Chiral preference of cutinase in the reaction with phosphonate inhibitors.

Introduction Lipases are lipolytic enzymes originating from a wide variety of sources, such as plants, microorganisms and higher animals. The most distinctive property of lipases is their rapid hydrolysis of ester bonds at an oil-water interface, whereas activity on monomeric substrates is generally low [l]. The first structures of lipases [2,3] already gave insight into their general architecture and mechanism of action. A common feature of all lipases is their a/p-hydrolase fold [4], and their active sites contain a 'classical' Ser-His-Asp (or Glu) catalytic triad together with an oxyanion hole, comprising backbone NH groups and sometimes side chain NHand OHgroups [S]. Hence the catalytic mechanism of lipases is similar to that of serine proteases in many respects. Organophosphonates are known to block serine hydrolases by reacting with the active-site serine, and the covalent product has been suggested to mimic the transition state of the acylation step in ester hydrolysis [6]. The X-ray structures of all phosphonate-inhibited lipases show that the phosphoryl oxygen is positioned in the oxyanion hole [7-111, in agreement with the proposed mechanism of ester hydrolysis. However, until now no kinetic evidence for the importance of oxyanion stabilization in the inhibition reaction of lipases with phosphonates has been established. Recently, we reported the synthesis of triacylglycerol analogues in which two ester bonds were replaced by a carbamoyl function, while the third ester bond was replaced by an O-p-nitrophenylalkyl phosphonate [ 121. These organophosphonates appeared to be good lipase inhibitors. In addition to the stereoselectivity for the glycerol backbone, cutinase was also found to be highly stereoselective at the phosphorus chiral centre of these inhibitors. The latter stereoselectivity is high when a diacylglycerol substituent is attached to the phosphorus but is considerably lower for a small methoxy substituent. Such enantioselectivity at phosphorus