Mechanism of monofunctional and bifunctional alkylation of DNA by mitomycin C

The relative amounts of monofunctional and bifunctional alkylation products of DNA with mitomycin C (MC) depend on whether one or both masked alkylating functions of MC are activated reductively; adduct 8 is the result of one function and adducts 7 and 9, formed as a pair, are the result of both functions being activated [Tomasz, M., Lipman, R., Chowdary, C., Pawlak, J., Verdine, G. L., & Nakanishi, K. (1987) Science (Washington, D.C.) 235, 1204-1208]. To determine the mechanism governing this differential reactivity of MC with DNA, MC-Micrococcus luteus DNA complexes formed under varying conditions in vitro were digested to nucleosides and adducts. Adduct distribution, analyzed by high-performance liquid chromatography, served as the measure of monofunctional and bifunctional activation. H2/PtO2 and xanthine oxidase/reduced nicotinamide adenine dinucleotide (NADH) activated MC mostly monofunctionally, and Na2S2O4 activated the drug bifunctionally under comparable conditions. Excess MC selectively suppressed, but excess PtO2 selectively promoted, bifunctional activation by H2/PtO2; excess xanthine oxidase and/or NADH also had promoting effects. O2 tested in the Na2S2O4 system was inhibitory. 10-Decarbamoyl-MC acted strictly monofunctionally under all conditions. Monoadducts bound to DNA were converted to bis adducts upon rereduction. A mechanism with the following features was derived: (i) Activation of MC at C-1 and C-10 is sequential (C-1 first). (ii) A one-time reduction is sufficient for both. (iii) Activation of the second function may be selectively inhibited by kinetic factors or O2. (iv) 7 and 9 are coproducts of bifunctional activation; their ratio depends on the DNA base sequence. (v) Activation of the second function involves an iminium intermediate. Direct applications to the action of MC in vivo are discussed.     

Recognition of specific DNA sequences by mitomycin C for alkylation.

Synthetic oligodeoxyribonucleotides were reacted with mitomycin C (MC) under conditions which restricted MC to monofunctional alkylating activity. The yields of monofunctional alkylation of oligonucleotides with variable sequence were determined by enzymatic digestion of the reaction mixture to unreacted nucleosides and the product of alkylation, a MC-deoxyguanosine adduct (2), followed by quantitative analysis by HPLC. The relative yields of 2 reflected relative monoalkylation reactivities. They were compared in a series of oligonucleotides having the sequence 5'-NGN' in which the 5'-base was varied while the 3'-base was kept constant as T. Under Na2S2O4 activation conditions a striking enhancement of the yield was observed at the 5'-CG sequence: 36%, compared to 2% at 5'-AG and 4.1% at 5'-TG. The 5'-GG sequence also showed enhanced reactivity although to a lesser extent (14.7%). The enhancements were specific to the duplex state of the oligonucleotides. Using NADPH:cytochrome c reductase as the reducing agent gave similar results. MC activated by acidic pH also displayed 5'-CG alkylation specificity. 10-Decarbamoyl-MC activated by Na2S2O4 showed the same 5'-CG specificity as MC. Replacement of deoxyguanosine by deoxyinosine in the opposite strand at a 5'-CG site abolished the enhancement of alkylation. Such replacement at a 5'-GG site had a similar effect. It was found that the base 3' to the guanine had only a relatively modest modulating effect on the enhanced reactivity of the G at the 5'-CG sequence. This 3'-base effect appeared to be independent of the 5'-base of the 5'-NGN' triplet. The order of reactivity is 3'-(C greater than T greater than A).(ABSTRACT TRUNCATED AT 250 WORDS)     

Reductive alkylation of proteins

Amino groups of proteins can be alkylated by reaction with a variety of aldehydes or ketones in the presence of several different mild reducing agents. Because reductive alkylation occurs under mild conditions and has relatively minor effects on most proteins, it is becoming one of the more important methods for protein modification. This report discusses some of the recent applications of this reaction.     

