Chemoselective Ligation and Modification Strategies for Peptides and Proteins

The investigation of biological processes by chemical methods, commonly referred to as chemical biology, often requires chemical access to biologically relevant macromolecules such as peptides and proteins. Building upon solid‐phase peptide synthesis, investigations have focused on the development of chemoselective ligation and modification strategies to link synthetic peptides or other functional units to larger synthetic and biologically relevant macromolecules. This Review summarizes recent developments in the field of chemoselective ligation and modification strategies and illustrates their application, with examples ranging from the total synthesis of proteins to the semisynthesis of naturally modified proteins.     

Semisynthesis of a Post‐Translationally Modified Protein by Using Chemical Cleavage and Activation of an Expressed Fusion Polypeptide

Herein, we describe a new semisynthetic strategy of a post‐translationally modified protein in which the middle region is glycosylated. We designed a single‐plasmid coding for a fusion polypeptide, which can provide both an N‐terminal α‐thioester and a C‐terminal cysteine peptide of a target glycoprotein by using chemical‐cleavage and activation methods. The use of these resultant peptide derivatives resulted in the successful synthesis of N‐glycosylated‐interleukin 13.     

Semisynthesis of a Homogeneous Glycoprotein Enzyme: Ribonuclease C: Part 1

Seven in one blow : The efficient formation of mixed disulfides on the thiol‐rich fusion protein A followed by subsequent intein cleavage gave the fragment B with all seven cysteines protected against oxidation. The native chemical ligation of B with synthetic glycopeptide thioesters provides glycoproteins.

     

Semisynthesis of a Homogeneous Glycoprotein Enzyme: Ribonuclease C: Part 2

Active RNase glycoprotein from three pieces : The glycoprotein enzyme ribonuclease C, which contains a complex saccharide N‐glycan, was synthesized by sequential native chemical ligation. An optimized ligation and isolation protocol allowed the efficient assembly and refolding of the 124 amino acid enzyme.

     

Synthesis of Cyclic Peptides in SPPS with Npb-OH Photolabile Protecting Group

Significant efforts have been made in recent years to identify more environmentally benign and safe alternatives to side-chain protection and deprotection in solid-phase peptide synthesis (SPPS). Several protecting groups have been endorsed as suitable candidates, but finding a greener protecting group in SPPS has been challenging. Here, based on the 2-(o-nitrophenyl) propan-1-ol (Npp-OH) photolabile protecting group, a structural modification was carried out to synthesize a series of derivatives. Through experimental verification, we found that 3-(o-Nitrophenyl) butan-2-ol (Npb-OH) had a high photo-release rate, high tolerance to the key conditions of Fmoc-SPPS (20% piperidine DMF alkaline solution, and pure TFA acidic solution), and applicability as a carboxyl-protective group in aliphatic and aromatic carboxyl groups. Finally, Npb-OH was successfully applied to the synthesis of head–tail cyclic peptides and side-chain–tail cyclic peptides. Moreover, we found that Npb-OH could effectively resist diketopiperazines (DKP). The α-H of Npb-OH was found to be necessary for its photosensitivity in comparison to 3-(o-Nitrophenyl)but-3-en-2-ol (Npbe-OH) during photolysis-rate verification.     

An Efficient One‐Pot Four‐Segment Condensation Method for Protein Chemical Synthesis

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.     

Eco‐Friendly Combination of the Immobilized PGA Enzyme and the S‐Phacm Protecting Group for the Synthesis of Cys‐Containing Peptides

Enzyme‐labile protecting groups have emerged as a green alternative to conventional protecting groups. These groups introduce a further orthogonal dimension and eco‐friendliness into protection schemes for the synthesis of complex polyfunctional organic molecules. S‐Phacm, a Cys‐protecting group, can be easily removed by the action of a covalently immobilized PGA enzyme under very mild conditions. Herein, the versatility and reliability of an eco‐friendly combination of the immobilized PGA enzyme and the S‐Phacm protecting group has been evaluated for the synthesis of diverse Cys‐containing peptides.     

2‐Nitroveratryl as a Photocleavable Thiol‐Protecting Group for Directed Disulfide Bond Formation in the Chemical Synthesis of Insulin

Chemical synthesis of peptides can allow the option of sequential formation of multiple cysteines through exploitation of judiciously chosen regioselective thiol‐protecting groups. We report the use of 2‐nitroveratryl (oNv) as a new orthogonal group that can be cleaved by photolysis under ambient conditions. In combination with complementary S‐pyridinesulfenyl activation, disulfide bonds are formed rapidly in situ. The preparation of Fmoc‐Cys(oNv)‐OH is described together with its use for the solid‐phase synthesis of complex cystine‐rich peptides, such as insulin.     

