Postglycosylation acetylation of sialic acid imparts unique roles to sialoglycoconjugates in mammalian immune response making structural and functional understanding of these analogues important. Five partially O-acetylated Neu5Ac analogues have been synthesized. Reaction of per-O-silylated Neu5Ac ester with AcOH and Ac2O in pyridine promotes regioselective silyl ether/acetate exchange in the following order: C4 (2°) &gt; C9 (1°) &gt; C8 (2°) &gt; C2 (anomeric). Subsequent hydrogenolysis affords the corresponding sialic acid analogues as useful chemical biology tools

Gervay-Hague, Jacquelyn

Park, Simon S

English

PubMed

eScholarship - University of California

UC DavisUC Davis Previously Published WorksTitleSynthesis of Partially O-Acetylated N‑Acetylneuraminic Acid Using Regioselective Silyl Exchange TechnologyPermalinkhttps://escholarship.org/uc/item/3jc3h4m8JournalOrganic Letters, 16(19)ISSN1523-7060AuthorsPark, Simon SGervay-Hague, JacquelynPublication Date2014-10-03DOI10.1021/ol502389g Peer reviewedeScholarship.org Powered by the California Digital LibraryUniversity of CaliforniaSynthesis of Partially O-Acetylated N‑Acetylneuraminic Acid UsingRegioselective Silyl Exchange TechnologySimon S. Park and Jacquelyn Gervay-Hague*Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, California 95616, United States*S Supporting InformationABSTRACT: Postglycosylation acetylation of sialic acid imparts unique roles to sialoglycoconjugates in mammalian immuneresponse making structural and functional understanding of these analogues important. Five partially O-acetylated Neu5Acanalogues have been synthesized. Reaction of per-O-silylated Neu5Ac ester with AcOH and Ac2O in pyridine promotesregioselective silyl ether/acetate exchange in the following order: C4 (2°) > C9 (1°) > C8 (2°) > C2 (anomeric). Subsequenthydrogenolysis affords the corresponding sialic acid analogues as useful chemical biology tools.In nature, sialic acids are found in more than 50 forms.1 Theseimportant carbohydrates are nine carbon keto-aldonic acidstypically attached to the terminal ends of glycolipids andglycoproteins in vertebrates and various pathogenic bacteria(Table 1).2 The most common form of sialic acid is Neu5Ac(Table 1),3 which plays critical roles in many biological andphysiological functions such as signal transduction,3 cell−cellrecognition and growth,4 and immunology.5 The structures ofsialoglycoconjugates are further diversified by O-acetylation(Table 1).6 These derivatives are products of sialate O-acetyltransferases (SOATs) that selectivelyO-acetylate at variouspositions of Neu5Ac. O-Acetylation influences the biology ofmammalian cells by altering the ligand properties anddegradation pathways of sialoglycoconjugates.7,8 In bacteria, O-acetylation can lead to inhibition of the host immune response,thereby serving as a masking system that enables pathogenicfunctions.9Historically, it has been suggested that O-acetylation canpotentially serve as a clue to mammalian evolutionaryphenomena.10 However, to date, only sialate-4-O-acetyltransfer-ase (4-SOAT) has been identified in mammals,11 and isolationand cloning 4-SOAT have not yet been successful.There is sufficient evidence documenting the presence of 4-O-acetyl containing Neu5Ac analogues (Table 1); however, fullcharacterization and biological understanding of these derivativesis lacking and the limitations of current extraction methods makesynthesis of these analogues important.While naturally occurringsialic acids found in mammalian cells are often conjugated toother sugars, partially acetylated monomers have been isolatedfrom natural sources (Table 1). Moreover, synthetic standardshave proven useful in monitoring