Programmable oligomers for minor groove DNA recognition

The four Watson-Crick base pairs of DNA can be distinguished in the minor groove by pairing side-by-side three five-membered aromatic carboxamides, imidazole (Im), pyrrole (Py), and hydroxypyrrole (Hp), four different ways. On the basis of the paradigm of unsymmetrical paired edges of aromatic rings for minor groove recognition, a second generation set of heterocycle pairs, imidazopyridine/pyrrole (Ip/Py) and hydroxybenzimidazole/pyrrole (Hz/Py), revealed that recognition elements not based on analogues of distamycin could be realized. A new set of end-cap heterocycle dimers, oxazole-hydroxybenzimidazole (No-Hz) and chlorothiophene-hydroxybenzimidazole (Ct-Hz), paired with Py-Py are shown to bind contiguous base pairs of DNA in the minor groove, specifically 5'-GT-3' and 5'-TT-3', with high affinity and selectivity. Utilizing this technology, we have developed a new class of oligomers for sequence-specific DNA minor groove recognition no longer based on the N-methyl pyrrole carboxamides of distamycin.     

Expanding the repertoire of heterocycle ring pairs for programmable minor groove DNA recognition

The discrimination of the four Watson-Crick base pairs by minor groove DNA-binding polyamides have been attributed to the specificity of three five-membered aromatic amino acid subunits, 1-methyl-1H-imidazole (Im), 1-methyl-1H-pyrrole (Py), and 3-hydroxy-1H-pyrrole (Hp) paired four different ways. The search for additional ring pairs that demonstrate DNA-sequence specificity has led us to a new class of 6-5 fused bicycle rings as minor groove recognition elements. The affinities and specificities of the hydroxybenzimidazole/pyrrole (Hz/Py) and hydroxybenzimidazole/benzimidazole (Hz/Bi) pairs for each of the respective Watson-Crick base pairs within the sequence context 5'-TGGXCA-3' (X = A, T, G, C) were measured by quantitative DNaseI footprinting titrations. The Hz/Py and Hz/Bi distinguish T.A from A.T. Hairpin polyamides containing multiple Hz/Py pairs were examined and were shown to mimic the Hp/Py pair with regard to affinity and specificity. Therefore, the Hz/Py pair may be considered a second-generation replacement for the Hp/Py pair.     

Influence of β‐Alanine on Hairpin Polyamide Orientation in the DNA Minor Groove

Antiparallel polyamides containing 1H‐pyrrole, 1H‐imidazole, and 3‐hydroxy‐1H‐pyrrole amino acids display a preference for minor‐groove binding oriented N? C with respect to the 5′‐3′ direction of the DNA helix. We find that replacement of a central Py/Py pair with a β/β pair within a ten‐ring hairpin relaxes the orientation preference and, for some DNA sequences, causes the polyamide to prefer the opposite C? N orientation. Substitution of the achiral γ‐aminobutanoic acid (γ) with either (R)(or S)‐2‐(acetylamino)‐4‐aminobutanoic acid moderates the orientation preference of the 2‐β‐2‐hairpin.     

Discrimination of A/T sequences in the minor groove of DNA within a cyclic polyamide motif

Eight-ring cyclic polyamides containing pyrrole (Py), imidazole (Im), and hydroxypyrrole (Hp) aromatic amino acids recognize predetermined six base pair sites in the minor groove of DNA. Two four-ring polyamide subunits linked by (R)-2,4-diaminobutyric acid [(R)H2Ngamma] residue form hairpin polyamide structures with enhanced DNA binding properties. In hairpin polyamides, substitution of Hp/Py for Py/Py pairs enhances selectivity for T. A base pairs but compromises binding affinity for specific sequences. In an effort to enhance the binding properties of polyamides containing Hp/Py pairings, four eight ring cyclic polyamides were synthesized and analyzed on a DNA restriction fragment containing three 6-bp sites 5'-tAGNNCTt-3', where NN = AA, TA, or AT. Quantitative footprint titration experiments demonstrate that contiguous placement of Hp/Py pairs in cyclo-(gamma-ImPyPyPy-(R)H2Ngamma-ImHpHpPy-) (1) provides a 20-fold increase in affinity for the 5'-tAGAACTt-3' site (Ka = 7.5 x 10(7)M(-1)) relative to ImPyPyPy-(R)H2Ngamma-ImHpHpPy-C3-OH (2). A cyclic polyamide of sequence composition cyclo-(gamma-ImHpPyPy-(R)H2Ngamma-ImHpPyPy-) (3) binds a 5'-tAGTACTt-3' site with an equilibrium association constant KA= 3.2 x 10(9)M(-1), representing a fivefold increase relative to the hairpin analogue ImHpPyPy-(R)H2Ngamma-ImHpPyPy-C3-OH (4). Arrangement of Hp/Py pairs in a 3'-stagger regulates specificity of cyclo-(gamma-ImPyHpPy-(R)H2Ngamma-ImPyHpPy-) (5) for the 5'-tAGATCTt-3' site (Ka = 7.5 x 10(7)M(-1)) threefold increase in affinity relative to the hairpin analogue ImPyHpPy-(R)H2Ngamma-ImPyHpPy-C3-OH (6), respectively. This study identifies cyclic polyamides as a viable motif for restoring recognition properties of polyamides containing Hp/Py pairs.     

