This review focuses on the concept, criteria, and methods of an orthogonal amide ligating strategy suitable for syntheses of peptides, peptide mimetics, and proteins. Utilizing unprotected peptides or proteins derived from chemical or biosynthetic sources, this ligation strategy has been shown to be general and exceptionally mild. Its orthogonality in ligating two unprotected segments with free N‐terminal (NT)‐amines at a specific NT‐amine is achieved through a chemoselective capture step and then an intramolecular acyl transfer reaction. Both coupling reagents for enthalpic activation and protection schemes therefore become unnecessary. More than a dozen orthogonal ligation methods based on either imine or thioester captures have been developed to afford native and unusual amino acids at ligation sites of linear, branched, or cyclic peptides. Because unprotected peptides and proteins of different sizes and forms can be obtained from either chemical or recombinant sources, orthogonal ligation removes the size limitation imposed on the chemical synthesis of a protein with a native or non‐native structure. Furthermore, by using building blocks from biosynthetic sources, orthogonal ligation provides a unifying operational concept for both total and semisynthesis of peptides and proteins. © 2000 John Wiley & Sons, Inc. Biopoly 51: 311–332, 1999

This paper describes an orthogonal and stereospecific method for ligating free peptide segments to form a monosubstituted pseudoproline bond with a hydroxymethyl moiety at the C2 carbon. The pseudoproline ligation, comprising both the oxaproline and thiaproline ligations, initially involves an imine capture of a peptidyl glycoaldehyde ester with an N-terminal cysteine, serine, or threonine peptide segment and then two spontaneous cyclization reactions. The thiazolidine or oxazolidine ester formed in the first cyclization undergoes an O,N-acyl transfer to form an pseudoproline bond. The thiaproline ligation can be carried out exclusively with unprotected peptide segments in both aqueous and nonaqueous pyridine-acetic acid conditions. However, the oxaproline ligation is best performed in a nonaqueous pyridine-acetic acid mixture with unprotected peptide segments except for those containing N-terminal nucleophilic amino acids such as Cys, His, and Trp. Pseudoproline ligation is not only regioselective but also stereospecific. 2D 1 H NMR studies of dipeptide models, Z-Xaa-ψPro-OMe, indicate that the newly created C2 stereocenter of the pseudoproline ring affords only an R-epimer and the C2-hydroxymethyl-substituted pseudoproline exhibits high preference for cis conformation. Three of the model peptides have more than 50% cis isomers. Finally, this novel method has been used successfully in ligating two segments of 24 and 35 amino acids under mild conditions to synthesize three analogues of bactenecin 7, an antimicrobial peptide containing 59 amino acid residues.

To prepare multipartite peptides with several functional cargoes including a cell-permeable sequence or transportant for intracellular delivery, tandem ligation of peptides is a convenient convergent approach with the fewest synthetic steps. It links three or four unprotected segments forming two or more regiospecific bonds consecutively without a deprotection step. This paper describes a tandem ligation strategy to prepare multipartite peptides with normal and branched architectures carrying a novel transportant peptide that is rich in arginine and proline to permit their cargoes to be translocated across membranes to affect their biological functions in cytoplasm. Our strategy consists of three ligation methods specific for amino terminal cysteine (Cys), serine/threonine (Ser/Thr), and N(alpha)-chloroacetylated amine to afford Xaa-Cys, Xaa-OPro (oxaproline) and Xaa-psiGly (pseudoglycine) at the ligation sites, respectively. Assembly of single-chain peptides from three different segments was achieved by the tandem Cys/OPro ligation to form two amide bonds, an Xaa-Cys and then an Xaa-OPro. Assembly of two- and three-chain peptides with branched architectures from four different segments was accomplished by tandem Cys/psiGly/OPro ligation. These NT-specific tandem ligation strategies were successful in generating cell-permeable multipartite peptides with one-, two-, and three-chain architectures, ranging in size from 52 to 75 residues and without the need of a protection or deprotection step. In addition, our results show that there is considerable flexibility in architectural design to obtain cell-permeable multipartite peptides containing a transportant sequence.

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.

A facile method has been developed to synthesize linear and cyclic dehydropeptides from unprotected peptide precursors. This method exploits an N-terminal Cys for a Cys-thioester ligation to generate an unprotected peptide and as a precursor for conversion to DeltaAla by beta-elimination under mild conditions.