Didier Boturyn

Oxime ligation: a chemoselective click-type reaction for accessing multifunctional biomolecular constructs.

There is a growing need for biocompatible click reactions in order to prepare multifunctional conjugates, which are valuable molecules for innovative biomedical applications. In this context, we review the recent advances in the implementation of oxime ligation for the synthesis of multivalent or multicomponent systems. The value of these products is emphasized by their use in cell targeting, imaging, synthetic vaccines, and surface modifications.

Tumor Targeting with RGD Peptide Ligands-Design of New Molecular Conjugates for Imaging and Therapy of Cancers

Development of molecular devices endowed with tumor-targeting functions and carrying cytotoxic components should enable the specific delivery of chemotherapeutics to malignant tissues, thus increasing their local efficacy while limiting their peripheral toxicity. Such molecular vectors can pave the way for the development of new classes of therapeutics, fighting against protagonists of neoplastic development. In line with this concept, peptide ligands containing the Arginine-Glycine-Aspartate (RGD) triad, which display a strong affinity and selectivity to the alpha(V)beta(3) integrin, have been developed to target the tumor-associated cells expressing the alpha (V)beta (3) receptors. Among the validated ligands, the leader compound is the cyclic pentapeptide c[-RGDf(NMe)V-] (Cilengitide) developed by kessler et al. (J. Med. Chem., 1999, 42, 3033-3040). This compound has entered phase II clinical trials as an anti-angiogenic agent. Further studies have been directed to develop molecular conjugates of the parent c[-RGDfK-] with conventional chemotherapeutics or with labels for non-invasive imaging technologies. More recently, multimeric RGD containing compounds have been exploited to improve the targeting potential as well as cell-membrane breaching, through receptor-mediated endocytosis. The latter have been constructed on various scaffolds (polylysines or polyglutamates, liposomes, nanoparticles...). Our group has developed a chemical system combining all these properties where multivalent RGD targeting functions are associated with functional molecules through a cyclopeptide template. The latter represents a relevant non-viral vector for tumor targeting, imaging and therapy. This review describes the considerations for the design of the diverse RGD ligands developed so far and reports an overview of the main applications of these structures in cancer research.

In vivo optical imaging of integrin αV-β3 in mice using multivalent or monovalent cRGD targeting vectors

BackgroundThe cRGD peptide is a promising probe for early non-invasive detection of tumors. This study aimed to demonstrate how RAFT-c(-RGDfK-)4, a molecule allowing a tetrameric presentation of cRGD, improved cRGD-targeting potential using in vivo models of αVβ3-positive or negative tumors.ResultsWe chose the human embryonic kidney cells HEK293(β3) (high levels of αVβ3) or HEK293(β1) (αVβ3-negative but expressing αV and β1) engrafted subcutaneously (s.c.) in mice. Non-invasive in vivo optical imaging demonstrated that as compared to its monomeric cRGD analogue, Cy5-RAFT-c(-RGDfK-)4 injected intravenously had higher uptake, prolonged retention and markedly enhanced contrast in HEK293(β3) than in the HEK293(β1) tumors. Blocking studies further demonstrated the targeting specificity and competitive binding ability of the tetramer.ConclusionIn conclusion, we demonstrated that Cy5-RAFT-c(-RGDfK-)4 was indeed binding to the αVβ3 receptor and with an improved activity as compared to its monomeric analog, confirming the interest of using multivalent ligands. Intravenous injection of Cy5-RAFT-c(-RGDfK-)4 in this novel pair of HEK293(β3) and HEK293(β1) tumors, provided tumor/skin ratio above 15. Such an important contrast plus the opportunity to use the HEK293(β1) negative control cell line are major assets for the community of researchers working on the design and amelioration of RGD-targeted vectors or on RGD-antagonists.

Highly efficient synthesis of peptide-oligonucleotide conjugates: chemoselective oxime and thiazolidine formation.

A convergent strategy for the synthesis of peptide-oligonucleotide conjugates (POC) is presented. Chemoselective ligation of peptide to oligonucleotide was accomplished by oxime and thiazolidine formation. Oxime conjugation was performed by treating an oxyamine-containing peptide with an aldehyde-containing oligonucleotide or vice versa. Ligation by thiazolidine formation was achieved by coupling a peptide, acylated with a cysteine residue, to an oligonucleotide that was derivatised by an aldehyde function. For both approaches, the conjugates were obtained in good yield without the need for a protection strategy and under mild aqueous conditions. Moreover, the oxime ligation proved useful for directly conjugating duplex oligonucleotides. Combined with molecular biology tools, this methodology opens up new prospects for post-functionalisation of high-molecular-weight DNA structures.

Multivalent RGD synthetic peptides as potent αVβ3 integrin ligands

We study herein the multivalency effect of a cluster of alphaVbeta3-ligands held on a cyclodecapeptide template. An array of RAFT(c[-RGDfK-])n derivatives containing from one to sixteen clustered RGD motifs were synthesized and comparatively assayed in vitro on alphaVbeta3-expressing cells. Efficient inhibition of the alphaVbeta3-specific 23C6 monoclonal antibody fixation was observed with ligands displaying three and four copies of the cyclo[-RGDfK-] peptide.

