Propargyloxyproline Regio- and Stereoisomers for Click-Conjugation of Peptides: Synthesis and Application in Linear and Cyclic Peptides

Abstract

The use of the click reaction for the introduction of conjugate groups, such as affinity or fluorescent labels, to a peptide for the study of peptide biochemistry and pharmacology is widespread. However, the nature and location of substituted 1,2,3-triazoles in peptide sequences may markedly affect conformation or binding as compared with native sequences. We have examined the preparation and application of propargyloxyproline (Pop) residues as a precursor to such peptide conjugates. Pop residues are available in a range of regio- and stereoisomers from hydroxyproline precursors and are readily prepared in Fmoc-protected form. They can be incorporated routinely in peptide synthesis and broadly retain the conformational properties of the parent proline containing peptides. This is exemplified by the preparation of biotin- and fluorophore-labelled peptides derived from linear and cyclic peptides.

Identification of a Cyanine-Dye Labeled Peptidic Ligand for Y1R and Y4R, Based upon the Neuropeptide Y C-Terminal Analogue, BVD-15

“…24 This prompted us to examine such analogues with Pro 3 as a point of conjugation and we have recently described propargyloxyproline containing Lys 4 -and Arg 4 -BVD-15 that could incorporate rhodamine B and 7-aminocoumarin fluorophores. 25 The tyrosine at the 5-position has been also shown to be capable of replacement with a conjugate group. …”

“…Labelling was then achieved by solution phase CuAAC reaction using the alkyne-containing RhB-2 and RhB-3, in presence of CuSO4 and sodium ascorbate as the catalysts (Method 3). 25 The synthesised analogues with their analytical data are summarised in Table 1. …”

4R- and 4S-iodophenyl hydroxyproline, 4R-pentynoyl hydroxyproline, and S-propargyl-4-thiolphenylalanine: conformationally biased and tunable amino acids for bioorthogonal reactions

“…5a, 18 The resulting protected 4 S -hydroxyproline 9 was then subjected to a second Mitsunobu reaction with 4-iodophenol to generate the doubly inverted 4 R -hydroxyproline iodophenyl ether. This second Mitsunobu reaction with iodophenol required ADDP to proceed in reasonable yield, as was also the case in the synthesis of the 4 S -iodophenyl hydroxyproline (Scheme 5).…”

“…Most other commercially available or readily synthesized alkyne derivatives are relatively flexible or lacking in fine conformational control. 5g In prior work, we demonstrated that the pentynoate of 4 R -hydroxyproline exhibits a strong bias for an exo proline ring pucker and trans amide bonds, suggesting its application when this conformation is preferred (Scheme 3, Figure 2, Table 1). 7e In addition, we have previously developed methods for the synthesis of the amino acid 4-thiolphenylalanine, and derivatives thereof, including the S-allyl substitution (Scheme 4).…”

“…Azidoprolines and aminoxyprolines are the best-developed examples of amino acids that both introduce bioorthogonal functional groups and impose inherent structural control. 5 Proline residues are particularly well suited as sites of modification, as prolines are often solvent-exposed at surface loops and turns in proteins. 6 In addition, proline plays important structural roles due to the conformational constraint of the cyclic amino acid.…”