Acyl transfer irreversible

The kinetic resolution of primary alcohols (RCH2OH) by enzymatic acylation is often demanding because of the remote position of the asymmetric center firom the reaction site. Moreover, special attention is needed to keep the acyl transfer irreversible and to prevent hydrolysis of the acylated product. 

There are only a few examples of low-temperature conditions reported to lead to a species behaving as an o-QM. All of these, except for our O-acyl transfer methods that will be discussed later, use a fluoride ion to trigger the formation of the o-QM in an almost instantaneous manner. In these examples, a high concentration of the intended nucleophile is necessary to prevent any side reactions with the o-QM, because given the low-temperature conditions its formation is usually irreversible. 

Prochiral Compounds. The enantiodifferentiation of prochi-ral compounds by lipase-catalyzed hydrolysis and transesterification reactions is fairly common, with prochiral 1,3-diols most frequently employed as substrates. Recent reports of asymmetric hydrolysis include diesters of 2-substituted 1,3-propanediols and 2-0-protected glycerol derivatives. The asymmetric transesterification of prochiral diols such as 2-0-benzylglycerol and various other 2-substituted 1,3-propanediol derivatives is also fairly common, most frequently with Vinyl Acetate as an irreversible acyl transfer agent. 

The asymmetric transesterification of cyclic me o-diols, usually with vinyl acetate as an irreversible acyl transfer agent, includes monocyclic cycloalkene diol derivatives, bicyclic diols, such as the ej o-acetonide in eq 12, bicyclic diols of the norbomyl type, andorganometallic l,2-bis(hydroxymethyl)ferrocenepossessing planar chirality. 

Similar to enol esters, oximes can also be used as irreversible acyl transfer agents for lipase catalysis. Thus, instead of a di-enol ester, Athavale et al. [67] polymerized diols with bis(2,3-butane dione monoxime) alkanedioate using Lipozyme IM-20. The results obtained by activation with enol-esters and their corresponding oximes were comparable. No attempts were made to analyze the end-group of the polyester. 

Faber, K., Riva, S. (1992). Enzyme-catalysed Irreversible Acyl Transfer. Synthesis, 895. 

In contrast to hydrol3dic reactions, where the nucleophile (water) is always in excess (55 mol/L), the concentration of the foreign nucleophile in acyl transfer reactions (such as another alcohol) is always hmited. As a result, trans- and interesterification reactions involving normal esters are generally reversible in contrast to the irreversible nature of a hydrolytic reaction. This leads to a slow reaction rate and can cause a severe depletion of the selectivity of the reaction for kinetic reasons (Sect. 2.1.1). 

Enol esters such as vinyl or isopropenyl esters liberate unstable enols as coproducts, which tautomerize to give the corresponding aldehydes or ketones [151,152] (Scheme 3.4). Thus, the reaction becomes completely irreversible and this ensures that all the benefits with regard to a rapid reaction rate and a high selectivity are accrued. Acyl transfer using enol esters has been shown to be about only ten times slower than hydrolysis (in aqueous solution) and about 10-100 times faster than acyl-transfer reactions using activated esters. In contrast, when nonactivated esters such as ethyl acetate were used, reaction rates of about lO -lO of that of the hydrolytic reaction are observed (Table 3.5) [153]. 

Acid Anhydrides. Another useful method of achieving completely irreversible acyl-transfer reactions is the use of acid anhydrides (Scheme 3.4) [165]. The selectivities achieved are usually high and the reaction rates are about the same as with enol esters. One of the advantages of this technique is that no aldehydic byproducts are formed and the enzyme is not acylated under the conditimis employed, making its reuse possible. 

Besides the more often-used acyl donors mentioned above, others which would also ensure an irreversible type of reaction have been investigated [170]. Bearing in mind that most of the problems of irreversible enzymatic acyl transfer arise from the formation of unavoidable byproducts, emphasis has been put on finding acyl donors that possess cyclic structures, which would not liberate any byproducts at all. However, with candidates such as lactones, lactams, cyclic anhydrides (e.g., succinic acid anhydride [171]), enol lactones (e.g., diketene [172, 173]), and oxazolin-5-one derivatives [174], the drawbacks often outweighed their merits. 