degradation products ofNeu5Ac lyase during sialoglycoconjugate isolation and otherbiochemical assays.12With growing interest in Neu5Ac analogues and glycosidesynthesis, methodologies that allow regioselective functionaliza-tion of carbohydrates in an efficient manner are of great utility tosynthetic chemists. However, Neu5Ac contains several hydroxylReceived: August 11, 2014Published: September 23, 2014Table 1. Acetylated Sialic Acids: Natural Occurrence andStructural Divergencecompd name abbreviation occurrence5-N-acetylneuraminic acid Neu5Ac V, E, Ps, Pz, F, B5-N-acetyl-4-O-acetylneuraminic acid Neu4,5Ac2 V5-N-acetyl-4,9-di-O-acetylneuraminicacidNeu4,5,9Ac3 V5-N-acetyl-4,7,9-tri-O-acetylneuraminic acidNeu4,5,7,9Ac4 V5-N-acetyl-4,7,8,9-tetra-O-acetylneuraminic acidNeu4,5,7,8,9Ac5 V5-N-acetyl-7-O-acetylneuraminic acid Neu5,7Ac2 V, Pz, B5-N-acetyl-9-O-acetylneuraminic acid Neu5,9Ac2 V, E, Pz, F, BAbbreviations used: V, vertebrates; E, echinoderms; Ps, protostomes(insects and mollusks); Pz, protozoa; F, fungi; B, bacteria.Letterpubs.acs.org/OrgLett© 2014 American Chemical Society 5044 dx.doi.org/10.1021/ol502389g | Org. Lett. 2014, 16, 5044−5047Terms of Usegroupswith similar reactivities that are challenging to control, andthere is evidence that intramolecular hydrogen bonding createsfurther complexity.13 To avoid these issues, traditional chemicalmethods have utilized multiple protection−deprotection steps,and although enzymatic approaches do not require protectinggroup manipulations these methods are applicable to a limitednumber of substrates.14Only a few chemical syntheses of partially O-acetylatedNeu5Ac have appeared in the literature. In 1990, Hasegawaand co-workers first reported the preparation of Neu4,5(Ac)2using isopropylidene protection of the C8 and C9 of Neu5Acthioglucosides followed by kinetically controlled acetylation.15More recently, Clarke and co-workers synthesized a series ofmonoacetylated Neu5Ac12 with an improved adaptation of theHasegawa approach using free Neu5Ac instead of preparingNeu5Ac thioglucosides. The overall yields of both approacheswere comparable.Previously in our laboratory, selective acetylation of aldosesugars was achieved using regioselective silyl-exchange technol-ogy (ReSET).16,17 Readily available per-O-silylated sugars weredissolved in pyridine and acetic anhydride, and upon addition ofacetic acid the silyl protecting groups exchanged with acetate in apredictable manner, depending upon the structure of the aldose.Although Neu5Ac is a keto-aldonic sugar rather than an aldose,we were hopeful that the methodology would prove equallysuccessful. With growing interest in step economy synthese-s,18a−c we endeavored to apply ReSET toward the synthesis ofpartially O-acetylated Neu5Ac natural products.The research began with sialic acid benzyl ester formationusing K2CO3 and BnBr in DMF to afford 1 in 85% yield (Scheme1). Esterification minimized solubility issues associated with theNeu5Ac carboxylic acid. After benzyl ester formation, our focusturned to the preparation of per-O-TMS Neu5Ac benzyl ester(2). Attempts were made to prepare 2 using publishedprotocols;19,20 however, we found that Neu5Ac benzyl esterwas only partially silylated under these conditions. Gratifyingly,an ether silylation method reported by Sweeley and co-workers,using hexamethyldisilazane (HMDS) and chlorotrimethylsilane(TMSCl) in pyridine, successfully afforded 2 in 85% yield(Scheme 1).21ReSET studies were initiated by diluting 2 in dry aceticanhydride and pyridine and 3 equiv of glacial acetic acid(≥99.85%) were added. The reaction mixture was stirred at rtovernight to afford a distribution of acetylated Neu5Ac analogues(3−6) of which 6 was