DNA alkylation by pyrrole-imidazole seco-CBI conjugates with an indole linker: sequence-specific DNA alkylation with 10-base-pair recognition through heterodimer formation

The sequence-specific DNA alkylation by conjugates 4 and 5, which consist of N-methylpyrrole (Py)-N-methylimidazole (Im) polyamides and 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI) linked with an indole linker, was investigated in the absence or presence of partner Py-Im polyamide 6. High-resolution denaturing polyacrylamide gel electrophoresis revealed that conjugate 4 alkylates DNA at the sequences 5'-(A/T)GCCTA-3' through hairpin formation, and alkylates 5'-GGAAAGAAAA-3' through an extended binding mode. However, in the presence of partner Py-Im polyamide 6, conjugate 4 alkylates DNA at a completely different sequence, 5'-AGGTTGTCCA-3'. Alkylation of 4 in the presence of 6 was effectively inhibited by a competitor 7. Surface plasmon resonance (SPR) results indicated that conjugate 4 does not bind to 5'-AGGTTGTCCA-3', whereas 6 binds tightly to this sequence. The results suggest that alkylation proceeds through heterodimer formation, indicating that this is a general way to expand the recognition sequence for DNA alkylation by Py-Im seco-CBI conjugates.     

Evaluation of the DNA Alkylation Properties of a Chlorambucil-Conjugated Cyclic Pyrrole-Imidazole Polyamide

Hairpin pyrrole-imidazole polyamides (hPIPs) and their chlorambucil (Chb) conjugates (hPIP-Chbs) can alkylate DNA in a sequence-specific manner, and have been studied as anticancer drugs. Here, we conjugated Chb to a cyclic PIP (cPIP), which is known to have a higher binding affinity than the corresponding hPIP, and investigated the DNA alkylation properties of the resulting cPIP-Chb using the optimized capillary electrophoresis method and conventional HPLC product analysis. cPIP-Chb conjugate 3 showed higher alkylation activity at its binding sites than did hPIP-Chb conjugates 1 and 2 . Subsequent HPLC analysis revealed that the alkylation site of conjugate 3 , which was identified by capillary electrophoresis, was reliable and that conjugate 3 alkylates the N3 position of adenine as do hPIP-Chbs. Moreover, conjugate 3 showed higher cytotoxicity against LNCaP prostate cancer cells than did conjugate 1 and cytotoxicity comparable to that of conjugate 2 . These results suggest that cPIP-Chbs could be novel DNA alkylating anticancer drugs.     

Sequence-specific gene silencing in mammalian cells by alkylating pyrrole-imidazole polyamides

Gene silencing was examined by sequence-specific alkylation of DNA by N-methylpyrrole (Py)-N-methylimidazole (Im) hairpin polyamides. Polyamides ImImPyPygammaImImPyLDu86 (A) and ImImPyPygammaImPyPyLDu86 (B) selectively alkylated the coding regions of the renilla and firefly luciferases, respectively, according to the base pair recognition rule of Py-Im polyamides. Two different plasmids, encoding renilla luciferase and firefly luciferase, were used as vectors to examine the effect of alkylation on gene silencing. Transfection of the alkylated luciferase vectors-by polyamide A or B-into HeLa, 293, and NIH3T3 cells demonstrated that these sequence-specific DNA alkylations lead to selective silencing of gene expression. Next, the vectors were cotransfected into HeLa cells and the cells were treated with polyamide A or B. Selective reduction of luciferase activities was caused by both polyamides. On the basis of this sequence-specific alkylation and gene silencing activity, these alkylating Py-Im polyamides thus have potential as antitumor drugs to target specific gene expression in human cells.     

Sequence-specific alkylation by Y-shaped and tandem hairpin pyrrole-imidazole polyamides

To extend the target DNA sequence length of the hairpin pyrrole-imidazole (Py-Im) polyamide 1, we designed and synthesized Y-shaped and tandem hairpin Py-Im polyamides 2 and 3, which possess 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI) as DNA-alkylating moieties. High-resolution denaturing polyacrylamide gel electrophoresis by using 5'-Texas-Red-labeled 465 base pair (bp) DNA fragments revealed that conjugates 2 and 3 alkylated the adenine of the target DNA sequences at nanomolar concentrations. Conjugate 2 alkylated adenine N3 at the 3' end of two 8 bp match sequences, 5'-AATAACCA-3' (site A) and 5'-AAATTCCA-3' (site C), while conjugate 3 recognized one 10 bp match sequence, 5'-AGAATAACCA-3' (site A) in the 465 bp DNA fragments. These results demonstrate that seco-CBI conjugates of Y-shaped and tandem hairpin polyamides have extended their target alkylation sequences.     

