Letter pubs.acs.org/macroletters
Straightforward Synthesis of N‑Glycan Polymers from Free Glycans via Cyanoxyl Free Radical-Mediated Polymerization Jinshan Tang,† Evgeny Ozhegov,‡ Yang Liu,§ Dan Wang,‡ Xinsheng Yao,† and Xue-Long Sun*,‡ †
Institute of Traditional Chinese Medicine & Natural Products, College of Pharmacy, Jinan University, West 601, Huangpu Avenue, Guangzhou, People’s Republic of China ‡ Department of Chemistry, Chemical and Biomedical Engineering and Center for Gene Regulation in Health and Disease (GRHD), Cleveland State University, 2121 Euclid Avenue, Cleveland, Ohio 44115, United States § Key Laboratory of Structure-Based Drugs Design and Discovery of Ministry of Education, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang, People’s Republic of China S Supporting Information *
ABSTRACT: We report a straightforward synthesis of N-glycan polymers from free glycans via glycosylamine intermediates followed by acrylation and polymerization via cyanoxyl-mediated free radical polymerization (CMFRP) in one-pot fashion. No protection and deprotection were used in either glycomonomer or glycopolymer synthesis. A typical synthetic procedure for N-glycan polymers from free monosaccharide and disaccharide, Glc, Gal, Man, GlcNAc, and Lac, was demonstrated. In addition, enzymatic sialylation of the Laccontaining N-glycan polymers and their anti-influenza virus hemagglutination activities were investigated.
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processing enzymes, antibody profiling, biomarkers, and pathogen and toxin identification applications. Therefore, design and synthesis of glycopolymers has become very important research field, where significant research efforts are highly needed to develop advanced glyco-polymeric architectures with improved performance.4 The precision in the design of synthetic glycopolymer, including different chain compositions, monomer sequences, and architectures, has vital importance when it comes to mimicking the chemical and biological functions of glycoproteins, as well as providing enhanced biological properties. In addition, the glycan attachment to the polymer backbone should be very essential for its performance but has been paid less attention so far. Most glycans were attached to the polymer backbone through O-linked spacer or N-reductive aminationlinked spacer, however, neither is a native glycan-amino acid linkage on glycoproteins, which may be a reason for the lower performance of glycopolymers. The N-glycans are mostly found in natural glycoproteins, where the sugar molecule is attached to a nitrogen atom of asparagine (Asn) residue of a protein. NGlycoside linkage between 2-acetamido-2-deoxy-D-glucopyranosylamine (GlcNAc) and L-Asn is the most commonly found, but D-galactose (Gal) and D-glucose (Glc) attached to Asn with
arbohydrate recognitions are crucial events in many biological processes. For example, cell surface carbohydrates, existing as glycoproteins, glycolipids, or proteoglycans, are involved in cell−cell signaling, immune recognition events, pathogen/host interactions, tumor metastasis, tissue growth and repair as well as other cellular events.1 Therefore, carbohydrate recognition has come to the forefront of biological scientific research aiming to uncover the molecular mechanisms of many physiological and pathological processes. In addition, it provides abundant opportunity to discover potential therapeutic targets or diagnostic mechanisms for various diseases. Consequently, reconstitution of carbohydrate epitopes and mimicry of their authentic compositions and presentations have become important goals in glycoscience and biomedical science. It has been known that the carbohydrate− protein interactions are significantly enhanced with multivalent carbohydrate ligands, referred to as the “cluster glycosidic effect”.2 In the past decades, glycopolymers, namely, polymers with glycan pendant groups, have been extensively explored as multivalent carbohydrate ligands for studying on carbohydrate−protein interactions and for important biomedical applications.3 For example, glycopolymers can act as agonists or antagonists for understanding the molecular mechanisms of many biological processes, and also provide tremendous opportunities for therapeutic applications. In addition, glycopolymers can serve as potential receptors for biochip/biosensor development, which can be used for understanding carbohydrate−protein interaction, substrate specificity of carbohydrate© XXXX American Chemical Society
