Synthesis, chemical properties, and biological evaluation of CC-1065 and duocarmycin analogues incorporating the 5-methoxycarbonyl-1,2,9,9a-tetrahydrocyclopropa

The synthesis of 5-methoxycarbonyl-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (C5-CO2Me-CBI), a substituted CBI derivative bearing a C5 methoxycarbonyl group, and its corresponding 5-hydroxymethyl derivative are described in efforts to establish substituent electronic effects on the agents' functional reactivity and the resulting effect this has on their rate of DNA alkylation. Resolution of an immediate C5-CO2Me-CBI precursor and its incorporation into both enantiomers of 16 and 17, analogues of the duocarmycins, are also detailed. A study of the solvolysis reactivity and regioselectivity of N-BOC-C5-CO2Me-CBI (12) revealed that the introduction of a C5 methyl ester modestly slowed the rate of solvolysis (1.8x, pH 3) without altering the inherent reaction regioselectivity (>20:1). The comparison of the X-ray structures of the N-CO2Me derivatives of C5-CO2Me-CBI and CBI revealed correlations with the reaction regioselectivity and the relative reactivity of the compounds. The latter correlated well with the less reactive C5-CO2Me-CBI exhibiting a shortened N2-C2a bond length (1.386 vs 1.390 A) and smaller chi1 dihedral angle (8.1 degrees vs 21.2 degrees ) indicative of greater vinylogous amide conjugation and was accompanied by a diminished (cross-conjugated) cyclopropane conjugation (shorter bond lengths). Establishment of the DNA alkyation properties revealed that C5-CO2Me-CBI-based agents retained the identical alkylation selectivity of the natural products. More importantly, the C5 methyl ester was found to decrease the rate (0.77x) of DNA alkylation relative to CBI, consistent with its inherent lower reactivity. These results indicate that the previously observed increase in the rate of DNA alkylation for C7-substituted CBI analogues including CCBI (7-cyano-CBI) is contrary to expectations based on their inherent reactivities. Unlike 17, in which the C5 methyl ester does not bind in the minor groove, the C7 substituent lies in the minor groove extending the rigid length of the agents, further enhancing the DNA binding-induced conformational change responsible for activation toward nucleophilic attack and catalysis of the DNA alkylation reaction.     

Theoretical study of the interaction between a high-valent manganese porphyrin oxyl-(hydroxo)-Mn(IV)-TMPyP and double-stranded DNA

Cationic porphyrin derivatives such as meso-tetrakis(4-N-methylpyridinium)porphyrin, TMPyP, have been shown to interact with double-stranded DNA. The manganese derivative, Mn(III)-TMPyP, activated by an oxygen donor like potassium monopersulfate, provides an efficient DNA-cleaving system. Previous experimental work1 has shown that DNA cleavage by the Mn(III)-TMPyP/KHSO(5) system was due to an oxidative attack, within the minor groove of B-DNA, at the C5' or C1' carbons of deoxyribose units. The aim of this study was to use molecular modeling to elucidate the specificity of the interactions between the transient active species oxyl-Mn(IV)-TMPyP and the DNA target. Geometric parameters, charges, and force field constants consistent with the AMBER 98 force field were calculated by DFT methods. Molecular modeling (mechanics and dynamic simulations) were performed for oxyl-(hydroxo)-Mn(IV)-TMPyP bound in the minor groove of the dodecamer d(5'-TCGTCAAACCGC)-d(5'-GCGGTTTGACGA). Geometry, interactions, and binding energy of the metalloporphyrin located at the A.T triplet region of the dodecamer were analyzed. These studies show no significant structural change of the DNA structure upon ligand binding. Mobility of the metalloporphyrin in the minor groove was restrained by the formation of a hydrogen bond between the hydroxo ligand trans to the metal-oxyl and a DNA phosphate, restricting the access of the oxyl group to the (pro-S) H atom at C5'.     