Imidazopyridine/Pyrrole and hydroxybenzimidazole/pyrrole pairs for DNA minor groove recognition

The DNA binding properties of fused heterocycles imidazo[4,5-b]pyridine (Ip) and hydroxybenzimidazole (Hz) paired with pyrrole (Py) in eight-ring hairpin polyamides are reported. The recognition profile of Ip/Py and Hz/Py pairs were compared to the five-membered ring pairs Im/Py and Hp/Py on a DNA restriction fragment at four 6-base pair recognition sites which vary at a single position 5'-TGTNTA-3', where N = G, C, T, A. The Ip/Py pair distinguishes G.C from C.G, T.A, and A.T, and the Hz/Py pair distinguishes T.A from A.T, G.C, and C.G, affording a new set of heterocycle pairs to target the four Watson-Crick base pairs in the minor groove of DNA.     

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.     

On the pairing rules for recognition in the minor groove of DNA by pyrrole-imidazole polyamides

Background: Cell-permeable small molecules that target predetermined DNA sequences with high affinity and specificity have the potential to control gene expression. A binary code has been developed to correlate DNA sequence with side-by-side pairings between N-methylpyrrole (Py) and N-methylimidazole (lm) carboxamides in the DNA minor groove. We set out to determine the relative energetics of pairings of Im/Py, Py/Im, Im/Im, and Py/Py for targeting G·C and A·T base pairs. A key specificity issue, which has not been previously addressed, is whether an Im/Im pair is energetically equivalent to an Im/Py pair for targeting G·C base pairs.Results: Equilibrium association constants were determined at two five-base-pair sites for a series of four six-ring hairpin polyamides, in order to test the relative energetics of the four aromatic amino-acid pairings opposite G·C and A·T base pairs in the central position. We observed that a G·C base pair was effectively targeted with Im/Py but not Py/Im, Py/Py, or Im/Im. The A·T base pair was effectively targeted with Py/Py but not Im/Py, Py/Im, or Im/Im.Conclusions: An Im/Im pairing is energetically disfavored for the recognition of both A·T and G·C. This specificity will create important limitations on undesirable slipped motifs that are available for unlinked dimers in the minor groove. Baseline energetic parameters will thus be created which, using the predictability of the current pairing rules for specific molecular recognition of double-helical DNA, will guide further second-generation polyamide design for DNA recognition.     

脂肪链在吡咯-咪唑聚酰胺及其结合物与B-DNA键合中的作用

吡咯-咪唑聚酰胺是一类人工合成的能够在B-DNA小沟特异性识别碱基序列的有机小分子,并且能通过细胞膜进入细胞,调控基因的表达。它主要由五元杂环化合物N-甲基吡咯(Py)、N-甲基咪唑(Im)、N-甲基-3-羟基吡咯(Hp)芳香氨基酸及脂肪链氨基酸组成。在这种小分子对生物大分子识别事件中, 脂肪链^作为构建聚酰胺及其结合物的一部分, 在聚酰胺特异性识别DNA、延长DNA识别序列、连接分子荧光标记、对DNA指定位点的烷基化及基因调控等方面都起着非常重要的作用。本文阐述了脂肪链在上述诸方面应用的研究进展,并简要地分析了存在的问题和应用前景。     

