Fully unprotected peptide o-aminoanilides can be efficiently activated by NaNO₂ in aqueous solution to furnish peptide thioesters for use in native chemical ligation. This finding enables the convergent synthesis of proteins from readily synthesizable peptide o-aminoanilides as a new type of crypto-thioesters. The practicality of this approach is shown by the synthesis of histone H2B from five peptide segments. Purification or solubilization tags, which are sometimes needed to improve the efficiency of protein chemical synthesis, can be incorporated into the o-aminoanilide moiety, as demonstrated in the preparation of the cyclic protein lactocyclicin Q.

Successive peptide ligation using a one-pot method can improve the efficiency of protein chemical synthesis. Although one-pot three-segment ligation has enjoyed widespread application, a robust method for one-pot four-segment ligation had to date remained undeveloped. Herein we report a new one-pot multisegment peptide ligation method that can be used to condense up to four segments with operational simplicity and high efficiency. Its practicality is demonstrated by the one-pot four-segment synthesis of a plant protein, crambin, and a human chemokine, hCCL21.

Blockade of the protein–protein interaction between the transmembrane protein programmed cell death protein 1 (PD-1) and its ligand PD-L1 has emerged as a promising immunotherapy for treating cancers. Using the technology of mirror-image phage display, we developed the first hydrolysis-resistant D-peptide antagonists to target the PD-1/PD-L1 pathway. The optimized compound ^DPPA-1 could bind PD-L1 at an affinity of 0.51 μM in vitro. A blockade assay at the cellular level and tumor-bearing mice experiments indicated that ^DPPA-1 could also effectively disrupt the PD-1/PD-L1 interaction in vivo. Thus D-peptide antagonists may provide novel low-molecular-weight drug candidates for cancer immunotherapy.

Disulfide-rich peptides containing three or more disulfide bonds are promising therapeutic and diagnostic agents, but their preparation is often limited by the tedious and low-yielding folding process. We found that a single cystine-to-diaminodiacid replacement could significantly increase the folding efficiency of disulfide-rich peptides and thus improve their production yields. The practicality of this strategy was demonstrated by the synthesis and folding of derivatives of the μ-conotoxin SIIIA, the preclinical hormone hepcidin, and the trypsin inhibitor EETI-II. NMR and X-ray crystallography studies confirmed that these derivatives of disulfide-rich peptide retained the correct three-dimensional conformations. Moreover, the cystine-to-diaminodiacid replacement enabled structural tuning, thereby leading to an EETI-II derivative with higher bioactivity than the native peptide.

Fully unprotected peptide o-aminoanilides can be efficiently activated by NaNO₂ in aqueous solution to furnish peptide thioesters for use in native chemical ligation. This finding enables the convergent synthesis of proteins from readily synthesizable peptide o-aminoanilides as a new type of crypto-thioesters. The practicality of this approach is shown by the synthesis of histone H2B from five peptide segments. Purification or solubilization tags, which are sometimes needed to improve the efficiency of protein chemical synthesis, can be incorporated into the o-aminoanilide moiety, as demonstrated in the preparation of the cyclic protein lactocyclicin Q.

Successive peptide ligation using a one-pot method can improve the efficiency of protein chemical synthesis. Although one-pot three-segment ligation has enjoyed widespread application, a robust method for one-pot four-segment ligation had to date remained undeveloped. Herein we report a new one-pot multisegment peptide ligation method that can be used to condense up to four segments with operational simplicity and high efficiency. Its practicality is demonstrated by the one-pot four-segment synthesis of a plant protein, crambin, and a human chemokine, hCCL21.

Blockade of the protein–protein interaction between the transmembrane protein programmed cell death protein 1 (PD-1) and its ligand PD-L1 has emerged as a promising immunotherapy for treating cancers. Using the technology of mirror-image phage display, we developed the first hydrolysis-resistant D-peptide antagonists to target the PD-1/PD-L1 pathway. The optimized compound ^DPPA-1 could bind PD-L1 at an affinity of 0.51 μM in vitro. A blockade assay at the cellular level and tumor-bearing mice experiments indicated that ^DPPA-1 could also effectively disrupt the PD-1/PD-L1 interaction in vivo. Thus D-peptide antagonists may provide novel low-molecular-weight drug candidates for cancer immunotherapy.