Synthesis of Oligonucleotides containing the cis-Interstrand Crosslink Produced by Mitomycins in their Reaction with DNA

Mitomycin C (MC) an antitumor drug and decarbamoylmitomycin C (DMC), a derivative of MC lacking the carbamoyl moiety, are DNA alkylating agents which can form DNA interstrand crosslinks (ICLs) between deoxyguanosine residues located on opposing DNA strands. MC forms primarily deoxyguanosine adducts with a 1“-R stereochemistry at the guanine-mitosene bond (1”-α, trans) whereas DMC forms mainly adducts with a 1“-S stereochemistry (1”-β, cis). The crosslinking reaction is diastereospecific: trans-crosslinks are formed exclusively at CpG sequences, while cis-crosslinks are formed only at GpC sequences. Until now, oligonucleotides containing 1“-β-deoxyguanosine adducts or ICL at a specific site could not be synthesized, thus limiting the investigation of the role played by the stereochemical configuration at C1′′ in the toxicity of these compounds. Here, a novel biomimetic synthesis to access these substrates is presented. Structural proof of the adducted oligonucleotides and ICL were provided by enzymatic digestion to nucleosides, high resolution mass spectral analysis, CD spectroscopy and UV melting temperature studies. Finally, a virtual model of the 25-mer 1”-β ICL synthesized was created to explore the conformational space and structural features of the crosslinked duplex.     

Observation of a single-stranded DNA/pyrrolobenzodiazepine adduct

Pyrrolobenzodiazepine (PBD) antitumor agents have, to date, only been observed to bind to duplex DNA, apparently requiring a minor groove environment for covalent bond formation between their C11-position and the C2-NH(2) functionality of a guanine base. Using an HPLC/MS assay we have now observed and isolated for the first time PBD adducts with single-stranded DNA fragments. Surprisingly, these adducts could only be formed through dissociation of duplex DNA adducts and not by direct interaction of PBDs with single-stranded DNA. They were sufficiently stable for characterization by MALDI-TOF-MS and remained intact after storing at -20 °C for at least 20 days, although the PBD became detached from the DNA within 7 days if stored at room temperature. Furthermore, addition of a complementary strand allowed the duplex adduct to reform. The relative stability of single-stranded PBD/DNA adducts despite a complete loss of minor groove structure was further confirmed by CD spectroscopic analysis. The CD signal induced by the presence of a PBD molecule in the single-stranded adducts remained prominent despite heating for 2 h at 50-60 °C, thus indicating their relatively robust nature.     

FR900482 class of anti-tumor drugs cross-links oncoprotein HMG I/Y to DNA in vivo

BACKGROUND: Overexpression of the high-mobility group, HMG I/Y, family of chromatin oncoproteins has been implicated as a clinical diagnostic marker for both neoplastic cellular transformation and increased metastatic potential of several human cancers. These minor groove DNA-binding oncoproteins are thus an attractive target for anti-tumor chemotherapy. FR900482 represents a new class of anti-tumor agents that bind to the minor groove of DNA and exhibit greatly reduced host toxicity compared to the structurally related mitomycin C class of anti-tumor drugs. We report covalent cross-linking of DNA to HMG I/Y by FR900482 in vivo which represents the first example of a covalent DNA-drug-protein cross-link with a minor groove-binding oncoprotein and a potential novel mechanism through which these compounds exert their anti-tumor activity. RESULTS: Using a modified chromatin immunoprecipitation procedure, fragments of DNA that have been covalently cross-linked by FR900482 to HMG I/Y proteins in vivo were polymerase chain reaction-amplified, isolated and characterized. The nuclear samples from control cells were devoid of DNA fragments whereas the nuclear samples from cells treated with FR900482 contained DNA fragments which were cross-linked by the drug to the minor groove-binding HMG I/Y proteins in vivo. Additional control experiments established that the drug also cross-linked other non-oncogenic minor groove-binding proteins (HMG-1 and HMG-2) but did not cross-link major groove-binding proteins (Elf-1 and NFkappaB) in vivo. Our results are the first demonstration that FR900482 cross-links a number of minor groove-binding proteins in vivo and suggests that the cross-linking of the HMG I/Y oncoproteins may participate in the mode of efficacy as a chemotherapeutic agent. CONCLUSIONS: We have illustrated that the FR class of anti-tumor antibiotics, represented in this study by FR900482, is able to produce covalent cross-links between the HMG I/Y oncoproteins and DNA in vivo. The ability of this class of compounds to cross-link the HMG I/Y proteins in the minor groove of DNA represents the first demonstration of drug-induced cross-linking of a specific cancer-related protein to DNA in living cells. We have also demonstrated that FR900482 cross-links other minor groove-binding proteins (HMG-1 and HMG-2 in the present study) in vivo; however, since HMG I/Y is the only minor groove-binding oncoprotein presently known, it is possible that these non-histone chromatin proteins are among the important in vivo targets of this family of drugs. These compounds have already been assessed as representing a compelling clinical replacement for mitomycin C due to their greatly reduced host toxicity and superior DNA interstrand cross-linking efficacy. The capacity of FR900482 to cross-link the HMG I/Y oncoprotein with nuclear DNA in vivo potentially represents a significant elucidation of the anti-tumor efficacy of this family of anticancer agents.     

