Non-natural Nucleoside Research Articles

Synthesis of Nonnatural Nucleoside Diphosphate Sugars

AbstractRecently, we reported an efficient chemical method for the synthesis of a variety of naturally occurring nucleoside diphosphate (NDP) sugars. This method, which is based on the cycloSal approach, can also be used, in principle, for the preparation of rare or even nonnatural NDP sugars. Herein, the syntheses of sulfoquinovose‐, glucose‐6‐sulfate‐, L‐galactose‐, and 2‐fluoroglycopyranoside‐containing NDP sugars are presented, as well as the synthesis of NDP sugars with non‐natural nucleosides. The reactions described gavestereoisomerically defined NDP sugars in high yields and short reaction times.

Adenosine-1,3-diazaphenoxazine Derivative for Selective Base Pair Formation with 8-Oxo-2′-deoxyguanosine in DNA

The selective detection of 8-oxo-2'-deoxyguanosine (8-oxo-dG) in DNA without chemical or enzymatic treatment is an attractive tool for genomic research. We designed and synthesized the non-natural nucleoside analogue, the adenosine-1,3-diazaphenoxazine (Adap) derivative, for selective recognition of 8-oxo-dG in DNA. This study clearly showed that Adap has a highly selective stabilizing effect on the duplex containing the Adap-8-oxo-dG base pair. Furthermore, the fluorescent property of Adap was shown to be useful for the selective detection of 8-oxo-dG in the duplex DNA. To the best of our knowledge, this is the first successful demonstration of a non-natural nucleoside with a high selectivity for 8-oxo-dG in DNA.

Generation of chemically engineered ribosomes for atomic mutagenesis studies on protein biosynthesis

The protocol describes the site-specific chemical modification of 23S rRNA of Thermus aquaticus ribosomes. The centerpiece of this 'atomic mutagenesis' approach is the site-specific incorporation of non-natural nucleoside analogs into 23S rRNA in the context of the entire 70S ribosome. This technique exhaustively makes use of the available crystallographic structures of the ribosome for designing detailed biochemical experiments aiming at unraveling molecular insights of ribosomal functions. The generation of chemically engineered ribosomes carrying a particular non-natural 23S rRNA residue at the site of interest, a procedure that typically takes less than 2 d, allows the study of translation at the molecular level and goes far beyond the limits of standard mutagenesis approaches. This methodology, in combination with the presented tests for ribosomal functions adapted to chemically engineered ribosomes, allows unprecedented molecular insight into the mechanisms of protein biosynthesis.

ChemInform Abstract: Nonnatural Nucleosides Based on 1,2,4‐Triazolo[1,5‐a]pyrimidin‐7‐ones.

AbstractTwo methods for the synthesis of non‐natural nucleosides are developed.

A Novel Non-Natural Nucleoside That Influences P-Glycoprotein Activity and Mediates Drug Resistance

Multidrug resistance during cancer chemotherapy is commonly acquired by overexpression of the ATP binding cassette transporter, P-glycoprotein (P-gp). As such, inhibitors that target P-gp activity represent potential therapeutic agents against this form of drug resistance. This study evaluated the ability of various non-natural nucleosides that mimic the core structure of adenosine to modulate drug resistance by inhibiting the ATPase activity to P-gp. Of several analogues tested, only one novel non-natural nucleoside, 5-cyclohexylindolyl-2'-deoxyribose (5-CHInd), behaves as a P-gp inhibitor. Although 5-CHInd is an adenosine analogue that should block the binding of ATP, the non-natural nucleoside surprisingly stimulates the ATPase activity of P-gp in vitro. However, 5-CHInd is not an exportable substrate for P-gp as it is not transported across an MDCK-MDR1 monolayer. In addition, 5-CHInd differentially modulates MDR by decreasing or increasing the cytotoxicity of several chemotherapeutic agents. Although 5-CHInd displays variable activity in modulating the efflux of various drugs by P-gp, there is a correlation between changes observed in the drug-stimulated ATPase catalytic efficiency induced by 5-CHInd and its effect on drug efflux. The paradoxical behavior of 5-CHInd is rationalized within the context of contemporary models of P-gp function. In addition, the data are used to develop a predictive in vitro model for rapidly identifying potential drug-drug interactions with P-gp.

Lactobacillus reuteri 2'-deoxyribosyltransferase, a novel biocatalyst for tailoring of nucleosides.

