Antennapedia and other homeoproteins have the unique ability to efficiently translocate across biological membranes, a property that is mediated by the third helix of the homeodomain. To analyze the effects of sequence divergence in the homeodomain, we have compared the cellular uptake efficiencies and interaction properties in a membrane-mimicking environment of four peptides corresponding to the third helix sequence of Antennapedia, Engrailed-2, HoxA-13, and Knotted-1. NMR studies revealed that these peptides adopt helical conformations in SDS micelles. Their localization with respect to the micelle was investigated using Mn(2+) as a paramagnetic probe. Peptides are positioned parallel to the micelle surface, but subtle differences in the depth of immersion were observed. Using a recently developed method for quantification of CPP cellular uptake based on MALDI-TOF mass spectrometry, all of these peptides were found to translocate into cells but with large differences in their uptake efficiencies. The peptide with the highest uptake efficiency was found to be the least deeply inserted within the micelle, indicating that electrostatic surface interactions may be a major determinant for membrane translocation. A new cell-penetrating peptide derived from Knotted-1 homeodomain with improved uptake properties compared to penetratin is introduced here.

The dermaseptins S are closely related peptides with broad-spectrum antibacterial activity that are produced by the skin of the South American hylid frog, Phyllomedusa sauvagei. These peptides are polycationic (Lys-rich), alpha-helical, and amphipathic, with their polar/charged and apolar amino acids on opposing faces along the long axis of the helix cylinder. The amphipathic alpha-helical structure is believed to enable the peptides to interact with membrane bilayers, leading to permeation and disruption of the target cell. We have identified new members of the dermaseptin S family that do not resemble any of the naturally occurring antimicrobial peptides characterized to date. One of these peptides, designated dermaseptin S9, GLRSKIWLWVLLMIWQESNKFKKM, has a tripartite structure that includes a hydrophobic core sequence encompassing residues 6-15 (mean hydrophobicity, +4.40, determined by the Liu-Deber scale) flanked at both termini by cationic and polar residues. This structure is reminiscent of that of synthetic peptides originally designed as transmembrane mimetic models and that spontaneously become inserted into membranes [Liu, L., and Deber, C. M. (1998) Biopolymers 47, 41-62]. Dermaseptin S9 is a potent antibacterial, acting on gram-positive and gram-negative bacteria. The structure of dermaseptin S9 in aqueous solution and in TFE/water mixtures was analyzed by circular dichroism and two-dimensional NMR spectroscopy combined with molecular dynamics calculations. Dermaseptin S9 is aggregated in water, but a monomeric nonamphipathic alpha-helical conformation, mostly in residues 6-21, is stabilized by the addition of TFE. These results, combined with membrane permeabilization assays and surface plasmon resonance analysis of the peptide binding to zwitterionic and anionic phospholipid bilayers, demonstrate that spatial segregation of hydrophobic and hydrophilic/charged residues on opposing faces along the long axis of a helix is not essential for the antimicrobial activity of cationic alpha-helical peptides.

This paper deals with the cytotoxic chaetoglobosins obtained from culture on maize of the strains ATCC 26115 and MRC 2654 of the fungus Phomopsis leptostromiformis. Both strains produced, in more or less important amounts, the known chaetoglobosins M (1) and N (2). In addition, the new chaetoglobosin O (3), was isolated from the ATCC 26115 strain and characterized by HRMS and 2D NMR experiments. With the MRC 2654 strain, a fungal metabolite 4, more polar than the chaetoglobosins M and N, was extracted and purified. Spectroscopic and chromatographic data of metabolite 4 led to its identification to the bridged chaetoglobosin named compound S, which previously has been obtained by intramolecular Michael reaction on the chaetoglobosin M. Moreover, a methylation under Williamson conditions of the chaetoglobosin 4 resulted in the formation of the trimethylated chaetoglobosin 5.

