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.     

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.     

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 a terminal formamido group on the sequence recognition of DNA by polyamides

Pyrrole (Py)-imidazole (Im)-containing polyamides bind in the minor groove of DNA and can recognize specific sequences through a stacked antiparallel dimer. It has been proposed that there are two different low energy ways to form the stacked dimer and that these are sensitive to the presence of a terminal formamido group: (i) a fully overlapped stacking mode in which the N-terminal heterocycles of the dimer stack on the amide groups between the two heterocycles at the C-terminal and (ii) a staggered stacking mode in which the N-terminal heterocycles are shifted by approximately one unit in the C-terminal direction (Structure 1997, 5, 1033-1046). Two different DNA sequences will be recognized by the same polyamide stacked in these two different modes. Despite the importance of polyamides as sequence specific DNA recognition agents, these stacking possibilities have not been systematically explored. As part of a program to develop agents that can recognize mismatched base pairs in DNA, a set of four polyamide trimers with and without terminal formamido groups was synthesized, and their interactions with predicted DNA recognition sequences in the two different stacking modes were evaluated. Experimental difficulties in monitoring DNA complex formation with polyamides were overcome by using surface plasmon resonance (SPR) detection of the binding to immobilized DNA hairpin duplexes. Both equilibrium and kinetic results from SPR show that a terminal formamido group has a pronounced effect on the affinity, sequence specificity, and rates of DNA-dimer complex formation. The formamido polyamides bind preferentially in the staggered stacking mode, while the unsubstituted analogues bind in the overlapped mode. Affinities for cognate DNA sequences increase by a factor of around 100 when a terminal formamido is added to a polyamide, and the preferred sequences recognized are also different. Both the association and the dissociation rates are slower for the formamido derivatives, but the effect is larger for the dissociation kinetics. The formamido group thus strongly affects the interaction of polyamides with DNA and changes the preferred DNA sequences that are recognized by a specific polyamide stacked dimer.     

Toward an Understanding of the Chemical Etiology for DNA Minor‐Groove Recognition by Polyamides

Crescent‐shaped polyamides composed of aromatic amino acids, i.e., 1‐methyl‐1H‐imidazole Im , 1‐methyl‐1H‐pyrrole Py , and 3‐hydroxy‐1H‐pyrrole Hp , bind in the minor groove of DNA as 2 : 1 and 1 : 1 ligand/DNA complexes. DNA‐Sequence specificity can be attributed to shape‐selective recognition and the unique corners or pairs of corners presented by each heterocycle(s) to the edges of the base pairs on the floor of the minor groove. Here we examine the relationship between heterocycle structure and DNA‐sequence specificity for a family of five‐membered aromatic amino acids. By means of quantitative DNase‐I footprinting, the recognition behavior of polyamides containing eight different aromatic amino acids, i.e., 1‐methyl‐1H‐pyrazole Pz , 1H‐pyrrole Nh , 5‐methylthiazole Nt , 4‐methylthiazole Th , 3‐methylthiophene Tn , thiophene Tp , 3‐hydroxythiophene Ht , and furan Fr , were compared with the polyamides containing the parent‐ring amino acids Py, Im , and Hp for their ability to discriminate between the four Watson? Crick base pairs in the DNA minor groove. Analysis of the data and molecular modeling showed that the geometry inherent to each heterocycle plays a significant role in the ability of polyamides to differentiate between DNA sequences. Binding appears sensitive to changes in curvature complementarity between the polyamide and DNA. The Tn / Py pair affords a modest 3‐fold discrimination of T?A vs. A?T and suggests that an S‐atom in the thiophene ring prefers to lie opposite T not A.     

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.     

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.     

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.     

