Christiane Garbay-Jaureguiberry

Tight binding of the antitumor drug ditercalinium to quadruplex DNA

The structural selectivity of the DNA‐binding antitumor drug ditercalinium was investigated by competition dialysis with a series of nineteen different DNA substrates. The 7H‐pyridocarbazole dimer was found to bind to double‐stranded DNA with a preference for GC‐rich species but can in addition form stable complexes with triplex and quadruplex structures. The preferential interaction of the drug with four‐stranded DNA structures was independently confirmed by electrospray mass spectrometry and a detailed analysis of the binding reaction was performed by surface plasmon resonance (SPR) spectroscopy. The BIAcore SPR study showed that the kinetic parameters for the interaction of ditercalinium with the human telomeric quadruplex sequence are comparable to those measured with a duplex sequence. Slow association and dissociation were observed with both the quadruplex and duplex structures. The newly discovered preferential binding of ditercalinium to the antiparallel quadruplex sequence d(AG3[T2AG3]3) provides new perspectives for the design of drugs that can bind to human telomeres.

Improved synthesis of [p-phosphono and p-sulfo]methylphenylalanine. Resolution of [p-phosphono-, p-sulfo-, p-car☐y-and p-N-hydroxycar☐amido-]methylphenylalanine.

Abstract To analyse the role of post-translational phosphorylation and sulfation of tyrosine in peptides and proteins, we recently synthesized the new amino acids, p(CH 2 PO 3 H 2 )Phe and p(CH 2 SO 3 H)Phe (D,L mixtures) as chemically and enzymatically stable analogs of (O-phospho) and (O-sulfo)-tyrosine. Here, we report improved syntheses of these amino acids and of their N-Boc protected derivatives for use in solid-phase peptide synthesis. A protocol for their enzymatic resolution by use of subtilisin Carlsberg esterase is developed and extended to the resolution of the modified amino acids p(CH 2 CO 2 H)-D,L-Phe and p(CH 2 CONHOH)-D,L-Phe. The optical purity of the resolved amino acids was tested by chiral phase HPLC. Physical constants of these amino acids and their N-Boc derivatized forms including their characterization under PTC form (phenylthiocarbamyl) are given.

Synthesis of constrained 4-(phosphonomethyl)phenylalanine derivatives as hydrolytically stable analogs of O-phosphotyrosine

Abstract In order to elucidate the role of protein tyrosine phosphorylation involved in various intracellular signaling pathways, peptides containing O -phosphotyrosine have been developed. However, in order to improve the stability of the phosphorylated amino acid, we have designed some years ago a hydrolytically stable analogue, the 4-(phosphonomethyl)phenylalanine (Pmp). Introduced in peptide sequences, this residue, which is resistant to phosphatase action, was shown also able to inhibit substrate recognition by protein targets. With the aim to design peptidomimetics endowed with improved affinity and selectivity, we report in this study the synthesis of five new sterically hindered amino acids derived from Pmp. These modifications include α-methyl, β-methyl, β,β-dimethyl substitutions, α,β-cyclization of Pmp and methyl substitution on the phosphomomethyl group of Pmp.

ENANTIOSELECTIVE SYNTHESIS OF N-FMOC PROTECTED DI-TERT-BUTYL 4-PHOSPHONOMETHYL-L-PHENYLALANINE: A HYDROLYTICALLY STABLE ANALOGUE OF O-PHOSPHOTYROSINE

Abstract Fmoc-L-Pmp(tBu) 2 -OH was prepared with high enantiomeric purity by an asymmetric synthetic pathway, using a camphor sultam as chiral auxiliary.

15N spin-lattice relaxation study of linear peptides: Preliminary results on Leu-enkephalin and Tyr-Gly-Gly-Phe

Abstract The ability of 15 N relaxation measurements in conformational analysis of linear peptides was studied using Leu-enkephalin: Tyr-★Gly-★Gly-★Phe-★Leu and and related tetrapeptide Tyr-★Gly-★Gly-★Phe 95 % 15 N enriched. 15 N spin-lattice relaxation times measured at different temperatures in Me 2 SO solution indicate the presence of highly preferential folded structures in both peptides. A marked dependence of T 1 upon the motional effects (segmental rather than anisotropic overall) was observed, while hydrogen bonding affects weakly the relaxation times. From a comparison of 15 N relaxation parameters it appears that the tetrapeptide exhibits a more rigid structure than Leu-enkephalin, in accordance with previous 1 H NMR studies. This paper provides evidence for the usefulness of 15 N T 1 as a mobility probe (independent from 13 C) in the investigation of the conformational dynamics of peptides.

Increase of neutral endopeptidase-24.11 with cellular density and enzyme modulation with an inhibitor on human Reh6 cell line.

