Synthesis and Substrate Evaluation of (E)-5-[(3-Selenophene-2-Carboxamido)Prop-1-en-1-yl]-Uridine-5'-O-Triphosphate for RNA Polymerase.

Design, synthesis and T7 RNA polymerase substrate evaluation of (E)-5-[(3-selenophene-2-carboxamido)prop-1-en-1-yl]-uridine-5'-O-triphosphate is reported. The title compound is shown to be a good substrate for RNA polymerase by RNA labeling through in vitro transcription. pTRI-plasmid DNA with ß-actin gene sequence (∼300 base pairs) with T7 promoter was used as a template for the in vitro transcription. Transcribed product is characterized for incorporation by gel assay and for integrity, full length and size by bioanalyzer. The title compound will be very useful in biophysical techniques to obtain information on dynamics and recognition properties in real time as well as 3D structure of nucleic acids.

Synthesis of 1-(ß-D-Galactopyranosyl)Thymine-6'-O-Triphosphate - A Potential Probe to Generate Reactive Dialdehyde for DNA-Enzyme Cross-Linking.

Concise, facile, and efficient synthesis of 1-(ß-D-galactopyranosyl)thymine-6'-O-triphosphate, a potential probe that can generate reactive dialdehyde for DNA-enzyme cross-linking applications, was described starting from O,O'-bis(trimethylsilyl)thymine. Stannic chloride promoted glycosylation of 1,2,3,4,6-penta-O-acetyl-α-D-galactopyranose with O,O'-bis(trimethylsilyl)thymine, resulting in the formation of 1-(2,3,4,6-O-tetraacetyl-ß-D-galactopyranosyl)thymine in 91% yield. Acetyl deprotection using methanolic ammonia afforded 1-(ß-D-galactopyranosyl)thymine in 98% yield. The modified one-pot methodology was used to convert 1-(ß-D-galactopyranosyl)thymine into 1-(ß-D-galactopyranosyl)thymine-6'-O-triphosphate in 72% yield, which involves the formation of 1-(ß-D-galactopyranosyl)thymine dichlorophosphoridate using POCl3 as the reagent at the monophosphorylation step followed by reaction with tributylammonium pyrophosphate and hydrolysis of resulting cyclic intermediate.

Fluorous-assisted synthesis of (E)-5-[3-aminoallyl]-uridine-5'-O-triphosphate.

An efficient, reliable method for the chemical synthesis of (E)-5-[3-aminoallyl]-uridine-5'-O-triphosphate (AA-UTP), starting from 5-iodouridine, is described. This new strategy features the involvement of one-pot triphosphate formation and fluorous solid-phase extraction (F-SPE). The one-pot synthesis involves the mono phosphorylation of fluorous-tagged uridine, followed by the reaction with pyrophosphate to afford the fluorous-tagged AA-UTP. The F-SPE is achieved by installing a fluorous-tag onto the uridine prior to triphosphate formation, purification via F-SPE, and cleavage of the fluorous-tag. It is worth mentioning that this protocol produces AA-UTP in high yield and purity using one simple F-SPE; no conventional column chromatography is involved.

Concise and efficient synthesis of 3'-O-tetraphosphates of 2'-deoxyadenosine and 2'-deoxycytidine.

We describe concise and efficient synthesis of biologically very important 3'-O-tetraphosphates namely 2'-deoxyadenosine-3'-O-tetraphosphate (2'-d-3'-A4P) and 2'-deoxycytidine-3'-O-tetra-phosphate (2'-d-3'-C4P). N(6)-benzoyl-5'-O-levulinoyl-2'-deoxyadenosine was converted into N(6)-benzoyl-5'-O-levulinoyl-2'-deoxyadenosine-3'-O-tetraphosphate in 87% yield using a one-pot synthetic methodology. One-step concurrent deprotection of N(6)-benzoyl and 5'-O-levulinoyl groups using concentrated aqueous ammonia resulted 2'-d-3'-A4P in 74% yield. The same synthetic strategy was successfully employed to convert N(4)-benzoyl-5'-O-levulinoyl-2'-deoxycytidine into 2'-d-3'-C4P in 68% yield.

Short and straightforward synthesis of triphosphates of artificial nucleobase pairs displaying unconventional pairing scheme.

