Efficient Palladium‐Assisted One‐Pot Deprotection of (Acetamidomethyl)Cysteine Following Native Chemical Ligation and/or Desulfurization To Expedite Chemical Protein Synthesis

The acetamidomethyl (Acm) moiety is a widely used cysteine protecting group for the chemical synthesis and semisynthesis of peptide and proteins. However, its removal is not straightforward and requires harsh reaction conditions and additional purification steps before and after the removal step, which extends the synthetic process and reduces the overall yield. To overcome these shortcomings, a method for rapid and efficient Acm removal using Pd^II complexes in aqueous medium is reported. We show, for the first time, the assembly of three peptide fragments in a one‐pot fashion by native chemical ligation where the Acm moiety was used to protect the N‐terminal Cys of the middle fragment. Importantly, an efficient synthesis of the ubiquitin‐like protein UBL‐5, which contains two native Cys residues, was accomplished through the one‐pot operation of three key steps, namely ligation, desulfurization, and Acm deprotection, highlighting the great utility of the new approach in protein synthesis.     

Sugar-assisted glycopeptide ligation

The chemical synthesis of glycopeptides and glycoproteins from readily available materials presents an attractive route to homogeneous products for structural and functional studies. Chemical synthesis of glycopeptides and glycoproteins based on native chemical ligation represents one of the useful methods for the synthesis of natural glycopeptide structures. Here we describe a method that allows for the synthesis of glycopeptides from cysteine-free peptides. This method utilizes a peptide thioester and a glycopeptide in which the sugar moiety is modified with a thiol handle at the C-2 position. Upon completion of the ligation reaction, the thiol handle can be reduced with H2/metal to the acetamide moiety, furnishing the unmodified glycopeptides. Together, this sequence of reactions displays an attractive potential in glycopeptides and glycoproteins synthesis.     

Extended sugar-assisted glycopeptide ligations: development, scope, and applications

Recently, we reported the development of sugar-assisted ligation (SAL), a novel peptide ligation method for the synthesis of glycopeptides. After screening a large number of glycoprotein sequences in a glycoprotein database, it became evident that a large proportion (approximately 53%) of O-glycosylation sites contain amino acid residues that will not undergo SAL reactions. To overcome these inherent limitations and broaden the scope of the method we report here the development of an extended SAL method. Glycopeptides containing up to six amino acid extensions N-terminal to the glycosylated residue were shown to facilitate ligation reactions with peptide thioesters, and these products were isolated in good yields. Kinetic analysis was used to show that as glycopeptides were extended by further amino acid residues, ligation reactions became slower. This finding was rationalized by molecular dynamics simulations using AMBER9. These studies suggested a general trend whereby the proximal distance between the reactive sites of the thioester intermediate (the N-terminal amine and the carbonyl carbon of the thioester) increased as glycopeptides were extended, thus slowing down the ligation rate. Each of the extended SAL methods showed broad tolerance to a number of different amino acid combinations at the ligation junction. Re-evaluation of the glycoprotein database suggested that 95% of the O-linked glycosylation sites can now be utilized to facilitate SAL or extended SAL reactions. As such, this method represents an extremely valuable tool for the synthesis of naturally occurring glycopeptides and glycoproteins. To demonstrate the applicability of the method, extended SAL was successfully implemented in the synthesis of the starting unit of the cancer-associated MUC1 glycoprotein.     

Sugar-assisted ligation of N-linked glycopeptides with broad sequence tolerance at the ligation junction

A novel method for the synthesis of N-linked glycopeptides using the sugar-assisted ligation strategy from cysteine free peptides is presented. The ligation junction tolerates a variety of amino acids, favoring less hindered amino acids and those with side chains that could serve as a general base in the ligation pathway. Since our approach allows the ligation of difficult junctions, the method could be applied to the synthesis of large peptides by enzymatic removal of the sugar moiety. Alternatively, more complex glycopeptides can be synthesized using glycosyltransferases. Together, this sequence of reactions should be amenable to the synthesis of glycopeptides and glycoproteins and their deglycosylated products.     

