Covalent adducts, formation

The experimentally observed pseudo-first order rate constant k is increased in the presence of DNA (18,19). This enhanced reactivity is a result of the formation of physical BaPDE-DNA complexes the dependence of k on DNA concentration coincides with the binding isotherm for the formation of site I physical intercalative complexes (20). Typically, over 90% of the BaPDE molecules are converted to tetraols, while only a minor fraction bind covalently to the DNA bases (18,21-23). The dependence of k on temperature (21,24), pH (21,23-25), salt concentration (16,20,21,25), and concentration of different buffers (23) has been investigated. In 5 mM sodium cacodylate buffer solutions the formation of tetraols and covalent adducts appear to be parallel pseudo-first order reactions characterized by the same rate constant k, but different ratios of products (21,24). Similar results are obtained with other buffers (23). The formation of carbonium ions by specific and general acid catalysis has been assumed to be the rate-determining step for both tetraol and covalent adduct formation (21,24). 

The and spectroscopy of a solution of 2-chloro-3,5-dinitropyridine in liquid ammonia at-40°C showed the formation of the C-6 adduct (10). This adduct is rather stable, since after 1 hr standing, no change in the spectrum was observed. It is interesting that at a somewhat lower temperature (-60°C) the addition takes place at C-4, i.e., formation of (9). Apparently one deals with the interesting concept of kinetically and thermodynamically controlled covalent adduct formation. At -60°C the addition is kinetically controlled, and at -40°C the addition is thermodynamically favored. The higher stability of the C-6 adduct compared to the C-4 adduct is probably due to the more extended conjugate resonance system (Scheme II.9). 

Generally, DNMT inhibitors can be divided in two big dasses. One group consists of base analogs which are incorporated into DNA and act as suidde substrates for DNMT via a covalent adduct formation. The other group acts on the free enzyme in the same way as classic enzyme inhibitors do. 

To model the crucial step of covalent adduct formation, adducts resulting from quenching of lO-azaBaP-4,5-epoxide with cytosine via the exocyclic amino group were computed, and their geometrical features and relative energies were compared (Fig. 24). The most stable stereoisomer was the one with the cytosine moiety trans to the hydroxyl, with both groups in pseudoequatorial conformation. Two structures... 

Munns AJ, De Voss JJ, Hooper WD, et al. Bioactivation of phenytoin by human cytochrome P450 characterization of the mechanism and targets of covalent adduct formation. Chem Res Toxicol 1997 10 1049 1058. 

The active site of MADH is relatively hydrophilic and located at the end of a hydrophobic channel between the a and (3 subunits. The C6 carbonyl of TTQ is exposed to solvent at the active site, and is the site of covalent adduct formation with the substrate (Huizinga ei ah, 1992). The two indole rings which comprise the TTQ structure are not coplanar but at a dihedral angle of approximately 38 (Chen et ah, 1998). Whereas the C6 carbonyl of TIQ is present in the active site, the edge of the second indole ring which does not contain the quinone, is exposed at the surface of MADH. 

Studies of nitroalkane oxidation by n-amino acid oxidase (55) and glucose oxidase 49, 56) have provided strong evidence both for intermediate substrate carbanions and for subsequent covalent adduct formation between these and the N position of the flavin nucleus. The rationale for using nitroalkanes can be seen in the following reaction stoichiometries for D-amino acid oxidase (55) ... 

The mechanism discussed above for the deprotonation of alkylaromatic radical cations, involving a bimolecular reaction between the radical cation and the base (B), leading to a carbon centered neutral radical and the conjugated acid of the base (BH" ") as described in Scheme 28, has been recently questioned by Parker who provided evidence for an alternative mechanism in proton-transfer reactions between methylanthracene radical cations and pyridine bases [154] this involved reversible covalent adduct formation between the radical cation and the base followed by elimination of BH+ (Scheme 36). 

Kretz-Rommel, A. Boelsterli, U.A. "Mechanism of Covalent Adduct Formation of Diclofenac to Rat Hepatic Microsomal Proteins. Retention of the Glucuronic Acid Moiety in the Adduct, Drug Metab Dispos. 22, 956-961 (1994). 

