Neoglycoconjugates Based on Cyclodextrins and Calixarenes

SEPTEMBER/OCTOBER 2001 Volume 12, Number 5 © Copyright 2001 by the American Chemical Society

REVIEWS Neoglycoconjugates Based on Cyclodextrins and Calixarenes David A. Fulton and J. Fraser Stoddart* Department of Chemistry and Biochemistry, University of California, Los Angeles, 405 Hilgard Avenue, Los Angeles, California 90095. Received March 21, 2001 INTRODUCTION

Recent years have witnessed not only a growing understanding of the molecular basis of carbohydrateprotein interactions (1), but also a deeper appreciation of their importance in many biological processes. On cell surfaces, carbohydrate-protein interactions are involved in the mediation of important biological processes in which tissue cells, bacteria, and viruses recognize one another. Embryogenesis, cancer metastasis, inflammation, and bacterial and viral infection are all examples of biological events which involve the selective recognition of carbohydrates by proteins. However, isolated carbohydrate-protein interactions are typically very weak (2) with Kd values of the order of 10-3 to 10-6 M. Nature compensates for the weakness of these isolated interactions by tending to cluster together multiple copies of carbohydrate ligands and their receptors (3) in order to allow for stronger cooperative binding to take placesthe so-called multivalent effect (4). This biological phenomenon is an important one to consider when planning how to capitalize on carbohydrate-protein interactions when developing a potentially new class of carbohydrate-based therapeutics. In view of the important roles played by carbohydrateprotein interactions in disease states, it is hardly surprising that the exploitation of these interactions to develop a new generation of carbohydrate-based therapeutics is already well under way. For example, it is known (5) that the Shiga and cholera toxins, which have an AB5 structure (6), gain entry to mammalian cells when the B5 pentamer recognizes carbohydrates on the cell surface. * To whom correspondence should be addressed. Tel: (310) 206 7078. Fax: (310) 206 1843. E-mail: [email protected].

The recent demonstration by Bundle and co-workers (7) of a pentavalent carbohydrate ligand capable of inhibiting the binding of the B5 subunit of the Escherichia coli O157: H7 Shiga-like toxin I with subnanomolar activity, is one of the most impressive applications of glycoscience to have been described in the literature to date. The use of carbohydrates to inhibit a particular lectinmediated process is not the only method by which carbohydrates can find therapeutic applicationssindeed, carbohydrate-based cancer vaccines which are based on tumor-associated carbohydrate antigens, are an active area of research at present (8). Another area of contemporary interest is the carbohydrate-mediated delivery of drugs (9). Since cell-surface lectins are often displayed in a very cell-specific manner, it may be possible to use a carbohydrate, which is specifically recognized by a lectin found on the surface of a specific cell type, to direct an existing covalently attached drug molecule to the desired tissues in a selective manner. For example, an earlier study (10) has shown that the antiinflammatory, naproxen, when covalently attached to galactose-derivatized human serum albumin, can be delivered selectively to the liversin preference to the kidneyssin rat models. Recent years have witnessed the emergence of another approach by which the power of carbohydrates to target cell surfaces can be exploited in the selective delivery of drugs to their target tissues. Supramolecular chemists have long recognized (11) the ability of certain macrocyclic hosts to complex with complementary guests, including biologically important molecules (12). The macrocyclic hosts that have been studied traditionally by supramolecular chemists almost invariably lack a biologically recognizable component, however. So even if we discount the biological disadvantages associated with

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656 Bioconjugate Chem., Vol. 12, No. 5, 2001

many macrocycles, such as their lack of aqueous solubility and potential toxicities, they are, at best, limited in their biological potential. The possibility does exist, however, to modify many of these macrocyclic hosts chemically by grafting biorecognizable unitsssuch as carbohydratessonto their reactive functional groups. Thus, such a unique class of glycoconjugates would possess the potential to sequester a drug molecule noncovalently within their macrocyclic cavities and then rely upon their appended carbohydrate units to direct the glycoconjugate-drug complex to a specific tissue type, dictated by the choice of carbohydrate units displayed on the periphery of the macrocycle. This approach would dispense with one of the problems associated with conventional receptor-mediated glycotargetingsnamely, the molecular structure of the drug molecule may have to be modified to allow it to be covalently attached to the glycoprotein, which could have serious consequences on the drug’s potency. Even if it is not necessary to alter the structure of the drug molecule, covalently attaching the drug to a linker could still affect the drug’s potency. Once the glycoconjugate-drug complex had located the specific target, the drug molecule would be released slowly into the cell membrane and adsorbed by the cell wherein it could fulfill its pharmacological purpose. It is the prospect of this kind of scenario that has brought about a union of the disciplines practiced by supramolecular and carbohydrate chemists where the aim has been one of designing, synthesizing, and then determining the supramolecular and biochemical properties of these macrocyclic-based “intelligent” drug-delivery systems. This review will concentrate on recent developments in this relatively new field of glycoscience. Design Considerations. The choice of macrocyclic hostswhich will become not only the host moiety but a scaffold for the attachment of the carbohydrate residues alsosis an extremely important decision that has to be made at the outset by any researcher entering the field. Ideally, the starting macrocycle should be readily available and have appropriately positioned functional groups to allow for the facile attachment of carbohydrate residues. The macrocycle itself should display low pharmacological activity, and ideally, a high degree of biocompatability. It is also advantageous if there is a welldeveloped literature dealing with the efficient chemical modification of the macrocycle. Another very important point for consideration is that the macrocycle should display a reasonably predictable propensity to complex with a wide range of guest molecules. The design of the macrocycle-based glycoconjugate must avoid the situation where its carbohydrate appendage(s), or any other part of the molecule for that matter, becomes bound inside the macrocyclic cavity in either an intramolecular or intermolecular sense, thus severely reducing the macrocycles’ ability to complex with guest molecules. Indeed, weak complexation of a drug molecule by the macrocycle could result in the two becoming completely dissociated before the complex reaches its target. Therefore, it is advisable for chemists to design macrocyclic-based glycoconjugates which possess suitably strong association constants with their intended guest molecules. The choice of carbohydrates to be covalently attached to the macrocycle will be determined by the desired lectin target of the neoglycoconjugate, whether for in vivo applications or simply model studies. Many of the compounds described in this review have been designed such that their interactions with plant lectins can be studied. Additionally, an emphasis has been placed by many researchers on producing homogeneous compounds which

Fulton and Stoddart

Figure 1. (a) Condensed structural formulas of R-, β-, and γ-cyclodextrin, with the primary and secondary faces indicated. (b) Full structural formula of R-cyclodextrin.