Received: December 5, 2016 Accepted: January 9, 2017
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DOI: 10.1021/acsmacrolett.6b00928 ACS Macro Lett. 2017, 6, 107−111
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ACS Macro Letters
Scheme 1. Straightforward Synthesis of N-Glycans Polymers from Free Glycans via Glycosylamine Intermediates Followed by Acrylation and Polymerization via Cyanoxyl Free Radical-Mediated Polymerization
Scheme 2. Facile Syntheses of Glycosylamines from Free Sugars via Likhosherstov Method and Their N-Acryloyl Derivatives as Glycomonomers
N-glycosidic linkage were also reported.5 Sugar analogues containing N-glycosidic linkage were widely prepared for glycopeptides and glycoproteins synthesis and functional study.6 In this study, we proposed N-glycan containing polymers that closely mimic the natural N-glycan conjugates. The synthesis of glycopolymers is still challenge since it often requires multistep synthetic steps. A major synthetic route to the glycopolymers starts from the synthesis of glycomonomers with a polymerizable group at the anomeric position of the saccharide followed by polymerization. Alternatively, attaching of glycosyl derivatives to the presynthesized polymer is often used. Protection and deprotection of hydroxyl groups on the saccharide moieties are often necessary in either glycomonomer or glycopolymer synthesis. Therefore, it is often laborious and requires multistep synthesis and purification processes and thus costly. Herein, we report a straightforward synthesis of Nglycan polymers from free saccharides via glycosylamine intermediates followed by acrylation and polymerization via cyanoxyl-mediated free radical polymerization (CMFRP) in one-pot fashion (Scheme 1). No protection and deprotection were used in either glycomonomer or glycopolymer synthesis. A typical synthetic procedure of glycopolymers from free saccharide, Glc, Gal, mannose (Man), GlcNAc, and lactose (Lac) was demonstrated. In addition, enzymatic sialylation of the Lac-containing N-glycan polymers and their anti-influenza virus hemagglutination activities were investigated. Glycosylamines are important compounds in the chemical synthesis of glycoproteins and glycopolymers;7−9 therefore, they are valuable intermediates for the synthesis of N-glycan polymers. Numerous strategies for the synthesis of glycosylamines involving glycosyl azides, glycals, and unprotected reducing oligosaccharides as precursors have been developed.10 Direct amination of unprotected reducing sugar is an easy way to synthesize glycosylamines and thus our choice to synthesize
the glycomonomers. Two direct methods to glycosylamines have been explored so far. First, the Kochetkov method, with the use of a saturated solution of ammonium bicarbonate in ammonium hydroxide for amination of unprotected watersoluble carbohydrates, is widely carried out.11 This strategy was often used for complex carbohydrate glycopeptide syntheses pioneered by Danishefsky.12 However, this method often needs a prolonged and labor-consuming procedure for the evaporation of aqueous solutions to remove considerable amounts of the volatile salt restricts. Moreover, glycosylamines are unstable in aqueous solutions and undergo fast hydrolysis at pH 1.5−9.0 and produce diglycosylamines in concentrated solutions, which often occur upon the isolation of glycosylamines and thus reduce the yield and purity of the resulting glycosylamines. Recently, a practical protocol for the selective amination of unprotected sugar derivatives was introduced by Likhosherstov et al., which used ammonium carbamate in methanol.13 In this method, the carbamic