Molecular mechanics calculations on conformers of Ac-HGly-NHMe, Ac-beta(2) -HAla-NHMe and Ac-beta(3) -HAla-NHMe indicate that low-energy conformations of the beta-amino acids backbone, corresponding to gauche rotamers around the Calpha-Cbeta bond, may overlap canonical backbone conformers observed for alpha-amino acids. Therefore, Substance P (SP) was used as a model peptide to analyse the structural and biological consequences of the substitution of Phe7 and Phe8 by (R)-beta(2) -HPhe and of Gly9 by HGly (R)-beta(2) -HAla or (S)-beta(3) -HAla. [(R)-beta(2) -HAla9]SP has pharmacological potency similar to that of SP while [HGly9]SP and [(S)-beta(3) -HAla9]SP show a 30- to 50-fold decrease in biological activities. The three analogues modified at position 9 are more resistant to degradation by angiotensin converting enzyme than SP and [Ala9]SP. NMR analysis of these SP analogues suggest that a beta-amino acid insertion in position 9 does not affect the overall backbone conformation. Altogether these data suggest that [HGly9]SP, [(S)-beta(3) -HAla9]SP and [(R)-beta(2) -HAla9]SP could adopt backbone conformations similar to that of SP, [Ala9]SP and [Pro9]SP. In contrast, incorporation of beta(2) -HPhe in position 7 and 8 of SP led to peptides that are almost devoid of biological activity. Thus, a beta-amino acid could replace an alpha-amino acid within the sequence of a bioactive peptide provided that the additional methylene group does not cause steric hindrance and does not confine orientations of the side chain to regions of space different from those permitted in the alpha-amino acid.

Molecular mechanics calculations on conformers of Ac‐HGly‐NHMe, Ac‐β2‐HAla‐NHMe and Ac‐β3‐HAla‐NHMe indicate that low‐energy conformations of the β‐amino acids backbone, corresponding to gauche rotamers around the Cα–Cβ bond, may overlap canonical backbone conformers observed for α‐amino acids. Therefore, Substance P (SP) was used as a model peptide to analyse the structural and biological consequences of the substitution of Phe7 and Phe8 by (R)‐β2‐HPhe and of Gly9 by HGly (R)‐β2‐HAla or (S)‐β3‐HAla. [(R)‐β2‐HAla9]SP has pharmacological potency similar to that of SP while [HGly9]SP and [(S)‐β3‐HAla9]SP show a 30‐ to 50‐fold decrease in biological activities. The three analogues modified at position 9 are more resistant to degradation by angiotensin converting enzyme than SP and [Ala9]SP. NMR analysis of these SP analogues suggest that a β‐amino acid insertion in position 9 does not affect the overall backbone conformation. Altogether these data suggest that [HGly9]SP, [(S)‐β3‐HAla9]SP and [(R)‐β2‐HAla9]SP could adopt backbone conformations similar to that of SP, [Ala9]SP and [Pro9]SP. In contrast, incorporation of β2‐HPhe in position 7 and 8 of SP led to peptides that are almost devoid of biological activity. Thus, a β‐amino acid could replace an α‐amino acid within the sequence of a bioactive peptide provided that the additional methylene group does not cause steric hindrance and does not confine orientations of the side chain to regions of space different from those permitted in the α‐amino acid.

Molecular mechanics calculations on conformers of Ac-HGly-NHMe, Ac-beta2-HAla-NHMe and Ac-beta3-HAla-NHMe indicate that low-energy conformations of the beta-amino acids backbone, corresponding to gauche rotamers around the Calpha-Cbeta bond, may overlap canonical backbone conformers observed for alpha-amino acids. Therefore, Substance P (SP) was used as a model peptide to analyse the structural and biological consequences of the substitution of Phe7 and Phe8 by (R)-beta2-HPhe and of Gly9 by HGly (R)-beta2-HAla or (S)-beta3-HAla. [(R)-beta2-HAla9]SP has pharmacological potency similar to that of SP while [HGly9]SP and [(S)-beta3-HAla9]SP show a 30- to 50-fold decrease in biological activities. The three analogues modified at position 9 are more resistant to degradation by angiotensin converting enzyme than SP and [Ala9]SP. NMR analysis of these SP analogues suggest that a beta-amino acid insertion in position 9 does not affect the overall backbone conformation. Altogether these data suggest that [HGly9]SP, [(S)-beta3-HAla9]SP and [(R)-beta2-HAla9]SP could adopt backbone conformations similar to that of SP, [Ala9]SP and [Pro9]SP. In contrast, incorporation of beta2-HPhe in position 7 and 8 of SP led to peptides that are almost devoid of biological activity. Thus, a beta-amino acid could replace an alpha-amino acid within the sequence of a bioactive peptide provided that the additional methylene group does not cause steric hindrance and does not confine orientations of the side chain to regions of space different from those permitted in the alpha-amino acid.