possess a well-defined structure and a precise number of carbohydrate residues. The extra effort required to produce homogeneous compounds is probably worthwhile, since it can be difficult to reproduce accurately the composition of a heterogeneous mixture and evaluate it biologically. When all the requirements and limitations are taken into consideration, it is perhaps hardly surprising that chemists in this area have chosen “tried-and-tested” macrocycles as the initial compounds for the construction of glycoconjugates. Since cyclodextrins, calixarenes, and calixresorcarenes all have rich and extremely welldocumented synthetic and supramolecular chemistries, they have, thus far, been the macrocycles of choice for the construction of these glycoconjugates. This review will cover the research reported for each of these three classes of macrocycles in turn. CyclodextrinssVersatile Receptor Molecules. Cyclodextrins (CDs) (13) are cyclic oligosaccharides, composed of six, seven, or eight R-D-glucose units, and giving rise to R-, β-, and γ-CD, respectively. They have an overall shape reminiscent of a truncated cone. On account of their relatively hydrophobic interiors, CDs have the ability to form inclusion complexes with a wide range of substrates in aqueous solution. As CDs are obtained on an industrial scale by the enzymatic degradation of starch by bacterial microorganisms, they are readily available and relatively affordable. This important feature, combined with their inherently low toxicities and pharmacological activities, has meant that CDs are already finding applications in the pharmaceutical industry (14). The formation of inclusion complexes with drug molecules helps to enhance their solubilities, stabilities, and bioavailabilitiessfor example, to improve its solubility, the antiinflammatory drug, piroxicam, is commonly marketed in many European countries as its β-CD complex. The physical properties of CDs can be altered upon their chemical modification. CDs contain two classes of free hydroxyl groupssthe primary 6-OH groups are situated around the circumference of the narrower “primary face”, and the secondary 2- and 3-OH groups are located on the circumference of the wider “secondary face” (Figure 1). It is these hydroxyl groups, and their associated differences in reactivity, which provide the fundamental basis for the regioselective modification of CDs. There are a limited number of examples of the chemical modification of CDs with bioactive molecules in an attempt to use the characteristics of the CD to improve the potency of a bioactive molecule. For example, the antitumor sulfonylurea LY237868 (1) has been attached (15) (Scheme 1) covalently to an R-CD derivative 2. The

Reviews

Bioconjugate Chem., Vol. 12, No. 5, 2001 657

Scheme 1. Reaction of the Antitumor Sulfonyl Urea 1 with the Modified CD Core 2 To Form the Cluster Compound 3 as a Mixture of Structural Isomers

resulting conjugate 3 is a mixture of isomers which contain approximately 14 sulfonylurea groups per CD molecule. Importantly, the attachment of these sulfonylurea groups to the R-CD core greatly increases the bulkiness of the conjugate, and it is thus less likely to pass through cell membranes. As a consequence, it has been possible to prove that the antitumor sulfonylurea blocks NADH oxidase activity on the external plasma membrane surface of HeLa cells and not in the interior of the cells. However, it is the prospect of utilizing recent advances in glycobiology which has done much to shape current research in this area. It is the possibility of employing CDs adorned with carbohydrate units to target cell surfaces selectively and thus to guide an included drug molecule to a desired site, which has been receiving the attention of many researchers of late. Cyclodextrins Appended with One Carbohydrate. CDs adorned on their primary faces with one or more carbohydrate units have been known (16) since the mid 1960s. These branched CDs, which consist of an R-Dglucopyranosyl unit or a (1f4)-R-D-glucan connected to the 6-position of one or more of the R-D-glucopyranosyl units of the CD torus, are minor products formed during the action of Bacillus macerans cycloamylose glucotransferase on starch during the formation of native CDs. Researchers at the time were interested in these branched CDs, as they potentially offered advantages over native CDs in terms of solubility or substrate complexing ability, as well as being useful in studies of the enzymatic degradation of CDs. However, the tedious purification

procedures required to isolate these compounds prompted (17) the first chemical synthesis of the branched CD derivative 4. Shortly thereafter, several research groups reported (18) the synthesis of the thio analogues of 4s namely the 6-S-R-D-glucopyranosyl-6-thiocyclomaltoheptaose derivative 5. Although the ability of saccharide-branched CDs to complex with guest molecules had been investigated (19) sometime ago, it was not until the early 1990s that researchers recognized the potential of utilizing the carbohydrate appendage of a CD torus in order to direct the glycoconjugate toward a specific biological site. Realizing the potential saccharide-branched CDs could have for the “vectorized transport of drugs”, Parrot-Lopez et al. began to develop synthetic methods to link carbohydrate units onto CD cores. However, the French group recognized an important limitation of the naturally occurring saccharide-branched CDs. Compounds such as 4, where the carbohydrate appendage is connected directly to the CD torus through an O-glycosidic linkage, will probably not possess sufficient flexibility for the appendage to interact with the receptor. To circumvent this potential pitfall, a saccharide-branched CD was prepared (20) which relies upon a flexible C9 spacer to attach the carbohydrate unit to the CD core. Thus, a carbohydrate derivative 6 (Scheme 2), containing a β-Nglucosidic linkage and a C9 linker, terminated by a free carboxylic acid function, was prepared. Standard amide bond-forming chemistry (DCC/HOBt) was then used to attach 6 to mono-6-amino-β-CD (7), affording the target compound 8 in a 40% yield. 1H NMR spectroscopic analyses of 8 revealed very little conformational perturbation of the CD toroidal protons in either DMSO-d6 or D2O as solvents, indicating that there is no interaction between the C9 spacer arm and the hydrophobic cavity of the CD. Compound 8 was also found to have a much improved solubility (200 g L-1) in water relative to that (19 g L-1) of native β-CD. 1H NMR spectroscopic evidence suggested that 8 can form an inclusion complex with the antihypertensive drug, nicardipine, although no association constant was reported for the complex. The same group also subsequently reported (21) the synthesis of β-N-galactoside, β-N-fucoside, and R-N-mannoside derivatives of 8. Various biological experiments were then performed using some of these compounds. They indicated (22) that the β-N-galactosyl derivatives possess lower hemolytic properties than does the native β-CD, and that the β-N-galactosyl, β-N-fucosyl, and β-N-acetylglucosylamine derivatives display a certain cytotoxicity toward some human tumor cells. Later work by Parrot-Lopez and co-workers (23) highlighted the fact that saccharide-branched CDs, with spacer arms between the appended carbohydrate units


Fulton and Stoddart

Scheme 2. Synthesis of a CD Derivative Appended with One N-glucoside Unit at the End of an Alkyl Chain Spacer Arm

Scheme 3. Chemical-Enzymatic Synthesis of Mono6-[Gal-β-1,4-GlcNAc-β-(1,6′)-hexyl]amido-6-deoxy-β-CD 11

and the CD torus, could be prepared efficiently, using a chemical-enzymatic strategy. The incorporation of an enzymatic step into the overall synthetic scheme would arguably allow access to these saccharide-branched CDs on a large scale, an issue which would have to be addressed should these compounds ever become of commercial interest. Peptide coupling (Scheme 3) of the GlcNAc derivative 9, which contains a hexanoic acid

spacer arm, to mono-6-amino-β-CD (7) yields the product 10 in 28% yield. Enzymatic coupling of UDP-galactose to 10 in the presence of 4-β-galactosyltransferase affords mono-6-[Gal-β-1,4-GlcNAc-β-(1,6′)-hexyl]amido-6-deoxyβ-CD (11) in 68% yield. Biological assays indicate that 11 can be recognized by the galactose-specific Kluveromyces bulgaricus cell wall lectin (KbCWL), whereas the simpler GlcNAc derivative 10 is not recognized at all by this lectin. The effects of the lengths of the spacer arm on the ability of monosaccharide-branched CDs to recognize lectins has been investigated by numerous researchers. Parrot-Lopez and co-workers (24) prepared a series of β-CD derivatives 12, appended with a single N-β-D-

galactosyl unit at the end of a spacer arm of varying length (n ) 3-6 or 9). The ability of these compounds to be recognized by the KbCWL in a flocculation assay was then demonstrated. The results suggest that those compounds with short spacer arms are not recognized by KbCWL as well as those compounds with long spacer arms. Another study, which deals briefly with the effects of spacer arm lengths on lectin recognition, has been performed by Hattori and co-workers (25). In this work, the technique of surface plasmon resonance (SPR) has been used to measure the rates of association and dissociationsinformation which allows the direct calculation of Kdsof various saccharide-branched CD derivatives with the lectin Concanavalin A (Con A), which is immobilized on a biosensor chip. One important conclusion from this investigation is that the R-glucosylgluconoamide-β-CD 13 exhibits a significantly stronger interac-

