C3 Position Research Articles

Studies on the Oxidative Damage of the Wobble 5-Methylcarboxymethyl-2-Thiouridine in the tRNA of Eukaryotic Cells with Disturbed Homeostasis of the Antioxidant System

We have previously shown that 2-thiouridine (S2U), either as a single nucleoside or as an element of RNA chain, is effectively desulfurized under applied in vitro oxidative conditions. The chemically induced desulfuration of S2U resulted in two products: 4-pyrimidinone nucleoside (H2U) and uridine (U). Recently, we investigated whether the desulfuration of S2U is a natural process that also occurs in the cells exposed to oxidative stress or whether it only occurs in the test tube during chemical reactions with oxidants at high concentrations. Using different types of eukaryotic cells, such as baker’s yeast, human cancer cells, or modified HEK293 cells with an impaired antioxidant system, we confirmed that 5-substituted 2-thiouridines are oxidatively desulfurized in the wobble position of the anticodon of some tRNAs. The quantitative LC-MS/MS-MRMhr analysis of the nucleoside mixtures obtained from the hydrolyzed tRNA revealed the presence of the desulfuration products of mcm5S2U: mcm5H2U and mcm5U modifications. We also observed some amounts of immature cm5S2U, cm5H2U and cm5U products, which may have indicated a disruption of the enzymatic modification pathway at the C5 position of 2-thiouridine. The observed process, which was triggered by oxidative stress in the living cells, could impair the function of 2-thiouridine-containing tRNAs and alter the translation of genetic information.

Synthesis and Characterization of a Novel Intrinsically Fluorescent Analog of Cholesterol with Improved Photophysical Properties.

Live-cell imaging of cholesterol trafficking depends on suitable cholesterol analogs. However, existing fluorescent analogs of cholesterol either show very different physicochemical properties compared to cholesterol or demand excitation in the ultraviolet spectral region. We present a strategy to synthesize two novel intrinsically fluorescent sterol probes with a close resemblance of cholesterol. The analogs contain four conjugated double bonds in the ring system and either a keto group (probe 5) or a hydroxy group (probe 6) in the C3 position. The emission of 5 is in the visible range of the spectrum, i.e., red-shifted by 150 nm compared to the widely used dehydroergosterol. Together with its high multiphoton absorption, this allows for imaging of 5 on conventional microscopes, including multicolor 3D and time-lapse microscopy. Molecular dynamics simulations and nuclear magnetic resonance spectroscopy reveal that 5 can condense the fatty acyl chains of phospholipids in model membranes. In giant unilamellar vesicles, 5 partitions equally into the liquid-ordered and disordered phases. In contrast, 6 emits in the ultraviolet range and is unstable in solution, preventing its use in live-cell imaging applications. The good photophysical properties of 5 make it a suitable analogue for improved live-cell imaging of sterol transport.

Bioinspired synthesis of cucurbalsaminones B and C.

Cucurbalsaminones B (1) and C (2) are two abeo-cucurbitane triterpenoids with a unique 5/6/3/6/5-fused ring system and exhibited potent multidrug resistance (MDR)-reversing activity. Herein, we report the first synthesis of these two natural products, both of them were accomplished in 14 steps from commercially available inexpensive resource compound lanosterol. Key features of this synthesis include a biomimetic tandem Wagner-Meerwein type lanostane-to-cucurbitane rearrangement followed by a bioinspired photochemical oxa-di-π-methane (ODPM) rearrangement to complete the skeleton construction and an Eosin Y photoinduced Barton-McCombie deoxygenation to realize the challenging oxidation state adjustment of the sterically hindered C11 position.

Bioinspired synthesis of cucurbalsaminones B and C

Cucurbalsaminones B (1) and C (2) are two abeo‐cucurbitane triterpenoids with a unique 5/6/3/6/5‐fused ring system and exhibited potent multidrug resistance (MDR)‐reversing activity. Herein, we report the first synthesis of these two natural products, both of them were accomplished in 14 steps from commercially available inexpensive resource compound lanosterol. Key features of this synthesis include a biomimetic tandem Wagner‐Meerwein type lanostane‐to‐cucurbitane rearrangement followed by a bioinspired photochemical oxa‐di‐π‐methane (ODPM) rearrangement to complete the skeleton construction and an Eosin Y photoinduced Barton‐McCombie deoxygenation to realize the challenging oxidation state adjustment of the sterically hindered C11 position.