Concise, facile and efficient synthesis of 5'-O-triphosphates of 6-amino-5-nitro-3-(1'-ß-D-2'-deoxyribofuranosyl)-2(1H)-pyridone (dZ) and its Watson-Crick complement 2-amino-8-(1'-ß-D-2'-deoxyribofuranosyl)-imidazo[1,2a]-1,3,5-triazin-4(8H)-one (dP) is reported using a one-pot synthetic procedure.

Facile protection-free one-pot chemical synthesis of nucleoside-5'-tetraphosphates.

A new, straightforward, reliable, and convenient protection-free one-pot method for the synthesis of 2'-deoxynucleoside-5'-tetraphosphate and ribonucleoside-5'-tetraphosphate is reported. The present synthetic strategy involves the monophosphorylation of a nucleoside followed by reaction with tris-(tri-n-butylammonium) triphosphate and subsequent hydrolysis of the putative cyclic tetrametaphosphate intermediate to provide nucleoside-5'-tetraphosphate in moderate yield with high purity. A plausible mechanism is proposed to account for the formation of product.

The association of the triglyceride-to-HDL cholesterol ratio with insulin resistance in White European and South Asian men and women.

INTRODUCTION: There is recent interest surrounding the use of the triglyceride-to-HDL cholesterol ratio as a surrogate marker of insulin resistance in clinical practice, as it may identify people at high risk of developing diabetes or its complications. However, it has been suggested using this lipid ratio may not be appropriate for measuring insulin resistance in African-Americans, particularly women. We investigated if this inconsistency extended to South Asian women in a UK multi-ethnic cohort of White Europeans and South Asians. METHODS: Cross-sectional analysis was done of 729 participants from the ADDITION-Leicester study from 2005 to 2009. The association between tertiles of triglyceride-to-HDL cholesterol ratio to fasting insulin, homeostatic model of assessment for insulin resistance (HOMA1-IR), quantitative insulin sensitivity check index (QUICKI) and glucose: insulin ratio was examined with adjustment for confounding variables. RESULTS: Incremental tertiles of the triglyceride-to-HDL cholesterol ratio demonstrated a significant positive association with levels of fasting insulin, HOMA1-IR, glucose: insulin ratio and a negative association with QUICKI in White European men (n = 255) and women (n = 250) and South Asian men (n = 124) (all p<0.05), but not South Asian women (n = 100). A significant interaction was demonstrated between sex and triglyceride-to-HDL cholesterol ratio tertiles in South Asians only (p<0.05). The area under the receiver operating characteristic curve for triglyceride-to-HDL cholesterol ratio to detect insulin resistance, defined as the cohort HOMA1-IR ≥ 75(th) percentile (3.08), was 0.74 (0.67 to 0.81), 0.72 (0.65 to 0.79), 0.75 (0.66 to 0.85) and 0.67 (0.56 to 0.78) in White European men and women, South Asian men and women respectively. The optimal cut-points for detecting insulin resistance were 0.9-1.7 in mmol/l (2.0-3.8 in mg/dl) for the triglyceride-to-HDL ratio. CONCLUSION: In South Asian women the triglyceride-to-HDL cholesterol ratio was not associated with insulin resistance; therefore there may be limitations in its use as a surrogate marker in this group.

A new chalcone derivative (E)-3-(4-methoxyphenyl)-2-methyl-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one suppresses prostate cancer involving p53-mediated cell cycle arrests and apoptosis.

Previous studies suggested chalcones as antineoplastic drug candidates. We synthesized a new chalcone derivative (E)-3-(4-methoxyphenyl)-2-methyl-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one, (CHO27) with an up to 1000-fold increased cytotoxic potency relative to its parent compound in cell culture assays. CHO27 at low nanomolar levels, inhibited prostate cancer (PCa) cell growth through cell cycle arrest and caspase-dependent apoptosis. Activation of p53 accounted for, at least in part, the growth inhibition by CHO27 in vitro. Furthermore, i.p. administration of CHO27 suppressed the growth of established PCa 22Rv1 xenograft tumors accompanied with p53 and p21(Cip1) induction. CHO27 may be a lead for development of new therapeutic agents for PCa.

An efficient synthesis of pyrimidine specific 2'-deoxynucleoside-5'-tetraphosphates.

