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Sudhakara S.,Laboratory of Bioorganic Chemistry | Chadha A.,Laboratory of Bioorganic Chemistry | Chadha A.,Indian Institute of Technology Madras
Journal of Biotechnology | Year: 2015

We present an FTIR based assay to monitor the whole cell mediated oxidation of aryl alcohols by measuring the characteristic IR absorption of the hydroxyl group [OH] of the substrate and the carbonyl group [CO] of the corresponding oxidized product. This method expedites the analysis of whole cell mediated catalysis which is usually done by GC and/or HPLC. The FTIR assay had linearity with R2≥0.980 and sensitivity up to 10μM. The accuracy and precision of FTIR assay was found ≥81% and ≥94%, respectively. This assay was validated by GC which exhibited ≥82% accuracy and ≥79% precision. The time of analysis taken by this assay was 2-3min per sample in comparison with 20-40min by GC. © 2015 Elsevier B.V.

Zhao C.,Laboratory of Bioorganic Chemistry | Hoppe T.,Laboratory of Biochemistry and Genetics | Setty M.K.H.G.,Center for Biologics Evaluation and Research | Murray D.,Laboratory of Immunoregulation | And 3 more authors.
Nature Communications | Year: 2014

The remarkable stability of peptide nucleic acids (PNAs) towards enzymatic degradation makes this class of molecules ideal to develop as part of a diagnostic device. Here we report the development of chemically engineered PNAs for the quantitative detection of HIV RNA at clinically relevant levels that are competitive with current PCR-based assays. Using a sandwich hybridization approach, chemical groups were systematically introduced into a surface PNA probe and a reporter PNA probe to achieve quantitative detection for HIV RNA as low as 20 copies per millilitre of plasma. For the surface PNA probe, four cyclopentane groups were incorporated to promote stronger binding to the target HIV RNA compared with PNA without the cyclopentanes. For the reporter PNA probe, 25 biotin groups were attached to promote strong signal amplification after binding to the target HIV RNA. These general approaches to engineer PNA probes may be used to detect other RNA target sequences. © 2014 Macmillan Publishers Limited.

Hu J.,Laboratory of Bioorganic Chemistry | Hu K.,Laboratory of Bioorganic Chemistry | Liu T.,Laboratory of Bioorganic Chemistry | Stern M.K.,Laboratory of Bioorganic Chemistry | And 4 more authors.
Journal of Biological Chemistry | Year: 2013

ClassAGprotein-coupled receptors (GPCRs) are able to form homodimers and/or oligomeric arrays. We recently proposed, based on bioluminescence resonance energy transfer studies with the M3 muscarinic receptor (M3R), a prototypic class A GPCR, that the M3R is able to form multiple, structurally distinct dimers that are probably transient in nature (McMillin, S. M., Heusel, M., Liu, T., Costanzi, S., and Wess, J. (2011) J. Biol. Chem. 286, 28584-28598). To provide more direct experimental support for this concept, we employed a disulfide cross-linking strategy to trap various M3R dimeric species present in a native lipid environment (transfected COS-7 cells). Disulfide cross-linking studies were carried out with many mutant M3Rs containing single cysteine (Cys) substitutions within two distinct cytoplasmic M3R regions, the C-terminal portion of the second intracellular loop (i2) and helix H8 (H8). The pattern of cross-links that we obtained, in combination with molecular modeling studies, was consistent with the existence of two structurally distinct M3R dimer interfaces, one involving i2/i2 contacts (TM4-TM5-i2 interface) and the other one characterized by H8-H8 interactions (TM1-TM2-H8 interface). Specific H8-H8 disulfide cross-links led to significant impairments in M3R-mediated G protein activation, suggesting that changes in the structural orientation or mobility of H8 are critical for efficient receptor-G protein coupling. Our findings provide novel structural and functional insights into the mechanisms involved inM3Rdimerization (oligomerization). Because theM3Rshows a high degree of sequence similarity with many other class A GPCRs, our findings should be of considerable general interest. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

Dix A.V.,Laboratory of Bioorganic Chemistry | Moss S.M.,Laboratory of Bioorganic Chemistry | Phan K.,Laboratory of Bioorganic Chemistry | Hoppe T.,Laboratory of Biochemistry and Genetics | And 6 more authors.
Journal of the American Chemical Society | Year: 2014

A programmable ligand display system can be used to dissect the multivalent effects of ligand binding to a membrane receptor. An antagonist of the A 2A adenosine receptor, a G-protein-coupled receptor that is a drug target for neurodegenerative conditions, was displayed in 35 different multivalent configurations, and binding to A2A was determined. A theoretical model based on statistical mechanics was developed to interpret the binding data, suggesting the importance of receptor dimers. Using this model, extended multivalent arrangements of ligands were constructed with progressive improvements in binding to A2A. The results highlight the ability to use a highly controllable multivalent approach to determine optimal ligand valency and spacing that can be subsequently optimized for binding to a membrane receptor. Models explaining the multivalent binding data are also presented. © This article not subject to U.S. Copyright. Published 2014 by the American Chemical Society.

Takao K.,Laboratory of Bioorganic Chemistry | Miyashiro T.,Laboratory of Bioorganic Chemistry | Sugita Y.,Laboratory of Bioorganic Chemistry
Chemical and Pharmaceutical Bulletin | Year: 2015

A series of piperic acid amides (4-24, 29, 30) were synthesized and their 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging and α-glucosidase inhibitory activities were evaluated. Among the synthesized compounds, the amides 11, 13 and 15, which contain o-methoxyphenol, catechol or 5-hydroxyindole moieties, showed potent DPPH free radical scavenging activity (11: EC50 140 μM; 13: EC50 28 μM; 15: EC50 20 μM). The amides 10, 18 and 23 showed higher inhibitory activity of α-glucosidase (10: IC50 21 μM; 18: IC50 21 μM; 23: IC50 12 μM). These data suggest that the hydrophobicity of the conjugated amines is an important determinant of α-glucosidase inhibitory activity. In addition, the amides 13 and 15 showed both potent DPPH free radical scavenging activity and α-glucosidase inhibitory activity (13: IC50 46 μM; 15: IC50 46 μM). This is the first report identifying the DPPH free radical scavenging and α-glucosidase inhibitory activities of piperic acid amides and suggests that these amides may serve as lead compounds for the development of novel αglucosidase inhibitors with antioxidant activity. © 2015 The Pharmaceutical Society of Japan.

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