Thymidine Kinase-Independent Click Chemistry DNADetect Probes for DNA Proliferation Assessment in Malaria Parasites.

Metabolic chemical probes are small-molecule reagents that utilize naturally occurring biosynthetic enzymes for in situ incorporation into biomolecules of interest. These reagents can be used to label, detect, and track important biological processes within living cells including protein synthesis, protein glycosylation, and nucleic acid proliferation. A limitation of current chemical probes, which have largely focused on mammalian cells, is that they often cannot be applied to other organisms due to metabolic differences. For example, the thymidine derivative 5-ethynyl-2'-deoxyuridine (EdU) is a gold standard metabolic chemical probe for assessing DNA proliferation in mammalian cells; however, it is unsuitable for the study of malaria parasites due to Plasmodium species lacking the thymidine kinase enzyme that is essential for metabolism of EdU. Herein, we report the design and synthesis of new thymidine-based probes that sidestep the requirement for a thymidine kinase enzyme in Plasmodium. Two of these DNADetect probes exhibit robust labeling of replicating asexual intraerythrocytic Plasmodium falciparum parasites, as determined by flow cytometry and fluorescence microscopy using copper-catalyzed azide-alkyne cycloaddition to a fluorescent azide. The DNADetect chemical probes are synthetically accessible and thus can be made widely available to researchers as tools to further understand the biology of different Plasmodium species, including laboratory lines and clinical isolates.

Identification and characterization of two drug-like fragments that bind to the same cryptic binding pocket of Burkholderia pseudomallei DsbA.

Disulfide-bond-forming proteins (Dsbs) play a crucial role in the pathogenicity of many Gram-negative bacteria. Disulfide-bond-forming protein A (DsbA) catalyzes the formation of the disulfide bonds necessary for the activity and stability of multiple substrate proteins, including many virulence factors. Hence, DsbA is an attractive target for the development of new drugs to combat bacterial infections. Here, two fragments, bromophenoxy propanamide (1) and 4-methoxy-N-phenylbenzenesulfonamide (2), were identified that bind to DsbA from the pathogenic bacterium Burkholderia pseudomallei, the causative agent of melioidosis. The crystal structures of oxidized B. pseudomallei DsbA (termed BpsDsbA) co-crystallized with 1 or 2 show that both fragments bind to a hydrophobic pocket that is formed by a change in the side-chain orientation of Tyr110. This conformational change opens a `cryptic' pocket that is not evident in the apoprotein structure. This binding location was supported by 2D-NMR studies, which identified a chemical shift perturbation of the Tyr110 backbone amide resonance of more than 0.05 p.p.m. upon the addition of 2 mM fragment 1 and of more than 0.04 p.p.m. upon the addition of 1 mM fragment 2. Although binding was detected by both X-ray crystallography and NMR, the binding affinity (Kd) for both fragments was low (above 2 mM), suggesting weak interactions with BpsDsbA. This conclusion is also supported by the crystal structure models, which ascribe partial occupancy to the ligands in the cryptic binding pocket. Small fragments such as 1 and 2 are not expected to have a high energetic binding affinity due to their relatively small surface area and the few functional groups that are available for intermolecular interactions. However, their simplicity makes them ideal for functionalization and optimization. The identification of the binding sites of 1 and 2 to BpsDsbA could provide a starting point for the development of more potent novel antimicrobial compounds that target DsbA and bacterial virulence.

Synthesis of 5-Alkynyl Substituted 2'-Arabinosyl 2'-Halogenated Uridine Nucleosides.

This unit describes the detailed preparation of 5-alkynyl-2'-halogenated arabinosyl uridine nucleosides (2'-halo-ara-EdU) from uridine. These compounds were synthesized as prospective chemical probes for the detection of DNA synthesis in proliferating cells. Currently, this is the only synthetic methodology reported to access these compounds. The key to success of the synthetic approach was to employ a 3-N-nitro-protecting group to stabilize the required 2'-triflate nucleoside precursor toward nucleophilic substitution. Several synthetic challenges were overcome to accommodate the combination of a 5-alkyne and 3-N-nitro functional group, including facile introduction and removal of the N-nitro group, and removal of the sugar acetyl groups under acidic conditions. © 2019 by John Wiley & Sons, Inc.

