Studies in glycopeptide synthesis

This short report describes the preliminary results of the synthesis of glycopeptides bearing complex type biantennary sialylglycans. Using the latest efficient auxiliary for peptide-peptide coupling reactions, we successfully synthesized glycopeptides.


Introduction
Carbohydrates and their oligomers, glycans, play important roles in many biological events such as cell-cell interactions, immune system regulation, and protein activity stabilization. 1 Glycans are roughly divided into three groups, as glycoproteins, glycolipids, and glucosaminoglycans.Glycoproteins have asparagine-linked N-glycans and threonine-or serine-linked O-glycans. 2The N-linked glycans are further divided into three subgroups such as complex type, high-mannose type, and hybrid type.The biosynthesis of glycoproteins begins in the endoplasmic reticulum, where high-mannose type glycans are attached to the proteins.Depending on their maturation processes in the second organelle (Golgi apparatus), high-mannose type glycans are converted into complex type sialylglycans.Hybrid type glycans possess parts of both the high-mannose type and complex-type in a glycan structure.
However, glycan sequences made in the biosynthetic pathway are not regulated by the gene.Enzymes such as glycosyltransferases and glycosidases regulate the construction of glycan structures.However, the enzymatic regulation results in considerable heterogeneity in the glycan structure. 3,4Therefore, glycans on the cell surface and in body fluid show considerable structural diversity, and therefore, we could not identify which glycans are essential for the individual biological events.
In addition to this, a small amount of homogeneous glycans can be isolated from a natural source, but this amount is not enough for extensive investigation of glycobiology.As a result, the study of glycan functions has been hindered.][7][8][9][10][11][12][13] In particular, the Kunz group has extensively studied the synthesis of glycopeptides over a long period of time. 14They efficiently synthesized Fmoc-protected sugar amino acid for solid phase glycopeptide synthesis 15 and studied protecting group such as allyloxycarbonyl group 16 and 3-(3-pyridyI)allyloxycarbonyl group. 17Furthermore, they developed a special linker, Hycron linker, which detaches glycopeptides from resins under the neutral conditions. 18This efficient system yielded acid-labile glycopeptides.
Our group has also studied glycopeptide synthesis (Figure 1).However, the synthesis of oligosaccharides was difficult for us to provide a suitable amount for SPPS and therefore our group employed a semi-synthetic method of N-glycans.Fortunately, we isolated a homogeneous complex type biantennary N-linked sialylglycan from egg yolk. 19After peptidase digestion, asparagine was protected with an Fmoc group, and two carboxylic acids of sialic acids were efficiently protected with benzyl esters to give Fmoc-Asn-(glycan)-OH 1.We then used 1 for glycopeptide synthesis (Figure 1). 20We performed SPPS toward PEGA resin having acidlabile linker and then added Fmoc-Asn-(glycan)-OH 1.After glycopeptide synthesis was complete, the target glycopeptide 2 was detached from the resins by acid-cleavage.We found that the sialic acid groups were stable even under strongly acidic conditions due to the benzylesterification.However, this SPPS method has a drawback.Since we could not use an excess amount of the valuable Fmoc-Asn-(glycan)-OH 1, the total synthetic yield from coupling the first amino acid to the resin was not high.In addition to low yields, aspartimide derivative was formed from Fmoc-Asn-(glycan)-OH 1 during the glycopeptide coupling. 21Despite, these disadvantages, we did manage to use this method for practical glycopeptide syntheses.Based on a highly supportive and inspiring discussion with Prof. Kunz at a scientific conference, we began work that could finally be published. 20n the past decade, glycopeptide syntheses has dramatically improved 2,22 and recently, glycopeptide syntheses are used for glycoprotein syntheses. 22These researches use efficient peptide-peptide coupling reactions.Peptide-peptide coupling is an essential reaction and native chemical ligation (NCL) has been used for the synthesis of proteins. 23However NCL requires cysteine residue at the NCL site.Therefore, supplementary methods have been used to provide cysteine surrogates, such as β-mercaptoamino acids and auxiliary systems. 24nder these circumstances, Prof Seitz group developed an excellent auxiliary for peptide-peptide coupling reactions (Figure 2). 25,26We had an interest in whether his robust auxiliary could couple bulky Asn-(glycan)-OH and peptide.This short paper reports the preliminary results of this new reaction.

