Florian Le Joubioux

The control of Novozym® 435 chemoselectivity and specificity by the solvents in acylation reactions of amino-alcohols

The current work describes the differential behaviour of Novozym® 435 (immobilized Candida antarctica lipase B) in O-acylation and N-acylation catalysis of bifunctional amino-alcohols acyl acceptors. We performed acylation experiments on three amino-alcohols (alaninol, 4-amino-1-pentanol and 6-amino- 1-hexanol) using myristic acid as an acyl donor. Two organic solvents (tert-amyl alcohol and n-hexane) and one ionic liquid (1-butyl-3-methylimidazolium hexafluorophosphate: Bmim [PF6]) were used to determine the effect of the solvent. The influence of the amino-alcohol carbon chain length between the alcohol and amino groups on chemoselectivity C (kcat,app O-acylation/kcat,app N-acylation) was highlighted. N-acylation is improved using alaninol, a short chain amino-alcohol (no mono-O-acylation in tert-amyl alcohol and C = 0.12 in n-hexane) whereas O-acylation is improved using 4-amino-1-pentanol and 6- amino-1-hexanol which are amino-alcohols with longer chain (C = 10.5 in tert-amyl alcohol and C = 539 in n-hexane). On the other hand, the production of the acylated amino-alcohols after 96 h of reaction was shown to be strongly affected by the solvent nature and the amino-alcohol structure: starting from alaninol as an acyl acceptor, the yield of amide synthesis reaches up to 98% in tert-amyl alcohol using 0.7 equivalents of myristic acid while the yield of amido-ester synthesis reaches up to 88% in Bmim [PF6] using 1.75 equivalents of myristic acid.

Ultrasonic-assisted preparation of a low molecular weight heparin (LMWH) with anticoagulant activity.

Low molecular weight heparin (LMWH) is currently used as an anticoagulant agent and constitutes an alternative to unfractionated heparin, which is the cause of serious adverse drug reaction such as heparin-induced thrombocytopenia (HIT). Commercially available LMWH is produced by enzymatic depolymerization that is costly or by chemical methods that are generally carried out under conditions that could imply side reactions that reduce final product efficiency and yields. In this work, we present the use of a physicochemical method for the production of LMWH. This method consists in the use of hydrogen peroxide-catalyzed radical hydrolysis assisted by ultrasonic waves. LMWH that are produced using this physicochemical method have an average molecular weight and anticoagulant properties (Anti-Xa and Anti-IIa) that are comparable to some of commercial LMWH that are currently used. Ultrasonic-assisted radical depolymerization of heparin leads to products with a remarkably low polydispersity index. Moreover, in comparison to other LMWH such as those produced by enzymatic β-elimination, this physicochemical depolymerization of heparin induces fewer oligosaccharides with less than five monosaccharide units. This contributes to the better preservation of the ATIII pentasaccharide binding sequence, which results in a high Anti-Xa/Anti-IIa ratio (1.86). However, LMWH obtained using this physicochemical method have a lower degree of sulfation than other LMWH, which seems to be the cause of a lower Anti-Xa and Anti-IIa activity (143.62±5.42 and 77.07±4.4, respectively).

Proton Shuttle Mechanism in the Transition State of Lipase‐Catalyzed N‐Acylation of Amino Alcohols

An increased reaction rate for lipase‐catalyzed N‐acylation of amino alcohols relative to that of monofunctionalized amines can be explained by a hydrogen shuttling mechanism that avoids nitrogen inversion in the transition state. The mechanism does not involve acyl migration from an ester intermediate that would be formed first, an explanation that permeates the literature. Our suggested reaction mechanism is dependent on the preference of amino alcohols to form intramolecular hydrogen bonds and the capability of the enzyme to accommodate and exploit the specific hydrogen bonding pattern provided by the ligand during catalysis. Our proposed proton shuttle mechanism involves the transfer of two protons in the transition state concomitant with a nucleophilic attack on the acyl enzyme and provides an explanation for the high reaction rate and chemoselectivity for lipase‐catalyzed N‐acylation of amino alcohols. Moreover, the proton shuttle mechanism explains the increased reaction rate for the enzyme‐catalyzed N‐acylation of diamines and of methoxy‐2‐propylamine, for which O‐ to N‐acyl migration is impossible. A linear free‐energy relationship analysis based on the experimental results showed that all of our investigated difunctionalized amine substrates afforded a substrate‐assisted rate acceleration of the N‐acylation by the same reaction mechanism. Furthermore, the results of the analysis were consistent with partial proton transfer in the rate‐limiting transition state, which further supports our suggested proton shuttle mechanism.