Tethered imidazole mediated duplex stabilization and its potential for aptamer stabilization

Previous investigations of the impact of an imidazole-tethered thymidine in synthetic DNA duplexes, monitored using UV and NMR spectroscopy, revealed a base context dependent increase in thermal stability of these duplexes and a striking correlation with the imidazolium pK(a). Unrestrained molecular dynamics (MD) simulations demonstrated the existence of a hydrogen bond between the imidazolium and theHoogsteen side of a nearby guanosine which, together with electrostatic interactions, form the basis of the so-called pK(a)-motif responsible for these duplex-stabilizing and pK(a)-modulating properties. Here, the robustness and utility of this pK(a)-motif was explored by introducing multiple imidazoletethered thymidines at different positions on the same dsDNA duplex. For all constructs, sequence based expectations as to pK(a)-motif formation were supported by MD simulations and experimentally validated using NOESY. Based on the analysis of the pK(a) values and melting temperatures, guidelines are formulated to assist in the rational design of oligonucleotides modified with imidazoliumtethered thymidines for increased thermal stability that should be generally applicable, as demonstrated through a triply modified construct. In addition, a proof-of-principle study demonstrating enhanced stability of the L-argininamide binding aptamer modified with an imidazole-tethered thymidine in the presence and absence of ligand, demonstrates its potential for the design of more stable aptamers

Identification of a pKa-regulating motif stabilizing imidazole-modified double-stranded DNA

The predictable 3D structure of double-stranded DNA renders it ideally suited as a template for the bottom-up design of functionalized nucleic acid-based active sites. We here explore the use of a 14mer DNA duplex as a scaffold for the precise and predictable positioning of catalytic functionalities. Given the ubiquitous participation of the histidine-based imidazole group in protein recognition and catalysis events, single histidine-like modified duplexes were investigated. Tethering histamine to the C5 of the thymine base via an amide bond, allows the flexible positioning of the imidazole function in the major groove. The mutual interactions between the imidazole and the duplex and its influence on the imidazolium pKa(H) are investigated by placing a single modified thymine at four different positions in the center of the 14mer double helix. Using NMR and unrestrained molecular dynamics, a structural motif involving the formation of a hydrogen bond between the imidazole and the Hoogsteen side of the guanine bases of two neighboring GC base pairs is established. The motif contributes to a stabilization against thermal melting of 6 degrees C and is key in modulating the pKa(H) of the imidazolium group. The general features, prerequisites and generic character of the new pKa(H)-regulating motif are described

Trifluoromethylated Proline Surrogates as Part of "Pro-Pro" Turn-Inducing Templates

Proline is often found as a turn inducer in peptide or protein domains. Exploitation of its restricted conformational freedom led to the development of the d-Pro-l-Pro (corresponding to (R)-Pro-(S)-Pro) segment as a "templating" unit, frequently used in the design of beta-hairpin peptidomimetics, in which conformational stability is, however, inherently linked to the cis-trans isomerization of the prolyl amide bonds. In this context, the stereoelectronic properties of the CF3 group can aid in conformational control. Herein, the impact of alpha-trifluoromethylated proline analogues is examined for the design of enhanced beta-turn inducers. A theoretical conformational study permitted the dipeptide (R)-Pro-(R)-TfmOxa (TfmOxa: 2-trifluoromethyloxazolidine-2-carboxylic acid) to be selected as a template with an increased trans-cis rotational energy barrier. NMR spectroscopic analysis of the Ac-(R)-Pro-(R)-TfmOxa-(S)-Val-OtBu beta-turn model, obtained through an original synthetic pathway, validated the prevalence of a major trans-trans conformer and indicated the presence of an internal hydrogen bond. Altogether, it was shown that the (R)-Pro-(R)-TfmOxa template fulfilled all crucial beta-turn-inducer criteria

Characterisation of magnesium ascorbyl phosphate, a raw material in cell therapy

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The ancient CYP716 family is a major contributor to the diversification of eudicot triterpenoid biosynthesis

Triterpenoids are widespread bioactive plant defence compounds with potential use as pharmaceuticals, pesticides and other high-value products. Enzymes belonging to the cytochrome P450 family have an essential role in creating the immense structural diversity of triterpenoids across the plant kingdom. However, for many triterpenoid oxidation reactions, the corresponding enzyme remains unknown. Here we characterize CYP716 enzymes from different medicinal plant species by heterologous expression in engineered yeasts and report ten hitherto unreported triterpenoid oxidation activities, including a cyclization reaction, leading to a triterpenoid lactone. Kingdom-wide phylogenetic analysis of over 400 CYP716s from over 200 plant species reveals details of their evolution and suggests that in eudicots the CYP716s evolved specifically towards triterpenoid biosynthesis. Our findings underscore the great potential of CYP716s as a source for generating triterpenoid structural diversity and expand the toolbox available for synthetic biology programmes for sustainable production of bioactive plant triterpenoids

