To determine the angiotensin-I converting enzyme (ACE) inhibitory activity of marine cryptides, different methods were tested. ACE inhibition was measured using two synthetic substrates, (N-[3-(2-furyl) acryloyl]-Phe-Gly-Gly (FAPGG) and N-hippuryl-His-Leu hydrate salt (HHL)), and a natural one, angiotensin-I. The IC50 value (defined as the concentration of inhibitory molecule needed to inhibit 50% of the ACE activity) of the reference synthetic inhibitor captopril was in the nanomolar range (1.79-15.1 nM) when synthetic substrates were used, whereas it exhibited IC50 of micromolar range (16.71 μM) with angiotensin-I. We chose losartan, an antagonist of angiotensin-II receptor as negative control for the ACE inhibition. Losartan was also able to inhibit ACE whatever the substrate tested, with IC50 of micromolar range (17.13-146 μM). We defined this value as a limit above which molecules are not showing in vitro ACE inhibitory activity. Val-Trp (VW), Val-Tyr (VY), Lys-Tyr (KY), Lys-Trp (KW), Ile-Tyr (IY), Ala-Pro (AP), Val-Ile-Tyr (VIY), Leu-Lys-Pro (LKP), Gly-Pro-Leu (GPL), Ala-Lys-Lys (AKK), and Val-Ala-Pro (VAP) were tested as inhibitors of ACE with synthetic and natural substrates. IC50 displayed were substrate-dependent. With FAPGG as substrate, IW, VAP, KY, IY, AP, AKK, and VIY show IC50 values over the IC50 value of losartan and should not be considered as inhibitors of ACE. VY, VW, KW, and LKP exhibited IC50 value lower than the IC50 value of losartan for all substrates tested and were thus considered as good candidates for effectively decreasing hypertension. It appears that the comparison of IC50 is not consistent when IC50 values are obtained with different substrates and different methods. In vitro ACE inhibitory activity assays should always include various ACE substrates and references such as captopril and a negative control to obtain data reliable to discriminate ACE inhibitory peptides.

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.

a b s t r a c tThe selective acylation of multifunctional compounds exhibiting both alcohol and amino groups gives interesting products with many applications in food, cosmetic and pharmaceutical industries, but it is real challenge. The current work describes the different behavior shown by Candida antarctica lipase B (Novozym 435) when catalyzing the O-acylation and N-acylation of bifunctional acyl acceptors. The acylation of three amino-alcohols (alaninol, 4-amino-1-pentanol and 6-amino-1-hexanol) was studied using myristic acid as an acyl donor. To achieve this, a structure-reactivity study was performed in tertamyl alcohol as a solvent, comparing the three amino-alcohols as acyl acceptors and a series of structurally related amines, namely (R)-sec-butylamine, 1-methoxy-2-propylamine and 1,2-diaminopropane. These substrates were designed to investigate the effect of the group located in ␤-position of the amino group on the acyl acceptor: the more nucleophilic the group, the more the apparent maximal velocity (V max,app ) of N-acylation increases. Moreover, the crucial role of the carbon chain length between the alcohol and amino groups on the chemoselectivity was also demonstrated. The chemoselectivity for the N-acylation was improved when the carbon chain included two carbons (alaninol) whereas the chemoselectivity for the O-acylation was improved when the carbon chain included four carbons or more (4-amino-1-pentanol and 6-amino-1-hexanol).These results provided new insights for the selective synthesis of amides or esters produced from the acylation of bifunctional substrates.