biomimetics

Review Bioisosteres of Carbohydrate Functional Groups in Glycomimetic Design

Rachel Hevey

Molecular Pharmacy, Department Pharmaceutical Sciences, University of Basel, Klingelbergstr. 50, 4056 Basel, Switzerland; [email protected]



Received: 30 June 2019; Accepted: 26 July 2019; Published: 28 July 2019


Abstract: The aberrant presentation of carbohydrates has been linked to a number of diseases, such as cancer metastasis and immune dysregulation. These altered glycan structures represent a target for novel therapies by modulating their associated interactions with neighboring cells and molecules. Although these interactions are highly specific, native carbohydrates are characterized by very low affinities and inherently poor pharmacokinetic properties. Glycomimetic compounds, which mimic the structure and function of native glycans, have been successful in producing molecules with improved pharmacokinetic (PK) and pharmacodynamic (PD) features. Several strategies have been developed for glycomimetic design such as ligand pre-organization or reducing polar surface area. A related approach to developing glycomimetics relies on the bioisosteric replacement of carbohydrate functional groups. These changes can offer improvements to both binding affinity (e.g., reduced desolvation costs, enhanced metal chelation) and pharmacokinetic parameters (e.g., improved oral bioavailability). Several examples of bioisosteric modifications to carbohydrates have been reported; this review aims to consolidate them and presents different possibilities for enhancing core interactions in glycomimetics.

Keywords: bioisostere; carbohydrate; drug design; glycomimetic; lectin

1. Introduction The interaction of carbohydrates with proteins and cells is well established to play important roles in biology, from the maintenance of healthy tissue to the progression of diseases. The majority of cell types have a thick layer of carbohydrates at their surface which is approximately 10–100 Å thick, referred to as the glycocalyx [1,2]. This layer acts as a primary point of contact for neighboring cells and biomolecules. These interactions are critical for communicating whether a cell is ‘self’ or ‘foreign’, and play an important role in immune activation and regulation. The composition of carbohydrates often differs between healthy and diseased cells as a result of changes in the expression levels of glycosidases and glycosyltransferases which generates unique carbohydrate patterns such as truncated or hypersialylated structures [3–5]. Carbohydrate-binding proteins can be classified into three major groups: (i) enzymes; (ii) glycosaminoglycan (GAG)-binding proteins; and (iii) lectins. Carbohydrate-binding enzymes include glycosidases and glycosyltransferases, which play important roles in post-translational glycosylation processes. GAGs are a constituent of proteoglycans and can interact with a number of GAG-binding proteins, most notably the interaction of heparin with antithrombin which contributes to regulation of the coagulation pathway [6]. Lectins are abundant in all species and play integral roles in infection and disease, and have therefore become desirable therapeutic targets. Although highly specific for their carbohydrate targets, they are also characterized by low affinities to their native ligands (high micromolar to millimolar Kd values), except for a handful of exceptions [7–9].

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The low affinity of lectins arises from their inherent structural properties. Ligands engage the protein through only weak interactions, such as H-bonding, metal chelation, salt bridges, and weak hydrophobic interactions. Combined, these are typically insufficient in strength to compensate for the high enthalpic desolvation penalties associated with such polar substrates and shallow, solvent-exposed binding sites. High affinity ligand binding often relies heavily on hydrophobic interactions, which are clearly lacking in polyhydroxylated, polar carbohydrate substrates. Carbohydrate–lectin interactions instead rely primarily on H-bonding, where native ligands can have difficulty competing with H-bonding from bulk solvent. To compensate for these weak interactions with native substrates, lectins typically rely on multivalency to enhance affinity in which multiple carbohydrate-binding domains engage simultaneously with multiple ligands on complementary surfaces [10–13]. Multivalent interactions are collectively much stronger than the sum of their individual interactions, due to different multivalency effects which in cooperation afford enhanced affinities. Entropy is a major contributor to multivalency: once an initial binding event has occurred, subsequent ligand interactions suffer from a reduced entropic binding penalty. In addition, multivalent presentation can effectively increase the local concentration of ligand in the proximity of the receptor. As mentioned previously, aberrant carbohydrate patterns have been associated with a number of diseases, including cancer and immune dysregulation. Even carbohydrate structures present on healthy cells play a role in disease as they are important for immune regulation and suppression, and invading organisms are known to mimic host structures to avoid immune activation. Surface carbohydrates can also be targeted by pathogens and their toxins to immobilize onto and/or gain entry into cells [14,15]. These characteristic carbohydrates present novel targets for therapeutic development but methods to improve their binding affinities must first be established. Native carbohydrates are also susceptible to hydrolysis in both the acidic pH of the gut or by endogenous glycosidases, and methods to slow down their metabolic degradation will improve their suitability as therapeutics. Due to their high polar surface area, native carbohydrate ligands are unable to passively permeate the intestinal enterocyte layer and therefore are not orally bioavailable compounds. Features of passively permeable compounds typically conform to the Lipinski and Veber rules, such that compounds should have a low molecular weight, reduced polar surface area, and few H-bonding partners [16,17]. In addition, potentially therapeutic carbohydrates are further hindered by poor binding kinetics (fast koff rates leading to short residence times) and rapid renal clearance from the circulation.

1.1. General Strategies for Glycomimetic Synthesis In efforts to overcome the inherently poor properties of carbohydrates as therapeutics, much interest has been focused on designing ‘glycomimetics’ which can imitate the structure and function of native glycans [18,19]. Glycomimetics contain structural modifications which render improvements in both affinity and selectivity. Several successful examples already exist of glycomimetics developed as potential therapeutics targeting both lectins and carbohydrate-binding enzymes, for example the neuraminidase inhibitors oseltamivir (Tamiflu) and zanamivir (Relenza) have already shown great success in treating influenza infection [20–22]. There are multiple approaches to glycomimetic design, and these have been covered in detail in a recent review [23]. Strategies to improve binding affinity include deoxygenation, in which the OH groups deemed non-essential for binding can be removed to reduce polar surface area, thereby reducing the desolvation costs associated with binding and potentially generating new hydrophobic interactions with the protein surface. Glycomimetics can also introduce adjoining fragments that target new interactions with the protein surface peripheral to the primary binding site, most commonly targeting hydrophobic aromatic or aliphatic residues. The addition of hydrophobic fragments is also associated with reduced polar surface area, and further enhances affinity by reducing associated desolvation penalties. Conformational pre-organization has also been incorporated into glycomimetic design as it can significantly reduce entropic penalties associated with ligand binding; it has been successfully applied in several glycomimetics [24–27] and additionally tends to reduce polar surface Biomimetics 2019, 4, 53 3 of 23 area since the stabilizing intramolecular interactions typically involve polar functional groups which then become shielded from bulk solvent. In carbohydrate-binding enzymes, pre-organization has also been largely successful through efforts toward transition state mimetics, such as the iminoglycosides where ring distortion has been utilized to mimic the planarity of oxocarbenium transition states [8]. Additionally, multivalency has been used to successfully mimic the multivalent presentation of native ligands, which can enhance affinity through chelation, statistical rebinding effects, or clustering of binding partners [28–31]. Apart from binding affinity, strategies in glycomimetic design have also targeted improvements in pharmacokinetic properties such as better bioavailability and longer serum half-lives. As carbohydrates are easily degraded, the hydrolysis can be minimized by replacing the O-glycoside atom with a more stable surrogate atom (e.g., C, S) or alternatively by adding electronegative substituents to the glycan ring which destabilizes the oxocarbenium transition state required for degradation [32–34]. Owing to their large polar surface area, carbohydrates are unable to passively permeate the intestinal membrane and are therefore poorly absorbed into circulation. To improve passive permeation, the pro-drug approach has been used in which polar functionalities (e.g., OH, CO2−) are temporarily masked by hydrophobic groups (e.g., alkyl or aryl esters) which are then cleaved by endogenous enzymes after uptake into circulation (e.g., Tamiflu) [20,21]. Biomimetic groups can also be used to replace functional groups that are prone to degradation or are associated with rapid clearance from the serum. With regards to enhancing affinity through chemical modification, a vast majority of the efforts in glycomimetics have been focused on targeting peripheral regions of the binding site to generate novel interactions, for example the well-studied FimH antagonists which target a ‘tyrosine gate’ [35–38]. Although this approach has proven successful, it is worthwhile to also consider bioisosteric changes that can be made to carbohydrate functional groups (OH, NHAc, etc.) and how these can be used to improve PK/PD properties. Bioisosteric changes can potentially improve both binding affinity (e.g., reduced desolvation costs, enhanced metal chelation) and pharmacokinetic parameters (e.g., modification of groups prone to metabolic degradation, improved oral bioavailability). Examples of bioisosteric modifications are scattered throughout the literature, and this review aims to consolidate them to present a range of possibilities for enhancing core interactions in glycomimetics. A summary of the strategies outlined in this review is presented in Table1.

Table 1. A summary of the different bioisosteres of carbohydrate functional groups discussed in this review, including the rationale behind the selected substitution.

Bioisosteric Functional Group Rationale Disadvantages Replacement O-Glycosides can be susceptible Enhanced flexibility (larger O-Glycosidic linkage N-, C-, S-, Se-Glycosides to chemical/enzymatic atom, loss of anomeric effect) hydrolysis in vivo Enhance stability; Reduce polar Imino-, thio-, Changes in pyranoside surface area; Iminosugars can Endocyclic O atom carbasugars, phostones, conformation; Loss of mimic charged oxocarbenium phostines anomeric effect transition state Reduce polar surface area; Potentially disrupts critical OH Deoxygenation Increase hydrophobic contacts ligand-protein interactions; with protein Disrupts ligand pre-organization Similar polarity and size; H-bond acceptor ability; Reduce OH Deoxyfluorination Removes H-bond donor ability polar surface area; Destabilize oxocarbenium transition state Removes H-bond donor ability; OH Methyl etherification Reduce polar surface area Potential steric incompatibilities Reduce polar surface area Larger atoms; Longer OH SH/SeH substitution (enhanced atom polarizability); bonds/altered bond angles; Enhance π-interactions Weaker H-bond donors Biomimetics 2019, 4, 53 4 of 23

Table 1. Cont.

