P-Nitrobenzyl ester

The o-nitrobenzyl ester, used in this example to protect penicillin precursors, can be cleaved by irradiation (H20/dioxane, pH 7). Reductive cleavage of benzyl or p-nitrobenzyl esters occurred in lower yields. 

N-[N-(benzyloxycarbonyl)-L-aspart-l-oyl-(L-alanyl-L-threonine p-nitrobenzyl ester)-4-oyl]-... 

N-[N-(benzyloxycarbonyl)-L-aspart-l-oyl-(L-alanyl-L-threonyl-L-leucyl-L-alanine p-nitrobenzyl ester)-4-oyl]-N-[N-(benzyloxycarbonyl)-L-aspart-l-oyl-(L-alanyl-L-threonyl-L-leucyl-L-alanyl-L-serine p-nitrobenzyl ester)-4-oyl]-N-[N-(benzyloxycarbonyl)-L-aspart-1 -oyl-(glycine ethyl ester)-4-oyl -N-[N-(benzyloxycarbonyl)-L-aspart-l-oyl-(glycyl-L-serine methyl ester)-4-oyl]-... 

E Guibe-J ampel, M Wakselman. Selective cleavage of p-nitrobenzyl ester with sodium dithionite. Syn Commun 12, 219, 1982. 

This minor product has been now isolated and converted into the corresponding pentacyclo[5.3.0.0 .0 .0 ]decanedicarboxylic acid via intramolecular [2 - - 2] photocyclization. The material thereby obtained was converted into the corresponding cage di(p-nitrobenzyl ester) derivative via the method shown in Scheme 12. The structure of the resulting diester was established unequivocally as 40 via application of X-ray crystallographic methods (see Fig. 3.2) [28]. 

Nitrobeozyl alcohol, 882 m-Nitrobenzyl alcohol, 881 p-Nitrobenzyl bromide, 961 p-Nitrobenzyl cyanide, 751, 763 p-Nitrobenzyl esters, 362 ... 

Method 2 (p-nitrobenzyl ester). To the residue are added 3 ml of ethanol and a 20-fold excess of 1 -p-nitrobenzyl-3-p-tolyltriazene [43]. The contents are mixed, loosely covered and heated at a gentle reflux for 1 h. The solution is cooled and an aliquot portion is subjected to chromatography. The derivatives are non-polar compared to the reagent and the parent fatty acid, and may be separated on silica gel with non-polar solvents such as hexane-diethyl ether. HPLC should also be useful with a system similar to that used for the benzyl esters. The limits of detection of the p-nitrobenzyl derivatives should be significantly lower than those of the benzyl esters. 

The sample (2-3 mg) is placed in a 5-ml tapered reaction vial. 3 ml of ethanol and 50 mg of the PNBTT reagent are added. The vial is covered loosely (nitrogen is evolved during the reaction, so the vial should not be sealed tightly) and heated in a bath at 65 °C until derivatization is complete (generally 1 h or less). The vial is then sealed and cooled to room temperature. The solution may be chromatographed directly or cleaned up as described above. The p-nitrobenzyl esters are non-polar compared with the parent acids and the by-products of reaction. For chromatography on silica gel, a non-polar solvent is used. 

For some purposes protection of the carboxyl group by conversion into p-phenylphenacyl, p-bromophenacyl, and p-nitrobenzyl esters is useful. General procedures for their formation are described in Section 9.6.15, p. 1261. 

A. Drop 1 g of sodium into 10 ml of methanol in a small flask provided with a small water condenser heat the mixture until all the sodium has dissolved. Cool, and add 1 g of the ester and 0.5 ml of water. Frequently the sodium salt of the acid will be deposited either at once or after boiling for a few minutes. If this occurs, filter off the solid at once, wash it with a little methanol and convert it into the p-bromophenacyl ester, p-nitrobenzyl ester or S-benzyl isothiouronium salt (for experimental details, see Section 9.6.15, p. 1261). If no solid separates, continue the boiling for 30-60 minutes, boil off the alcohol, allow to cool, render the product just neutral to phenolphthalein with dilute sulphuric or hydrochloric acid, convert the sodium salt present in solution into a crystalline derivative (Section 9.6.15, p. 1261) and determine its melting point. 

Eli Lilly (Indianapolis/IN, USA) needed a p-nitrobenzyl esterase to hydrolyze the p-nitrobenzyl ester of a )3-lactam compound in DMF. The purely organic route with zinc salts and large amounts of organic solvent was unattractive, so an enzyme was sought which could hydrolyze the ester. One with rather low activity was found which served as a starting point for directed evolution. As nature never had to hydrolyze an antibiotic in DMF, this approach seemed very promising. 

Fig. 7. Molecular model of the pNB esterase showing positions of antibiotic p-nitrobenzyl ester substrate (white CPK structure), catalytic residues (S189, E310, and H399), and beneficial mutations accumulated during directed evolution. Mutations at positions 322 and 370 are believed to improve expression, while the remaining six substitutions improve specific activity [2]. Arrows indicate the position of new mutations found after DNA shuffling...

---