A novel type II nucleoside 2'-deoxyribosyltransferase from Lactobacillus reuteri (LrNDT) has been cloned and overexpressed in Escherichia coli. The recombinant LrNDT has been structural and functionally characterized. Sedimentation equilibrium analysis revealed a homohexameric molecule of 114 kDa. Circular dichroism studies have showed a secondary structure containing 55% alpha-helix, 10% beta-strand, 16% beta-sheet, and 19% random coil. LrNDT was thermostable with a melting temperature (T(m)) of 64 degrees C determined by fluorescence, circular dichroism, and differential scanning calorimetric studies. The enzyme showed high activity in a broad pH range (4.6 to 7.9) and was also very stable between pH 4 and 7.9. The optimal temperature for activity was 40 degrees C. The recombinant LrNDT was able to synthesize natural and nonnatural nucleoside analogues, improving activities described in the literature, and remarkably, exhibited unexpected new arabinosyltransferase activity, which had not been described so far in this kind of enzyme. Furthermore, synthesis of new arabinonucleosides and 2'-fluorodeoxyribonucleosides was carried out.

Non-natural nucleic acids for synthetic biology

Genetic manipulation is an important facet of synthetic biology but can be complicated by undesired nuclease degradation. Incorporating non-natural nucleic acids into a gene could convey resistance to nucleases and promote expression. The compatibility of non-natural nucleosides with polymerases is reviewed with a focus on results from the past two years. Details are provided about how the different systems could be useful in synthetic biology.

Chromatin-associated proteins HMGB1/2 and PDIA3 trigger cellular response to chemotherapy-induced DNA damage

The identification of new molecular components of the DNA damage signaling cascade opens novel avenues to enhance the efficacy of chemotherapeutic drugs. High-mobility group protein 1 (HMGB1) is a DNA damage sensor responsive to the incorporation of nonnatural nucleosides into DNA; several nuclear and cytosolic proteins are functionally integrated with HMGB1 in the context of DNA damage response. The functional role of HMGB1 and HMGB1-associated proteins (high-mobility group protein B2, HMGB2; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; protein disulfide isomerase family A member 3, PDIA3; and heat shock 70 kDa protein 8, HSPA8) in DNA damage response was assessed in human carcinoma cells A549 and UO31 by transient knockdown with short interfering RNAs. Using the cell proliferation assay, we found that knockdown of HMGB1-associated proteins resulted in 8-fold to 50-fold decreased chemosensitivity of A549 cells to cytarabine. Western blot analysis and immunofluorescent microscopy were used to evaluate genotoxic stress markers in knocked-down cancer cells after 24 to 72 hours of incubation with 1 micromol/L of cytarabine. Our results dissect the roles of HMGB1-associated proteins in DNA damage response: HMGB1 and HMGB2 facilitate p53 phosphorylation after exposure to genotoxic stress, and PDIA3 has been found essential for H2AX phosphorylation (no gamma-H2AX accumulated after 24-72 hours of incubation with 1 micromol/L of cytarabine in PDIA3 knockdown cells). We conclude that phosphorylation of p53 and phosphorylation of H2AX occur in two distinct branches of the DNA damage response. These findings identify new molecular components of the DNA damage signaling cascade and provide novel promising targets for chemotherapeutic intervention.

Non-natural nucleosides based on 1,2,4-triazolo[5,1-c][1,2,4]triazin-4(6H)-ones

Non-natural nucleosides based on 1,2,4-triazolo[5,1-c][1,2,4]triazin-4(6H)-ones

Synthesis and DNA duplex recognition of a triplex-forming oligonucleotide with an ureido-substituted 4-phenylimidazole nucleoside

A 4-(3- n-butylureidophenyl)imidazole nucleoside was successfully incorporated into a triplex-forming oligonucleotide (TFO). Binding affinity and base pair selectivity of the TFO containing this non-natural nucleoside were studied with various duplex targets containing all four possible Watson–Crick base pairs opposite the nucleoside analog in the third strand. Triplex thermal stabilities indicate that the synthetic nucleoside acts as a universal base in binding to all four possible Watson–Crick base pairs with moderate affinity but poor selectivity. Based on an analysis of its binding thermodynamics, this can be rationalized by the absence of strong specific interactions and more favorable entropic contributions upon triplex formation.

An intact ribose moiety at A2602 of 23S rRNA is key to trigger peptidyl-tRNA hydrolysis during translation termination

Peptide bond formation and peptidyl-tRNA hydrolysis are the two elementary chemical reactions of protein synthesis catalyzed by the ribosomal peptidyl transferase ribozyme. Due to the combined effort of structural and biochemical studies, details of the peptidyl transfer reaction have become increasingly clearer. However, significantly less is known about the molecular events that lead to peptidyl-tRNA hydrolysis at the termination phase of translation. Here we have applied a recently introduced experimental system, which allows the ribosomal peptidyl transferase center (PTC) to be chemically engineered by the introduction of non-natural nucleoside analogs. By this approach single functional group modifications are incorporated, thus allowing their functional contributions in the PTC to be unravelled with improved precision. We show that an intact ribose sugar at the 23S rRNA residue A2602 is crucial for efficient peptidyl-tRNA hydrolysis, while having no apparent functional relevance for transpeptidation. Despite the fact that all investigated active site residues are universally conserved, the removal of the complete nucleobase or the ribose 2′-hydroxyl at A2602, U2585, U2506, A2451 or C2063 has no or only marginal inhibitory effects on the overall rate of peptidyl-tRNA hydrolysis. These findings underscore the exceptional functional importance of the ribose moiety at A2602 for triggering peptide release.

