The structure of a formamidopyrimidine (FAPY) adduct arising from imidazole ring opening of the initially formed trans-8, 9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 adduct under basic conditions and positioned in the 5'-d(CTATFAPYGATTCA)-3'*5'-d(TGAATCATAG)-3' oligodeoxynucleotide was determined. The FAPY adduct may be a major progenitor of aflatoxin B1-induced mutations in DNA. The freshly prepared sample showed biphasic melting, with transitions at 28 and 56 degreesC. NMR initially showed multiple subspectra. Over a period of several days at 4 degreesC, the sample converted to a single species with a Tm of 56 degreesC, 15 degrees C greater than the unmodified duplex. The deoxyribose was in the beta configuration about the anomeric carbon, evidenced by NOEs between FAPYG5 H3', H2', H2", and H1'. FAPY formation resulted in the loss of the guanine H8 proton, and the introduction of the formyl proton, which showed NOEs to FAPYG5 H1' and A6 N6Ha. A total of 31 NOEs from AFB1 to DNA protons were observed, mostly to the 5'-neighboring base, T4 in the modified strand. Sequential NOEs were interrupted between T4 and FAPYG5 in the modified strand, between C16 and A17 in the complementary strand, and between T4 N3H and FAPYG5 N1H. An NOE between FAPYG5 N1H and C16 N4H showed intact hydrogen bonding at FAPYG5*C16. Upfield chemical shifts were observed for T4 H6 and A17 H8. Molecular dynamics calculations converged with pairwise rmsd differences of <0.9 A. The sixth root residual was 8.7 x 10(-2). The AFB1 moiety intercalated from the major groove between FAPYG5 and T4*A17, and stacked with T4 and FAPYG5 and partially stacked with A17. The base step between T4*A17 and FAPYG5*C16 was increased from 3.4 to 7 A. The duplex unwound by about 15 degrees. The FAPY formyl group was positioned to form a hydrogen bond with A6 N6Ha. Strong stacking involving the AFB1 moiety, and this hydrogen bond explains the thermal stabilization of four base pairs by this adduct, and may be a significant factor in its processing.

Aflatoxin B 1 (AFB) epoxide forms an unstable N7 guanine adduct in DNA. The adduct undergoes base-catalyzed ring opening to give a highly persistent formamidopyrimidine (FAPY) adduct which exists as a mixture of forms. Acid hydrolysis of the FAPY adduct gives the FAPY base which exists in two separable but interconvertible forms that have been assigned by various workers as functional, positional, or conformational isomers. Recently, this structural question became important when one of the two major FAPY species in DNA was found to be potently mutagenic and the other a block to replication [

Calcitonin gene-related peptide (CGRP) has been implicated in the pathogenesis of migraine. Herein we describe optimization of CGRP receptor antagonists based on an earlier lead structure containing a (3R)-amino-(6S)-phenylcaprolactam core. Replacement of the phenylimidazolinone with an azabenzimidazolone gave stable derivatives with lowered serum shifts. Extensive SAR studies of the C-6 aryl moiety revealed the potency-enhancing effect of the 2,3-difluorophenyl group, and trifluoroethylation of the N-1 amide position resulted in improved oral bioavailabilities, ultimately leading to clinical candidate 38 (MK-0974).

The palladium-catalyzed Suzuki-Miyaura reaction has been utilized as one of the most powerful methods for C-C bond formation. However, Suzuki reactions of electron-deficient 2-heterocyclic boronates generally give low conversions and remain challenging. The successful copper(I) facilitated Suzuki coupling of 2-heterocyclic boronates that is broad in scope is reported. Use of this methodology affords greatly enhanced yields of these notoriously difficult couplings. Furthermore, mechanistic investigations suggest a possible role of copper in the catalytic cycle.

Aflatoxin (AF) B(1) exo-8,9-epoxide hydrolysis yields AFB(1) dihydrodiol, which undergoes base-catalyzed rearrangement to, and is in equilibrium with, AFB(1) dialdehyde. We investigated the reaction of AFB(1) dialdehyde with albumin to generate a Lys adduct, previously characterized by others [Sabbioni, G., Skipper, P. L., Büchi, G., and Tannenbaum, S. R. (1987) Carcinogenesis8, 819-824; Sabbioni, G. (1990) Chem.-Biol. Interact. 75, 1-15]. Pronase digestion of bovine albumin serum treated with AFB(1) dialdehyde and HPLC yielded the adduct, identified by its characteristic UV and mass spectra. The structure of the Lys-AFB(1) dialdehyde adduct is concluded to be (S)-alpha-amino-2,3-dihydro-2-oxo-4-(1,2,3,4-tetrahydro-7-hydroxy-9-methoxy-3,4-dioxocyclopenta[c][1]benzopyran-6-yl)-1H-pyrrole-1-hexanoic acid, structure B of the former paper and 8 of the latter, based on work with the methylamine adduct described in the following paper in this issue [Guengerich, F. P., Voehler, M., Williams, K. M., Deng, Z., and Harris, T. M. (2002) Chem. Res. Toxicol. 15, 793-798]. The time course of product formation at varying concentrations of AFB(1) dialdehyde could be described by complexation with albumin with a K(d) of 1.5 mM and a first-order reaction rate with the N6-amino group of Lys of 0.033 min(-)(1). The reaction of AFB(1) dialdehyde with N(2)-acetylLys was monitored by UV spectroscopy and yielded a final spectrum similar to that of the described Lys adduct. Kinetic analysis of the changes at pH 7.2 was best described with a scheme involving equilibrium of the dialdehyde with dihydrodiol and a rate-limiting reaction of AFB(1) dialdehyde with the N6 atom of N(2)-acetylLys, with an apparent second-order rate constant of 2.6 x 10(3) M(-)(1) min(-)(1), followed by putative carbinolamine formation and rearrangement, collectively described by a first-order rate constant of 7.6 min(-)(1). Competition experiments with the hydrolysis of AFB(1) exo-8,9-epoxide indicate that N2-acetylLys also reacts with the epoxide at pH 7.2 (k = 350 M(-)(1) min(-)(1)) and 9.5 (k = 1.8 x 10(3) M(-)(1) min(-)(1)). This reaction might contribute to the formation of protein Lys adducts, depending upon the local concentration of free or protein Lys. Mass spectral analysis of trypsin digests of bovine serum albumin modified with AFB(1) dialdehyde indicated selective modification of Lys455 and Lys548. Collectively, these results provide more insight into the mechanism of formation of AFB(1) dialdehyde-protein adducts and indicate that the formation of Lys adducts is a moderately efficient process. The binding of AFB(1) dialdehyde to albumin or the protonation of the N6-amino group retards the reaction with Lys residues.