An efficient chemical synthesis of pyrimidine specific 2'-deoxynucleoside-5'-tetraphosphates, such as 2'-deoxycytidine-5'-tetraphosphate (dC4P) and thymidine-5'-tetraphosphate (T4P) is described. The present three-step synthetic strategy involves monophosphorylation of 2'-deoxynucleoside using phosphorous oxychloride, conversion of 5'-monophosphate into the corresponding imidazolide salt, followed by reaction with tris[tributylammonium] triphosphate leading to the 2'-deoxynucleoside-5'-tetraphosphate in good yields.

Inhibition of mitogen activated protein kinases increases the sensitivity of A549 lung cancer cells to the cytotoxicity induced by a kava chalcone analog.

We are interested in investigating the biological activity of chalcones, a major class of compounds found in the beverage kava, in order to develop potent and selective chemopreventive candidates. Consumption of kava in the South Pacific Islands is inversely correlated with cancer incidence, even among smokers. Accordingly, chalcones have anti-cancer activities in animal and cell culture models. To investigate signaling pathways that affect chalcone action we studied a potent analog, (E)-3-(3-hydroxy-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (chalcone-24). Chalcone-24 was selected from a series of chalcone analogs that were synthesized based on the structures derived from flavokawain compounds found in kava, and screened in A549 lung cancer cells for induction of cytotoxicity and inhibition of NF-κB, a transcription factor associated with cell survival. Incubation of A549 cells with chalcone-24 resulted in a dose-dependent inhibition of cell viability, inhibition of NF-κB, activation of caspases, and activation of extracellular signal regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK); ERK1/2 and JNK are mitogen activated protein kinases that play central roles in regulating cell fate. Pharmacological inhibitors of ERK1/2 or JNK increased the sensitivity of A549 cells to chalcone-24-induced cytotoxicity, without affecting NF-κB or caspase activity. These results will help refine the synthesis of chalcone analogs to maximize the combination of actions required to prevent and treat cancer.

Independent effect of ethnicity on glycemia in South Asians and white Europeans.

OBJECTIVE: HbA(1c) levels are higher in most ethnic groups compared with white Europeans (WEs) independent of glycemic control. This comparison has not been performed between South Asians (SAs) and WEs. We analyzed the independent effect of ethnicity on HbA(1c) and fasting and 2-h plasma glucose (FPG and 2 hrPG, respectively) between these groups. RESEARCH DESIGN AND METHODS: Analysis of the ADDITION-Leicester study, in which 4,688 WEs and 1,352 SAs underwent oral glucose tolerance testing, HbA(1c), and other risk factor measurements. RESULTS: Significant associations with HbA(1c) included ethnicity, FPG, 2 hrPG, and homeostasis model assessment of ß-cell function (P < 0.001); age and sex (P < 0.01); and fasting insulin and potassium (P < 0.05). After adjusting for these and other risk factors, SAs demonstrated higher HbA(1c) (6.22 and 6.02%, mean difference 0.20%, 0.10-0.30, P < 0.001), FPG (5.15 and 5.30 mmol/L, mean difference 0.15 mmol/L, 0.09-0.21, P < 0.001), and 2 hrPG (5.82 and 6.57 mmol/L, mean difference 0.75 mmol/L, 0.59-0.92, P < 0.001) compared with WEs, respectively. CONCLUSIONS: HbA(1c), FPG, and 2 hrPG levels were higher in SAs independent of factors affecting glycemic control.

Gram-scale chemical synthesis of 2'-deoxynucleoside-5'-o-triphosphates.

A simple, straightforward, reliable, and efficient method for the chemical synthesis of sodium salt of 2'-deoxynucleoside-5'-O-triphosphates (dNTPs), starting from the corresponding nucleoside, is described. This improved "one-pot, three-step" synthetic strategy involves the monophosphorylation of nucleoside, followed by reaction with tributylammonium pyrophosphate and hydrolysis of the resulting cyclic intermediate to provide the corresponding dNTP in good yields (65% to 70%). It is noteworthy that the protocol holds good for both the purine deoxynucleotides, such as 2'-deoxyguanosine-5'-O-triphosphate (dGTP) and 2'-deoxyadenosine-5'-O-triphosphate (dATP), and pyrimidine deoxynucleotides, such as 2'-deoxycytidine-5'-O-triphosphate (dCTP), thymidine-5'-O-triphosphate (TTP), and 2'-deoxyuridine-5'-O-triphosphate (dUTP).