Disulfide-rich peptides containing three or more disulfide bonds are promising therapeutic and diagnostic agents, but their preparation is often limited by the tedious and low-yielding folding process. We found that a single cystine-to-diaminodiacid replacement could significantly increase the folding efficiency of disulfide-rich peptides and thus improve their production yields. The practicality of this strategy was demonstrated by the synthesis and folding of derivatives of the μ-conotoxin SIIIA, the preclinical hormone hepcidin, and the trypsin inhibitor EETI-II. NMR and X-ray crystallography studies confirmed that these derivatives of disulfide-rich peptide retained the correct three-dimensional conformations. Moreover, the cystine-to-diaminodiacid replacement enabled structural tuning, thereby leading to an EETI-II derivative with higher bioactivity than the native peptide.

As a unique and unappreciated protein posttranslational modification, arginine N-glycosylation was recently discovered to play an important role in the process that bacteria counteract host defenses. To provide chemical tools for further proteomic and biochemical studies on arginine N-glycosylation, we report the first general strategy for a rapid and cost-effective synthesis of glycopeptides carrying single or multiple arginine N-GlcNAcyl groups. These glycopeptides were successfully utilized to generate the first antibodies that can specifically recognize arginine N-GlcNAcylated peptides or proteins in a sequence-independent manner.

Sortase-mediated hydrazinolysis of proteins with hydrazine or its derivatives was developed for the production of recombinant protein hydrazides. This process provides an alternative approach for protein semisynthesis through the use of recombinant protein hydrazides as thioester surrogates. It also provides an alternative method for C-terminal modification of proteins with functional units as well as for the preparation of C-to-C fusion proteins.

Sortase-mediated hydrazinolysis of proteins with hydrazine or its derivatives was developed for the production of recombinant protein hydrazides. This process provides an alternative approach for protein semisynthesis through the use of recombinant protein hydrazides as thioester surrogates. It also provides an alternative method for C-terminal modification of proteins with functional units as well as for the preparation of C-to-C fusion proteins.

A novel method for the synthesis of non-natural L- and D-amino acids by a Ni-catalyzed reductive cross-coupling reaction is described. This strategy enables the racemization-free cross-coupling of serine/homoserine- derived iodides with aryl/acyl/alkyl halides. It provides convenient access to varieties of enantiopure and functionalized amino acids, which are important building blocks in bioactive compounds and pharmaceuticals.

A copper-catalyzed reductive cross-coupling reaction of nonactivated alkyl tosylates and mesylates with alkyl and aryl bromides was developed. It provides a practical method for efficient and cost-effective construction of aryl–alkyl and alkyl–alkyl CC bonds with stereocontrol from readily available substrates. When used in an intramolecular fashion, the reaction enables convenient access to various substituted carbo- or heterocycles, such as 2,3-dihydrobenzofuran and benzochromene derivatives.

Palladium- and nickel-catalyzed cross-coupling recations of unactivated alkyl bromides with diboron reagents have been developed as practical methods for the synthesis of primary and secondary alkylboronic esters. These reactions extend the concept and utility of Pd- and Ni-catalyzed cross-coupling of aliphatic electrophiles. They also show different substrate selectivity and ligand dependence as compared to the recently reported Cu-catalyzed borylation reaction.

