Synthesis of S‐Adenosyl‐L‐homocysteine Capture Compounds for Selective Photoinduced Isolation of Methyltransferases

Abstract

Understanding the interplay of different cellular proteins and their substrates is of major interest in the postgenomic era. For this purpose, selective isolation and identification of proteins from complex biological samples is necessary and targeted isolation of enzyme families is a challenging task. Over the last years, methods like activity-based protein profiling (ABPP) and capture compound mass spectrometry (CCMS) have been developed to reduce the complexity of the proteome by means of protein function in contrast to standard approaches, which utilize differences in physical properties for protein separation. To isolate and identify the subproteome consisting of S-adenosyl-L-methionine (SAM or AdoMet)-dependent methyltransferases (methylome), we developed and synthesized trifunctional capture compounds containing the chemically stable cofactor product S-adenosyl-L-homocysteine (SAH or AdoHcy) as selectivity function. SAH analogues with amino linkers at the N6 or C8 positions were synthesized and attached to scaffolds containing different photocrosslinking groups for covalent protein modification and biotin for affinity isolation. The utility of these SAH capture compounds for selective photoinduced protein isolation is demonstrated for various methyltransferases (MTases) acting on DNA, RNA and proteins as well as with Escherichia coli cell lysate. In addition, they can be used to determine dissociation constants for MTase-cofactor complexes.

Protein Arginine Allylation and Subsequent Fluorophore Targeting

“…Compound 2, [9] N-Bocg-tosyl-homoserine-tert-butylester, [10] and all tetrazole compounds [18] were synthesized as previously described in literature. The silica gel used for flash column chromatography was received from Yantai Yuanbo Silica Gel Co.,C hina.…”

“…[9] In parallel, N-Boc-g-tosyl-l-homoserine-tert-butylester was produced from Boc-Asp-tBu according to al iterature procedure. [10] In situ cleavage of the acetylthio functionality of thioester 2 with fresh CH 3 ONa in MeOH under O 2 -free conditions was followed by coupling with NBoc-g-tosyl-l-homoserine-tert-butylester to give 3.T here was as trong tendency for disulfide formation in the presence of O 2 . [9,10] The reactiont ime andt he basicity of solution should also be carefullyc ontrolled to avoid aM eOH-involved transesterification product, as it could render the downstream deprotection process problematic.…”

Mass spectrometry approaches to monitor protein–drug interactions

“…The actual selectivity towards target proteins is achieved by an interchangeable third function which reversibly interacts with a particular target protein or target class [101]. Trifunctional probes displaying pantarget class selectivity have been described for kinases [102], cAMP-binding proteins [103], and methyltransferases [104].…”

Mass Spectrometry-Based Chemoproteomic Approaches

“…In this strategy, small molecules generating selectivity for individual enzyme classes are attached to a common building block containing reactive group and sorting function (i.e., biotin) that is common to all probes. Recent reports demonstrated examples for profi ling of cAMP-binding proteins ( 42 ) , kinases ( 43 ) , and methyltransferases ( 44 ) .…”