Residue Leu10 of substance P (SP) is critical for NK-1 receptor recognition and agonist activity. In order to probe the bioactive conformation of this residue, cis- and trans-3-substituted prolinoleucines were introduced in position 10 of SP. The substituted SP analogues were tested for their affinity to human NK-1 receptor specific binding sites (NK-1M and NK-1m) and their potency to stimulate adenylate cyclase and phospholipase C in CHO cells transfected with the human NK-1 receptor. [trans-3-prolinoleucine10]SP retained affinity and potency similar to SP whereas [cis-3-prolinoleucine10]SP shows dramatic loss of affinity and potency. To analyze the structural implications of these biological results, the conformational preferences of the SP analogues were analyzed by NMR spectroscopy and minimum-energy conformers of Ac-cis-3-prolinoleucine-NHMe, Ac-trans-3-prolinoleucine-NHMe and model dipeptides were generated by molecular mechanics calculations. From NMR and modeling studies it can be proposed that residue Leu10 of SP adopts a gauche(+) conformation around the chi1 angle and a trans conformation around the chi2 angle in the bioactive conformation. Together with previously published results, our data indicate that the C-terminal SP tripeptide should preferentially adopt an extended conformation or a PPII helical structure when bound to the receptor.

Blocking angiogenesis is an attractive strategy to inhibit tumor growth, invasion, and metastasis. We describe here the structure and the biological action of a new cyclic peptide derived from vascular endothelial growth factor ( VEGF). This 17-amino acid molecule designated cyclopeptidic vascular endothelial growth inhibitor (cyclo-VEGI, CBO-P11) encompasses residues 79 - 93 of VEGF which are involved in the interaction with VEGF receptor-2. In aqueous solution, cyclo-VEGI presents a propensity to adopt a helix conformation that was largely unexpected because only beta-sheet structures or random coil conformations have been observed for macrocyclic peptides. Cyclo-VEGI inhibits binding of iodinated VEGF(165) to endothelial cells, endothelial cells proliferation, migration, and signaling induced by VEGF(165). This peptide also exhibits anti-angiogenic activity in vivo on the differentiated chicken chorioallantoic membrane. Furthermore, cyclo-VEGI significantly blocks the growth of established intracranial glioma in nude and syngeneic mice and improves survival without side effects. Taken together, these results suggest that cyclo-VEGI is an attractive candidate for the development of novel angiogenesis inhibitor molecules useful for the treatment of cancer and other angiogenesis- related diseases.

In this paper, we describe the enantiospecific synthesis and the complete characterization of the two hexacoordinated ruthenium(II) monocations [Ru(bpy)(2)ppy](+) and [Ru(bpy)(2)quo](+) (bpy = 2,2'-bipyridine, ppy = phenylpyridine-H+, quo = 8-hydroxyquinolate) in their enantiomeric Delta and Delta forms. The corresponding enantiomeric excesses (ee's) are determined by H-1 NMR using pure Delta-Trisphat (tris(tetrachlorobenzenedialato)phosphate(V) anion) as a chiral H-1 NMR shift reagent. A complete H-1 and C-13 NMR study has been carried out on rac-[Ru(bPY)(2)ppy]PF6 and rac-[Ru(bpy)(2)quo]PF6. Additionally, the X-ray molecular structure of rac-[Ru(bpy)(2)quo]PF6 is reported; this latter species crystallizes in the monoclinic C2/c space group (a = 22.079 Angstrom, b = 16.874 Angstrom, c = 17.533 Angstrom, (alpha = 90degrees, beta = 109.08degrees, gamma = 90degrees).

Dermaseptins are antimicrobial peptides from frog skin that have high membrane-lytic activity against a broad spectrum of microorganisms. The structure of dermaseptin B2 in aqueous solution, in TFE/water mixtures, and in micellar and nonmicellar SDS was analyzed by CD, FTIR, fluorescence, and NMR spectroscopy combined with molecular dynamics calculations. Dermaseptin B2 is unstructured in water, but helical conformations, mostly in segment 3-18, are stabilized by addition of TFE. SDS titration showed that dermaseptin 132 assumes nonhelical structures at SDS concentrations far below the critical micellar concentration and helical structures at micellar concentrations. Dermaseptin B2 bound to SDS micelles (0.4 mM peptide, 80 mM SDS) adopts a well-defined amphipathic helix between residues 11-31 connected to a more flexible helical segment spanning residues 1-8 by a flexible hinge region around Va19 and Gly10. Experiments using paramagnetic probes showed that dermaseptin B2 lies near the surface of SDS micelles and that residue Trp3 is buried in the SDS micelle, but close to the surface. A slow exchange equilibrium occurs at higher peptide/SDS ratios (2 mM peptide, 80 mM SDS) between forms having distinct sets of resonances in the N-terminal 1-11 segment. This equilibrium could reflect different oligomeric states of dermaseptin B2 interacting with SDS micelles. Structure-activity studies on dermaseptin B2 analogues showed that the N-terminal 1-11 segment is an absolute requirement for antibacterial activity, while the C-terminal 10-33 region is also important for full antibiotic activity.

