molecules Review Anomeric O-Functionalization of Carbohydrates for Chemical Conjugation to Vaccine Constructs Simon S. Park † ID , Hsiao-Wu Hsieh † ID and Jacquelyn Gervay-Hague *,† Department of Chemistry, University of California, Davis, CA 95616, USA; [email protected] (S.S.P.); [email protected] (H.-W.H.) * Correspondence: [email protected]; Tel.: +1-530-754-9577 † These authors contributed equally to this work. Academic Editors: Paul Kovac, Peng Xu and Helene Pfister Received: 25 June 2018; Accepted: 13 July 2018; Published: 17 July 2018 Abstract: Carbohydrates mediate a wide range of biological interactions, and understanding these processes benefits the development of new therapeutics. Isolating sufficient quantities of glycoconjugates from biological samples remains a significant challenge. With advances in chemical and enzymatic carbohydrate synthesis, the availability of complex carbohydrates is increasing and developing methods for stereoselective conjugation these polar head groups to proteins and lipids is critically important for pharmaceutical applications. The aim of this review is to provide an overview of commonly employed strategies for installing a functionalized linker at the anomeric position as well as examples of further transformations that have successfully led to glycoconjugation to vaccine constructs for biological evaluation as carbohydrate-based therapeutics. Keywords: carbohydrates; glycosylation; glycosides; glycoconjugates; anomeric functionalization 1. Introduction 1.1. Emergence of Glycomics Nucleic acids, proteins and carbohydrates are three important classes of biomolecules. DNA, RNA and the proteins they encode have relatively confined connectivity and predictable chemistries owing to the limited number of ways the nucleic acid and amino acid building blocks can be combined. In contrast, carbohydrates have multiple similarly reactive hydroxyl groups that connect with varying region- and stereochemistries giving rise to a complex set of structures that have no corresponding genetic blueprint [1,2]. With the launch of the Human Genomic Project in the beginning of this century [3,4], efforts have shifted toward understanding structure/function relationships of post-translational modifications of proteins. Glycosylation is a prominent form of post-translational modification occurring in a majority of eukaryotic proteins [5–7]. Other biomolecules such as glycolipids and glycosylphosphatidylinositols (GPI anchor) display hydrophilic carbohydrate moieties that participate in ligand-receptor binding, cell-to-cell interactions and pathogenic processes such as bacterial and viral infection as well as cancer metastasis [8,9]. Deciphering the “sugar codes” created by specific sequences of oligosaccharides linked to lipid and protein anchors is an emerging area of glycomics, which like proteomics has the underlying goal of connecting chemical structures to biological functions. Understanding how structure gives rise to function is critical for the development of carbohydrate-based therapeutics and expedient access to synthetic materials is a significant challenge for researchers in this area. Like most natural products, glycoconjugates are typically difficult to isolate and may only occur as heterogenic mixtures in scarce amounts. Common isolation techniques often Molecules 2018, 23, 1742; doi:10.3390/molecules23071742 www.mdpi.com/journal/molecules Molecules 2018, 23, x 2 of 23 Molecules 2018, 23, 1742 2 of 23 isolation techniques often require enzymatic digestion, detergent extraction and multiple purifications, which may degrade the sugars; although recent methods to extract O-glycans using requirebleach hold enzymatic promise digestion, for commercial detergent use extraction [10]. To address and multiple these limitations, purifications, synthetic which mayplatforms degrade that theafford sugars; large-scale although recentproduction methods of topure extract andO -glycanschemically using defined bleach holdglycoconjugates promise for commercialare under usedevelopment. [10]. To address Chemoenzymatic these limitations, methods synthetic offer platforms a complementary that afford large-scaleapproach; productionhowever, ofenzyme pure andavailability, chemically substrate defined glycoconjugatesspecificity and arescalability under development.can hinder product Chemoenzymatic diversity. methodsIn either offer case, a complementaryachieving the desired approach; stereochemic however, enzymeal specificity availability, and substratemultiplicity specificity of ligation and scalability products canwith hinder high productpurity remains diversity. a challenge In either [11–13]. case, achieving One approach the desired to conjugating stereochemical carbohydrates specificity to andbinding multiplicity partners ofof ligationinterest products(i.e., protein with or high lipid) purity in remainsenantiomer a challengeically pure [11 –form13]. is One through approach the tostereoselective conjugating carbohydratesinstallment of tolinkers binding with partners functionalizable of interest handles, (i.e., protein which or can lipid) be elaborated in enantiomerically to generate pure complex form isglycans through and/or the stereoselectivemultivalent displays. installment of linkers with functionalizable handles, which can be elaborated to generate complex glycans and/or multivalent displays. 