Ryo Okamoto

Uncovering a latent ligation site for glycopeptide synthesis.

Glycosylation, one of the most important posttranslational modifications, plays an important role in a variety of biological events. Oligosaccharides on glycoproteins exhibit structural heterogeneity, which makes it difficult to elucidate the relationship between the oligosaccharide structure and the function of the glycoprotein. Chemical synthesis is one of the powerful approaches for obtaining homogeneous glycoproteins. We have already reported the synthesis of a glycoprotein with a homogeneous N-linked complex-type oligosaccharide. This synthesis employed native chemical ligation (NCL) to perform peptide-segment coupling. NCL relies on the thiol-exchange reaction between a peptide with an a-thioester group at the C terminus and another peptide with a cysteine residue at the N terminus and on the subsequent intramolecular acyl transfer. However, occasionally, the cysteine residue is not properly located or does not exist in the target protein. To take this potential difficulty into consideration, a long glycopeptide sequence that is 30–50 amino acids from one cysteine site to another cysteine site occasionally needs to be synthesized for glycoprotein synthesis by the NCL method. The synthesis of such a glycopeptide with an N-linked glycopeptide is not easy to perform and requires an appropriate amount of N-linked complex-type oligosaccharides; therefore, there is greater difficulty in glycoprotein synthesis than in simple protein synthesis. To examine NCL without a cysteine residue in a long target peptide, reduction methods changing the sulfhydryl group of cysteine to a hydrogen atom after NCL and utilizing an auxiliary group have been developed. In the latter method, the amino acid sequence at the ligation site is limited for performance. For the development of a widely usable method in glycopeptide synthesis, we have also explored suitable NCL approaches; this endeavor enabled us to find a new ligation position at the serine site in the consensus sequence NXS (X: any amino acid except for proline), by which an asparagine residue is generally incorporated in an Nlinked oligosaccharide. This sequence is found in glycoproteins along with the NXT sequence. In order to use the serine site for a new NCL, we have examined the new concept and attempted the conversion of a cysteine residue into a serine residue after NCL. For such a technique, it was necessary to explore concise reaction sequences. As a result, we found the possibility of using a CNBr cleavage method at a methylcysteine site, which could be obtained by specific methylation of cysteine. Herein, we report a new chemical ligation approach at serine sites, which relies on the conversion of a cysteine residue into a serine residue after NCL. The strategy is shown in Scheme 1. After NCL (product A), the conversion of cysteine into serine was performed by the following reactions: S methylation of cysteine with methyl 4-nitrobenzenesulfonate (product B) and intramolecular rearrangement by activation with CNBr in 80% HCOOH solution followed by an Oto N-acyl shift. Activation of the S-methyl group by CNBr results in intramolecular attack by the neighboring carbonyl oxygen atom on the b-carbon atom of the methylcysteine residue and generates an O-ester peptide intermediate (product C). This intermediate can be converted into the desired peptide (productD) through the Oto N-acyl shift under slightly basic conditions (pH 7–8). In order to examine this strategy, we first demonstrated the utility of the reaction by means of a model tetrapeptide with a cysteine residue (Table 1, entry 1). As shown in entry 1 in Table 1, in the case of tetrapeptide Ac-ACGL-OH, we could achieve the conversion of cysteine into serine in moderate yield. To confirm the optical purity of the peptide thus prepared, we compared it with authentic peptide samples containing d-amino acids, such as Ac-ASGL-OH, AcASGL-OH, Ac-ASGL-OH, and Ac-ASGL-OH, by HPLC and NMR analysis (Figure 1 and the Supporting Information). These results showed that product 2 is identical to Ac-ASGL-OH and the conversion method did not cause any epimerization in the peptide. We also examined this method by using octaand undecapeptides (Table 1, entries 2 and 3), and each of the conversion reactions was found to afford the desired peptides in moderate yield. It is known that CNBr has also been used for cleavage at the methionine site in proteins. In order to distinguish methionine from methylcysteine residues, we introduced methionine in the sulfoxide form. Due to the fact that the sulfoxide form of methionine is inactive for the CNBr reaction, we expected that an oxidation/reduction protocol would enable us to use this new approach for the synthesis of peptides with methionine residues. We examined the strategy by using pentapeptide 7, which contained cysteine and the sulfoxide form of methionine. S methylation of this pentapeptide afforded 9. Conversion of S-methylcysteine to a serine residue and subsequent reduction of the sulfoxide group by NH4I, SMe2, and trifluoroacetic acid (TFA) were performed as a one-pot reaction and afforded the desired pentapeptide 8 in good yield (73% yield of [*] R. Okamoto, Prof. Dr. Y. Kajihara International Graduate School of Arts and Sciences Yokohama City University 22-2, Seto, Kanazawa-ku, Yokohama, 236-0027 (Japan) Fax: (+81)45-787-2413 E-mail: kajihara@yokohama-cu.ac.jp