The photoadduct formed by photolysis of the [Bapa0, (pBzl)Phe5, Met(O2)11]SP/NK-1 complex localised within the T173MP175 domain of the NK-1 receptor cannot be cleaved by CNBr on the C-side of methionine; an unusual rearrangement of the intermediate sulfonium instead occurred. The reactivity of 1,1-diphenyl-2-methylsulfinyl ethanol 10 towards CNBr treatment and the stability of the 1,1-diphenyl oxirane 14 were analysed by NMR and mass spectrometry. 1,1-Diphenyl ethylene 13 can be formed from epoxide 14 even in slightly acidic conditions and during positive DCI/NH3 mass spectrometry analysis. Altogether, these results suggest that if a covalent linkage between the [Bapa0, (pBzl)Phe5, Met(O2)11] and the NK-1 receptor occurred on the CγH2 of methionine-174, CNBr treatment will lead to an epoxide/ketone and an ethylenic compound.

Substance P (SP) interacts with the neurokinin-1 (NR-1) G-protein-coupled receptor, which has been cloned in several species. In the present study, the domains of the NK-1 receptor involved in the binding of SP and SP-(7-11) C-terminal fragment have been analyzed using two peptide analogs containing the photoreactive amino acid para-benzoylphenylalanine ((p-Bz)Phe) in position 8 of their sequence. This study was carried out with [BAPA-Lys(6),(p-Bz)Phe(8),Pro(9),Met(O-2)(11)]SP-(7-11) and [BAPA(0),(p-Bz)Phe(8)]SP on both rat and human NK-1 receptors expressed in CHO cells. Combined trypsin and endo-GluC enzymatic complete digestions and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis led to the identification of the same domain of covalent interaction, (TMPSR177)-T-173, for the two photoactivatable peptides. Further digestion of this fragment with carboxypeptidase Y led to the identification of (TMP175)-T-173 in the second extracellular loop (E2) of the NK-1 receptor as the site of covalent attachment. Models of the conformation of this E2 loop in the human NLK-1 receptor were generated using two different strategies, one based on homology with bovine rhodopsin and the other based on the solution conformation preferences of a synthetic peptide corresponding to the E2 loop.

Two binding sites NK-1M (major, more abundant) and NK-1m (minor) are associated with the neurokinin-1 receptor. For the first time with a bioactive peptide, the Calpha methylation constraint, shown to be a helix stabiliser in model peptides, was systematically used to probe the molecular requirements of NK-1M and NK-1m binding sites and the previously postulated bioactive helical conformation of substance P (SP). Seven Calpha methylated analogues of the undecapeptide SP (from position 5-11) have been assayed for their affinities and their potencies to stimulate second messenger production. The consequences of Calpha methylation on the structure of SP have been analysed by circular dichroism and nuclear magnetic resonance combined with restrained molecular dynamics. The decreased potencies of six out of these seven Calpha methylated SP analogues do not allow the identification of any clear-cut differences in the structural requirements between the two binding sites. Strikingly, the most active analogue, [alphaMeMet5]SP, leads to variable subnanomolar affinity and potency when interacting with the NK-1m binding site. The conformational analyses show that the structural consequences associated with Calpha methylation of SP are sequence dependent. Moreover, a single Calpha methylation is not sufficient by itself to drastically stabilize a helical structure even pre-existing in solution, except when Gly9 is substituted by an alpha-aminoisobutyric acid. Furthermore, Calpha methylation of residues 5 and 6 of SP in the middle of the postulated helix does not stabilize, but decreases (to different extents) the stability of the helical structure previously observed in the 4-8 domain of other potent SP analogues.