Orthogonally Positioned Diamino Pyrrole- and Imidazole-Containing Polyamides: Synthesis of 1-(3-Substituted-propyl)-4-nitropyrrole-2-carboxylic Acid and 1-(3-Chloropropyl)-4-nitroimidazole-2-carboxylic Acid

Pyrrole- and imidazole-containing polyamides can be tailored to recognize the DNA 6–8 base pair sequence. We found that adding a second amino group via the N1-position of pyrrole or imidazole in polyamides could enhance their DNA binding affinity and water solubility while retaining sequence specificity. Synthesis of the key 1-substituted-4-nitropyrrole (and imidazole)-2-carboxylic acid building blocks are described.

[Supplementary materials are available for this article. Go to the publisher's online edition of Synthetic Communications® for the following free supplemental resource(s): Full experimental and spectral details.]     

Base pair recognition of the stereochemically alpha-substituted gamma-turn of pyrrole/imidazole hairpin polyamides

Recognition of the sequences 5'-NGCACA-3' (N = T, A, C, G) by pyrrole/imidazole polyamides with (R/S)-alpha-hydroxyl/alpha-amino-substituted gamma-aminobutyric acid as a gamma-turn was investigated. Four novel polyamides, 2, 3, 4, and 5, including (R)-alpha-hydroxyl-gamma-aminobutyric acid (gammaRO), (S)-alpha-hydroxyl-gamma-aminobutyric acid (gammaSO), (R)-alpha,gamma-diaminobutyric acid (gammaRN), and (S)-alpha,gamma-diaminobutyric acid (gammaSN) residues, respectively, were synthesized, and their binding affinity to T.A, A.T, G.C, and C.G base pairs at turn position was studied by the surface plasmon resonance (SPR) technique. SPR data revealed that polyamide 3, AcImbetaImPy-gammaSO-ImPybetaPy-beta-Dp, with a gammaSO turn, possesses a marked binding preference for T.A over A.T with a 25-fold increase in specificity, despite low binding affinity relative to 2, with a gammaRO turn. Similarly, AcImbetaImPy-gammaSN-ImPybetaPy-beta-Dp (5), with a gammaSN-turn, gives rise to a 8.7-fold increase in specificity for T.A over A.T. Computer-assisted molecular modeling suggests that 3 binds more deeply in the minor groove of the T.A base pair relative to the A.T base pair, allowing hydrogen bonding to O2 of the thymine at the turn position, which explains the SPR results. These results suggest that gammaSO and gammaSN may function as T-recognition units at the turn position, as well as a gamma-turn in the discrimination of polyamides.     

Surface-enhanced Raman study of the interactions between tripodal cationic polyamines and polynucleotides

Raman and surface-enhanced Raman spectra of new DNA/RNA-binding compounds consisting of three imidazole (Im) and three pyridine (Py) rings connected by tripodal polyaminomethylene linkages were obtained by the near-infrared excitation at 1064 nm. Study of interactions of Im and Py polyamines with single-stranded RNA polynucleotides (poly?A, poly?G, poly?C, poly?U), double-stranded DNA polynucleotides (poly?dAdT-poly?dAdT, poly?dGdC-poly?dGdC) and calf thymus DNA (ct-DNA) by surface-enhanced Raman spectroscopy (SERS) reveals unambiguous enhancement of the Raman scattering from the small molecules as well as appearance of new bands in spectra associated mainly with nucleobases. The SERS experiments point toward comparable interactions of Im and Py polyamines with single-stranded purine and pyrimidine polynucleotides. Furthermore, SERS experiments with double stranded polynucleotides reveal the base-pair dependent selectivity of Im and Py, whereby interactions within both, major and minor groove are indicated for poly?dAdT-poly?dAdT, at variance to preferred binding of Im and Py to only major groove of poly?dGdC-poly?dGdC. SERS spectra of Im and Py with ct-DNA imply that protonated amino groups of these compounds preferentially interact with N7 atoms (adenine, guanine) while nitrogen in aromatic rings of polyamines might be attracted to C6-NH(2) (adenine), all sites being located at the major groove of the DNA helix. Wavenumber downshift of the imidazole (Im) and pyridine (Py) ring vibrations supports aromatic stacking interactions of imidazole and pyridine aromatic moieties with DNA base-pairs.     