Circular dichroism and redox properties of high redox potential ferredoxins

The circular dichroism (CD) spectra of 13 examples of high-potential iron-sulfur proteins (HiPIPs), a class of [4Fe-4S] ferredoxins, have been determined. In contrast to the proposal of Carter [Carter, C. W., Jr. (1977) J. Biol. Chem. 252, 7802-7811], no strict correlation between visible CD features and utilization of the [4Fe-4S]2+/[4Fe-4S]3+ oxidation levels was found. Although most HiPIPs have these features, the model requires their presence in all species. There is also no simple relationship between CD spectral features and the presence of conserved tyrosine-19. In addition, no apparent correlation between CD properties and oxidation-reduction potential could be detected. However, amino acid side chains in close contact to the iron-sulfur cluster appear to be important in modulating spectral and oxidation-reduction properties. In particular, the negative shoulder at 290 nm and negative maximum at 230 nm correlate with the presence of Trp-80 (Chromatium vinosum numbering). Two HiPIPs that do not have Trp at this position have positive bands at 290 and 230 nm. These bands in the Ectothiorhodospira halophila HiPIPs are apparently associated with Trp-49, which is located on the opposite side of the effective mirror plane of the cluster from Trp-80. The effect of pH on circular dichroism and redox potential in Thiocapsa roseopersicina HiPIP, which has a histidine at position 49, is consistent with the interaction of the side chain with the cluster. Despite specific differences in their CD spectra, the various HiPIPs studied show general similarity consistent with structural homology within this class of iron-sulfur proteins.     

Effects of alkylation by dimethyl sulfate, nitrogen mustard, and mitomycin C on DNA structure as studied by the ethidium binding assay.

The extent of alkylation of DNA by dimethyl sulfate, nitrogen mustard, and the antibiotic mitomycin C is related to the resulting decrease in the fluorescence of intercalated ethidium. The fluorescence losses due to the first two types of reagents show a marked pH dependence, with greater losses of fluorescence being observed at alkaline pH values. At pH 11.6 the fluorescence shows a slow recovery, so that with low levels of methylation (4% deoxyguanosine residues modified) one observes complete return of fluorescence. We postulate that these phenomena are due to conversion of 7-methyldeoxyguanosine to the zwitterionic form, and partial denaturation of the DNA duplex with loss of ethidium binding sites. Hydroxide-ion-catalyzed imidazole ring opening, and the removal of the positive charge permits reannealing with concomitant return of the ethidium intercalation sites. This conclusion is substantiated by enzymatic hydrolysis of 14C-labelled methylated DNA and identifiions of the ethidium assay. The distinctly different behavior of mitomycin C confirms previous conclusions that its alkylation, preferentially on guanine, does not take part at the N-7 position.     

Efficient DNA alkylation by a pyrrole-imidazole CBI conjugate with an indole linker: sequence-specific alkylation with nine-base-pair recognition

Conjugates 7, 8, and 10 of N-methylpyrrole (Py)-N-methylimidazole (Im) polyamides and 1,2,9,9a-tetrahydrocyclopropa[1,2-c]benz[1,2-e]indol-4-one (CBI) with a 5-amino-1H-indole-2-carbonyl linker were synthesized by Fmoc solid-phase synthesis and a subsequent liquid-phase coupling procedure. The DNA alkylating abilities of conjugates 7, 8, 6b, and 10 were examined using Texas Red-labeled PCR fragments and high-resolution denaturing gel electrophoresis. CBI conjugates 7 and 8 exhibited highly efficient sequence-specific DNA alkylation comparable with previous CBI conjugates with a vinyl linker. In particular, conjugate 10, with a 10-ringed hairpin Py-Im polyamide, alkylated at the adenine of 5'-ACAAATCCA-3'. Introduction of an indole linker greatly facilitated the synthesis of sequence-specific alkylating Py-Im polyamides.     

Reductive alkylation of lysine residues in subtilisin DY

Only lysine epsilon-amino groups (and the N-terminal alpha-amino group) in native subtilisin DY were reductively alkylated by glyceraldehyde in the presence of sodium cyanoborohydride. The modified protein molecule was cleaved by TosPheCH2Cl-trypsin or cyanogen bromide and the two sets of peptides obtained were fractionated and purified by gel filtration and HPLC. For determination of the degree of modification of each lysine residue, selected peptides were subjected to sequence analysis combined with quantitative estimation of the containing PTH-Lys and PTH-epsilon-DHP-Lys. The data obtained showed that the lysine residues in positions 12, 15, 27, 43, 136, 141, 265 were entirely modified, those in positions 170, 184, 237 were partially modified, and Lys22 and Lys94 were unaccessible for the reagent. The caseinolytic activity decreased by 23% when the maximum number of lysine residues (8.6 of the total 12 residues) in subtilisin DY were modified. The CD-spectra of native and modified enzyme showed only slight differences. Both these experiments suggest that the lysine residues do not take part directly in the catalytic reaction but are responsible for maintaining the native three-dimensional enzyme structure. The data obtained for the accessibility of the different lysine residues in subtilisin DY correlated very well with the positions of these residues in a video model of the structure of subtilisin Carlsberg, thus suggesting that the spatial structures of these two enzymes are very similar.     

A new high potential redox transition for cytochrome aa3.

