Cell-Specific Chemical Delivery Using a Selective Nitroreductase-Nitroaryl Pair.

The utility of small molecules to probe or perturb biological systems is limited by the lack of cell-specificity. "Masking" the activity of small molecules using a general chemical modification and "unmasking" it only within target cells overcomes this limitation. To this end, we have developed a selective enzyme-substrate pair consisting of engineered variants of E. coli nitroreductase (NTR) and a 2-nitro- N-methylimidazolyl (NM) masking group. To discover and optimize this NTR-NM system, we synthesized a series of fluorogenic substrates containing different nitroaromatic masking groups, confirmed their stability in cells, and identified the best substrate for NTR. We then engineered the enzyme for improved activity in mammalian cells, ultimately yielding an enzyme variant (enhanced NTR, or eNTR) that possesses up to 100-fold increased activity over wild-type NTR. These improved NTR enzymes combined with the optimal NM masking group enable rapid, selective unmasking of dyes, indicators, and drugs to genetically defined populations of cells.

Chemoenzymatic probes for detecting and imaging fucose-α(1-2)-galactose glycan biomarkers.

The disaccharide motif fucose-α(1-2)-galactose (Fucα(1-2)Gal) is involved in many important physiological processes, such as learning and memory, inflammation, asthma, and tumorigenesis. However, the size and structural complexity of Fucα(1-2)Gal-containing glycans have posed a significant challenge to their detection. We report a new chemoenzymatic strategy for the rapid, sensitive detection of Fucα(1-2)Gal glycans. We demonstrate that the approach is highly selective for the Fucα(1-2)Gal motif, detects a variety of complex glycans and glycoproteins, and can be used to profile the relative abundance of the motif on live cells, discriminating malignant from normal cells. This approach represents a new potential strategy for biomarker detection and expands the technologies available for understanding the roles of this important class of carbohydrates in physiology and disease.

End-functionalized glycopolymers as mimetics of chondroitin sulfate proteoglycans.

Glycosaminoglycans are sulfated polysaccharides that play important roles in fundamental biological processes, such as cell division, viral invasion, cancer and neuroregeneration. The multivalent presentation of multiple glycosaminoglycan chains on proteoglycan scaffolds may profoundly influence their interactions with proteins and subsequent biological activity. However, the importance of this multivalent architecture remains largely unexplored, and few synthetic mimics exist for probing and manipulating glycosaminoglycan activity. Here, we describe a new class of end-functionalized ring-opening metathesis polymerization (ROMP) polymers that mimic the native-like, multivalent architecture found on chondroitin sulfate (CS) proteoglycans. We demonstrate that these glycopolymers can be readily integrated with microarray and surface plasmon resonance technology platforms, where they retain the ability to interact selectively with proteins. ROMP-based glycopolymers are part of a growing arsenal of chemical tools for probing the functions of glycosaminoglycans and for studying their interactions with proteins.

Identification of the plasticity-relevant fucose-alpha(1-2)-galactose proteome from the mouse olfactory bulb.

Fucose-alpha(1-2)-galactose [Fucalpha(1-2)Gal] sugars have been implicated in the molecular mechanisms that underlie neuronal development, learning, and memory. However, an understanding of their precise roles has been hampered by a lack of information regarding Fucalpha(1-2)Gal glycoproteins. Here, we report the first proteomic studies of this plasticity-relevant epitope. We identify five classes of putative Fucalpha(1-2)Gal glycoproteins: cell adhesion molecules, ion channels and solute carriers/transporters, ATP-binding proteins, synaptic vesicle-associated proteins, and mitochondrial proteins. In addition, we show that Fucalpha(1-2)Gal glycoproteins are enriched in the developing mouse olfactory bulb (OB) and exhibit a distinct spatiotemporal expression that is consistent with the presence of a "glycocode" to help direct olfactory sensory neuron (OSN) axonal pathfinding. We find that expression of Fucalpha(1-2)Gal sugars in the OB is regulated by the alpha(1-2)fucosyltransferase FUT1. FUT1-deficient mice exhibit developmental defects, including fewer and smaller glomeruli and a thinner olfactory nerve layer, suggesting that fucosylation contributes to OB development. Our findings significantly expand the number of Fucalpha(1-2)Gal glycoproteins and provide new insights into the molecular mechanisms by which fucosyl sugars contribute to neuronal processes.