Synthesis of Oligonucleotides Containing Trans Mitomycin C DNA Adducts at N6 of Adenine and N2 of Guanine

Mitomycin C, (MC), an antitumor drug, is a DNA alkylating agent currently used in the clinics. Inert in its native form, MC is reduced to reactive mitosenes, which undergo nucleophilic attack by guanine or adenine bases in DNA to form monoadducts as well as interstrand crosslinks (ICLs). Although ICLs are considered the most cytotoxic lesions, the role of each individual adduct in the drug's cytotoxicity is still not fully understood. Synthetic routes have been developed to access modified oligonucleotides containing dG MC-monoadducts and dG-MC-dG ICL at a single position of their base sequences to investigate the biological effects of these adducts. However, until now, oligonucleotides containing monoadducts formed by MC at the adenine base had not been available, thus preventing the examination of the role played by these lesions in the toxicity of MC. Here, we present a route to access these substrates. Structural proof of the adducted oligonucleotides were provided by enzymatic digestion to nucleosides and high-resolution mass spectral analysis. Additionally, parent oligonucleotides containing a dG monoadduct and a dG-MC-dG ICL were also produced. The stability and physical properties of all substrates were compared via CD spectroscopy and UV melting temperature studies. Finally, virtual models were created to explore the conformational space and structural features of these MC-DNA complexes.     

Study on the Interaction of Mitomycin C with ct-DNA by Pd-Porphin Room Temperature Phosphorescence Probe

Study of the interaction of drugs with calf thymus DNA (ct-DNA) in molecular level1-4 is helpful to understand the fundamental aspects of activation of anticancer drugs and provide the valuable information for designing and developing new antitumor agents. Mitomycin C (MMC) is an anticancer drugs being widely used in clinic with high activity and effectiveness for many cancers. DNA has been proved to be the main target molecule of MMC in the body. The activated MMC can hinder the rep…     

Reactivity of guanine at m5CpG steps in DNA: evidence for electronic effects transmitted through the base pairs.

BACKGROUND: Mitomycin C (MC), a DNA cross-linking and alkylating agent, targets guanines in the m5CpG sequence with 2-3-fold preference over guanines in unmethylated CpG. Benzo[a]pyrenediolepoxide (BPDE) and several other aromatic carcinogens form guanine adducts with an identical selectivity for m5CpG, and in certain cancers G to T transversion mutation 'hotspots' in the p53 tumor suppressor gene are more frequent at this sequence than at guanines in other sequences. MC appears suitable to probe the general mechanism of this selectivity. RESULTS: A 162-bp DNA fragment containing C, m5C or f5C (5-fluoro cytosine) at all cytosine positions was cross-linked by MC at guanines in CpG steps. The extent of cross-linking increased in the order f5C < C < m5C. Monoalkylation or cross-linking of duplex 12-mer oligonucleotides containing a single CpG, f5CpG or m5CpG step gave yields of adducts that increased in the same order. The rates showed a correlation with the Hammett sigma constant of the methyl and fluoro substituents of the cytosine. Only the base-pair cytosine substituent influenced reactivity of guanine. CONCLUSIONS: The 2-amino group of guanine in the m5CpG sequence of DNA has a greater nucleophilic reactivity with mitomycin than CpG. Evidence is presented for a novel mechanism: transmission of the electron-donating effect of the 5-methyl substituent of the cytosine to guanine through H-bonding of the m5C.G base pair. The results explain the enhanced reaction of BPDE at m5CpG in DNA and the origin of G-T mutational hotspots in the p53 gene in cancer.     