An improved protection-free one-pot chemical synthesis of 2'-deoxynucleoside-5'-triphosphates.

A facile, straightforward, reliable, and an efficient method for the gram-scale chemical synthesis of both purine deoxynucleotides such as 2 '-deoxyguanosine-5 '-triphosphate (dGTP) and 2 '-deoxyadenosine-5 '-triphosphate (dATP) and pyrimidine deoxynucleotides such as 2 '-deoxycytidine-5 '-triphosphate (dCTP), thymidine-5 '-triphosphate (TTP), and 2 '-deoxyuridine-5 '-triphosphate (dUTP) starting from the corresponding nucleoside is described. This improved "one-pot, three step" Ludwig synthetic strategy involves the monophosphorylation of nucleoside followed by reaction with tributylammonium pyrophosphate and hydrolysis of the resulting cyclic intermediate to provide the corresponding dNTP in good yields (65%-70%).

Structure-activity relationship and molecular mechanisms of ethyl 2-amino-6-(3,5-dimethoxyphenyl)-4-(2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (CXL017) and its analogues.

Multidrug resistance (MDR) in cancer is a phenomenon in which administration of a single chemotherapeutic agent causes cross-resistance of cancer cells to a variety of therapies even with different mechanisms of action. Development of MDR against standard therapies is a major challenge in the treatment of cancer. Previously we have demonstrated a unique ability of CXL017 (5) to selectively target MDR cancer cells and synergize with mitoxantrone (MX) in HL60/MX2 MDR cells. Here we expand its scope and demonstrate that 5 can synergize with both vincristine and paclitaxel in three different MDR cell lines (HL60/DNR, K562/HHT300, and CCRF-CEM/VLB100). We also demonstrate that 5 has potent cytotoxicity in the NCI-60 panel of cell lines with an average IC(50) of 1.04 µM. In addition, 5 has a unique mechanism of action in comparison with standard agents in the NCI database based on COMPARE analysis. Further structure-activity relationship study led to the development of a more potent analogue, compound 7d, with an IC(50) of 640 nM in HL60/MX2. Additionally, one enantiomer of 5 is 13-fold more active than the less active enantiomer. Taken together, our study has led to the discovery of a series of analogues that selectively target drug-resistant cancer cells with the potential for the treatment of drug-resistant cancers.

A three-layer competition-based giant magnetoresistive assay for direct quantification of endoglin from human urine.

This study presents a three-layer competition-based assay for ultrasensitive detection and quantification of endoglin from unprocessed human urine samples using a giant magnetoresistive (GMR) sensor and high-moment magnetic nanoparticle-based biosensing technology. This biosensing platform detects as few as 1000 copies of endoglin at concentrations as low as 83 fM with high detection specificity and has a three-order dynamic range. The results reveal that endoglin levels in urine have the potential to predict for the presence of prostate cancer and to distinguish between prostate cancers of different grades.

Chalcone-based inhibitors against hypoxia-inducible factor 1--structure activity relationship studies.

Many tumor tissues are under hypoxic conditions. Activating hypoxia-inducible factor 1 (HIF-1), a transcription factor, is a major mechanism for tumor cells to survive and even to evade other tissues. Therefore inhibiting HIF-1 is a potential strategy to help improve cancer treatment. Chalcone is a promising template to develop HIF-1 inhibitor because quite a few of chalcone-based compounds reveal moderate HIF-1 inhibitory activity and many chalcone-based compounds demonstrate promising anticancer activities in various animal models. However, there are no reports about the structure-activity relationship of chalcone compounds with respect to HIF-1 inhibition. This study reports the HIF-1 inhibitory activities of a panel of chalcones, identifies a few lead candidates of single-digit micromolar potency, and determines important structural modifications.

Should glycated haemoglobin (HbA1c) be used to detect people with type 2 diabetes mellitus and impaired glucose regulation?

