Nitriles, detection hydrolysis

Nitration. Whereas aryl nitriles have been mononitrated by conventional methods, dinitration has not been achieved because the forcing conditions required effect hydrolysis (and oxidation) of the nitrile group. In contrast, mono- and dinitration can be carried out with nitronium telrafluoroborate in anonaqueous, acid-free system where the only acid originates from the proton elimination during nitration. The solvent used, tetramethylene sulfone or acetonitrile (both water-soluble), is sufficiently basic to keep the acid at a concentration below the level needed to effect detectable hydrolysis (or oxidation). 

B). Many nitriles when treated with hydrogen peroxide in warm alkaline solution undergo hydrolysis to amides which can thus be readily obtained in high yield. Insoluble liquid nitriles can be treated directly in the aqueous suspension, but for insoluble solid nitriles the addition of a suitable organic solvent to give a complete solution may be desirable, although the completion of the hydrolysis may not then be so readily detected. 

Peptide nitriles are reversible inhibitors of cysteine proteases. 1,2 Peptide nitrile reacts with the active site thiol group to form an imidothioate, a dead-end product that does not undergo hydrolysis to an amide.134 This imidothioate derivative has been detected by NMR spectroscopic studies.P 5 The inhibition of papain, a cysteine protease, by a peptide nitrile proved to be reversible in a dialysis experiment. 3 Peptide nitriles are weaker inhibitors of cysteine proteases than the corresponding aldehydes. 61 Most peptide nitriles show poor inhibition toward serine proteases, however those nitriles with proper peptide sequences are potent inhibitors of serine proteases. 7-9 ... 

At intervals (1 hr), small aliquots (0.2-0.3 mL) of the reaction mixture are withdrawn, acidified with a few drops of coned HCI (35%), and extracted with diethyl ether (2 mL). Then they are analyzed by GC (DB5 capillary column) both the amide and the acid derived from the reacting nitrile are present. After 4.5 hr, the product is 99% acid 3 (2-phenylpropionic acid). The checkers followed the hydrolysis by TLC analysis SiC>2 plates 4 1 hexane ethyl acetate short-wave UV detection R (acid) = 0.43, Rf (amide) = 0.61. 

With methylamine, no intermediate was detected and it is inferred that further oxidation proceeds readily in this instance. Subsequent reactions involve the oxidation of Ru to Ru followed by hydrolysis (with cyclohexylimine l or dehydrogenation to form the corresponding nitrile complexes. It is noted that the chemistry of imine complexes in water is severely restricted by their ease of hydrolysis. 

Figure 5. NMR spectra obtained for supernatants ofE. coli whole cell cultures expressing the Pseudomonas gene, catalyzing the hydrolysis of hydrocinnamonitrile. (A) Initial time point, only the nitrile substrate is observed, (B) At 6 hours, nitrile, acid and amide are observed. Triplets at 2.987ppm and 2.497ppm are assigned to hydrocinnamic acid triplets at 2,944 ppm and 2.604 ppm are assigned to the hydrocinnamide. (C) and (D) At 18 and 40 hours, both the products are detected.

This work has demonstrated the utility of high throughput H NMR for the discovery of novel enzymes from genomic libraries. The use of NMR to detect biotransformation products enabled the unexpected observation of multiple product formation for the nitrile hydrolyzing enzyme. This observation resulted in the identification and proposal of a novel mechanism. Additional NMR was used to elucidate the enzymatic activity. Furthermore, it was shown that hydrolysis of the substrate was not due to a combination of nitrile hydratase and amidase. 

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