Alternative heterocycles for DNA recognition: the benzimidazole/imidazole pair

Boc-protected benzimidazole-pyrrole, benzimidazole-imidazole, and benzimidazole-methoxypyrrole amino acids were synthesized and incorporated into DNA binding polyamides, comprised of N-methyl pyrrole and N-methyl imidazole amino acids, by means of solid-phase synthesis on an oxime resin. These hairpin polyamides were designed to determine the DNA recognition profile of a side-by-side benzimidazole/imidazole pair for the designated six base pair recognition sequence. Equilibrium association constants of the polyamide-DNA complexes were determined at two of the six base pair positions of the recognition sequence by quantitative DNase I footprinting titrations on DNA fragments each containing matched and single base pair mismatched binding sites. The results indicate that the benzimidazole-heterocycle building blocks can replace pyrrole-pyrrole, pyrrole-imidazole, and pyrrole-hydroxypyrrole constructs while retaining relative site specifities and subnanomolar match site affinities. The benzimidazole-containing hairpin polyamides represent a novel class of DNA binding ligands featuring tunable target recognition sequences combined with the favorable properties of the benzimidazole type DNA minor groove binders.     

特异性识别DNA的吡咯-咪唑多聚酰胺的研究进展

吡咯-咪唑多聚酰胺为一类人工合成的主要由五元杂环化合物N-甲基吡咯(Py)、N-甲基咪唑(Im)和N-甲基3-羟基吡咯(Hp)芳香氨基酸组成的,经酰胺键连接的人工小分子配体.它们具有与天然DNA结合蛋白相媲美的DNA特异性识别和结合能力.近20年来,对此类化合物的研究取得了重要进展,确定了简单的氨基酸对识别碱基对的规则,研究了多种方式连接的吡咯-咪唑多聚酰胺与DNA小沟结合模式,合成了多种双功能吡咯-咪唑多聚酰胺,且吡咯-咪唑多聚酰胺能穿过细胞膜,具有在体内外调节基因表达的作用。     

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.     

Fmoc solid phase synthesis of polyamides containing pyrrole and imidazole amino acids

[structure: see text]. Polyamides containing N-methylimidazole (Im) and N-methylpyrrole (Py) amino acids are synthetic ligands that have an affinity and specificity for DNA comparable to those of many naturally occurring DNA binding proteins. A machine-assisted Fmoc solid phase synthesis of polyamides has been optimized to afford high stepwise coupling yields (>99%). Two monomer building blocks, Fmoc-Py acid and Fmoc-Im acid, were prepared in multigram scale. Cleavage by aminolysis followed by HPLC purification affords up to 200 mg quantities of polyamide with purities and yields greater than or equal to those reported using Boc chemistry. A broader set of reaction conditions will increase the number and complexity of minor groove binding polyamides which may be prepared and help ensure compatibility with many commercially available peptide synthesizers.     

Synthesis and properties of new halogen‐substituted polyamides from 5‐halo‐m‐phenylenediamines and both aliphatic and aromatic dicarboxylic acids

New halogen‐substituted aromatic–aliphatic and wholly aromatic polyamides with high inherent viscosities were synthesized by the direct polycondensation of 5‐halo‐m‐phenylenediamines, where the halogens were Cl, Br, and I, with both aliphatic and aromatic dicarboxylic acids in N‐methyl‐2‐pyrrolidone with a mixture of triphenyl phosphite and pyridine as a condensing agent. The solubility of the halogen‐substituted polyamides was much higher than that of the parent polyamides derived from m‐phenylenediamine. The glass‐transition temperatures of the substituted aromatic–aliphatic polyamides increased in the order Cl < Br < I, whereas the temperatures of 10% weight loss in air decreased in the reverse order. The limiting oxygen index values, as an indication of flammability, increased for the substituted aromatic–aliphatic polyamides in the order Cl < Br < I. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 38: 3911–3918, 2000     

Highly Sensitive Functionalized Conducting Copolypyrrole Film for DNA Sensing and Protein‐resistant

