Phosphodiester Substrates for Incorporation of Nucleotides in DNA Using HIV-1 Reverse Transcriptase

In previous research we demonstrated that some amino acid derivatives of deoxyadenosine 5'-O-monophosphate act as substrates for incorporation into DNA by HIV-1 reverse transcriptase while retaining the canonical base-pair selectivity for all natural bases. Thus, some amino acids mimic the pyrophosphate group in the polymerization process with this enzyme. Herein we extended this study to the evaluation of a range of potential new leaving groups with aromatic and aliphatic structures carrying one or two carboxylic acid functions. Out of this series, the isophthalic acid derivative of deoxyadenosine 5'-O-monophosphate gave single-nucleotide incorporation results similar to those obtained with the L-aspartic acid phosphoramidate of deoxyadenosine monophosphate. The glycolic acid analogue is a better substrate than the glycine congener, supporting the good leaving group properties of a phosphodiester linkage. These investigations provide new insight into the structural requirements for leaving groups that can mimic the pyrophosphate moiety of nucleoside triphosphates.status: publishe

Bidirectional Graphormer for Reactivity Understanding: neural network trained to reaction atom-to-atom mapping task

This work introduces GraphormerMapper – a new algorithm for reactions atom-to-atom mapping (AAM) based on a distance-aware BERT neural network. In benchmarking studies with IBM RxnMapper, the best AAM algorithm according to our previous study, we demonstrate that our AAM algorithm is superior on our “Golden” benchmarking dataset. The mapper is implemented in Chython [https://github.com/chython/chython] and Chytorch [https://github.com/chython/chytorch, https://github.com/chython/chytorch-rxnmap] Python packages which are freely available for out-the-box use. Chython is a cheminformatics library with a simple interface for processing reaction and molecular data. The key features of Chython are: chemical functional groups standardization, checking atom valence errors, substructure search, and advanced reaction manipulation, for example, generating products from reactants and reaction atom-to-atom mapping. Chytorch provides a PyTorch-like interface for graph-based neural networks developed specifically for chemical tasks

Contribution of dihydrouridine in folding of the D-arm in tRNA

Posttranscriptional modifications of transfer RNAs (tRNAs) are proven to be critical for all core aspects of tRNA function. While the majority of tRNA modifications were discovered in the 1970s, their contribution in tRNA folding, stability, and decoding often remains elusive. In this work an NMR study was performed to obtain more insight in the role of the dihydrouridine (D) modification in the D-arm of tRNA iMet from S. pombe. While the unmodified oligonucleotide adopted several undefined conformations that inter-convert in solution, the presence of a D nucleoside triggered folding into a hairpin with a stable stem and flexible loop region. Apparently the D modification is required in the studied sequence to fold into a stable hairpin. Therefore we conclude that D contributes to the correct folding and stability of D-arm in tRNA. In contrast to what is generally assumed for nucleic acids, the sharp ‘imino’ signal for the D nucleobase at 10 ppm in 90% H2O is not indicative for the presence of a stable hydrogen bond. The strong increase in pKa upon loss of the aromatic character in the modified nucleobase slows down the exchange of its‘imino’proton significantly, allowing its observation even in an isolated D nucleoside in 90% H2O in acidic to neutral conditions.crosscheck: This document is CrossCheck deposited related_data: Supplementary Information copyright_licence: The Royal Society of Chemistry has an exclusive publication licence for this journal copyright_licence: The accepted version of this article will be made freely available after a 12 month embargo period history: Received 28 January 2015; Accepted 20 March 2015; Advance Article published 27 March 2015; Version of Record published 22 April 2015status: publishe

NMR study on the interaction of the conserved CREX 'stem-loop' in the hepatitis E virus genome with a naphthyridine-based ligand

A 2-amino-1,8-naphthyridine derivative that is described to bind single guanine bulges in RNA-DNA and RNA-RNA duplexes was synthesized and its interaction with the single G bulge in the conserved CREX of the Hepatitis E Virus (HEV) genome was explored by NMR and molecular modeling. Results indicate that the ligand intercalates in the internal loop, though none of the expected hydrogen bonds with the single G in the bulge could be demonstrated.crosscheck: This document is CrossCheck deposited related_data: Supplementary Information copyright_licence: The Royal Society of Chemistry has an exclusive publication licence for this journal copyright_licence: The accepted version of this article will be made freely available after a 12 month embargo period history: Received 7 July 2015; Accepted 5 August 2015; Accepted Manuscript published 5 August 2015; Advance Article published 12 August 2015; Version of Record published 16 September 2015status: publishe

Influence of the nucleobase and anchimeric assistance of the carboxyl Acid groups in the hydrolysis of amino Acid nucleoside phosphoramidates

