MicroBiopharm Japan Co., Ltd.

Biaryl coupling reactions are pivotal in the synthesis of complex therapeutic compounds, such as michelline B, vancomycin and arylomycin A2 derivatives. Synthesizing macrocycles, particularly the 2,2'-disubstituted biaryl-bridged peptide in arylomycin derivatives, present significant challenges, including low yields and the requirement for high transition metal loadings. Recent advances in DNA sequencing and enzyme engineering have facilitated the exploration of biocatalytic transformations. By leveraging enzyme engineering and substrate modifications, we report the development of a biocatalytic process using engineered cytochrome P450 enzymes for the oxidative carbon-carbon bond formation, yielding the biphenolic macrocycles present in arylomycin derivatives, at gram scale. This work underscores the transformative potential of P450 enzymes in synthetic organic chemistry, paving the way for novel pharmaceutical advancements.

The conserved N-linked glycan at the Fc domain of recombinant monoclonal antibodies is an attractive target for site-specific payload conjugation for preparation of homogenous antibody-drug conjugates (ADCs). Here, we report a novel ADC constructing strategy, named "ez-ADiCon", that is achieved by one-step enzymatic transglycosylation of a payload-preloaded bi-antennary glycan oxazoline onto a deglycosylated antibody. In this method, a mixture of different glycoforms of the Fc-glycan is replaced with a pre-defined payload-linked glycan. Since two payloads are linked on each donor glycan substrate, efficient conjugation results in a highly homogenous ADC with mostly-four drug molecules per antibody, facilitating hydrophobic interaction chromatography analysis and purification. We validated this conjugation strategy using Monomethyl auristatin E (MMAE) and an anti-Human epidermal growth factor receptor 2 (anti-Her2) antibody as the model ADC components and demonstrated its target-specific in vitro cytotoxicity. Our novel conjugation strategy, ez-ADiCon, provides a new approach for the preparation of next generation ADCs.

An enzyme belonging to glycoside hydrolase family 68 (GH68) from Beijerinckia indica subsp. indica NBRC 3744 was expressed in Escherichia coli. Biochemical characterization showed that the enzyme was identified to be a β-fructosyltransferase (BiBftA). Crystallization of a full-length BiBftA was initially attempted, but no crystals were obtained. We constructed a variant in which 5 residues (Pro199-Gly203) and 13 residues (Leu522-Gln534) in potentially flexible regions were deleted, and we successfully crystallized this variant BiBftA. BiBftA is composed of a five-bladed β-propeller fold as in other GH68 enzymes. The structure of BiBftA in complex with fructose unexpectedly indicated that one β-fructofuranose (β-Fruf) molecule and one β-fructopyranose molecule bind to the catalytic pocket. The orientation of β-Fruf at subsite −1 is tilted from the orientation observed in most GH68 enzymes, presenting a second structure of a GH68 enzyme in complex with the tilted binding mode of β-Fruf.