• glycopathology

Microwave Application for Chemical Synthesis of Glycopeptide

illustration

Glycopeptides are molecules having glycans and peptide chains, which play an important role in biological functions and pharmacological actions. Since most of animal functional proteins are glycosylated, these substructures have attracted much attention in the fields of vaccines, anticancer drugs, and diagnostic agents. In nature, glycans on glycoproteins are not homogeneous, and it is difficult to separate and purify them.

Therefore, it is not easy to obtain a glycopeptide with a single glycan chain from nature. It is then necessary to synthesize glycopeptides which required combination of two different synthetic methods, peptide synthesis and glycosylation. For example, peptide chains are synthesized by peptide synthesis method, while sugar moieties are chemically synthesized on the amino acid derivatives in advance. Chemoenzymatic synthesis, where monosaccharide peptides are chemically synthesized followed by enzymatic elongation and rearrangement of the sugar chains, is an effective method. Here, an overview of the current status of microwave-assisted solid-phase synthesis of glycopeptides is described with an emphasis on the synthesis of glycopeptides in an automated peptide synthesizer.

Automated synthesizers based on solid-phase synthesis are commercially available for peptide synthesis. Solid-phase peptide synthesis is a synthetic method in which peptides are obtained by stretching amino acids one by one onto a solid phase (resin), and excess reagents and by-products derived from reagents can be removed by simple washing of the solid phase. After the syntheses, the target peptide on the solid phase is excised and purified by HPLC to obtain the pure product. Since separation and purification are not performed after each reaction, a “reaction yield close to 100%” is required at each step. If we intend to synthesize a thirty amino acid residual peptide and a conjugation of one amino acid can be carried out with 90% yield, we will perform 29 times conjugation to obtain the peptide and then the total yield will be only 4% as 0.90 times 29. If a yield of elongation of one amino acid is achieved at 96% yield, then the total yield will be 31%. A large excess amount (e.g., three to ten equivalents) of amino acid synthons, therefore, is generally used for amino acid elongation steps. In addition, microwave has recently been used to improve yields, and equipment is commercially available (CEM, Biotage, etc.). In these devices, microwave irradiation rapidly heats to about 100℃ to complete reactions in a few minutes, resulting in high yields, shortening the total synthesis time, and obtaining long-chain peptides. About forty residual peptide can be synthesized by an automated synthesizer. However, the high temperature may induce side reactions for introduction of His or Cys, use of certain protecting groups, depending on sequence and so on, in which case it is recommended to limit the reaction temperature up to about 50℃.

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Fig.1. Concept of solid phase synthesis of peptides with Fmoc strategy
1) deprotection of the Fmoc groups of the amino acids on the resin and 2) conjugation of the next Fmoc-protected amino acid or glycoamino acid. After synthesis of the desired glycopeptide on the resin, the product is separated from the resin and amino acid side chain protecting groups are removed (typically, the synthetic scheme is designed to perform both reactions with an acid, such as TFA) followed by deprotection of the hydroxy groups of the sugars (treatment with base such as NaOMe to deprotect acetyl groups). AA0,...AAz: amino acids. PG: protecting group for the side chain of amino acids.

Theoretically, glycopeptide synthesis can be achieved by using glyco-amino acid synthons, but practically, it is not easy because glyco-amino acid synthons have multifunctional group and are unstable under heating, acids and bases. In addition, commercially available glyco-amino acid synthons are expensive (Fmoc-Ser[GalNAc(Ac)3-α-ᴅ]-OH is commercially available from Sigma-Aldrich for $1,070.00 as of Dec. 2024). For example, in the case of 0.1 mmol scale synthesis, 10 equivalents is 1 mmol, which is 656 mg of Fmoc-Ser[GalNAc(Ac)3-α-ᴅ]-OH synthon. Then, one million yen is required for only one residue of glycol-amino acid extension. Thus, the cost of synthesizing glycopeptides with multiple sugars or oligosaccharides using an automated synthesizer would be enormous.