the major product (Table 2, entry 1).Delighted with this result, we then attempted to reduce thereaction time by subjecting the reaction mixture to microwaveirradiation in a commercial CEM-microwave reactor at 60 °C and30 W power for 30 min, which afforded 3−6 in a slightly loweroverall yield (Table 2, entry 2). Reducing the amount of aceticacid to 2 equiv and heating the reaction to 70 °Cwith 40Wpowerfor 30min gave 3−6 in the most even distribution (Table 2, entry3). To increase the scale of the reaction, the amount of 2 wasnearly doubled and set up with 2 equiv of acetic acid at 58 °C and30W power for 18 min to afford 3−6 with noticeably increasedamounts of 5 and 6 (Table 2, entry 4). Likewise, we were to ableto optimize for the production of 3 and 4 by reducing the amountof acetic acid to 1 equiv while running the reaction at 55 °C and30 W power (Table 2, entry 5). Optimizing conditions for theproduction of compound 4 was especially important because it isa precursor to analogue 15 (see Scheme 3). To further shortenthe synthesis, attempts were made to directly apply ReSET to 1;however, per-O-acetylated Neu5Ac was the only productobserved after 10 min. This result illustrates the importance ofthe silyl protecting groups in achieving regioselective exchange.Each ReSET product was analyzed by heteronuclear multiplebond correlation (HMBC) and heteronuclear single quantumcoherence (HSQC)NMRexperiments to determine the positionof the acetyl protecting groups. The HMBC NMR experimentswere critical to observe the correlation between the sugarbackbone C−H protons to the carbonyl carbon of the acetylprotecting groups to determine the position of the acetylprotecting group (Figure 1). A four-bond HMBC NMRexperiment was performed to observe correlation betweenmethyl protons of the acetate to the sugar carbon to characterize6 since the anomeric carbon ofNeu5Ac does not bear a proton forthree-bond HMBC.Once the products of the reactions were identified, we wereable to determine the order of acetate exchange using TLC datathat had been collected during the course of the reaction. The firstspot to form below the starting material (2) was 3 then 4 and 5.The last spot to form on the TLC was compound 6. The C9,bearing the primary OTMS group, was expected to be the first toexchange as observed in our previous work with aldohexoses;17instead, the secondary hydroxyl group (C4) next to the NHAcScheme 1. Benzylation and Silylation of Neu5AcTable 2. Various Conditions of ReSET To Afford 3-6entry scale (mg) time (min) T (°C) power (W) AcOH (equiv) 3 (%) 4 (%) 5 (%) 6 (%)1 113 overnight rt no 3 4 11 20 432 207 30 60 30 3 5 13 22 243 234 30 70 40 2 11 20 17 284 470 18 58 30 2 13 8 32 465 196 45 55 30 1 28 31 16 11Organic Letters Letterdx.doi.org/10.1021/ol502389g | Org. Lett. 2014, 16, 5044−50475045was most reactive. Upon introduction of the C4 acetate, silylexchange next occurred at the primary C9, as evidenced byformation of 4 on the TLC. Once the C9 acetate was introduced,the C8 was acetylated in favor of exchange of the anomeric ether.Thus, the order by which regioselective silyl exchange occurredwas as follows: C4 (2°) > C9 (1°) >C8 (2°) > C2 (anomeric). TheC-7 TMS ether did not exchange under these conditions (Figure2).Neu5Ac ReSET revealed completely different regioselectivitythan previous work with pyranose sugars.16,17 In aldohexoses, theprimary C6 typically exchanges first followed by the anomeric C1.After C1 exchange, C2 is usually next to react then furtherexchange occurs in a sequential manner around the pyranose ring.Witschi and co-workers also performed ReSET on N-acetylglucosamine (GlcNAc), which is an aldose sugar structurallysimilar to Neu5Ac in terms of