Chemical Synthesis of Proteins with Non‐Strategically Placed Cysteines Using Selenazolidine and Selective Deselenization

Although native chemical ligation has enabled the synthesis of hundreds of proteins, not all proteins are accessible through typical ligation conditions. The challenging protein, 125‐residue human phosphohistidine phosphatase 1 (PHPT1), has three cysteines near the C‐terminus, which are not strategically placed for ligation. Herein, we report the first sequential native chemical ligation/deselenization reaction. PHPT1 was prepared from three unprotected peptide segments using two ligation reactions at cysteine and alanine junctions. Selenazolidine was utilized as a masked precursor for N‐terminal selenocysteine in the middle segment, and, following ligation, deselenization provided the native alanine residue. This approach was used to synthesize both the wild‐type PHPT1 and an analogue in which the active‐site histidine was substituted with the unnatural and isosteric amino acid β‐thienyl‐l ‐alanine. The activity of both proteins was studied and compared, providing insights into the enzyme active site.     

Synthesis of Disulfide Surrogate Peptides Incorporating Large‐Span Surrogate Bridges Through a Native‐Chemical‐Ligation‐Assisted Diaminodiacid Strategy

The use of synthetic bridges as surrogates for disulfide bonds has emerged as a practical strategy to obviate the poor stability of some disulfide‐containing peptides. However, peptides incorporating large‐span synthetic bridges are still beyond the reach of existing methods. Herein, we report a native chemical ligation (NCL)‐assisted diaminodiacid (DADA) strategy that enables the robust generation of disulfide surrogate peptides incorporating surrogate bridges up to 50 amino acids in length. This strategy provides access to some highly desirable but otherwise impossible‐to‐obtain disulfide surrogates of bioactive peptide. The bioactivities and structures of the synthetic disulfide surrogates were verified by voltage clamp assays, NMR, and X‐ray crystallography; and stability studies established that the disulfide replacements effectively overcame the problems of disulfide reduction and scrambling that often plague these pharmacologically important peptides.     

Efficient Solid‐Phase Synthesis of Sulfotyrosine Peptides using a Sulfate Protecting‐Group Strategy

Double protection : Efficient Fmoc‐based solid‐phase synthesis (SPPS) of sulfotyrosine (sY) peptides is achieved by incorporating the sY residue(s) as a dichlorovinyl‐protected (DCV) sulfodiester(s) and using 2‐methylpiperidine for Fmoc removal. After removal of the other protecting groups, the DCV group could be cleaved by mild hydrogenolysis giving the sY peptides in good yield.

     

New Protecting Group Strategies for the Solid-Phase Synthesis and Modification of Peptides,Oligonucleotides, and Oligosaccharides

Blocking and activation can be achieved with protecting groups such as the ortho-nitrobenzenesulfonyl group (see picture). In solid-phase peptide synthesis, this group can be used for temporary protection (path A) as well as the activation and selective N-alkylation (path B) of an amino group.     

Total Synthesis of Human Hepcidin through Regioselective Disulfide‐Bond Formation by using the Safety‐Catch Cysteine Protecting Group 4,4′‐Dimethylsulfinylbenzhydryl

A safety‐catch cysteine protecting group, S‐4,4′‐dimethylsulfinylbenzhydryl (Msbh), was designed and developed to expand the capabilities of synthetic strategies for the regioselective formation of disulfide bonds in cysteine‐rich peptides. The directed regioselective synthesis of human hepcidin, which contains four disulfide bonds, was undertaken and led to a high‐resolution NMR structure under more physiologically relevant conditions than previously. Conversely, hepcidin synthesized with the formerly assigned vicinal disulfide‐bond connectivity displayed significant conformational heterogeneity under similar conditions. The two synthetic forms of human hepcidin induced ferroportin internalization with apparent EC₅₀ values of 2.0 (native fold, 1 ) and 4.4 nM (non‐native fold, 2 ), with 2 undergoing isomerization to 1 in the presence of ferroportin expressing cells.     

N-Amino-1,8-Naphthalimide is a Regenerated Protecting Group for Selective Synthesis of Mono-N-Substituted Hydrazines and Hydrazides

A new route to synthesis of various mono-N-substituted hydrazines and hydrazides by involving in a new C−N bond formation by using N-amino-1,8-naphthalimide as a regenerated precursor was invented. Aniline and phenylhydrazines are reproduced upon reacting these individually with 1,8-naphthalic anhydride followed by hydrazinolysis. The practicality and simplicity of this C−N dihalo alkanes; developed a synthon for bond formation protocol was exemplified to various hydrazines and hydrazides. N-amino-1,8-naphthalimide is suitable synthon for transformation for selective formation of mono-substituted hydrazine and hydrazide derivatives. Those are selective mono - amidation of hydrazine with acid halides; mono-N-substituted hydrazones from aldehydes; synthesis of N-aminoazacycloalkanes from acetohydrazide scaffold and inserted to hydroxy derivatives; distinct synthesis of N,N-dibenzylhydrazines and N-benzylhydrazines from benzyl halides; synthesis of N-amino-amino acids from α-halo esters. Ecofriendly reagent N-amino-1,8-naphthalimide was regenerated with good yields by the hydrazinolysis in all procedures.