Sugar-assisted ligation for the synthesis of glycopeptides

Chemical synthesis of glycoproteins from readily available materials is a powerful method for obtaining a pure product with full control of its atomic structure. Sugar-assisted ligation (SAL) is an emerging approach that allows the synthesis of a large glycopeptide from two unprotected fragments. Contrary to other ligation methods that are limited to the use of a cysteine residue or depend on external auxiliary, SAL takes advantage of the existing sugars in glycopeptides to promote proximity between the two peptides to facilitate an amide bond formation.     

Sugar-assisted glycopeptide ligation with complex oligosaccharides: scope and limitations

We have previously shown sugar-assisted ligation (SAL) to be a useful method for the convergent construction of glycopeptides. However to date SAL has only been carried out on systems where the thiol auxiliary is attached to a monosaccharide. For SAL to be truly applicable to the construction of fully elaborated glycopeptides and glycoproteins, it must be possible to carry out the reaction when the thiol auxiliary is attached to more elaborate sugars, as these are frequently what are observed in nature. Here we examine the effects of glycosylation at C-3, C-4, and C-6 of the C-2 auxiliary-containing glycan. Model glycopeptides where synthesized chemoenzymatically and reacted with peptide thioesters used in our previous work. These studies reveal that SAL is sensitive to extended glycosylation on the auxiliary-containing sugar. While it is possible to carry out SAL with extended glycosylation at C-4 and C-6, the presence of glycosylation at C-3 prevents the ligation from occurring. Additionally, with glycosylation at C-4 the ligation efficiency is affected by the identity of the N-terminal AA, while the nature of the C-terminal residue of the peptide thioester does not appear to affect ligation efficiency. These studies provide useful guidelines in deciding when it is appropriate to use SAL in the synthesis of complex glycopeptides and glycoproteins and how to choose ligation junctions for optimal yield.     

Efficient Chemical Protein Synthesis using Fmoc‐Masked N‐Terminal Cysteine in Peptide Thioester Segments

We report an operationally simple method to facilitate chemical protein synthesis by fully convergent and one‐pot native chemical ligations utilizing the fluorenylmethyloxycarbonyl (Fmoc) moiety as an N‐masking group of the N‐terminal cysteine of the middle peptide thioester segment(s). The Fmoc group is stable to the harsh oxidative conditions frequently used to generate peptide thioesters from peptide hydrazide or o‐aminoanilide. The ready availability of Fmoc‐Cys(Trt)‐OH, which is routinely used in Fmoc solid‐phase peptide synthesis, where the Fmoc group is pre‐installed on cysteine residue, minimizes additional steps required for the temporary protection of the N‐terminal cysteinyl peptides. The Fmoc group is readily removed after ligation by short exposure (<7 min) to 20 % piperidine at pH 11 in aqueous conditions at room temperature. Subsequent native chemical ligation reactions can be performed in presence of piperidine in the same solution at pH 7.     

A novel ring opening reaction of peptide N-terminal thiazolidine with 2,2′-dipyridyl disulfide (DPDS) efficient for protein chemical synthesis

In the protein chemical synthesis via native chemical ligation (NCL) method with three peptide segments, the N-terminal cysteine residue of middle segment is generally protected by thiazolidine ring. In this paper, we show the novel method for thiazolidine ring opening using 2,2′-dipyridyl disulfide (DPDS). The N-terminal thiazolidine was converted into S-pyridylsulfenylated cysteine residue with DPDS under acidic conditions, and this N-terminally Cys peptide protected with disulfide was applicable to NCL reaction without purification and deprotection steps. DPDS treatment did not remove other Cys protecting groups generally used for regioselective disulfide bond formation reactions. These results indicate that this thiazolidine ring opening reaction is quite useful for the protein chemical synthesis with three-segment NCL strategy.     

Selective desulfurization of cysteine in the presence of Cys(Acm) in polypeptides obtained by native chemical ligation

Increased versatility for the synthesis of proteins and peptides by native chemical ligation requires the ability to ligate at positions other than Cys. Here, we report that Raney nickel can be used under standard conditions for the selective desulfurization of Cys in the presence of Cys(Acm). This simple and practical tactic enables the more common Xaa-Ala junctions to be used as ligation sites for the chemical synthesis of Cys-containing peptides and proteins. [reaction: see text].     