Reviews

tion with Con A than either of the simpler R-glucosyl- or R-maltosyl-β-CDs 4 and 14. This result indicates that the carbohydrate appendage has to be spatially far enough removed from the CD core in order to enter into a strong interaction with Con A. However, it also indicates that the crucial terminal carbohydrate appendage must additionally have a certain degree of conformational freedom to help it interact with the Con A, which would explain why the R-glucosylgluconoamide-β-CD 13 experiences a much stronger interaction with Con A than does the R-maltosyl-β-CD 14, which has a conformationally more restricted terminal R-glucopyranosyl residue. In the study of multivalent carbohydrate-protein interactions, the spacer arm length and relative spatial orientation of the appended carbohydrate ligand is of fundamental importance. Recent investigations (26) have focused upon the effect of these structural characteristics in a more systematic and detailed manner. It is probably fair to conclude that, in the area of saccharide-branched CDs, and indeed saccharide-branched macrocycles, the research carried out to date only begins to scratch the surface of understanding the subtleties of the relative spatial orientation of carbohydrate ligands, relative to their scaffolds and to each other.

Research performed (27) by the groups of Fernańdez and Defaye has resulted in the development of a route for the attachment of N-glycopeptide entities onto CD cores, utilizing thiourea formation. The glucosylamino acid derivative 15 shown in Scheme 4 was prepared in six steps from 1,2-O-isopropylidene-R-D-glucofuranose. Reaction of the free amino function in 15 with mono-6isothiocynanato-β-CD 16, followed by removal of the Bocprotecting group, affords the target compound 17 in high yield. The authors hope that this synthetic strategy will be efficient enough to allow the attachment of other more complex N-glycopeptide structures units onto CD cores. Another interesting example of monofunctionalized CD derivatives has recently been published (28) by the same group. In this investigation, instead of functionalizing the CD core with a single carbohydrate appendage, it has been functionalized with a single dendritic wedge containing up to six carbohydrate units. As these compounds contain multiple copies of their carbohydrate ligands, they now possess the potential to exploit the so-called “multivalent” or “glycocluster” effects (4). Simpler compounds such as 18 and 19, consisting of β-CD cores and monosubstituted with diantennary galactosyl and mannosyl moieties 19 had previously been described by Driguez and co-workers (29). The synthesis and use of glycodendrimers designed to exploit these effects, have been an active area of research in recent years. This topic has been reviewed elsewhere in detail (30). The synthetic

Bioconjugate Chem., Vol. 12, No. 5, 2001 659 Scheme 4. The Attachment N-glycopeptide to a CD Core

of

a

Simple

strategy of attaching carbohydrate-containing dendritic wedges to CD cores has several advantages. It allows the synthesis of a CD-based cluster containing multiple displays of carbohydrate units, without having to resort to more synthetically challenging perfunctionalizations of the CD core. Additionally, the binding ability of the CD core is also less likely to be perturbed relative to that of the corresponding native β-CD. The Franco-Spanish group have developed an efficient method for the construction of dendrons, substituted with mannosyl residues on their peripheries. Their synthesis, which uses the efficient and clean reaction of amines with isothiocyanates to form thioureas, allows the construction, in a convergent manner, of several mannose-containing dendrons, such as 20 shown in Scheme 5, with an isothiocyanate function at their focal points. Reaction of 20 with mono-6-amino-β-CD (7), followed by de-O-acetylation, affords the target compound 21. A series of compounds containing various numbers (1-4 or 6) of mannosyl residues were prepared in a similar manner, and their

660 Bioconjugate Chem., Vol. 12, No. 5, 2001 Scheme 5 The Attachment of a Dendritic Wedge Containing Six Mannosyl Residues at Its Periphery to a CD Core

Fulton and Stoddart Scheme 6. The First Reported Synthesis of CDs Persubstituted on Their Primary Faces with Galactosyl Residues

24 affords (Scheme 6) the persubstituted products 25 and 26 in 50% and 80% yields, respectively. Shortly afterward, Djedaı¨ni-Pilard and co-workers (32) reported a more detailed account of the synthesis and host-guest binding properties of CDs, persubstituted with carbohydrate appendages. The French group was initially interested in comparing the water solubilities of CDs, persubstituted with carbohydrates, relative to those of monosaccharide-branched CDs and native CDs. Increased aqueous solubility could endow the modified CDs with an increased ability to bind drug molecules. Utilizing a strategy similar to that reported independently by Defaye (18a) and Driguez (18b) for the synthesis of 5, the per-6-thio-R- and per-6-thio-β-D-glucopyranosyl derivatives 27 and 28 of CD were prepared.

abilities to interact with Con A were determined by an enzyme-linked lectin assay (ELLA). Although the results of this assay are not disclosed by the authors, they do mention that “IC50 values for inhibition of Con A-yeast mannan binding reflected the expected amplification of lectin-binding strength for the higher-valent representatives”. It was also observed that these mannosyl-coated β-CD-dendrimer constructs had an abilityswhich was similar to that observed for monobranched CDssto solubilize the anticancer drug Taxote´re. Cyclodextrins Appended with Many Carbohydrates. The previously described example highlights the potential advantages that molecules displaying multiple carbohydrate ligands may havesnamely the ability to interact with lectins with much stronger affinities than their monovalent counterparts. Research in the field of multivalent CDs to date has largely focused on the synthesis of persubstituted CDs, where each glucosyl residue of the CD torus has an appended carbohydrate ligand. Although CD derivatives of this type retain their inherent Cn symmetries, their perfunctionalization can pose very formidable synthetic problems. The first chemical syntheses of CDs, persubstituted with carbohydrate appendages, were performed by Driguez co-workers (31) in 1994. The synthetic approach they utilized expands upon that used by them previously (18b) in the synthesis of the mono-branched CDs 5si.e., simple nucleophilic displacement of a suitable leaving group in the 6-position(s) of β-CD with a thiolate anion. Reaction of the sodium salts 22 and 23 of both 1-thio-R- and 1-thioβ-D-galactopyranose, respectively, with per-6-bromo-β-CD

Solubility studies indicated that these persubstituted CDs were more soluble than their monobranched counterparts, with the R-anomers being considerably more soluble than the corresponding β-anomers. An extensive NMR spectroscopic study of inclusion complexes formed between both the monosubstituted and persubstituted CD hosts and a series of drug molecules, was then performed. This investigation allowed the determination of association constants (Ka values) and binding orientations of the guests within the CD cavitiessand, therefore, an estimation of the effect that the appended carbohydrate moieties have on the complexation ability of the CD cavitysand produced some initially unexpected results. Interestingly, it was found that p-nitrophenol, a well-known guest for β-CD, was found to form an exceptionally weak inclusion complex with 27. This result was explained by the fact that β-CD prefers (33) to bind p-nitrophenol through its narrower primary face, suggesting that the bulky carbohydrate groups on the primary face of 27 present enough steric hindrance to prevent the complexation of the guest altogether. A different scenario is found in the complexation of the steroid, prednisolone, where 2D-ROSEY experiments suggest that it is complexed principally by 27 from the secondary face of its CD torus. However, the measured Ka value for the association of prednisolone with 27 in D2O was only 200 M-1, a value which is considerably