In order to exploit the applications of polypyrrole (PPy) derivatives in biosensors and bioelectronics, the different immobilization mechanisms of biomolecules onto differently functionalized conducting PPy films are investigated. Pyrrole and pyrrole derivatives with carboxyl and amino groups were copolymerized with ω‐(N‐pyrrolyl)‐octylthiol self‐assembled on Au surface by the method of the chemical polymerization to form a layer of the copolymer film, i.e., poly[pyrrole‐co‐(N‐pyrrolyl)‐caproic acid] (poly(Py‐co‐PyCA)) and poly[pyrrole‐co‐(N‐pyrrolyl)‐hexylamine] (poly(Py‐co‐PyHA)), in which the carboxyl groups in poly(Py‐co‐PyCA) were activated to the ester groups. Based on the structure characteristics, the immobilization/hybridization of DNA molecules on PPy, poly(Py‐co‐PyCA) and poly(Py‐co‐PyHA) were surveyed by cyclic voltammograms measurements. For differently functionalized copolymers, the immobilization mechanisms of DNA are various. Besides the electrochemical properties of the composite electrodes of PPy and its copolymers being detected before and after bovine serum albumin (BSA) adsorption, the kinetic process of protein binding was determined by surface plasmon resonance of spectroscopy. Since few BSA molecules could anchor onto the PPy and its copolymers surfaces, it suggests this kind of conducting polymers can be applied as the protein‐resistant material.     

Thermal isomerization of ruthenium hydride compounds containing asymmetric bidentate pyrrole‐imine ligands

A series of ruthenium hydride compounds containing substituted bidentate pyrrole‐imine ligands were synthesized and characterized. Reacting RuHCl(CO)(PPh₃)₃ with one equivalent of [C₄H₃NH(2‐CH=NR)] in ethanol in the presence of KOH gave compounds {RuH(CO)(PPh₃)₂[C₄H₃N(2‐CH=NR)]} where trans‐Py‐Ru‐H 1, R = CH₂CH₂C₆H₉; cis‐Py‐Ru‐H 2, R = Ph‐2‐Me; and cis‐Py‐Ru‐H 3, R = C₆H₁₁. Heating trans‐Py‐Ru‐H 1 in toluene at 70°C for 12 hr resulted a thermal conversion of the trans‐Py‐Ru‐H 1 into its cis form, {RuH(CO)(PPh₃)₂[C₄H₃N(2‐CH=NCH₂CH₂C₆H₉)]} (cis‐Py‐Ru‐H 1) in very high yield. The ¹H NMR spectra of trans‐Py‐Ru‐H 1, cis‐Py‐Ru‐H 2, cis‐Py‐Ru‐H 3, and cis‐Py‐Ru‐H 1 all show a typical triplet at ca. δ–11 for the hydride. The trans and cis form indicate the relative positions of pyrrole ring and hydride. The geometries of trans‐Py‐Ru‐H 1, cis‐Py‐Ru‐H 1, and cis‐Py‐Ru‐H 3 are relatively similar showing typical octahedral geometries with two PPh₃ fragments arranged in trans positions.     

Cooperative Hydrogen Bonding in Glyco–Oligoamides: DNA Minor Groove Binders in Aqueous Media

A strategy to create cooperative hydrogen‐bonding centers by using strong and directional intramolecular hydrogen‐bonding motifs that can survive in aqueous media is presented. In particular, glyco–oligoamides, a family of DNA minor groove binders, with cooperative and non‐cooperative hydrogen‐bonding donor centers in the carbohydrate residues have been designed, synthesized, and studied by means of NMR spectroscopy and molecular modeling methods. Indeed, two different sugar moieties, namely, β‐D ‐Man‐Py‐γ‐Py‐Ind ( 1 ; Ind=indole, Man=mannose, Py=pyrrole) and β‐D ‐Tal‐Py‐γ‐Py‐Ind ( 2 ; Tal=talose), were chosen according to our design. These sugar molecules should present one‐ or two‐directional intramolecular hydrogen bonds. The challenge has been to study the conformation of the glyco–oligoamides at low temperature in physiological media by detecting the exchangeable protons (amide NH and OH resonances) by means of NMR spectroscopic analysis. In addition, two more glyco–oligoamides with non‐cooperative hydrogen‐bonding centers, that is, β‐D ‐Glc‐Py‐γ‐Py‐Ind ( 3 ; Glc=glucose), β‐D ‐Gal‐Py‐γ‐Py‐Ind ( 4 ; Gal=galactose), and the model compounds β‐D ‐Man‐Py‐NHAc ( 5 ) and β‐D ‐Tal‐Py‐NHAc ( 6 ) were synthesized and studied for comparison. We have demonstrated the existence of directional intramolecular hydrogen bonds in 1 and 2 in aqueous media. The unexpected differences in terms of stabilization of the intramolecular hydrogen bonds in 1 and 2 relative to 5 and 6 promoted us to evaluate the influence of CH—π interactions on the establishment of intramolecular hydrogen bonds by using computational methods. Initial binding studies of 1 and 2 with calf‐thymus DNA and poly(dA‐dT)₂ by NMR spectroscopic analysis and molecular dynamics simulations were also carried out. Both new sugar–oligoamides are bound in the minor groove of DNA, thus keeping a stable hairpin structure, as in the free state, in which both intramolecular hydrogen‐bonding and CH—π interactions are present.     

