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.     

Synthesis of an adenine N-3 substituted CBI adduct by alkylation of adenine with a 1-iodomethylindoline seco-CBI precursor

The synthesis of a 1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (CBI)-adenine adduct via regioselective N-3 alkylation of adenine with a 1-(iodomethyl)-2,3-dihydro-1H-benzo[e]indol-5-ol (I-seco-CBI)-containing precursor is described. Spectroscopic analyses of the unlabeled and adenine-C8 carbon-13 labeled adducts utilizing ROESY NMR techniques allowed structural assignment of the alkylation product as the adenine N-3 substituted regioisomer. A stable-isotopically labeled version of the N-3 adduct incorporating six carbon-13 labels was also prepared by this method for use as an LC-MS internal standard. The cyclized CBI-containing compound was also found to alkylate adenine at elevated temperatures to produce the N-3 adduct albeit at a significantly slower rate than that observed for the I-seco-CBI precursor. Adenine alkylation with an I-seco-CBI precursor offers scalable and facile access to a CBI-adenine adduct facilitating its use as an efficacy marker for the development of duocarmycin-based ADCs.     

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.     

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.     

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.     

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.     

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-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.     

Regioselective alkylation of the exocyclic nitrogen of adenine and adenosine by the Mitsunobu reaction

A novel synthetic route to N⁶-substitution of adenine is presented, employing the Mitsunobu reaction as the key step. A range of primary and secondary alcohols all coupled in very good to excellent yields within 30 min at 45 °C, offering a milder alternative to the traditional nucleophilic aromatic substitution of 6-chloropurine. The utility of this protocol is further demonstrated by its application to the syntheses of N⁶,N⁹-di-substituted adenines, including the potent and selective A₁ adenosine receptor agonist N⁶-cyclopentyladenosine.     

Generation of oligonucleotide conjugates via one-pot diselenide-selenoester ligation–deselenization/alkylation

A breadth of strategies are needed to efficiently modify oligonucleotides with peptides or lipids to capitalize on their therapeutic and diagnostic potential, including the modulation of in vivo chemical stability and for applications in cell-targeting and cell-permeability. The chemical linkages typically used in peptide oligonucleotide conjugates (POCs) have limitations in terms of stability and/or ease of synthesis. Herein, we report an efficient method for POC synthesis using a diselenide-selenoester ligation (DSL)-deselenization strategy that rapidly generates a stable amide linkage between the two biomolecules. This conjugation strategy is underpinned by a novel selenide phosphoramidite building block that can be incorporated into an oligonucleotide by solid-phase synthesis to generate diselenide dimer molecules. These can be rapidly ligated with peptide selenoesters and, following in situ deselenization, lead to the efficient generation of POCs. The diselenide within the oligonucleotide also serves as a flexible functionalisation handle that can be leveraged for fluorescent labelling, as well as for alkylation to generate micelles.

An efficient and versatile approach for the late-stage generation of oligonucleotide conjugates by diselenide-selenoester ligation (DSL)–deselenization/alkylation was developed.     

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 DNA Alkylation Targeting for Kras Codon 13 Mutation by Pyrrole–Imidazole Polyamide seco‐CBI Conjugates

Hairpin N‐methylpyrrole‐N‐methylimidazole polyamide seco‐CBI conjugates 2 – 6 were designed for synthesis by Fmoc solid‐phase synthesis, and their DNA‐alkylating activities against the Kras codon 13 mutation were compared by high‐resolution denaturing gel electrophoresis with 225 base pair (bp) DNA fragments. Conjugate 5 had high reactivity towards the Kras codon 13 mutation site, with alkylation occurring at the A of the sequence 5′‐ACGTCACC A ‐3′ (site 2), including minor 1 bp‐mismatch alkylation against wild type 5′‐ACG C CACC A ‐3′ (site 3). Conjugate 6 , which differs from conjugate 5 by exchanging one Py unit with a β unit, showed high selectivity but only weakly alkylated the A of 5′‐ACGTCACC A ‐3′ (site 2). The hairpin polyamide seco‐CBI conjugate 5 thus alkylates according to Dervan′s pairing rule with the pairing recognition which β/β pair targets T–A and A–T pairs. SPR and a computer‐minimized model suggest that 5 binds to the target sequence with high affinity in a hairpin conformation, allowing for efficient DNA alkylation.     

Specific and sensitive detection of nucleic acids and RNases using gold nanoparticle-RNA-fluorescent dye conjugates

Gold nanoparticles were modified with RNA and utilized to detect specific DNA sequences and various RNA nucleases.     

Synthesis of N-substituted indoles by extractive alkylation

A method was developed for the N-alkylation of indole and its derivatives by alkyl halides in the presence of sodium hydroxide and catalytic amounts of trimethylbenzylammonium chloride.Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 2, pp. 191–193, February, 1976.The authors thank Prof. A. N. Kost for his critical remarks and advice during a discussion of the results of our research on the alkylation of N-carbomethoxy-3-indolylcyanoacetic ester