ACCERBATIN, a small molecule at the intersection of auxin and reactive oxygen species homeostasis with herbicidal properties

The volatile two-carbon hormone ethylene acts in concert with an array of signals to affect etiolated seedling development. From a chemical screen, we isolated a quinoline carboxamide designated ACCERBATIN (AEX) that exacerbates the 1-aminocyclopropane-1-carboxylic acid-induced triple response, typical for ethylene-treated seedlings in darkness. Phenotypic analyses revealed distinct AEX effects including inhibition of root hair development and shortening of the root meristem. Mutant analysis and reporter studies further suggested that AEX most probably acts in parallel to ethylene signaling. We demonstrated that AEX functions at the intersection of auxin metabolism and reactive oxygen species (ROS) homeostasis. AEX inhibited auxin efflux in BY-2 cells and promoted indole-3-acetic acid (IAA) oxidation in the shoot apical meristem and cotyledons of etiolated seedlings. Gene expression studies and superoxide/hydrogen peroxide staining further revealed that the disrupted auxin homeostasis was accompanied by oxidative stress. Interestingly, in light conditions, AEX exhibited properties reminiscent of the quinoline carboxylate-type auxin-like herbicides. We propose that AEX interferes with auxin transport from its major biosynthesis sites, either as a direct consequence of poor basipetal transport from the shoot meristematic region, or indirectly, through excessive IAA oxidation and ROS accumulation. Further investigation of AEX can provide new insights into the mechanisms connecting auxin and ROS homeostasis in plant development and provide useful tools to study auxin-type herbicides

Design and systematic study of imidazole based DNAzymes : an integrated NMR and molecular dynamics approach

Proteins are known as the workhorses of the cell and fulfill numerous tasks essential for the cell’s survival. One of the most important tasks is their involvement in catalysis. From a thermodynamic point of view, this is made possible by the stabilisation of the transition state, a high-energy reaction state between reactants and products. This stabilization is made possible due to a combination of a wide variety of chemical functionalities inherent to the twenty amino acid building blocks. This variety observed with proteins stands in sharp contrast to the limited structural diversity of DNA and RNA. These biomolecules are optimized for hydrogen bonding and the formation of complementary, predictable helix-like structures. It thus seemed highly unlikely DNA and RNA could ever fulfill the same catalytic functions as proteins. The discovery and subsequent development of natural and synthetic catalytic RNA and DNA (deoxy)ribozymes has overturned this belief. Mainly identified by means of in vitro selection and evolution experiments, RNA/DNA-based catalysts employ a variety of catalytic mechanisms. Nevertheless, despite the numerous successes of the top-down in vitro approach, structural insight in how these RNA- and DNAzymes ultimately achieve catalysis is still lacking and can be considered as one of the main drawbacks for their further development. Inspired by these developments and limitations, the current research project aims to develop DNA-based hydrolases via a more bottom-up approach. Here the stable and predictable nature of the DNA duplex is employed to position one or more imidazole-bearing thymine nucleotide building blocks (TIm) using standard phosphoramidite solid phase chemistry. Via systematic studies using UV-VIS thermal melting experiments, NMR and molecular dynamics simulations, the mutual impact of the modification and the DNA scaffold could be identified. This approach was applied in two major systematic studies focussing on both single and multiple TIm modified DNA systems. In case of the single modified systems a so-called pKaH-regulating motif (figure) has been uncovered where the imidazole modification at position n in the DNA duplex engages in a persistent hydrogen bond interaction with the carbonyl groups of guanine bases at positions n+1 and n+2 residing in the DNA major groove. This interaction in turn contributes in a significant thermal stabilisation of ±6°C and increase of over 1 pKaH unit with respect to other non-interacting modified systems. In addition to its identity and overall features, the possible sequential permutations of this motif were explored as well. Given that a single TIm functionality alone is unlikely to generate any meaningful catalytic activity, the second systematic study focused on the mutual impact of multiple imidazole residues residing in the same system, both in the presence and absence of the interaction motif. Furthermore these systems allowed to confirm that the motif is tolerated when multiple imidazoles are present and relative positioning of the pKaH-regulating motif with respect to the non-interacting imidazole allows to regulate the pKaH value of the second non-interacting imidazole functionality within certain limits as well. The observations and guidelines obtained during these studies should allow to gradually develop more intricate systems that are ultimately able to cleave ester and/or amide bonds in a stereo selective fashion