Carbohydrate Microarrays and Fluorous-Phase Synthesis - American

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Carbohydrate Microarrays and Fluorous-Phase Synthesis: Interfacing Fluorous-Phase Tags with the Direct Formation of Glycoarrays Nicola L . Pohl Department of Chemistry and the Plant Sciences Institute, Iowa State University, Ames, IA 50011

Phase switching, such as the use of fluorocarbon tags, is a common technique to facilitate purification of intermediates in iterative biopolymer synthesis. After a review of the application of fluorous tags in carbohydrate synthesis, a new method to directly array sugars via noncovalent fluorousfluorous interactions is discussed. Light-fluorous tags now can serve not only to simplify iterative oligosaccharide synthesis, but also to directly pattern sugars on surfaces for biological screening.

Introduction Carbohydrates interact specifically with proteins to mediate biological processes that include inflammatory responses, pathogen invasion, cell differentiation, cell-cell communication, cell adhesion and development, and tumor cell metastasis (7-5). Information about these interactions would help illuminate the role of carbohydrates in the life cycles of organisms as well as foster the development of sugar-based therapeutics such as vaccines that intervene in these carbohydrate-protein interactions. Unfortunately, the molecular basis for many of these sugar-protein interactions is not understood, © 2007 American Chemical Society

In Current Fluoroorganic Chemistry; Soloshonok, V., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

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262 in part because homogeneous well-defined carbohydrates are extremely difficult to obtain (3). Whereas pure peptides and nucleic acids are readily commercially available by automated synthesis, an analogous commercial process for carbohydrate synthesis is still missing. The difficulty of carbohydrate synthesis also limits the range of structures that can be incorporated into microarrays for screening of carbohydrate-protein interactions.


Phase-Switching and Fluorous-Phase Carbohydrate Synthesis Iterative biopolymer synthesis is often facilitated by the use of soluble or solid-phase supports to simplify the purification of intermediates (Figure 1). For example, solid-phase carbohydrate synthesis allows excess reagents required for reaction completion to be washed off between steps in a process that has been automated (4). Alternatively, to avoid poor reaction kinetics inherent to biphasic systems, soluble tags with unique physical properties can be attached to the growing chain to aid in purification of intermediates by tag precipitation, extraction into a liquid phase, or affinity chromatography/solid-phase extraction (5). Because tag precipitation is not quantitative, tags for extraction methods are attractive options. protected carbohydrate building block

1) couple '2) filter, precipitate or extract linker K

phase-switching group (solid phase, soluble oligomer, light or heavy fluorous tag)

1) deprotect 2) filter, precipitate or extract

t

protecting group

Figure 1. A basic scheme for phase-switching approaches to iterative carbohydrate synthesis. Soluble fluorocarbon tags have been employed in the synthesis of a variety of carbohydrates. Fluorocarbons w i l l phase separate from aqueous or conventional organic solvents. Several fluorocarbon chains can be incorporated into a protecting group to allow extraction of the compound containing the "heavy" fluorous tag into a liquid fluorocarbon layer or a single fluorocarbon chain, a "light" fluorous tag, can capture the tagged molecule by fluorousderivatized silica gel in a solid-phase extraction process (6-8). Both "heavy" and


263 "light" fluorous tags have been developed specifically for the challenges of oligosaccharide synthesis.


Fluorous Tags for Liquid-Liquid Extractions in Carbohydrate Synthesis Nonenzymatic carbohydrate synthesis usually relies on protecting groups to permanently mask some hydroxyl and other functional groups and to temporarily block future reaction sites. These protecting groups are perfect locations for the introduction of fluorocarbon tags that allow liquid-liquid extraction of the reaction product away from excess reagents. The first protecting group introduced for this purpose to aid sugar synthesis was a variation of the commonly-used benzyl group. Three fluorocarbon chains attached to a silicon modify a benzyl group (Bnf, Figure 2) and allow extraction of molecules protected with the group into perfluorohexanes (9). After a monomer addition reaction to a triply Bnf-protected glucal, disaccharide 1 could be isolated by extraction into a liquid fluorocarbon phase. Two variations of the commonly-employed ester protecting group have also been designed for the synthesis of disaccharides 2 and 3 (Figure 2) with facile purification of intermediates by liquid-liquid extraction to create up to pentasaccharides (1012). A benzoyl variation has also been reported recently (13).

Figure 2. Fluorocarbon hydroxyl protecting groups for the synthesis of carbohydrates with liquid-liquid extraction of intermediates.