bearing an NHAc group. In thatcase, the first exchange also occurred at the primaryC6 rather thanthe anomeric position, which was proximal to the amide.16The presence of NHAc in 2 presumably pulls electron densityfrom the C4 O−Si bond, which allows for exchange to occur firstat C4 in favor of the primary C9 position. Moreover, the presenceof methylene protons at C3 assures a less sterically hinderedenvironment than what is found in common pyranose sugars.Once C9 is acetylated, C8 is the next to react. Again, the electroniceffect of the C9 ester group makes the C8 O−Si bond mostsusceptible to attack. The observation of C8 exchange in favor ofthe anomeric silyl ether group indicates that the quaternarycenter is not readily accessible. These experimental findingsfurther illustrate the remarkable balance between steric andelectronic effects of ReSET (Figure 2).17In targeting naturally occurring 7 and 8, our plan was to usemethanolysis to deprotect the TMS silyl ethers first22,23 and thenremove the benzyl ester. However, upon methanolysis, weobserved slow reaction times in addition to transesterification. Toavoid these complications, 3−6 were subjected to hydrogenationto first remove the benzyl ester. Fortuitously, the TMS groupswere also deprotected under these conditions. While 3 and 4readily reacted in a mixture of ethyl acetate, methanol and water,analogues 5 and 6were sluggish in this solvent system. It is knownthat protic solvents enhance hydrogenation in comparison toaprotic organic solvents (e.g., ethyl acetate, acetonitrile), whichcan coordinate with the palladium metal reducing hydrogenadsorption.24 The combination of 2-propanol and methanol ledto increased efficiency for TMSdeprotection of 5 requiring only 4h compared to 19 h when reacted in an ethyl acetate/methanol/water mixture. With this global deprotection protocol, weobtained the naturally occurring Neu4,5(Ac)2 (7) in 92% yield,Neu4,5,9(Ac)3 (8) quantitatively, and Neu4,5,8,9(Ac)4 (9) in88% yield (Scheme 2).In pursuit of the synthesis of Neu4,5,7,8,9(Ac)5 (15),compound 4 was selectively deprotected to expose the C7 andC8 diol (11, Scheme 3). The anomeric silyl protecting groupremained in tact presumably due to steric hindrance. Subjecting11 to 1.5 equiv acetic anhydride gave selective acetylation of C7(12), while excess acetic anhydride gave 13 (Scheme 3). Uponhydrogenolysis of 12, acylmigration from the 7-O-acetyl to theC8position occurred affording compound 9. Attempts to avoidmigration using various catalysts including palladium (98%),palladium hydroxide, platinum(IV) oxide, and Raney nickel wereunsuccessful. C7 to C8 acyl migration occurred under allconditions, suggesting the C-8 acetate is a thermodynamic sink.Meanwhile, 13 was subjected to hydrogenation to remove theanomeric silyl and benzyl groups to afford naturally occurring 15in 92% yield. This route allowed for an alternative synthesis of 15,which had been previously synthesized.25Figure 1. Key HMBC signals for characterization.Figure 2.Preferred silyl ether/acetate exchange ofNeu5Ac: C4 (2°) >C9(1°) > C8 (2°) > C2 (anomeric).Scheme 2. Deprotection of TMS and Bn GroupsOrganic Letters Letterdx.doi.org/10.1021/ol502389g | Org. Lett. 2014, 16, 5044−50475046These experimental findings show unique expansion of ReSETtoward structurally complex sugars like Neu5Ac while demon-strating a facile preparation of naturally occurring O-acetylatedNeu5Ac analogues in the highest yields to date (Table 3). Inaddition to reducing the chemical reaction steps, time efficiencywas increased by >75% compared to previous reports and thechemicals used for this reaction were reduced to approximately50% (Table 3). Given our efforts to conserve valuable resourcesin the chemical research setting, this study validates ReSET as astep-, chemical-, and time- economical synthetic methodology.Analogues from ReSET may be used for future carbohydrate-based drug designs or as suitable standards that can expand theunderstanding of biology, immunology, and chemical functions.