Ashok J. Chavan

Chemical Attachment of Organic Functional Groups to Single-walled Carbon Nanotube Material

We have subjected single-walled carbon nanotube materials (SWNTMs) to a variety of organic functionalization reactions. These reactions include radioactive photolabeling studies using diradical and nitrene sources, and treatment with dichlorocarbene and Birch reduction conditions. All of the reactions provide evidence for chemical attachment to the SWNTMs, but because of the impure nature of the staring materials, we are unable to ascertain the site of reaction. In the case of dichlorocarbene we are able to show the presence of chlorine in the SWNT bundles, but as a result of the large amount of amorphous carbon that is attached to the tube walls, we cannot distinguish between attachment of dichlorocarbene to the walls of the SWNTs and reaction with the amorphous carbon.

Site-specific photobiotinylation of immunoglobulins, fragments and light chain dimers

Herein we report a new method to rapidly photoinsert biotin into a specific and highly conserved site on the Ig structure using a mild photochemical activation step. This site resides in the Fv fragment and involves invariant residues which provide base stacking interactions to the purine ring of ATP (Rajagopalan et al. (1996) Proc. Natl. Acad. Sci. USA 93, 6019-6024). Biotin was coupled to either the phosphate or the ribose of the 8-azidopurine nucleotide or nucleoside photoaffinity probe and shown to insert into the affinity site efficiently. Several monoclonal and polyclonal antibodies, as well as enzymatic and recombinant antibody fragments and light chain dimers were photoaffinity biotinylated and used in ELISA, FACS and Western blots. The selectivity of this site-specific biotinylation method also allows for biotinylation of antibodies in culture supernatants and immune sera without prior purification. Because the biotinylation takes place under physiological conditions and within a short time period, photobiotinylation would be the preferred method for antibodies which are easily damaged by classical non-site specific random biotinylation chemistry.

Photoaffinity labeling of rat liver microsomal steroid 5α-reductase by 2-azido-NADP

Abstract Preincubation of female rat liver microsomal preparations with [2′- 32 P]2N 3 -NADP + followed by photolysis with UV lighjt (254 nm) and analysis by SDS-PAGE/autoradiography showed incorporation of 32 P into at least 3 major protein bands in the molecular weight range of 14–197 Kd. Labeling of a 26 Kd band, the apparent molecular weight of 5α-reductase in liver microsomes, was accompanied by a loss of enzyme activity, consistent with its covalent modification. The inclusion of 20-fold excess NADP + (100μM) completely inhibited the incorporation of [2′- 32 P]2N 3 -NADP + and preserved the enzyme activity, whereas excess NAD + (100μM) failed to protect 5α-reductase (5αR) activity. Similar results were obtained with the detergent-solubilized form of 5αR. Polyethylene glycol (PEG) fractionation of detergent-solubilized with the detergent-solubilized preparations of 5αR showed that all the 5αR activity could be recovered in the 6.5%, pellet with a 3—4-fold increase in the specific activity. Photolysis of this fraction with [2′- 32 P]2N 3 -NADP + resulted in ∼ 2-fold increase in 32 P labeling of the 5αR band. Increasing photolysis time and concentration of the [2′- 32 P]2N 3 -NADP + indicated that the half-life for photoincorporation and the apparent K d were 1.0 min and 2 μM, respectively. Theser results suggest that 2N 3 -NADP + is an effective probe of the NADP(H) binding site of 5αR, and is a useful marker during purification of the enzyme.

Thiazolidine and thiazoline derivatives of 3-aryl-3-trifluoromethyldiazirines for the preparation of fluorescent or 35S-radiolabeled photoaffinity probes

Abstract The condensation of cysteine with 3-(4-formylphenyl) or 3-(4-cyanophenyl)-3-trifluoromethyldiazirine furnished thiazolidine and thiazoline derivatives in good yield. These heterocycles provide convenient access to photoaffinity probes an 35 S radiolabel or a fluorescent dansyl group.