Noninvasive Optical Imaging of Ovarian Metastases Using Cy5-labeled RAFT-c(-RGDfK-)4

Our group has developed a new molecular tool based on the use of a regioselectively addressable, functionalized template (RAFT) scaffold, where four cyclic (Arg-Gly-Asp) (cRGD) peptide motifs were grafted. The aim of this study was to determine whether RAFT-c(-RGDfK-)4 combined with optical imaging could allow noninvasive detection of deep ovarian metastases. Human ovarian adenocarcinoma IGROV1 cells expressing low levels of integrin αvβ3 (the main receptor for the cRGD peptide) were used for in vitro and in vivo assays in combination with Cy5-labeled RAFT-c(-RGDfK-)4, cRGD, or RAFT-c(-RβADfK-)4. In vivo fluorescence imaging was performed on subcutaneous (SC) tumors and intraperitoneal IGROV1 metastases in nude mice. The accumulation of RGD-Cy5 conjugates in cultured cells or in tumor tissues was examined using confocal laser scanning microscopy. RAFT-c(-RGDfK-)4 exhibited stronger staining in vitro, enhanced tumor-to-background ratio for SC tumors, and allowed early detection of 1- to 5-mm large intraabdominal nodules using noninvasive optical imaging. Histological study revealed that RAFT-c(-RGDfK-)4 accumulated into tumor neovasculature but also into tumor cells. Our data demonstrate that a Cy5-labeled RAFT-c(-RGDfK-)4 is an efficient optical probe for early and noninvasive tumor detection.

One-Pot Approach to Well-Defined Biomolecular Assemblies by Orthogonal Chemoselective Ligations†

Click-click cyclopeptides: Well-defined biomolecular assemblies are synthesized using orthogonal oxime bond formation and copper(I)-mediated alkyne-azide cycloaddition reactions in a stepwise or in a one-pot approach. To illustrate this strategy, regioselective ligation of biologically relevant peptides onto a cyclopeptidic scaffold was performed.

Activatable fluorescent probes for tumour-targeting imaging in live mice.