Using cyano-complexes of iron, tungsten, and molybdenum and a platinum working electrode, we have been able to attain and hold voltages in the range of 400 to 900 mV (vs. standard hydrogen electrode) in an aqueous medium. With this system we have obtained additional information in support of an earlier conclusion that cytochrome a3 has a high Em transition (i.e. greater than 460 mV) in addition to its Em in the 180-200 mV range (Hendler, R. W., K. V. S. Reddy, R. I. Shrager, and W. S. Caughey. 1986. Biophys. J. 49:717-729; Reddy, K. V. S., and R. W. Hendler. 1986. Biophys. J. 49:693-703). The proposed new transition has an Em near 770 mV and an n value greater than 1. The reduced form of the high-potential species of cytochrome a3 does not bind CO, in contrast to the reduced form of the low-potential species which does. A possible reaction scheme for cytochrome aa3 which incorporates the new information is presented.     

Reductive activation of mitomycin C and mitomycin C metabolites catalyzed by NADPH-cytochrome P-450 reductase and xanthine oxidase

Under anaerobic conditions and with proper electron donors, NADPH-cytochrome P-450 reductase (EC 1.6.2.4) and xanthine oxidase (EC 1.2.3.2) similarly reductively metabolized mitomycin C. Reversed phase high performance liquid chromatography was used to separate, detect, and isolate several metabolites. Three metabolites were identified by mass spectrometry and thin layer chromatography as 1,2-cis- and trans-2,7-diamino-1-hydroxymitosene and 2,7-diaminomitosene. Three metabolites were phosphate-dependent, and two of them were identified to be 1,2-cis- and trans-2,7-diaminomitosene 1-phosphate. The amounts of the five identified metabolites generated during the reduction of mitomycin C varied with pH and nucleophile concentration. At pH 6.5, 2,7-diaminomitosene was essentially the only metabolite formed, whereas from pH 6.8 to 8.0, trans- and cis-2,7-diamino-1-hydroxymitosene increased in quantity as 2,7-diaminomitosene decreased. The disappearance of mitomycin C and the production of metabolites were enzyme and mitomycin C concentration-dependent. Substrate saturation was not reached for either enzyme up to 5 mM mitomycin C. Electron paramagnetic resonance studies demonstrated the formation of mitomycin C radical anion as an intermediate during enzymatic activation. Our results indicate that either enzyme catalyzed the initial activation of mitomycin C to a radical anion intermediate. Subsequent spontaneous reactions, including the elimination of methanol and the opening of the aziridine ring, generate one active center at C-1 which facilitates nucleophilic attack. Simultaneous generation of two reactive centers was not observed. All five primary metabolites were metabolized further by either flavoenzyme. The secondary metabolites exhibited similar changes in their absorbance spectra and were unlike the primary metabolites, suggesting that a second alkylating center other than C-1 was generated during secondary activation. We propose that secondary activation of monofunctionally bound mitomycin C is probably a main route for the bifunctional binding of mitomycin C to macromolecules and that the cytotoxic actions of mitomycin C result from multiple metabolic activations and reactions.     

A fluorescent redox sensor with tuneable oxidation potential

A fluorescent redox sensor was prepared by attachment of hydroquinones to the fluorophore rhodamine B; fluorescence is reversibly modulated by hydroquinone-centered chemical redox reactions, and oxidation potential of the sensor is tuneable by variation of hydroquinone structure.     

Reductive alkylation of ribosomes as a probe to the topography of ribosomal proteins

Escherichia coli ribosomes were treated with a number of different aldehydes of various sizes in the presence of NaBH(4). After incorporation of either (3)H or (14)C, the ribosomal proteins were separated by two-dimensional polyacrylamide-gel electrophoresis and the extent of alkylation of the lysine residues in each protein was measured. The same pattern of alkylation was observed with the four reagents used, namely formaldehyde, acetone, benzaldehyde and 3,4,5-trimethoxybenzaldehyde. Every protein in 30S and 50S subunits was modified, although there was considerable variation in the degree of alkylation of individual proteins. A topographical classification of ribosomal proteins is presented, based on the degree of exposure of lysine residues. The data indicate that every protein of the ribosome has at least one lysine residue exposed at or near the surface of the ribonucleo-protein complex.     

Selenoprotein K form an intermolecular diselenide bond with unusually high redox potential

Selenoprotein K (SelK) is a membrane protein involved in antioxidant defense, calcium regulation and the ER-associated protein degradation pathway. We found that SelK exhibits a peroxidase activity with a rate that is low but within the range of other peroxidases. Notably, SelK reduced hydrophobic substrates, such as phospholipid hydroperoxides, which damage membranes. Thus, SelK might be involved in membrane repair or related pathways. SelK was also found to contain a diselenide bond—the first intramolecular bond of that kind reported for a selenoprotein. The redox potential of SelK was −257 mV, significantly higher than that of diselenide bonds in small molecules or proteins. Consequently, SelK can be reduced by thioredoxin reductase. These finding are essential for understanding SelK activity and function.     