Specific adenine alkylation by pyrrole-imidazole CBI conjugates

We examined DNA alkylation by pyrrole (Py)-imidazole (Im) hairpin polyamides, which possess 1,2,9,9a-tetrahydrocyclopropa[1,2-c]benz[1,2-e]indol-4-one (CBI) or cyclopropapyrroloindole (CPI) as DNA alkylating moieties. High-resolution denaturing gel electrophoresis revealed that alkylation by CBI conjugates 2 and 4 occurred specifically at adenines (A) in matched sequences, whereas CPI conjugates 1 and 3 alkylated both A and guanines (G) in matched sequences. The origin of the different reactivity of CBI and CPI conjugates is discussed in relation to the electrophilicity of the cyclopropane moiety. The high selectivity of the CBI conjugate gives additional sequence specificity relative to CPI conjugates that would be useful for the biological applications.     

Sequence specific alkylation of DNA by hairpin pyrrole-imidazole polyamide conjugates

BACKGROUND: Pyrrole-imidazole polyamides are synthetic ligands that recognize predetermined sequences in the minor groove of DNA with affinities and specificities comparable to those of DNA-binding proteins. As a result of their DNA-binding properties, polyamides could deliver reactive moieties for covalent reaction at specific DNA sequences and thereby inhibit DNA-protein interactions. Site-specific alkylation of DNA could be a useful tool for regulating gene expression. As a minimal first step, we set out to design and synthesize a class of hairpin polyamides equipped with DNA alkylating agents and characterize the specificity and yield of covalent modification. RESULTS: Bis(dichloroethylamino)benzene derivatives of the well-characterized chlorambucil (CHL) were attached to the gamma turn of an eight-ring hairpin polyamide targeted to the HIV-1 promoter. We found that a hairpin polyamide-CHL conjugate binds and selectively alkylates predetermined sites in the HIV promoter at subnanomolar concentrations. Cleavage sites were determined on both strands of a restriction fragment containing the HIV-1 promoter, revealing good specificity and a high yield of alkylation. CONCLUSIONS: The ability of polyamide-CHL conjugates to sequence specifically alkylate double-stranded DNA in high yield and at low concentrations sets the stage for testing their use as regulators of gene expression in cell culture and ultimately in complex organisms.     

Sequence‐Specific DNA Alkylation by Tandem Py–Im Polyamide Conjugates

Tandem N‐methylpyrrole? N‐methylimidazole (Py? Im) polyamides with good sequence‐specific DNA‐alkylating activities have been designed and synthesized. Three alkylating tandem Py? Im polyamides with different linkers, which each contained the same moiety for the recognition of a 10 bp DNA sequence, were evaluated for their reactivity and selectivity by DNA alkylation, using high‐resolution denaturing gel electrophoresis. All three conjugates displayed high reactivities for the target sequence. In particular, polyamide 1 , which contained a β‐alanine linker, displayed the most‐selective sequence‐specific alkylation towards the target 10 bp DNA sequence. The tandem Py? Im polyamide conjugates displayed greater sequence‐specific DNA alkylation than conventional hairpin Py? Im polyamide conjugates ( 4 and 5 ). For further research, the design of tandem Py? Im polyamide conjugates could play an important role in targeting specific gene sequences.     

Selective intercalation of charge neutral intercalators into GG and CG steps: implication of HOMO-LUMO interaction for sequence-selective drug intercalation into DNA

We have synthesized naphthopyranone epoxide 4 from D-isoascorbic acid together with its three diastereoisomers. DNA alkylation of ODNs containing 5'XGT3' and 5'TGY3' by 4 (11R, 13R), where X and Y are any nucleotide bases, occurred at all G residues except at G of the 5'TGC3' sequence. In contrast, the three other diastereoisomers of 4 showed only weak G alkylation activity. Differential (1)H NMR NOE of the 4-G adduct confirmed the G-N7 alkylation at the epoxide carbon of 4 with concomitant S(N)2 ring opening of the epoxide. Quantitative HPLC analysis of G alkylation efficiency for 4 showed the order of G alkylation susceptibility as TGGT approximately CGT > TGA > AGT > TGT > TGC. The order was fully consistent with those reported for aflatoxin B(1) oxide and kapurimycin A(3), suggesting that the sequence selectivity observed for these DNA alkylating agents is not structure dependent but most likely due to the intrinsic property of DNA sequences. We found that the order of G alkylation susceptibility obtained for 4 completely matched the calculated HOMO energy level of G-containing sequences. These results underscore that 4 is a unique molecular probe for ranking the HOMO level of G-containing sequences by well-known G alkylation chemistry and suggests that the intercalation of charge neutral intercalators is a HOMO-controlled process.     

