Intermediate Methyl Research Articles

DNA methylation subgroups in melanoma are associated with proliferative and immunological processes.

BackgroundDNA methylation at CpG dinucleotides is modified in tumorigenesis with potential impact on transcriptional activity.MethodsWe used the Illumina 450 K platform to evaluate DNA methylation patterns of 50 metastatic melanoma tumors, with matched gene expression data.ResultsWe identified three different methylation groups and validated the groups in independent data from The Cancer Genome Atlas. One group displayed hypermethylation of a developmental promoter set, genome-wide demethylation, increased proliferation and activity of the SWI/SNF complex. A second group had a methylation pattern resembling stromal and leukocyte cells, over-expressed an immune signature and had improved survival rates in metastatic tumors (p < 0.05). A third group had intermediate methylation levels and expressed both proliferative and immune signatures. The methylation groups corresponded to some degree with previously identified gene expression phenotypes.ConclusionsMelanoma consists of divergent methylation groups that are distinguished by promoter methylation, proliferation and content of immunological cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12920-015-0147-4) contains supplementary material, which is available to authorized users.

Epigenetic analysis of regulatory T cells using multiplex bisulfite sequencing.

This work was supported by Wellcome Trust Grant 096388, JDRF Grant 9-2011-253, the National Institute for Health Research Cambridge Biomedical Research Centre (BRC) and Award P01AI039671 (to LSW. and JAT.) from the National Institute of Allergy and Infectious Diseases (NIAID). CW is supported by the Wellcome Trust (089989). The content of this article is solely the responsibility of the authors and does not necessarily represent the official views of NIAID or the National Institutes of Health. The Cambridge Institute for Medical Research is in receipt of Wellcome Trust Strategic Award 100140. We gratefully acknowledge the participation of all NIHR Cambridge BioResource volunteers. We thank the Cambridge BioResource staff for their help with volunteer recruitment. We thank members of the Cambridge BioResource SAB and Management Committee for their support of our study and the National Institute for Health Research Cambridge Biomedical Research Centre for funding. We thank Fay Rodger and Ruth Littleboy for running the Illumina MiSeq in the Molecular Genetics Laboratories, Addenbrooke's Hospital, Cambridge. This research was supported by the Cambridge NIHR BRC Cell Phenotyping Hub. In particular, we wish to thank Anna Petrunkina Harrison, Simon McCallum, Christopher Bowman, Natalia Savinykh, Esther Perez and Jelena Markovic Djuric for their advice and support in cell sorting. We also thank Helen Stevens, Pamela Clarke, Gillian Coleman, Sarah Dawson, Jennifer Denesha, Simon Duley, Meeta Maisuria-Armer and Trupti Mistry for acquisition and preparation of samples.

Abstract 828: Methylation status of HPV16 E2-binding sites identifies subtypes of HPV-associated oropharyngeal squamous cell carcinomas