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Unfortunately, the necessity to include multiple fluorocarbon tails for efficient liquid-liquid extraction also limits the solubility of the compounds in the nonfluorocarbon solvents required for a range of reaction types. Additionally, the large protecting groups can complicate spectral interpretation for characterization and a substantial amount of the molecular weight of the intermediates is accounted for by the fluorocarbon tags. Nonetheless, the tags allow iterative carbohydrate synthesis with minimal chromatography and with the benefits of solution phase reaction kinetics and reaction monitoring not possible by solid-phase approaches.

Fluorous Tags for Solid-Phase Extraction in Carbohydrate Synthesis The addition of only one fluorocarbon chain to a protected carbohydrate renders the molecule separable from nontagged compounds not by liquid-liquid extraction but by solid-phase extraction (SPE) instead. The reaction mixture is loaded on fluorous silica gel, untagged compounds are eluted, and then a change of solvent allows elution of the pure tagged compound (7). Several carbohydrate protecting groups as well as an anomeric activating group for glycosylation reactions have been designed with single fluorous tags to simplify purification schemes. Addition of a fluorous tag to a thiol anomeric activating group creates a glycosylation building block 4 (Figure 3) that could easily be purified by SPE (14). The thiol byproduct after glycosylation could be readily removed by SPE and recycled after reduction of any disulfide formed. In addition to the benefits of purification ease, the fluorous tag also rendered the thiol less repugnant.

Figure 3. A single fluorous tagfor a recyclable activating group for glycosylotions. Fluorous protecting groups have also been used to facilitate iterative carbohydrate synthesis by solid-phase extraction of growing chain intermediates. A fluorous version of a silicon protecting group (15) was used to protect the anomeric position of a glucosamine building block and build up the Lewis a trisaccharide 5 (Figure 4) with intermediates purified by fluorous SPE (16-17). However, unlike tags with multiple fluorocarbon chains, the fluorous group did not prevent the use of standard chromatography methods if necessary. A related fluorous silyl group has been used not in iterative synthesis but to cap oligosaccharides made on solid-phase for isolation of tagged sequences by SPE (18). More recently, a fluorous version of a carbamate nitrogen protecting group



265 was developed and applied to the synthesis of a disaccharide 6 (Figure 4 ) (19). The group can be synthesized in three steps and removed with exchange to an acetyl group using zinc in acetic anhydride with triethylamine. Unfortunately, the new protecting group contains a stereogenic center, but at least in the cases reported this center does not overly complicate spectra. Finally, a fluorous version of the allyl protecting group has also been developed for facile purification of intermediates in the synthesis of polymannosides such as 7, for example (Figure 4 ) (20-21). The fluorous allyl group allows fluorous SPE purification of intermediates and can be removed using standard palladiummediated deallylation conditions.

AcO B n O - ^N O OH H BnO BnO 0 5

OAc

B

n

^ 0

^ \

0

BnO BnO o-^-^S

Ac£> AcO

6

C F 8

17

7

Figure 4. Protecting groups with single fluorous tags for the synthesis of carbohydrates with fluorous solid-phase extraction of intermediates.

Direct Formation of Fluorous-based Carbohydrate Microrrays Like solid-phase approaches, fluorous-phase methods are showing promise in simplifying the iterative synthesis of carbohydrates with the added benefits of standard solution-phase reaction monitoring, reduced building block requirements at each coupling cycle, and the possibility of using other purification techniques as needed with intermediates. However, synthetic carbohydrates are still extremely precious and therefore, ideally, would be used sparingly to allow multiple bioassays. Microarrays such as D N A chips require minimal sample usage and therefore have spurred development of a range of technologies for protein-detection assays on glass slides (22-23). Some of these technologies have been applied to the synthesis of carbohydrate chips (24-33), but most of these microarray methods rely on covalent attachment of a compound to the slide and therefore require unique functional handles. Recently, a simpler method that relies on noncovalent fluorous-fluorous interactions has been developed that allows the direct formation of carbohydrate microarrays


266


using a single fluorocarbon chain that can also facilitate oligosaccharide synthesis (Figure 5 ) (34).

Figure 5. A combined strategy for use of a single fluorous tag to both facilitate iterative carbohydrate synthesis and allow direct formation of microarrays for biological screening. (Adapted with permission from reference 34. Copyright 2005 American Chemical Society.)

