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A new class of positional isomeric pairs of -Boc protected oligopeptides comprised of alternating nucleoside derived β-amino acid (β-Nda-) and L-amino acid residues (alanine, valine, and phenylalanine) have been differentiated by both positive and negative ion electrospray ionization ion-trap tandem mass spectrometry (ESI-MS). The protonated dipeptide positional isomers with β-Nda- at the N-terminus lose CHOH, NH, and CHO, whereas these processes are absent for the peptides with L-amino acids at the N-terminus. Instead, the presence of L-amino acids at the N-terminus results in characteristic retro-Mannich reaction involving elimination of imine. A good correlation has been observed between the conformational structure of the peptides and the abundance of y and b ions in MS spectra. In the case of tetrapeptide isomers that are reported to form helical structures in solution phase, no y and b ions are observed when the corresponding amide -NH- participates in the helical structures. In contrast, significant y and b ions are formed when the amide -NH- is not involved in the H-bonding. In the case of tetra- and hexapeptides, it is observed that abundant b ions are formed, presumably with stable oxazolone structures when the C-terminus of the b ions possessed L-amino acid and the β-Nda- at the C-terminus appears to prevent the cyclization process leading to the absence of corresponding b ions. [ABSTRACT FROM AUTHOR]

AGS62P1 is an antibody drug conjugate (ADC) composed of a human IgG1κ monoclonal antibody against FLT3 (FMS-like tyrosine kinase 3) with a p-acetyl phenylalanine (pAF) residue inserted at position 124 of each heavy chain linked to the proprietary microtubule disrupting agent AGL-0182-30 via an alkoxyamine linker that forms an oxime upon conjugation to the antibody. AGS62P1 is currently in Phase I human clinical trials for acute myelogenous leukemia (AML). The identified primary metabolite of an oxime-linked ADC is presented for the first time. AGS62P1 metabolism was assessed in xenograft tumor-bearing mice and rats treated with the ADC using liquid chromatography and mass spectrometry-based methods described herein. In this study, we identified the metabolite of AGS62P1 as pAF-AGL-0185-30, which contains a fragment resulting from the catabolism of the antibody component of the ADC and hydrolysis of the terminal amide portion of the linker-payload. We demonstrated that the metabolite of AGS62P1 is tolerated in rats above 1.5 mg/kg and above 0.334 mg/kg in cynomolgus monkeys when given as a single dose. Furthermore, we established in vitro that pAF-AGL-0185-30 does not significantly inhibit hERG or cytochrome P450 family enzymes (CYPs).

The molecular recognition of carbohydrates plays a fundamental role in many biological processes. However, the development of carbohydrate-binding reagents for biomedical research and use poses a challenge due to the generally poor affinity of proteins toward sugars in aqueous solution. Here, we describe the effective molecular recognition of pyranose monosaccharides (in particular, galactose and mannose) by a rationally designed protein receptor based on the human lipocalin scaffold (Anticalin). Complexation relies on reversible covalent cis-diol boronate diester formation with a genetically encoded l-boronophenylalanine (Bpa) residue which was incorporated as a non-natural amino acid at a sterically permissive position in the ligand pocket of the Anticalin, as confirmed by X-ray crystallography. Compared with the metal-ion and/or avidity-dependent oligovalent lectins that prevail in nature, our approach offers a novel and promising route to generate tight sugar-binding reagents both as research reagents and for biomedical applications.