Partial resolution of the rac-[Ru(bpy)(2)(ppy)](+) monocation into [Delta -Ru(bpy)(2)(ppy)]PF6 (1 Delta) (ee = 44%) and [Lambda -Ru(bpy)(2)(pPY)]PF6 (I Lambda) (ee = 50%) enantiomers was achieved by an unprecedented resolving process which involves the diastereoselective preparation of optically active networks of the type ([Lambda -Ru(bpy)(2)(ppy)][Mn-Lambda -Co(ox)(3)])(n) (2 Lambda) and ([Delta -Ru(bpy)(2)(ppy)][Mn-Delta -Co(ox)(3)])(n) (2 Delta). In these optically active inorganic polymers, the counter-monocation [Ru(bpy)(2)(ppy)](+) of appropriate configuration occupies the cavities while the excess [Ru(bpy)(2)(ppy)](+), of opposite configuration, remains in solution and is thus recovered. Anion metathesis of the optically active [Ru(bpy)(2)(ppy)]PFB (1 Delta) (ee = 44%) and (1 Lambda) (ee = 50%) with [1-cinchonidinium][Delta -trisphat] [trisphat = tris (tetrachlorobenzenediolato)-phosphate (V)] provides the related diastereomers [Delta -Ru(bpu)(2)(ppy)][Delta -trisphat] (3 Delta) (de= 44%) and [Lambda -Ru(bpy)(2)(ppu) ][Delta -trisphat] (3 Lambda) (de = 50%) in 60% yield. Their diastereomeric excesses were assessed by H-1 NMR analysis. All products were fully characterized and the absolute configuration was determined by circular dichroism techniques in solution or in the solid state. Additionally the X-say structure of [Ru(bpy)(2)(PPY)]PF6 (1) Was determined.

Two binding sites NK‐1M (major, more abundant) and NK‐1m (minor) are associated with the neurokinin‐1 receptor. For the first time with a bioactive peptide, the Cα methylation constraint, shown to be a helix stabiliser in model peptides, was systematically used to probe the molecular requirements of NK‐1M and NK‐1m binding sites and the previously postulated bioactive helical conformation of substance P (SP). Seven Cα methylated analogues of the undecapeptide SP (from position 5–11) have been assayed for their affinities and their potencies to stimulate second messenger production. The consequences of Cα methylation on the structure of SP have been analysed by circular dichroism and nuclear magnetic resonance combined with restrained molecular dynamics. The decreased potencies of six out of these seven Cα methylated SP analogues do not allow the identification of any clear‐cut differences in the structural requirements between the two binding sites. Strikingly, the most active analogue, [αMeMet5]SP, leads to variable subnanomolar affinity and potency when interacting with the NK‐1m binding site. The conformational analyses show that the structural consequences associated with Cα methylation of SP are sequence dependent. Moreover, a single Cα methylation is not sufficient by itself to drastically stabilize a helical structure even pre‐existing in solution, except when Gly9 is substituted by an α‐aminoisobutyric acid. Furthermore, Cα methylation of residues 5 and 6 of SP in the middle of the postulated helix does not stabilize, but decreases (to different extents) the stability of the helical structure previously observed in the 4–8 domain of other potent SP analogues.

The ability of BPAC(8), a member of the bis-pyridinium aldehyde (BPA) family with a linear octamethylene chain linking the two charged pyridinium moieties, to covalently bond with a guanine residue at a 5'-CpG-3' site is studied. Three oligomers including a central CpG step with different conformations are studied. By H-1 NMR spectroscopy and gel analysis it is established that the covalent reaction occurs for the decamer CRE, d(ATGACGTCAT); and to a lesser extent for the methylated dodecamer, d(GAAAAmeCGTTTTC), while there is no reaction for the sequence d(GAAAACGTTTTC). The ease of reaction with BPA is correlated with both the geometry of CpG and the malleability of the oligomer. When the CpG structure is stiffened by a rigid nucleotide environment, such as A-T tracts, the reaction with BPA is inhibited.

The original use of shifted laminar pulses to excite selectively several hyperfine‐shifted signals spread over a spectral window as large as 100 ppm is presented. As an application of such a multi‐site selective excitation we developed a multiNOE difference experiment which permits one to detect dipole–dipole couplings arising from various well separated hyperfine‐shifted signals within a single one‐dimensional experiment. Although typical information on a 1D NOE experiment is lost when several signals are simultaneously irradiated, the experiment can be useful for collecting selective relaxation rates from NOE build‐up curves. This will provide a significant gain in experimental time with respect to a series of individual NOE build‐up experiments. Such experiments can also be used as a building block for multi‐dimensional experiments such as multiNOE‐NOESY, which maintains the selective information of 1D NOE and can, therefore, be used for assignment purposes. This constitutes the first application of a chemical shift‐based filter as a complement to relaxation‐based filters, generally used when dealing with paramagnetic systems. The application of multiple selective pulses in paramagnetic systems is expected to be particularly useful when dealing with double and triple resonance experiments on isotopically enriched samples. Coherence transfer pathways involving only resonances affected by the hyperfine interactions could be selected by this approach. Copyright © 2000 John Wiley & Sons, Ltd.