Alkylation of inorganic oxo compounds and insights on preventing DNA damage

Metabolism of food- and tobacco-borne procarcinogens results in the exposure of DNA to toxic alkylating agents. These assaults can bring about DNA alkylation damage, mutations, and cancer. Dietary inorganic compounds such as selenium and vanadium are known to prevent cancer, possibly by reacting directly with alkylating agents, thereby preventing DNA damage. To understand potential interactions between oxo species and alkylating toxins, we reacted a series of alkylating agents with varied classes of oxo compounds (i.e., vanadates, selenate, phosphate, sulfate, acetate, nitrate, and nitrite). A new organic-soluble selenate, [(C6H5)4P]3(O3SeOCH2OSeO3)(HSeO4), was synthesized and characterized for these studies. Vanadates were found to convert ethylating agents into ethanol, whereas other anions formed esters upon alkylation. General trends show that oxo anions of the greatest charge density were the most reactive. These studies suggest that the design of new compounds for cancer prevention should incorporate reactive oxo groups with high anionic charge density.     

Enantioselective DNA alkylation by a pyrrole-imidazole S-CBI conjugate

Conjugates 12S and 12R of N-methylpyrrole (Py)-N-methylimidazole (Im) seven-ringed hairpin polyamide with both enantiomers of 1,2,9,9a-tetrahydrocyclopropa[1,2-c]benz[1,2-e]indol-4-one (CBI) were synthesized, and their DNA alkylating activity was examined. High-resolution denaturing gel electrophoresis revealed that 12S selectively and efficiently alkylated at one match sequence, 5'-TGACCA-3', in 450-bp DNA fragments. The selectivity and efficiency of the DNA alkylation by 12S were higher than those of the corresponding cyclopropapyrroloindole (CPI) conjugate, 11. In sharp contrast, another enantiomer, 12R, showed very weak DNA alkylating activity. Product analysis of the synthetic decanucleotide confirmed that the alkylating activity of 12S was comparable with 11 and that 12S had a significantly higher reactivity than 12R. The enantioselective reactivity of 12S and 12R is assumed to be due to the location of the alkylating cyclopropane ring of the CBI unit in the minor groove of the DNA duplex. Since the CBI unit can be synthesized from commercially available 1,3-naphthalenediol, the present results open up the possibility of large-scale synthesis of alkylating Py-Im polyamides for facilitating their use in future animal studies.     

Molecular design of a pyrrole-imidazole hairpin polyamides for effective DNA alkylation

New hairpin polyamide-CPI (CPI = cyclopropylpyrroloindole) conjugates, compounds 12-14, were synthesized and their DNA-alkylating activities compared with the previously prepared hairpin polyamide, compound 1, by high-resolution denaturing gel electrophoresis with 450 base pair (bp) DNA fragments and by HPLC product analysis of the synthetic decanucleotide. In accord with our previous results, alkylation by compound 1 occurred predominantly at the G moiety of the sequence 5'-AGTCAG-3' (site 3). However, compound 12, in which the structure of the alkylating moiety of compound 1 is replaced with segment A of duocarmycin A DU-86 (CPI), did not show any DNA alkylating activity. In clear contrast, the hairpin CPI conjugate 13, which differs from compound 1 in that it lacks one Py unit and possesses a vinyl linker, alkylated the A of 5'-AGTCAG-3' (site 3) efficiently at nanomolar concentrations. Alkylation by compound 14, which has a vinyl linker, occurred at the A of 5'-AGTCCA-3' (site 6) and at several minor alkylation sites, including mismatch alkylation at A of 5'-TCACAA-3' (site 2). The significantly different reactivity of the alkylating hairpin polyamides 1, 12, 13, and 14 was further confirmed by HPLC product analysis by using a synthetic decanucleotide. The results suggest that hairpin polyamide--CPI conjugate 13 alkylates effectively according to Dervan's pairing rule, and with a new mode of recognition in which the Im-vinyl linker (L) pair targets G-C base pairs. These results demonstrate that incorporation of the vinyl-linker pairing with Im dramatically improves the reactivity of hairpin polyamide--CPI conjugates.     