Au(I)-Catalyzed Regioselective Hydrogen Isotope Labeling of Indoles.

The gold(I)-catalyzed hydrogen isotope exchange reaction on indoles and related heterocycles is described under mild conditions and low catalyst loadings, using CD3OD and D2O as readily available deuterium sources. C3-unsubstituted indoles are labeled at the C3 position with exquisite regioselectivity, while C3-substituted indoles are labeled at the C2 position. The method is also applicable to the regioselective tritiation of indoles. Mechanistic studies revealed the involvement of aurated indoles as key intermediates.

Synthetic strategy for polyhydroxylated indolizidine iminosugar from sugar-derived HWE precursor

The diversity of polyhydroxylated indolizidine (PI) iminosugars is ever-expanding due to the wide range of methods developed and substrate choice during synthesis. This study used an HWE precursor derived from d-glucose to extend the chain length at the C1 position. A double reductive amination and dihydroxylation of the resulting olefin, followed by intramolecular cyclization, enabled the successful synthesis of a new PI. In addition, the precursor intermediate for Mitsunobu reaction was utilized in synthesis of a new iminononulol.

Synthesis, Structure, and In Vitro Biological Evaluation of Semi-Synthetic Derivatives of Betulin

Betulin and α-lipoic acid are naturally occurring substances with different biological properties. Combining two phytochemical units into a conjugate is a frequently used method to obtain new compounds with better pharmacokinetic parameters. This research concerned the preparation of lipoate derivatives of betulin using the Steglich method. Experimental lipophilicity values were determined for target compounds 6–10 by reversed-phase thin-layer chromatography. In silico methods were used to calculate the physicochemical parameters and lipophilicity of new derivatives and to determine the probable directions of biological activity. α-Lipoic acid, betulin, and lipoate derivatives 6–10 were tested for antiproliferative activity against MV4-11, A549, MCF-7, PC-3, HCT116, MiaPaca-2, and Hs294T cancer cells. 3-(5-(1,2-Dithiolan-3-yl)pentanoyl))betulin 10 showed moderate anticancer activity against MV4-11, PC-3, and HCT116, with IC50 values in the range of 39.8–76.7 µM. The introduction of a dithiolate substituent at the C3 position in 28-acetylbetulin gave compound 9 the highest activity (IC50 = 37.9 µM), in the ratio of biphenotypic B myelomonocytic leukemia cells (MV4-11). All lipoate derivatives were inactive towards normal cells.

Abiraterone and Galeterone, Powerful Tools Against Prostate Cancer: Present and Perspective

Due to the high prostate cancer incidence worldwide, the development of different methods of treatment continues to be a hot research topic. Since its first clinical application at the beginning of the 2010s, abiraterone in the form of prodrug abiraterone acetate continues to be the most used hormone derivative in the treatment of castration-resistant prostate cancer. This is the reason behind the publication of many scientific results regarding its synthesis, biological activity, metabolism, novel designed steroid derivatives based on its structure, etc. A similar steroid compound with a heterocycle in the C17 position, called galeterone, also designed to treat prostate cancer, continues to be in clinical studies, which provides further proof of the importance of these steroid derivatives. Besides prostate cancer treatment, abiraterone showed indications for possible clinical application in the treatment of breast, ovarian, lung, kidney, salivary gland, and adrenocortical cancer, congenital adrenal hyperplasia, Cushing’s syndrome, and COVID-19, while galeterone is investigated for its use against prostate, pancreatic, and breast cancer. Herein, we report a review comprising methods of synthesis, possible clinical applications, and mechanisms of action, as well as structures and bioactivities of derivatives of these two important steroids.

A Novel A105Y Mutant of CYP17A1 Exhibits Almost Perfect Regioselectivity in the Production of 17α-Hydroxyprogesterone.