Analysis of noncovalent complexes between human telomeric DNA and polyamides containing N-methylpyrrole and N-methylimidazole by using electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) was used to investigate noncovalent complexes formed between four novel polyamides containing N-methylpyrrole (Py) and N-methylimidazole (Im), and human telomeric DNA. Of the four polyamides investigated, PyPyPygammaImImImbetaDp (3) had the highest binding affinity towards the duplex d(TTAGGGTTAGGG/CCCTAACCCTAA) (D1). Results of competition analysis showed that the polyamides had binding affinities with D1 in the order PyPyPygammaImImImbetaDp (3)>PyPyPyPygammaPyImImPybetaDp (4)>PyPyPybetaImImImbetaDp (2)>ImImImbetaDp (1). MS/MS spectra confirmed that binding between D1 and the hairpin polyamides is more stable than that with the three-ring polyamides. By contrast, in the case of single-stranded d(TTAGGGTTAGGG)(D2), the binding order changes to ImImImbetaDp (1)>PyPyPygammaImImImbetaDp (3)>PyPyPybetaImImImbetaDp (2).     

Discrimination of hairpin polyamides with an alpha-substituted-gamma-aminobutyric acid as a 5'-TG-3' reader in DNA minor groove

Pyrrole-imidazole (Py-Im) polyamides containing stereospecifically alpha-amino- or alpha-hydroxyl-substituted gamma-aminobutyric acid as a 5'-TG-3' recognition element were synthesized by machine-assisted Fmoc solid-phase synthesis. Their binding properties to predetermined DNA sequences containing a core binding site of 5'-TGCNCA-3'/3'-ACGN'GT-5' (N.N' = A.T, T.A, G.C, and C.G) were then systematically studied by surface plasmon resonance (SPR). SPR results revealed that the pairing of stereospecifically alpha-amino-/alpha-hydroxyl-substituted gamma-aminobutyric acids, (R or S)-alpha,gamma-diaminobutyric acid (gammaRN or gammaSN) and (R or S)-alpha-hydroxyl-gamma-aminobutyric acid (gammaRO or gammaSO), side-by-side with beta-alanine (beta) in such polyamides significantly influenced the DNA binding affinity and recognition specificity of hairpin polyamides in the DNA minor groove compared with beta/beta, beta/gamma, and gamma/beta pairings. More importantly, the polyamide Ac-Im-gammaSO-ImPy-gamma-ImPybetaPy-beta-Dp (beta/gammaSO) favorably binds to a hairpin DNA containing a core binding site of 5'-TGCNCA-3'/3'-ACGN'GT-5' (N.N' = A.T) with dissociation equilibrium constant (K(D)) of 1.9 x 10(-)(7) M over N.N' = T.A with K(D) = 3.7 x 10(-)(6) M, with a 19-fold specificity. By contrast, Ac-Im-gammaSN-ImPy-gamma-ImPybetaPy-beta-Dp (beta/gammaSN) binds to the above sequence with N.N' = A.T with K(D) = 8.7 x 10(-)(7) M over N.N' = T.A with K(D) = 8.4 x 10(-)(6) M, with a 9.6-fold specificity. The results also show that the stereochemistry of the alpha-substituent, as well as the alpha-substituent itself may greatly alter binding affinity and recognition selectivity of hairpin polyamides to different DNA sequences. Further, we carried out molecular modeling studies on the binding by an energy minimization method, suggesting that alpha-hydroxyl is very close to N3 of the 3'-terminal G to induce the formation of hydrogen bonding between hydroxyl and N3 in the recognition event of the polyamide Ac-Im-gammaSO-ImPy-gamma-ImPybetaPy-beta-Dp (beta/gammaSO) to 5'-TGCNCA-3'/3'-ACGN'GT-5' (N.N' = A.T). Therefore, SPR assays and molecular modeling studies collectively suggest that the (S)-alpha-hydroxyl-gamma-aminobutyric acid (gammaSO) may act as a 5'-TG-3' recognition unit.     

