Bidirectional Tandem Pseudoproline Ligations of Proline-Rich Helical Peptides

Abstract

We have developed a bidirectional ligation strategy for preparing proline-rich peptides that couples three unprotected segments in tandem to form two pseudoproline bonds (thia-or oxaproline) without the need for a protection scheme. Ligation in the CfN direction exploits the regioselectivity of an amino terminal (NT)-Cys in forming a thiaproline bond over an NT-Ser or NT-Thr peptide in forming an oxaproline bond with a peptide that bears a carboxyl terminal (CT)-glycoaldehyde ester. Thus, successive ligations of three unprotected segments in a predetermined order formed a thiaproline and then an oxaproline bond. However, ligation through the NfC direction is flexible. An NT-Cys, NT-Ser, or NT-Thr segment bearing a CT-glycerol ester as a masked CT-glycoaldehyde was used to form a pseudoproline bond with another CT-glycoaldehyde ester segment. Oxidative activation of the glycerol ester product to a CT-glycoaldehyde ester effected another round of pseudoproline ligation with an NT-Ser, NT-Thr, or NT-Cys segment. This sequential process could be extended for ligating three or more segments. Optimized conditions for this bidirectional strategy were applied successfully to syntheses of five analogues of a proline-rich helical antimicrobial peptide, the 59residue bactenecin 7 (Bac 7), using three segments containing 24, 14, and 21 amino acids, respectively. CD spectra showed that Bac 7 and its analogues displayed typical polyproline II helical structures in phosphate buffers. Furthermore, the ψPro-containing analogues exhibited antibacterial activity similar to Bac 7.

Chemoselective Peptide Ligation: A Privileged Tool for Protein Synthesis

“…This reaction was applied in the assembly of proline-rich analogs of the 59-residue helical antibacterial peptide bactenecin 7, which showed comparable biological activities to the natural product [113]. In this aspect it is important to note that a three-segment condensation was used, in which the first junction was constructed by the formation of a thiazolidine of the Cys peptide -a faster step than the formation of the corresponding oxazolidine of the Thr peptide.…”

Mimicking Reverse Protein Splicing by Three-Segment Tandem Peptide Ligation

“…The O,N-acyl transfer reaction provides a proline mimetic that retains the amide backbone structure of an α-peptide. In contrast to thiaproline ligation [13][14][15] which is usually performed in aqueous buffers at pH 4 to 7, oxaproline ligation was performed under nearly nonaqueous conditions containing pyridine and acetic acid [14,21,42]. It was also shown that NT-amino acids such as NT-Cys, NTTrp, and NT-His can be ligated under such a condition [34].…”

“…Even before the mechanism of protein splicing was fully elucidated [10], proximity-driven ligation methods had been developed for ligating unprotected peptide segments [11][12][13][14][15]. For our purpose, we call the unassisted ligation reaction orthogonal ligation because its mechanism in forming an amide bond involves a specific N-terminal (NT) amino acid that is generally orthogonal to other NT-amino acids [16].…”

“…1). * Over the past decade, various types of orthogonal ligation methods have been reported [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27]. They share a common feature of having a di-nucleophile at the N-terminus of a reactant peptide.…”

“…The ester intermediates spontaneously undergo a proximity-driven acyl rearrangement to produce a peptide bond. Examples of dinucleophilic NT-amino acids include cysteine (Cys), selenocysteine (SeCys), homocysteine (Hcy), serine (Ser), threonine (Thr), tryptophan (Trp) and histidine (His), forming Cys [17,18], SeCys [19], methionine [20], thiaproline [13][14][15], oxaproline [13,21], tryptophan [22] and histidine [23] bonds at the ligation site, respectively. The repertoire of ligation methods also include NT-amino substituted auxiliary groups as Cys mimetics that require a follow-up deprotection step [24,25] as well as Staudinger ligation [26][27] that requires an NT-azido moiety.…”