Abstract Background: The viral E2 protein is a transcriptional repressor of the HPV oncogenes E6/E7 and loss of E2 function is considered a key step in carcinogenesis. Integration of HPV into the host genome may disrupt the E2 gene. Furthermore, methylation of CpG dinucleotides in E2 binding sites (E2BSs) in the HPV upstream regulatory region (URR) may interfere with transcriptional repression of E6 and E7 by the E2 protein. We hypothesized that CpG methylation of the E2BS is found in a proportion of HPV16-associated oropharyngeal squamous cell cancers (OPSCC) in association with E2 gene integrity and viral integration. Methods: Methylation of 10 CpGs within the URR, encompassing E2BSs 1, 2, 3 and 4, was quantitatively analyzed by bisulfite pyrosequencing in 57 HPV16-associated OPSCC. E2 status was analyzed by gene amplification and quantitative real-time RT-PCR. Viral integration was determined by integration-specific PCR methods. Results: Three subgroups with differential E2BSs3 and 4 methylation were identified: a) complete methylation (&amp;gt;80%) associated with presence of integrated HPV genomes with intact E2 gene, b) intermediate methylation levels (20-80%) with predominantly episomal HPV genomes with intact E2, and c) no methylation (&amp;lt;20%) with integrated HPV with disrupted E2. Patients with complete and without methylation had poor 5-year overall survival compared to patients with intermediate methylation (stage-adjusted hazard ratio 3.8 (95%CI 1.3-11.2) and 3.1 (95%CI 1.14-8.28); p = 0.03). Conclusions: E2BSs3 and 4 methylation in oropharyngeal cancers with episomal HPV and integrated HPV with intact E2 gene might explain deregulated viral oncogene expression in the presence of E2. The methylation level appears to be of prognostic significance for patients with HPV-associated OPSCC. Citation Format: Ernst-Jan M. Speel, Miriam Reuschenbach, Christian U. Huebbers, Elena-Sophie Prigge, Justo L. Bermejo, Simon Kalteis, Simon Preuss, Jutta Kolligs, Nadine C. Olthof, Bernd Kremer, Steffen Wagner, Jens P. Klussmann, Svetlana Vinokurova, Magnus von Knebel Doeberitz. Methylation status of HPV16 E2-binding sites identifies subtypes of HPV-associated oropharyngeal squamous cell carcinomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 828. doi:10.1158/1538-7445.AM2015-828

DNA methylation signature in peripheral blood reveals distinct characteristics of human X chromosome numerical aberrations.

BackgroundAbnormal sex chromosome numbers in humans are observed in Turner (45,X) and Klinefelter (47,XXY) syndromes. Both syndromes are associated with several clinical phenotypes, whose molecular mechanisms are obscure, and show a range of inter-individual penetrance. In order to understand the effect of abnormal numbers of X chromosome on the methylome and its correlation to the variable clinical phenotype, we performed a genome-wide methylation analysis using MeDIP and Illumina’s Infinium assay on individuals with four karyotypes: 45,X, 46,XY, 46,XX, and 47,XXY.ResultsDNA methylation changes were widespread on all autosomal chromosomes in 45,X and in 47,XXY individuals, with Turner individuals presenting five times more affected loci. Differentially methylated CpGs, in most cases, have intermediate methylation levels and tend to occur outside CpG islands, especially in individuals with Turner syndrome. The X inactivation process appears to be less effective in Klinefelter syndrome as methylation on the X was decreased compared to normal female samples. In a large number of individuals, we verified several loci by pyrosequencing and observed only weak inter-loci correlations between the verified regions. This suggests a certain stochastic/random contribution to the methylation changes at each locus. Interestingly, methylation patterns on some PAR2 loci differ between male and Turner syndrome individuals and between female and Klinefelter syndrome individuals, which possibly contributed to this distinguished and unique autosomal methylation patterns in Turner and Klinefelter syndrome individuals.ConclusionsThe presented data clearly show that gain or loss of an X chromosome results in different epigenetic effects, which are not necessary opposite.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-015-0112-2) contains supplementary material, which is available to authorized users.

The synthesis of some sulfonamides based on 2,3-dihydro-1H-pyrrolo[3,4-c]pyridine

Synthetic protocols for the preparation of 1-oxo-, 3-oxo-, and 1,3-dioxopyrrolo[3,4- c ]pyridines which contain sulfonamide groups linked to the pyrrole nitrogen atom of the pyrrolo[3,4- c ]pyridine cycle by a flexible (CH2)6 aliphatic linker were developed. 1,3-Dioxopyrrolo[3,4- c ]pyridines were obtained from 3,4-pyridinedicarboxylic acid by condensation of intermediate methyl 3-(chlorocarbonyl)isonicotinate with amines. 3-Oxopyrrolo[3,4- c ]pyridines were prepared by regioselective reduction of 1,3-dioxopyrrolo[3,4- c ]pyridines with a tin dust followed by treatment with triethylsilane. 1-Oxopyrrolo[3,4- c ]pyridines were prepared by a synthetic sequence consisting of the treatment of methyl isonicotinate with N -bromosuccinimide and the following condensation of the obtained ethyl 3-bromomethylisonicotinate with primary amines. How to Cite Ikaunieks, M.; Bjorkling, F.; Loza, E. Chem. Heterocycl. Compd. 201 5 , 51 , 658. [ Khim. Geterotsikl. Soedin. 2015 , 51 , 658.] For this article in the English edition see DOI 10.1007/s10593-015-1753-y