Reviews Scheme 7. Attachment of Seven N-Galactosyl Residues to a CD Core Using Amide Bond Formation as the Key Step

smaller than that (2000 M-1) for the complexation of native β-CD with prednisolone. Molecular modeling of the native β-CD-prednisolone complex, using NMR-derived distance constraints, reveals that hydrogen bonding between the 6-hydroxyl groups of β-CD and the C-3 carbonyl group of prednisolone plays an important role in the stabilization of the complex. The absence of this interaction in the 27/prednisolone complex may therefore account for the decrease in the Ka value. Parrot-Lopez and co-workers (24) have expanded their synthetic studies on monobranched CDs such as 8 to investigations of persubstituted β-CD derivatives, adorned with seven N-β-D-galactosyl units at the end of a spacer arm. Reaction (Scheme 7) of 8.4 equiv of 29 with per-6amino-β-CD (30), under standard amide bond-forming conditions, yields the fully substituted cluster compound 31. Our own research group has also used (34) amide bond formation as the key step in the synthesis of the Sglucosyl- and S-lactosyl CD-based clusters 32 and 33,


respectively. The lactose cluster 33 was shown to bind the nonsteroidal antiinflammatory, naproxen, in phosphate-buffered saline solution more weakly (Ka ) 165 M-1) than does β-CD (Ka ) 374 M-1) under identical conditions. Another research group (35) in the US has utilized amide bond formation as the key step in the attachment of carbohydrate units to CD cores. However, in this research the carboxylic acid functions are located on the primary face of the CD and the amine functions on the appended carbohydrate units. Mixtures containing an average of five carbohydrates per CD and four carbohydrates per CD for the cases of R-CD and β-CD, respectively, were reported. The clean and efficient reaction between amines and isothiocyanates to form thioureas, has been applied (36) by the research groups of Fernańdez and Defaye in the preparation of persubstituted CDs. The readily prepared carbohydrate isothiocyanates 34 and 35 (Scheme 8) react readily with per-6-amino-β-CD 30 to afford the fully substituted glycoclusters 36 and 37, respectively, in good yields. The conditions required for this coupling reaction (Me2CO/H2O/NaHCO3) result in partial deacetylation of the products, which are then fully deacetylated in a further step to afford the free cluster compounds. Research groups led (37) by Roy and Santoyo-Gonza´lez have performed excellent and extensive investigations into both the syntheses and lectin-binding affinities of perglycosylated β-CD derivatives. In this research, perglycosylated β-CDs, containing four distinct linkage structures between the appended carbohydrate unit and the CD core have been prepared. The first set of CDs synthesized in this study are identical to 28. The investigation was extended to the synthesis of the corresponding per-β-galactosides, per-β-N-acetylglucosamines and per-R-mannosides derivatives, all prepared by slightly differing methods to those described previously. To investigate further, the link between spacer arm length on the lectin binding abilities of the glycoconjugates, the second set of CDs were designed to incorporate a short CH2CONH linker arm between the CD torus and the appended carbohydrate units. Reaction (Scheme 9) of per6-amino-β-CD (30) with chloroacetic anhydride affords the heptachloro-β-CD derivative 38 in a near quantitative yield. When 38 is treated with the isothiouronium-β-Dglucoside 39, the desired glycocluster 40 is obtained in excellent yield after de-O-acetylation. This synthetic methodology was repeated to obtain the β-galactosyl, β-Nacetylglucosamine, and R-mannosyl analogues of 40. The third and fourth set of derivatives utilize para-substituted phenylmannosides only as the carbohydrate units.

Scheme 8. Preparation of CD-Based Clusters, Using the Reaction of Isothiocyanates with Primary Amines to Form Thioureas as the Key Step in the Attachment of the Carbohydrate Units to the CD Core


Fulton and Stoddart

Scheme 9. Reaction of Isothiouronium 39sWhich Acts as a Latent Sulfur Heptachloro-β-CD 38 to Afford, after Deprotection, the Persubstituted Cluster 40

Nucleophileswith

the

Scheme 10. Synthesis of r-Mannosyl Clusters with Spacer Arms of Varying Lengths

Reaction of 41 (Scheme 10a) with per-6-iodo-β-CD (42), or reaction (Scheme 10b) of the isothiocyanate 43 with the extended β-CD core 44, affords the two mannosyl derivatives 45 and 46, respectively, with the obvious structural difference between these two compounds being the longer spacer arms present in 46. Various assays were then performed on these compounds. They indicated that (1) the cluster compounds are more effective inhibitors than the corresponding simple monosaccharide in competitive inhibition assays, (2) those clusters with longer spacer arms are more effective inhibitors than those with no spacer arms in competitive inhibition assays, and (3) the clusters can act as lectin cross-linking agents as a consequence of their multivalent nature. Unfortunately, the host-guest prop-

erties of none of these carbohydrate clusters have been, to our knowledge, reported by the authors yet. More recently, Roy and co-workers (38) have described the preparation of the protected persialylated β-CDs 47, 48 and 49, using similar synthetic methods to those described above. Nucleophilic displacement reactions have also proven to be a very effective method for the attachment of carbohydrate units onto CDs. Research groups lead by Scheme 11. Reaction of the N-Glucosyl Isothiouronium 50sWhich Acts as a Latent Sulfur Nucleophileswith the Heptaiodo-β-CD 51 To Afford the Persubstituted Cluster 52

Reviews

Santoyo-Gonza´lez and Vargas-Berenguel have utilized (39) N-glycosyl isothiouronium derivativesswhich act as latent thiolate nucleophilessin the synthesis of CD-based cluster N-glycosides. Reaction (Scheme 11) of the isothiouronium derivative 50 with per-2,3-diacetyl-per-6-iodoβ-CD (51) in the presence of cesium carbonate affords, after reacetylation, the persubstituted derivative 52. Standard deacetylation is then employed to afford the final deprotected cluster. This synthetic strategy was employed to prepare the corresponding N-galactosyl, 2-acetamido-2-deoxy-N-glucosyl and N-mannosyl derivatives. A variation of this nucleophilic displacement strategy has been used (40) recently to construct clusters, such as the galactosyl cluster 53 which contains longer spacer arms bridging the gap between the CD core and its attached carbohydrate residues. Glucosylamino, lactosyl, and lactosylamino derivatives of this compound were also prepared and their abilities to inhibit the hemagglutination of blood cells by lectins was also reported in this investigation.

Bioconjugate Chem., Vol. 12, No. 5, 2001 663 Scheme 12. Perfunctionalization of Both the Primary and Secondary Faces of a β-CD Core with 14 β-S-Glucosyl Residues

for the binding of doxorubicin with 57 is a consequence of the galactosyl appendages lowering significantly the rate of dissociation of doxorubicin from the CD cavity. All CD-based carbohydrate clusters synthesized to date have involved the attachment of carbohydrate units onto the primary face of CDs, probably as a consequence of the relative ease of carrying out CD primary face synthetic modifications. The attachment of carbohydrate residues to the secondary face or both faces of CDs could have some potential advantages relative to their primary face-substituted counterparts. In addition, they present a considerable challenge in the preparation of chemically modified CDs. We have devised (41) a method for the perfunctionalization with carbohydrate residues of (1) the primary face, (2) the secondary face, and (3) both the primary and secondary faces, simultaneously, of β-CD. For the key step, our strategy relies upon (1) the wellknown photoaddition of thiols to allyl ethers in an antiMarkovnikov fashion to yield thioethers, and (2) the fact that allyl ethers can be attached selectively to either or both the primary and secondary faces of CDs. The power of this methodology is demonstrated by the fact that the reaction (Scheme 12) of per-2,6-diallyl-β-CD (54) with an excess of β-D-thioglucose (55) in the presence of UV light from an Hg lamp affords, after deprotection, the fully substituted cluster compound 56 in a 70% yield. Derivatives persubstituted on either their primary or secondary faces only were also prepared in this straightforward manner. Hattori and co-workers (42) have synthesized a persubstituted galactosyl cluster 57 and its monovalent analogue 58. Using SPR-based techniques, they have estimated the binding constants of these two compounds with peanut lectin and found that the multivalent galactosyl cluster has an association constant (Ka ) 130000 M-1) with the lectin approximately 16 times higher than that (Ka ) 8100 M-1) of its monovalent analogue. SPR was also used to measure the association constants of the anticancer drug, doxorubicin with 58 (Ka ) 3100 M-1) and with 57 (Ka ) 62000 M-1). The Japanese group have suggested that the greatly increased Ka value

