Enhanced Chemical Stability of AdoMet Analogues for Improved Methyltransferase-Directed Labeling of DNA

Abstract

Methyltransferases catalyze specific transfers of methyl groups from the ubiquitous cofactor S-adenosyl-l-methionine (AdoMet) to various nucleophilic positions in biopolymers like DNA, RNA, and proteins. We had previously described synthesis and application of AdoMet analogues carrying sulfonium-bound 4-substituted but-2-ynyl side chains for transfer by methyltransferases. Although useful in certain applications, these cofactor analogues exhibited short lifetimes in physiological buffers. Examination of the reaction kinetics and products showed that their fast inactivation followed a different pathway than observed for AdoMet and rather involved a pH-dependent addition of a water molecule to the side chain. This side reaction was eradicated by synthesis of a series of cofactor analogues in which the separation between an electronegative group and the triple bond was increased from one to three carbon units. The designed hex-2-ynyl moiety-based cofactor analogues with terminal amino, azide, or alkyne groups showed a markedly improved enzymatic transalkylation activity and proved well suitable for methyltransferase-directed sequence-specific labeling of DNA in vitro and in bacterial cell lysates.

Methyltransferase‐Directed Labeling of Biomolecules and its Applications

“…The molecule is inherently unstable towards nucleophilic attack and the methyl group readily participates in substitution reactions 3. This results in a half‐life of 17 hours for AdoMet in the M. Hha I reaction buffer at pH 7.4, 37 °C,4 and this susceptibility to nucleophilic attack is instrumental to the function of MTases. A mechanism for C5 cytosine methylation by the Hha I methyltransferase (M. Hha I), which results in 5‐methyl cytosine (5mC), was first described by Santi et al 5.…”

“…For example, incorporation of different transferrable amine moieties ( 21 ,4, 18 22 ,4, 19 23 ,4, 19b Table S2) can be followed by a coupling reaction with the N ‐hydroxysuccinimide (NHS) ester of an amine‐reactive probe 18. In addition, several examples exist on the application of AdoMet analogues with a transferrable terminal alkyne ( 5 ,20 Scheme 6; 20 ,20g,20h 24 ,20b,20c,20g and 25 ,4, 19b Table S2) or azide ( 26 g ,20, 21 27 ) 4. The transferred alkynes and azides can be further functionalized by using the biocompatible and highly‐efficient azide–alkyne cycloaddition reaction (one of the “click” series of reactions).…”

“…The transferred alkynes and azides can be further functionalized by using the biocompatible and highly‐efficient azide–alkyne cycloaddition reaction (one of the “click” series of reactions). However, some of these analogues, such as compound 20 (Table S2), which features a terminal alkyne, are highly unstable 4, 20g,20h. Other AdoMet analogues, such as the AdoEnyYn compound ( 5 , Scheme 6), are more stable, thus making them more suitable for use in bioorthogonal ligations 20b–20h…”

“…4, 24 Reversible racemization (route a) to the R  diastereomer of AdoMet is also common and can result in the formation of an inactive AdoMet analogue. The presence of the sulfonium center activates the adjacent carbon atoms towards decomposition reactions.…”

“…The partial positive charge at the 1′′‐C increases, thereby making it more susceptible to nucleophilic attack (route d). When electronegative groups (e.g., Y=NH 2 ) are in close proximity to the triple bond, this can lead to a higher electron deficiency at both 4′′‐C and 1′′‐C, which enables the addition of water (route e) to both to give the hydrated compound, which shows almost no reactivity towards most DNA MTases and protein MTases 4, 24…”