Imidazole-promoted ligation of peptide phenyl esters was recently found to be a complementary method for protein chemical synthesis. Theoretical calculations have been carried out to understand the detailed mechanism of this particular ligation process. It is found that both the reaction of the phenyl ester with imidazole and the reaction of the acyl imidazole intermediate with cysteine proceed through an addition-elimination mechanism. The cleavage of the CO bond in the reaction between the phenyl ester and imidazole is the rate-limiting step of the overall ligation process. Interestingly, although the imidazole-promoted phenyl ester ligation has a higher free energy barrier than the conventional thiophenol-promoted native chemical ligation for a sterically less hindered C-terminal amino acid (e.g. gylcine), for a sterically hindered C-terminal amino acid (e.g. proline) the imidazole-promoted phenyl ester ligation is calculated to be more favorable than the conventional thiophenol-promoted native chemical ligation.

The development of efficient chemical methods for total synthesis or semisynthesis of integral membrane proteins is an important challenge at the interface between chemistry and biology. This review outlines the recent advances in the synthesis of integral membrane proteins, with particular focus on the methods for difficult peptide synthesis, purification, and enhancement of peptide solubility under the ligation conditions. The applications of these methods to the synthesis of integral membrane proteins with one or multiple transmembrane domains are also described.

Quantitative nucleophilicity scales are fundamental to organic chemistry and are usually constructed on the basis of Mayr’s equation [log k=s(N+E)] by using benzhydrylium ions as reference electrophiles. Here an ab initio protocol was developed for the first time to predict the nucleophilicity parameters N of various π nucleophiles in CH₂Cl₂ through transition-state calculations. The optimized theoretical model (BH&HLYP/6-311++G(3df,2p)//B3LYP/6-311+G(d,p)/PCM/UAHF) could predict the N values of structurally unrelated π nucleophiles within a precision of ca. 1.14 units and therefore may find applications for the prediction of nucleophilicity of compounds that are not readily amenable to experimental characterization. The success in predicting N parameters from first principles also allowed us to analyze in depth the electrostatic, steric, and solvation energies involved in electrophile–nucleophile reactions. We found that solvation does not play an important role in the validity of Mayr’s equation. On the other hand, the correlations of the E, N, and log k values with the energies of the frontier molecular orbitals indicated that electrostatic/charge-transfer interactions play vital roles in Mayr’s equation. Surprising correlations observed between the electrophile–nucleophile CC distances in the transition state, the activation energy barriers, and the E and N parameters indicate the importance of steric interactions in Mayr’s equation. A method is then proposed to separate the attraction and repulsion energies in the nucleophile–electrophile interaction. It was found that the attraction energy correlated with N+E, whereas the repulsion energy correlated to the s parameter.

The mechanism of a recently discovered intramolecular Heck-type coupling of oximes with aryl halides (Angew. Chem. Int. Ed. 2007, 46, 6325) was systematically studied by using density functional methods enhanced with a polarized continuum solvation model. The overall catalytic cycle of the reaction was found to consist of four steps: oxidative addition, migratory insertion, β-H elimination, and catalyst regeneration, whereas an alternative base-promoted CH activation pathway was determined to be less favorable. Migratory insertion was found to be the rate determining step in the catalytic cycle. The apparent activation barrier of migratory insertion of the (E)-oxime was +20.5 kcal mol⁻¹, whereas the barrier of (Z)-oxime was as high as +32.7 kcal mol⁻¹. However, (Z)-oxime could isomerize to form the more active (E)-oxime with the assistance of K₂CO₃, so that both the (E)- and (Z)-oxime substrates could be transformed to the desired product. Our calculations also indicated that the Z product was predominant in the equilibrium of the isomerization of the imine double bond, which constituted the reason for the good Z-selectivity observed for the reaction. Furthermore, we examined the difference between the intermolecular Heck-type reactions of imines and of olefins. It was found that in the intermolecular Heck-type coupling of imines, the apparent activation barrier of migratory insertion was as high as +35 kcal mol⁻¹, which should be the main obstacle of the reaction. The analysis also revealed the main problem for the intermolecular Heck-type reactions of imines, which was that the breaking of a CN π bond was much more difficult than the breaking of a CC π bond. After systematic examination of a series of substituted imines, (Z)-N-amino imine and N-acetyl imine were found to have relatively low barriers of migratory insertion, so that they might be possible substrates for intermolecular Heck-type coupling.