Boronic acids protease activity

The peptide boronic acids form adducts with the active site serine of serine proteases)1... 

Transition-state inhibitors stably mimic the transition state of the enzymatic reaction, and thereby interact with the substrate-bin-ding and catalytic machinery of the enzyme in a low-energy conformation. Transition-state analogs are competitive, reversible inhibitors, although some have extremely low Kj s and very slow off-rates. All proteases activate a nucleophile to attack a carbonyl, which leads to the formation of a tetrahedral intermediate that then collapses to form the enzyme products—two peptides. Thus, synthetic small molecules that mimic the tetrahedral intermediate of the protease reaction are attractive transition-state analogs. A classic class of protease transition-state inhibitors uses a boronic acid scaffold (4, 10). Boronic acid adopts a stable tetrahedral conformation in the protease active site that is resistant to nucleophilic attack. Boronic acid inhibitors, which are derivatized with different specificity elements, have been developed against every class of protease... 

Figure 7 Various transition-state protease inhibitors. Bortezomib is an approved drug for the treatment of multiple myeloma. It is a boronic acid analog that inhibits the proteosome, a threonine protease. The boronic acid moiety can adopt a tetrahedral conformation in the active site. Pepstatin is a peptidyl aspartic acid inhibitor. The reactive statine group binds to the catalytic machinery, and the chiral hydroxyl group of the statine mimics the tetrahedral geometry of the transition state. Idinavir is an approved HIV 1 Protease inhibitor that binds to the active site via a hydroxyethylene transition state isostere. Aldehydes are also transition state analogs, which are susceptible to nucleophilic attack. In cysteine, serine and threonine proteases, this results in a covalent, reversible inhibition mechanism.

Support for this concept is provided by H NMR studies which have identified a downfield resonance of the hydrogen-bonded proton in this pair at 18 ppm in ch3miotrypsinogen and chymotrypsin at low pH and at 14.9-15.5 ppm at high pH values.Similar resonances are seen in the a-lytic protease, in sub-tilisin, in adducts of serine proteases with boronic acids or peptidyl trifluoromethyl ketones, in alkylated derivative of the active site histidine, and in molecular complexes that mimic the Asp-His pair in the active sites of serine pro teases. 

Subtilisin [11] and cholesterol esterase [12, 13] are also inhibited by a range of aliphatic and aromatic boronic acids. Work on inhibiting a-CHT and cell replication with a variety of boronic acids concluded that the a-CHT activity was associated with chromatin in normal and tumorous tissue of mice [14]. Certain boronic acids were demonstrated to inhibit protease activity in rat liver chromatin [15]. It was concluded that good boronate inhibitors of CHT, like PEBA, inhibit cell replication and that this effect is expected to be higher in rapidly proliferating cancer cells than in normal tissue [14]. 

Subtilisin (from Bacillus subtilis) [9014-01-1] Mr 27,000 (sedimentation equilibrinm) [EC 3.4.21.62], This alkaline protease is purified 211-fold by affinity chromatography using 4-(4-aminophenylazo)phenylarsonic acid complex to activated CH-Sepharose 4B. It is inhibited by 2-phenylethane boronic acid, PMSF, 3,4-dichloroisocoumarin, acetone and benzamide. [Chandraskaren Dhar Anal Biochem 150 141 1985, Schomburg Schomburg Springer Handbook of Enzymes 2nd Edn vol 7 p 286 2002.]... 

Physically safer as a mechanistic criterion is reversible inhibition by boronic acids, RB(0H)2, which add the active site serine to form tetrahedral species [RB(0H)20Ser] , which mimic the tetrahedral intermediate/transition state. They also mimic the tetrahedral intermediates in aspartic protease action, however, and are therefore not as definitive. 

Boronic acid derivatives of proteases have recently been shown to combine with active-site nucleophiles in the manner shown below, both by X-ray and infrared spectroscopy. The pH dependence of binding is also consistent with this view, according to which these analogs are considered to resemble tetrahedral intermediates in the formation and breakdown of covalent acyl-enzyme intermediates in double displacement. ... 

As discussed earlier, free boronic acids can only be used to bind to one side of the binding pocket of a protease, which does not allow for the maximal use of the active site to achieve the best affinity and specificity. Moreover, the reversible nature of boronic acid complex formation makes it hard to incorporate functional groups on... 

In the synthesis of amino boronic acid dipeptides 7 (Scheme 11.37) as inhibitors of the serine protease dipeptidyl peptidase IV (DPPIV), Kelly and co-workers have reported an elegant preparation of the optically active proline boronic acid (boroPro) by a direct lithiation of N-Boc-pyrrolidine and a quench with B(OMe)3. The subsequent hydrolysis and resolution with (-1-)-pinanediol provided rapid access to a key chiral intermediate in the synthesis of these dipeptides. The direct a-functionalisation approach avoided a catal5Aic hydrogenation step required in their original synthesis starting from IV-Boc-pyrrole. ... 

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