The hydration properties of the non-palindromic duplex d(CTACTGCTTTAG). d(CTAAAGCAGTAG) were investigated by NMR spectroscopy. The oligonucleotide possesses a heterogeneous B-DNA structure. The H2(n)-H1'(m+1) distances reflect a minor groove narrowing within the TTT/AAA segment (approximately 3.9A) and a sudden widening at the T10:A15 base-pair (approximately 5.3A), the standard B-DNA distance being approximately 5A. The facing T10pA11 and T14pA15 steps at the end of the TTTA/AAAT segment have completely different behaviors. Only A15 ending the AAA run displays NMR features comparable to those shown by adenines of TpA steps occupying the central position of TnAn (n> or =2) segments. These involve particular chemical shifts and line broadening of the H2 and H8 protons. Positive NOESY cross-peaks were measured between the water protons and the H2 protons of A15, A16 and A17 reflecting the occurrence of hydration water molecules with residence times longer than 500 picoseconds along the minor groove of the TTT/AAA segment. In contrast no water molecules with long residence times were observed neither for A3, A20 and A23 nor for A11 ending the 5'TTTA run. We confirm thus that the binding of water molecules with long residence time to adenine residues correlates with the minor groove narrowing. In contrast, the widening of the minor groove at the A11:T14 base-pair ending the TTTA/TAAA segment, likely associated to a high negative propeller twist value at this base-pair, prevents the binding of a water molecule with long residence time to A11 but not to A15 of the preceding T10:A15 base-pair. Thus, in our non-palindromic oligonucleotide the water molecules bind differently to A11 and A15 although both adenines are part of a TpA step. The slower motions occurring at A15 compared to A11 are also well explained by the present results.

The hydration properties of the non-palindromic duplex d(CTACTGCTTTAG). d(CTAAAGCAGTAG) were investigated by NMR spectroscopy. The oligonucleotide possesses a heterogeneous B-DNA structure. The H2(n)-H1'(m+1) distances reflect a minor groove narrowing within the TTT/AAA segment (approximately 3.9A) and a sudden widening at the T10:A15 base-pair (approximately 5.3A), the standard B-DNA distance being approximately 5A. The facing T10pA11 and T14pA15 steps at the end of the TTTA/AAAT segment have completely different behaviors. Only A15 ending the AAA run displays NMR features comparable to those shown by adenines of TpA steps occupying the central position of TnAn (n> or =2) segments. These involve particular chemical shifts and line broadening of the H2 and H8 protons. Positive NOESY cross-peaks were measured between the water protons and the H2 protons of A15, A16 and A17 reflecting the occurrence of hydration water molecules with residence times longer than 500 picoseconds along the minor groove of the TTT/AAA segment. In contrast no water molecules with long residence times were observed neither for A3, A20 and A23 nor for A11 ending the 5'TTTA run. We confirm thus that the binding of water molecules with long residence time to adenine residues correlates with the minor groove narrowing. In contrast, the widening of the minor groove at the A11:T14 base-pair ending the TTTA/TAAA segment, likely associated to a high negative propeller twist value at this base-pair, prevents the binding of a water molecule with long residence time to A11 but not to A15 of the preceding T10:A15 base-pair. Thus, in our non-palindromic oligonucleotide the water molecules bind differently to A11 and A15 although both adenines are part of a TpA step. The slower motions occurring at A15 compared to A11 are also well explained by the present results.

Gel analysis, MALDI-TOF mass spectrometry and NMR spectroscopy show that a tethered pyridinium aldehyde, from a new class of DNA-interacting agents, binds to the self-complementary [d(ATGACGTCAT)]2 decamer. The reaction proceeds via a two-step pathway: fast non-covalent outside association (complex formation) is followed by slow covalent addition (adduct formation) upon temperature increase. Both interactions occur mainly at the central CpG in the oligonucleotide, the latter leading finally to the reversible formation of an aminal through nucleophilic attack of the exocyclic guanine amino group in the minor groove of the helix.