In this Phase I project, a subset of human milk oligosaccharides (HMOs) ranging from disaccharides to pentasaccharides will be prepared on scales ranging from several grams to kilograms. Specifically, N-acetyl-lactosamine will be chemically prepared in kilogram quantities, six HMOs will be chemically prepared on multi-gram scales (10-100 g), and several HMOs will be extended on smaller scales (1-10 g) with N-acetyl-neuraminic acid using a CMP-sialic acid donor and commercially available sialyltransferases. The primary aims of the project are to chemically prepare key HMO building block molecules on large scales, and basic HMO units on modest scales, and to demonstrate the commercial practicality of extending several of the latter basic HMOs to larger sialyl-containing structures va enzymic modification. All syntheses will be conducted under cGLP conditions; existing Omicron documents, procedures, SOPs, methods and laboratory policies developed in prior cGMP projects will be modified for use in this work. For each oligosaccharide product, high- resolution 1H (600 MHz) and 13C (150 MHz) NMR data will be obtained to confirm structure and purity. The NMR characterization will include an effort to assign the 1H and 13C signals as completely as possible, with assistance from 2D homo- and heteronuclear datasets. For each oligosaccharide product, high-resolution mass spectral (HRMS) data will also be obtained to confirm structural assignments made by NMR, and high-performance liquid chromatography (HPLC) will be used to confirm chemical purity. A major tangible outcome of this project will be the identification and implementation of synthetic methods capable of producing small HMOs on large scales for future pre-clinical trials.
In this Phase I project, a subset of human milk oligosaccharides (HMOs) ranging from disaccharides to pentasaccharides will be prepared in multi-gram quantities using concise and reliable methods that are conducive to scale-up to support future pre-clinical trials. The primary aims of the project are (1) to chemically prepare key building block molecules on large (kilogram) scales, (2) to chemically prepare basic HMO units on modest (10-100 g) scales, and (3) to demonstrate the commercial practicality of extending the latter basic HMOs to larger sialyl-containing structures via enzymic modification on small (1-10 g) scales.