17α-Hydroxyprogesterone (17α-OHP) is a steroid hormone with significant biological activity that can be obtained by catalyzing progesterone (PROG), the main product of sitosterol, through CYP17A1. However, increasing the catalytic specificity of HCYP17A1 for C17 hydroxylation of progesterone (PROG) poses a formidable challenge due to the close proximity of the C16 and C17 positions. In this study, a rational design was utilized to alter the spatial configuration of the substrate channel, leading to the complete abolition of its 16-hydroxylation activity. Subsequent molecular dynamics simulations revealed that the A105Y mutation heightened the rigidity of the G95-I112 region of CYP17A1, consequently regulating the direction of the entry of PROG into the catalytic pocket. Moreover, the establishment of hydrogen bonding between Y105 and R239, coupled with Pi-stacking of A105Y with F114, effectively immobilizes the substrate PROG in a fixed position, explaining the practically perfect regioselectivity observed in A105Y. Finally, a multifaceted enzymatic cascade system, incorporating A105Y, cytochrome P450 reductase (CPR), and glucose-6-phosphate dehydrogenase (ZWF) for NADPH cofactor regeneration, was constructed in Pichia pastoris GS115. The resulting biocatalyst produced 106 ± 3.2 mg L-1 17α-OHP, a 4.6-fold increase compared with A105Y alone. Thus, this study provides valuable insights for improving the regioselectivity and activity of P450 enzymes.

Structures of a DNA Polymerase Caught while Incorporating Responsive Dual-Functional Nucleotide Probes.

Functionalizing nucleic acids using DNA polymerases is essential in biophysical and biotechnology applications. This study focuses on understanding how DNA polymerases recognize and incorporate nucleotides with diverse chemical modifications, aiming to develop advanced nucleotide probes. We present the crystal structures of ternary complexes of Thermus aquaticus DNA polymerase (KlenTaq) with C5-heterocycle-modified environment-sensitive 2'-deoxyuridine-5'-triphosphate (dUTP) probes. These nucleotides include SedUTP, BFdUTP and FBFdUTP, which bear selenophene, benzofuran and fluorobenzofuran, respectively, at the C5 position of uracil, and exhibit high conformational sensitivity. SedUTP and FBFdUTP serve as dual-app probes, combining a fluorophore with X-ray anomalous scattering Se or 19F NMR labels. Our study reveals that the size of the heterocycle influences how DNA polymerase families A and B incorporate these modified nucleotides during single nucleotide incorporation and primer extension reactions. Remarkably, FBFdUTP's responsiveness enabled real-time monitoring of the binary complex formation and polymerase activity through fluorescence and 19F NMR. Comparative analysis of incorporation profiles, fluorescence, 19F NMR data, and crystal structures of ternary complexes highlights the enzyme's plasticity. Key insights are provided into the role of gatekeeper amino acids (Arg660 and Arg587) in accommodating and processing these modified substrates, offering a structural basis for next-generation nucleotide probe development.

Structures of a DNA Polymerase Caught while Incorporating Responsive Dual‐Functional Nucleotide Probes

Functionalizing nucleic acids using DNA polymerases is essential in biophysical and biotechnology applications. This study focuses on understanding how DNA polymerases recognize and incorporate nucleotides with diverse chemical modifications, aiming to develop advanced nucleotide probes. We present the crystal structures of ternary complexes of Thermus aquaticus DNA polymerase (KlenTaq) with C5‐heterocycle‐modified environment‐sensitive 2′‐deoxyuridine‐5′‐triphosphate (dUTP) probes. These nucleotides include SedUTP, BFdUTP and FBFdUTP, which bear selenophene, benzofuran and fluorobenzofuran, respectively, at the C5 position of uracil, and exhibit high conformational sensitivity. SedUTP and FBFdUTP serve as dual‐app probes, combining a fluorophore with X‐ray anomalous scattering Se or 19F NMR labels. Our study reveals that the size of the heterocycle influences how DNA polymerase families A and B incorporate these modified nucleotides during single nucleotide incorporation and primer extension reactions. Remarkably, FBFdUTP's responsiveness enabled real‐time monitoring of the binary complex formation and polymerase activity through fluorescence and 19F NMR. Comparative analysis of incorporation profiles, fluorescence, 19F NMR data, and crystal structures of ternary complexes highlights the enzyme's plasticity. Key insights are provided into the role of gatekeeper amino acids (Arg660 and Arg587) in accommodating and processing these modified substrates, offering a structural basis for next‐generation nucleotide probe development.