The use of 1,3-dipolar cycloadditions is perhaps an unusual choice of reaction to use as the key synthetic step in the attachment of carbohydrate units to CD cores. Nonetheless, Santoyo-Gonza´lez and co-workers (43) have prepared a galactosyl nitrile oxide derivative 59, which, when reacted (Scheme 13) with the per-6-propargyl-βCD derivative 60 in refluxing toluene, affords the cluster compound 61 in 79% yield. The isoxazole rings are formed in a regioselective manner, allowing the isolation of the isomerically pure compound 61. Calixarenes Appended with Carbohydrates. Calixarenes (44) are macrocyclic compounds commonly derived from the base-induced reaction of phenols with formaldehyde. They can exist in a conformation reminiscent of a basket or vasesindeed, the name calixarene is derived from a type of Greek vase called a calix. Although phenolderived calixarenes have [1n]metacyclophane constitutions (Figure 2) and are commonly composed of four, six, or eight arene units, compounds containing other numbers of arene units are also known. All calixarenes discussed within this review contain four arenes per macrocycle and are therefore, broadly referred to as calix[4]arenes. Substituents located on the “wider rim” of the


Fulton and Stoddart Scheme 14. The Use of the Mitsonobu Reaction in the Formation of Calix[4]arenes Mono- and Disubstituted with Protected Mannosyl Residues

Figure 2. (a) Condensed structural formulas for the calix[n]arenes with the upper and lower rims of the bowl-shaped macrocycle indicated. (b) The complete structural representation of a calix[4]arene. Scheme 13. The Use of 1,3-Dipolar Cycloaddition Reactions To Prepare a Persubstituted CD Cluster

Scheme 15. Preparation Calix[4]arene Cluster

calixarene are said to be situated on the “upper rim” of the macrocycle, while those on the “narrow rim” are said to be situated on the “lower rim” of the macrocycle. They possess an extensive host-guest chemistry, a property which makes them of potential interest to those researchers involved in developing molecular delivery vehicles. A multitude of metal cations, simple arenes, small neutral molecules and even C60 are all known examples of guests in both the solution and solid statessfor complexation by calixarenes of varying sizes and shapes and with a range of functionalities. The literature of calixarenes has been reviewed extensively, and the reader should consult the monographs and reviews listed in ref 44 for more details. In a pioneering study, Dondoni, Ungaro and co-workers (45) have developed methods for the attachment of carbohydrates onto calixarenes to form the so-called “calixsugars”. They anticipated that the attachment of carbohydrate substituents would induce water solubility upon the calixarene derivatives, thus opening up the possibility of being able to explore the supramolecular chemistry of calixarenes in water. They also realized that these compounds may have some useful role to play in the study of carbohydrate recognition processes. In their investigations, the Italian consortium developed synthetic routes to O-glycosyl calix[4]arene derivatives, examining how carbohydrates could be attached to both the upper and lower rims of calixarenes. The approach they have developed for the attachment of carbohydrate units to the lower rim of calix[4]arene 62 takes advantage of the

of

a

Tetragalactosyl

fact that, even although the phenolic hydroxyl groups are poor nucleophiles, they do possess acidic protons. This feature opens up the possibility to couple carbohydratess unprotected at their anomeric centerssto the phenolic hydroxyl groups using Mitsonobu conditions. Thus, reaction (Scheme 14) of 62 with R-D-mannofuranose diacetonide 63 under Mitsonobu conditions affords, in a stereospecific manner, the β-anomers of the mono- or disubstituted products 64 or 65, depending on the number of equivalents of the acetonide used. Unfortunately, it was not possible to remove the protecting groups cleanly in order to afford the free mannofuranosyl derivatives. Glycosylation of 62 with tetra-O-acetyl-R,βD-glucopyranose under similar conditions produced a complex mixture of diastereoisomeric mono- and bisglycosylated calixarenessindicating clearly the limitations of using Mitsonobu conditions for the preparation of calixsugars. In an attempt to glycosylate the upper rim of calixarene derivatives, the Italian consortium have chosen calix[4]arene derivatives which are locked into

Reviews


Scheme 16. The Formation of Tri- and Tetragalactosyl Calix[4]arenes by Wittig Reactions

Scheme 17. Reaction of β-Glucosyl Isothiocyanates with a Diaminocalix[4]arene To Form the Dithiourea Glucosyl Cluster

cone conformations by four O-propyl groups on their lower rims. Hydroxymethyl substituents on the upper rims of these conformationally locked calix[4]arenes act as the glycosyl acceptors for thioglycoside glycosyl donors. After preparing bisglycoside-calix[4]arenes initiallysonly to find that these deprotected calixsugars were not water solublesthe consortium turned their attention to the synthesis of tetrakisglycoside-calix[4]arenes. Reaction (Scheme 15) of the thiogalactoside tetraacetate 66 with the protected calix[4]arene 67 affords, after deprotection, the tetrakis-O-galactosyl calix[4]arene 68. This compound was found to be not only water soluble but also to act as a host toward charged guests such as D-glucosamine hydrochloride and the dihydrogenphosphate anion. Dondoni and co-workers (46) have also extended their synthetic methodology to prepare upper rim functionalized O-ketopyranosyl calix[4]arenes. Concerned about the limitations imposed upon these calixarene-based carbohydrate clusters by the potentially acid labile glycosidic bonds linking the carbohydrate units to the macrocycle, Dondoni and co-workers (47) have developed another synthetic route in which the carbohydrate appendages are linked to the calixarene core by carbon-carbon bonds. Reaction (Scheme 16) of the diacetonegalactose phosphonium salt 69 with BuLi forms the corresponding phosphorane, which is then reacted with the tetraformylcalix[4]arene 70 to afford, after hydrogenation of the olefins, the tris- and tetragalactosides 71 and 72, respectively, in a 1:1 ratio. Removal of the protecting groups in 72 by acid hydrolysis affords the free

Scheme 18. The Use of Suzuki Cross-Coupling Reactions To Prepare a Di-β-glucosyl Calix[4]arene

carbohydrate cluster 73, which, however, is not soluble in watersfurther highlighting the problems of trying to modify calixarenes with appended carbohydrates to induce water solubility. Ribose analogues of these galactosyl clusters were also synthesized by the Italian group. Santoyo-Gonza´lez and co-workers (48) have used the much tried-and-tested thiourea forming reaction for the attachment of carbohydrate residues to the lower rim of calixarenes. Reaction (Scheme 17) of the diamino-calix[4]arene 74 with the glucosyl isothiocyanate 34 affords, after deprotection, the bisglycosylthiourea-calix[4]arene 75. This simple synthesis has been extended to the R-Dmannosyl, β-D-galactosyl, and D-lactosyl derivatives as well. No comments were made on the water-solubility of these particular carbohydrate clusters. Another interesting route to calixarene-based carbohydrate clusters has been reported by Parrot-Lopez and co-workers (49). It relies upon Suzuki-type coupling reactions to attach the carbohydrate units to the upper