The UV spectrum of quinazoline in cyclohexane shows three n->-n transitions with maxima at 311 (3.32), 267 (3.49) and 220 (4.61) nm with an inflection point at 330 nm of low intensity (2.30) assigned to n->7i. The 4-position in quinazoline is highly electrophilic the electrophilicity is accentuated in the protonated species. Covalent adduct formation between quinazoline or 4-unsub-stituted quinazolines and water occurs in aqueous acidic solutions. The hydroxyl group enters the... 

The carbonyl functional group provides an electrophilic site for nucleophilic attack. As none of the naturally occurring amino acids contains a ketone or aldehyde moiety, it is not possible for a protein-bound carbonyl group to be present at an enzyme active site, unless it is provided by a cofactor. The most commonly seen reactive carbonyl group in enzymes is that provided by the pyridoxal phosphate cofactor which is derived from vitamin B6." The carbonyl serves as a site for covalent adduct formation with a nucleophilic substrate, often an amine or the amino group of amino acid substrates. An alternative strategy for introduction of a reactive carbonyl at an enzyme active sight has been described for a class of enzymes that possess a covalent pyruvoyl cofactor. 

The reductive half-reaction of methylamine dehydrogenase is shown in Scheme 10. The methylamine substrate initiates a nucleophilic attack on the quinone carbon at the C6 position of the TTQ cofactor displacing the oxygen to form a substrate-TTQ Schiff base adduct (29). The reactivity of the C6 position was demonstrated by covalent adduct formation at this position by hydrazines which are inactivators of methylamine dehydrogenase. Deprotonation of the substrate-derived carbon of 29 by an active-site amino acid residue results in reduction of the cofactor and yields an intermediate in which the Schiff base is now between the nitrogen and substrate-derived carbon (30). Hydrolysis of 30 releases the formaldehyde product and yields the aminoquinol form of the cofactor with the substrate-derived amino group still covalently bound (31). 

JV-Alkyl(or Aryl)phthalazinium Salts, Betaines, or Ylides Covalent Adduct Formation... 

Almost all of the proposed tests have to do with changes at the DNA or chromosome level. These DNA alterations (mutations, covalent adduct formation, etc.) or chromosomal modifications (sister chromatid exchange, micronucleus test, etc.) are proposed to be predictive of carcinogens. It is not claimed that they have any predictive value for non-neoplastic diseases. 

Quenching can also occur between covalently linked fluorophore-quencher pairs. One common exanqde is the formation of exciplexes by covalently linked aronudc hydrocarbons and amines. Another example is the covalent adduct formation by indole and acrylamide. For exan le, the lifetime ofN-acetyltryptamineisnearS.l ns. When the acetyl group is replaced by an acr oyl poiq>, the lifetime is reduced to 31 ps (Hguie 834). Similarly, covalent attadiment of s toa iuq)hUialene deriva-... 

Acrylamide quenching. 238. 239, 244-246. 250,251,252 absorption spectra. 249 bimolecular quenching constants. 252 covalent adduct formation, 257 intensity decays, 282,283 NATA, 285 jHx>teins, 463... 

Since the introduction of chemical ionization by Munson and Field in 1966 (10), ion-molecule reactions have been studied extensively for the structural analysis of unknown compounds. In brief, the reagent gas is allowed into the ion source at pressures significantly higher than that of the analyte. The reagent gas is ionized by electron impact and a variety of ion-molecule reactions can occur between charged and neutral gas molecules. These products, in turn, may ionize the analyte by one of five mechanisms. The most important by far is proton transfer, which is the only positive ion Cl reaction that has seen broad acceptance for analytical purposes. Three that apply to the case of acetonitrile are proton transfer, charge transfer, and assodation (also called addition or covalent adduct formation). Acetonitrile Cl is unique in providing very selective analytical information as a result of adduct formation. 

Notley et al. (2002) investigated the P450 forms responsible for covalent drug-protein adduct formation and the possibility that covalent adduct formation might occur via alternative pathways to catechol formation. Recombinant P450 3A4 catalysed... 

---