Synthesis, biological evaluation, and modeling of dimeric PPI analogues as novel DNA minor groove binders

A series of symmetrical dimeric proton pump inhibitor (PPI) analogues, designed as novel type DNA minor groove binders, was synthesized and evaluated for anti-tumor activity. Some of these new compounds showed IC(50) values below 10 microM in an in vitro anti-tumor test. A molecular modeling study was performed to confirm the sequence selectivity of these compounds towards AT base pairs in DNA. Two effective compounds were selected and docked into the minor groove of DNA. The snug binding may be responsible for their cytotoxic and anti-tumor effects.     

Photoinduced DNA Interstrand Cross-Linking by 1,1′-Biphenyl Analogues: Substituents and Leaving Groups Combine to Determine the Efficiency of Cross-Linker

Two series of 1,1′-biphenyl analogues with various leaving groups (L=OAc, OCH₃, OCHCH=CH₂, OCH₂Ph, SPh, SePh, and Ph₃P⁺) were synthesized. Their reactivity towards DNA and the reaction mechanism were investigated by determining DNA interstrand cross-link (ICL) efficiency, radical and carbocation formation, and the cross-linking reaction sites. All compounds induced DNA ICL formation upon 350 nm irradiation via a carbocation that was generated from oxidation of the corresponding free radicals. The ICL efficiency and the reaction rate strongly depended on the combined effect of the leaving group and the substituent. Among all compounds tested, the high ICL efficiency (30–43 %) and fast reaction rate were observed with compounds carrying a nitrophenyl group and acetate ( 2 a ), ether ( 2 b and 2 c) , or triphenylphosphonium salt ( 2 g ) as leaving groups. Most compounds with a 4-methoxybenzene group showed similar DNA ICL efficiency (≈30 %) with a slow DNA cross-linking reaction rate. Both cation trapping and free radical trapping adducts were detected in the photo activation process of these compounds, which provided direct evidence for the proposed mechanism. Heat stability study in combination with sequence study suggested that these photo-generated benzyl cations alkylate DNA at dG, dA, and dC sites.     

Differential solvation and tautomer stability of a model base pair within the minor and major grooves of DNA

2-(2'-Hydroxyphenyl)benzoxazole (HBO) may be used as a model base pair to study solvation, duplex environment, and tautomerization within the major and minor groves of DNA duplexes. In its ground state, HBO possesses an enol moiety which may be oriented syn or anti relative to the imino nitrogen of the benzoxazole ring. In the absence of external hydrogen-bond donors and acceptors HBO exists as the internally hydrogen-bonded syn-enol, a mimic of the rare base pair tautomer found in DNA, which may be photoinduced to tautomerize and form the keto tautomer, a mimic of the dominant base pair tautomer. Previously, we demonstrated that when incorporated into DNA such that the enol moiety is positioned in the major groove, HBO is not solvated, exists exclusively as the internally hydrogen-bonded syn-enol which is efficiently photoinduced to tautomerize, and the corresponding keto tautomer is preferentially stabilized. In stark contrast, we now show that when HBO is incorporated in DNA such that the enol moiety is positioned in the minor groove, the enol tautomer is preferentially stabilized. Molecular dynamics simulations suggest that this results from the formation of a stable hydrogen-bond between the HBO enol and the O4' atom of an adjacent nucleotide, an H-bond acceptor that is only available in the minor groove. The differential stabilization of the enol and keto tautomers in the major and minor grooves may reflect the functions for which these environments evolved, including duplex replication, stability, and recognition.     