666 Bioconjugate Chem., Vol. 12, No. 5, 2001 Scheme 19. Reaction of 2,3,4,5,6-Pentaacetyl-D-gluconyl Chloride 80 with the Diaminocalix[4]arene 74 To Afford, after Deacetylation, the Disubstituted Cluster 82

rim of the calixarene scaffold and, at the same time, increase the cavity size of the calixarene. The diboronic acid calix[4]arene 77 can be prepared (Scheme 18) from the dibromide 76 by reaction with n-BuLi and B(OMe)3 in THF. Cross-coupling reaction of 77 (2 equiv) with the 4-bromophenyl glycoside 78 using Pd(PPh3)4 as the catalyst afforded the bisglycosylated product 79 in 32% yield after deprotection. Removal of the acetate protecting groups proved to be troublesome, resulting in a yield of only 11% in the deprotection step. The same research group have also developed a “one-pot” synthesis of an analogous calix[4]arene, bearing one maltosyl unit. The water-solubilities of the calixarene-based carbohydrate clusters prepared in this study have not been discussed by the authors. Lhota´k and co-workers (50) have prepared some calixarene-based carbohydrate clusters with the aim of developing new molecular receptors, which, they claim, could have interesting properties on account of their chiral polyhydroxylated carbohydrate appendages. Their initial attempts to attach carbohydrate units to the lower rim of calix[4]arenes through amide bond formation

Fulton and Stoddart

proved successful. Removal of the carbohydrate protecting groups, however, was not so easy. To circumvent this problem, they tried an alternative synthesis. 2,3,4,5,6Penta-O-acetyl-D-gluconyl chloride 80, which was prepared from sodium gluconate in two steps, is reacted (Scheme 19) with 74 to afford the protected compound 81 with two amide bonds in a 49% yield. Deacetylation (NaOMe/MeOH) afforded the deprotected compound 82 in an almost quantitative yield. Interestingly, 82 was found to dimerize in solution (Kdimerization ) 6300 M-1 in CDCl3), a process which presumably is occurring through intermolecular hydrogen bonding between carbohydrate residues. The complexing ability of 82 was investigated in CDCl3 using 1-O-octyl-R-D-glucopyranoside (Ka ) 650 M-1), 1-O-octyl-β-D-glucopyranoside (Ka ) 1050 M-1), and 1-S-octyl-β-D-glucopyranoside (Ka ) 1000 M-1) as guest molecules. Job plots indicated that 1:1 binding occurs between these guests and the host, although the dimerization of 82 must hamper a precise evaluation of the binding data presented. The authors are of the opinion that these binding constants are similar enough to discount any selectivity of the host toward these guests. Roy and co-workers have also developed methods for the preparation of calixarene-based carbohydrate clusters. Realizing the lack of a suitable spacer arm between the calixarene scaffold and the carbohydrate appendages, and poor water solubility, in the calixarene-based clusters described so far, the Canadian group have designed compounds which circumvent these problems. In their initial communication (51), the Canadian group synthesized, in six steps, the calix[4]arene derivative 83, which contains four spacer arms linked to the phenolic oxygen atoms of the calix[4]arenes’ lower rim. The N-chloroacetyl groups present in 83 have been shown in Roy’s laboratory to act as efficient substrates for the attachment of thiolated carbohydrates. Recalling their previous success in the synthesis of sialoside-coated glycodendrimers, they chose to attach R-thiolated sialosides onto the calixarene scaffold. Reaction (Scheme 20) of the thiol 84 with 83 afforded the protected cluster compound 85 in 65% yield. 1 H NMR spectroscopic evidence supports the hypothesis that the calixarene was completely substituted with four sialoside units. Deprotection of 85 afforded the final compound 86 in a quantitative yield. Fortunately, 86 was found to be fairly soluble in water (3 mg/mL). A turbidimetric analysis was performed, which indicated that 86 has a strong cross-linking ability with the lectin, wheat germ agglutinin. The amphiphilic nature of calixarene-based carbohydrate clusters has been a hindrance to researchers in this

Scheme 20. Preparation of a Calix[4]arene-Based Tetrasialic Acid Cluster

Reviews Scheme 21. Attachment of Four r-N-Acetylgalactosyl Residues to the Lower Rim of a Tetraacid Chloride Calix[4]arene To Form the Tetra-r-N-acetylgalactosyl Cluster

field since it confers upon these compounds, at the very best, poor water solubility, and, more often than not, no solubility in water at all. However, Roy and co-workers (52) have taken advantage of the amphiphilic nature of calixarene-based carbohydrate clusters to prepare a new series of compounds which, on account of their hydrophobic upper rims, can be adsorbed onto the hydrophobic well surfaces in polystyrene microtiter plates. This observation means that amphiphilic carbohydrate cluster compounds of this particular design can be used as antigens in ELISA-type biological assays. The model carbohydrate 87, chosen in this study, was based on the TN antigen (GalNAcR1 f O-Ser/Thr) without the O-Ser/ Thr aglycon. The TN antigen corresponds to one of the

Bioconjugate Chem., Vol. 12, No. 5, 2001 667 Scheme 22. Preparation galactosyl Cluster Cluster

of

an

Octa-r-N-acetyl-

immunodominant epitopes found in human adenocarcinoma mucinssa class of large, heavily glycosylated glycoproteins associated with some cancerous tumors. Reaction (Scheme 21) of 87 with the readily prepared tetrachlorocalix[4]arene 88 afforded after deprotection, the tetravalent cluster 89. A semiconvergent approach was then used to synthesize calixarenes with higher valencies. Reaction (Scheme 22) of the bromide 90s prepared in four synthetic steps from 87swith the extended calixarene core 91 yields the dendritic octameric cluster 92. The synthesis has been expanded in an impressive manner to afford the hexadecamer cluster 93. In all cases, the homogeneities of the cluster compounds were demonstrated by mass spectrometry or by NMR spectroscopysa technique which was also used to verify that the compounds exist with the calixarene moieties


Figure 3. Condensed structural formula for the calix[4]resorcarenes with the upper and lower rims of the bowl-shaped macrocycle indicated. A complete structural representation of a calix[4]resorcarene is also shown.

in their cone conformations. The relative lectin-binding properties of 89, 92, and 93 were measured with the lectin Vicia villosa agglutinin (VVA) which is known to bind R-D-GalNAc derivatives. In a solid-phase competitive inhibition experiment, compound 93 was found to be a very effective inhibitor (IC50 ) 13.4 µMscf., a monovalent analogue with IC50 ) 158.3 µM) of the binding of VVA with asialoglycophorin, which is a natural human blood group serotype. Calixresorcarenes Appended with Carbohydrates. Calixresorcarenes are a subdivision of calixarenes. Since the body of work concerning calixresorcarene-based carbohydrate clusters is unique, however, it merits having a separate subsection devoted to it. Calixresorcarenes are cyclo-oligomers (Figure 3) formed from the condensation of resorcinol with aldehydes to form macrocycles with comparable structural and host-guest properties to those of calixarenes. Calixresorcarenes exhibit similar binding and conformational properties to those of calixarenes. Consequently, they have been intensely studied (45, 53) receptors. Calix[4]resorcarenes possess eight resorcinic hydroxyl groups located on their upper rims. They serve as useful handles for the further chemical modification of these receptors. Additionally, they possess four “feet” attached to their lower rims. These feet are determined by the choice of aldehyde utilized in the synthesis of the receptor. These structural features, among others, make calixresorcarenes a useful and readily available choice of receptor for further elaboration with carbohydrate appendages.

