Hydroxynitrile mechanism

CRYSTAL STRUCTURES OF HYDROXYNITRILE LYASES AND MECHANISM OF CYANOGENESIS... 

Dreveny, I., Kratky, C. and Gruber, K. (2002) The active site of hydroxynitrile lyase from Prunus amygdalus modeling studies provide new insights into the mechanism of cyanogenesis. Protein Science A Publication of the Protein Society, 11, 292-300. 

Wagner, U.G., Hasslacher, M., Griengl, H. et al. (1996) Mechanism of cyanogenesis the crystal structure of hydroxynitrile lyase from Hevea brasiliensis. Structure (London, England 1993), 4, 811-822. 

This structural feature is in agreement with the proposed Uni Bi mechanism for hydroxynitrile lyases [25,40], which suggests that reagents enter the active site in a sequential fashion. The residues involved in the catalytic action of the active site and their positive assignment are discussed in Sect. 3.1. 

K. Gruber, G. Gartler, B. Krammer, H. Schwab, C. Kratky, Reaction mechanism of hydroxynitrile lyases of the x/ 3-... 

M. Hasslacher, C. Kratky, H. Griengl, H. Schwab, S. D. Kohlwein, Hydroxynitrile lyase from Hevea hrasiliensis, molecular characterization and mechanism of enzyme catalysis. Proteins 1997, 27, 438-449. 

Although many biochemical reactions take place in the bulk aqueous phase, there are several, catalyzed by hydroxynitrile lyases, where only the enzyme molecules close to the interface are involved in the reaction, unlike those enzyme molecules that remain idly suspended in the bulk aqueous phase [6, 50, 51]. This mechanism has no relation to the interfacial activation mechanism typical of lipases and phospholipases. Promoting biocatalysis in the interface may prove fruitful, particularly if substrates are dissolved in both aqueous phases, provided that interfacial stress is minimized. This approach was put into practice recently for the enzymatic epoxidation of styrene [52]. By binding the enzyme to the interface through conjugation of chloroperoxidase with polystyrene, a platform that protected the enzyme from interfacial stress and minimized product hydrolysis was obtained. It also allowed a significant increase in productivity, as compared to the use of free enzyme, and simultaneously allowed continuous feeding, which further enhanced productivity. 

In cases of promiscuous activity, one species in the pathway often acts as a common intermediate for both mechanisms leading to different activities. An enam-ine between PLP and the substrate branches off into a decarboxylation or a transamination, a TPP-bound intermediate can react either to decarboxylation or to carboligation, and the triad Ser-His-Asp in hydroxynitrile lyase is responsible for both the main function, oxynitrilation, and the subordinate function, ester hydrolysis. It can be assumed that many more promiscuous functionalities will be discovered in the coming years. 

Despite acetonitrile s feeble acidity (pATa ca 29) compared with enolizable aldehydes (67, pA s 16-17), the combination of a simple ruthenium complex, [RuCp(PPli3)2]+, and diazabicycloundecane (DBU) brings about a nitrile-selective deprotonation to give /I-hydroxynitriles (68).274 A mechanism is proposed in which DBU, aldehyde, and acetonitrile can displace triphenylphosphines, with the metal centre activating acetonitrile to convert it to an NC-CFU- ligand (proposed intermediate, 69). A nickel-diarylamidodiphosphine complex (70) also catalyses this transformation in the presence of DBU.275... 

The hydroxynitrile lyase (HNL) class of enzymes, also referred to as oxynitrilases, consists of enzymes that catalyze the formation of chiral cyanohydrins by the stereospecific addition of hydrogen cyanide (HCN) to aldehydes and ketones (Scheme 19.36).275 279 These chiral cyanohydrins are versatile synthons, which can be further modified to prepare chiral a-hydroxy acids, a-hydroxy aldehydes and ketones, acyloins, vicinal diols, ethanolamines, and a- and P-amino acids, to name a few.280 Both (R)- and (.S )-selective HNLs have been isolated, usually from plant sources, where their natural substrates play a role in defense mechanisms of the plant through the release of HCN. In addition to there being HNLs with different stereo-preferences, two different classifications have been defined, based on whether the HNL contains a flavin adenine dinucleotide (FAD) co-factor. 

In the first step, cyanogenic glucosides are cleaved by p-glucosidases. The resulting a-hydroxynitriles are unstable and dissociate to produce a carbonyl compound and HCN. In many plants, the dissociation of the cyanohydrins is accelerated by an HNL. In plants that accumulate diglucosidic compounds, the hydrolysis of these substances can be achieved by either a sequential or a simultaneous mechanism (Kuroki et al, 1984) (Fig. 3.5). 

Oxynitrilases or hydroxynitrile lyases (HNL) constitute a group of enzymes that catalyze the reversible addition of HCN to ketones and aldehydes. The natural role of these enzymes is a defence mechanism of higher plants against herbivores, whereby HCN is liberated from cyanoglucosides such as prunasin (almond, cherry, apple) by the action of a glycosidase and a hydroxynitrile lyase. 

The cyclization reactions of dideuterated hydroxynitrile (10) with n-BuLi forming the Af-metalated nitrile, or with i-PrMgCl giving the C-metalated nitrile, that form trans- and cis-decalin, respectively. Scheme 11, have shown that both reactions occur exclusively by an 5 mechanism. In fact, this mechanism is found for all the cyclizations of primary allylic chlorides. Secondary and trisubstituted allylic chlorides also react with n-BuLi or j-PrMgCl giving either the trans- and c -decalin by an 5 or an mechanism. Yields for the cyclization range from 34 to 89%. 

The negatively charged intermediate formed in the first step in the mechanism is highly reactive and quickly reacts with an H+ ion (from HCN, from dilute acid or from water present in the reaction mixture). This forms the 2-hydroxynitrile product. 

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