Bioisosteric Functional Group Rationale Disadvantages Replacement Similar size and hydrophobicity; Alters electron-density in H Fluorination Chemically inert; Destabilize neighboring substituents oxocarbenium transition state Enhance metal chelation; Potentially introduces steric NHAc C-, N-derivatives Introduce novel functionalities incompatibilities or for bioconjugation (e.g., ketone) charged substituents Reduce polar surface area; Amide, sulfonate, Disrupts critical CO Enhance charged 2− phosphonate carboxylate–protein interactions protein interactions

1.2. Affinity Enhancement Versus Enzyme Activity It is important to emphasize beforehand that using biomimetic functional groups to enhance binding affinities can have variable biological consequences, especially in enzymatic carbohydrate-binding proteins. For interactions where no chemical transformation occurs upon binding, such as those involving lectins, an enhancement in target binding affords lower Kd values and typically stronger biological responses. In glycomimetics serving as antigens in carbohydrate-based conjugate vaccines, non-native ligands can afford enhanced immunogenicity over their native counterparts, resulting in elevated antibody titers and improved vaccine constructs [32,39–41]. Alternatively, biomimetic functional groups have been shown to have drastically different effects on enzymes. Although binding to a target can be enhanced by biomimetic replacement, its influence on the transition state of the enzymatic process can dictate whether it behaves as a substrate or an inhibitor of the enzyme. Inhibitors can be generated by directly modifying the functional group involved in the enzymatic transformation so that the ligand binds but no longer has the functionality required for the reaction. In glycosidases and related enzymes, the addition of an electron-withdrawing substituent can have profound effects on the enzymatic rate due to its destabilization of the oxocarbenium intermediate, where the position of the electron-withdrawing substituent can influence the degree of destabilization as inductive effects quickly diminish with distance [32–34].

2. Modifications to the O-Glycoside Linkage Native O-glycan structures are sensitive to both chemical and enzymatic hydrolysis in vivo which can limit their oral bioavailability and lifetime in circulation, and subsequently their use in therapeutics. A common strategy used in glycomimetics to improve stability has been to replace the oxygen atom of the glycosidic linkage with a less labile atom or group, such as nitrogen, carbon, sulfur, or selenium [42–51]. Although these analogues impart enhanced stability, they can also introduce conformational changes which increase the entropic barrier of binding causing losses in affinity; often a desirable balance between stability and activity must be established. These conformational changes can arise from a combination of larger atom (e.g., S, Se) or loss of the exo-anomeric effect due to replacement with a less electronegative substituent (e.g., C). This conformational flexibility can potentially be reduced through the addition of steric bulk that constrains the molecule to a particular conformation [52–54], or by the addition of electronegative atoms such as fluorine to C-glycosides to reestablish the electron-withdrawing character of the anomeric substituent [51,55–58]. Aside from the more conventional isosteres listed above, the glycosidic linkage has also been replaced by other functionalities (sulfones, sulfoxides), or the linkage position has been varied to non-natural analogues which has enhanced stability [59–62]. As this has been a very well covered area of glycomimetics with many prominent examples, it has not been covered further in this review. Biomimetics 2019, 4, 53 5 of 23

3. Replacement of the Endocyclic O Atom In modifying glycan acetals the endocyclic oxygen atom can also be exchanged, for example, to form iminosugars, carbasugars, or thiosugars [44,63–70]. In fact, iminosugars are found naturally and widespread among different plants and microorganisms [71,72]. Hemiaminals can be unstable and therefore the lack of a C-1 oxygen-bearing substituent has been useful for improving the structural stability of these derivatives. On account of the endocyclic N atom which becomes positively charged at physiological pH, this class of molecules can mimic the charged oxocarbenium transition state observed in glycan-processing enzymes. Deoxynojirimycin is a well-studied naturally abundant iminosugar from which several drugs have been developed, including miglitol (for type II diabetes) and miglustat (N-butyl-1-deoxynojirimycin; for lysosomal storage disease) [72,73]. BiomimeticsAn endocyclic- 2019, 4, x FORS PEER-containing REVIEW derivative of α-GalCer (KRN7000) was synthesized for use5 as of an22 adjuvant, and was observed to have a stronger immunostimulatory effect on IFN-γ production invariant natural killer T cells as compared to its endocyclic-O-containing counterpart [74]. In in invariant natural killer T cells as compared to its endocyclic-O-containing counterpart [74]. combination with perfluoroalkyl lipid modification, a selective modulation of the immune response In combination with perfluoroalkyl lipid modification, a selective modulation of the immune could be achieved where the glycomimetic 5-thio-α-galactopyranosyl-N-perfluorooctanoyl response could be achieved where the glycomimetic 5-thio-α-galactopyranosyl-N-perfluorooctanoyl phytosphingosine was shown to selectively stimulate TH1 cytokine production, while its phytosphingosine was shown to selectively stimulate TH1 cytokine production, while its perfluorotetradecanoyl derivative elicited a TH2 cytokine profile. perfluorotetradecanoyl derivative elicited a T 2 cytokine profile. 5-Thiopyranosyl rings were also usedH in the preparation of a series of di-, tri-, and 5-Thiopyranosyl rings were also used in the preparation of a series of di-, tri-, and tetrasaccharide tetrasaccharide β-(1→3)-glucan analogues (Figure 1) [75]. Upon evaluation of their binding to β-(1 3)-glucan analogues (Figure1)[ 75]. Upon evaluation of their binding to complement receptor 3 complement→ receptor 3 (CR3) and dectin-1 via an inhibitory assay, it was observed that the (CR3) and dectin-1 via an inhibitory assay, it was observed that the trisaccharide derivative of 3 was trisaccharide derivative of 3 was most optimally sized. This was a surprising observation given that most optimally sized. This was a surprising observation given that the native substrate (1) requires the native substrate (1) requires much larger oligomers for effective binding. This enhancement in much larger oligomers for effective binding. This enhancement in affinity is believed to arise from the affinity is believed to arise from the more hydrophobic nature of S as compared to O, which enhances more hydrophobic nature of S as compared to O, which enhances hydrophobic interactions with the hydrophobic interactions with the protein. Although desirably more hydrophobic in nature, C–S protein. Although desirably more hydrophobic in nature, C–S bonds are also longer and therefore can bonds are also longer and therefore can result in steric mismatch of the binding partners. The related result in steric mismatch of the binding partners. The related hydroxylamine analogues (2) were also hydroxylamine analogues (2) were also synthesized and showed comparable affinity to the 5- synthesized and showed comparable affinity to the 5-thiopyranosyl derivatives [75,76]. The di-and thiopyranosyl derivatives [75,76]. The di-and tri-hydroxylamine mimetics were both shown to inhibit tri-hydroxylamine mimetics were both shown to inhibit CR3 and dectin-1 binding, which was again CR3 and dectin-1 binding, which was again attributed to the increase in hydrophobic nature resulting attributed to the increase in hydrophobic nature resulting from the more polarizable hydroxylamine from the more polarizable hydroxylamine moiety and lack of a C-2 substituent [76–78], which could moiety and lack of a C-2 substituent [76–78], which could then interact more strongly with hydrophobic then interact more strongly with hydrophobic residues in the binding site (e.g., Trp221 and His223 in residues in the binding site (e.g., Trp221 and His223 in dectin-1 [79]). dectin-1 [79]).

OH OH OH O O O HO HO HO OH HO O O OH OH OH n 1

OH OH OH

HO O HO O HO OH HO N N N n 2

OH OH OH HO S HO S HO S HO S S OH OH OH OH n 3 Figure 1. Oligosaccharide analogues to mimic β-(1 3)-glucan were synthesized as inhibitors of → complementFigure 1. Oligosaccharide receptor 3 (CR3) analogues and dectin-1, to mimic targeting β-(1 the→3)-glucan regulation were of phagocytosis synthesized [75as, 76inhibitors]. of complement receptor 3 (CR3) and dectin-1, targeting the regulation of phagocytosis [75,76]. Cyclic phosphonates (‘phostones’) and phosphinates (‘phostines’) have also been used as glycomimetics,Cyclic phosphonates with the phosphinolactone (‘phostones’) and group phosphin (O–P=O)ates functioning (‘phostines’) as a bioisosterehave also ofbeen hemiacetals used as (O–C–OH).glycomimetics, Phostones with the have phosphinolactone been used as group mimetics (O–P=O) of both functioning furanosides as a and bioisostere pyranosides, of hemiacetals where a (O–C–OH). Phostones have been used as mimetics of both furanosides and pyranosides, where a phosphorous atom replaces the anomeric carbon (4–8; Figure 2) [80,81]. Similar to phostones, phostines can additionally impart an enhanced stability owing to their C-glycoside-character, and have been used to generate substrates that were antiproliferative against rat glioma tumor cells or used as antidepressants [82,83]. Phosphinolactone is a good metal chelator and H-bond acceptor; evidence supports that 6-membered rings containing phosphorus adopt stereoelectronic-dependent interactions comparable to the anomeric effect [84].

Biomimetics 2019, 4, 53 6 of 23

phosphorous atom replaces the anomeric carbon (4–8; Figure2)[ 80,81]. Similar to phostones, phostines can additionally impart an enhanced stability owing to their C-glycoside-character, and have been used to generate substrates that were antiproliferative against rat glioma tumor cells or used as antidepressants [82,83]. Phosphinolactone is a good metal chelator and H-bond acceptor; evidence supports that 6-membered rings containing phosphorus adopt stereoelectronic-dependent interactions BiomimeticsBiomimetics 2019, 4, x 2019FOR, PEER4, x FOR REVIEW PEER REVIEW 6 of 22 6 of 22 Biomimeticscomparable 2019 to, 4 the, x FOR anomeric PEER REVIEW effect [84]. 6 of 22

