Chemical Glycobiology - American Chemical Society

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Chapter 12

Chemical Approaches to Glycobiology

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Nicholas J. Agard Department of Pharmaceutical Chemistry, University of California at San Francisco, San Francisco, CA 94158-2552

Post-translational modifications play fundamental roles in regulating protein structure, localization, and function. Presented ubiquitously on eukaryotic proteins, these modifications provide a layer of structural and functional complexity beyond what is available from the genome. Despite their essential roles, post-translational modifications remain challenging to study in large part because researchers cannot apply traditional genetic manipulations to these non-templated structures. This lack of direct genetic control has driven researchers to pursue chemical approaches to modify and characterize these modifications. Here I will discuss one chemical method, the bioorthogonal chemical reporter strategy, which has been used extensively to study the most common post-translational modification, glycosylation. I will focus first on ways to metabolically introduce monosaccharides bearing unnatural functionality into cellular glycoconjugates. This will be followed by a survey of chemical methods used to modify these unnatural glycans. Lastly, I will highlight recent applications of these chemical ligations in the profiling, proteomic identification, and fluorescence imaging of glycosylation.

© 2008 American Chemical Society

In Chemical Glycobiology; Chen, X., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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Introduction Glycosylation is a ubiquitous post-translational modification affecting nearly 50% of all proteins and greater than 90% of those that traffic through the secretory pathway (/, 2). Glycans mediate both intra- and intercellular interactions and regulate diverse biological processes, including development (5), homeostasis (4\ and immunity (5). Cell surface carbohydrates are a primary marker of a cell's physiological state. Changes in glycosylation are known to correlate with inflammatory (tf), pathogenic (7, 8), and carcinogenic signals via the dynamic action of membrane recycling and glycan biosynthetic enzymes (i.e., glycosidases and glycosyltransferases) (9, 10). Thus, it is possible for a cell to remodel its glycoconjugates in the absence of de novo translation. Despite the paramount importance of glycans in cellular regulation, details of their function remain poorly understood in part because carbohydrate structures are not directly genetically encoded. In eukaryotes, carbohydrates (with the exception of the β-0-GlcNAc modification) are attached to protein scaffolds as they traffic through the endoplasmic reticulum and the Golgi apparatus (//). Glycosyltransferases and glycosidases act sequentially on these scaffolds, but the addition of each monosaccharide relies on stochastic binding events. The glycans that emerge from this ordered, non-uniform process are heterogeneous, yet they contain common underlying structures. This nontemplated biosynthesis has complicated elucidation of carbohydrate function in two important ways. First, heterogeneity in glycan expression has frustrated attempts to establish direct structure-function relationships involving individual carbohydrate epitopes. Second, glycans are not amenable to the direct genetic manipulations that facilitate protein purification and labeling. As a result, it remains difficult to visualize glycoconjugates or inventory their expression in living organisms. The detection and purification of glycoconjugates has been addressed through the development of a two-step strategy, termed the bioorthogonal chemical reporter strategy (12, 13). In this approach, a monosaccharide bearing an unnatural functional group (the chemical reporter) is metabolically incorporated into glycoconjugates (Figure 1) (14). The chemical reporter is then covalently tagged by exogenous reagents appended to detection or affinity probes. Successful labeling depends on both the capacity of biosynthetic enzymes to accept the unnatural substrate and the functional group tolerance and specificity of the subsequent reaction. Enzymes involved in monosaccharide activation and glycan biosynthesis are highly specific and will tolerate only very slight modifications to the native structure. This specificity prohibits direct incorporation of fluorescent molecules or epitope tags and limits the size of the chemical reporter to simple functional groups. In addition, the covalent chemistry used must rapidly and exclusively label the unnatural glycans under

In Chemical Glycobiology; Chen, X., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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conditions compatible with the target glycoconjugates. In the most restricted systems, living cells and animals, this means the reactions must take place at 37 °C in aqueous solvent and in the presence of competing functionalities, including amines, thiols, and molecular oxygen. Chemical reporters that successfully traverse biosynthetic pathways and react selectively under physiological conditions despite competing functionality are termed "bioorthogonal"

Figure 1. The bioorthogonal chemical reporter strategy. First, unnatural monosaccharides are metabolically incorporated into glycoconjugates. Nex the unnatural functionality (black square) is covalently tagged using appropriately derivatized reagents (white square).

Here I will review the bioorthogonal chemical reporter strategy. First, I will outline glycan biosynthetic pathways and their tolerance for unnatural monosaccharides. Next, I will discuss the corresponding chemical methods for their derivatization, including hydrazone/oxime formation, thiol alkylation, Staudinger ligation, and [3+2] azide-alkyne cycloadditions (both coppercatalyzed and strain-promoted). Last, I will outline the current applications of the bioorthogonal chemical reporter strategy.