Neutral endopeptidase (EC 3.4.24.11, NEP) is an ectoenzyme, identified as the common acute lymphoblastic leukemia antigen (CALLA, CD10). This enzyme is involved in the inactivation of regulatory peptides such as enkephalins and atrial natriuretic peptide and its expression on the cell surface is therefore essential. NEP levels have been measured under different conditions on leukemic cell lines. NEP activity per cell was found to increase during the cell growth of Reh6 and CEM cells, a cell-cell contact mechanism being suggested by experiments using Transwell cell chambers. The same process was not observed with ICIG-7 fibroblasts. The numbers of enzymatic sites was also found to be selectively modulated by treatment with 0.1 microM N-[3-(R,S)-[(hydroxyamino)carbonyl]-2-benzyl-1-oxopropyl]glycine (HACBOGly), a potent (Ki = 1.4 nM) and specific inhibitor of NEP. A maximal 13% decrease in sites was observed after 8 hr incubation, this effect disappearing after 12 hr. This weak but specific negative modulation was not observed with a compound, chemically related to HACBOGly, which has a 10,000-fold lower inhibitory potency. The modulation was inhibited by low temperature or monensin treatment and could be brought about by an internalization of the enzyme, compensated for by an increased biosynthesis or by the sequestration of NEP in a non-membranous compartment.

Asymmetry and dynamics in bis-intercalated DNA

The bis-intercalator ditercalinium (NSC 366241), composed of two 7 H-pyridocarbazoles linked by a bis(ethylpiperidinium), binds to DNA with a binding constant greater than 10(7) M-1. One distinctive aspect of the 3-D X-ray structure of a DNA-ditercalinium complex is its asymmetry. We propose here that the activity of ditercalinium may be related to structural polymorphism and dynamic conversion between conformers. It was previously reported that activity is closely related to linker composition. Activity increases with increasing conformational restraints of the linker. We suggest these conformational restraints can lead to asymmetry in DNA complexes and that this asymmetry results directly in structural polymorphism. Using the Cambridge Structural Database (CSD) as a source of information about chemical fragments that are analogous to the linker of ditercalinium, we have explored the conformational space available to ditercalinium. The results indicate that the linker is highly constrained and that the DNA complex is intrinsically asymmetric. We propose a reasonable mechanism of ring reversal that is consistent with the conformations of analogous fragments within the CSD.

A new and improved synthesis of a potential antitumour 7H-pyrido[4,3-c]carbazole analog

Abstract The 6-ethyl-10-methoxy-7H-pyrido[4,3-c ]carbazole is synthesized via a novel and convenient method involving the condensation of the 2-lithio-5-methoxyindole with the appropriate alkyl pyridyl ketone. Quaternarization of this compound by means of a rigid bis(ethyl-piperidyl) linking chain leads to a new potential antitumour dimer.

Correlation of 15N and 13C relaxation data as a mobility probe for linear peptides. A theoretical and experimental study using enkephalins and related models

Abstract The potential of 15N NMR relaxation data for conformational studies of small linear peptides is discussed, using 15N enriched enkephalin derivatives as follows: Tyr-∗Gly-∗Gly-∗Phe, I; Boc-Tyr-∗Gly-∗Gly-∗Phe-OCH3, II; and Tyr-∗Gly-∗Gly-∗Phe-∗Leu, III. 15N relaxation data (T1/NOE) are interpreted in terms of molecular motion using the simplified two-spin system assumption for the NH bonds. In that way, two different approaches are developed: (i) the comparison of the temperature variation of T1 for 15N and for 13Cα which shows the occurrence of concerted motion for linked 15N and 13C, nuclei along the peptide backbone; (ii) the correlation of 15N relaxation data pairs (T1/NOE) by means of different molecular motion models to check which best fits with the experimental results. The isotropic overall motion model with internal libration appears most appropriate. The 15N “antisymmetric” T1 obtained with an INEPT-derived pulse sequence provide useful parameters to study the conformational preferences in free linear peptides, because of the sensitivity of these quantities to intramolecular exchange processes which are distance dependent. All the results support folded conformations for the free peptides I and III and a random one for the diprotected peptide II.

[125I]azido-DTLET as a tool for selective covalent labeling of δ-opioid receptors in rat brain sections

The binding kinetics and pharmacological selectivity of the photoaffinity delta-opioid ligand [125I]azido-DTLET (Tyr-D-Thr-Gly-Phe(pN3)-Leu-Thr) were investigated in serial frozen sections from rat neostriatum prior to ultraviolet irradiation (i.e., in conditions of reversibility). Scatchard analysis of saturation binding experiments indicated that [125I]azido-DTLET binds to both a high (KD = 5.04 nM) and a low (KD = 38 nM) affinity site. Binding to the low-affinity site was no longer detectable in the presence of unlabeled [D-Ala2,N-MePhe4,Gly-Ol5]enkephalin (DAGO), suggesting that this site corresponds to mu-opioid receptors. This interpretation was further supported by the dose-dependent inhibition of the binding of [3H]DAGO by non-radioactive azido-DTLET. Binding to the high-affinity site was totally inhibited, in a dose-dependent fashion, by a variety of opioid drugs among which delta-opioid ligands showed the highest order of potency. It is concluded that, in the nanomolar range, [125I]azido-DTLET constitutes a highly selective tool for covalent labeling of delta-opioid receptors in rat brain sections.