The effect of N-substitution was examined for the enantioselective lipase-catalyzed ring-opening reaction of racemic 4-phenylazetidin-2-one with methanol in dry organic solvents. Marked differences in the reactivity of various N-protected 4-phenylazetidin-2-ones were observed. Preparativescale reactions with Candida antarctica lipase B (Novozym 435 preparation) yielded N-acylated methyl (R)-3-amino-3-Introduction β-Amino acids and their derivatives, including β-lactams (azetidin-2-ones), are a group of pharmaceutically important compounds and intermediates of synthetic products. [1] Various chemo-and enzyme-catalyzed synthetic routes for the preparation of these compounds in enantiomeric form have been the target of intensive studies. For instance, the transition-metal-catalyzed asymmetric hydrogenation of β-dehydroamino acid derivatives and the Mannich reaction with silyl enolates along with organocatalytic approaches have been reported. [1a-1c,1e,1f] From a biocatalytic perspective, the lipase-catalyzed (EC 3.1.1.3) kinetic resolution of racemic β-lactams by an enantioselective ring-opening reaction represents a viable and extensively used approach for the preparation of β-amino acid, β-amino ester, and β-dipeptide enantiomers, leaving behind the less reactive β-lactam enantiomer.[1d,1g,2] The formation of poly(β-alanine) has even been described by the lipase-catalyzed ring-opening of azetidin-2-one.[2d] The kinetic resolution of N-hydroxymethylated β-lactams by lipase-catalyzed O-acylation followed by the removal of the N-methanol tail from the resolved product has allowed the simultaneous preparation of both β-lactam enantiomers. [1d,1g,3] The cascade reaction that is catalyzed by nitrile hydratase and amidase enzymes in Rhodococcus erythropolis AJ270 whole cells represents more recent advances in the biocatalytic preparation of [a] β-amino acids and derivatives from the corresponding nitriles.[4]Lipase enzymes, belonging to the serine hydrolase family, hydrolyze lipids in nature. The ability of lipases to cleave an amide bond is rare, [5] whereas serine proteases may undergo a reaction at both the amide and ester bonds. Previous studies have explained the difference between the two types of serine hydrolases by using the mechanistic details of amide hydrolysis, that is, lipases lack the transition-state stabilizing interaction between the active site and the amide nitrogen atom, which is pivotal in the amide hydrolysis by serine proteases.[6] Some lipases such as Candida antarctica lipase B (CAL-B, the Novozym 435 preparation) and Burkholderia cepacia lipase (lipase PS-D preparation) have been successfully used to cleave a β-lactam ring in a highly effective and enantioselective manner, [1d,1g,2] which can be explained by the fact that the amide bond in a β-lactam ring is less stable than a normal resonance stabilized peptide bond. However, the use of elevated temperatures or structural activation has been a prerequisite with certain β-lactam structures. Thus, in the first lipase-catalyzed β-lactam ring...