Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX
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Synthesis and Glycan−Protein Interaction Studies of Se-Sialosides by 77Se NMR Tatsuya Suzuki,‡ Chieka Hayashi,‡ Naoko Komura,† Rie Tamai,‡ Jun Uzawa,∥ Junya Ogawa,‡ Hide-Nori Tanaka,† Akihiro Imamura,‡ Hideharu Ishida,†,‡ Makoto Kiso,‡,§ Yoshiki Yamaguchi,∥,⊥ and Hiromune Ando*,†,§
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†
Center for Highly Advanced Integration of Nano and Life Sciences (G-CHAIN), Gifu University, 1-1, Yanagido, Gifu-shi, Gifu 501-1193, Japan ‡ Department of Applied Bioorganic Chemistry, Gifu University, 1-1, Yanagido, Gifu-shi, Gifu 501-1193, Japan § Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida Ushinomiya-cho, Sakyo-ku, Kyoto, Japan ∥ Structural Glycobiology Team, Systems Glycobiology Research Group, RIKEN Global Research Cluster, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan S Supporting Information *
ABSTRACT: To expand the potential of Se-carbohydrates for multifunctional mimicry of sugars, herein we addressed the synthesis of the highly challenging and biologically significant Se-glycosides of sialic acid (Se-sialosides). An α-sialyl selenolate anion generated in situ smoothly reacted with electrophiles to give α-Se-sialosides as single stereoisomers. A Se-sialoside was sequentially incorporated with selenium, producing a triseleno-sialoside. This molecule was used as a 77 Se NMR-active handle for studying glycan−protein interaction, revealing different binding profiles of sialic acid binding proteins.
T
novel Se-sialoglycan containing multiple selenium atoms to gain insight into carbohydrate−protein interactions. The preparation of Se-sialosides with an α configuration is highly challenging, and the methods available in the literature suffer from a limited substrate scope.6 To establish an interresidual Se-glycoside of sialic acid (Se-sialosides) with an α configuration, we applied the in situ glycosyl selenolate anion methodology developed by our group,7 which allows the convenient preparation of Se-glycosides as single stereoisomers via retention of the anomeric configuration of the glycosyl selenolate anion. In this study, the p-methylbenzoylselenyl derivative 2 was used as the precursor for the generation of an αsialyl selenolate anion. The synthesis of 2 was attempted using the β-chloride 1 as the starting material (Scheme 1).8 For selenium incorporation, we evaluated the use of potassium pmethylselenobenzoate 39 as a selenating agent according to a reported protocol, which provided a complex reaction mixture. Mass spectrometric analysis revealed a mixture composed of the desired product 2, the elimination glycal impurity, sialyl pmethylbenzoyldiselenide, and sialyl p-methylbenzoate derivatives. This complex reaction mixture might result from the reaction of 1 with the impurities contained in 3. These
he chemical synthesis of seleno-glycosides (Se-glycosides) has been receiving increasing attention due to their versatile biochemical potential.1 Se-Carbohydrates, containing Se-glycosides, have prime medicinal relevance and serve as key bioisosteres of ligands to carbohydrate-binding proteins.2 Their use as phasing molecules in the X-ray crystallographic analysis is a vital tool for studying carbohydrate−protein complexes.3 In addition, the utility of Se-glycosides and glycosyldiselenides as NMR-active handles and reporters for studying carbohydrate conformations and carbohydrate−lectin interactions by NMR spectroscopy has been recently demonstrated.4 However, to fully harness the potential of this class of carbohydrates, the scope of available synthetic methods needs to be expanded to allow the preparation of complex and biologically significant Secarbohydrates. In addition, the development of methodologies for the incorporation of multiple selenium atoms into glycans is of prime importance to elucidate carbohydrate−protein interactions involving multiple interactions between their functional groups. Sialic acid containing glycans (sialoglycans) are well-known for their varied bioactivities,5 and the present study addresses the synthesis of Se-sialoglycans, which have never been synthesized. We report herein the synthesis of Sesialoglycans via stepwise incorporation of a selenium atom into a Se-sialoglycan. A 77Se NMR study was conducted using the © XXXX American Chemical Society
Received: July 3, 2019
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DOI: 10.1021/acs.orglett.9b02303 Org. Lett. XXXX, XXX, XXX−XXX
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Organic Letters
Scheme 2. Synthesis of Triseleno-sialyl Galactose Derivativea
Scheme 1. Stereospecific Introduction of Tolylselenium Group at the Anomeric Carbon of Sialic Acida
a
Abbreviations: DMF, N,N-dimethylformamide; DMA, N,N-dimethylacetamide.
Table 1. Formation of Se-Sialosides via in Situ Generation of Sialyl Selenolate Aniona
a
Abbreviations: TFAc, trifluoroacetyl; DBU, diazabicylo[5.4.0]undec7-ene; MS, molecular sieves; TBAF, tetra-n-butylammonium fluoride.