HO OHHO OH O O O O HO O O HO HOOH HO OHHO OH OP O P O OMe OMe HO HO OEt OEt HOHO HOP P HO OOH O P OMe HO HO O O HO OEt HO P HO HOO P O HO P O HO HO O HO 4 O 4 P HO7 7 4 O O 7 HO O HOO O OMe OMe HO OHHO OH HOHO HOO O O O O O OMe HO HOOOH HO OH OH OP O P HO O O HO HO OEt OEt HO HOPO P 6 OH6 P HO O HO OEt HO P 6 OMe OMe HO HO 5 5 OMe HO8 8 5 8 Figure 2.FigureFigure A series 2. ofAA seriesphostone series of of phostone phostone analogues analogues analogues have been have have evaluated b beeneen evaluated evaluated as glycomimetics as glycomimetics [80,81]. [80,81]. [80,81]. Figure 2. A series of phostone analogues have been evaluated as glycomimetics [80,81]. CarbasugarsCarbasugarsCarbasugars have have also alsohave been beenalso developed developedbeen developed as asglycan glycan as mimetics,glycan mimetics, mimetics, where where thewhere the OO-endocyclic -endocyclicthe O-endocyclic atom atom is atom is is Carbasugars have also been developed as glycan mimetics, where the O-endocyclic atom is replacedreplacedreplaced by by a aC Cbyatom atom a C[69]. [atom69 ].5a’-Carbamaltoses 5a’-Carbamaltoses[69]. 5a’-Carbamaltoses were were synthesized were synthesized synthesized where where awhere aCH CH2 group2a groupCH 2 replacedgroup replaced replaced the the the replaced by a C atom [69]. 5a’-Carbamaltoses were synthesized where a CH2 group replaced the endocyclicendocyclicendocyclic oxygen, oxygen, oxygen, with with the the with derivatives derivatives the derivatives glycosidically glycosidically glycosidically linked linked via linked via imino, imino, via ether, imino, ether, or ether, orsulfide sulfide or linkagessulfide linkages linkages [85]. [85 ]. [85]. endocyclic oxygen, with the derivatives glycosidically linked via imino, ether, or sulfide linkages [85]. TheThe derivatives derivativesThe derivatives were basedbased were on onbased the theα-glucosidase onα-glucosidase the α-glucosidase inhibitor inhibitor methyl inhibitor methyl acarviosin methylacarviosin and acarviosin the andN-linked the and N glycoside-linked the N -linked The derivatives were based on the α-glucosidase inhibitor methyl acarviosin and the N-linked glycosideshowedglycoside showed moderate showed moderate inhibition moderate inhibition of yeast inhibition αof-glucosidase yeast of α -glucosidaseyeast [85 α,86-glucosidase]. Cellotriose [85,86]. [85,86].Cellotriose and cellotetraose Cellotriose and cellotetraose and derivatives cellotetraose glycoside showed moderate inhibition of yeast α-glucosidase [85,86]. Cellotriose and cellotetraose derivativeswere alsoderivatives were synthesized also were synthesized wherealso synthesized one where of the one ringswhere of hadthe one rings been of the replacedhad rings been had byreplaced abeen cyclohexene replaced by a cyclohexene by moiety, a cyclohexene in moiety, efforts tomoiety, derivatives were2 also synthesized where one of the rings had been replaced by a cyclohexene moiety, inmimic effortsin the toefforts spmimic-hybridized to themimic sp2-hybridized the transition sp2-hybridized state transition of cellulase transition state hydrolysisof statecellul ofase cellul [ 87hydrolysis]. Thease hydrolysis cyclohexene [87]. The [87]. mimeticscyclohexene The cyclohexene were in efforts to mimic the sp2-hybridized transition state of cellulase hydrolysis [87]. The cyclohexene mimeticsindeedmimetics shownwere indeed to were effectively shownindeed bind toshown effectiv an endoglucanaseto elyeffectiv bindely an bind (NCE5)endoglucanase an fromendoglucanaseHumicola (NCE5) insolens (NCE5)from Humicolaand from were Humicola insolens observed insolens mimetics were indeed shown to effectively bind an endoglucanase (NCE5) from Humicola insolens andto bindwereand strongerobserved were observed than to bind their tostronger native bind stronger pyranose than their than analogues. native their pyranose native pyranose analogues. analogues. and were observed to bind stronger than their native pyranose analogues. 4. 4.Replacement Replacement4. Replacement of ofOH OH Functional of Functional OH Functional Groups Groups Groups 4. Replacement of OH Functional Groups BioisostericBioisostericBioisosteric replacement replacement replacement of functionalfunctional of functional groups groups is groups commonlyis commonly is commonly used used in medicinal in used medicinal in chemistry medicinal chemistry to improvechemistry to to Bioisosteric replacement of functional groups is commonly used in medicinal chemistry to improvethe desiredimprove the desired properties the desiredproperties of a molecule.properties of a molecule. Biomimeticof a molecule. Biom functionalimetic Biom functionalimetic groups functional shouldgroups havegroupsshould similar shouldhaveelectronic similar have similar improve the desired properties of a molecule. Biomimetic functional groups should have similar electronicand stericelectronic and properties steric and properties tosteric the originalproperties to the group, originalto the but original oftengroup, di ffbutgroup,erent often bioisostericbut different often different groupsbioisosteric are bioisosteric appropriate groups groupsare for are electronic and steric properties to the original group, but often different bioisosteric groups are appropriatedifferentappropriate situations for different for depending different situations onsituations thedepending key depending interactions on the keyon involved theinteractions key (Table interactions 2involved). Which involved (Table bioisostere 2).(Table Which to use2). Which appropriate for different situations depending on the key interactions involved (Table 2). Which bioisosteredependsbioisostere onto theuse requirements dependsto use depends on the of thatrequirementson the particular requirements of functional that particularof th groupat particular functional in that specificfunctional group binding ingroup that event specificin that with specific bioisostere to use depends on the requirements of that particular functional group in that specific bindingregardsbinding event to, for with event example, regards with steric regardsto, for restrictions example, to, for example, orster H-bondic restrictions ster donorsic restrictions /oracceptors. H-bond or H-bond donors/acceptors. donors/acceptors. binding event with regards to, for example, steric restrictions or H-bond donors/acceptors. Table 2. Common bioisosteres of different functional groups. Table 2. TableCommon 2. Common bioisosteres bioisosteres of different of different functional functional groups. groups. Table 2. Common bioisosteres of different functional groups. OriginalOriginalOriginal Group Group Potential Potential Potential Replacements Replacements Replacements Original Group Potential Replacements H H D FD F H D F

OH OH F SHF SeHSH SeHNH2 NH2 OH F SH SeH NH2

O OS S O S

O O O O O O O O O O O O S S OH OH OH OH NH2 NH2S NH OH OH 2

CommonCommon bioisosteres bioisosteres of the OH of thegroup OH include group includeF, SH, SeH, F, SH, and SeH, NH and2, although NH2, although on alkyl on scaffolds alkyl scaffolds Common bioisosteres of the OH group include F, SH, SeH, and NH2,, although although on on alkyl alkyl scaffolds scaffolds the latterthe becomes latter becomes charged charged at physiological at physiological pH and pH therefore and therefore poorly mimicspoorly2 mimicsthe neutral the neutralOH group. OH group. the latter becomes charged at at physiological pH pH and and therefore therefore poorly poorly mimics mimics the the neutral neutral OH OH group. group. OH groupsOH groupsinvolved involved in metal in chelation metal chelation can be replacedcan be replaced with functionalities with functionalities that act thatas improved act as improved OH groups involved in metal chelation can be replaced with functionalities that act as improved metal ligands,metal ligands, which canwhich be canused be to used enhance to enhance binding binding affinity. affinity. Several Severaldifferent different bioisosteric bioisosteric metal ligands, which can be used to enhance binding affinity. Several different bioisosteric replacementsreplacements have been have used been in usedthe generation in the generation of glycomimetics of glycomimetics and are anddiscussed are discussed in the context in the contextof of replacements have been used in the generation of glycomimetics and are discussed in the context of this review.this review. this review. It is importantIt is important to keep toin keepmind in that mind the that modifi thecation modifi ofcation carbohydrates of carbohydrates with functional with functional groups groups It is important to keep in mind that the modification of carbohydrates with functional groups that are thatmore are electron-withdrawing more electron-withdrawing (e.g., OH (e.g., to F, OH CO to2− F,in COuronic2− in acids),uronic regardlessacids), regardless of position, of position, has has that are more electron-withdrawing (e.g., OH to F, CO2− in uronic acids), regardless of position, has been shownbeen shownto increase to increase stability stabil of andity ofpreference and preference for the forα-anomer the α-anomer [88]. Although [88]. Although somewhat somewhat been shown to increase stability of and preference for the α-anomer [88]. Although somewhat counter-intuitive,counter-intuitive, since electron-withdrawing since electron-withdrawing groups groupsreduce electronreduce electron density densityon the endocyclic-on the endocyclic-O- O- counter-intuitive, since electron-withdrawing groups reduce electron density on the endocyclic-O- atom andatom thereby and therebywould reducewould thereduce strength the strength of the endo-anomeric of the endo-anomeric effect, it effect, is suggested it is suggested that these that these atom and thereby would reduce the strength of the endo-anomeric effect, it is suggested that these

Biomimetics 2019, 4, 53 7 of 23

OH groups involved in metal chelation can be replaced with functionalities that act as improved metal ligands, which can be used to enhance binding affinity. Several different bioisosteric replacements have been used in the generation of glycomimetics and are discussed in the context of this review. It is important to keep in mind that the modification of carbohydrates with functional groups that are more electron-withdrawing (e.g., OH to F, CO2− in uronic acids), regardless of position, has been shown to increase stability of and preference for the α-anomer [88]. Although somewhat counter-intuitive, since electron-withdrawing groups reduce electron density on the endocyclic-O-atom and thereby would reduce the strength of the endo-anomeric effect, it is suggested that these electron-withdrawing substituents can reduce steric gauche interactions and enhance favorable Coulombic interactions between the C-1 and C–H ring substituents [88].

4.1. Deoxygenation The deoxygenation of OH moieties that are non-essential for binding has shown to be a very useful method for enhancing binding affinities. Deoxygenation removes a polar functional group, which reduces polar surface area and its associated desolvation penalty, and in addition can generate new hydrophobic contacts with the protein. Studies support that at least two H-bonds to an OH group are required for a favorable binding free energy in shallow, solvent-exposed binding sites, in which case the gain in enthalpy afforded by ligand interactions becomes stronger than the enthalpic cost associated with desolvation of the polar OH group [89,90]. As deeply buried binding sites tend to be more hydrophobic, the strength of single H-bonds in these systems is enhanced. Deoxygenation at the C-6 position of pyranoses is even more favorable as it increases hydrophobicity of the molecule, and also removes a degree of freedom which lowers the entropic cost of binding. The substitution of an OH group for H has other effects on the ligand properties. In general, deoxygenation removes an electron-withdrawing substituent and therefore increases electron density on the glycan scaffold, which can make other OH substituents more nucleophilic and potentially strengthen (or weaken) neighboring interactions such as metal coordination or H-bonding. OH groups that are not in direct contact with the protein can also be involved in pre-organization of the ligand; if this pre-organization is disturbed, then it can increase the entropic penalty of binding and therefore it may be more beneficial to retain the substituent or replace it with an alternative biomimetic group (e.g., fluorine). Diverse methods have been used for synthesizing deoxygenated mimetics [91]. Deoxygenated derivatives have also been widely used in activity studies to gain structural information on the importance of different OH groups in receptor binding [92,93].