Nucleoside phosphoramidates (NPs) are a class of nucleotide analogues that has been developed as potential antiviral/antitumor prodrugs. Recently, we have shown that some amino acid nucleoside phosphoramidates (aaNPs) can act as substrates for viral polymerases like HIV-1 RT. Herein, we report the synthesis and hydrolysis of a series of new aaNPs, containing either natural or modified nucleobases to define the basis for their differential reactivity. Aqueous stability, kinetics, and hydrolysis pathways were studied by NMR spectroscopy at different solution pD values (5-7) and temperatures. It was observed that the kinetics and mechanism (PN and/or PO bond cleavage) of the hydrolysis reaction largely depend on the nature of the nucleobase and amino acid moieties. Aspartyl NPs were found to be more reactive than Gly or β-Ala NPs. For aspartyl NPs, the order of reactivity of the nucleobase was 1-deazaadenine>7-deazaadenine>adenine>thymine≥3-deazaadenine. Notably, neutral aqueous solutions of Asp-1-deaza-dAMP degraded spontaneously even at 4 °C through exclusive PO bond hydrolysis (a 50-fold reactivity difference for Asp-1-deaza-dAMP vs. Asp-3-deaza-dAMP at pD 5 and 70 °C). Conformational studies by NMR spectroscopy and molecular modeling suggest the involvement of the protonated N3 atom in adenine and 1- and 7-deazaadenine in the intramolecular catalysis of the hydrolysis reaction through the rare syn conformation.status: publishe

Effect of ethylene glycol and glycerol fructosides on the activity and product specificity of bacterial and plant fructosyltransferases

Fructosyltransferases (FTs) are key enzymes in plants and bacteria to synthesize fructans. To gain insight on the specificity of the hexose subsites in the active site of FTs, ethylene glycol fructoside (EGF) and glycerol fructoside (GF), containing fructose in the furanose configuration, were synthesized in vitro and used as substrates to study the effect on the activity of bacterial levansucrase (BsLS), chicory root sucrose:sucrose 1-fructosyltransferase (1-SST) and fructan:fructan 1-fructosyltransferase (1-FFT). The results demonstrated that EGF and GF, at physiologically relevant concentrations, were efficient acceptor substrates for BsLS and 1-FFT, but not for 1-SST. EGF and GF cannot be used as donor substrates for BsLS, 1-SST and 1-FFT. A model is proposed to explain the subsite specificity differences between the three FTs involved in this study.status: publishe

Global Reactivity Models are Impactful in Industrial Synthesis Applications

Artificial Intelligence is revolutionizing many aspects of the pharmaceutical industry. Deep learning models are now routinely applied to guide drug discovery projects leading to faster and improved findings, but there are still many tasks with enormous unrealized potential. One such task is the reaction yield prediction. Every year more than one fifth of all synthesis attempts result in product yields which are either zero or too low. This equates to chemical and human resources being spent on activities which ultimately do not progress the programs, leading to a triple loss when accounting for the cost of opportunity in time wasted. In this work we pre-train a BERT model on more than 16 million reactions from 4 different data sources, and fine tune it to achieve an uncertainty calibrated global yield prediction model. This model is an improvement upon state of the art not just from the increase in pre-train data but also by introducing a new embedding layer which solves a few limitations of SMILES and enables integration of additional information such as equivalents and molecule role into the reaction encoding, the model is called BERT Enriched Embedding (BEE). The model is benchmarked on an open-source dataset against a state-of-the-art synthesis focused BERT showing a near 20-point improvement in r2 score. The model is fine-tuned and tested on an internal company data benchmark, and a prospective study shows that the application of the model can reduce the total number of negative reactions (yield under 5%) ran in Janssen by at least 34%. Lastly, we corroborate the previous results through experimental validation, by directly deploying the model in an on-going drug discovery project and showing that it can also be used successfully as a reagent recommender due to its fast inference speed and reliable confidence estimation, a critical feature for industry application

Sedoheptulose accumulation under CO2 enrichment in leaves of Kalanchoë pinnata: a novel mechanism to enhance C and P homeostasis?

In contrast to the well documented roles of its mono- and bisphosphate esters, the occurrence of free sedoheptulose in plant tissues remains a matter of conjecture. The present work sought to determine the origin of sedoheptulose formation in planta as well as its physiological importance. Elevated CO2 and sucrose induction experiments were used to study sedoheptulose metabolism in the CAM plants Kalanchoë pinnata and Sedum spectabile. Experimental evidence suggests that sedoheptulose is produced from the oxidative pentose phosphate pathway intermediate sedoheptulose-7-phosphate, by a sedoheptulose-7-phosphate phosphatase. Carbon flux through this pathway is stimulated by increased triose-P levels (elevated CO2, compromised sink availability, sucrose incubation of source leaves) and attenuated by ADP and Pi. The accumulation of free sedoheptulose is proposed to act as a mechanism contributing to both C and P homeostasis by serving as an alternative carbon store under elevated CO2 or compromised sink capacity to avoid sucrose accumulation, depletion of inorganic phosphate and suppression of photosynthesis. It remains to be established whether this acclimation avoiding mechanism is confined to CAM plants, which might be especially vulnerable to Pi imbalances, or whether some C3 and C4 plants also dispose of the genetic capacity to induce and accelerate sedoheptulose synthesis upon CO2 elevation.status: publishe

Synthesis and biological evaluation of an (123)I-labeled bicyclic nucleoside analogue (BCNA) as potential SPECT tracer for VZV-tk reporter gene imaging

An iodine-123 labeled bicyclic nucleoside analogue ([(123)I]-4) has been synthesized and evaluated as a potential single photon emission tomography (SPECT) reporter probe for the non-invasive imaging of expression of the varicella zoster virus thymidine kinase (VZV-tk) reporter gene. In vitro enzymatic assays revealed that the non-radioactive mono-iodo derivative 4 has good affinity for VZV-TK (IC(50): 4.2muM). Biodistribution of [(123)I]-4 was examined in normal mice. Evaluation of [(123)I]-4 in HEK-293T cells showed 1.74-fold higher accumulation in VZV-TK-expressing cells compared to control cells.status: publishe