Fluorescent Dynamic Covalent Polymers for DNA Complexation and Templated Assembly.

Dynamic covalent polymers (DCPs) offer opportunities as adaptive materials of particular interest for targeting, sensing and delivery of biological molecules. In this view, combining cationic units and fluorescent units along DCP chains is attractive for achieving optical probes for the recognition and delivery of nucleic acids. Here, we report on the design of acylhydrazone-based DCPs combining cationic arginine units with π-conjugated fluorescent moieties based on thiophene-ethynyl-fluorene cores. Two types of fluorescent building blocks bearing neutral or cationic side groups on the fluorene moiety are considered in order to assess the role of the number of cationic units on complexation with DNA. The (chir)optical properties of the building blocks, the DCPs, and their complexes with several types of DNA are explored, providing details on the formation of supramolecular complexes and on their stability in aqueous solutions. The DNA-templated formation of DCPs is demonstrated, which provides new perspectives on the assembly of fluorescent DCP based on the nucleic acid structure.

Generation of Multicomponent Molecular Cages using Simultaneous Dynamic Covalent Reactions.

Cage compounds are very attractive structures for a wide range of applications and there is ongoing interest in finding effective ways to access such kinds of complex structures, particularly those possessing dynamic adaptive features. Here we report the accessible synthesis of new type of organic cage architectures, possessing two different dynamic bonds within one structure: hydrazones and disulfides. Implementation of three distinct functional groups (thiols, aldehydes and hydrazides) in the structure of two simple building blocks resulted in their spontaneous and selective self-assembly into aromatic cage-type architectures. These organic cages contain up to ten components linked together by twelve reversible covalent bonds. The advantage provided by the presented approach is that these cage structures can adaptively self-sort from a complex virtual mixture of polymers or macrocycles and that dynamic covalent chemistry enables their deliberate disassembly through controlled component exchange.

Degradable hybrid materials based on cationic acylhydrazone dynamic covalent polymers promote DNA complexation through multivalent interactions.

The design of smart nonviral vectors for gene delivery is of prime importance for the successful implementation of gene therapies. In particular, degradable analogues of macromolecules represent promising targets as they would combine the multivalent presentation of multiple binding units that is necessary for achieving effective complexation of therapeutic oligonucleotides with the controlled degradation of the vector that would in turn trigger drug release. Toward this end, we have designed and synthesized hybrid polyacylhydrazone-based dynamic materials that combine bis-functionalized cationic monomers with ethylene oxide containing monomers. Polymer formation was characterized by (1) H and DOSY NMR spectroscopy and was found to take place at high concentration, whereas macrocycles were predominantly formed at low concentration. HPLC monitoring of solutions of these materials in aqueous buffers at pH values ranging from 5.0 to 7.0 revealed their acid-catalyzed degradation. An ethidium bromide displacement assay and gel electrophoresis clearly demonstrated that, despite being dynamic, these materials are capable of effectively complexing dsDNA in aqueous buffer and biological serum at N/P ratios comparable to polyethyleneimine polymers. The self-assembly of dynamic covalent polymers through the incorporation of a reversible covalent bond within their main chain is therefore a promising strategy for generating degradable materials that are capable of establishing multivalent interactions and effectively complexing dsDNA in biological media.

Dipeptides as leaving group in the enzyme-catalyzed DNA synthesis.

Conjugates of 2'-deoxyadenosine monophosphate with dipeptides have been synthesized and tested as substrates for several polymerases. Although the incorporation efficiency is not very high, it demonstrates that some of these dipeptides can be accommodated in the active site of polymerases and function as leaving groups in the enzymatic synthesis of DNA.

Iminodipropionic acid as the leaving group for DNA polymerization by HIV-1 reverse transcriptase.

Previous studies have demonstrated that some selected amino monoacids and amino diacids can function as leaving groups in the polymerase-catalyzed incorporation of deoxynucleotides into DNA. Among these, the iminodiacetic acid phosphoramidate of deoxyadenosine monophosphate (IDA-dAMP) represents an interesting example, as it could overcome some of the problems observed when using L-aspartic acid as the leaving group, that is, poor chain elongation. We have now synthesized and evaluated a series of IDA-dAMP analogues that bear either an extended aliphatic chain in the amino acid function, or a phosphonic acid moiety (substituting for the carboxylic acid function). Among these compounds, the nucleotide with an iminodipropionic acid leaving group (IDP-dAMP) was identified as the best substrate; the excellent single incorporation (91 % conversion to a P+1 strand at 50 µM) was at a substrate concentration ten times lower than that used for IDA-dAMP). This nucleotide also presented improved kinetics and elongation capability compared to IDA-dAMP. The analogues with T, G, and C base moieties were also investigated for their incorporation ability with HIV-1 RT. The incorporation efficiency was found to decrease in the order A>T>G>C. The properties of the iminodipropionic acid as the leaving group surpass those of previously evaluated leaving groups; this acid will be a prime candidate for in vivo testing.