Photochemical generation of oligodeoxynucleotide containing a C4'-oxidized abasic site and its efficient amine modification: dependence on structure and microenvironment

Bleomycin-induced oxidative DNA damage under limited oxygen conditions results in the formation of the C4'-oxidized abasic site (1). We synthesized the oligodeoxynucleotides (ODN) 5, which contains 4'-o-nitrobenzyloxythymidine (3), and 6, which contains 2-nitrobenzyloxy-4'-methoxy-2'-deoxy-d-ribofuranoside (4), as the caged precursors of 7, an ODN containing 1, to study its reactivity with amines. Photoirradiation of the single- and double-stranded 5 led to the formation of 7. Uncaging of the duplex was faster and the yield of 7 was higher with the double-stranded than with the single-stranded ODN. It was suggested that a low dielectric environment of the o-nitrobenzyloxy group in the minor groove of the duplex might accelerate the uncaging rate. Similarly, 6 and its duplex yielded 7 by photoirradiation. However, the yields of 7 were lower than those of 5, and duplex formation slowed the uncaging rate. Reaction of the obtained 7 with an amine resulted in the formation of the lactam 2b in good yield in both single- and double-stranded forms, showing that amine modification of biomolecules by an ODN containing 1 is possible under physiologic conditions.     

Direct observation of essential DNA dynamics: melting and reformation of the DNA minor groove

The dynamics of bound water and ions present in the minor groove of a dodecamer DNA has been decoupled from that of the long-range twisting/bending of the DNA backbone, using the minor groove binder Hoechst 33258 as a fluorescence reporter in the picosecond-resolved time window. The bound water and ions are essential structural components of the minor groove and are destroyed with the destruction of the minor groove when the dodecamer melts at high temperatures and reforms on subsequent cooling of the melted DNA. The melting and rehybridization of the DNA has been monitored by the changes in secondary structure using circular dichroism (CD) spectroscopy. The change in the relaxation dynamics of the DNA has been studied with picosecond resolution at different temperatures, following the temperature-dependent melting and rehybridization profile of the dodecamer, using time-resolved emission spectra (TRES). At room temperature, the relaxation dynamics of DNA is governed by a 40 ps (30%) and a 12.3 ns (70%) component. The dynamics of bound water and ions present in the minor groove is characterized by the 40 ps component in the relaxation dynamics of the probe bound in the minor groove of the dodecamer DNA. Analyses of the TRES taken at different temperatures show that the contribution of this component decreases and ultimately vanishes with the destruction of the minor groove and reappears again with the reformation of the groove. The dynamical behavior of bound water molecules and ions of a genomic DNA (from salmon testes) at different temperatures is also found to be consistent with that of the dodecamer. The longer component of approximately 10 ns in the DNA dynamics is found to be associated with the long-range bending/twisting of the DNA backbone and the associated counterions. The transition from bound water to free water at the DNA surface, indicative of the change in the hydration number associated with each base pair, has also been ascertained in the case of the genomic DNA at different temperatures by employing densimetric and acoustic techniques.     

The inefficiency of incisions of ecteinascidin 743-DNA adducts by the UvrABC nuclease and the unique structural feature of the DNA adducts can be used to explain the repair-dependent toxicities of this antitumor agent.