4.2. Deoxyfluorination Fluorine is a commonly utilized bioisostere of the OH group, due to its polarity and similarity in size. The van der Waals radius of F (1.47 Å) is between that of H (1.2 Å) and O (1.57 Å) making it a suitable mimic of both [94]. Fluorine is also more hydrophobic than oxygen, and as a bioisostere of OH groups it reduces the polar surface area and binding-associated desolvation penalties and can also enhance oral bioavailability by improving passive permeation through the intestinal membrane. Although hydrophobic, it still generates a polar C–F bond that can undergo polar interactions with the protein, although typically of weaker strength. The substitution of F for OH has been widely studied, both in structural analyses of binding sites (to evaluate H-bond donor and acceptor requirements) and in the synthesis of therapeutic ligands [64,95–98]. Given the significant electronegativity of fluorine, its inductive ability can strongly destabilize oxocarbenium transition states in carbohydrate-binding enzymes and significantly slow enzymatic rates such that fluorinated glycomimetics act as excellent inhibitors of these enzymes. This was exemplified in the synthesis of 4-fluorinated derivatives of GlcNAc 1-phosphate (and related per-acetylated derivatives) which were used to reduce N-glycan expression on human prostate (PC-3) cancer cells and inhibit cell-growth, although the two effects appeared to arise from inhibition of two different Biomimetics 2019, 4, 53 8 of 23 pathways [99]. In the field of diagnostics, [18F]-fluorinated derivatives have shown wide applicability in positron emission tomography (PET), both in the detection of cancer as well as in newer applications such as the imaging of infections [100–102]. 2-Deoxy-2-fluoro derivatives of two Phlorizin analogues were prepared and evaluated as inhibitors of the sodium-glucose co-transporters (SGLT1 and SGLT2) in the search for a novel therapy against type II diabetes [95]. SGLT2 is a glucose transporter located in the renal proximal tubule [103,104] which plays an important role in glucose reabsorption—by inhibiting the transporter more glucose is eliminated instead of reabsorbed, thereby decreasing glucose levels in circulation [105]. Phlorizin is a natural product isolated from apple tree bark, which inhibits both SGLT1 (causing adverse effects [106,107]) and SGLT2 and is also prone to hydrolysis, therefore improved mimetics are desirable. 2-Fluorinated analogues of SGLT2 inhibitors were evaluated for their activity and improved resistance to hydrolysis [95]; although a substrate was shown to be selective for SGLT2, it was surprisingly found to be metabolized much more quickly, presumably due to its higher lipophilicity which made it a better substrate for metabolic enzymes. Deoxyfluorination has also been widely used in the synthesis of haptens for the preparation of carbohydrate-based conjugate vaccines. Non-natural epitopes can often be more immunogenic than native glycans, and given the similarity between the F and OH groups the antibodies elicited by non-natural epitopes are often cross-reactive with the natural targets. A small library of fluorinated trisaccharide antigens based on lipophosphoglycan from Leishmania donovani were synthesized in efforts to develop a vaccine against leishmaniasis [108]. Different fluorinated derivatives of β-Gal-(1 4)-[α-Man-(1 2)]-α-Man were evaluated to identify an appropriate fluorination pattern → → capable of improving hydrolytic stability and enhancing immunogenicity, yet still generating antibodies cross-reactive with the native glycan. A conjugate vaccine based on mucins also utilized deoxyfluorination—although the native glycopeptides were shown to have good immunogenicity, they were rapidly degraded in vivo and therefore an improvement in stability was necessary [109]. Glycomimetic derivatives with fluorine at C-6, C-20, or C-60 of Tn, TF, and 6-sialyl-TF antigens were synthesized and incorporated into MUC1 sequences containing a functional handle for subsequent incorporation into conjugate vaccines. Fluorinated haptens have been used in a number of other examples to improve both immunogenicity and hydrolytic stability [110–112]. Interestingly, one study on fluorinated GM4 derivatives reported a significant change in biological effects between fluorinated and non-fluorinated analogues [113]. GM4 is a sialosylgalactosylceramide ganglioside which is a major constituent of myelin sheath. Biological studies indicated that the natural substrate had no effect on cell viability in myelin basic protein (MBP+) oligodendrocytes, while the fluorinated derivative surprisingly upregulated oligodendrocyte differentiation and is being further evaluated as a therapeutic agent to inhibit demyelination, potentially offering a therapy for re-myelination of nerves in individuals affected with multiple sclerosis.

4.3. Methyl Etherification Monomethyl ethers have also been used as mimics of the OH group, and widely used in activity studies to assess the binding requirements for ligand interactions. For example, each of the different monomethyl ethers of β-lactoside were synthesized and evaluated as ligands for both ricin and agglutinin, two galactoside-specific lectins isolated from Ricinus communis seeds [114]. In another study, a library of double-modified xyloside analogues were synthesized [115]. The library was comprised of the different combinations of monomethyl etherified, epimerized, deoxygenated, and deoxyfluorinated substitutions at the C-2 and C-4 positions with an anomeric β-linked naphthyl group, and were evaluated for their galactosyltransferase activity. As mentioned previously, the sodium glucose co-transporter 2 (SGLT2) is a novel target for lowering glucose levels in diabetic patients since inhibitors have been shown to potentially lower the renal reabsorption of glucose and reduce the risk of hyperglycemia [116,117]. In a recent study, a library of Sergliflozin-A (9) derivatives were prepared with various substitutions on the glucose fragment Biomimetics 2019, 4, 53 9 of 23

(methyl etherification, deoxygenation, deoxyfluorination) (Figure3)[ 118]. The 4-OMe derivative (10) displayed similar potency to Sergliflozin-A (73% of the inhibition) in a rat urinary glucose excretion experiment, and with significantly improved pharmacokinetic stability. The 6-deoxy analogue 12 was Biomimetics 2019, 4, x FOR PEER REVIEW 9 of 22 also shown to be a mild inhibitor, while derivatives with substitutions at C-2 and C-3 were inactive.

OMe OMe

HO HO O O HO O MeO O HO HO OH OH

910

OMe OMe

MeO O O HO HO O HO O HO OH OH

11 12 Figure 3. Sergliflozin-A (9) is an inhibitor of sodium glucose co-transporter 2 (SGLT2) but is ineffective as Figurea therapeutic 3. Sergliflozin-A against diabetes (9) is an due inhibitor to its poor of sodium metabolic glucose stability. co-transporter A library of 2 Sergliflozin-A(SGLT2) but is derivativesineffective asidentified a therapeutic the 4-O -methylatedagainst diabetes derivative due to10 itsas havingpoor metabolic similar potency stability. to Sergliflozin-AA library of Sergliflozin-A but enhanced derivativespharmacokinetic identified stability the [4-118O-methylated]. derivative 10 as having similar potency to Sergliflozin-A but enhanced pharmacokinetic stability [118]. Although potency was retained for the above Sergliflozin-A derivative, methyl etherification of carbohydrateAlthough hydroxyl potency groups was retained is typically for the not above the best Sergliflozin-A choice of bioisosteric derivative, replacement. methyl etherification The CH3 group of carbohydrateis much larger hydroxyl than an H groups and can is introduce typically unfavorablenot the best steric choice clashes, of bioisosteric and in addition replacement. the methyl The etherCH3 group isis unablemuch tolarger act as than an H-bond an H and donor can in introduce ligand–protein unfavorable interactions. steric One clashes, study and which in demonstratesaddition the methylthis incompatibility ether group is was unable aiming to act to synthesizeas an H-bond inhibitors donor ofin polypeptide-ligand–proteinα-GalNAc-transferase, interactions. One study an whichenzyme demonstrates which catalyzes this theincompatibility first step of O was-glycan aimi biosynthesisng to synthesize [119]. inhibitors Derivatives of of polypeptide- UDP-GalNAcα- whichGalNAc-transferase, were methylated an atenzyme either thewhich 3-, 4-,catalyzes or 6-position the first were step not of substrates O-glycan forbiosynthesis the enzyme [119]. and Derivativesdisplayed only of veryUDP-GalNAc weak activity which as competitivewere methylated inhibitors. at either the 3-, 4-, or 6-position were not substrates for the enzyme and displayed only very weak activity as competitive inhibitors. 4.4. SH and SeH Substitution 4.4. SHThiosugars and SeH Substitution have been reported as naturally occurring products, although few examples of free thiol-containingThiosugars structureshave been exist reported such as thenaturally antibiotics occu rhodonocardinrring products, A although and B isolated few examples from a Nocardia of free thiol-containingspecies [120]. Both structures S and Se atomsexist such are commonly as the antibiotics used bioisosteres rhodonocardin of O. Although A and sharingB isolated comparable from a Nocardiaelectronic species properties, [120]. S andBoth Se S areand both Se atoms larger are atoms common whichly results used bioisosteres in longer C–S of/Se O. bonds Although and disharingfferent comparablebond angles. electronic These changes properties, can have S and a negativeSe are both impact larger on atoms steric which matching results with in thelonger native C–S/Se substrates. bonds Althoughand different atom bond size angles. varies, theThese larger changes size and can polarizability have a negative of S andimpact Se atomson steric results matching in an enhancedwith the nativelipophilicity substrates. which Although can improve atom hydrophobic size varies, interactionsthe larger size with and the polarizability protein and reduceof S and desolvation Se atoms resultsrequirements in an enhanced of ligand binding.lipophilicity These which mimetics can improve are also hydrophobic worse H-bond interactions donors as comparedwith the protein to OH andgroups, reduce but non-covalentdesolvation requirementsπ-interactions of are ligand typically binding. stronger These with mimetics S than with are O also [121 worse,122]. H-bond donorsThiol as compared or selenol replacementto OH groups, of but carbohydrate non-covalent OH π functional-interactions groups are typically is not entirely stronger common, with S likely than withgiven O the [121,122]. difficulty in synthesis and isolation of these compounds caused by their propensity to dimerize into disulfidesThiol or selenol and diselenides. replacement Examples of carbohydrate of both dimers OH functional and alkylated groups thio- is/selenoether not entirely derivatives common, likelyhave beengiven presented the difficulty in the in literaturesynthesis andand studiedisolation for of variousthese compounds biological caused applications, by their such propensity as their toadjuvant dimerize/immunostimulatory into disulfides and abilities, diselenides. carcinostatic Examples activities, of both dimers and antioxidant and alkylated properties thio-/selenoether [123–125]. derivativesSeveral have methods been havepresented been establishedin the literature to synthesize and studied thiosugars for various to obtain biological glycomimetics applications, such such as 13as andtheir14 adjuvant/immunostimulatory(Figure4)[ 126]. As it was previously abilities, reportedcarcinostatic that activities,S-linked pseudodisaccharidesand antioxidant properties bound [123–125].better to the plant lectin concanavalin A (ConA) than their respective O-derivatives [60], isothermal titrationSeveral calorimetry methods (ITC)have andbeen saturation established transfer to synthesize difference thio (STD)sugars NMRto obtain experiments glycomimetics were usedsuch as to 13evaluate and 14 the (Figure binding 4) [126]. of these As2-thioglycosides it was previously to reported ConA. Surprisingly, that S-linked the pseudodisaccharides 2-deoxy-2-thio-mannoside bound better to the plant lectin concanavalin A (ConA) than their respective O-derivatives [60], isothermal titration calorimetry (ITC) and saturation transfer difference (STD) NMR experiments were used to evaluate the binding of these 2-thioglycosides to ConA. Surprisingly, the 2-deoxy-2-thio-mannoside derivative 14 was observed to bind ConA much better than Man itself [126,127], although STD NMR experiments suggested that this enhancement in affinity is actually the result of a slightly altered binding mode.