Synthetic Glycoforms Reveal Carbohydrate‐Dependent Bioactivity of Human Saposin D

The main glycoforms of the hydrophobic lysosomal glycoprotein saposin D (SapD) were synthesized by native chemical ligation. An approach for the challenging solid‐phase synthesis of the fragments was developed. Three SapD glycoforms were obtained following a general and robust refolding and purification protocol. A crystal structure of one glycoform confirmed its native structure and disulfide pattern. Functional assays revealed that the lipid‐binding properties of three SapD glycoforms are highly affected by the single sugar moiety of SapD showing a dependency of the size and the type of N‐glycan.     

Chemical synthesis of homogeneous human glycosyl-interferon-β that exhibits potent antitumor activity in vivo

Chemical synthesis of homogeneous human glycoproteins exhibiting bioactivity in vivo has been a challenging task. In an effort to overcome this long-standing problem, we selected interferon-β and examined its synthesis. The 166 residue polypeptide chain of interferon-β was prepared by covalent condensation of two synthetic peptide segments and a glycosylated synthetic peptide bearing a complex-type glycan of biological origin. The peptides were covalently condensed by native chemical ligation. Selective desulfurization followed by deprotection of the two Cys(Acm) residues gave the target full-length polypeptide chain of interferon-β bearing either a complex-type sialyl biantennary oligosaccharide or its asialo form. Subsequent folding with concomitant formation of the native disulfide bond afforded correctly folded homogeneous glycosyl-interferon-β. The chemically synthesized sialyl interferon-β exhibited potent antitumor activity in vivo.     

Palladium‐Mediated Direct Disulfide Bond Formation in Proteins Containing S‐Acetamidomethyl‐cysteine under Aqueous Conditions

One of the applied synthetic strategies for correct disulfide bond formation relies on the use of orthogonal Cys protecting groups. This approach requires purification before and after the deprotection steps, which prolongs the entire synthetic process and lowers the yield of the reaction. A major challenge in using this approach is to be able to apply one‐pot synthesis under mild conditions and aqueous media. In this study, we report the development of an approach for rapid disulfide bond formation by employing palladium chemistry and S‐acetamidomethyl‐cysteine [Cys(Acm)]. Oxidation of Cys(Acm) to the corresponding disulfide bond is achieved within minutes in a one‐pot operation by applying palladium and diethyldithiocarbamate. The utility of this reaction was demonstrated by the synthesis of the peptide oxytocin and the first total chemical synthesis of the protein thioredoxin‐1. Our investigation revealed a critical role of the Acm protecting group in the disulfide bond formation, apparently due to the generation of a disulfiram in the reaction pathway, which significantly assists the oxidation step.     

Chemical synthesis of a glycoprotein having an intact human complex-type sialyloligosaccharide under the Boc and Fmoc synthetic strategies

The chemical synthesis of complex glycoproteins is an ongoing challenge in protein chemistry. We have examined the synthesis of a single glycoform of monocyte chemotactic protein-3 (MCP-3), a CC-chemokine that consists of 76 amino acids and one N-glycosylation site. A three-segment native chemical ligation strategy was employed using unprotected peptides and glycopeptide. Importantly, the synthesis required the development of methods for the generation of sialylglycopeptide-alphathioesters. For the sialylglycopeptide-alphathioester segment, we examined and successfully implemented approaches using Fmoc-SPPS and Boc-SPPS. To avoid use of hydrogen fluoride, the Boc approach utilized minimal side chain protection and direct thiolysis of the resin bound peptide. Using these strategies, we successfully synthesized a glycoprotein having an intact and homogeneous complex-type sialyloligosaccharide.     

Oxidative Deselenization of Selenocysteine: Applications for Programmed Ligation at Serine

Despite the unique chemical properties of selenocysteine (Sec), ligation at Sec is an under‐utilized methodology for protein synthesis. We describe herein an unprecedented protocol for the conversion of Sec to serine (Ser) in a single, high‐yielding step. When coupled with ligation at Sec, this transformation provides a new approach to programmed ligations at Ser residues. This new reaction is compatible with a wide range of functionality, including the presence of unprotected amino acid side chains and appended glycans. The utility of the methodology is demonstrated in the rapid synthesis of complex glycopeptide fragments of the epithelial glycoproteins MUC5AC and MUC4 and through the total synthesis of the structured, cysteine (Cys)‐free protein eglin C.     

