Isaac Krauss

Methionine aminopeptidase (MAP) is useful in chemical biology research for the N-terminal processing of peptides and proteins and in medicine as a potential therapeutic target. These technologies can benefit from a precise understanding of the enzyme's substrate specificity profiled over a wide chemical space, including not just natural substrates, peptides containing N-terminal Met, but also unnatural peptide substrates containing N-terminal Met analogues that are also cleaved by MAP like homopropargylglycine (HPG) and azidohomoalanine (AHA). A few studies have profiled substrate specificity for cleavage of N-terminal Met, but none have systematically done so using N-terminal Met analogues. Therefore, we devised a high-throughput profiling experiment based on mRNA display and next-generation sequencing to probe MAP's substrate specificity using N-terminal HPG. From subgroup analysis of either single residues or two-residue combinations, we could establish the impact of residue identity at various positions downstream from the cleavage site. To validate the selection results, a collection of short peptides was chemically synthesized and assayed for cleavage efficiency, where we observed reasonable agreement with the selection data. Results generally followed previously reported trends using N-terminal Met, the strongest trend being that the second residue (P1' position) had the greatest impact on MAP cleavage efficiency with moderate impacts discerned for residues further downstream, which could be rationalized through modeling the enzyme-substrate interaction.

In vitro selection is typically limited to discovery of peptides, proteins, and nucleic acids. Given the importance of carbohydrate-protein interactions in diverse areas of biology including cell adhesion/recognition, immunoregulation and host-pathogen interactions, directed-evolution-based methods for discovery of potent glycoligands are greatly needed. We have previously reported a method for in vitro selection of glycopeptides that combines mRNA display, alkynyl amino acid incorporation, and CuAAC "click" glycosylation. Herein, we describe extensions of this method that incorporate chemical cyclization, removal of N-terminal glycosylation sites, and next-generation sequencing; as an approach to HIV immunogen design, we have used this method to develop mimics of the High Mannose Patch (HMP), the region on HIV envelope protein gp120 most commonly targeted by HIV broadly neutralizing antibodies (bnAbs). We prepared libraries of 1012-14 glycopeptides about 50 amino acids in length, containing variable placement of high mannose (Man9GlcNAc2) glycans and cyclization sites. From selection, we obtained binders to HIV bnAbs PGT128, PGT122, and gl-PGT121, a germline precursor of PGT122, and chemically synthesized numerous glycopeptide hits. Several glycopeptides bound very tightly to their target HIV bnAb, e.g., with a K D as low as 0.5 nM for PGT128. These glycopeptides are of interest as immunogens and tools for HIV vaccine design.

In vitro selection is typically limited to discovery of peptides, proteins and nucleic acids. Given the importance of carbohydrate-protein interactions in diverse areas of biology including cell adhesion/recognition, immunoregulation and host-pathogen interactions, directed-evolution-based methods for discovery of potent glycoligands are greatly needed. We have previously reported a method for in vitro selection of glycopeptides that combines mRNA display, alkynyl amino acid incorporation, and CuAAC "click" glycosylation. Herein, we describe extensions of this method that incorporate chemical cyclization, removal of N-terminal glycosylation sites and next-generation sequencing; as an approach to HIV immunogen design, we have then used this method to develop mimics of the High Mannose Patch (HMP), which is the region on HIV envelope protein gp120 most commonly targeted by HIV broadly neutralizing antibodies (bnAbs). We prepared libraries of 10^(12-14) glycopeptides about 50 amino acids in length, containing variable numbers of high mannose (Man9GlcNAc2) glycans and cyclization at varied sites. We performed selections to obtain binders of HIV bnAbs PGT128, PGT122, and gl-PGT121, a germline precursor of PGT122, and prepared numerous glycopeptide hits by chemical synthesis. Selected glycopeptides in some cases bound very tightly to their target HIV bnAb, e.g., with a KD as low as 0.5 nM for PGT128. These glycopeptides are of interest as immunogens and tools for HIV vaccine design.

SFM4-3, KOD DGLNK, and Therminator polymerase are investigated for their compatibility with SELection with Modified Aptamers (SELMA), an aptamer discovery method that enables incorporation of large nucleobase modifications such as glycans. We demonstrated that with suitable modifications to the primer design and protocol, these enzymes are compatible with SELMA, enabling 2'-fluoro or 2'-methoxy ribose modifications at all positions. In the case of 2'-fluoro modifications, Therminator exhibits cleaner incorporation of an alkyne-modified nucleobase for click chemistry.

Access to homogeneous high-mannose glycans in high-mg quantities is necessary for carbohydrate-based HIV vaccine development research. We have used directed evolution to design highly antigenic oligomannose clusters that are recognized in low-nM affinity by HIV antibodies. Herein we report an optimized large-scale synthesis of Man9GlcNAc2 including improved building block synthesis and a fully stereoselective 5 + 6 coupling, yielding 290 mg of glycan. We then use this glycan to study the effect of the GlcNAc2 core on the antigenicity of an evolved 2G12-binding glycopeptide, 10F2.

An efficient multigram synthesis of alkynyl amino acid Fmoc-l-homopropargylglycine-OH is described. A double Boc protection is optimized for high material throughput, and the key Seyferth-Gilbert homologation is optimized to avoid racemization. Eighteen grams of the enantiopure (>98% ee) noncanonical amino acid was readily generated for use in solid phase synthesis to make peptides that can be functionalized by copper-assisted alkyne-azide cycloaddition.

Carbohydrate binding proteins (CBPs) are attractive targets in medicine and biology. Multivalency, with several glycans binding to several binding pockets in the CBP, is important for high-affinity interactions. Herein, we describe a novel platform for design of multivalent carbohydrate cluster ligands by directed evolution, in which serum-stable 2'-fluoro modified RNA (F-RNA) backbones evolve to present the glycan in optimal clusters. We have validated this method by the selection of oligomannose (Man) glycan clusters from a sequence pool of ∼10 that bind to broadly neutralizing HIV antibody 2G12 with 13 to 36 nM affinities.