Matsushita et al. achieved the synthesis of a twenty amino acid residual glycopeptide, a MUC1 repeating unit with five of trisaccharides, by heating the reaction temperatures to 50℃ by microwave irradiation. They compared three reaction conditions, namely at room temperature (25℃), at 50℃ by conventional heating and at 50℃ by microwave, then microwave heating at 50℃ gave the best results, total yield 44% and total reaction time 7 hours. The synthesis under totally same conditions with the case of using microwave but heated by conventional heating at 50℃, the total reaction time was indeed 7 hours but the total yield was only 15%. They discussed that these results indicated some kind of microwave unique effect (1,2). Despite their discussion, the data obtained might not be to reflect the microwave effect because their experiments were not performed on the same scale and with the same equipment. Ohki et al. developed a microwave synthesizer with a cooling unit that could investigate the microwave effect (3). Yokoe et al. demonstrated the synthesis of a five amino acid residual peptide. The overall yield was increased under microwave irradiation and they also reported that only 1.3 equivalents of glyco-amino acid synthons, which are usually used in large excess, were required (3). Thus, the advantages of microwave irradiation are not only to obtain high yields but also to reduce of the essential amount of reagents.

Shimizu et al. have successfully synthesized complex glycopeptides containing galactosamine monosaccharide, Tn antigen, Sia-Gal-GalNAc trisaccharide, ST antigen, and Sia-Gal-(Sia)GalNAc tetrasaccharide, diSiaT antigen and they are now developing drug discovery research (unpublished). Hojo et al. introduced benzyl-protected diSiaT tetrasaccharide amino acid at room temperature, and performed further extension for a few residues by microwave heating to 90℃ (4). Although sialic acid-containing glyco-amino acid synthons are considered to be extremely unstable, they are relatively heat stable when incorporated into peptide chains.

Although the mechanism and details of how microwave can provide non-thermal effects are still under study, Nagashima et al. investigated the microwave effect by changing the frequency of irradiated microwaves for enzymatic hydrolysis (5). Microwave heating is mainly caused by the dielectric effect of microwaves on water molecules which are dipole molecules, but they discussed that this enzymatic reaction was accelerated by the conductive effect, i.e. microwaves selectively affected ions. They discussed that one of the microwave effects was the molecular/ion selective affection. The author also considers that the contribution of microwaves to hydrogen bonding networks for substrates and/or enzymes may also produce the microwave effect.

Some of the academic reports for glycopeptide syntheses have tended to be qualitative or at the sub-mg level due to the difficulty in obtaining reagents, and there have been very few reports discussing details of reaction conditions and stability studies. On the other hand, the need for glycopeptide synthesis is expected to increase in the research fields of protein science and glycoscience, as well as in drug discovery. It has become clear that the use of microwaves, even at low temperatures, offers advantages, and the synthesis of O-glycan glycopeptides such as the diSiaT series, which are complex and difficult to synthesize, is now becoming more feasible. Microwaves are expected to be one of the ways to break through this situation.


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Fig. 2. Effect of microwave irradiation for glycopeptide synthesis
Microwave irradiation provides advantages such as reaction activation and reduction of reagents required even at low temperatures, which enables the synthesis of complex glycopeptides.

Hiroki Shimizu
(Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST))

References
(1) Matsushita T, Hinou H, Kurogochi M, Shimizu H, Nishimura SI: Rapid Microwave-Assisted Solid-Phase Glycopeptide Synthesis. Org. Lett. 7, 877-880, 2005
(2) Matsushita T, Hinou H, Fumoto M, Kurogochi M, Fujitani N, Shimizu H, Nishimura SI: Construction of Highly Glycosylated Mucin-Type Glycopeptides Based on Microwave-Assisted Solid-Phase Syntheses and Enzymatic Modifications. J. Org. Chem. 71, 3051-3063, 2006
(3) Yokoe T, Ohki Y, Nagashima I, Takahashi N, Shimizu H: Study of Glycopeptide Synthesis under Microwave Irradiation at Low Temperatures towards Automated Synthesiser. JEMEA J. 3, 32-39, 2019
(4) Nagashima K, Ito S, Takei T, Takao T, Kiyozumi D, Hojo H: Synthesis and Structural Analysis of NICOL with O-Linked Glycosylation. The 61st Japanese Peptide Society, P-023, 2024
(5) Nagashima I, Sugiyama Ji, Shimizu H: Study of 400 MHz microwave conduction loss effect for a hydrolysis reaction by thermostable β-Glucosidase HT1. Biosci. Biotechnol. Biochem. 87, 158-162, 2023

Mar. 3, 2025

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