Fluorescence study of DNA alkylation by epoxides

A simple fluorescence assay was devised to measure alkylation of guanine. The assay was tested with simple epoxides: propylene oxide, glycidol, epichlorohydrin, trichloropropylene oxide and styrene oxide, which are known to vary considerably in their mutagenic potency. The order of reactivity parallelled the mutagenic potency, trichloropropylene oxide being the most reactive alkylating agent. Each epoxide alkylated deoxyguanosine faster than single-stranded DNA, at equal concentrations of guanine. Single-stranded DNA was alkylated substantially faster than was double-stranded DNA. The reaction products with each substrate were analysed by thin-layer chromatography and exhibited similar Rf-values. It was concluded that polymers, particularly double-stranded DNA, reacted slower than deoxyguanosine due to the properties of polymers in solution rather than the unavailability of reactive sites for alkylation.     

Reductive alkylation of hyaluronic acid for the synthesis of biocompatible hydrogels by click chemistry

Hyaluronan (HA) based hydrogels have been synthesized combining chemical modification of the polysaccharide by partial oxidation, reductive amination and 'click chemistry'. HA was oxidized by 4-acetamido-TEMPO-mediated reaction, using sodium hypochlorite as primary oxidant and NaBr in buffered pH, so that the produced aldehyde moieties (hemiacetals) were trapped in situ by adding primary amines containing azide or alkyne-terminal groups. The structure of the reaction products, oxidized-HA and primary amines bonded to HA, was elucidated using 2D NMR spectroscopy. SEC-MALLS analysis of the modified substrates showed a negligible degradation of the polysaccharide using this procedure. Furthermore, azido- and alkynyl derivatives underwent cross-linking by click chemistry into hydrogels, which were characterized by NMR, FT-IR, swelling degree and mechanical properties. Possible application of the material as scaffold for tissue engineering was tested by seeding and proliferation of chondrocytes for up to 15 days.     

Interactions of surface-confined DNA with electroreduced mitomycin C comparison with acid-activated mitomycin C

The anticancer activity of the antineoplastic drug mitomycin C (MC) was investigated using transfer stripping cyclic voltammetry (TSCV) with single-stranded DNA-modified hanging mercury drop electrode (HMDE). Reductive activation of MC is necessary for drug covalent binding to DNA, and we have found that some potential-controlled interactions of MC with DNA occur at the electrode, i.e. MC can be activated by electroreduction. Acid and electroreductive MC activations were compared and different adducts were subsequently generated, suggesting that the drug can bind to DNA in more than one way. Under conditions of acid activated MC, a monofunctional adduct between C-1 of MC and N-7 of guanine was formed on the electrode surface, reduced at - 0.44 V (vs. SCE). However, when the DNA-modified electrode was immersed in a MC solution and potentials corresponding to the quinone moiety reduction (- 0.3 V or more negative vs. SCE) were applied, an intrastrand bifunctional adduct between C-1 and C-10 of MC and two N-7 of a pair of adjacent guanines in ssDNA were formed at the electrode, reduced at - 0.49 V, i.e. 50 mV more negative than the monoadduct. The results presented in this paper show for the first time electrochemical detection of DNA-MC adducts at the hanging mercury drop electrode.     

Photocontrollable sequence-specific DNA alkylation by a pyrrole-imidazole polyamide seco-CBI conjugate

We designed and synthesized a Py-Im polyamide seco-CBI conjugate protected by a photocleavable group and demonstrated that it was selectively activated by UV irradiation both in vitro and in vivo. Sequence-specific alkylating Py-Im polyamides containing photolabile linkers may be useful for developing novel chemical- or enzyme-activated anticancer agents and may facilitate spatiotemporal control of gene expression.     

Reductive alkylation with oxidized nucleotides. Use in affinity labeling or affinity chromatography

A study has been made of the products of a reaction of oxidized ribonucleotides with a primary amine. As a model reaction, periodate-oxidized adenosine was combined with glycine in the presence of NaCNBH3. The purified major product of this reaction, adenine 9,2'-(4'-carboxymethyl-6'-hydroxymethylmorpholine), was characterized by 13C and 1H NMR spectroscopy, ultraviolet spectroscopy, and thin layer chromatography. When used to generate affinity columns, oxidized adenosine or oxidized ATP formed stable products with immobilized diaminohexane when treated with NaCNBH3. Failure to treat with NaCNBH3 yielded an unstable affinity matrix. These results are used in the interpretation of differing results when oxidized nucleotides have been used as affinity labels for different proteins.