A new covalent mitomycin C-DNA adduct (4) was isolated from DNA exposed to reductively activated mitomycin C (MC) in vitro. The MC-treated DNA was hydrolyzed enzymatically under certain conditions, and the new adduct was isolated from the hydrolysate by HPLC. Its structure was determined by ultraviolet and circular dichroism spectroscopy and chemical and enzymatic transformations conducted on microscale. In the structure, a single 2" beta, 7"-diaminomitosene residue is linked bifunctionally to two guanines in the dinucleoside phosphate d(GpG). The guanines are linked at their N2 atoms to the C1" and C10" positions of the mitosene, respectively. A key to the structure was a finding that removal of the mitosene from the adduct by hot piperidine yielded d(GpG); another was that the adduct was slowly converted to the known interstrand cross-link adduct 3 by snake venom diesterase and alkaline phosphatase. Adduct 4 represents an intrastrand cross-link in DNA formed by MC. Of the two possible strand-polarity isomers of 4, 4a in which the mitosene 1"-position is linked to the 3'-guanine of d(GpG) is designated as the proper structure, on the basis of the mechanism of the cross-linking reaction. The same adduct 4 was isolated from poly(dG).poly(dC), synthetic oligonucleotides containing the GpG sequence, and Micrococcus luteus and calf thymus DNAs. The relative yields of interstrand and intrastrand cross-links (3 and 4) were determined under first-order kinetic conditions; an average 3.6-fold preference for the formation of 3 over that of 4 was observed. An explanation for this preference is proposed.(ABSTRACT TRUNCATED AT 250 WORDS)

Mitomycin C (MC; la) has been shown previously to bind covalently to DNA upon reductive activation, forming three different major adducts with deoxyguanosine residues. Two of these adducts have been fully characterized before as products of monofunctional and bifunctional alkylation of N2-positions of guanines by MC. The third, unknown adduct was now isolated in sufficient quantities for determination of its structure. Reaction of Micrococcus luteus DNA with either MC or 10-decarbamoyl-MC (DMC; lc) in the presence of Na2S204 followed by enzymatic digestion of the DNA-drug complex released this adduct. Its structure was shown to be A2-) 10"-decarbamoyl-2"6,7"-diaminomitosen-1 "a-yl)-2'-deoxyguanosine (3a) by using microscale spectroscopic techniques previously developed for the study of adducts in this series, featuring second derivative Fourier transform infrared spectroscopy, circular dichroism for assessing the C-l" stereochemistry, and proton NMR for detection of coupling between the N2-proton of guanine and the 1 "-proton of the mitosene residue. Independent proof was obtained by converting the known 10"-carbamoyl homologue to 7, the acetylation product of 3a, by an authentic route. This completes the identification of the three major adducts of reduced MC and DNA. In contrast to MC, the biologically active analogue DMC yields 3a, but no bifunctional alkylation adduct ("DNA cross-link"), as its sole product with DNA.The antibiotic mitomycin C (la; MC) is useful in the treatment of numerous types of cancers.

This study demonstrates that small organic and organometallic molecules are capable of detecting differences in amyloid fibril structure and/or amyloid protein sequence. Molecules of this type could have utility as neuropathological probes or imaging agents, since they are much easier to prepare and functionalize than antibodies and are specific for the fibrillar form of the amyloid proteins.

The mitomycins are a group of antitumor antibiotics that covalently bind to DNA upon reductive activation. Mitomycin A (lb; MA) is more toxic than its clinically useful cousin, mitomycin C (la; MC).The greater toxicity of mitomycin A has been previously attributed to its higher reduction potential. In this report, the DNA alkylation products of reductively activated MA were isolated and characterized by conversion to the known 7-amino mitosene-deoxyguanosine adducts. The three major adducts formed were identified as a monoadduct, 7V2-(2"/3-amino-7"-methoxymitosen-l"a-yl)-2,-deoxyguanosine (5), a decarbamoyl monoadduct, A2-(2///3-amino-10"-decarbamoyl-7"-methoxymitosen-1 "a-yl)-2'-deoxyguanosine (6), and a bisadduct, /V2-(2"/3-amino-10//-deoxyguanosin-Á2-yl-7-methoxymitosen-1 "a-yl)-2'-deoxyguanosine (7). Under all reductive activation conditions employed, MA selectively alkylated the 2-amino group of guanine in DNA, like MC. In addition, both MA and MC alkylated DNA and cross-linked oligonucleotides to a similar extent. However, variations in the reductive activation conditions (H2/Pt02, Na2S204, or enzymatic) affected the distribution of the three major MA adducts in a different manner than the distribution of MC adducts was affected. A mechanism is proposed wherein the 7-methoxy substituent of MA allows initial indiscriminate activation of either of the drugs' two electrophilic sites. While oxygen inhibited cross-linking by MC, similar aerobic conditions exhibited little influence on the cross-linking ability of MA. Hence, the greater toxicity of MA may be influenced by increased and nonselective activation and cross-link formation in both aerobic and anaerobic cells. This effect is a direct consequence of the higher redox potential of MA as compared to MC. Kohn et al., Hoey et al. (1988)]. Finally, structures of the major alkylation products between reduced MC and DNA, two monoadducts (2 and 3) (Tomasz et al., 1986a(Tomasz et al., ,b, 1988a) and one bisadduct (4), (Tomasz et al., 1987) have been determined. The latter was shown to constitute the interstrand cross-links of DNA by MC (Borowy-Borowski et al., 1990a). Studies into the forces governing the relative ratios of these adducts have demonstrated that the reduction conditions influence the cross-link to monoadduct ratio (Tomasz et al., 1987(Tomasz et al., , 1988b.

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We have developed an automated serial chromatographic technique for screening a library of compounds based upon their relative affinity for a target molecule. A "target" column containing the immobilized target molecule is set in tandem with a reversed-phase column. A combinatorial peptide library is injected onto the target column. The target-bound peptides are eluted from the first column and transferred automatically to the reversed-phase column. The target-specific peptide peaks from the reversed-phase column are identified and sequenced. Using a monoclonal antibody (3E-7) against beta-endorphin as a target, we selected a single peptide with sequence YGGFL from approximately 5800 peptides present in a combinatorial library. We demonstrated the applicability of the technology towards selection of peptides with predetermined affinity for bacterial lipopolysaccharide (LPS, endotoxin). We expect that this technology will have broad applications for high throughout screening of chemical libraries or natural product extracts.

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