Biomimetics 2019, 4, 53 10 of 23 derivative 14 was observed to bind ConA much better than Man itself [126,127], although STD NMR Biomimetics 2019, 4, x FOR PEER REVIEW 10 of 22 experiments suggested that this enhancement in affinity is actually the result of a slightly altered

Biomimeticsbinding mode. 2019, 4, x FOR PEER REVIEWHO HO SH 10 of 22 HO O HO O HO OMe HO HO HO SH SH O OOMe HO OMe HO HO 13 HO 14 SH OMe 13 14 Figure 4. 2-Thiocarbohydrates were evaluated as ligands of the plant lectin ConA [126]. Figure 4. 2-Thiocarbohydrates were evaluated as ligands of the plant lectin ConA [126]. InFigure another 4. 2-Thiocarbohydrates study, 6-thio and were 6-seleno evaluated derivatives as ligands of of ManNAc the plant lectinwere ConAsynthesized [126]. and evaluated as inhibitorsIn another of study,human 6-thio N-acetylmannosamine and 6-seleno derivatives kinase of (MNK) ManNAc (Figure were 5) synthesized [128]. It was and hypothesized evaluated as In another study, 6-thio and 6-seleno derivatives of ManNAc were synthesized and evaluated thatinhibitors these analogues of human Nwould-acetylmannosamine still bind to the kinaseenzyme, (MNK) yet could (Figure not5 be)[ 128phosphorylated]. It was hypothesized and therefore that as inhibitors of human N-acetylmannosamine kinase (MNK) (Figure 5) [128]. It was hypothesized wouldthese analogues act as inhibitors would still of bindthe native to the enzyme,substrate. yet 6- couldO-Ac-ManNAc not be phosphorylated was previously and thereforereported wouldas an that these analogues would still bind to the enzyme, yet could not be phosphorylated and therefore inhibitoract as inhibitors of the ofenzyme the native [122,129,130] substrate. but 6-O its-Ac-ManNAc ester is easily was hydrolyzed previously reportedin vivo making as an inhibitor it a poor of would act as inhibitors of the native substrate. 6-O-Ac-ManNAc was previously reported as an therapeuticthe enzyme option. [122,129 In,130 synthesizing] but its ester the is 6-substituted easily hydrolyzed ligands,in vivo the highmaking oxidation it a poor potential therapeutic of selenols option. inhibitor of the enzyme [122,129,130] but its ester is easily hydrolyzed in vivo making it a poor preventedIn synthesizing isolation the 6-substituted of the monomer ligands, and as the a highresult oxidation only diselenide potential dimer of selenols 17 could prevented be evaluated isolation [128]; of therapeutic option. In synthesizing the 6-substituted ligands, the high oxidation potential of selenols forthe the monomer thiol derivative, and as a result both only monomer diselenide 15 and dimer dimer17 could 16 could be evaluated be isolated [128 and]; for evaluated. the thiol derivative, Both the prevented isolation of the monomer and as a result only diselenide dimer 17 could be evaluated [128]; presenceboth monomer of Se 15andand the dimer dimerization16 could itself be isolated provided and enhancements evaluated. Both in theactivity; presence the diselenide of Se and the17 for the thiol derivative, both monomer 15 and dimer 16 could be isolated and evaluated. Both the inhibiteddimerization MNK itself inprovided the low enhancementsμM range and in upon activity; treatment the diselenide of Jurkat17 inhibited cells a reduction MNK in thein lowsurfaceµM presence of Se and the dimerization itself provided enhancements in activity; the diselenide 17 sialylationrange and uponwas also treatment observed. of Jurkat cells a reduction in surface sialylation was also observed. inhibited MNK in the low μM range and upon treatment of Jurkat cells a reduction in surface sialylation was also observed. HO S HO Se HS NHAc NHAc NHAc O HO S O HO Se O HO NHAc NHAc HO HO OSHO HO OSeHO HS NHAc HO NHAc HO NHAc O HO S O OH HO Se O OH HO OH NHAc NHAc HO 15 HO O HO16 HO O HO17 HO HO OH OH OH Figure 5.5. 6-6-SS-- and and 6- 156-SeSe-derivatives-derivatives of ManNAcofHO ManNAc16 were were synthesized synthesi andzed HO evaluatedand evaluated17 as inhibitors as inhibitors of human of humanN-acetylmannosamine N-acetylmannosamine kinase (MNK).kinase (MNK). The high The oxidation high oxidation potential potential of Se preventedof Se prevented isolation isolation of its Figure 5. 6-S- and 6-Se-derivatives of ManNAc were synthesized and evaluated as inhibitors of ofmonomer its monomer [128]. [128]. human N-acetylmannosamine kinase (MNK). The high oxidation potential of Se prevented isolation 4.5. Other Modifications 4.5. Otherof its monomerModifications [128]. Propylamycin (18) is a 4 -deoxy-4 -alkyl paromomycin which is demonstrably effective against 4.5. OtherPropylamycin Modifications (18 ) is a 40′-deoxy-4′0-alkyl paromomycin which is demonstrably effective against a broad selection of ESKAPE pathogens (Figure6)[ 131]. Early studies had demonstrated that a broad selection of ESKAPE pathogens (Figure 6) [131]. Early studies had demonstrated that 4′-O- 4 -O-ethylationPropylamycin of paromomycin(18) is a 4′-deoxy-4 afforded′-alkyl enhanced paromomycin ribosomal which selectivity is demonstrably and reduced effective ototoxicity against ethylation0 of paromomycin afforded enhanced ribosomal selectivity and reduced ototoxicity in ain broad antibiotic selection treatment of ESKAPE [132], withpathogens the latter (Figure being 6) one[131]. of Early the major studies negative had demonstrated side effects correlatedthat 4′-O- antibiotic treatment [132], with the latter being one of the major negative side effects correlated with withethylation aminoglycoside of paromomycin therapies. afforded Although enhanced less ototoxic, ribosomal this selectivity modification and was reduced also correlated ototoxicity with in aminoglycoside therapies. Although less ototoxic, this modification was also correlated with a loss in antibiotica loss in activity. treatment In [132], contrast, with 4 the-deoxygenated latter being one paromomycin of the major maintained negative side antibacterial effects correlated activity with but activity. In contrast, 4′-deoxygenated0 paromomycin maintained antibacterial activity but had no aminoglycosidehad no enhancement therapies. in ribosomal Although selectivity. less ototoxic, Based this onmodification these results, was it also was correlated hypothesized with a that loss the in enhancement in ribosomal selectivity. Based on these results, it was hypothesized that the alkyl activity.alkyl derivatization In contrast, with4′-deoxygenated an electron-donating paromomycin substituent maintained could aantibacterialfford both the activity desired but selectivity had no derivatization with an electron-donating substituent could afford both the desired selectivity while whileenhancement maintaining in ribosomal antibacterial selectivity. activity. Based Indeed, on these replacement results, it of was the 4hypothesized-OH group withthat the a propyl alkyl maintaining antibacterial activity. Indeed, replacement of the 4′-OH group0 with a propyl chain derivatizationchain interfered with with an protein electron-donating synthesis in substituen bacteria andt could also reducedafford both inner the ear desired toxicity selectivity in a guinea while pig interfered with protein synthesis in bacteria and also reduced inner ear toxicity in a guinea pig model maintainingmodel [131]. Thisantibacterial activity isactivity. believed Indeed, to arise fromreplacement the electron-donating of the 4′-OH alkyl group substituent with a propyl which makeschain [131]. This activity is believed to arise from the electron-donating alkyl substituent which makes the interferedthe pyranose with ring protein more synthesis electron-rich in bacteria and its and endocyclic also reduced O atom inner more ear toxicity nucleophilic in a guinea and therefore pig model a pyranose ring more electron-rich and its endocyclic O atom more nucleophilic and therefore a better [131].better This H-bond activity acceptor, is believed enhancing to arise the from ‘pseudo’ the electron-donating base pair interaction alkyl with substituent A1408 upon which binding. makes the H-bond acceptor, enhancing the ‘pseudo’ base pair interaction with A1408 upon binding. pyranose ring more electron-rich and its endocyclic O atom more nucleophilic and therefore a better

H-bond acceptor, enhancing the ‘pseudo’ base pairOH interaction with A1408 upon binding. O HO OH NH2 H2N OO O NH2 HOHO O O NH2 OH H2N O O NH2 H2N OHHO O O O O OH OH H2N OH HO ONH2 O OH 18

HO NH2 Figure 6. Propylamycin (18) is a glycomimetic that18 has shown excellent antibiotic efficacy against several important drug-resistant Gram-negative pathogens. The propyl substituent is believed to Figure 6. Propylamycin (18) is a glycomimetic that has shown excellent antibiotic efficacy against several important drug-resistant Gram-negative pathogens. The propyl substituent is believed to

Biomimetics 2019, 4, x FOR PEER REVIEW 10 of 22

HO HO SH HO O HO O HO OMe HO SH OMe 13 14

Figure 4. 2-Thiocarbohydrates were evaluated as ligands of the plant lectin ConA [126].