Fulton and Stoddart

Researchers led by Yasuhiro Aoyama have been the pioneers in the study of resorcarene-based carbohydrate clusters. In their first communication (54), this Japanese group reported the synthesis of an octagalactose calixresorcarene-based cluster by reaction (Scheme 23) of the octaamine 94 with lactonolactone (95) to afford the galactosyl cluster 96 in a 79% yield. This compounds despite its amphiphilic naturesis soluble in water (>0.1 M). Dynamic light scattering experiments indicate that, in aqueous solution, it is monomeric at concentrations of 0.1 mM, while it aggregates at concentrations of ∼1 mM. When a quartz plate was dipped into an aqueous solution of 96, electronic adsorption spectroscopy indicated that the galactosyl cluster was adsorbed onto its surface with a packing density of 3.5 nm2 per molecule. Additionally, it was also found that compound 96 cannot be rinsed off the plate by repeated washings with water: bases, pyridine, or amines, such as ethanolamine, are required in order to desorb the galactosyl cluster from the quartz plate. These results, supported by other experiments, indicate that compound 96 must be binding to the quartz surface through hydrogen bonding between the hydroxyl functions in the cluster and the silanol and silyl ether functionalities of the quartz surface. Another additional important experimental observation is that galactose and other carbohydrate compoundsswhen present in a large excessscannot either inhibit the binding of the galactosyl cluster 96 to the quartz surface, or promote its desorption from the quartz surface. This result indicates that compound 96 must be binding to the quartz surface in a multivalent fashion, i.e., there exists a highly cooperative binding interaction between the hydroxy functions of the carbohydrate appendages of 96 and the many hydrogen bond donor/acceptor sites present on the quartz surface, resulting in the strong binding of the galactosyl cluster to the quartz surface. Additionally, compound 96 was found to form a stable 1:1 complex (Ka ) 2.2 × 105 M-1) in aqueous solution with 8-anilinonaphthalene-1-sulfonate (ANS) which is located within the resorcarene cavity of 96. Although ANS is not readily adsorbed onto a quartz surface, it is adsorbed onto this surface in the presence of compound 96. Experiments have indicated that there is a 1:1 ratio of both ANS and 96 present on the surface. This evidence suggests that

Scheme 23. Formation of an Octagalactosyl Calix[4]resorcarene-Based Cluster 96 from the Octaamino Calix[4]resorcarene 94 and Lactonolactone 95. Structural Formula for the Glucosyl Derivative (97) of 96 Is Also Shown, as Is the Cartoon Representation of Both of These Cluster Compounds

Reviews


Figure 4. Schematic representation of the galactosyl cluster 96, immobilized on a hydrophobic surface-plasmon-resonance (SPR) chip. When galactose-binding lectins are passed through the flow cell, they can bind to the immobilized 96, inducing a refractive index change of the SPR chip.

compound 96 acts as a molecular delivery vehicle and has the ability to deliver guest molecules, with little or no affinity for a quartz surface, onto that same surface. However, since it is possible to remove the guest from the quartz surface by repeated washings, it is likely that the guest molecule is not completely encapsulated between the adsorbed galactosyl cluster and the surface. The hydrophobicity of the alkyl chains present in the galactosyl cluster has been used (55) to form (Figure 4) a monolayer of compound 96 on a hydrophobic SPR sensor chip. Thus, the interaction of 96 with various compounds, such as lectins and other proteins, can be detected when a solution of these compounds is passed over the 96-immobilized SPR chip. A strong interaction was detected between the galactosyl cluster and peanut agglutinin (PNA), a lectin which selectively binds galactosyl residues. A very weak interaction was, however, detected between the cluster and Con A, a lectin which selectively binds both mannosyl and glucosyl residues. Conversely, the analogous glucosyl cluster 97 was found to bind Con A but not PNA. The interaction of the clusters 96 and 97 with various monolayers has also been studied (56) using a quartz crystal microbalance. From this work, it has been reported that the clusters have a more favorable interaction with anionic and carbohydratebased monolayers compared with cationic, zwitterionic, and nonionic monolayers. Aoyama and co-workers (57) have also shown how these cluster compounds can be used to deliver guests to an immobilized Con A-Sepharose gel, purely as a consequence of carbohydrate-lectin interactions. Compound 96 and its glucosyl analogue 97 both form 1:1 complexes with the dye, eosin Y (98) with association constants of Ka ) 7.5 × 105 M-1 and Ka ) 1.8 × 105 M-1, respectively. In a simple series of experiments, the Japanese group have shown (Figure 5) that only the glucosyl cluster 97 can direct the adsorption of the guest dye 98 onto the surface of the Con A-Sepharose gel whereas the galactosyl cluster 96 cannot. Although, in reality, there is a certain amount of nonspecific binding interactions between the clusters and guest with the sepharose, the carbohydrate-lectin mediated binding is sufficiently strong to allow for a significant percentage of the guest in solution to be delivered to the Con A-Sepharose gel surface. The Fukuoka group (58) have taken the concept of carbohydrate-directed delivery one step further and, in a most impressive piece of work, they have shown how their calixresorcarene-based carbohydrate clusters can be used to deliver guest molecules to cells. Although

Figure 5. Schematic representation of the use of the glucosyl cluster 97 to deliver the dye eosin Y 98 to the surface of sepharose immobilized Con A. The 97‚98 complex forms a ternary complex with the Con A-immobilized on the sepharose. However, as the eosin Y is not completely encapsulated in the ternary complex, it can dissociate from the ternary complex, leaving the ConA‚97 complex.

carbohydrate-cell surface protein interactions are very specific, cells can also show high nonspecific affinities toward hydrophobic molecules. This problem is one which must be overcome if carbohydrate-protein interactions are to be the sole driving force for a delivery system. The Japanese group chose to study molecular delivery to hepatocyte cells (liver cells), which are known to contain a cell surface lectinsthe asialoglycoprotein receptors which specifically binds terminal galactose residues. As potential delivery vehicles, they chose the galactosyl and glucosyl clusters 96 and 97, and as a suitable guest molecule, the fluorescent dye phloxine B (99). Both clusters were shown to form 1:1 complexes with 99 in phosphate-buffered saline solution with association constants of 2.0 × 105 M-1 and 2.1 × 105 M-1, respectively, for 96 and 97. When a solution of the dye 99 was added to rat hepatoma (liver cancer) cells, these cells were found to fluoresce when viewed under a fluorescence microscope, indicating that the dye has been captured by the


cell as a consequence of nonspecific binding interactions. However, when a solution of 99 and the glucosyl cluster

97 is added to the cell (at a concentration where ∼100% of the guest is bound by 97), no fluorescence is detected from the cells, indicating that (1) the dye is so strongly bound by 97 that it is not available for nonspecific binding with the cell, and (2) that the glucosyl cluster is also not bound by the cell in a nonspecific manner. This hypothesis is further supported by the fact that the 96‚99 complex is bound by the cells as a consequence of the interaction between the terminal galactosyl residues present in the cluster 96 and the asialoglycoprotein receptor on the cell surfaces, causing the cells to fluoresce when viewed under the fluorescence microscope. If mouse spleen cellsscells which lack the asialoglycoprotein receptorsare used in the experiment, then the galactosyl cluster 96 cannot deliver the dye 98 to the cells. Thus, it can be concluded that (1) the clusters 96 and 97 do not bind with the cells through nonspecific interactions, and that (2) the cluster 96 can bind with the hepatocyte cells through interaction with the cell-surface asialoglycoprotein receptor. This work highlights the important role that carbohydrates can play in the masking of hydrophobicitysin compounds such as 96, it is only because its carbohydrate moieties mask the hydrophobicity of the calixresorcarene core sufficiently well that the carbohydrate-directed delivery of the guest dye to the cell surface is allowed to occur.

