), SalL (2.10 mm), DTT (1.00 mm)a nd BSA (1.00 mg mL À1 ), potassiumphosphate buffer (100 mm,p H6.8), 24 h, 37 8 8C.…”

“…[22][23][24][25][26] Furthermore,S AM analogues are inherently unstable in buffered solution (t 1/2 942 min for SAM at pH 8). [34] ClDAi sashelf-stable,a tom-economical adenosine source for such aprocess catalyzed by SalL compared to ATP, which is asubstrate for SAM production by methionine adenosyltransferase (MAT). [15,29,30] One example of this one-pot process is the generation of cofactor analogues in situ from either ClDAo rA TP and (m)ethionine, [15,[29][30][31][32][33] followed by C-(m)ethyl transfer catalyzed by aM Tase (Figure 1b).…”

“…[15,29,30] One example of this one-pot process is the generation of cofactor analogues in situ from either ClDAo rA TP and (m)ethionine, [15,[29][30][31][32][33] followed by C-(m)ethyl transfer catalyzed by aM Tase (Figure 1b). [34] ClDAi sashelf-stable,a tom-economical adenosine source for such aprocess catalyzed by SalL compared to ATP, which is asubstrate for SAM production by methionine adenosyltransferase (MAT). [15,27,29,35,36] Although in-depth knowledge of the substrate promiscuity of C-MTases has been garnered from structural and mutagenesis studies, [17,19,20,37] little is known about how the structural features of the SAM cofactor itself influences the yield and scope of C-alkylation.…”

An ortho C-methylation/O-glycosylation motif on a hydroxy-coumarin scaffold, selectively installed by biocatalysis

“…4,30 NovO accepts a broad spectrum of donor and acceptor substrates and it was previously shown that it methylates 1 site-selectively at the desired C-8 position. 22,31 However, a matching GT for ortho Oglucosylation still had to be identified. Although glucosylation of a broad range of ( poly)phenolic acceptors by various promiscuous GTs was reported, 13,19,20,32 the selective modification of a single hydroxyl group of a polyphenolic substrate remains a particular challenge.…”