The direct and noninvasive in vivo visualisation of molecular processes such as ligand–receptor interaction, enzymatic activity, or gene expression, is the goal of molecular imaging. Optical imaging appears as a new complementary modality to the traditional nuclear or MRI (magnetic resonance imaging) techniques because of its low cost and fewer constraints. 4] Thus, the need for new optical probes with increased targeting and imaging capabilities arises. 6] Tung et al. introduced the concept of “smart” or activatable probes for imaging the activity of proteases over-expressed in mice tumours, such as MMP2 or cathepsin D. In these activatable probes, Cy5.5 dyes are grafted to peptide branching arms of a co-polymer, in such a way that they auto-quench initially. Whenever the proteases specifically cut the peptide sequence, the Cy5.5 fluorescence is recovered. In the present work, the concept of activatable probes is extended to the imaging of different molecular events, in the expectation of improving the image contrast in comparison to that obtained with classical targeting agents. The molecular structures we propose can be used for imaging enzymatic activity, specific targeting, and molecular processes triggered by ligand–receptor interaction. Moreover, their nonpolymeric nature can be very well controlled and characterised for pharmacology and medical applications. These activatable probes are built on a cyclodecapeptide template named RAFT (Regioselectively Addressable Functionalized Template), a new molecular vector for targeted drug delivery and molecular imaging of tumours and metastasis. This new class of molecules differs from other systems by the topological separation of two independent functional domains, which can be addressed in a spatially controlled manner: a cell targeting domain, and a therapeutic or imaging domain (Scheme 1). Clustered RGD-containing markers are used for cell recognition by the aVb3 integrin, [16–19] a vitronectin receptor over-expressed on the surface of endothelial cells of growing blood vessels, and therefore a cardinal feature of many malignant tumours. A multimeric presentation of the RGD motif is essential for integrin-mediated internalisation of the probe. 22–24] In the activatable probes we designed, the imaging function is composed of the cyanine 5 fluorescent dye (Cy5), a cleavable bond, and a quencher (Q). The cleavable bond is used to follow the cellular internalisation of the probe induced by binding to its receptor. Disulfide bridges are known to be reduced enzymatically by thioredoxines in the cytosol, and have been used to improve the efficiency of targeted drug delivery. The redox potential within the endosomal system could also be reducing. Therefore, -Cysteine-S S-Cysteine(abbreviated as S–S below) is chosen as a cleavable bond, to follow the internalisation of the fluorescent probe into the cells after its RGD-mediated binding on their surface. Demonstrating targeting can be crucial for screening and comparing the recognition efficiency of different biological markers. Moreover, demonstrating internalisation is also very important to estimate the ability of a molecule, such as the RAFT, to be used as a vector for targeted drug delivery. The self-quenching of the cyanine dyes allows the use of the Cy5-S S-Cy5 group as an activatable unit (Scheme 1). Alternatively, a diarylrhodamine derivative quencher (QSY21) can be used in place of one of the Cy5 units. Cy5 fluorescence is markedly inhibited in the Cy5-S S-QSY21 group (Table 1). This dynamic and static quenching in aqueous buffer can be accounted for by the hydrophobic nature and opposite charges of the molecules, which favour their stacking. In this context, the RAFT-(cRGD)4 Cy5-S S-Cy5 1, RAFT-(cRGD)4 Cy5-S SQSY21 2, and RAFT-(cRGD)4 QSY21-S S-Cy5 3 have been synthesised (Scheme 1). The peptide moiety of the molecule is prepared by a combination of solid and solution phase synthesis using a Fmoc/tBu strategy, followed by a chemoselective assembling of ligand moieties and template, as reported elsewhere. Npys-protected cysteine is chosen to graft the fluorescent unit to the RAFT. This allows a regioselective disulfide bond formation. Cy5 and QSY21, available in the activated ester form, are then attached successively to the macromolecule, providing, after RP-HPLC purification, the desired compounds in satisfying overall yields (13–18%). The activation of the fluorescent probes is demonstrated first in vitro. Figure 1A displays the absorption spectra of the RAFT-(cRGD)4 Cy5-S S-QSY21 2 before and 2 h after addition of 2-mercaptoethanol (2-MCE). The spectrum after cleavage matches that of the sum of Cy5 and QSY21, whereas for 2, the electronic interaction between the dye and quencher is evidenced by an intense absorption band at 600 nm. Similar results are obtained for 1: the 600 nm absorption band in this case is indicative of the presence of Cy5 dimers, whereas the 647 nm band is attributed to the dye monomer. Fluorescence measurements (Figure 1B) show the initial Cy5 fluorescence quenching and its 100% recovery upon the S S bond [a] Dr. J. Razkin, Dr. D. Boturyn, Prof. P. Dumy LEDSS, UMR CNRS 5616, 301 rue de la chimie, BP 53, 38041 Grenoble Cedex 9 (France) Fax: (+33)476-635-540 E-mail : Didier.Boturyn@ujf-grenoble.fr [b] Dr. I. Texier LETI/DTBS CEA Grenoble, 17 rue des martyrs, 38054 Grenoble Cedex 9 (France) Fax: (+33)438-785-787 E-mail : isabelle.texier-nogues@cea.fr [c] Dr. Z.-h. Jin, Prof. M. Favrot, Dr. J.-L. Coll INSERM U578, Institut Albert Bonniot, 38706 La Tronche (France) [d] Dr. V. Josserand ANIMAGE—CREATIS, B=timent CERMEP, 59 Boulevard Pinel, 69677 Bron Cedex (France) Supporting information for this article is available on the WWW under http://www.chemmedchem.org or from the author.

In vivo noninvasive optical imaging of receptor-mediated RGD internalization using self-quenched Cy5-labeled RAFT-c(-RGDfK-)(4).

We reported that regioselectively addressable functionalized template (RAFT)-c(-RGDfK-)4 presenting four cyclic (Arg-Gly-Asp) (cRGD) peptides targets integrin aVb3 with an improved specificity compared with monomeric cRGD. In this study, we improved this vector by creating a “stealth” molecule in which a fluorescence quencher (Q) is linked to Cy5 via a disulfide bond (-SS-). RAFT-c(-RGDfK-)4-Cy5-SS-Q fluorescence is quenched unless activated by reduction during internalization. RAFT-c(-RGDfK-)4-Cy5-SS-Q fluorescence was negligible when compared with the control but totally recovered after cleavage of the disulfide bridge. Confocal microscopy showed that only the intracellular Cy5 signal could be detected using RAFT-c(-RGDfK-)4-Cy5-SS-Q, confirming that uncleaved extracellular molecules are not visible. Whole-body imaging of mice bearing subcutaneous tumors injected intravenously with RAFT-c(-RGDfK-)4-Cy5-SS-Q showed a very significant enhancement of the fluorescent contrast in tumors compared with the unquenched molecule. Histology of the tumor confirmed the intracellular accumulation of Cy5. These results demonstrate that the presence of a labile disulfide bridge between the targeting vector and a drug mimetic is an efficient way to deliver a dye, or a drug, intracellularly. In addition, this quenched RAFT-c(-RGDfK-)4-Cy5-SS-Q probe is a very powerful vector for imaging tumor masses and investigating in vivo RGD-mediated internalization.

Synthesis of fluorescent probes for the detection of abasic sites in DNA

Abstract The three luminescent molecules 1–3 were prepared as highly sensitive and specific probes for the quantification of a major DNA lesion, the abasic site. These molecules incorporate in their structure the reactive oxyamino function linked to the Dansyl or Lissamine-Rhodamine fluorophores through amido or polyether chains.