Phase I/II Study of Continuous-Infusion Troxacitabine in Refractory Acute Myeloid Leukemia

Troxacitabine is a non-natural nucleoside analog with unique structural and metabolic features. Bolus intravenous (IV) troxacitabine regimens have shown significant activity in patients with refractory acute myeloid leukemia (AML) and preclinical data suggest that administration via continuous infusion may result in enhanced antitumor activity. Patients with refractory AML initially received troxacitabine 10.1 mg/m2 by continuous IV infusion (CIVI) for 48 hours. Infusion duration and daily dose were increased in subsequent patient cohorts. Forty-eight patients, median age 58 years (range, 21 to 81 years), were treated. Dose-limiting toxicities were mucositis and hand-foot syndrome, and 12.0 mg/m2/d for 5 days was established as the maximum-tolerated dose. Seven patients (15%) achieved complete remission (CR) or CR with incomplete platelet recovery (CRp), with a median survival among responders of 12 months. Steady-state concentrations of troxacitabine were found to be linearly and inversely proportionally related to calculated creatinine clearance at doses of 10.1 and 12.0 mg/m2/d. All patients responding to troxacitabine had steady-state serum drug concentration of more than approximately 80 ng/mL. In 27 patients achieving target troxacitabine plasma concentrations (ie, approximately 80 ng/mL) the CR + CRp rate was 26%. Troxacitabine administered as a CIVI allows a significant increase in dose-intensity in comparison to IV bolus regimens, has antileukemic activity, and warrants additional investigation in patients with refractory AML. The recommended phase II study dose is 12.0 mg/m2 daily CIVI for 5 days.

Synthesis and DNA triplex formation of an oligonucleotide containing an urocanamide

An urocanamide nucleoside designed and previously tested as its protected ribose derivative in aprotic solvents for binding a cytosine–guanine (CG) Watson–Crick base pair was successfully incorporated into a triplex forming oligonucleotide. Binding affinity and specificity of this nonnatural nucleoside were studied in a triple helix with duplex targets containing all four possible Watson–Crick base pairs opposite the nucleoside analog in the third strand. UV melting experiments indicate the formation of a well-defined triplex with specific binding of the urocanamide analog to a CG inversion of the homopurine–homopyrimidine target. However, binding affinities in the triplex are weak and much lower when compared to the canonical base triads.

Chemical engineering of the peptidyl transferase center reveals an important role of the 2'-hydroxyl group of A2451

The main enzymatic reaction of the large ribosomal subunit is peptide bond formation. Ribosome crystallography showed that A2451 of 23S rRNA makes the closest approach to the attacking amino group of aminoacyl-tRNA. Mutations of A2451 had relatively small effects on transpeptidation and failed to unequivocally identify the crucial functional group(s). Here, we employed an in vitro reconstitution system for chemical engineering the peptidyl transferase center by introducing non-natural nucleosides at position A2451. This allowed us to investigate the peptidyl transfer reaction performed by a ribosome that contained a modified nucleoside at the active site. The main finding is that ribosomes carrying a 2′-deoxyribose at A2451 showed a compromised peptidyl transferase activity. In variance, adenine base modifications and even the removal of the entire nucleobase at A2451 had only little impact on peptide bond formation, as long as the 2′-hydroxyl was present. This implicates a functional or structural role of the 2′-hydroxyl group at A2451 for transpeptidation.

Prevalence of drug resistance mutations among antiretroviral drug-naive HIV-1-infected patients in China

To collect background information on drug-resistant HIV-1 strains in various regions before the start of nation-wide antiretroviral therapy in China. Twenty percent of the 2,000 blood samples from antiretroviral therapy naive patients collected for the 2nd national HIV molecular epidemiology survey (NHMES) in 2002 were randomly sampled for this study. The entire protease gene and 20-230 amino acids of the reverse transcriptase gene were amplified by PCR from provirus DNA and sequenced. The results were analyzed with HIV db-Drug Resistance Algorithm and genotypic resistance mutations were determined to particular anti-HIV drugs. Totally 164 protease gene sequences and 138 reverse transcriptase gene sequences were obtained from patients; 0.61% of 164 sequences displayed primary resistance mutations in the protease gene, whereas 99.39% carried 1 or more secondary mutations. Genotypic resistance to at least one nucleoside reverse transcriptase inhibitors (NRTI) was present in 5.80%,and resistance to at least one non-nucleo side reverse transcriptase inhibitors (NNRTI) was present in 1.45% of samples. The prevalence of genotypic drug resistance is very low in drug-naive HIV infected patients from 21 provinces of China tested in this study. Laboratories participated in the NHMES have organized a network to provide drug resistance monitoring service in the current nation-wide antiviral treatment program in China.

