Oligosaccharide Mimetics Obtained by Novel, Rapid Screening of Carboxylic Acid Encoded Glycopeptide Libraries

Abstract

Glycopeptides that mimic the action of oligosaccharides have been rapidly identified through the implementation of combinatorial library methodology combined with a novel, easy, screening and analysis method. A glycopeptide library containing three different glycosyl amino building blocks, Fmoc-Asn(β-Ac3GlcNAc)-OPfp (5), Fmoc-Thr(α-Ac4Man)-OPfp (6), and Fmoc-Thr[α-Ac4Man(1→3)α-2-O-Bz-4,6-Ac2Man]-OPfp (7), was synthesized by the portion-mixing method on PEGA solid support. The library was designed to facilitate rapid and unambiguous analysis of the active glycopeptides detected during the high throughput-screening step. Consequently, the library was synthesized using the ladder synthesis approach and linked to the solid support via a photolabile linker. The glycosyl amino acids were labeled with carboxylic acid tags to allow unambiguous identification of the glycan moiety. Photolytic release of active glycopeptide from the resin was induced by irradiation of the bead with the MALDI-TOF-MS laser, and analysis of the resulting spectrum presenting the ladder of glycopeptide fragments yielded the sequence of the active glycopeptide. Glycopeptide ligands were identified for the C-type lectin from Lathyrus odoratus by screening the fluorescent-labeled protein in a solid-phase binding assay of the PEGA resin-bound glycopeptide library. Of the several glycopeptide ligands detected, most contained Man or GlcNAc, glycans that display specificity for the lectin in hemagglutination assays. The most active glycopeptides detected from the library screening were T(α-d-Man)ALKPTHV, LHGGFT(α-d-Man)HV, T(α-d-Man)EHKGSKV, GT(α-d-Man)FPGLAV, and T(α-d-Man)LFKGFHV.

Small molecule analysis by MALDI mass spectrometry

“…Siuzdak and Lewis have demonstrated that peptides and carbohydrates, covalently linked to a polymeric support through a photolabile linker, can be directly identified by MALDI in a single step that requires no pretreatment of the sample to induce cleavage from the support [42]. In another application [43], MALDI was used for photolytic release and determination of an active glycopeptide from the resin support. The resulting mass spectral data contained the ladder of glycopeptide fragments and yielded the active glycopeptide sequence.…”

Genetically Encoded Fragment-Based Discovery of Glycopeptide Ligands for Carbohydrate-Binding Proteins

“…12 We pursued an approach in which we aim to identify peptides that can serve as a moiety that synergizes with glycans in binding to lectins (Figure 1), rather than acting as a neutral linker or standalone recognition element. Indeed, synthetic conjugates of peptides and carbohydrates are known to yield effective inhibitors of carbohydrate–protein interactions, 13 but the throughput of synthesis of these ligands is limited. Here, we employ genetically encoded fragment-based discovery (GE-FBD) to identify peptide fragments (Figure 1) capable of forming a synergistic interaction together with a glycan moiety by taking advantage of the immense diversity of a genetically encoded peptide library.…”

A Dual Photochemical Ring-Opening/Cleavage Approach for the Synthesis and Decoding of One-Bead-One-Compound Cyclic Peptide Libraries

“…These results suggest that the adduct product is modified into the dehydrated specie when exposed to the laser during ionisation in the MALDI instrument. The ability of the MALDI's Nd:YAG laser to induce photochemical reaction has been shown and used by a few groups for synthesis monitoring and structural elucidation of biomolecules [10,11]. MS/MS analysis of the dehydrated product molecular ion yielded high-quality spectra from which the linearized peptide 4 could be unambiguously sequenced manually and by using de novo sequencing with the Peaks software (Figure 3b) [12].…”