There is a need to simplify screening tests for type 2 diabetes mellitus (T2DM) so patients can be identified earlier and more efficiently. Glycated haemoglobin (HbA1c) has been recommended by some international organisations as a diagnostic tool for detecting T2DM and impaired glucose regulation (IGR, also termed prediabetes and includes impaired fasting glucose and/or impaired glucose tolerance). The HbA1c cut-point of ≥6.5% (48 mmol/mol) has been selected as diagnostic for T2DM, while the cut-points for IGR are debated by the different international organisations: an International Expert Committee has suggested using HbA1c 6.0-6.4% (42-46 mmol/mol); however, the American Diabetes Association has recommended using HbA1c 5.7-6.4% (39-46 mmol/mol). Some countries will adopt a new method of reporting HbA1c values in millimoles per mole (mmol/mol). Use of HbA1c has some logistical advantages over using an oral glucose tolerance test (OGTT). As patients do not need to fast, appointments do not need to be limited to the morning. The HbA1c result reflects longer term glycaemia and is less affected by recent physical/emotional stress. However, there is some debate as to whether HbA1c should replace fasting plasma glucose or the OGTT. As the two tests detect different people, some individuals with diabetes detected on OGTT will no longer be classified as having T2DM using HbA1c ≥6.5% criteria. Furthermore, some medical conditions can result in HbA1c assay measurements not reflecting glycaemic control over the last 2-3 months; these include haematological disorders, renal failure, and chronic excess alcohol consumption.

The potential impact and optimal cut-points of using glycated haemoglobin, HbA1c, to detect people with impaired glucose regulation in a UK multi-ethnic cohort.

INTRODUCTION: Recommended diagnostic cut-points to detect impaired glucose regulation (IGR, also termed prediabetes: impaired fasting glucose and/or impaired glucose tolerance based on WHO 1999 criteria) are HbA1c 6.0-6.4% and 5.7-6.4% from an International Expert Committee and American Diabetes Association, respectively. We investigated the impact on prevalence/phenotype from using these criteria compared to IGR detected on oral glucose tolerance testing (OGTT) and determined optimal HbA1c cut-points for IGR in a multi-ethnic cohort. METHODS: Analysis of 8696 participants in the LEADER study of primary care individuals aged 40-75 years without diabetes, in Leicestershire (UK) who underwent OGTT and had HbA1c measured. RESULTS: Use of OGTT detected less people with IGR (n=1407, 16.2%) compared to HbA1c 6.0-6.4% (n=1610, 18.5%) and HbA1c 5.7-6.4%(n=3904, 44.9%), a 1.1- and 2.8-fold increase in prevalence, respectively. There were 930 (10.7%) and 534 (6.1%) people with IGR on OGTT not detected using HbA1c 6.0-6.4% and 5.7-6.4%, respectively. From ROC curve analysis, the optimal cut-point for detecting IGR in white Europeans was HbA1c>or=5.8%, sensitivity/specificity 61.5%/67.9%, but in south Asians HbA1c>or=6.0%, sensitivity/specificity 63.8%/69.4%. CONCLUSION: Recommended HbA1c cut-points to detect IGR significantly increase numbers detected, however introduce a change in people identified. Using HbA1c 6.0-6.4% lacks sensitivity in white Europeans, but is a reasonable option in south Asians.

Nanomagnetic competition assay for low-abundance protein biomarker quantification in unprocessed human sera.

A novel giant magnetoresistive sensor and uniform high-magnetic-moment FeCo nanoparticles (12.8 nm)-based detecting platform with minimized detecting distance was developed for rapid biomolecule quantification from body fluids. Such a system demonstrates specific, accurate, and quick detection and quantification of interleukin-6, a low-abundance protein and a potential cancer biomarker, directly in 4 muL of unprocessed human sera. This platform is expected to facilitate the identification and validation of disease biomarkers. It may eventually lead to a low-cost personal medical device for chronic disease early detection, diagnosis, and prognosis.

A new and facile method for measurement of apparent density of monodisperse polymer beads.

The apparent density, an intrinsic physical property of polymer beads, plays an important role in the application of beads in micro-total analysis systems and separation. Here we have developed a new, facile and milligram-scale method to describe the motion of beads in aqueous solution and further detect the apparent density of beads. The motion of beads in solutions is determined by the viscosity of solutions and the density difference between beads and solutions. In this study, using various glycerol aqueous solutions with certain viscosities and densities, the motion time (i.e. floating or sedimentation time) of hybrid polymer beads was experimentally measured and theoretically deduced, and consequently, the apparent density of monodisperse beads can be quickly and easily calculated. The results indicated that the present method provided a more precise way to predict the movement of hybrid beads in aqueous solution compared with the approach for commercial use. This new method can be potentially employed in flow cytometry, suspension stability, and particle analysis systems.