Table 2. Sialyl Galactose Derivatives Used for Biolayer Interferometry and Their Binding Affinity to Sialic Acid Binding Lectins
Kd value (μM) entry
fixed ligand
WGA
SSA
1 2
20 21
62.7 83.7
2.14 1.51
selenobenzoic anhydride 410 upon treatment with piperidine dramatically improved the product profile, affording 2 in 70% yield. We were pleased to find that this method delivered the product as a single α-anomer and allowed the scalable preparation of 2 in a sufficient quantity for the next experiments. The anomeric configuration of 2 was determined by the empirical rule of 1H NMR of sialoside.11 The spectrum of 2 showed the signal of H-4 at 4.97 ppm and the signal of H-7 with the coupling constant between H-7 and H-8 with 5.9 Hz, indicating the anomeric configuration as α. Having successfully prepared 2, we examined the formation of α-Se-sialosides (Table 1). The Se-sialic acid derivative 2 was reacted with various electrophiles upon activation with piperazine in the presence of Cs2CO3 in DMF. In all reactions, the α-configuration of the sialic acid was completely retained to
a
Abbreviations: SE, 2-(trimethylsiyl)ethyl; Bz, benzoyl; MP, 4methoxyphenyl.
impurities formed during the large-scale preparation of 3 and were inseparable during purification. In contrast, the in situ generation of a p-methylselenobenzoate anion from p-methylB
DOI: 10.1021/acs.orglett.9b02303 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters
Figure 1. 77Se NMR analysis of the interaction between triseleno-sialyl galactose 19 and lectins. (A) 77Se NMR spectra of 19 before and after the addition of lectins: WGA (upper panel) and SSA (lower panel). 77Se chemical shift (ppm) and line width (Hz) are indicated. (B) Changes in line width of 77Se signals upon addition of lectins: WGA (green bar) and SSA (orange bar). (C) 77Se chemical shift changes upon addition of lectins: WGA (green bar) and SSA (orange bar).
fluoride anion,14,15 and was swiftly reacted with 3-methylselenenylpropyl tosylate 17 to furnish 18 in a high yield. Finally, global deprotection under basic conditions delivered triselenosubstituted sialyl-α(2,6)galactoside 19 in 86% yield. We also evaluated the O-glycosylation of the Se-sialyl galactosyl imidate 14 without affecting the Se-glycosidic linkage. To investigate the binding affinity of the Se-sialoside to lectins, the amine-terminated 21 and the O-glycoside 20 were synthesized (see Supporting Information) and their binding affinities were evaluated. The sialoside-binding lectins (WGA: Wheat-germ agglutinin)16 bind to the sialic acid, and SSA (Sambucus sieboldiana agglutinin)17 binds to sialyl-α(2,6)galactose; the immobilized 20 and 21 were analyzed by the biolayer interferometry (BLI).18 To make generally weak carbohydrate−protein interactions detectable by BLI, the carbohydrates were immobilized on biosensors. As summarized in Table 2, the Se-sialoside 21 was shown to bind to the lectins with similar affinities to those of the O-glycoside 20. To WGA, the Se-glycoside 21 showed a slightly weaker Kd value (83.7 μM) than that of the O-glycoside 20 (62.7 μM). In the case of SSA, the order of Kd values of 20 and 21 was reversed and the difference was again marginal. These results indicated the Sesialoside 21 served as a mimic of a carbohydrate ligand. Although O- and Se-glycosides are different in their geometric properties (e.g., C−X bond length and C−X−C angle), the more flexible Se-glycoside due to the weak stereoelectronic effect of Se might allow it to be arrested in a conformation that would be energetically favored in the interaction with the protein counterpart. In addition, Se−π interaction between the Secarbohydrate and aromatic amino acid residues might contribute to enhancing the affinity of Se-carbohydrate more than the natural counterpart.4a Next, we examined the interaction of the triseleno-sialyl galactoside 19 with the lectins by 77Se NMR (Figure 1). In the absence of lectin, the three selenium atoms of 19 resonated at 40, 256, and 413 ppm in 77Se NMR spectroscopy, respectively.4c Upon addition of lectin (WGA and SSA), changes to the 77Se NMR spectra were observed. Addition of WGA broadened the signals of the sialyl selenium at 413 ppm and the galactosyl selenium at 256 ppm by 18.8 and 9.1 Hz, respectively, and downshifted all signals by 0.03 to 0.17 ppm. On the other hand,