BACKGROUND: Ecteinascidin 743 (Et 743), a natural product derived from a marine tunicate, is a potent antitumor agent presently in phase II clinical trials. Et 743 binds in the minor groove of DNA and alkylates N2 of guanine via a unique mechanism involving catalytic activation. The sequence selectivity of Et 743 is governed by different patterns of hydrogen-bonding to DNA, which results in differential reversibility of the covalent adducts. As determined by nuclear magnetic resonance spectroscopy, the preferred sequences 5'-PuGC and 5'-PyGG are stabilized by a hydrogen-bonding network, while the non-preferred sequences 5'-NG(A/T) are much less stabilized due to the lack of a key hydrogen bond to the GC base pair on the 3'-side of the alkylated guanine. RESULTS: Mammalian cell lines (XPB, XPD, XPF, XPG, and ERCC1) deficient in the nucleotide excision repair (NER) gene products show resistance to Et 743. The recognition and subsequent incision of Et 743-DNA adducts by the bacterial multisubunit endonuclease UvrABC were used to evaluate DNA repair-mediated toxicity as a rationale for the resistance of NER-defective cell lines and the antitumor activity of Et 743. The Et 743-DNA adducts are indeed recognized and incised by the UvrABC repair proteins; however, the pattern of incision indicated that the non-preferred, and less stable, sequences (i.e. 5'-NG(A/T)) modified with Et 743 are generally incised at a much higher efficiency than the preferred, more stable sequences (i.e. 5'-PuGC or 5'-PyGG). In addition, within the same Et 743 recognition sequence, the level of incision varies, indicating that flanking regions also contribute to the differential incision frequency. CONCLUSIONS: The inefficient repair incision by the UvrABC nuclease of Et 743-DNA adducts provides a basis for rationalizing the observed repair-dependent cytotoxicities of these DNA adducts, if other associated structural properties of Et 743-DNA adducts are taken into account. In particular, the wedge-shaped Et 743, which forces open the minor groove of DNA, introducing a major groove bend, and the extrahelical protrusion of the C-subunit of Et 743 provide unique characteristics alongside the hydrogen-bonding stabilization of a covalent DNA adduct, which we propose traps an intermediate in NER processing of Et 743-DNA adducts. This trapped intermediate protein-Et 743-DNA adduct complex can be considered analogous to a poisoned topoisomerase I- or topoisomerase II-DNA complex. In the absence of an intact NER nuclease complex, this toxic lesion is unable to form, and the Et 743-DNA adducts, although not repaired by the NER pathway, are less toxic to cells. Conversely, elevated levels of either of these nucleases should lead to enhanced Et 743 toxicity.     

Solid-phase synthesis of positively charged deoxynucleic guanidine (DNG) tethering a Hoechst 33258 analogue: triplex and duplex stabilization by simultaneous minor groove binding

Deoxynucleic guanidine (DNG), a DNA analogue in which positively charged guanidine replaces the phosphodiester linkages, tethering to Hoechst 33258 fluorophore by varying lengths has been synthesized. A pentameric thymidine DNG was synthesized on solid phase in the 3' --> 5' direction that allowed stepwise incorporation of straight chain amino acid linkers and a bis-benzimidazole (Hoechst 33258) ligand at the 5'-terminus using PyBOP/HOBt chemistry. The stability of (DNA)(2).DNG-H triplexes and DNA.DNG-H duplexes formed by DNG and DNG-Hoechst 33258 (DNG-H) conjugates with 30-mer double-strand (ds) DNA, d(CGCCGCGCGCGCGAAAAACCCGGCGCGCGC)/d(GCGGCGCGCGCGCTTTTTGGGCCGCGCGCG), and single-strand (ss) DNA, 5'-CGCCGCGCGCGCGAAAAACCCGGCGCGCGC-3', respectively, has been evaluated by thermal melting and fluorescence emission experiments. The presence of tethered Hoechst ligand in the 5'-terminus of the DNG enhances the (DNA)(2).DNG-H triplex stability by a DeltaT(m) of 13 degrees C. The fluorescence emission studies of (DNA)(2).DNG-H triplex complexes show that the DNG moiety of the conjugates bind in the major groove while the Hoechst ligand resides in the A:T rich minor groove of dsDNA. A single G:C base pair mismatch in the target site decreases the (DNA)(2).DNG triplex stability by 11 degrees C, whereas (DNA)(2).DNG-H triplex stability was decreased by 23 degrees C. Inversion of A:T base pair into T:A base pair in the center of the binding site, which provides a mismatch selectively for DNG moiety, decreases the triplex stability by only 5-6 degrees C. Upon hybridization of DNG-Hoechst conjugates with the 30-mer ssDNA, the DNA.DNG-H duplex exhibited significant increase in the fluorescence emission due to the binding of the tethered Hoechst ligand in the generated DNA.DNG minor groove, and the duplex stability was enhanced by DeltaT(m) of 7 degrees C. The stability of (DNA)(2).DNG triplexes and DNA.DNG duplexes is independent of pH, whereas the stability of (DNA)(2).DNG-H triplexes decreases with increase in pH.     