Stereoselective Synthesis of Highly Functionalized Arabinosyl Nucleosides through Application of an N-Nitro Protecting Group.

2'-Deoxy-2',5-disubstituted arabinosyl uridine derivatives bearing a halogen (Cl, Br or I) at C2' and an ethynyl group at C5 have been synthesized in 6 steps from 2',3',5'-tri- O-acetyl-5-iodo-uridine in overall yields of 61% (compound 3, Cl), 47% (compound 4, Br), and 19% (compound 5, I). Stabilization of a 2'- O-triflyl leaving group intermediate to overcome spontaneous intramolecular 2,2'-anhydro uridine formation was pivotal to the synthesis. Specifically, to favor SN2 reaction with a halogen nucleophile over intramolecular cyclization, the nucleophilicity of O-2 oxygen was reduced by incorporation of an adjacent electron withdrawing nitro substituent at N-3. The introduction of the 3- N-nitro group proceeded rapidly (nitronium trifluoroacetate, 1 min) and in quantitative yield. A one-pot method to remove the 3- N-nitro group by reductive nitration (zinc metal in acetic acid, 5 min) and the silyl protecting groups of the alkyne and 3',5' hydroxyls (fluoride reagent, 16 h) was established as the final synthetic step. This application of the 3- N-nitro protecting group addresses the significant shortfalls of the conventional approach to synthesis of 2' modified nucleosides, wherein condensation of a 2' modified sugar fragment with a pyrimidine base provides poor stereocontrol of N-glycosylation, low yields and incompatibility with 2' iodo sugars.

Development of ethynyl-2'-deoxyuridine chemical probes for cell proliferation.

A common method of evaluating cellular proliferation is to label DNA with chemical probes. 5-Ethynyl-2'-deoxyuridine (EdU) is a widely utilized chemical probe for labeling DNA, and upon incorporation, EdU treatment of cells is followed by a reaction with a small molecule fluorescent azide to allow detection. The limitations when using EdU include cytotoxicity and a reliance on nucleoside active transport mechanisms for entry into cells. Here we have developed six novel EdU pro-labels that consist of EdU modified with variable lipophilic acyl ester moieties. This pro-label:chemical probe relationship parallels the prodrug:drug relationship that is employed widely in medicinal chemistry. EdU and EdU pro-labels were evaluated for their labeling efficacy and cytotoxicity. Several EdU pro-label analogues incorporate into DNA at a similar level to EdU, suggesting that nucleoside transporters can be bypassed by the pro-labels. These EdU pro-labels also had reduced toxicity compared to EdU.

Labeling of Cellular DNA with a Cyclosal Phosphotriester Pronucleotide Analog of 5-ethynyl-2'-deoxyuridine.

DNA synthesis is a fundamental biological process central to all proliferating cells, and the design of small molecule probes that allow detection of this DNA is important for many applications. 5-Ethynyl-2'-deoxyuridine, known as EdU, has become a workhorse for metabolic labeling of DNA in mammalian cells, followed by bioconjugation to a small molecule fluorescent azide using copper-catalyzed azide-alkyne cycloaddition (CuAAC), click chemistry, to allow detection. In this study, we demonstrate that a cyclosal phosphotriester pronucleotide analog of EdU is suitable for metabolic incorporation into DNA of proliferating cells and subsequent labeling by CuAAC. This analog has two advantages over EdU; first, by delivering EdU with a preinstalled 5'-monophosphate moiety, it bypasses the need for thymidine kinase processing, and second, the increased lipophilicity compared to EdU may enable passive diffusion across the cell membrane and may circumvent the reliance on nucleoside active transport mechanisms for cellular uptake. These advantages pave the way for the development of additional novel pronucleotides to widen experimental opportunities for future bioconjugation applications involving cellular DNA.