■ ASSOCIATED CONTENT*S Supporting InformationGeneral experimental procedures, NMR, and mass spectra. Thismaterial is available free of charge via the Internet at http://pubs.acs.org.■ AUTHOR INFORMATIONCorresponding Author*Fax: (530) 754-6915. Tel: (530) 754-9557. E-mail:jgervayhague@ucdavis.edu.NotesThe authors declare no competing financial interest.■ ACKNOWLEDGMENTSThis work is supported by the National Institutes of Health, NIHGrant No. R01GM090262. NSF CRIF program (CHE -9808183), NSF Grant No. OSTI 97-24412, and NIH GrantNo. RR11973 provided funding for theNMR spectrometers usedon this project. We thank Dr. Jerry Dallas (University ofCalifornia, Davis) for help with the long-range HMBC NMRexperiments and 2D NMR experiments.■ REFERENCES(1) Byrne, G.; Donohoe, G.; O’Kennedy, R. Drug Discov. Today 2007,12, 319−326.(2) Chen, X.; Varki, A. ACS Chem. Biol. 2010, 5, 163−179.(3) Kiefel, M.; Von Itzstein, M. Chem. Rev. 2002, 102, 439−469.(4) Fukuda, M. In Molecular and Cellular Glycobiology; OxfordUniversity Press: New York, 2000; pp 33−44.(5) Feizi, T. Immunol. Rev. 2000, 173, 79−88.(6) Angata, T.; Varki, A. Chem. Rev. 2002, 102, 471−490.(7) Kelm, S.; Schauer, R. Int. Rev. Cytol. 1997, 175, 137−240.(8) Engstler, M.; Schauer, R.; Brun, R. Acta. Trop. 1995, 59, 117−129.(9) Schauer, R.; Srinivasan, G.; Wipfler, D.; Kniep, B.; Schwartz-Albiez,R. Adv. Exp. Med. Biol. 2011, 705, 525−548.(10) Schauer, R. Zoology 2004, 107, 49−64.(11) Iwersen, M.; Dora, H.; Kohla, G.; Gasa, S.; Schauer, R. Biol. Chem.2003, 384, 1035−1047.(12) Clarke, P.; Mistry, N.; Thomas, G. Org. Biomol. Chem. 2012, 10,529−535.(13) De Meo, C.; Priyadarshani, U. Carbohydr. Res. 2008, 1540−1552.(14) Khedri, Z.; Muthana, M.; Li, Y.; Muthana, S.; Yu, H.; Cao, H.;Chen, X. Chem. Commun. 2012, 48, 3357−3359.(15) Hasegawa, A.; Murase, T.; Ogawa, M.; Ishida, H.; Kiso, M. J.Carbohydr. Chem. 1990, 9, 415.(16) Witschi, M.; Gervay-Hague, J. Org. Lett. 2010, 12 (19), 4312−4315.(17) Hsieh, H.-W.; Schombs, M.; Witschi, M.; Gervay-Hague, J. J. Org.Chem. 2013, 78, 9677−9688.(18) (a) Wender, P.; Verma, V.; Paxton, T.; Pillow, T. Acc. Chem. Res.2008, 41, 40−49. (b) Newhouse, T.; Baran, P.; Hoffmann, R. Chem. Soc.Rev. 2009, 38, 3010−3021. (c) Wender, P.; Miller, B. Nature 2009, 460,197−201.(19) Bhat, A.; Gervay-Hague, J. Org. Lett. 2001, 3, 2081−2084.(20) Schombs, M.; Park, F.; Du, W.; Kulkarni, S.; Gervay-Hague, J. J.Org. Chem. 2010, 75, 4891.(21) Sweeley, C.; Bentley, R.; Makita, M.; Wells, W. J. Am. Chem. Soc.1963, 85, 2497−2507.(22) Du, W.; Kulkarni, S.; Gervay-Hague, J. Chem. Commun. 2007,2336−2338.(23) Davis, R.; Lin, C.; Gervay-Hague, J. Chem. Commun. 2012, 48,9083−9085.(24) Ikawa, T.; Sajiki, H.; Hirota, K. Tetrahedron 2004, 6901−6911.(25) Kenkyusho, N. Japanese patent 09-249682, Sep 22, 1997.Scheme 3. Alternative Synthetic Route to Neu4,5,7,8,9(Ac)5Table 3. Summary of Previous and Current Syntheses ofNaturally Occurring O-Acetylated Neu5AcOrganic Letters Letterdx.doi.org/10.1021/ol502389g | Org. Lett. 2014, 16, 5044−50475047

Synthesis of Partially O-Acetylated N‑Acetylneuraminic Acid Using Regioselective Silyl Exchange Technology

http://pubs.acs.org/page/policy/authorchoice_termsofuse.html)

Synthesis of partially O-acetylated N-acetylneuraminic acid using regioselective silyl exchange technology.

Abstract

Similar works

Full text

Available Versions

eScholarship - University of California