Along the same lines, the remarkable synthetic potential of enzyme-catalyzed irreversible acyl transfer in nearly anhydrous organic solvents can be demonstrated particularly weU by the transformation of alcoholic substrates (such as organo-metallics or cyanohydrins) which are prone to decomposition reactions in an aqueous medium and thus cannot be transformed via enzyme-catalyzed hydrolysis reactions. 

When A -nucleophiles such as ammonia, amines or hydrazine are subjected to acyl-transfer reactions, the corresponding Af-acyl derivatives - amides or hydrazides - are formed through interception of the acyl-enzyme intermediate by the iV-nucleophile (Schemes 2.1 and 3.21) [262]. Due to the pronounced difference in nucleophiU-city of the amine (or hydrazine) as compared to the leaving alcohol (R -OH) (Scheme 3.21), aminolysis reactions can be regarded as quasi-irreversible. Any type of serine hydrolase which forms an acyl-enzyme intermediate (esterases, lipases, and most proteases) is able to catalyze these reactirms. Among them, proteases such as subtilisin and peniciUin acylase and lipases from Candida antarctica and Pseudomonas sp. have been used most often. 

Despite the fact that thioesters of optically active amino acids have long been known to racemize promptly even in the presence of rather weak bases [48], and that they are able to act as irreversible acyl donors in enzyme-mediated acyl transfers [49], it was only in 1995 that they were reported to be good substrate candidates for DKR, where they are continuously racemized by an organic base while a hydrolytic enzyme acts preferentially on one of the two enantiomers [50]. In later works, the same authors reported on a more detailed and systematic study of the same reaction system applied to a broader array of substrates. The reactions were ran in a biphasic water/toluene mixture in the presence of trioctylamine as a hydrophobic organic base, so that the substrate racemization takes place in the organic phase while the hydrolysis product is continuously extracted into the water phase. Under racemizing conditions, the authors obtained almost complete conversion and significantly better optical purity (Scheme 8.8) [51], which is attributable to the continue depletion of the unreactive substrate enantiomer. 

Mechanistically, NCL is a two-step process, which is initiated by a reversible transthioesterification step followed by a spontaneous, irreversible S- to N-acyl transfer (Fig. 1). The reactivity is affected by several factors such as concentration of the reactants, temperature, pH, and choice of an appropriate thiol. Also the C-terminally thioes-terified amino acid at the ligation site has to be considered carefully [3]. In feet, NCL relies basically on an observation ofWieland et al., who first demonstrated the transfer of a valyl residue from a reactive thioester onto a cysteine under slightly basic conditions followed by a rearrangement and yielding a Val-Cys dipeptide [4]. 

Sakai and co-workers, reported the synthesis of a biomimetic trifunctional organocatalyst 56 that mimics the active site of serine proteases. It shows a high acceleration for the acyl transfer reaction with highly active, irreversible vinyl trif-luoroacetate (55) up to almost 4 10 -fold vs. background (Scheme 7.13) [59]. The proposed mechanism is shown in Scheme 7.14. 

Fig. 8.33 DYKAT of 1,3-diols via lipase-catalyzed acyl-transfer in combination with Ru-catalyzed epimerization of hydroxyl groups. G=chiral carbon, convertible for equilibration and acyl migration, but not for the irreversible step H=chiral carbon, convertible for equilibration, acyl migration and the irreversible step l=chiral carbon, convertible for acyl migration, stable chirality. (From J. Steinreiber, K. Faber, H. Griengl, De-racemization of enantiomers versus de-epimerization of diastereomers-chssification of dynamic kinetic asymmetric transformations (DYKAT), Chemistry 14 (2(X)8), 8060. Copyright 2008 Wiley).

The lipase-catalyzed resolution of chiral acids can be accomplished via the reaction of their mixed carboxylic-carbonic anhydrides [128]. This irreversible acyl transfer reaction may proceed in a highly enantioselective manner in some cases [129], as shown in Scheme 22. 

