Alkylated acyl-enzyme derivative

The acyl-enzyme can eliminate the 4-chlorine atom to generate this reactive intermediate that can then react with a nearby nucleophile such as His57 to give an alkylated acyl-enzyme derivative in which the inhibitor moiety is bound to the enzyme by two covalent bonds (Scheme 11.5). Inhibition is irreversible.59 The mechanism has been confirmed by X-ray structural analysis of protease-isocoumarin complexes. There is a cross-link between the inhibitor and the Serl95 and His57 residues of PPE.60 Human leukocyte elastase is also very efficiently inactivated.61... 

The 4-piperazinyl nitrogen in pipemidic acid (69) has been alkylated, acylated and sulfonylated in the search for enhanced antibacterial activity, whilst the 3-pyrrolidinyl position in piromidic acid (68) is hydroxylated metabolically and enzymically to (74) (75MI21503), from which acyloxy derivatives have been prepared. 

A different strategy for preparing enzymes for immobilization is to introduce vinyl groups by alkylating or acylating enzymes with activated vinyl monomers [40]. The modified enzymes are then polymerized with mono- and bifunctional acrylamide derivatives to yield elastic particles of irregular shape after crushing of the formed polymer blocks. Such copolymerization processes have yielded stable industrial biocatalysts for pharmaceutical application which are especially suited for stirred tank applications [41]. 

Acetylsucrose [63648-81-7] has been prepared in 40% yield by direct acetylation of sucrose using acetic anhydride in pyridine at 40° C (36). The 6-ester has subsequently been obtained in greater than 90% yield, by way of 4,6-cycHc orthoacetate. Other selective methods for the 6-acylated derivatives include the use of alkyl tin reagents such as dibutyl tin oxide (37) and of dibutyl stannolane derivatives (38). Selective acetylation of sucrose by an enzymic process has also been described. Treatment of sucrose with isopropenyl acetate in pyridine in the presence of Lipase P Amano gave, after chromatography, 6-0-acetylsucrose (33%) and 4/6-di-O-acetylsucrose (8%). The latter compound has been obtained in 47% yield by the prolonged treatment (39). 

A number of examples of monoacylated diols produced by enzymatic hydrolysis of prochiral carboxylates are presented in Table 3. PLE-catalyzed conversions of acycHc diesters strongly depend on the stmcture of the substituent and are usually poor for alkyl derivatives. Lipases are much less sensitive to the stmcture of the side chain the yields and selectivity of the hydrolysis of both alkyl (26) and aryl (24) derivatives are similar. The enzyme selectivity depends not only on the stmcture of the alcohol, but also on the nature of the acyl moiety (48). 

The role of protein kinase C in many neutrophil functions is undisputed and has been recognised for some time. For many years it was believed that the source of DAG, the activator of protein kinase C, was derived from the activity of PLC on membrane phosphatidylinositol lipids. Whilst this enzyme undoubtedly does generate some DAG (which may then activate protein kinase C), there are many reasons to indicate that this enzyme activity is insufficient to account for all the DAG generated by activated neutrophils. More recently, experimental evidence has been provided to show that a third phospholipase (PLD) is involved in neutrophil activation, and that this enzyme is probably responsible for the majority of DAG that is formed during cell stimulation. The most important substrate for PLD is phosphatidylcholine, the major phospholipid found in neutrophil plasma membranes, which accounts for over 40% of the phospholipid pool. The sn-1 position of phosphatidylcholine is either acyl linked or alkyl linked, whereas the sn-2 position is invariably acyl linked. In neutrophils, alkyl-phosphatidylcholine (1-0-alky 1-PC) represents about 40% of the phosphatidylcholine pool (and is also the substrate utilised for PAF formation), whereas the remainder is diacyl-phosphatidylcholine. Both of these types of phosphatidylcholine are substrates for PLD and PLA2. 

There remains model 4, and MacQuarrie and Bernhard 175) have utilized the full-site reactivity by iodoacetamide and half-site reactivity by FAP to provide support for this model. Thus, di(2-furylacryloyl) enzyme was prepared, and the two remaining sites were blocked with iodoacetate. Acyl groups were then removed from this derivative by arsenolysis, and the resulting dialkyl enzyme was tested for stoichiometry with FAP. Only one acyl group could be incorporated into the dialkyl enzyme. This result cannot be explained in terms of an induced asymmetry model, and indeed, can only be explained by a preexisting asymmetry model if there is a subunit rearrangement. In addition, alkylation of the enzyme with varying quantities of iodoacetate, followed by acylation of these derivatives with FAP, showed a 2 1 ratio of alkylation to acylation, independent... 

Amide-modified farnesylcysteine (AMFC) analogs were synthesized and evalnated as snbstrates or inhibitors of the methyltransferase. Acyl substitutions bearing small alkyl groups are substrates however, a pivaloyl-FC derivative has neither substrate nor inhibitor activity. A benzolyl-FC series was also investigated and similarly lacked activity however, introducing a flexible linker between the benzolyl and FC groups restored substrate activity. These data indicate that bulky substitutions at the amine group of the FC are not well tolerated by the enzyme. 

Alkyl halides are even less reactive than acyl halides, as indicated by the compilation of reaction rates of thiolate anions with various types of alkyl halides (282). Nevertheless, potentially useful affinity labels have been synthesized with alkyl halide substituents and have been shown to specifically inactivate several enzymes, albeit slowly the low reactivity of the alkyl halides may minimize nonspecific reaction. Adenosine 5 -(2-bromoethyl)phosphate has been characterized and reported to inactivate NAD -dependent isocitrate dehydrogenase (283). The 2 - and 3 -(2-bromoethyl)-AMP labels have also been synthesized, and model reactions of the bromoethyl-AMPs with cysteine, lysine, histidine, and tyrosine have been studied (284). More recently, esters of adenosine 5 -monophosphate have been prepared with ethyl, propyl, or hexyl moieties and bromo or chloro substituents at the w position (285). Yeast alcohol dehydrogenase exhibited enhanced inactivation by the hexyl derivative, but inactivation rates of other dehydrogenases were unremarkable. Two iodopropyl derivatives of cAMP have been described, namely, 1, A -(3-iodopropyleno)adenosine 3, 5 -cyclic monophosphate and 3 -0-(2-iodo-3-hydroxypropyl)adenosine 3, 5 -cyclic monophosphate the latter inactivates cAMP phosphodiesterase from human platelets, with a pseudo-first-order rate constant of 0.147 hr" (286). 

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