acid salt of glycosylamine formed first, from which the free glycosylamine is easily generated by base treatment or under high vacuum. In our study, both Kochetkov and Likhosherstov method were used to synthesize the glycosylamines (Scheme 2). We found that the Likhosherstov method showed the general applicability for a series of glycosylamines of monosaccharide, Glc, Gal, Man, and GlcNAc, while the Kochetkov method provides a higher yield for Lactoglycosylamine derivatives. In particular, with ammonium carbamate, carbamic acid salt of glycosylamine formed as white precipitates from the reaction solution, which allows easy purification from a crude carbohydrate preparation. In addition, this carbamic salt formation prevents the hydrolysis and glycosylamine-dimer formation. With all glycosylamine salts obtained from free sugars, the N-acryloyl group was introduced by adding acryloyl chloride to the glycosylamine salts in methanol−water and in the presence of Na2CO3 at 0 °C, 108
DOI: 10.1021/acsmacrolett.6b00928 ACS Macro Lett. 2017, 6, 107−111
Letter
ACS Macro Letters
Figure 1. 1H NMR spectrum of β-D-Gal(1−4)-β-D-Glc-N-glycopolymer (N-Lac-P, 4e), α-D-Neu5Ac(2−6)-β-D-Gal(1−4)-β-D-Glc-N-glycopolymer (N-SAα2,6Lac-P, 5), and α-D-Neu5Ac(2-,3)-β-D-Gal(1−4)-β-D-Glc-N-glycopolymer (N-SAα2,3Lac-P, 6) in D2O at 400 MHz. 109
DOI: 10.1021/acsmacrolett.6b00928 ACS Macro Lett. 2017, 6, 107−111
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ACS Macro Letters Scheme 3. Enzymatic Synthesis of Sialyl N-Glycan Glycopolymer from N-Lac Copolymer
Overall, the N-glycan copolymers with acrylamide (AA) were obtained in good conversion yield (around 60%) and low polydispersity (1.6 < Mw/Mn < 3.0). The enzymatic glycosylation was reported as an efficient synthesis of oligosaccharides, without protection and in high anomeric control, in the past decade.20 Enzymatic sialylation was explored to synthesize various sialyloligosaccharides, in which the glycosidic linkage between the sialic acid and the acceptor carbohydrate is extremely controlled by the type of sialyltranferase selected.21 In the present study, the transfer of sialic acid residue from CMP-Neu5Ac to 3- and 6-positions of terminal Gal of N-Lac copolymer (4e), by enzymes α-2,6sialyltransferase and α-2,3-sialyltransferase, were investigated to afford sialylactose-containing N-glycopolymer α2,6SGP (5) and α2,3SGP (6), respectively (Scheme 3). The resultant SGP sialyloligosaccharides were characterized by 1H NMR spectra as well (Figure 1). The successful sialyation of N-Lac copolymer (4e) was confirmed by the signals of protons from Neu5Ac (1.95 ppm, CH3-Neu5NAc and 2.68 ppm, H3eq-Neu5Ac), the degree of sialyation and the polymer length as well were calculated also using the 1H NMR spectra by comparing the integration value of proton signals from aromatic protons (7.31 and 7.16 ppm), anomeric protons (4.98 and 4.40 ppm) of Gal and Glc, C3-equatorial proton (2.70 pm) of Neu5Ac and methyl and methylene protons of polymer backbone as shown in Figure 1. As a result, more than 90% enzymatic sialylation was obtained for both α-2,3sialylation and α-2,6-sialylation of N-Lac copolymer (4e). Sialic acid-terminated oligosaccharides expressed on the respiratory tract epithelial cell surface are involved in influenza virus infection in both virus attaching and detaching processes.22,23 Therefore, synthetic sialic acid-containing oligosaccharides may serve as anti-influenza virus agents as receptor mimetics. Several sialyloligosaccharide-containing glycopolymers have been reported as influenza virus hemagglutinin (HA) receptors and inhibitors of hemagglutination by binding to viral HA.24,25 In the present study, we investigated the binding activities of the sialo-N-glycan copolymers to influenza virus by hemagglutination inhibition (HI) assay (Table 1). As a result, N-SAα2,6Lac polymer (5) and N-
