Orthogonal Ligation of Unprotected Peptide Segments through Pseudoproline Formation for the Synthesis of HIV-1 Protease Analogs^,

Abstract

We describe the total synthesis of three active HIV-1 protease analogs by an orthogonal ligation through thiaproline formation of two large, unprotected peptide segments. The central element of this strategy in achieving orthogonality of peptide bond formation is through proximity effect, and the key reaction is a side chain thiol initiated aldehyde capture to overcome the entropic problem of coupling between two large molecular weight peptide segments. The capture step also leads to specific entropic activation of the acyl segment because the respective termini are brought to close proximity, resulting in a spontaneous acyl rearrangement to form the amide bond. A general method using a thioester for introducing an aldehyde moiety to the C-terminus of an unprotected peptide segment was also developed. Three active analogs of HIV-1 protease were obtained in excellent yield by ligating two segments of 38 and 61 residues. Two analogs contained a thioproline residue at position 39, and the third contained a 38−39 non-peptide backbone. Efficient ligation at pH 4 was attained at peptide segment concentrations as low as 50 μM, a concentration which is not feasible with conventional convergent methods using protected peptide segments.

Synthesis of Native Proteins by Chemical Ligation

“…The original ligation chemistries (50,53,(59)(60)(61) (62). Unprotected peptide segments are reacted by means of reversible thiol/thioester exchange to give thioester-linked initial reaction products.…”

Designing hydrogel adhesives for corneal wound repair

“…46 This mild procedure involving a thiol and amine reacting with an aldehyde has been applied successfully to the synthesis of a variety of proteins. [58][59][60][61][62] These two crosslinking strategies have been used to repair a variety of corneal wounds, as described below.…”

“…Thus, we are investigating the use of a thiazolidine linkage which is formed between an N-terminal cysteine and an aldehyde; this reaction belongs to a family of peptide liga tion reactions. [58][59][60][61][62] For this approach to work, we need a dendritic polymer which contains 3 or more N-terminal cysteines and a PEG which contains at least two terminal aldehyde groups. Aldehyde-based adhesives have been explored for ophthalmic use including a condroitin sulfate and a PEG-PLL micelle systems as well as there is a commercial product used for cardiovascular surgery.…”

New Developments for the Site-Specific Attachment of Protein to Surfaces

“…13). Tam and coworkers 88,89 have further developed this reaction by using a glycol aldehyde ester (see Fig. 13A).…”