Lieselot Carrette

Multifunctionalized Sequence‐Defined Oligomers from a Single Building Block

Two decades of progress in the field of living and controlled polymerizations, combined with the elaboration of efficient conjugation reactions, greatly contributed to the elegant preparation of functionalized macromolecular architectures. However, these state-of-the-art methodologies, while providing a high degree of structural and topological control, are inadequate tools for controlling the polymer microstructure. Crucial parameters like primary structure (i.e. monomer sequence) and tacticity largely remain unmastered by current man-made approaches. Expectations for the next generation synthetic polymers include their performance as single chains, ability to fold and self-regulate, and to sense specific molecules and/or catalyze reactions. These precisely functionalized linear polymers should exhibit sharply defined and tailored structure-activity relationships, analogous to Nature’s delicately engineered macromolecules. Therefore, progress towards reliable sequence-controlled polymerization, enabling preprogrammed distribution of multiple functional groups along the backbone, is drawing attention in a growing number of research groups worldwide. Pioneering efforts to control the primary structure of functionalized polymers have been based on several approaches, such as different reactivity ratios of vinyl monomers, spatial prearrangement of monomers on a (macromolecular) template or, as recently demonstrated, the action of a small-molecule machine. Other attempts use (automated) sequential addition of building blocks on a solid or liquid support, leading to sequence control as a result of iterative coupling steps, thereby omitting the need for pre-organization. These protocols, established for peptide and oligonucleotide synthesis, present considerable drawbacks for sequence-controlled polymerization: they generally require the use of protecting groups and the restricted number of readily available building blocks (‘monomer alphabet’) equipped with the appropriate functional handle can further hamper the preparation of tailor-made functionalized sequences. The development of new chemical protocols for chain elongation, often on a solid support, resulting in sequence-defined (macro)molecular structures with unique backbones and side chain functionalities, or fragments thereof that could be combined to obtain sequence controlled polymers, is therefore highly desirable. We here report on a new coupling strategy for the controlled generation of sequence-defined multi-functionalized oligomers on solid support in a protecting group-free approach, inspired by the ‘submonomer’ synthetic protocol for the preparation of functionalized peptoids, via thiolactone-based chemistry. While the generated oligomers are small in size, reconstitution approaches could further allow the synthesis of larger chains, featuring designed and repetitive display of carefully selected and well-positioned functional entities.

Toxicity inspired cross-linking for probing DNA–peptide interactions

A cross-linking methodology for the study of DNA-protein interactions is described. The method is inspired by the metabolic activation of furans causing toxic DNA damage, including DNA-protein cross-links (DPC). The furan moiety, representing a latent functionality, is easily incorporated into oligonucleotides, and can be activated on demand to release a reactive aldehyde. Reaction with nucleophilic lysine side chains is shown to be distance-sensitive and allows for site-selective DPC formation.

Furan oxidation based cross-linking: a new approach for the study and targeting of nucleic acid and protein interactions

Furan mediated nucleic acid cross-linking, initially developed for DNA interstrand duplex cross-linking, has matured into a versatile tool for the study of protein and nucleic acid interactions, ready to face its applications. The methodology was initially developed for easy and clean chemical generation of DNA interstrand cross-linked duplexes, but has been further expanded for use with other probes, targets and triggers, now allowing mild biologically significant cross-linking with potential therapeutic benefit. It was shown that the methodology could be repurposed for RNA interstrand cross-linking, which is very relevant in todays antisense approaches or miRNA target identification endeavors. This further illustrates the furan oxidation methods generality and mildness, especially when using red light for oxidation. A complementary antigene approach has been explored through duplex targeting with furan modified triplex forming oligonucleotides (TFOs) and DNA binding proteins. Also targeting of peptides and proteins by furan-modified DNA and peptides has been explored. Thorough methodology examination exploring variable reaction conditions in combination with a series of different furan-modified building blocks and application of different activation signals resulted in a detailed understanding of the mechanisms involved and factors influencing the yield and selectivity of the reaction. In order to draw the bigger picture of the scope and limitations of furan-oxidation cross-linking, we here provide a unique side by side comparison and discussion of our published data, supplemented with unpublished results, providing a clear performance report of the currently established furan toolbox and its application potential in various biomacromolecular complexes.