Sequence-specific alkylation of double-strand human telomere repeat sequence by pyrrole-imidazole polyamides with indole linkers

We designed and synthesized pyrrole (Py)-imidazole (Im) hairpin polyamide 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI) conjugates 1 and 2, which target both strands of the double-stranded region of the human telomere repeat sequences, 5'-d(TTAGGG)(n)-3'/5'-d(CCCTAA)(n)-3'. High-resolution denaturing polyacrylamide gel electrophoresis demonstrated that conjugates 1 and 2 alkylated DNA at the 3' A of 5'-ACCCTA-3' and 5'-AGGGTTA-3', respectively. Cytotoxicities of conjugates 1 and 2 were evaluated using 39 human cancer cell lines; averages of log IC(50) values for conjugates 1 and 2 were -6.96 (110 nM) and -7.24 (57.5 nM), respectively. Conjugates 1 and 2 have potential as antitumor drugs capable of targeting telomere repeat sequence.     

Catalytic Reductive N‐Alkylations Using CO2 and Carboxylic Acid Derivatives: Recent Progress and Developments

N‐Alkylamines are key intermediates in the synthesis of fine chemicals, dyes, and natural products, and hence are highly valuable building blocks in organic chemistry. Consequently, the development of greener and more efficient procedures for their production continues to attract the interest of both academic and industrial chemists. Reductive procedures such as reductive amination or N‐alkylation through hydrogen autotransfer by employing carbonyl compounds or alcohols as alkylating agents have prevailed for the synthesis of amines. In the last few years, carboxylic/carbonic acid derivatives and CO₂ have been introduced as alternative and convenient alkylating sources. The safety, easy accessibility, and high stability of these reagents makes the development of new reductive transformations with them as N‐alkylating agents a useful alternative to existing procedures. In this Review, we summarize reported examples of one‐pot reductive N‐alkylation methods that use carboxylic/carbonic acid derivatives or CO₂ as alkylating agents.     

Hybrids of amino acids and acetylenic DNA-photocleavers: optimising efficiency and selectivity for cancer phototherapy

Hybrid agents which combine potent DNA-photocleavers with tunable amino acids or small peptides were designed to improve selectivity of Nature's most potent class of antibiotics towards cancer cells. The ability of these compounds to photocleave DNA is controlled by their incorporation into hybrid architectures with functional elements derived from natural amino acids. These conjugates are highly effective at inducing double-strand DNA cleavage and, in some cases, rival or even surpass both naturally occurring DNA cleavers and anticancer agents that are currently in clinical use. The possibility of triggering their activity in a photochemical and pH-sensitive fashion allows for a high degree of selectivity over activation. The conjugates were shown to penetrate cell membranes and induce efficient intracellular DNA cleavage. Initial in vitro tests against a variety of cancer cell lines confirm the potential of these compounds as anticancer agents at low nanomolar concentrations.     

Sequence-selective single-molecule alkylation with a pyrrole-imidazole polyamide visualized in a DNA nanoscaffold

We demonstrate a novel strategy for visualizing sequence-selective alkylation of target double-stranded DNA (dsDNA) using a synthetic pyrrole-imidazole (PI) polyamide in a designed DNA origami scaffold. Doubly functionalized PI polyamide was designed by introduction of an alkylating agent 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI) and biotin for sequence-selective alkylation at the target sequence and subsequent streptavidin labeling, respectively. Selective alkylation of the target site in the substrate DNA was observed by analysis using sequencing gel electrophoresis. For the single-molecule observation of the alkylation by functionalized PI polyamide using atomic force microscopy (AFM), the target position in the dsDNA (～200 base pairs) was alkylated and then visualized by labeling with streptavidin. Newly designed DNA origami scaffold named "five-well DNA frame" carrying five different dsDNA sequences in its cavities was used for the detailed analysis of the sequence-selectivity and alkylation. The 64-mer dsDNAs were introduced to five individual wells, in which target sequence AGTXCCA/TGGYACT (XY = AT, TA, GC, CG) was employed as fully matched (X = G) and one-base mismatched (X = A, T, C) sequences. The fully matched sequence was alkylated with 88% selectivity over other mismatched sequences. In addition, the PI polyamide failed to attach to the target sequence lacking the alkylation site after washing and streptavidin treatment. Therefore, the PI polyamide discriminated the one mismatched nucleotide at the single-molecule level, and alkylation anchored the PI polyamide to the target dsDNA.     

Synthesis of isochrysohermidin-distamycin hybrids

The synthesis of the alkylating subunit of the DNA cross-linking agent, isochrysohermidin (2), and its subsequent incorporation into conjugates with distamycin A (1) are described. The DNA binding properties of these agents were compared to that of distamycin A, using a fluorescence intercalator displacement (FID) assay.