(Quasi-)Racemic X-ray Structures of Glycosylated and Non-Glycosylated Forms of the Chemokine Ser-CCL1 Prepared by Total Chemical Synthesis

Our goal was to obtain the X-ray crystal structure of the glycosylated chemokine Ser-CCL1. Glycoproteins can be hard to crystallize because of the heterogeneity of the oligosaccharide (glycan) moiety. We used glycosylated Ser-CCL1 that had been prepared by total chemical synthesis as a homogeneous compound containing an N-linked asialo biantennary nonasaccharide glycan moiety of defined covalent structure. Facile crystal formation occurred from a quasi-racemic mixture consisting of glycosylated L-protein and non-glycosylated-D-protein, while no crystals were obtained from the glycosylated L-protein alone. The structure was solved at a resolution of 2.6-2.1u2005Å. However, the glycan moiety was disordered: only the N-linked GlcNAc sugar was well-defined in the electron density map. A racemic mixture of the protein enantiomers L-Ser-CCL1 and D-Ser-CCL1 was also crystallized, and the structure of the true racemate was solved at a resolution of 2.7-2.15u2005Å. Superimposition of the structures of the protein moieties of L-Ser-CCL1 and glycosylated-L-Ser-CCL1 revealed there was no significant alteration of the protein structure by N-glycosylation.

Total Chemical Synthesis and Biological Activities of Glycosylated and Non-Glycosylated Forms of the Chemokines CCL1 and Ser-CCL1†

CCL1 is a naturally glycosylated chemokine protein that is secreted by activated T-cells and acts as a chemoattractant for monocytes. Originally, CCL1 was identified as a 73 amino acid protein having one N-glycosylation site, and a variant 74 residue non-glycosylated form, Ser-CCL1, has also been described. There are no systematic studies of the effect of glycosylation on the biological activities of either CCL1 or Ser-CCL1. Here we report the total chemical syntheses of both N-glycosylated and non-glycosylated forms of (Ser-)CCL1, by convergent native chemical ligation. We used an N-glycan isolated from hen egg yolk together with the Nbz linker for Fmoc chemistry solid phase synthesis of the glycopeptide-(α) thioester building block. Chemotaxis assays of these glycoproteins and the corresponding non-glycosylated proteins were carried out. The results were correlated with the chemical structures of the (glyco)protein molecules. To the best of our knowledge, these are the first investigations of the effect of glycosylation on the chemotactic activity of the chemokine (Ser-)CCL1 using homogeneous N-glycosylated protein molecules of defined covalent structure.

Chemical synthesis of homogeneous glycopeptides and glycoproteins.

Oligosaccharides linked to proteins are known to play important roles in several biological events. However, oligosaccharides are heterogeneous, which has hindered detailed elucidation of oligosaccharide functions. In order to solve this problem, glycoproteins having homogeneous oligosaccharides have long been required. For this purpose, an efficient preparative method of complex-type oligosaccharides has been investigated from a natural source and this method was found to afford over 24 kinds of diverse complex-type oligosaccharides by use of chemical methods and branch-specific sequential glycosidase digestion. The sufficient amount of homogeneous complex type oligosaccharides obtained enabled us to examine the synthesis of homogeneous glycopeptides as well as glycoproteins by use of solid phase glycopeptide synthetic method and native chemical ligation. This review describes recent progress related to the efficient method of oligosaccharide preparation and synthesis of glycoproteins including bioactive erythropoietin.

Unique Self-Anhydride Formation in the Degradation of Cytidine-5′-monophosphosialic Acid (CMP-Neu5Ac) and Cytidine-5′-diphosphosialic Acid (CDP-Neu5Ac) and its Application in CMP-sialic Acid Analogue Synthesis

Sialyloligosaccharides are synthesised by various glycosyltransferases and sugar nucleotides. All of these nucleotides are diphosphate compounds except for cytidine-5-monophosphosialic acid (CMP-Neu5Ac). To obtain an insight into why cytidine-5-diphosphosialic acid (CDP-Neu5Ac) has not been used for the sialyltransferase reaction and why it is not found in biological organisms, the compound was synthesised. This synthesis provided the interesting finding that the carboxylic acid moiety of the sialic acid attacks the attached phosphate group. This interaction yields an activated anhydride between carboxylic acid and the phosphate group and leads to hydrolysis of the pyrophosphate linkage. The mechanism was demonstrated by stable isotope-labelling experiments. This finding suggested that CMP-Neu5Ac might also form the corresponding anhydride structure between carboxylic acid and phosphate, and this seems to be the reason why CMP-Neu5Ac is acid labile in relation to other sugar nucleotides. To confirm the role of the carboxylic acid, CMP-Neu5Ac derivatives in which the carboxylic acid moiety in the sialic acid was substituted with amide or ester groups were synthesised. These analogues clearly exhibited resistance to acid hydrolysis. This result indicated that the carboxylic acid of Neu5Ac is associated with its stability in solution. This finding also enabled the development of a novel chemical synthetic method for CMP-Neu5Ac and CMP-sialic acid derivatives.