In another study, 6-thio and 6-seleno derivatives of ManNAc were synthesized and evaluated as inhibitors of human N-acetylmannosamine kinase (MNK) (Figure 5) [128]. It was hypothesized that these analogues would still bind to the enzyme, yet could not be phosphorylated and therefore would act as inhibitors of the native substrate. 6-O-Ac-ManNAc was previously reported as an inhibitor of the enzyme [122,129,130] but its ester is easily hydrolyzed in vivo making it a poor therapeutic option. In synthesizing the 6-substituted ligands, the high oxidation potential of selenols prevented isolation of the monomer and as a result only diselenide dimer 17 could be evaluated [128]; for the thiol derivative, both monomer 15 and dimer 16 could be isolated and evaluated. Both the presence of Se and the dimerization itself provided enhancements in activity; the diselenide 17 inhibited MNK in the low μM range and upon treatment of Jurkat cells a reduction in surface sialylation was also observed.

HO S HO Se HS NHAc NHAc NHAc O HO S O HO Se O HO NHAc NHAc HO O HO O HO HO HO OH OH OH 15 HO 16 HO 17 Figure 5. 6-S- and 6-Se-derivatives of ManNAc were synthesized and evaluated as inhibitors of human N-acetylmannosamine kinase (MNK). The high oxidation potential of Se prevented isolation of its monomer [128].

4.5. Other Modifications Propylamycin (18) is a 4′-deoxy-4′-alkyl paromomycin which is demonstrably effective against a broad selection of ESKAPE pathogens (Figure 6) [131]. Early studies had demonstrated that 4′-O- ethylation of paromomycin afforded enhanced ribosomal selectivity and reduced ototoxicity in antibiotic treatment [132], with the latter being one of the major negative side effects correlated with aminoglycoside therapies. Although less ototoxic, this modification was also correlated with a loss in activity. In contrast, 4′-deoxygenated paromomycin maintained antibacterial activity but had no enhancement in ribosomal selectivity. Based on these results, it was hypothesized that the alkyl derivatization with an electron-donating substituent could afford both the desired selectivity while maintaining antibacterial activity. Indeed, replacement of the 4′-OH group with a propyl chain interfered with protein synthesis in bacteria and also reduced inner ear toxicity in a guinea pig model [131]. This activity is believed to arise from the electron-donating alkyl substituent which makes the Biomimeticspyranose 2019ring, 4 more, 53 electron-rich and its endocyclic O atom more nucleophilic and therefore a 11better of 23 H-bond acceptor, enhancing the ‘pseudo’ base pair interaction with A1408 upon binding.

OH O HO NH2 H2N O O NH2 HO O O OH

H2N OH O O OH

HO NH2 Biomimetics 2019, 4, x FOR PEER REVIEW 18 11 of 22 Figure 6.6. PropylamycinPropylamycin ( 18(18) is) is a glycomimetica glycomimetic that that has shownhas shown excellent excellent antibiotic antibiotic efficacy efficacy against against several enhance nucleophilicity of the endocyclic O atom and thereby enhance the interaction with its importantseveral important drug-resistant drug-resistant Gram-negative Gram-negative pathogens. pathogens. The propyl The substituentpropyl substituent is believed is believed to enhance to electrophilic binding partner [131]. nucleophilicity of the endocyclic O atom and thereby enhance the interaction with its electrophilic binding partner [131]. 5. Replacement of H Atoms 5. Replacement of H Atoms 5.1. Fluorination 5.1. Fluorination The small size, chemical inertness, and inherent hydrophobicity of F all contribute to it being a goodThe bioisostere small size, of chemicalH. Although inertness, sterically and similar, inherent its hydrophobicity strong inductive of F ability all contribute can substantially to it being influencea good bioisostere neighboring of H.functional Although groups. sterically For exampl similar,e, its F strongsubstitution inductive of a Neu5Ac ability can C-3 substantially proton was usedinfluence to inhibit neighboring sialyltransferase functional groups. activity, For sinc example,e its Finherent substitution electron-withdrawing of a Neu5Ac C-3 protoncapability was significantlyused to inhibit increases sialyltransferase electrophilicity activity, of since the itsC-2 inherent atom and electron-withdrawing destabilizes the oxocarbenium capability significantly transition state.increases This electrophilicity destabilization of hinders the C-2 atomits formation, and destabilizes significantly the oxocarbenium slowing the transitionenzymatic state. rate Thisand effectivelydestabilization reducing hinders tumor its formation, growth significantlyin vivo [133,134]. slowing theA C-2 enzymatic difluorinated rate and derivative effectively reducingof α-D- glucopyranosyl-1-phosphatetumor growth in vivo [133,134 ].was A C-2 difluorinatedalso synthesized derivative and of α-evaluatedd-glucopyranosyl-1-phosphate as a substrate wasof thymidylyltransferasealso synthesized and evaluated Cps2L and as a guanidylyltransferase substrate of thymidylyltransferase GDP-ManPP Cps2L [135]. and guanidylyltransferase GDP-ManPPThe CF3 group [135]. has been shown to be an excellent mimic of CH3 in Fuc and related derivatives (19; FigureThe CF 7).3 group The enhanced has been hydrophobicity shown to be an imparted excellent mimicby CF3 of reduces CH3 in desolvation Fuc and related costs derivatives associated with(19; Figure binding7). Theand enhancedcan enhance hydrophobicity hydrophobic imparted interactions by CF with3 reduces the protein desolvation surface. costs To associateddate, the synthesiswith binding of CF and3 derivatives can enhance has hydrophobicproven challenging interactions and typically with the requires protein very surface. lengthy To synthetic date, the sequencessynthesis of[136,137]. CF3 derivatives 6,6,6-Trifluoro- has provenα-fucose challenging has shown and typicallygood utility requires as a veryfucosylation lengthy syntheticinhibitor duringsequences the [ 136production,137]. 6,6,6-Trifluoro- of therapeuticα-fucose monoclonal has shown antibodies, good utility by binding as a fucosylation at an allosteric inhibitor site during and inhibitingthe production GDP-mannose of therapeutic 4,6-dehydratase monoclonal [137,138]. antibodies, With by reduced binding fucosylation at an allosteric on sitethe Asn297 and inhibiting glycan structuresGDP-mannose of IgG1 4,6-dehydratase antibodies, the [137 Fc ,region138]. With can bind reduced more fucosylation strongly to onFcγ theRIIIa Asn297 on effector glycan cells, structures which inducesof IgG1 antibodies,enhanced antibody-dependent the Fc region can bind cellular more stronglycytotoxicity. to Fc γFucosylationRIIIa on effector inhibitors cells, whichcould inducesalso be usefulenhanced in a antibody-dependentnumber of disease therapies, cellular cytotoxicity. including cancer Fucosylation [139], arthritis inhibitors [140], could and alsosickle be cell useful disease in a [141].number of disease therapies, including cancer [139], arthritis [140], and sickle cell disease [141].

OH

F C O 3 OH OH HO 19 Figure 7. 6,6,6-Trifluoro-6,6,6-Trifluoro-α-fucose-fucose ( (19)) was was synthesized synthesized and and utilized utilized as as an an inhibitor inhibitor of fucosylation on monoclonal antibodies [137] [137].. 5.2. Other Modifications 5.2. Other Modifications The replacement of H is also possible using other small, hydrophobic functional groups. For The replacement of H is also possible using other small, hydrophobic functional groups. For example, a small library of sialyl Lewis X derivatives were synthesized with the C-6 methyl group of example, a small library of sialyl Lewis X derivatives were synthesized with the C-6 methyl group of Fuc modified (alkyne, fluoro, hydroxy, and hydroxymethyl) [142]. The syntheses were achieved using Fuc modified (alkyne, fluoro, hydroxy, and hydroxymethyl) [142]. The syntheses were achieved a recombinant l-fucokinase/GDP-fucose pyrophosphorylase and an α-(1,3)-fucosyltransferase [143]. using a recombinant L-fucokinase/GDP-fucose pyrophosphorylase and an α-(1,3)-fucosyltransferase Sialyl Lewis X has a very rigid structure, due to an important interaction involving hydrophobic [143]. Sialyl Lewis X has a very rigid structure, due to an important interaction involving hydrophobic stacking of the Fuc and Gal residues, where the C-5 Fuc methyl forms important van der Waals stacking of the Fuc and Gal residues, where the C-5 Fuc methyl forms important van der Waals interactions with the hydrophobic face of Gal [144]. It was hypothesized that an alkynyl substitution interactions with the hydrophobic face of Gal [144]. It was hypothesized that an alkynyl substitution on C-6 Fuc would enhance this pre-organization since the alkynyl group would undergo even stronger van der Waals interactions [142]. The sialyl Lewis X derivatives were observed to bind to each of the E-, L-, and P-selectins and are reportedly undergoing further study. The Globo H epitope has been used in clinical trials of several carbohydrate-based conjugate vaccines targeting late stage breast cancer, ovarian cancer, and prostate cancer [145]. In order to improve efficacy, it was desired to create a glycomimetic which would be more immunogenic yet still elicit antibodies cross-reactive with the native antigen present on tumor cells. Globo H derivatives were prepared with modification at either C-6 of the reducing end Glc moiety (F, N3, OPh, O-p- nitrophenyl, NO2 derivatives) or on the C-6 methyl group of Fuc (OH, N3, F, alkyne derivatives) [145]. Upon synthesis, the glycomimetic analogues were conjugated to a diphtheria toxoid cross-reactive

Biomimetics 2019, 4, 53 12 of 23 on C-6 Fuc would enhance this pre-organization since the alkynyl group would undergo even stronger van der Waals interactions [142]. The sialyl Lewis X derivatives were observed to bind to each of the E-, L-, and P-selectins and are reportedly undergoing further study. The Globo H epitope has been used in clinical trials of several carbohydrate-based conjugate vaccines targeting late stage breast cancer, ovarian cancer, and prostate cancer [145]. In order to improve efficacy, it was desired to create a glycomimetic which would be more immunogenic yet still elicit antibodies cross-reactive with the native antigen present on tumor cells. Globo H derivatives were prepared with modification at either C-6 of the reducing end Glc moiety (F, N3, OPh, O-p-nitrophenyl, BiomimeticsNO derivatives) 2019, 4, x FOR or onPEER the REVIEW C-6 methyl group of Fuc (OH, N , F, alkyne derivatives) [145].12 Upon of 22 Biomimetics2 2019, 4, x FOR PEER REVIEW 3 12 of 22 synthesis, the glycomimetic analogues were conjugated to a diphtheria toxoid cross-reactive material material (CRM) 197 carrier protein, and then together with the adjuvant glycolipid C34 were injected (CRM)material 197 (CRM) carrier 197 protein, carrier protein, and then and together then together with the with adjuvant the adjuvant glycolipid glycolipid C34 were C34 were injected injected into into Balb/c mice. IgG antibodies elicited from several of the Glc derivatives as well as the 6-N3-Fuc intoBalb /Balb/cc mice. mice. IgG IgG antibodies antibodies elicited elicited from from several several of of the the Glc Glc derivatives derivatives as as well well asas thethe 6-N6-N33-Fuc derivative (21; Figure 8) were shown to be cross-reactive with Globo H and its related epitopes Gb5 derivative ( 21;; Figure 88)) werewere shownshown toto bebe cross-reactivecross-reactive withwith GloboGlobo HH andand itsits relatedrelated epitopesepitopes Gb5Gb5 and SSEAS4, as measured in a glycan array. The antibodies also recognized cell surface structures on and SSEAS4, as measured in a glycan array. The antibodies also recognized cell surface structures on Globo H-expressing MCF-7 tumor cells and could affect complement-dependent cytotoxic responses. Globo H-expressing MCF-7 tumor cells cells and and could could af afffectect complement-dependent complement-dependent cytotoxic cytotoxic responses. responses.

