Functional AdoMet Isosteres Resistant to Classical AdoMet Degradation Pathways

Abstract

S-adenosyl-l-methionine (AdoMet) is an essential enzyme cosubstrate in fundamental biology with an expanding range of biocatalytic and therapeutic applications. We report the design, synthesis, and evaluation of stable, functional AdoMet isosteres that are resistant to the primary contributors to AdoMet degradation (depurination, intramolecular cyclization, and sulfonium epimerization). Corresponding biochemical and structural studies demonstrate the AdoMet surrogates to serve as competent enzyme cosubstrates and to bind a prototypical class I model methyltransferase (DnrK) in a manner nearly identical to AdoMet. Given this conservation in function and molecular recognition, the isosteres presented are anticipated to serve as useful surrogates in other AdoMet-dependent processes and may also be resistant to, and/or potentially even inhibit, other therapeutically relevant AdoMet-dependent metabolic transformations (such as the validated drug target AdoMet decarboxylase). This work also highlights the ability of the prototypical class I model methyltransferase DnrK to accept non-native surrogate acceptors as an enabling feature of a new high-throughput methyltransferase assay.

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

“…Further enhancement was achieved using an excess of ClDAa nalogues (2 equiv) and l-Met (10 equiv). [27] Te trazole analogues 4c, 4k,a nd 4l produced significantly lower amounts of 5a relative to SAM. As identified in an earlier study,M TANw as added to the reaction mixture in order to degrade the SAH analogue,which inhibits NovO.…”

“…[18][19][20] Ahallmark of NovO is its substrate promiscuity (e.g., 1)and the ability to utilize S-alkylated analogues of SAM to form products such as 2 ( Figure 1a). [27,28] Am ore step-and atom-efficient strategy is to couple cofactor formation with C-alkyl transfer. [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).…”

“…[27,28] Am ore step-and atom-efficient strategy is to couple cofactor formation with C-alkyl transfer. [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. [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).…”

“…Replacing adenine with 7-deazaadenine (4d)r esulted in only 10 %conversion (Figure 3c), [27] whereas ahypoxanthine nucleobase did not form 4e.This suggests that the interaction between the N7 of adenine and Asn188 is critical for the catalytic function of SalL. High conversions to 4f-j were observed using analogues containing modifications to the 2and 6-position of adenine.C ombining 2,6-diamino or 2chloro-6-aminoadenine modifications with tet Met produced 4k and 4l in greater than 99 %a nd 41 %c onversion, respectively (Figure 3d).…”

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

“…At present, the broader applicability of small‐molecule MTs is hampered by the inherent instability of SAM (942 min at pH 8) as the corresponding methylating agent . Additionally, the synthesis of SAM by chemical methods results in the formation of a diastereomeric mixture; this increases the cost of the process and has the potential for undesirable C‐MT inhibition by the unwanted diastereomer …”