Glycomics is emerging as a new field for the biology of complex glycoproteins and glycoconjugates. The lack of versatile glycanlabeling methods has presented a major obstacle to visualizing at the cellular level and studying glycoconjugates.

Sulfatases, which cleave sulfate esters in biological systems, play a key role in regulating the sulfation states that determine the function of many physiological molecules. Sulfatase substrates range from small cytosolic steroids, such as estrogen sulfate, to complex cell-surface carbohydrates, such as the glycosaminoglycans. The transformation of these molecules has been linked with important cellular functions, including hormone regulation, cellular degradation, and modulation of signaling pathways. Sulfatases have also been implicated in the onset of various pathophysiological conditions, including hormone-dependent cancers, lysosomal storage disorders, developmental abnormalities, and bacterial pathogenesis. These findings have increased interest in sulfatases and in targeting them for therapeutic endeavors. Although numerous sulfatases have been identified, the wide scope of their biological activity is only beginning to emerge. Herein, accounts of the diversity and growing biological relevance of sulfatases are provided along with an overview of the current understanding of sulfatase structure, mechanism, and inhibition.

Developing tools for investigating the cellular activity of glycans will help to delineate the molecular basis for aberrant glycosylation in pathological processes such as cancer. Metabolic oligosaccharide engineering, which inserts sugar-reporting groups into cellular glycoconjugates, represents a powerful method for imaging the localization, trafficking, and dynamics of glycans and isolating them for glyco-proteomic analysis. Herein, we show that the alkyne-reporting group can be incorporated into cellular glycans. The alkyne group is a small, inert, bio-orthogonal handle that can be chemoselectively labeled by using the Cu(I) catalyzed [3 ؉ 2] azide-alkyne cycloaddition, or click chemistry. Alkynyl sugar monomers, based on fucose (Fuc) and N-acetylmannosamine (ManNAc), were incorporated into fucosylated and sialylated glycans in several cancer cell lines, allowing for cell surface and intracellular visualization of glycoconjugates, as well as, observation of alkyne-bearing glycoproteins. Similarly to our previous results with an azido Fuc/alkynyl probe system, we demonstrated that click-activated fluorogenic probes are practical tools for efficiently and selectively labeling alkynyl-modified glycans. Because Fuc and sialic acid are terminal glycan residues with a notably increased presence in many tumors, we hope that our method will provide useful information about their roles in cancer and ultimately can be used for diagnostic and therapeutic purposes.click chemistry ͉ fluorescent imaging ͉ fucose ͉ sialic acid

N-glycosylation of eukaryotic proteins helps them fold and traverse the cellular secretory pathway and can increase their stability, although the molecular basis for stabilization is poorly understood. Glycosylation of proteins at naïve sites (ones that normally are not glycosylated) could be useful for therapeutic and research applications, but currently results in unpredictable changes to protein stability. We show that placing a Phe residue two or three positions prior to a glycosylated Asn in distinct reverse turns facilitates stabilizing interactions between the aromatic side chain and the first N-acetylglucosamine (GlcNAc) of the glycan. Glycosylating this portable structural module, an “enhanced aromatic sequon”, in three different proteins stabilizes their native states by −0.7 to −2.0 kilocalories per mole and increases cellular glycosylation efficiency.

The folding energetics of the mono-N-glycosylated adhesion domain of the human immune cell receptor cluster of differentiation 2 (hCD2ad) were studied systematically to understand the influence of the N-glycan on the folding energy landscape. Fully elaborated N-glycan structures accelerate folding by 4-fold and stabilize the ␤-sandwich structure by 3.1 kcal/mol, relative to the nonglycosylated protein. The N-glycan's first saccharide unit accounts for the entire acceleration of folding and for 2/3 of the native state stabilization. The remaining third of the stabilization is derived from the next 2 saccharide units. Thus, the conserved N-linked triose core, ManGlcNAc 2, improves both the kinetics and the thermodynamics of protein folding. The native state stabilization and decreased activation barrier for folding conferred by N-glycosylation provide a powerful and potentially general mechanism for enhancing folding in the secretory pathway.glycoprotein stability ͉ N-glycan function ͉ N-glycosylation ͉ protein folding ͉ kinetics

The sulfonation (also known as sulfurylation) of biomolecules has long been known to take place in a variety of organisms, from prokaryotes to multicellular species, and new biological functions continue to be uncovered in connection with this important transformation. Early studies of sulfotransferases (STs), the enzymes that catalyze sulfonation, focused primarily on the cytosolic STs, which are involved in detoxification, hormone regulation, and drug metabolism. Although known to exist, the membrane-associated STs were not studied as extensively until more recently. Involved in the sulfonation of complex carbohydrates and proteins, they have emerged as central players in a number of molecular-recognition events and biochemical signaling pathways. STs have also been implicated in many pathophysiological processes. As a result, much interest in the complex roles of STs and in their targeting for therapeutic intervention has been generated. Progress in the elucidation of the structures and mechanisms of sulfotransferases, as well as their biological activity, inhibition, and synthetic utility, are discussed in this Review.

No abstract

A process is reported for efficient, enantioselective production of key intermediates for the common chiral side chain of statin-type cholesterol-lowering drugs such as Lipitor (atorvastatin) and Crestor (rosuvastatin). The process features a one-pot tandem aldol reaction catalyzed by a deoxyribose-5-phosphate aldolase (DERA) to form a 6-carbon intermediate with installation of two stereogenic centers from 2-carbon starting materials. An improvement of almost 400-fold in volumetric productivity relative to the published enzymatic reaction conditions has been achieved, resulting in a commercially attractive process that has been run on up to a 100-g scale in a single batch at a rate of 30.6 g͞liter per h. Catalyst load has been improved by 10-fold as well, from 20 to 2.0 wt % DERA. These improvements were achieved by a combination of discovery from environmental DNA of DERAs with improved activity and reaction optimization to overcome substrate inhibition. The two stereogenic centers are set by DERA with enantiomeric excess at >99.9% and diastereomeric excess at 96.6%. In addition, downstream chemical steps have been developed to convert the enzymatic product efficiently to versatile intermediates applicable to preparation of atorvastatin and rosuvastatin. Biocatalysis has received attention recently as a tool of enormous potential for the synthesis of pharmaceutical, industrial, and agricultural chemicals and intermediates (1-5). To capitalize further on this potential, enzymes must demonstrate not only superior performance characteristics such as high enantioselectivity and low catalyst loading but also costeffectiveness and scalability. A successful industrial biocatalytic process requires enzymes that can be produced cheaply in kilogram quantities and are stable and active at very high substrate and product concentrations, frequently in the presence of organic solvents. Although many remarkable biocatalytic transformations have been reported on the milligram-to-gram scale, especially in light of recent advances in directed evolution and high-throughput screening for novel enzymes (6-8), the number of processes that have been applied on an industrial scale remains limited (5, 9). This is especially true for biocatalytic processes involving formation of carbon-carbon bonds.The aldol reaction is an extremely useful transformation that allows the formation of new carbon-carbon bonds and the introduction of up to two new stereocenters into the product. Controlling the stereochemistry of aldol reactions can be challenging and has been an area of intense research. Although several elegant asymmetric methods have been developed (10-12), there remains a need for additional methods, and in recent years enzyme-catalyzed approaches using aldolases have received attention as alternatives to chemical methodologies (13).Several years ago, Wong and coworkers (14, 15) reported unprecedented one-pot tandem aldol reactions catalyzed by a deoxyribose-5-phosphate aldolase (DERA), in which 2 eq of acetaldehyde were added in sequen...