Chemoenzymatic synthesis of glycosides relies on efficient glycosyl donor substrates able to react rapidly and efficiently, yet with increased stability towards chemical or enzymatic hydrolysis. In this context, glycosyl thioimidates have previously been used as efficient donors, in the case of hydrolysis or thioglycoligation. In both cases, the release of the thioimidoyl aglycone was remotely activated through a protonation driven by a carboxylic residue in the active site of the corresponding enzymes. A recombinant glucosidase (DtGly) from Dictyoglomus themophilum, previously used in biocatalysis, was also able to use such glycosyl thioimidates as substrates. Yet, enzymatic kinetic values analysis, coupled to mutagenesis and in silico modelling of DtGly/substrate complexes demonstrated that the release of the thioimidoyl moiety during catalysis is only driven by its leaving group ability, without the activation of a remote protonation. In the search of efficient glycosyl donors, glycosyl thioimidates are attractive and efficient. Their utility, however, is limited to enzymes able to promote leaving group release by remote activation.
The methoxy group is generally considered as a poor leaving group for nucleophilic substitution reactions. This work verified the superior ability of the methoxy group in nucleophilic amination of arenes mediated by the sodium hydride and lithium iodide through experimental and computational approaches.
ABSTRACTPseudomonas aeruginosaarylsulfatase (PAS) hydrolyses sulfate and, promiscuously, phosphate monoesters. Enzyme-catalyzed sulfate transfer is crucial to a wide variety of biological processes, but detailed studies of the mechanistic contributions to its catalysis are lacking. We present an investigation based on linear free energy relationships (LFERs) and kinetic isotope effects (KIEs) of PAS and active site mutants that suggest a key role for leaving group (LG) stabilization. In LFERs wild type PAS has a much less negative Br0nsted coefficient (βleaving groupobs-Enz= −0.33) than the uncatalyzed reaction (βleavingroupobs= −1.81). This situation is diminished when cationic active site groups are exchanged for alanine. The considerable degree of bond breaking during the TS is evidenced by an18ObridgeKIE of 1.0088. LFER and KIE data for several active site mutants point to leaving group stabilization by active-site lysine K375, in cooperation with histidine H211.15N KIEs combined with an increased sensitivity to leaving group ability of the sulfatase activity in neat D2O (Δβleaving groupH-D= +0.06) suggest that the mechanism for S-Obridgebond fission shifts, with decreasing leaving group ability, from charge compensation via Lewis acid interactions towards direct proton donation.18OnonbridgeKIEs indicate that the TS for PAS-catalyzed sulfate monoester hydrolysis has a significantly more associative character compared to the uncatalyzed reaction, while PAS-catalyzed phosphate monoester hydrolysis does not show this shift. This difference in enzyme-catalyzed TSs appears to be the major factor favoring specificity toward sulfate over phosphate in this promiscuous hydrolase, since other features are either too similar (uncatalyzed TS) or inherently favor phosphate (charge).
A short review of the development of nucleofugality and electrofugality scales based on solvolysis rates of benzhydryl derivatives is presented. Accordingly, the rate of the heterolytic step in the SN1 displacement reaction and the leaving group ability (nucleofugality) in a given solvent are related with the special linear free-energy relationship (LFER) equation: log k = s
f + E
f). The impact of electronic effects in the leaving group (nucleofuge) on the overall SN1 reactivity of the substrate is given. The importance of inductivity, resonance, polarity and field effects in the leaving group moiety in the transition state is analyzed. Also, the effect of the negative hyperconjugation and the influence of other electronic effects in the leaving group on the height of the reaction intrinsic barrier are considered.1 Introduction2 Development of the Nucleofugality Scale3 Inductive and Resonance Effects4 Negative Hyperconjugation5 Intrinsic Barrier6 Conclusions