The most satisfactory method to carry out an irreversible transesterification is the reaction of acylation of an alcohol with vinyl acylates [130,131]. In this reaction the back reaction is prevented by the irreversible tautomerization of vinyl alcohol to acet dehyde. This latest product could cause the inhibition of the enzyme that has been hnmobilized to overcome this complication [132]. In some studies, however, a few cycles of reactions could be performed without affecting the enantioselectivity of the reaction [133]. Also oxime esters have been proposed as acyl transfer agents [134] for irreversible enzymatic transesterifications (Scheme 23). 

Scheme 22 Irreversible acyl transfer reaction by mixed anhydrides.

Both lipase-catalyzed hydrolyses in water and acyl transfer reactions in an organic medium are more or less reversible reactions that will be detrimental to the enantiomeric excess of the compound of interest. Therefore, many techniques used for enhancing the enantiomeric excess are based on shifting the thermodynamic equilibrium position toward an irreversible situation. 

Several techniques of displacing the reaction equilibrium to reach a quasi-irreversible situation have been used previously. For a review of these, see Faber and Riva [119]. The techniques of using activated acyl donors when resolving chiral alcohols afford a more or less irreversible acylation step in the reaction mechanism since the first product is designed to be a poor nucleophile or is supposed to tautomerize or otherwise leave the re tion system (Scheme 3). Some examples of acyl donors frequently used include 2-haloethyl, cyanomethyl, oxime, and enol esters. The rates of the acyl transfer reactions of racemic 2-octanol with various esters catalyzed by porcine pancreatic lipase were one to two orders of magnitude faster when activated esters were used compared with methyl or ethyl al-kanoates [120]. 

A number of lyases are known which, unlike the aldolases, require thiamine pyrophosphate as a cofactor in the transfer of acyl anion equivalents, but mechanistically act via enolate-type additions. The commercially available transketolase (EC 2.2.1.1) stems from the pentose phosphate pathway where it catalyzes the transfer of a hydroxyacetyl fragment from a ketose phosphate to an aldehyde phosphate. For synthetic purposes, the donor component can be replaced by hydroxypyruvate, which forms the reactive intermediate by an irreversible, spontaneous decarboxylation. 

The preceding experiments prove that there is an intermediate on the reaction pathway in each case, the measured rate constants for the formation and decay of the intermediate are at least as high as the value of kcat for the hydrolysis of the ester in the steady state. They do not, however, prove what the intermediate is. The evidence for covalent modification of Ser-195 of the enzyme stems from the early experiments on the irreversible inhibition of the enzyme by organo-phosphates such as diisopropyl fluorophosphate the inhibited protein was subjected to partial hydrolysis, and the peptide containing the phosphate ester was isolated and shown to be esterified on Ser-195.1516 The ultimate characterization of acylenzymes has come from x-ray diffraction studies of nonspecific acylenzymes at low pH, where they are stable (e.g., indolylacryloyl-chymotrypsin),17 and of specific acylenzymes at subzero temperatures and at low pH.18 When stable solutions of acylenzymes are restored to conditions under which they are unstable, they are found to react at the required rate. These experiments thus prove that the acylenzyme does occur on the reaction pathway. They do not rule out, however, the possibility that there are further intermediates. For example, they do not rule out an initial acylation on His-57 followed by rapid intramolecular transfer. Evidence concerning this and any other hypothetical intermediates must come from additional kinetic experiments and examination of the crystal structure of the enzyme. 

Irreversible inhibitors are effectively esteratic site inhibitors which, like true substrates, react with the hydroxyl group of serine at the catalytic active site. Such inhibitors, sometimes referred to as acid-transferring inhibitors, include the organophosphates, the organo-sulfonates, and the carbamates. All form acyl-enzyme complexes which, unlike substrate-enzyme intermediates, are relatively stable to hydrolysis. Indeed, the phosphorylated enzyme intermediates have half-lives from a few hours to several days (A12), whereas the sulfonated or carbamylated enzyme complexes have much shorter half-lives—several minutes to a few hours. Several strong lines of direct evidence point to the formation of an acyl complex—the isolation of phosphorylated serine from hydrolysates of horse cholinesterase (J2), complex formation and carbamylation (02), and the sulfonation of butyrylcholinesterase by methanesulfonyl fluoride in the presence of tubocurarine and eserine (P6). 

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