Traceless Nucleophile Strategy for C5-Selective C-H Sulfonylation of Pyridines.

The functionalization of pyridines is crucial for the rapid construction and derivatization of agrochemicals, pharmaceuticals, and materials. Conventional functionalization approaches have primarily focused on the ortho- and para-positions, while achieving precise meta-selective functionalization, particularly at the C5 position in substituted pyridines, remains a formidable challenge due to the intrinsic electronic properties of pyridines. Herein, we present a new strategy for meta- and C5-selective C-H sulfonylation of N-amidopyridinium salts, which employs a transient enamine-type intermediate generated through a nucleophilic addition to N-amidopyridinium salts. This process harnesses the power of electron donor-acceptor complexes, enabling high selectivity and broad applicability, including the construction of complex pyridines bearing valuable sulfonyl functionalities under mild conditions without the need for an external photocatalyst. The remarkable C5 selectivity, combined with the broad applicability to late-stage functionalization, significantly expands the toolbox for pyridine functionalization, unlocking access to previously unattainable meta-sulfonylated pyridines.

Traceless Nucleophile Strategy for C5‐Selective C−H Sulfonylation of Pyridines

AbstractThe functionalization of pyridines is crucial for the rapid construction and derivatization of agrochemicals, pharmaceuticals, and materials. Conventional functionalization approaches have primarily focused on the ortho‐ and para‐positions, while achieving precise meta‐selective functionalization, particularly at the C5 position in substituted pyridines, remains a formidable challenge due to the intrinsic electronic properties of pyridines. Herein, we present a new strategy for meta‐ and C5‐selective C−H sulfonylation of N‐amidopyridinium salts, which employs a transient enamine‐type intermediate generated through a nucleophilic addition to N‐amidopyridinium salts. This process harnesses the power of electron donor‐acceptor complexes, enabling high selectivity and broad applicability, including the construction of complex pyridines bearing valuable sulfonyl functionalities under mild conditions without the need for an external photocatalyst. The remarkable C5 selectivity, combined with the broad applicability to late‐stage functionalization, significantly expands the toolbox for pyridine functionalization, unlocking access to previously unattainable meta‐sulfonylated pyridines.

Palladium‐Catalyzed Regioselective C(4)−H Fluoroalkoxylation of Indoles Through Weak Chelation Assistance

AbstractInstalling fluoroalkyl motifs into biorelevant indoles is particularly interesting due to their ubiquitous presence in drug molecules. Herein, we demonstrate the regioselective C4 fluoroalkoxylation of indoles using fluoroalcohols via palladium‐catalyzed chelation‐assisted C─H activation. The weak chelating benzoyl moiety at the C3 position acts as a directing group for remote C(4)─H fluoroalkoxylation of diversely substituted indoles. This methodology demonstrates a high level of regioselectivity and tolerates a range of crucial functional groups, yielding diverse trifluoroalkoxylated indoles in moderate to good yields. Removal of directing/protecting groups and further functionalization established the synthetic utility of the methodology. A preliminary mechanistic investigation is conducted by isolating the palladacycle intermediate and performing the deuterium scrambling study.

