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Site-selective solid-phase synthesis of a CCR5 sulfopeptide library to interrogate HIV binding and entry.

ABSTRACT: Tyrosine (Tyr) sulfation is a common post-translational modification that is implicated in a variety of important biological processes, including the fusion and entry of human immunodeficiency virus type-1 (HIV-1). A number of sulfated Tyr (sTyr) residues on the N-terminus of the CCR5 chemokine receptor are involved in a crucial binding interaction with the gp120 HIV-1 envelope glycoprotein. Despite the established importance of these sTyr residues, the exact structural and functional role of this post-translational modification in HIV-1 infection is not fully understood. Detailed biological studies are hindered in part by the difficulty in accessing homogeneous sulfopeptides and sulfoproteins through biological expression and established synthetic techniques. Herein we describe an efficient approach to the synthesis of sulfopeptides bearing discrete sulfation patterns through the divergent, site-selective incorporation of sTyr residues on solid support. By employing three orthogonally protected Tyr building blocks and a solid-phase sulfation protocol, we demonstrate the synthesis of a library of target N-terminal CCR5(2-22) sulfoforms bearing discrete and differential sulfation at Tyr10, Tyr14, and Tyr15, from a single resin-bound intermediate. We demonstrate the importance of distinct sites of Tyr sulfation in binding gp120 through a competitive binding assay between the synthetic CCR5 sulfopeptides and an anti-gp120 monoclonal antibody. These studies revealed a critical role of sulfation at Tyr14 for binding and a possible additional role for sulfation at Tyr10. N-terminal CCR5 variants bearing a sTyr residue at position 14 were also found to complement viral entry into cells expressing an N-terminally truncated CCR5 receptor.

SUBMITTER: Liu X

PROVIDER: S-EPMC4168781 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Site-selective solid-phase synthesis of a CCR5 sulfopeptide library to interrogate HIV binding and entry.

Liu Xuyu X Malins Lara R LR Roche Michael M Sterjovski Jasminka J Duncan Renee R Garcia Mary L ML Barnes Nadine C NC Anderson David A DA Stone Martin J MJ Gorry Paul R PR Payne Richard J RJ

ACS chemical biology 20140724 9

Tyrosine (Tyr) sulfation is a common post-translational modification that is implicated in a variety of important biological processes, including the fusion and entry of human immunodeficiency virus type-1 (HIV-1). A number of sulfated Tyr (sTyr) residues on the N-terminus of the CCR5 chemokine receptor are involved in a crucial binding interaction with the gp120 HIV-1 envelope glycoprotein. Despite the established importance of these sTyr residues, the exact structural and functional role of th ...[more]

PMID: 24963694

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Project description:The promise of exploiting combinatorial synthesis for small molecule discovery remains unfulfilled due primarily to the "structure elucidation problem": the back-end mass spectrometric analysis that significantly restricts one-bead-one-compound (OBOC) library complexity. The very molecular features that confer binding potency and specificity, such as stereochemistry, regiochemistry, and scaffold rigidity, are conspicuously absent from most libraries because isomerism introduces mass redundancy and diverse scaffolds yield uninterpretable MS fragmentation. Here we present DNA-encoded solid-phase synthesis (DESPS), comprising parallel compound synthesis in organic solvent and aqueous enzymatic ligation of unprotected encoding dsDNA oligonucleotides. Computational encoding language design yielded 148 thermodynamically optimized sequences with Hamming string distance ? 3 and total read length <100 bases for facile sequencing. Ligation is efficient (70% yield), specific, and directional over 6 encoding positions. A series of isomers served as a testbed for DESPS's utility in split-and-pool diversification. Single-bead quantitative PCR detected 9 × 10(4) molecules/bead and sequencing allowed for elucidation of each compound's synthetic history. We applied DESPS to the combinatorial synthesis of a 75,645-member OBOC library containing scaffold, stereochemical and regiochemical diversity using mixed-scale resin (160-?m quality control beads and 10-?m screening beads). Tandem DNA sequencing/MALDI-TOF MS analysis of 19 quality control beads showed excellent agreement (<1 ppt) between DNA sequence-predicted mass and the observed mass. DESPS synergistically unites the advantages of solid-phase synthesis and DNA encoding, enabling single-bead structural elucidation of complex compounds and synthesis using reactions normally considered incompatible with unprotected DNA. The widespread availability of inexpensive oligonucleotide synthesis, enzymes, DNA sequencing, and PCR make implementation of DESPS straightforward, and may prompt the chemistry community to revisit the synthesis of more complex and diverse libraries.

Dataset Information

Site-selective solid-phase synthesis of a CCR5 sulfopeptide library to interrogate HIV binding and entry.

Publications

Site-selective solid-phase synthesis of a CCR5 sulfopeptide library to interrogate HIV binding and entry.

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