Minor groove binding of a novel tetracationic diviologen

Novel tetracationic diviologen compounds of the general formula CH3(CH2)nV2+(CH2)6V2+(CH2)nCH3 (where V2+ = 4,4'-bipyridinium and n = 5 or 11) were investigated as electrochemical reporters of DNA duplex formation. These compounds bind to both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) when the DNA is either present in solution or immobilized at electrode surfaces. Binding to thiolated ssDNA and dsDNA immobilized at Au electrodes was characterized using the electrochemical response for the reduction of the V2+ state to the V+ (viologen radical cation) state. An analysis of the charge for this reduction provided isotherms and binding constants for binding of these diviologens to both forms of immobilized DNA. Saturation of the binding is achieved at solution concentrations near 20 microM. For both the n = 5 and 11 diviologens, binding to ssDNA is driven by electrostatic charge neutralization. For the n = 11 case, the binding is cooperative. In the presence of dsDNA, the n = 11 diviologen exhibits a unique reduction potential for the V2+/+ redox couple that is shifted approximately 100 mV negative of that in the presence of ssDNA. This new electrochemical signature is attributed to the reduction of viologen groups bound in the minor groove of the DNA duplex. For dsDNA in solution, an increase in the thermal denaturation temperature (Tm) from 60 to 66 degrees C as a function of the n = 11 diviologen concentration confirmed its interaction with the duplex. Circular dichroism (CD) spectroscopy also was used to investigate the binding of both the V2+ and V+ redox states of the n = 11 diviologen to dsDNA in solution. For the V+ state, a CD signal was observed that is consistent with the presence of face-to-face pi dimers of the viologen groups. This unambiguously demonstrates the binding of this redox state of the diviologen in the dsDNA minor groove and the formation of such dimers in the minor groove.     

Inter- and intrastrand DNA crosslinks by 2-fluoro-substituted pyrrolobenzodiazepine dimers: stability, stereochemistry and drug orientation

A 2-fluoro-substituted pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimer with a 1,4-di-n-propyl piperazine linker was studied with respect to its binding and crosslinking capability towards double-helical DNA targets. Duplex thermal stabilizations upon drug binding as measured by UV melting experiments suggest that two guanine bases separated by four AT base pairs constitute the favorable binding site for the PBD dimer. Large stabilizations were observed for the self-complementary duplex d(AACAATTGTT)(2) as well as for the non-self-complementary duplex d(AAGAATTGTT)·d(AACAATTCTT) with both guanines located on the same strand. Formation of interstrand and intrastrand crosslinks by the covalent binding of both PBD moieties of the dimer to the exocyclic 2-amino group of the two guanine bases within the duplex minor groove was confirmed by NMR structural studies. In both the symmetric and non-symmetric DNA-PBD adducts the newly created stereogenic center at C11 of the tricyclic PBD subunits favors an S configuration. Different orientations of the PBD aromatic A-ring with respect to the covalently modified guanine as observed in the non-symmetric complex are shown to result in characteristic changes of PBD H11 and H11a proton chemical shifts. Based on a compilation of available NMR data on various PBD complexes, these differences may be used as valuable probes for the identification of PBD orientational preferences in DNA-PBD adducts.     

Sequence specific fluorescence detection of double strand DNA

Methods for the fluorescent detection of specific sequences of double strand DNA in homogeneous solution may be useful in the field of human genetics. A series of hairpin polyamides with tetramethyl rhodamine (TMR) attached to an internal pyrrole ring were synthesized, and the fluorescence properties of the polyamide-fluorophore conjugates in the presence and absence of duplex DNA were examined. We observe weak TMR fluorescence in the absence of DNA. Addition of >/=1:1 match DNA affords a significant fluorescence increase over equimolar mismatch DNA for each polyamide-TMR conjugate. Polyamide-fluorophore conjugates offer a new class of sensors for the detection of specific DNA sequences without the need for denaturation. The polyamide-dye fluorescence-based method can be used to screen in parallel the interactions between aromatic ring pairs and the minor groove of DNA even when the binding site contains a non-Watson-Crick DNA base pair. A ranking of the specificity of three polyamide ring pairs-Py/Py, Im/Py, and Im/Im-was established for all 16 possible base pairs of A, T, G, and C in the minor groove. We find that Im/Im is an energetically favorable ring pair for minor groove recognition of the T.G base pair.     