Identification of a 301 bp promoter core region of the SrUGT91D2 gene from Stevia rebaudiana that contributes to hormone and abiotic stress inducibility

BackgroundThe UDP-glucuronosyltransferase 91D2 (SrUGT91D2) gene is a crucial element in the biosynthetic pathway of steviol glycosides (SGs) and is responsible for creating 1,2-β-D glucosidic bonds at the C19 and C13 positions. This process plays a vital role in the synthesis of rebaudioside M (RM) and rebaudioside D (RD). The promoter, which regulates gene expression, requires functional analysis to understand gene expression regulation. However, investigations into the function of the promoter of SrUGT91D2 (pSrUGT91D2) have not been reported.ResultsThe pSrUGT91D2 was isolated from six S. rebaudiana lines, and subsequent multiple sequence comparisons revealed the presence of a 26 bp inDel fragment (pSrUGT91D2-B1188 type) in lines GP, GX, 110, 1114, and B1188 but not in the pSrUGT91D2 of line 023 (pSrUGT91D2-023 type). Bioinformatics analysis revealed a prevalence of significant cis-regulatory elements (CREs) within the promoter sequences, including those responsive to abscisic acid, light, anaerobic conditions, auxin, drought, low temperature, and MeJA. To verify the activity of pSrUGT91D2, the full-length promoter and a series of 5’ deletion fragments (P1–P7) and a 3’ deletion fragment (P8) from various lines were fused with the reporter β-glucuronidase (GUS) gene to construct the plant expression vector, pCAMBIA1300-pro∷GUS. The transcriptional activity of these genes was examined in tobacco leaves through transient transformation. GUS tissue staining analysis and enzyme activity assays demonstrated that both the full-length promoter and truncated pSrUGT91D2 were capable of initiating GUS expression in tobacco leaves. Interestingly, P8-pSrUGT91D2-B1188 (containing the inDel segment, 301 bp) exhibited enhanced activity in driving GUS gene expression. Transient expression studies of P8-pSrUGT91D2-B1188 and P8-pSrUGT91D2-023 in response to exogenous hormones (abscisic acid and indole-3-acetic acid) and light indicated the necessity of the inDel region for P8 to exhibit transcriptional activity, as it displayed strong responsiveness to abscisic acid (ABA), indole-3-acetic acid (IAA), and light induction.ConclusionsThese findings contribute to a deeper understanding of the regulatory mechanism of the upstream region of the SrUGT91D2 gene and provide a theoretical basis for future studies on the interaction between CREs of pSrUGT91D2 and related transcription factors.

Investigating the inhibition of xanthine oxidase by five catechins: Kinetic studies, spectroscopy, molecular docking, and dynamics simulations

Catechins compounds from tea have demonstrated significant inhibitory effects on xanthine oxidase (XOD). However, the precise inhibitory mechanisms of the main catechins on XOD remain to be fully elucidated. This study explored the inhibition mechanisms and binding characteristics of five catechins (GC, EGC, EC, EGCG, and ECG) on XOD through a combination of inhibition kinetics, multi-spectroscopy analysis, molecular docking, and dynamics simulations. Among the catechins, EGCG and ECG exhibited the most potent inhibitory activities against XOD. All five catechins were found to exhibit mixed inhibition, affecting the hydrophobic groups and secondary structure of XOD predominantly through hydrophobic interactions and hydrogen bonding. Molecular dynamics simulations revealed that a 3,4,5-trihydroxybenzoic acid moiety at C3 position significantly enhances the binding affinity of EGCG and ECG to XOD. Additionally, the decrease of β-sheet and random coil induced by EGCG and ECG was found to be crucial for enhancing inhibitory activity of XOD. In vitro cell experiments showed that EGCG and ECG significantly reduced high uric acid levels of BRL-3A cell. This study elucidates the inhibitory mechanisms of catechins on XOD, paving the way for their application as XOD inhibitors to combat hyperuricemia.

Fragment Addition-Based Design of Heteroaromatic-Biphenyl-DAPYs as Potent and Orally Available Non-nucleoside Reverse Transcriptase Inhibitors Featuring Significantly Enhanced Safety.