HO OH HO OH HO OH HO HO HO OHO OHO OHO HO O O O O O HO O O O NHAc HO O NHAc HOO OH R O OH O OH R OH O HO OH O O NH OH HO O OHO O 2 OH HO O O NH2 HO OH OH OHO HO 20 R=H OH 20 R=H OH 21 R=N3 21 R=N3 Figure 8. The chemoenzymatic synthesis of a 6-azido-Fuc Globo H derivative (21) afforded a Figure 8. TheThe chemoenzymatic chemoenzymatic synthesis synthesis of of a a 6-azido-Fuc 6-azido-Fuc Globo Globo H H derivative derivative ( (2121)) afforded afforded a glycomimetic which when incorporated into a conjugate vaccine elicited a strong IgG immune glycomimetic which when incorporated into a conjugate vaccine elicited a strong IgG immune response in mice. The elicited antibodies were cross-reactive with the native Globo H antigen and response in mice. The The elicited elicited antibodies antibodies were were cr cross-reactiveoss-reactive with with the native Globo H antigen and mediated complement-dependent cell cytotoxicity against MCF-7 tumor cells [145]. mediated complement-dependent cell cytotoxicity against MCF-7 tumor cells [[145]145].. 6. Replacement Replacement of NHAc Substituents 6. Replacement of NHAc Substituents 6.1. C-Derivatives 6.1. C-Derivatives A number of biomimetic approach approacheses have been used to to replace replace NN-acetyl-acetyl substituents substituents on on glycans, glycans, A number of biomimetic approaches have been used to replace N-acetyl substituents on glycans, with most methods involving a modificationmodification atat C-2. In an early example, the NHAc groups of GlcNAc with most methods involving a modification at C-2. In an early example, the NHAc groups of GlcNAc and GalNAc werewere replacedreplaced withwith a a ketone ketone bioisostere bioisostere in in an an attempt attempt at at glycan glycan metabolic metabolic engineering engineering of and GalNAc were replaced with a ketone bioisostere in an attempt at glycan metabolic engineering ofcell cell surfaces surfaces (22 (;22 Figure; Figure9)[146 9) [146–148].–148]. The The analogues analogues could could be fed be to fed growing to growing cells, cells, which which would would then of cell surfaces (22; Figure 9) [146–148]. The analogues could be fed to growing cells, which would thenbe incorporated be incorporated onto the onto cell the surface cell surface and could and be could used asbe aused handle as fora handle conjugation for conjugation to dyes, probes, to dyes, etc. then be incorporated onto the cell surface and could be used as a handle for conjugation to dyes, probes,by reaction etc. withby reaction aminooxy with or aminooxy hydrazide-bearing or hydrazid molecules.e-bearing Uponmolecules. treatment Upon of treatment cells, 2-C of-modified cells, 2- probes, etc. by reaction with aminooxy or hydrazide-bearing molecules. Upon treatment of cells, 2- CGalNAc-modified derivatives GalNAc werederivatives taken up were and detectedtaken up at and the surfacedetected of at CHO the cells,surface but unexpectedlyof CHO cells, were but C-modified GalNAc derivatives were taken up and detected at the surface of CHO cells, but unexpectedlynot taken up by were the not three taken human up cellby the lines three examined human [ 147cell]; lines the GlcNAcexamined derivative [147]; the was GlcNAc not observed derivative on unexpectedly were not taken up by the three human cell lines examined [147]; the GlcNAc derivative wasany ofnot the observed cell types. on any of the cell types. was not observed on any of the cell types.

HO HO O HO OH HO O HO OH HO O O 22 22 Figure 9.9. 2-Deoxy-2-acetonyl2-Deoxy-2-acetonyl derivatives derivatives were were synthesized synthesized for for use use in glycan in glycan metabolic metabolic engineering engineering [146]. Figure 9. 2-Deoxy-2-acetonyl derivatives were synthesized for use in glycan metabolic engineering [146]. [146]Mycothiol. is a 1-d-1-O-[2-N-acetamido-l-cysteinamido)-2-deoxy-α-d-glucopyranosyl]-myo-inositol whichMycothiol appears tois playa 1- anD-1- importantO-[2-N-acetamido- role in actinomycetesL-cysteinamido)-2-deoxy- in protectionα from-D-glucopyranosyl]- toxic agents andmyo in- Mycothiol is a 1-D-1-O-[2-N-acetamido-L-cysteinamido)-2-deoxy-α-D-glucopyranosyl]-myo- preventinginositol which on appears oxidative to play intracellular an important environment, role in actinomycetes similar to the in protection role of glutathione from toxic [agents149]. and The inositol which appears to play an important role in actinomycetes in protection from toxic agents and indisaccharide preventing 1- ond-1- oxidativeO-(2-acetamido-2-deoxy- intracellular environmenα-d-glucopyranosyl)-t, similar to themyo role-inositol of glutathione is an intermediate [149]. The in in preventing on oxidative intracellular environment, similar to the role of glutathione [149]. The disaccharide 1-D-1-O-(2-acetamido-2-deoxy-α-D-glucopyranosyl)-myo-inositol is an intermediate in disaccharide 1-D-1-O-(2-acetamido-2-deoxy-α-D-glucopyranosyl)-myo-inositol is an intermediate in Mycobacteria mycothiol production, and inhibitors of an N-deacetylase enzyme could potentially be Mycobacteria mycothiol production, and inhibitors of an N-deacetylase enzyme could potentially be used as a treatment against infection. Derivatives in which the NHAc group of the GlcNAc moiety used as a treatment against infection. Derivatives in which the NHAc group of the GlcNAc moiety was replaced by a C-2 substituent were synthesized and evaluated as inhibitors (Figure 10) [150]. was replaced by a C-2 substituent were synthesized and evaluated as inhibitors (Figure 10) [150]. Both 2-deoxy-2-C-(2′-hydroxypropyl)-D-glucoside (23) and 2-deoxy-2-C-(2′-oxopropyl)-D-glucose Both 2-deoxy-2-C-(2′-hydroxypropyl)-D-glucoside (23) and 2-deoxy-2-C-(2′-oxopropyl)-D-glucose (24) derivatives successfully inhibited the production of inositol-containing metabolites, as measured (24) derivatives successfully inhibited the production of inositol-containing metabolites, as measured by the incorporation of radiolabeled [3H]inositol into molecules, but unexpectedly did not affect the by the incorporation of radiolabeled [3H]inositol into molecules, but unexpectedly did not affect the growth of Mycobacterium smegmatis in vitro. growth of Mycobacterium smegmatis in vitro.

Biomimetics 2019, 4, 53 13 of 23

Mycobacteria mycothiol production, and inhibitors of an N-deacetylase enzyme could potentially be used as a treatment against infection. Derivatives in which the NHAc group of the GlcNAc moiety was replaced by a C-2 substituent were synthesized and evaluated as inhibitors (Figure 10)[150]. Both 2-deoxy-2-C-(20-hydroxypropyl)-d-glucoside (23) and 2-deoxy-2-C-(20-oxopropyl)-d-glucose (24) derivatives successfully inhibited the production of inositol-containing metabolites, as measured by the incorporation of radiolabeled [3H]inositol into molecules, but unexpectedly did not affect the Biomimeticsgrowth of 2019Mycobacterium, 4, x FOR PEER smegmatis REVIEW in vitro. 13 of 22

HO HO O O HO O HO O HO HO HO O OH O O OH O OH O OH HO OH HO OH HO HO HO OH HO OH 23 24 Figure 10.10.2-Deoxy-2- 2-Deoxy-2-C-alkylglucosidesC-alkylglucosides of myo of -inositolmyo-inositol were synthesized were synthesize as inhibitorsd as ofinhibitors a Mycobacterium of a Mycobacteriumsmegmatis de-N smegmatis-acetylase de- [150N].-acetylase [150].