8 Man

9 GlcNAc

10 Gal

11 Fuc

Figure 6. A series of fluorous-tagged carbohydrates for microarray formation. To test the feasibility of using noncovalent fluorous-fluorous interactions for surface patterning, a series of carbohydrates were synthesized with a fluorous-tagged allyl linker. The allyl linkers were then converted to more



267 flexible and less reactive alkyl linkers by hydrogenation. After synthesis of the requisite fluorous-tagged sugars (Figure 6), a suitable fluorinated surface was needed. Initial microarray experiments used a commercially available glass microscope slide coated with a Teflon/epoxy mixture for compound spotting with a standard robot used for D N A arraying. The spotted slide was incubated for 20 minutes with a solution of the fluorescein isothiocyanate-labeled jack bean lectin concanavalin A (FITC-ConA) rinsed repeatedly with assay buffer and distilled water, and then scanned with a standard fluorescent slide scanner. The scan clearly showed binding of FITC-ConA only to the mannose-containing spots. The anomeric position also could be distinguished as the beta-linked GlcNAc 9 was not recognized by the lectin. Although this initial lectin experiment demonstrated the ability of the CgFn tail to anchor the carbohydrates to the slide surface even after repeated washes, the intrinsic uneven fluorescence of the commercial slide at 488 n M , a wavelength that is commonly used to detect labeled analytes, necessitated another approach. A n optically and fluorescently clear surface for the formation of compound microarrays then was obtained by reaction of a glass microscope slide with a fluoroalkylsilane. The new microarray substrate also allowed spotting of fluorous-tagged sugars using an arraying robot, but after incubation with FITC-labeled lectins the protein-bound carbohydrates stood out clearly from the background in the fluorescent scan (Figure 7). A test of the ability of the array to withstand detergents often included in biological screens was carried out with the labeled plant lectin from the bush Erythhna cristagalli ( F I T C - E C A ) and the hydrocarbon detergent Tween-20. Because hydrocarbons phase separate from fluorocarbons, the noncovalent fluorous-based array would be expected to be more stable to regular detergents than a noncovalent array approach based on hydrocarbon-hydrocarbon interactions. Indeed, the fluorous-based array withstood the 20 minute incubation time and repeated rinsing with this detergent-containing buffer. Man (8)

GlcNAc (9)

Gal (10)

Fuc (11)

Figure 7. Fluorescence images of arrayed carbohydrates probed with FITClabeled lectins. Columns of 4 spots each of 2, 1, 0.5 and 0.1 mM carbohydrates were incubatedfor 20 min with FITC-ConA (top) or FITC-ECA with 1% TWEEN-20 detergent (bottom) with BSA. (Reproduced with permission from reference 34. Copyright 2005 American Chemical Society.)


268 These initial results now have been expanded to the production of carbohydrate arrays that include disaccharides as well as a charged amino sugar with equal success (20). Applications of this fluorous-based strategy for microarray formation to larger saccharides as well as other molecules such as nucleic acids can easily be envisioned.


Future Directions The ability to directly form compound microarrays with a fluorous-tail makes a fluorocarbon-based phase-switching approach to compound synthesis even more appealing. "Light" fluorous tags already have proven their utility in accelerating small molecule synthesis, including carbohydrate synthesis. Reactions can be monitored using traditional solution-phase techniques and, unlike solid-phase approaches, large excesses of reagents are not required for high yields. However, several challenges remain. Unlike solid-phase approaches, iterative fluorous-phase synthesis of any molecule class has never been automated. Automation is key to gaining the benefits of facile library synthesis seen with the automation of both peptide and nucleic acid synthesis. Chemistry amenable to automation for iterative oligosaccharide synthesis based on fluorous tags has been developed (21), but demonstration of its automation remains to be seen. The generality of using noncovalent fluorous-fluorous interactions for microarray formation also needs to be probed. Extensions to not only a variety of oligosaccharides but also other biopolymers and small molecules ultimately will test the robustness and utility of this new fluorousbased surface patterning approach.

References 1. 2. 3. 4. 5. 6. 7.

8.

Dube, D . H.; Bertozzi, C. R. Nat. Rev. Drug Discov. 2005, 4, 477-488. Rudd, P. M.; Wormald, M. R.; Dwek, R. A. Trends Biotechnol. 2004, 22, 524-530. Ratner, D . M.; Adams, E . W.; Disney, M. D . ; Seeberger, P. H . ChemBioChem, 2004, 5, 1375-1383. Plante, O. J.; Palmacci, E . E . ; Seeberger, P. H . Science, 2001, 291, 1523-1527. Ito, Y . ; Manabe, S. Chem. Eur. J. 2002, 8, 3077-3084 and references therein. Horváth, I. T. Acc. Chem. Res. 1998, 31, 641-650. Curran, D . P. Separations with Fluorous Silica Gel and Related Materials. In The Handbook of Fluorous Chemistry, Gladysz, J.; Horváth, I.; Curran, D . P.; Wiley-VCH: Weinheim, 2004; pp 101-127. Zhang, W. Curr. Opin. Drug Discov. Develop. 2004, 7, 784-797.