Non-Natural Nucleosides for the Specific Recognition of Watson-Crick Base Pairs

Non-Natural Nucleosides for the Specific Recognition of Watson-Crick Base Pairs

Oligodeoxynucleotides with extended zwitter-ionic internucleotide linkage

Two non-natural nucleoside analogues, N-(2-hydroxyethyl)-2′,5′-dideoxy-5′-aminothymidine (dTNH) and N-(2-hydroxyethyl)-N-methyl-2′,5′-dideoxy-5′-aminothymidine (dTNMe), have been prepared and used in the synthesis of oligodeoxythymidilates and mixed-sequence oligodeoxynucleotides, modified at internucleotide linkages. Both modified oligodeoxythymidilates and mixed-sequence oligodeoxynucleotides have been shown to form zwitter-ionic phosphate–amine pairs as evidenced by their decreased electrophoretic mobility in denaturing polyacrylamide gel.

Preparation of stilbene-tethered nonnatural nucleosides for use with blue-fluorescent antibodies.

The synthesis of the first examples of stilbene-tethered hydrophobic C-nucleosides is described. Compounds of this type are targeted for use with our recently reported "blue-fluorescent antibodies" with the aim of probing native and nonnatural DNA. The nucleophilic addition of aryl Grignard reagents to either a protected 2'-deoxy-1'-chloro-ribofuranose or a protected 2'-deoxy-ribonolactone was the key synthetic step and afforded C-nucleosides in good yields. Both routes resulted in a final product that was >/=90% of the beta-anomer. Amide- and ether-based linkers for attachment of trans-stilbene to the nucleobase were assessed for utility during synthesis and in binding of the ligands to a blue-fluorescent monoclonal antibody. X-ray structures of each complex were obtained and serve as a guideline for second-generation stilbene-tethered C-nucleosides. The development of these hydrophobic nucleosides will be useful in current native and nonnatural DNA studies and invaluable for investigations regarding novel, nonnatural genomes in the future.

Synthesis of Non-Natural Pyrimidine Nucleosides

Two new non-natural nucleosides bearing an amide (8) or an amidine (9) function have been synthesized. Their properties and the geometry of the exocyclic double bond have been studied.

Triple‐Helix Formation by Pyrimidine Oligonucleotides Containing Nonnatural Nucleosides with Extended Aromatic Nucleobases: Intercalation from the major groove as a method for recognizing C·G and T · A base pairs

AbstractThe sequence‐specific recognition of double‐helical DNA by oligonucleotide‐directed triple helix formation is limited primarily to purine tracts. To identify potential lead compounds which are able to extend the sequence repertoire of triple helical complexes, we designed two carbocyclic nucleosides with nucleobases attached via amide bonds. N5‐[(1R, 2S, 3R, 4R)‐3‐hydroxy‐4‐(hydroxymethyl)‐2‐methoxycyclopentyl]‐2‐{[(1H‐pyrrol‐2‐yl)carbonyl]‐amino}thiazole‐5‐carboxamide (L1) and 2‐benzamido‐N5‐[(1R, 2S, 3R, 4R)‐3‐hydroxy‐4‐(hydroxymethyl)‐2‐methoxycyclopentyl]thiazole‐5‐carboxamide (L2) were synthesized and incorporated into pyrimidine oligonucleotides. The 2‐(trimethylsilyl)ethoxymethyl (SEM) protecting group for the 1H‐pyrrole NH was found to be compatible with DNA solid‐phase synthesis of pyrimidine Oligonucleotides. By quantitative DNase I footprinting analysis, both nonnatural nucleosides L1 and L2 showed preferential binding of pyrimidine over purine bases: L1/2·(C·G) ≈ L1/2·(T · A) > L1/2·(G·C) ≈ L1/2·(A · T). Comparison with the previously reported nonnatural nucleosides with extended aromatic nucleobases 1‐(2‐deoxy‐β‐D‐ribofuranosyl)‐4‐(3‐benzamidophenyl)‐imidazole (D3) and N4‐[6‐(benzamido)pyridin‐2‐yl]‐2′‐deoxycytidine (bzM) suggests that the observed binding selectivity C · G ≈ T · A > G · C ≈ A · T for the nucleoside analogs L1, L2, D3, and bzM is derived from sequence‐specific intercalation with preferential stacking of their nucleobases over pyrimidine · purine Watson‐Crick base pairs.