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.     

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.     

Parallel synthesis of H-pin polyamides by alkene metathesis on solid phase

A small library of H-pin polyamides with variable aliphatic bridge lengths (CH(2))(n)(), where n = 4-8, connecting a central Py/Py pair was prepared via parallel synthesis with Ru-catalyzed alkene metathesis on solid phase as a complexity-generating cross-linking reaction. DNA binding affinities and sequence specificities were analyzed for each member of the library to determine the optimum linker length. An H-pin polyamide with a six-methylene bridge was found to have the highest affinity to its match site with high selectivity over a 1-bp mismatch site. The relationship between the number of methylenes in the linker (CH(2))(n)() and affinity is n = 6 > 4 > 7 > 5 > 8. These results indicate that 6 followed by 4 methylene-bridged polyamides represent the optimum spacer length for the H-pin motif in the DNA minor groove. Importantly, the H-pin is competitive with hairpin polyamides with respect to affinity and specificity. The metathesis-based convergent synthetic route to H-pin polyamides expands the scope of readily available DNA recognition motifs for small molecule-based gene regulation studies.     

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.     

Sequence‐Specific DNA Recognition by Cyclic Pyrrole–Imidazole Cysteine‐Derived Polyamide Dimers

Pyrrole–imidazole (PI) polyamides bind to the minor groove of the DNA duplex in a sequence‐specific manner and thus have the potential to regulate gene expression. To date, various types of PI polyamides have been designed as sequence‐specific DNA binding ligands. One of these, cysteine cyclic PI polyamides containing two β‐alanine molecules, were designed to recognize a 7 bp DNA sequence with high binding affinity. In this study, an efficient cyclization reaction between a cysteine and a chloroacetyl residue was used for dimerization in the synthesis of a unit that recognizes symmetrical DNA sequences. To evaluate specific DNA binding properties, dimeric PI polyamide binding was measured by using a surface plasmon resonance (SPR) method. Extending this molecular design, we synthesized a large dimeric PI polyamide that can recognize a 14 bp region in duplex DNA.     

Supramolecular dioxygen receptors composed of an anionic water-soluble porphinatoiron(II) and cyclodextrin dimers

Three types of per-O-methylated β-cyclodextrin dimers, Im2CD, Im3NHCD and Py3NHCD, were prepared as globin models. Im2CD was synthesized by the condensation reaction of mono(2(A)-amino)-per-O-methylated β-cyclodextrin with 3-(1H-imidazol-1-yl)pentanedioic acid. Im3NHCD and Py3NHCD were obtained through the S(N)2 reactions of mono(2(A),3(A)-epoxy)-per-O-methylated β-cyclodextrin with 3-(1H-imidazol-1-yl)pentane-1,5-diamine and 3,5-bis(aminomethyl)pyridine, respectively. These cyclodextrin dimers formed 1:1 supramolecular inclusion complexes of tetrakis(4-sulfonatophenyl)porphinatoiron(II) (Fe(II)TPPS) in aqueous solution. The supramolecular complexes bound dioxygen (O(2)), with the O(2) affinity of the Fe(II)TPPS/Im3NHCD complex (P(1/2)(O2) = 1.5 ± 0.1 Torr) being much higher than those of the Fe(II)TPPS/Im2CD (36 ± 2 Torr) and Fe(II)TPPS/Py3NHCD complexes (70 ± 5 Torr). On the basis of the results of the present study and previous results, it is concluded that the imidazole axial ligand at the linker attached at the 3- and 3'-positions of the cyclodextrin units causes higher O(2) affinity as compared with the imidazole ligand at the 2- and 2'-positions and the pyridine ligand at the 2,2'- or 3,3'-positions. The electron donating ability and orientation of the axial ligand may control the O(2) affinity of a supramolecular receptor.