269


9. Curran, D. P.; Ferrito, R.; Hua, Y. Tetrahedron Lett. 1998, 39, 49374940. 10. Miura, T.; Hirose, Y.; Ohmae, M.; Inazu, T. Org. Lett. 2001, 3, 39473950. 11. Miura, T.; Goto, K.; Hosaka, D.; Inazu, T. Angew. Chem. Int. Ed. 2003, 42, 2047-2051. 12. Miura, T.; Goto, K.; Waragai, H.; Matsumoto, H.; Hirose, Y.; Ohmae, M.; Ishida, H.-k.; Satoh, A.; Inazu, T. J. Org. Chem. 2004, 69, 53485353. 13. Miura, T.; Satho, A.; Goto, K.; Murakami, Y.; Imai, N.; Inazu, T. Tetrahedron: Asymmetry 2005, 16, 3-6.

14. 15. 16. 17. 18.

Jing, Y.; Huang, X.; Tetrahedron Lett. 2004, 45, 4615-4618. Röver, S.; Wipf, P. Tetrahedron Lett. 1999, 40, 5667-5670. Manzoni, L. Chem. Commun. 2003, 2930-2931. Manzoni, L.; Castelli, R. Org. Lett. 2004, 6, 4195-4198. Palmacci, E. E.; Hewitt, M. C .; Seeberger, P. H. Angew. Chem. Int. Ed. 2001, 40, 4433-4437. 19. Manzoni, L.; Castelli, R. Org. Lett. 2006, 8, 955-957. 20. Mamidyala, S. K.; Ko, K.-S.; Jaipuri, F. A.; Park, G.; Pohl, N. L. J. Fluor. Chem. 2006, 127, ASAP. th

21. Jaipuri, F. A.; Pohl, N. L. Abstracts of Papers. 230 National Meeting of the American Chemical Society, Washington, DC, Aug 28-Sept 1, 2005; American Chemical Society: Washington, DC, 2005, ORGN 185. 22. Tomizaki, K.; Usui, K.; Mihara, H. ChemBioChem 2005, 6, 782-799. 23. Uttamchandani, M.; Wang, J.; Yao, S. Q. Mol. BioSyst. 2006, 2, 58-68. 24. Dyukova, V. I.; Shilova, N. V.; Galanina, O. E.; Rubina, A. Y.; Bovin, N. V. Biochim. Biophys. Acta 2006, ASAP.

25. Huang, G. L.; Zhang, H. C.; Wang, P. G. Bioorg. Med. Chem. Lett. 2006, 16, 2031-2033. 26. Shin, I.; Lee, M. Angew. Chem. Int. Ed. 2005, 44, 2881-2884. 27. Biskup, M.; Muller, J. U.; Weingart, R.; Schmidt, R. R. ChemBioChem 2005, 6, 1007-1015. 28. Adams, E. W.; Ratner, D. M.; Bokesch, H. R.; McMahon, J. B.; O'Keefe, B. R.; Seeberger, P. H. Chem. Biol. 2004, 11, 875-881. 29. Bryan, M.; Fazio, F.; Lee, H.; Huang, C .; Chang, A.; Best, M.; Paulson, J. C .; Burton, D.; Wilson. L.; Wong, C.-H. J. Am. Chem. Soc. 2004, 126, 8640-8641. 30. Ratner, D. M.; Adams, E. W.; Su, J.; O'Keefe, B. R.; Mrksich, M.; Seeberger, P. H. ChemBioChem 2004, 5, 379-383. 31. Blixt, O.; Head, S.; Mondala, T.; Scanlan, C .; Huflejt, M. E.; Alvarez, R.; Bryan, M. C .; Fazio, F.; Calarese, D.; Stevens, J.; Razi, N.; Stevens, D. J.; Skehel, J. J.; van Die, I.; Burton, D. R.; Wilson, I. A.; Cummings, R.; Bovin, N.; Wong, C.-H.; Paulson, P. Proc. Natl. Acad. Sci., USA 2004, 707, 17033-17038.


270


32. Köhn, M.; Wacker, R.; Peters, C.; Schröder, H . ; Soulère, L . ; Breinbauer, R.; Niemeyer, C. M.; Waldmann, H . Angew. Chem. Int. Ed. 2003, 42, 5830-5834. 33. Schwarz, M.; Spector, L.; Gargir, A.; Shevti, A.; Gortler, M.; Altstock, R. T.; Dukler, A. A.; Dotan, N. Glycobiology 2003, 13, 749-754. 34. K o , K . - S . ; Jaipuri, F. A . ; Pohl, N. L. J. Am. Chem. Soc. 2005, 127, 13162-13163.


Carbohydrate Microarrays and Fluorous-Phase Synthesis - American

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