A synthetic oligonucleotide model for evaluating the oxidation and crosslinking propensities of natural furan-modified DNA

We have previously developed a crosslinking methodology for oligonucleotides based on the incorporation of furan moieties, which can be selectively oxidised to reactive intermediates that will quickly react with the opposite bases in DNA, forming toxic interstrand crosslinks (ICLs). Furan moieties also occur in natural DNA, as a result of oxidative stress. Moreover, the furan‐containing degradation product of this modified DNA—kinetin—has been found to display beneficial anti‐ageing effects. To investigate the apparent discrepancy between the effects of the synthetic and the natural furan modifications in DNA, a quick and easy postsynthetic method providing access to the natural modification in short synthetic oligonucleotides was developed. On checking for potential crosslinking propensity, we found that the furan moiety does indeed undergo oxidation, in this way functioning as an important scavenger for oxidative stress. The reactive intermediate, however, was shown to degrade without producing toxic crosslinked products.

Peptide scalpels for site-specific dissection of the DNA–protein interface

Current review highlights emerging strategies for the design of DNA-binding peptides and crosslinking techniques capable of interference at the DNA–protein interface with emphasis on bottom-up organic synthesis. Despite the obvious fundamental character, these inquiries could ultimately result in interesting biomedical applications as designed genome interfering agents or diagnostics. Recent awareness of the potential of these larger peptidomimetic constructs has risen within the medicinal society.

Triplex Crosslinking through Furan Oxidation Requires Perturbation of the Structured Triple-Helix

Short oligonucleotides can selectively recognize duplexes by binding in the major groove thereby forming triplexes. Based on the success of our recently developed strategy for furan‐based crosslinking in DNA duplexes, we here investigated for the first time the use of the furan‐oxidation crosslink methodology for the covalent locking of triplex structures by an interstrand crosslink. It was shown that in a triplex context, although crosslinking yields are surprisingly low (to nonexistent) when targeting fully complementary duplexes, selective crosslinking can be achieved towards mismatched duplex sites at the interface of triplex to duplex structures. We show the promising potential of furan‐containing probes for the selective detection of single‐stranded regions within nucleic acids containing a variety of structural motifs.

DNA Interstrand Cross‐Link Formation Using Furan as a Masked Reactive Aldehyde

This unit describes a method for interstrand cross‐linking between a furan‐modified oligonucleotide and its unmodified complement. The synthesis of two furan‐modified phosphoramidites, selected based on high cross‐linking yield versus improved cross‐linking selectivity, is described. The methods allow gram‐scale synthesis starting from stable and readily available furan derivatives. Cross‐linking requires selective oxidation of the furan moiety to an aldehyde. The masked nature of the latter avoids undesired and off‐target reactions, resulting in clean and high‐yield cross‐link formation. Curr. Protoc. Nucleic Acid Chem. 54:5.12.1‐5.12.16.

A thiolactone strategy for straightforward synthesis of disulfide-linked side-chain-to-tail cyclic peptides featuring an N-terminal modification handle

The development of straightforward and versatile peptide cyclisation methods is highly desired to meet the demand for more stable peptide‐based drugs. Herein, a new method for the synthesis of side‐chain‐to‐tail cyclic peptides with the simultaneous introduction of an N‐terminal handle, based on the introduction of an N‐terminal thiolactone building block, is described. A primary amine liberates a homocysteine analogue from the thiolactone building block, which further enables cyclisation of the peptide through disulfide‐bond formation with a C‐terminal cysteamine. Postcyclisation modification can be achieved by using small bifunctional amines. Alternatively, the synthesis of lipopeptides is demonstrated through direct thiolactone opening with long‐chain alkyl amines.