Efficient Chemical Protein Synthesis using Fmoc-Masked N-Terminal Cysteine in Peptide Thioester Segments

We report an operationally simple method to facilitate chemical protein synthesis by fully convergent and one-pot native chemical ligations utilizing the fluorenylmethyloxycarbonyl (Fmoc) moiety as an N-masking group of the N-terminal cysteine of the middle peptide thioester segment(s). The Fmoc group is stable to the harsh oxidative conditions frequently used to generate peptide thioesters from peptide hydrazide or o-aminoanilide. The ready availability of Fmoc-Cys(Trt)-OH, which is routinely used in Fmoc solid-phase peptide synthesis, where the Fmoc group is pre-installed on cysteine residue, minimizes additional steps required for the temporary protection of the N-terminal cysteinyl peptides. The Fmoc group is readily removed after ligation by short exposure (<7 min) to 20 % piperidine at pH 11 in aqueous conditions at room temperature. Subsequent native chemical ligation reactions can be performed in presence of piperidine in the same solution at pH 7.     

Chemistry and glycobiology

Current interests in glycobiology have stimulated the development of new tools for use to tackle major problems in the field, including, for example, glycoprotein synthesis, glycan array development and post-translational glycosylation monitoring. Recent advances in the synthesis of glycoproteins involve glycoprotein remodelling, native chemical ligation (NCL), expressed protein ligation (EPL), Staudinger ligation, sugar-assisted ligation and pathway engineering to effectively produce homogeneous glycoproteins with well defined glycans for structural and functional studies. Moreover, the development of glycan synthesis, such as one-pot, chemoenzymatic and solid-supported syntheses, has greatly simplified the process in creating various glycans for functional and array study. Glycan array requires little sample and is able to test and compare many carbohydrate-protein interactions simultaneously. Finally, the changes in post-translational glycosylation, which is an indicator of disease progression, can be monitored by bioorthogonal chemical reporters with the cell's metabolic machinery. The interdisciplinary cooperation in chemistry and biology has yielded new strategies and led to an explosion of research in this field.     

A route to cyclic peptides and glycopeptides by native chemical ligation using in situ derived thioesters

The synthesis of cyclic peptides and glycopeptides by native chemical ligation using in situ derived thioesters is described.     

Advances in glycoprotein synthesis

The development of chemical and enzymatic methods for the synthesis of homogeneous glycoproteins is a fascinating challenge at the interface between chemistry and biology. Discussed here are the currently available methods for preparation of homogeneous glycoproteins. These methods include (1) glycopeptide ligation; (2) glycoprotein remodeling; and (3) in vivo suppressor tRNA technology.     

Stereoselective N-glycosylation by Staudinger ligation

Stereoselective methods for the chemical synthesis of beta-N-glycosyl amides are needed to generate glycopeptides and glycoproteins. Here, we report that the Staudinger ligation can be used to form glycosylated asparagine derivatives. The reaction proceeds with high stereoselectivity, and a variety of glycosyl azides can function as substrates. Our results provide precedence for the use of this powerful amide-bond-forming reaction for N-glycopeptide synthesis. [reaction: see text]     

Recent Development in Ligation Methods for Glycopeptide and Glycoprotein Synthesis

Glycoproteins are produced by the post‐translational modification process of proteins and they play an important role in mediating various biological processes. Our understanding towards biochemical functions of individual glycoproteins has been seriously hampered due to the heterogeneous expression of carbohydrate parts in glycoproteins. Despite the advancement in recombinant expression and chromatographic techniques, the isolation of pure glycoforms remains nearly impossible. To obtain homogenous glycoproteins, tremendous efforts hves been spent in developing various ligation and glycosylation techniques. This minireview discusses selected methods for the preparation and ligation of glycopeptides. The importance of the development of new chemical synthesis method for glycoproteins has also been discussed, which would be one of the next directions in this field.