Metabolic Labeling of Unnatural Monosaccharides Glycoproteins are biosynthesized by the transfer of monosaccharides from activated nucleotide-sugar donors to target proteins (75, 16). Nearly all of the activated sugars can be synthesized de novofromglucose and glucosamine (77). However, salvage pathways can also recycle monosaccharides from degraded glycoproteins and glycolipids (18). In some cell lines, salvaged monosaccharides compose up to 80% of the total glycans on the cell surface (19). These salvage pathways can be taken advantage of to incorporate unnatural functionalities into glycans. Indeed, most unnatural monosaccharides are activated via homologous salvage pathways.

In Chemical Glycobiology; Chen, X., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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254 Salvage pathways process free sugars in a stepwise fashion to form activated nucleotide donors. For three sugars, galactose (Gal), fiicose (Fuc) and Nacetylgalactosamine (GalNAc), the salvage pathway begins by anomeric phosphorylation of the free sugar (18). Condensation of the phosphosugar with a nucleotide triphosphate gives the activated nucleotide sugar (Figure 2). Salvage of three additional monosaccharides, N-acetylglucosamine (GlcNAc), glucose (Glc), and mannose (Man), begins with kinase-mediated phosphorylation of the 6hydroxyl to give the 6-phosphosugar (20). This compound is in turn isomerized and condensed with the nucleotide triphosphate to give the activated donor sugar. In contrast to other monosaccharides, activated sialic acid donors are biosynthesized from TV-acetylmannosamine (ManNAc) or directly from sialic acids (Sia), including N-acetylneuraminic acid (NeuAc), via a more complex pathway (21). ManNAc is phosphorylated at the at the 6-hydroxyl group and condensed with phosphoenolpyruvate to give W-acetylneuraminic acid-9phosphate (NeuAc-9-P). Phosphate ester hydrolysis is followed by direct condensation with CTP to give CMP-NeuAc (Figure 3). Sialic acids can intercept this pathway directly via enzymatic reaction with CTP. Diverse unnatural monosaccharides are tolerated by these metabolic pathways (22-34). However, only molecules modified with bioorthogonal chemical reporters are capable of subsequent bioorthogonal ligation. Four functional groups have been used for this purpose: azides, alkynes, ketones, and thiols (25, 26, 32, 34, 35). While ketones and thiols occur naturally on the interior of the cell and are therefore not bioorthogonal in the strictest sense, these functionalities are not highly abundant on the cell surface. Thus, selective conjugation is possible in this location. Numerous bioorthogonal ly functionalized monosaccharides have been incorporated into cellular glycoconjugates, including derivatives of ManNAc, Sia, GalNAc, GlcNAc, and Fuc (Figure 4) (23, 25, 26, 28-30, 32-34, 36). The tolerance of each pathway to unnatural functionalized monosaccharides depends on its unique suite of enzymes. Introduction of unnatural monosaccharides into glycans relies on the promiscuity of the salvage pathway enzymes and glycosyltransferases. The efficiency of this incorporation has been determined both by glycan analysis, which determines what percentage of the total monosaccharide content is composed by the unnatural analog, and by in vitro kinetic analysis of the associated enzymes. Cellular sialic acid content is analyzed by fluorescence detection on a reversed-phase HPLC after liberation of the labile sialosides via mild acid hydrolysis and subsequent condensation with l,2-diamino-4,5-methylenedioxybenzene (DMB). Analysis of cell lines incubated with azide-derivatized ManNAc (W-azidoacetylmannosamine (ManNAz)) showed variable incorporation of the metabolized unnatural monosaccharide, ranging from 4-41% of the total sialosides (37). This high variability results from a number of cell-line-dependent factors, including the

In Chemical Glycobiology; Chen, X., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

In Chemical Glycobiology; Chen, X., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

X

OH

ATP

X = Η or OH

X OPO3

OH

xTP

H

ΗΝ

1

UDP-GalNAc

Ho!

OUDP

un I °OUDP UDP-Gal

HO OH [O ,.OH

HO-

1

HO OH [O ..OH

ή

UDP-GlcNAc

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OH

GDP-Fuc

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GDP

Figure 2. Monosaccharides are metabolized via salvage pathways to give activated nucleotide sugars. Man and Glc (not pictured) are activated via the same pathway as GlcNAc.

Gal, Fuc GalNAc

GlcNAc, Man, Glc

ATP

2

OPO3 -

Downloaded by UNIV OF MICHIGAN ANN ARBOR on February 18, 2015 | http://pubs.acs.org Publication Date: September 1, 2008 | doi: 10.1021/bk-2008-0990.ch012

In Chemical Glycobiology; Chen, X., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

Η

OH

Η OH

NeuAc