Compounds such as 96, and its larger analogous cluster 100, which contains 40 glucopyranosyl residues, have been shown (59) to bind various phosphate anions in water. Compound 96 also displays an interesting anioninduced agglutination. Phosphate salts such as Na2HPO4/ NaH2PO4, D-ribose-5-phosphate and guanosine-5′-monophosphate induce the agglutination of 96. 31P NMR Spectroscopy was used to estimate the ratio of host:bound salt. In the same investigation, dynamic light scattering

Fulton and Stoddart Scheme 24. Formation Cavitand-Based Cluster

of

a

Tetraglucosyl

has been utilized to demonstrate that Na2HPO4 can induce 96 to form aggregates which can grow up to µMsized particles in a matter of minutes. This aggregation phenomena is almost certainly a consequence of hydrogen bonding interactions between the polyhydroxylated carbohydrate appendages of the cluster compounds and the phosphate anions, which, on account of their ability to interact with more than one cluster molecule at a time, can act as a supramolecular “glue”, sticking the clusters together. The Fukuoka group propose that the extended polyhydroxyl appendages in compounds such as 96 and 100 can act as a macrosolvent for anions, allowing these salts to be extracted from water into the unique environment of the cluster compounds. Another carbohydrate cluster compound prepared by Aoyama and co-workers (60) has been constructed on a cavitand, i.e., a bowl-shaped macrocycle derived from calixresorcarenes. Reaction (Scheme 24) of the tetrathiol cavitand 101 with the bromide 102 affords, after deprotection, the tetraglucose cluster 103. Maltosyl and maltotriosyl analogues of 103 have been prepared in a similar manner. These three cluster compounds were shown to bind ANS and found to interact with the lectin Con A. CONCLUSIONS

The field of neoglycoconjugates based on cyclodextrin and calixarene cores issat least as judged by the sheer number of recent publicationssa blossoming one, with many challenges and opportunities available to researchers who can assume multidisciplinary mantles. To date, much effort has been directed toward the syntheses of neoglycoconjugates in feats which are by no means trivialsespecially when recognition is granted to synthetic chemists for doing the notoriously difficult synthetic chemistry that surrounds CDs and calixarenes. It appears, however, that the synthetic problems associated with preparing these neoglycoconjugates have, to a large extent, been successfully overcome, thanks to the many varied and ingenious methods of syntheses now available to the talented synthetic chemist. By contrast, investigations of the molecular recognition properties of these neoglycoconjugates, and particularly their abilities to interact with lectins and cells, both in vitro and in vivo, have been few and far between. Thus, it seems obvious that, if the field of CD- and calixarene-based neoglycoconjugates is to continue to blossom, the emphasis in research must now be directed toward the physical and, most importantly of all, the biological evaluations of these compounds. Only when the biological chemistry is as well understood as the synthetic chemistry will the potential of this unique class of bioconjugates be truly realized and appreciated. The rewards for researchers who are willing and able to tackle real scientific issues surrounding

Reviews

neoglycoconjugates in a multidisciplinary fashion are immense to say the very least. ACKNOWLEDGMENT

We thank Dr. Bruce Turnbull for helpful discussions and Dr. Norma Stoddart for proof-reading the final manuscript. We also thank Anthony Pease for the design of the cover art work. LITERATURE CITED (1) (a) Varki, A. (1993) Biological roles of oligosaccharides all of the theories are correct. Glycobiology 3, 97-130. (b) Essentials of Glycobiology (A. Varki, R. Cummings, J. Esko, H. Freeze, G. Hart and J. Marth, Eds.), Cold Spring Harbor Laboratory Press, New York, 1999. (2) Lee, Y. C., and Lee, R. T. (1995) Carbohydrate-protein interactions - basis of glycobiology. Acc. Chem. Res. 28, 321327. (3) Drickamer, K. (1995) Multiplicity of lectin-carbohydrate interactions. Nat. Struct. Biol. 2, 437-439. (4) Lee, Y. C., and Lee, R. T. (1994) Neoglycoconjugates: Preparation and Applications (R. T. Lee and Y. C. Lee, Eds.) pp 23-50, Academic Press, San Diego. (5) (a) Lindberg, A. A., Brown, J. E., Stromberg, N., Westlingryd, M., Schultz, J. E., and Karlsson, K. A. (1987) Identification of the carbohydrate receptor for shiga toxin produced by Shigella-Dysenteriae type-1. J. Biol. Chem. 262, 1779-1785. (b) Merrit, E. A., and Hol, W. G. J. (1995) AB5 Toxins. Curr. Opin. Struct. Biol. 5, 165-171. (6) In a Shiga toxin AB5 structure, five identical subunits are noncovalently bound in a doughnut shape (the B5 pentamer component), with a single subunit (the A component) attached to one face of the B5 pentamer. (7) Kitov, P. I., Sadowska, J. M., Mulvey, G., Armstrong, G. D., Ling, H., Pannu, N. S., Read, J., and Bundle, D. R. (2000) Shiga-like toxins are neutralized by tailored multivalent carbohydrate ligands. Nature 403, 699-672. (8) Toyokuni, T., and Singhal, A. K. (1995) Synthetic carbohydrate vaccines based on tumor-associated antigens. Chem. Soc. Rev. 231-242. (9) (a) Monsigny, M., Roche, A.-C., Midoux, P., and Mayer, R. (1994) Glycoconjugates as carriers for specific delivery of therapeutic drugs and genes. Adv. Drug Delivery Rev. 14, 1-24. (b) Wadhwa, M. S., and Rice, K. G. (1995) Receptor mediated targeting. J. Drug Targeting 3, 111-127. (10) Franssen, E. J. F., Jansen, R. W., Vaalburg, M., and Meijer, D. K. F. (1993) Hepatic and intrahepatic targeting of an antiinflammatory agent with human serum albumin and neoglycoproteins as carrier molecules. Biochem. Pharm. 45, 1215-1226. (11) (a) Lehn, J.-M. (1995) Supramolecular Chemistry; VCH: Weinheim. (b) Comprehensive Supramolecular Chemistry (J. L. Atwood, J. E. D. Davies, D. D. MacNicol, and F. Vo¨gtle, Eds.) Pergamon, Oxford, 1996, 11 vols. (12) The chemical literature contains many examples of synthetic receptors designed to bind biologically important molecules. For a few examples, see Volume 2, Comprehensive Supramolecular Chemistry; (J. L. Atwood, J. E. D. Davies, D. D. MacNicol, and F. Vo¨gtle, Eds.) Pergamon, Oxford, 1996. (13) The chemistry, structure and function of cyclodextrins has been reviewed extensivly in the chemical literature. A recent issue of Chemical Reviews was exclusivly devoted to cyclodextrin chemistry: (1998) Chem. Rev. 98, 1741-2076. Also see: Wenz, G. (1994) Cyclodextrins as building-blocks for supramolecular structures and functional units. Angew. Chem., Int. Ed. Engl. 33, 803-822. (14) Uekama, K., and Irie, T. (1996) Comprehensive Supramolecular Chemistry (J. L. Atwood, J. E. D. Davies, D. D. MacNicol, and F. Vo¨gtle, Eds.) pp 451-482, Pergamon, Oxford. (15) Kim, C., MacKellar, W. C., Cho, N., Byrn, S. R., and Morre´, D. J. (1997) Impermeant antitumor sulfonylurea conjugates

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BC0100410

Neoglycoconjugates Based on Cyclodextrins and Calixarenes

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