Ethyl 2‐[N‐(tert‐butyl­oxy­carbonyl)‐l‐alanyl­amino]‐4‐methyl‐1,3‐thia­zole‐5‐carboxyl­ate reveals a trans orientation of the preceding amide N—H with respect to the thia­zole‐ring sulfur

The title mol­ecule, C₁₅H₂₃N₃O₅S, was prepared as a synthetic precursor to 4‐methyl­thia­zole‐based DNA minor groove binders which would bear chiral amino acids in the sequence. The crystallographic evidence presented herein shows that the aromatic amide NH group preceding the thia­zole ring points away from the direction of sulfur. The mol­ecule is biplanar, with the dihedral angle between the N‐terminus peptide moiety and the thia­zole‐containing plane being 49.7 (5)°, with a bend at the Cα carbon.     

Chemoselective synthesis of polyamides containing hydroxyl and amino substituents by direct polycondensation

A convenient method for the synthesis of polyamides containing hydroxyl and amino substituents on the aromatic rings of the backbones was developed. These polymers were prepared readily by the chemoselective polycondensation of dicarboxylic acids with diamines with hydroxyl and amino functional groups via the activating agent diphenyl(2,3‐dihydro‐2‐thioxo‐3‐bezoxazolyl)phosphonate. The model reactions were studied in detail to demonstrate the feasibility of chemoselective polycondensation. The direct polycondensation of 5‐hydroxy or 5‐aminoisophthalic acid with 4,4′‐diamino‐4″‐hydroxytriphenylmethane proceeded smoothly under mild conditions and produced the desired polyamides with inherent viscosities up to 0.73 dL · g⁻¹. The polymers obtained were characterized by IR, ¹H NMR, and ¹³C NMR spectroscopies. The polymers were readily soluble in aprotic polar solvents such as N‐methyl‐2‐pyrrolidinone, N,N‐dimethyl formamide, and dimethyl sulfoxide. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 38: 3875–3882, 2000     

Synthesis and characterization of N‐benzoylated wholly aromatic polyamides from 4,4′‐oxydianilinobis(benzimidoyl) chloride and aromatic dicarboxylic acids

A novel type of polyamides, N‐benzoylated wholly aromatic polyamides, were synthesized by low‐temperature solution polycondensation of a new aromatic bis(imidoyl) chloride, 4,4′‐oxydianilinobis(benzimidoyl) chloride, with aromatic dicarboxylic acids, 4,4′‐oxydibenzoic acid and isophthalic acid. Compared with the conventional all aromatic polyamides and also N‐phenylated wholly aromatic polyamides, these N‐benzoylated aramides exhibit better solubility in organic solvents, lower glass transition temperatures and thermal stability.     

Synthesis and spectral study of a novel distamycin conjugate

The ¹H and ¹³C NMR resonances for a novel distamycin conjugate, 3‐[1‐methyl‐4‐[1‐methyl‐4‐[1‐methyl‐4‐[N¹‐[5‐methyl‐2,4(1H,3H)pyrimidinedione]acetylamino]pyrrole‐2‐carboxamido]pyrrole‐2‐carboxamido]pyrrole‐2‐carboxamido]propionamidine hydrochloride ( 1 ), were assigned, using the concerted application of one‐ and two‐dimensional NMR techniques including nuclear Overhauser effect difference, DEPT, HMQC and HMBC experiments. Copyright © 2003 John Wiley & Sons, Ltd.