Acyl fluorophosphate

Recently a very mild method was discovered for the synthesis of dinucleoside fluorophosphates in the conversion of a dinucleoside phosphite with iV V -oxalyldiimi-dazole into the corresponding phosphoric imidazolide, which is then treated with acyl fluoride [204] <202]... 

Such an intermediate is known to be formed in reactions catalyzed by trypsin, chymotrypsin, thrombin, other enzymes of the blood-clotting cascade (except angiotensinconverting enzyme, which is an aspartic protease). An acyl-serine intermediate is also formed in the acetylcholinesterase reaction. The active site serine of this enzyme and the serine proteases can be alkylated by diisopropyl-fluorophosphate. See also Active Site Titration... 

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. 

The serine proteases act by forming and hydrolyzing an ester on a serine residue. This was initially established using the nerve gas diisopropyl fluorophosphate, which inactivates serine proteases as well as acetylcholinesterase. It is a very potent inhibitor (it essentially binds in a 1 1 stoichiometry and thus can be used to titrate the active sites) and is extremely toxic in even low amounts. Careful acid or enzymatic hydrolysis (see Section 9.3.6.) of the inactivated enzyme yielded O-phosphoserine, and the serine was identified as residue 195 in the sequence. Chy-motrypsin acts on the compound cinnamoylimidazole, producing an acyl intermediate called cinnamoyl-enzyme which hydrolyzes slowly. This fact was exploited in an active-site titration (see Section 9.2.5.). Cinnamoyl-CT features a spectrum similar to that of the model compound O-cinnamoylserine, on denaturation of the enzyme in urea the spectrum was identical to that of O-acetylserine. Serine proteases act on both esters and amides. 

Ligands closely related to orthophosphate esters include acetylphosphate, acetylphenylphosphate and fluorophosphate. Alkaline hydrolysis of [Co OP(0)20COMe (NH3)5] occurs exclusively at the carbonyl centre, and the acceleration provided by cobalt(III) four atoms removed is minimal (10 times, equation This is a good comparative example of phosphoryl versus acyl hydrolysis,... 

Abbreviations used for amino acids and the designations of peptides follow the rules of the lUPAC-IUB Commision of Biochemical Nomenclature in J. Biol. Chem., 247 (1972) 977-983. The following additional abbreviations are used AGP, acyl carrier protein AM, aminomethyl (resin) Boc, tert.-butyloxycarbonyl BOP, benzotriazol-l-yl-oxy-tris(dimethylamino)phosphonium hexa-fluorophosphate Bu, -butyl DCC, N,N -di-cyclohexylcarbodiimide DEAE, diethyl-aminoethyl DIEA, N,N-diisopropylethyl-amine DIPCDI, N,N -diisopropylcarbodi-imide DMAP, 4-dimethylaminopyridine DMF, N,N-dimethylformamide Dts, dithia-succinoyl EtOAc, ethyl acetate EtOH, ethanol Fmoc, 9-fluorenylmethyloxycarbon-yl HOBt, 1-hydroxybenzotriazole HPLC, high-performance liquid chromatography IR, infrared IRAA, internal reference amino acid MBHA, 4-methylbenzhydrylamine (resin) Nle, norleucine NMR, nuclear mag-... 

Phosphonium benzotriazolyl A-oxytrisdimethylaminophosphonium hexa-fluorophosphate (BOP) (9), benzotriazol-l-ylox)rtris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP) (10), Md 7-azabenzotriazol-l-yloxytris(pyr-rolidino)phosphonium hexafluorophosphate (PyAOP) (11), and aminium salts of A-[(lff-benzotriazol-l-yl)(dimethylamino)methylene]-Af-methylmethana-minium hexafluorophosphate A-oxide (HBTU) (12), A-[(l//-benzotriazol-l-yl)(dimethylamino)methylene]-iV-methylmethanaminium tetrafluoroborate (raTU) (13), and A-[(dimethylamino)-lH-l,2,3-triazolo[4,5-fi]pyridin-l-ylmethylene]-iV-methylmethan aminium hexafluorophosphate N-oxide (HATU) (14) are used in conjunction with N -protected amino acids and a tertiary base. Recently, it was reported that the use of HOXt in HXTU- and PyXOP-mediated couplings did not significantly increase the yield and purity of a sequence (15). Since there are at least three components required for an efficient acylation, protocols are different than those used with carbodiimide... 

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