followed by removing excess acryloyl chloride and sodium carbonate to yield N-acryloyl-glycosylamines as glycomonomers for N-glycan polymers synthesis (Scheme 2). Several methods have been developed for the direct synthesis of glycopolymers, including ring opening metathesis polymerization (ROMP),14 cyanoxyl-mediated free radical polymerization (CMFRP),15 atom transfer radical polymerization (ATRP),16 and reversible addition−fragmentation chain transfer (RAFT) polymerization.17 Among these, CMFRP is a straightforward approach for synthesizing glycopolymers in high yield and with low polydispersity (PDI < 1.5).15,18,19 In particular, the polymerization can be conducted in aqueous solution and is tolerant of a broad range of functional groups including −OH, −NH2, −COOH, and SO3− moieties, which excludes protection/deprotection steps often used in other polymerization methods. In the present study, the N-glycan polymers were synthesized via CMFRP scheme in one-pot fashion (Scheme 1), in which, 4-chloroanaline was used as initiator for the copolymerization of N-acryloyl-glycosylamine and acrylamide. Initially, cyanoxyl radicals were generated by an electron-transfer reaction between cyanate anions from a sodium cyanate aqueous solution and aryl-diazonium salts prepared in situ through a diazotization reaction of arylamine in water. In addition to cyanoxyl persistent radicals, aryl-type active radicals were simultaneously produced, and only the latter species was capable of initiating chain growth. The saccharide density and average molecular weight of the resultant glycopolymers were determined by 1H NMR spectrum, in which the integration of protons of the terminal phenyl group and sugar anomeric and polymer backbone were easily assigned and calculated. For example, by comparing the integration value of proton signals from terminal phenyl protons (7.31 and 7.16 ppm), anomeric protons (4.98 and 4.40 ppm) of Gal and Glc (Figure 1A), there are 13 lactose moieties were in the lactosyl N-glycopolymer 4e. Further, by comparing the integration value of proton signals from phenyl protons (7.31 and 7.16 ppm), anomeric protons (4.98 and 4.40 ppm) of Gal and Glc, and methyl (2.20 ppm) and methylene protons (1.60 ppm) of polymer backbone, an average molecular weight of 11,100 was obtained for the lactosyl N-glycopolymer 4e. 110
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Table 1. HI Activities of the N-Glycan Polymers and Fetuin against Human Influenza Viruses compounds 4a, N-glucosyl polymer 4b, N-galactosyl polymer 4c, N-mannosyl polymer 4d, N-GlcNAc polymer 4e, N-lactosyl polymer 5, N-α-2,6-sialolactosyl polymer 6, N-α-2,3-sialolactosyl polymer fetuin
influenza A (H1N1) virus (g/mL)
influenza A (H3N2) virus (g/mL)
1.25 × 10−4 10−3 ND ND ND 6.25 × 10−5
5 × 10−4 ND 3.13 × 10−5 ND ND ND
3.13 × 10−5
ND
1.95 × 10−7
3.13 × 10−5
ACKNOWLEDGMENTS This work was partially supported by a Research Fund from the American Heart Association Grant-in-Aid (14GRANT20290002) and the Center for Gene Regulation in Health and Disease (GRHD) at Cleveland State University, supported by the Ohio Department of Development (ODOD). The authors appreciate the National Science Foundation MRI Grant (CHE-1126384) for the HD 400 NMR spectrometer for acquiring NMR data. J.T. and Y.L. appreciate the China Oversea Scholar Award from China Scholarship Council. The authors appreciate Professor Peng George Wang at Georgia State University for providing α-2,3-sialyltransferase and α-2,6sialyltransferase.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.6b00928.
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REFERENCES
(1) Carbohydrate Recognition, Biological Problems, Methods and Applications; Wang, B., Boons, G.-J., Eds.; Wiley, 2011. (2) Lee, Y. C.; Lee, R. T. Acc. Chem. Res. 1995, 28, 321−327. (3) Macro-Glycoligands: Methods and Protocols (Methods in Molecular Biology); Sun, X.-L., Ed.; Springer, 2015. (4) Narla, S. N.; Nie, H.; Li, Y.; Sun, X.-L. J. Carbohydr. Chem. 2012, 31, 67−92. (5) Dwek, R. A. Chem. Rev. 1996, 96, 683−720. (6) Miller, J. S.; Dudkin, V. Y.; Lyon, G. J.; Muir, T. W.; Danishefsky, S. J. Angew. Chem., Int. Ed. 2003, 42, 431−434. (7) Kobayashi, K.; Tsuchida, A. Macromolecules 1997, 30, 2016− 2020. (8) Zeng, X.; Murata, T.; Kawagishi, H.; Usui, T.; Kobayashi, K. Biosci., Biotechnol., Biochem. 