furnish the corresponding α-selenoglycosides as single stereochemical outcomes. With simple alkylating reagents such as EtBr and p-methoxybenzyl chloride, the reaction proceeded very swiftly to afford the corresponding Se-glycosides 5 and 6, respectively in high yields (entries 1 and 2). Similarly, the selenation at the C6 iodide of 6-deoxygalactoside 7 proceeded smoothly to afford the sialyl Se-galactoside 8 in 87% yield. However, the C3 iodide of the 3-deoxygalactoside 9 hardly reacted with the sialylselenolate. This result might be accounted for by applying the Richardson−Hough rules,12 which postulate a repulsive dipolar crush between the partially bonded Se−C bond and the adjacent axial electron-withdrawing acetoxy group in the developing SN2 transition state. To overcome this challenge, we employed the 4,6-benzylidenated triflate 11, to exploit the vicinal triflate effect recently proposed by Hale et al.,13 as a suitable moiety for the C3−SN2 displacement. The reaction using 11 proceeded to deliver the desired (2,3)-linked Se-sialyl galactoside 12, albeit in a poor yield. The yield of 12 was improved to 47% upon increasing the reaction temperature to 50 °C. This moderate yield was partially attributed to the steric repulsion between bulky selenolate and the axial group at the C4 position. The synthesized molecules 8 and 12 are analogs of naturally occurring O-glycosides. With the Se-glycoside analog 8 of the sialyl-α(2,6)galactose in hand, we attempted the incorporation of selenium at the anomeric center of the galactose residue and the external terminus of the aglycone moiety to obtain a highly selenated sialyl glycan, to evaluate its use as a handle for NMR studies on glycan−protein interaction (Scheme 2). First, the hemiacetal derivative 13 was prepared in high yield without affecting the Seglycoside by cleaving the 2-(trimethylsilyl)ethyl group of 8 by treatment with TFAcOH in CH2Cl2. The resulting compound 13 was then converted into a trichloroacetimidate derivative 14 in 92% yield. By following our method for the Se-glycoside formation via transacetalization,14 the imidate 14 reacted with a selenoacetal derivative 1514 in the presence of TMSOTf and molecular sieves AW-300 at 0 °C in CH2Cl2 to afford the β-2(trimethylsilyl)ethylselenoglyoside 16 in 84% yield in a stereoselective manner, without affecting the inter-residual Seglycosidic bond. The disaccharide 16 was transformed into the corresponding glycosylselenolate in situ, upon exposure to the C
DOI: 10.1021/acs.orglett.9b02303 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters
Initiative (WPI), Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.
upon SSA addition, conspicuous signal broadening was observed only at the sialyl selenium (Δ 5.1 Hz), and all the signals upshifted to a lesser extent. The signal broadening observed here will result from the shortened transverse relaxation (T2) and/or the exchange term (Tex) due to the binding to lectins. From these results, we suggested that both lectins were weakly interacting with the terminal selenium atoms so that they are tolerant to the aglycon moiety. The NMR experiments also demonstrated that 77Se chemical shifts were highly sensitive to the local chemical environment, which was influenced by chemical shift anisotropy. The different profile of 77 Se NMR spectroscopy between WGA and SSA clearly reflects the different binding modes in the two lectins; WGA binds only the Neu5Ac part,19 whereas SNA binds the galactose residue in addition to Neu5Ac.20 77Se NMR of multiply selenated glycans would help identify the moieties available for chemical modification without affecting the binding affinity to sugarbinding proteins. In conclusion, the 2-p-methylbenzoylselenyl sialic acid 2 enabled the syntheses of various seleno-sialosides, most importantly the selenoglycoside mimetics of the biologically significant sialyl-α(2,6) and α(2,3)galactose epitopes. Furthermore, the preparation of the triseleno-glycan for the first time was successfully achieved by the incorporation of selenium atoms into the seleno-sialyl galactose. The 77Se NMR study suggests that the multiseleno-glycan will serve as a reporter for carbohydrate−protein binding. Since sialyl galactoses are widely involved in glycan receptors for virus and other pathogens,5b their seleno-mimetics would be highly useful for detailing the glycan−pathogen interactions by X-ray crystallography and NMR to aid the development of glycan-based therapeutics.
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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/acs.orglett.9b02303. Experimental details and procedures, compound characterization data, and copies of 1H, 13C, and 77Se spectra for new compounds (PDF)
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REFERENCES
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Hide-Nori Tanaka: 0000-0001-5307-4909 Hiromune Ando: 0000-0002-0551-0830 Present Address ⊥
Laboratory of Pharmaceutical Physical Chemistry, Tohoku Medical and Pharmaceutical University, 4-4-1, Komatsushima, Aoba-ku, Sendai, Miyagi 981-8558, Japan.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported in part by JSPS KAKENHI Grant Numbers JP15K07409 (H.I.), JP15H04495 (H.A.), JP16H04758 (Y.Y.), and JP18H03942 (H.A.); JST CREST Grant Number JPMJCR18H2 (H.A.); and by the Mizutani Foundation for Glycoscience (Y.Y. and H.A.). The iCeMS is supported by a World Premier International Research Center D
DOI: 10.1021/acs.orglett.9b02303 Org. Lett. XXXX, XXX, XXX−XXX