A numbernumber of of NHAc NHAc biomimetics biomimetics have have also beenalso directedbeen directed at enhancing at enhancing interactions interactions with important with importantmetal ions metal present ions in thepresent binding in the site, binding in efforts site to, introducein efforts strongerto introduce chelating stronger functionalities. chelating Trypanosomafunctionalities. brucei Trypanosoma N-acetylglucosamine-phosphatidylinositol brucei N-acetylglucosamine-phosphatidylinositol (GlcNAc-PI) de- N(GlcNAc-PI)-acetylase is ade- zincN- acetylasemetalloenzyme is a zinc involved metalloenzyme in the biosynthesis involved in ofthe glycosylphosphatidylinositol biosynthesis of glycosylphosphatidylinositol [151]. T. brucei is [151]. rich T.in brucei GPI-anchored is rich in proteinsGPI-anchored and it proteins is believed and thatit is believed disrupting that their disrupting synthesis their may synthesis offer a may potential offer atherapy potential for therapy African for sleeping African sickness sleeping [ 151sickness,152]. [151,152]. A computational A computational homology homology model based model on based two onother two GlcNAc-PI other GlcNAc-PI de-N-acetylases de-N-acetylases supports supports a binding a modebinding where mode the where NHAc the group NHAc is involvedgroup is 2+ 2+ involvedin coordination in coordination of a Zn of iona Zn [1532+ ion]. [153]. In eff Inorts efforts to install to install a better a better Zn Zn-binding2+-binding group group at at the the C-2 C- 2position position of of GlcNAc, GlcNAc, a seriesa series of of analogues analogues were were prepared prepared and and evaluated evaluated for for their their ability ability to inhibit to inhibit the thede- Nde--acetylaseN-acetylase (Figure (Figure 11) 11)[151 [151,154].,154]. Both Both carboxylic carboxylic acid acid (25 ,(2529,, 2931,) 31 and) and hydroxamic hydroxamic acid acid (27 ,(2733,) 33derivatives) derivatives demonstrated demonstrated enzyme enzyme inhibition inhibition with moderatewith moderate IC50 values IC50 values (0.1–1.5 (0.1–1.5 mM) [ 151mM)]. A [151]. similar A similarapproach approach to enhanced to enhanced metal chelation metal chelation was used wa fors used developing for developing inhibitors inhibitors of the de- ofN the-acetylase de-N- acetylasePgaB, which PgaB, is which known is to known coordinate to coordinate to the 2-NHAc to the 2-NHAc and 3-OH and substituents3-OH substituents of GlcNAc of GlcNAc [155]. [155]. Both Boththe groups the groups at C-3 at C-3 (–OH, (–OH, –SH, –SH, –NH –NH2, and2, and –N –N3)3) and and C-2 C-2 (–NHC((–NHC(=S)CH=S)CH3),), –NHS(=O)(=O)CH –NHS(=O)(=O)CH3, 3–, –CHCH2P(=O)(–OH)OMe,2P(=O)(–OH)OMe, and and –CH –CH2S(=O)(=O)NH2S(=O)(=O)NH2) were2) were modified modified and screened and screened as inhibitors as inhibitors of de-N of- acetylasesde-N-acetylases which which play an play important an important role in role biofilm in biofilm maintenance maintenance [155]. [ 155The]. strongest The strongest inhibitor, inhibitor, 2,3- dideoxy-2-thionoacetamide-3-thio-2,3-dideoxy-2-thionoacetamide-3-thio-β-D-glucosideβ-d-glucoside displayed displayed a Ki aofK 2.9of ± 2.9 0.6 μ0.6M.µ M. dideoxy-2-thionoacetamide-3-thio-β-D-glucoside displayed a Ki of i2.9 ± 0.6± μM.

HO HO HO HO O O O O HO HO HO HO BzO HO BzO HO O O O O O--NHEt + O O O O O NHEt3 OH OH NHOH NHOH O P OC H OH OH NHOH NHOH HO O P OC18H37 25 26 27 28 HO HO O O HO O HO HO HO HO HO O O O O O HO O HO O HO O HO O HO OH HO HO SPh HO SPh HO HO HO HO NHOH NH 33 O O SPh O O OH OH OH NH OH OH OH NH2 29 30 31 32 2+ Figure 11. AA series series of of 2-deoxygenated 2-deoxygenated 2- C-modifed glucosidesglucosides containing ZnZn2+-chelating-chelating groups groups were were synthesized andand evaluated evaluated as inhibitors as inhibitors of Trypanosomabrucei of Trypanosoma N-acetylglucosamine-phosphatidylinositol brucei N-acetylglucosamine- phosphatidylinositolde-N-acetylase as a potential de-N-acetylase novel therapy as a potential for African novel sleeping therapy sickness for African [151 sleeping]. sickness [151]. 6.2. Other Substitutions 6.2. Other Substitutions As previously mentioned, glycomimetic antigens can elicit enhanced immune responses yet As previously mentioned, glycomimetic antigens can elicit enhanced immune responses yet still still elicit antibodies which recognize the native antigens. In efforts to improve immunogenicity, elicit antibodies which recognize the native antigens. In efforts to improve immunogenicity, a number of N-propionyl modified haptens have been studied based on polysialic acid, sTn, and GM3 [32,40,41,145].

7. Replacement of Charged Substituents Although carboxylates are present in several important glycan building blocks, such as − neuraminic acids and uronic acids, there are very few examples of CO2− bioisosteres used in glycomimetics. Based on traditional medicinal chemistry approaches, bioisosteres of the CO2H group include those such as CONH2 or SO3H, but these are not prominently featured in the design of glycan- based drugs. This likely results from the importance of the carboxylate interaction in effective binding. This is supported by structural evidence in several instances, for example in the mammalian

Biomimetics 2019, 4, 53 14 of 23 a number of N-propionyl modified haptens have been studied based on polysialic acid, sTn, and GM3 [32,40,41,145].

7. Replacement of Charged Substituents Although carboxylates are present in several important glycan building blocks, such as neuraminic acids and uronic acids, there are very few examples of CO2− bioisosteres used in glycomimetics. Based on traditional medicinal chemistry approaches, bioisosteres of the CO2H group include those such as CONHBiomimetics2 or 2019 SO, 43, H,x FOR but PEER these REVIEW are not prominently featured in the design of glycan-based drugs.14 Thisof 22 likely results from the importance of the carboxylate interaction in effective binding. This is supported bysialyltransferase structural evidence ST3Gal-I in several where instances,it was demonstrated for example that in theconversion mammalian of the sialyltransferase carboxylate (IC ST3Gal-I50: 0.047 wheremM) to it waseither demonstrated an amide (IC that50:conversion 3.3 mM) or of thehydroxymethylene carboxylate (IC50 :(IC 0.04750: 4.2 mM) mM) to either group an significantly amide (IC50: 3.3impaired mM) or ligand hydroxymethylene binding [156,157]. (IC50 :Instead 4.2 mM) of group the bioisosteric significantly repl impairedacement ligand of a bindingsingle functional [156,157]. Insteadgroup, carboxylate-containing of the bioisosteric replacement fragments of ahave single been functional used to group, mimic carboxylate-containingtheir analogous monosaccharide fragments havebuilding been blocks used to[24,124,158–160]. mimic their analogous monosaccharide building blocks [24,124,158–160]. In one one of of the the few few studies studies reported, reported, a series a series of phosphono of phosphono and andtetrazolyl tetrazolyl derivatives derivatives of 4- ofmethylumbelliferyl 4-methylumbelliferyl β-D-glucuronideβ-d-glucuronide were weresynthesized synthesized and andevaluated evaluated as inhibitors as inhibitors of ofβ- βglucuronidases-glucuronidases (Figure (Figure 1212))[ 161[161].]. The The phosphonate phosphonate analogue analogue35 was 35 observed was observed to be slowly to hydrolyzedbe slowly byhydrolyzed an E. coli byβ-glucuronidase, an E. coli β-glucuronidase, but was not abut substrate was not for a bovinesubstrate liver forβ bovine-glucuronidase. liver β-glucuronidase. In inhibitory experiments,In inhibitory experiments, strong binding strong was observedbinding was only ob forserved the native only carboxylatefor the native analogue carboxylate37 (IC analogue50: 0.2 µ M)37 and(IC50 only: 0.2 μ veryM) and weak only inhibition very weak for phosphonateinhibition for 38phosphonate(IC50: 2 mM). 38 (IC50: 2 mM). Other examples of bioisosteres of carbohydratecarbohydrate derivativesderivatives containing charged modificationsmodifications include the installation of a carboxylate group at C-1 of lipid A to mimicmimic thethe nativenative phosphoricphosphoric acid moiety [162[162].]. Furthermore, Furthermore, 6-bromophosphonate 6-bromophosphona analogueste analogues of glucose-6-phosphate of glucose-6-phosphate were synthesized were andsynthesized demonstrated and demonstrated to inhibit glucose-6-phosphatase to inhibit glucose-6-phosphatase [163]. Glucose-6-phosphatase [163]. Glucose-6-phosphatase is involved inis theinvolved regulation in the of regulation glucose metabolism of glucose andmetabolism is upregulated and is inupregulated type II diabetes in type [164 II, 165diabetes], inferring [164,165], that inhibitorsinferring that of the inhibitors enzyme of may the oenzymeffer a potentially may offer novel a potentially therapeutic. novel therapeutic.

N N O NaO O NH NaO NaO P N O O O HO O O O HO O O O HO O O O HO HO HO OH OH OH

34 35 36

O NaO O NaO NaO P HO O O O HO HO HO HO HO HO O H O H O H HO N N HO N N HO N N

O O O 37 38 39 Figure 12.12. PhosphonoPhosphono and tetrazolyl derivatives of 4-methylumbelliferyl4-methylumbelliferyl β-Dd-glucuronide were synthesized andand evaluated evaluated as βas-glucuronidase β-glucuronidase inhibitors. inhibitors. The phosphonate The phosphonate mimetics ofmimetics the carboxylate of the displayedcarboxylate a verydisplayed low level a very of activity,low level while of acti thevity, other while mimetics the other were mimetics inactive were [161 ].inactive [161].

8. Conclusions Although native carbohydrates have traditiona traditionallylly been viewed as poor therapeutic agents, advancements in the development of glycomimetics have been made which has enabled carbohydrate-protein interactionsinteractions toto bebe more activelyactively consideredconsidered asas drugdrug targets.targets. A main strategy for aaffinityffinity enhancementenhancement in in glycomimetics glycomimetics is tois reduceto reduce the ligand the ligand polar surfacepolar surface area, which area, can which be achieved can be throughachieved thethrough bioisosteric the bioisosteric replacement replacement of carbohydrate of carbohydrate core functional core groups. functional Both deoxygenationgroups. Both anddeoxygenation deoxyfluorination and deoxyfluorination of OH groups have of demonstratedOH groups broadhave successesdemonstrated in enhancing broad successes affinity over in nativeenhancing substrates, affinity and over have native become substrates, a major and area have of focus become in glycomimetic a major area development. of focus in Thisglycomimetic benefit is development. This benefit is even further evident at the C-6 position of pyranosides, where deoxygenation removes a rotatable bond and thereby concomitantly reduces the entropic penalty of binding. With regards to fluorination, CF3-containing fucose derivatives have also been very successful, yet their accessibility has been limited by the difficulty encountered in their syntheses. In efforts to enhance the enthalpic gains associated with ligand binding, only limited efforts have been reported on the bioisosteric replacement of chelating groups or improved H-bond donors/acceptors, and these approaches should be evaluated further.

Funding: This research received no external funding.

Conflicts of Interest: The author declares no conflict of interest.

Biomimetics 2019, 4, 53 15 of 23 even further evident at the C-6 position of pyranosides, where deoxygenation removes a rotatable bond and thereby concomitantly reduces the entropic penalty of binding. With regards to fluorination, CF3-containing fucose derivatives have also been very successful, yet their accessibility has been limited by the difficulty encountered in their syntheses. In efforts to enhance the enthalpic gains associated with ligand binding, only limited efforts have been reported on the bioisosteric replacement of chelating groups or improved H-bond donors/acceptors, and these approaches should be evaluated further.

Funding: This research received no external funding. Conflicts of Interest: The author declares no conflict of interest.

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