1998, 62, 1171−1178. (9) Wang, Y.; Kiik, K. J. Am. Chem. Soc. 2005, 127, 16392−16393. (10) Hackenberger, C. P. R.; O’Reilly, M. K.; Imperiali, B. J. Org. Chem. 2005, 70, 3574−3578 and references therein. (11) Likhosherstov, L. M.; Novikova, O. S.; Derevitskaja, V. A.; Kochetkov, N. K. Carbohydr. Res. 1986, 146, C1−C5. (12) Walczak, M. A.; Hayashida, J.; Danishefsky, S. J. J. Am. Chem. Soc. 2013, 135, 4700−4703. (13) Likhosherstov, L. M.; Novikova, O. S.; Zheltova, A. O.; Shibaev, V. N. Russ. Chem. Bull. 2004, 53, 709−713. (14) Mortell, K. H.; Gingras, M.; Kiessling, L. L. J. Am. Chem. Soc. 1994, 116, 12053−12054. (15) Grande, D.; Baskaran, S.; Baskaran, C.; Gnanou, Y.; Chaikof, E. L. Macromolecules 2000, 33, 1123−125. (16) Muthukrishnan, S.; Jutz, G.; Andre, A.; Mori, H.; Muller, A. H. E. Macromolecules 2005, 38, 9−18. (17) Albertin, L.; Stenzel, M. H.; Barner-Kowollik, C.; Foster, L. J. R.; Davis, T. P. Macromolecules 2005, 38, 9075−9084. (18) Sun, X.-L.; Houston, K. M.; Grande, D.; Chaikof, E. L. J. Am. Chem. Soc. 2002, 124, 7258−7259. (19) Hou, S.; Sun, X.-L.; Dong, C.-M.; Chaikof, E. L. Bioconjugate Chem. 2004, 15, 954−959. (20) Muthana, S.; Yu, H.; Huang, S.; Chen, X. J. Am. Chem. Soc. 2007, 129, 11918−11919. (21) Yu, C.-C.; Withers, S. G. Adv. Synth. Catal. 2015, 357, 1633− 1654. (22) Skehel, J. J.; Wiley, D. C. Annu. Rev. Biochem. 2000, 69, 531− 569. (23) Palese, P.; Tobita, K.; Ueda, M.; Compans, R. W. Virology 1974, 61, 397−410. (24) Narla, S. N.; Sun, X.-L. Biomacromolecules 2012, 13, 1675−1682. (25) Tanaka, T.; Ishitani, H.; Miura, Y.; Oishi, K.; Takahashi, T.; Suzuki, T.; Sohda, S-i.; Kimura, Y. ACS Macro Lett. 2014, 3, 1074− 1078. (26) Spiro, R. G. J. Biol. Chem. 1960, 235, 2860−2869. (27) Baenziger, J. U.; Fiete, D. J. Biol. Chem. 1979, 254, 789−795. (28) Edge, A. S.; Spiro, R. G. J. Biol. Chem. 1987, 262, 16135−16141. (29) Green, E. D.; Adelt, G.; Baenziger, J. U.; Wilson, S.; vanHalbeek, H. J. Biol. Chem. 1988, 263, 18253−18268.
SAα2,3Lac polymer (6) showed strong binding to influenza A/ PR/8/34 (H1N1) virus with the minimum concentrations of 3.13 × 10−5 and 6.25 × 10−5 g/mL, respectively. No activity was observed with N-Lac polymer (4e) that lacks a SA residue. Interestingly, N-Glc polymer (4a) also showed moderate binding to influenza A/PR/8/34 (H1N1) and influenza A X31, A/Aichi/68 (H3N2) virus with the minimum concentrations of 1.25 × 10−4 and 5 × 10−4 g/mL, respectively. In particular, N-Man polymer (4c) showed strong binding to Influenza A X-31 A/Aichi/68 (H3N2) virus with the minimum concentrations of 3.13 × 10−5 g/mL. Fetuin is a blood protein containing sialyloligosaccharides, having both SAα2,6Gal and SAα2,3Gal residues at the nonreducing ends of oligosaccharides,26−29 showing strong binding to influenza A/PR/8/34 (H1N1) and influenza A X-31, A/Aichi/68 (H3N2) virus with a minimum of 1.95 × 10−7 and 3.13 × 10−5 g/mL, respectively, as a positive HI control. These N-glycan polymers may serve as anti-influenza virus agents, and further studies with different sugar densities and polymer molecular weights are highly deserved for continuing research. In conclusion, we demonstrated a straightforward synthesis of N-glycan polymers via acryloyl-glycosylamine and direction polymerization from free saccharides all in aqueous condition without protection/deprotection steps. In addition, enzymatic sialylation of the N-glycan polymers and their anti-influenza virus hemagglutination activities were confirmed. The present simple and efficient synthetic method can be applied to synthesize glycomonomers and glycopolymers from any kinds of free saccharides, either purified natural N-linked and Olinked oligosaccharides or synthetic N-linked and O-linked oligosaccharides, for variety applications of glycopolymers.
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Letter
Experimental details and spectroscopic data (PDF).
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The authors declare no competing financial interest. 111
DOI: 10.1021/acsmacrolett.6b00928 ACS Macro Lett. 2017, 6, 107−111