Synthesis of poly(amino)ester dendrimers via active cyanomethyl ester intermediates.

A novel strategy for the synthesis of poly(amino)ester dendrimers was developed on the basis of active cyanomethyl ester intermediates and an iteration of four consecutive steps of deprotection, activation, transesterification, and scavenging.

Structural characterization of poly(amino)ester dendrimers and related impurities by electrospray tandem mass spectrometry.

An acid-terminated poly(amino)ester dendrimer was studied by electrospray ionization tandem mass spectrometry to establish its fragmentation pathways, with the aim of using them to investigate the structure of any defective molecules generated during the dendrimer synthesis. This poly(amino)ester dendrimer could be ionized in both polarities but the most structurally relevant dissociation pathways were found from the deprotonated molecule in negative ion mode. The dissociation pattern of this dendrimer is fully described and supported by accurate mass measurements. The main dissociation reactions of the negatively charged polyacidic dendrimer were shown to consist of (i) the release of carbon dioxide and ethene within a branch, which proceeds as many times as intact neutral branches are available; and (ii) the elimination of an entire dendrimer arm. Monitoring the occurrence of these reactions together with any deviation from these two main routes allowed six major dendritic impurities to be structurally characterized.

Biomolecular dynamic covalent polymers for DNA complexation and siRNA delivery.

Synthetic delivery systems that are described as smart are considered essential for the successful development of gene therapies. Dynamic covalent polymers (DCP) are dynamic and adaptive species that can expand and shorten their main chain in a reversible fashion. In particular, polyacylhydrazone DCPs are pH-sensitive and undergo hydrolytic dissociation at acidic pH, which is an interesting feature for gene delivery. Building upon our previous finding that cationic DCPs can complex DNA through multivalent interactions, we report here on a new generation of DCPs that incorporate modified amino acids. The covalent self-assembly through polycondensation was extended towards multifunctional DCPs combining different building blocks and different molecular dynamics. These biomolecular DCPs were found able to complex both long DNA and siRNA, and biological studies demonstrate that they are able to deliver functional siRNA in living cells. This straightforward and modular approach to the self-production of multifunctional and biomolecular DCPs as siRNA vectors can therefore constitute a stepping stone in smart gene delivery using dynamic and adaptive biodynamers.

Bioactive clusters promoting cell penetration and nucleic acid complexation for drug and gene delivery applications: from designed to self-assembled and responsive systems.

Bioactive low-molecular-weight compounds are actively pursued, as an alternative to macromolecules, for biomedical applications such as drug and gene delivery. However, achieving effective biomolecular surface recognition with small molecules is a considerable challenge. We review herein recent progresses that have been made in the identification of bioactive cationic clusters that promote cell penetration and nucleic acid complexation and vectorisation. We further emphasize the emerging use of self-assembly processes, based on supramolecular interactions and/or dynamic covalent chemistry, for generating bioactive cationic clusters. Interestingly, the introduction of molecular and/or supramolecular dynamics endows reversibility to the multivalent recognition processes, thereby paving the way toward the development of "smart" adaptive and responsive devices that emulate the behaviour of natural systems for the dynamic control of bioactivity.

Macrocycle-embedded ß-lactams as novel inhibitors of the Penicillin Binding Protein PBP2a from MRSA.

Assuming that bicyclic ß-lactams endowed with high conformational adaptability should more easily form acyl-enzyme complexes with PBP2a than the traditional antibiotics, we have prepared a series of bis-2-oxo-azetidinyl macrocycles as potential inhibitors. The compounds are formally "head-head" (HH) cyclodimers of 1-(ω-alkenoyl)-3-(S)-(ω'-alkenoylamino)-2-azetidinones, with various lengths of the alkene chains, obtained by two successive metathesis reactions using the Grubbs catalyst. All compounds behave as acylating inhibitors of PBP2a and one ß-lactam (5c), embedded into the largest ring (32 atoms), features an activity close to that of Ceftobiprole. Conformational analyses, theoretical reactivity models and docking experiments in PBP2a cavity allow to propose a novel pharmacophore, i.e. the 3-(S)-acylamino-1-acyl-2-azetidinone ring, with the syn-conformation of the imide function, associated to a flexible macrocycle favoring the opening of the active site.

Microwave assisted "click" chemistry for the synthesis of multiple labeled-carbohydrate oligonucleotides on solid support.

A versatile approach has been developed for the multiple labeling of oligonucleotides. First, three linkers as a H-phosphonate monoester derivative were condensed on a solid-supported T12 to introduce H-phosphonate diester linkages which were oxidized in the presence of propargylamine. Second, three galactosyl azide derivatives were conjugated to the solid-supported three-alkyne-modified T12 by a 1,3-cycloaddition so-called "click chemistry" in the presence of Cu(I) assisted by microwaves.