1.2. Overview of Glycosylation Principles 1.2. Overview of Glycosylation Principles Carbohydrate synthesis requires a crafty approach in selecting protecting groups, participating groups,Carbohydrate promoter synthesissystems, glycosyl requires donor a crafty and approach selectively in selecting deprotected protecting glycosyl groups, acceptors participating in order groups,to achieve promoter stereo-controlled systems, glycosyl glycosylation donor and in reasonable selectively deprotectedyields [14,15] glycosyl (Figure acceptors1A). The inreactivities order to achieveof the carbohydrate stereo-controlled and glycosylation aglycon partners in reasonable can be finely-tuned yields [14,15 ]by (Figure introducing1A). The different reactivities protecting of the carbohydrategroups. According and aglycon to the partners “arm-disarm” can be finely-tunedconcept introduced by introducing by Fraser-Reid, different protectingelectron releasing groups. Accordingether-type to(i.e., the silyl “arm-disarm” and benzyl) concept protecting introduced groups arm byFraser-Reid, the donor while electron inductively releasing withdrawing ether-type (i.e.,ester silyl (i.e., and acetyl benzyl) and protecting benzoyl) groups disarm arm the the dono donorr [16,17]. while inductivelyDonor leaving withdrawing groups can ester be (i.e., activated acetyl andusing benzoyl) various disarm promoters, the donor which [16,17 are]. Donorcommonly leaving Lewis groups acids can beadded activated in stoichiometric using various or promoters, catalytic whichamounts. are commonlyUpon departure Lewis acidsof the added anomeric in stoichiometric leaving group, or catalyticthe resulting amounts. oxocarbenium Upon departure ion is ofready the anomericfor coupling leaving with group, a glycosyl the resulting acceptor oxocarbenium (nucleophile) ion to is readyform forthecoupling corresponding with a glycosylglycosidic acceptor bond. (nucleophile)When the acceptor to form is the another corresponding carbohydrate, glycosidic oligosa bond.ccharide When synthesis the acceptor results. is another If the carbohydrate, acceptor is a oligosaccharidenon-carbohydrate synthesis aglycon, results. a glyc Ifoconjugate the acceptor is isformed. a non-carbohydrate The stereoselectivity aglycon, aat glycoconjugate the anomeric isposition formed. can The be stereoselectivity influenced by ster at theic hindrance, anomeric positionthe anomeric can be effect, influenced and internal by steric or hindrance,external group the anomericparticipation effect, (such and as internal neighbor-group or external participation group participation or solvent (such effect) as neighbor-group[18,19] (Figures 1B,C). participation NMR is oran solvent essential effect) tool [18used,19] (Figureto determine1B,C). NMRthe anomer is an essentialic stereoselectivity. tool used to According determine to the the anomeric Karplus α β stereoselectivity.equation, α/β-glycosides According are to thecharacterized Karplus equation, by the chemical/ -glycosides shift of are the characterized anomeric proton, by the as chemical well as shift of the anomeric proton, as well as the 3J and 1J coupling constants.[20] Concordantly, the the 3JH,H and 1JC,H coupling constants.[20] H,HConcordantly,C,H the ratio of products is measured by ratiointegration of products of the is anom measurederic proton by integration peaks. of the anomeric proton peaks. (A) Reactivity Regioselectivity Stereoselectivity Promoter PGO (Lewis acid) PGO O + O O O PGO LG HO OR PGO O OR Solvent Glycosyl donor Glycosyl acceptor LA PG: Protecting group O O HOR PGO LG PGO or LG: Leaving group LA: Lewis acid Oxocarbenium ion (B) H (C) LA HOR LA O O O O ACN O PGO LG PGO PGO H PGO LG PGO OR N H 3J = 2-4 Hz O H-H Solvent participation C HOR HOR PGO OR α-glycoside Me H LA 3 O O JH-H = 8-10 Hz O lp - σ* interaction PGO LG PGO β-glycoside O O R O O O R PGO Neighboring-group participation OR Anomeric effect: Stabilization Figure 1. (A) Glycosylation reaction at a glance. (B) Mechanistic overview of α-glycosides. (C) Figure 1. (A) Glycosylation reaction at a glance. (B) Mechanistic overview of α-glycosides. Mechanistic overview of β-glycosides. (C) Mechanistic