Long range 1,4 and 1,6-interstrand cross-links formed by a trinuclear platinum complex. Minor groove preassociation affects kinetics and mechanism of cross-link formation as well as adduct structure

Reported here is a comparison of the kinetics of the stepwise formation of 1,4- and 1,6-GG interstrand cross-links by the trinuclear platinum anticancer compound (15)N-[[trans-PtCl(NH(3))(2)](2)[mu-trans-Pt(NH(3))(2)(H(2)N(CH(2))(6)NH(2))(2)]](4+), (1,0,1/t,t,t (1) or BBR3464). The reactions of (15)N-1 with the self-complementary 12-mer duplexes 5'-[d(ATATGTACATAT)(2)] (I) and 5'-[d(TATGTATACATA)(2)] (II) have been studied at 298 K, pH 5.3 by [(1)H,(15)N] HSQC 2D NMR spectroscopy. The kinetic profiles for the two reactions are similar. For both sequences initial electrostatic interactions with the DNA are observed for 1 and the monoaqua monochloro species (2) and changes in the chemical shifts of certain DNA (1)H resonances are consistent with binding of the central charged [PtN(4)] linker unit in the minor groove. The pseudo first-order rate constants for the aquation of 1 to 2 in the presence of duplex I (3.94 +/- 0.03 x 10(-5) s(-1)), or II(4.17 +/- 0.03 x 10(-5) s(-1)) are ca. 40% of the value obtained for aquation of 1 under similar conditions in the absence of DNA. Monofunctional binding to the guanine N7 of the duplex occurs with rate constants of 0.25 +/- 0.02 M(-1) s(-1) (I) and 0.34 +/- 0.02 M(-1) s(-1) (II), respectively. Closure to form the 1,4- or 1,6-interstrand cross-links (5) was treated as direct from 3 with similar rate constants of 4.21 +/- 0.06 x 10(-5) s(-1) (I) and 4.32 +/- 0.04 x 10(-5) s(-1) (II), respectively. Whereas there is only one predominant conformer of the 1,6 cross-link, evidence from both the (1)H and [(1)H,(15)N] NMR spectra show formation of two distinct conformers of the 1,4 cross-link, which are not interconvertible. Closure to give the major conformer occurs 2.5-fold faster than for the minor conformer. The differences are attributed to the initial preassociation of the central linker of 1 in the minor groove and subsequently during formation of both the monofunctional and bifunctional adducts. For duplex I, molecular models indicate two distinct pathways for the terminal [PtN(3)Cl] groups to approach and bind the guanine N7 in the major groove with the central linker anchored in the minor groove. To achieve platination of the guanine residues in duplex II the central linker remains in the minor groove but 1 must diffuse off the DNA for covalent binding to occur. Clear evidence for movement of the linker group is seen at the monofunctional binding step from changes of chemical shifts of certain CH(2) linker protons as well as the Pt-NH(3) and Pt-NH(2) groups. Consideration of the (1)H and (15)N shifts of peaks in the Pt-NH(2) region show that for both the 1,4 and 1,6 interstrand cross-links there is a gradual and irreversible transformation from an initially formed conformer(s) to product conformer(s) in which the amine protons of the two bound [PtN(3)] groups exist in a number of different environments. The behavior is similar to that observed for the 1,4-interstrand cross-link of the dinuclear 1,1/t,t compound. The potential significance of preassociation in determining kinetics of formation and structure of the adducts is discussed. The conformational flexibility of the cross-links is discussed in relation to their biological processing, especially protein recognition and repair, which are critical determinants of the cytotoxicity of these unique DNA-binding agents.