Our previously disclosed biphenyl-DAPY 3 emerged as a potent inhibitor against WT HIV-1 and various mutant strains. Yet, its journey toward clinical application was thwarted by pronounced cytotoxicity and low selectivity (CC50 = 6 μM, SI = 3515). The safety improvement approach we employed in this work entailed the incorporation of diverse heteroaromatic substituents at the C5 position to exploit the tolerant regions of the NNRTIs' binding pocket through fragment addition-based drug design strategy, ultimately leading to the identification of a series of novel heteroaromatic-biphenyl-DAPYs. The exemplary compound 10d revealed a striking reduction in cytotoxicity (CC50 > 272.81 μM), nearly 45.5 times lower than 3, while showcasing 15-fold increase in selectivity (SI > 52632). This analog sustained exceptional anti-HIV-1 activity against both WT HIV-1 (EC50 = 5 nM) and various mutant strains. Compared to 3, a markedly slower rate of metabolism in human liver microsomes of 10d was observed. Its pharmacokinetic profile was equally captivating, featuring excellent oral bioavailability (F = 57.4%). Moreover, 10d exhibited a delicate sensitivity toward CYP, minimal inhibition of hERG, and no detectable acute toxicity in vivo. These enchanting findings illuminated the potential of 10d as a promising candidate for HIV-1 therapy.

Amino acid based natural deep eutectic solvents: An efficient catalyst and solvent for N-acetyl-d-glucosamine conversion into organonitrogen chemicals

Organonitrogen chemicals are widely used and in great demand in the food, pharmaceutical and chemical industries. Hence, it is important to develop green and efficient synthesis paths to organonitrogen chemicals for their use as new resources. Herein, we propose the preparation of organonitrogen chemicals from chitin biomass monomer N-acetyl-d-glucosamine (GlcNAc) promoted by natural deep eutectic solvents (NADESs). The selective generation of 3-acetamido-5-(1′,2′-dihydroxyethyl) furan (Chromogen III) and 3-acetamido-5-acetylfuran (3A5AF) from GlcNAc was achieved by the design of NADESs components. GlcNAc reacts in binary NADES proline-glycerol (Pro-Gly) to generate 26 % of Chromogen III and 39 % of 3A5AF in ternary NADES proline-glycerol-lactic acid (Pro-Gly-LA). In addition, the intermolecular hydrogen bonds in NADESs were confirmed through 1H diffusion-ordered spectroscopy (DOSY) NMR technology. And the results of 13C NMR chemical shift titration indicated interaction between NADESs and the C3 and C6 position of GlcNAc. Based on the NMR experiments a mechanism is proposed.

Computational Design of Ligands for the Ir-Catalyzed C5-Borylation of Indoles through Tuning Dispersion Interactions.

The indole moiety is ubiquitous in natural products and pharmaceuticals. C-H borylation of the benzenoid moiety of indoles is a challenging task, especially at the C5 position. We have combined computational and experimental studies to introduce multiple noncovalent interactions, especially dispersion, between the substrate and catalytic ligand to realize C5-borylation of indoles with high reactivity and selectivity. The successful computational predictions of new ligands should be suitable for ligand design in other transition-metal catalyzed reactions.

Development of non-sedating benzodiazepines with in vivo antischistosomal activity.

The neglected tropical disease schistosomiasis infects over 200 million people worldwide and is treated with just one broad-spectrum antiparasitic drug (praziquantel). Alternative drugs are needed in the event of emerging praziquantel resistance or treatment failure. One promising lead that has shown efficacy in animal models and a human clinical trial is the benzodiazepine meclonazepam, discovered by Roche in the 1970s. Meclonazepam was not brought to market because of dose-limiting sedative side effects. However, the human target of meclonazepam that causes sedation (GABAARs) is not orthologous to the parasite targets that cause worm death. Therefore, we were interested in whether the structure of meclonazepam could be modified to produce antiparasitic benzodiazepines that do not cause host sedation. We synthesized 18 meclonazepam derivatives with modifications at different positions on the benzodiazepine ring system and tested them for in vitro antiparasitic activity. This identified five compounds that progressed to in vivo screening in a murine model, two of which cured parasite infections with comparable potency to meclonazepam. When these two compounds were administered to mice that were run on the rotarod test, both were less sedating than meclonazepam. These findings demonstrate the proof of concept that meclonazepam analogs can be designed with an improved therapeutic index and point to the C3 position of the benzodiazepine ring system as a logical site for further structure-activity exploration to further optimize this chemical series.