Synthesis of auxiliary
The Seitz group reported a very efficient auxiliary for peptide coupling. 25,26Therefore, we examined the same auxiliary for glycopeptide synthesis.This aromatic auxiliary bears a thiol instead of a cysteine to assist NCL, but this auxiliary can be removed from peptides via radical reactions.The Seitz group extensively studied the mechanism of the auxiliary detaching from the peptides after NCL-type reactions.However, as the starting material for the synthesis of Seitz's auxiliary was difficult to obtain in Japan, we studied an alternative synthetic route that uses commercially available substrate 3 (Figure 3).Trityl protection toward the primary alcohol of 3 gave 4 in 92% yield.Then, mesylation and substitution with thioacetate yielded compound 5 in 72% yield (2 steps).After the acetate group was removed (yield = 84%), we found that the trityl group of 6 could migrate to the thiol group under acidic conditions, which successfully yielded primary alcohol 7 in 78% yield.Finally, Dess-Martin oxidation yielded the desired auxiliary substrate 8 in 70% yield.

Preparation of Asn-(complex type sialyl glycan)-OH
Because we could prepare the Seitz's auxiliary, next we examined the modification of Asn-glycan 9 20 with the auxiliary (Figure 4).For the modification, we explored various reductive-amination conditions and found that picoline borane conditions were more efficient than NaBH3CN conditions, because glycan dissolves in water, but not in organic solvent.Toward an amino group of Asn-(glycan)-OH 9, we added auxiliary under the reductive amination condition.Extensive optimizations enabled us to obtain the target 10 in moderate yield (55%).
We next examined peptide ligations between model peptides and auxiliary-bearing Asn-(glycan)-OH 11 (Figure 5).The trityl group of the auxiliary could be removed by trifluoroacetic acid/triisopropylsilane (TFA/TIPS) to yield 12. All peptide coupling reactions employed conventional NCL conditions. 23We prepared short three model peptide-thioesters 13-15 by the safe Boc-SPPS. 27These model experiments efficiently yielded ligation products 16-18, which were monitored by reverse phase HPLC.These results proved that Seitz's auxiliary works well for glycopeptide synthesis.
The next step of our glycopeptide synthesis was the coupling reaction of another peptide at the C-terminal of TrtS-auxiliary-Asn-(glycan-Pac)-amino acid 11.We first attempted to couple peptide-Asn-(glycan-Pac)-SR, a glycopeptide-thioester, with another peptide.However, peptide-Asn-(glycan-Pac)-SR formed aspartimide 20 before the coupling reaction could proceed.We could not optimize this reaction to avoid the aspartimide formation; therefore, we studied direct coupling with amino acid-hydrazide 19 as a model compound.We employed low-temperature conditions to avoid unexpected asparagine epimerization and aspartimide formation.This direct coupling was already demonstrated by the Unverzagt group. 28We confirmed that this direct coupling reaction could yield the desired TrtS-auxiliary-Asn-(glycan-Pac)-amino acid-NHNHBoc 21.Here, we used a mono amino acid hydrazide, but we confirmed that long peptide-hydrazides could be coupled at the C-terminal of Fmoc-Asn-(glycan)-OH.
Finally, we confirmed that the trityl group of the auxiliary, as well as the t-Bu and t-Boc groups, were removed by TFA/TIPS or TFA/H2O conditions to give the corresponding product 22 in moderate yield.As the Seitz group already demonstrated the detachment of the auxiliary by radical reactions, our data indicates that Seitz's auxiliary can be used for glycopeptide synthesis as well.

Conclusions
We investigated auxiliary-based glycopeptide synthesis using Seitz's auxiliary for efficient peptide coupling.We are currently optimizing the conditions for this method to be used for practical glycopeptide syntheses.

Experimental Section
General. 1 H and 13 C NMR spectra were recorded on a 400 MHz spectrometer (Bruker Avance III).All 1 H chemical shifts are reported in parts per million (ppm) relative to D2O (4.79 ppm).Mass spectra were recorded on a Bruker Esquire 3000 plus , or an amaZon ETD mass spectrometer.

Figure 4 .
Figure 4. Synthesis of complex type biantennary sialyloligosaccharide bearing the auxiliary.