Identification of an Intermediate Methyl Carrier and the Stereochemical Outcomes of H‐atom Abstraction and Methylthiolation by the Radical SAM Enzyme RimO

RimO is a radical S‐adenosylmethionine (RS) enzyme that catalyzes the insertion of a methylthio group (‐SCH3) at C3 of a conserved aspartyl residue of protein S12, a component of the 30S subunit of the bacterial ribosome. RimO contains a [4Fe‐4S] cluster to which S‐adenosylmethionine (SAM) binds, allowing for its reductive cleavage to methionine and a 5′‐deoxyadenosyl 5′‐radical. Unlike most RS enzymes, methylthiotransferases (MTTases) contain an additional [4Fe–4S] cluster that is believed to be the source of the sulfur atom of the inserted –SCH3 group. It was believed that the sequence of MTTase reactions involved initial sulfur insertion into the substrate followed by capping with a SAM‐derived methyl group. We show that RimO from Thermotoga maritima catalyzes the synthesis and subsequent insertion of a ‐SCH3 group by mediating methyl transfer from SAM to an acid/base labile acceptor on the protein in the absence of its substrate and the requisite reductant that triggers radical chemistry. Consistent with the assignment of the acceptor as an Fe‐S cluster (presumably the auxiliary [4Fe–4S] cluster), denaturation of the SAM‐treated protein with acid results in production of methanethiol (CH3SH). We show chemical competence of the methylated intermediate by using unlabeled and [methyl‐d3] SAM and demonstrate that RimO catalyzes multiple turnovers when CH3SH is supplied exogenously. Using deuterated isotopologs at C3 of aspartate and 1H NOESY NMR, we determined the stereospecificity of H‐atom abstraction and the absolute configuration of the chiral center created upon ‐SCH3 insertion by RimO.

Individual epigenetic status of the pathogenic D4Z4 macrosatellite correlates with disease in facioscapulohumeral muscular dystrophy.

BackgroundBoth forms of facioscapulohumeral muscular dystrophy (FSHD) are associated with aberrant epigenetic regulation of the chromosome 4q35 D4Z4 macrosatellite. Chromatin changes due to large deletions of heterochromatin (FSHD1) or mutations in chromatin regulatory proteins (FSHD2) lead to relaxation of epigenetic repression and increased expression of the deleterious double homeobox 4 (DUX4) gene encoded within the distal D4Z4 repeat. However, many individuals with the genetic requirements for FSHD remain asymptomatic throughout their lives. Here we investigated family cohorts of FSHD1 individuals who were either affected (manifesting) or without any discernible weakness (nonmanifesting/asymptomatic) and their unaffected family members to determine if individual epigenetic status and stability of repression at the contracted 4q35 D4Z4 array in myocytes correlates with FSHD disease.ResultsFamily cohorts were analyzed for DNA methylation on the distal pathogenic 4q35 D4Z4 repeat on permissive A-type subtelomeres. We found DNA hypomethylation in FSHD1-affected subjects, hypermethylation in healthy controls, and distinctly intermediate levels of methylation in nonmanifesting subjects. We next tested if these differences in DNA methylation had functional relevance by assaying DUX4-fl expression and the stability of epigenetic repression of DUX4-fl in myogenic cells. Treatment with drugs that alter epigenetic status revealed that healthy cells were refractory to treatment, maintaining stable repression of DUX4, while FSHD1-affected cells were highly responsive to treatment and thus epigenetically poised to express DUX4. Myocytes from nonmanifesting subjects had significantly higher levels of DNA methylation and were more resistant to DUX4 activation in response to epigenetic drug treatment than cells from FSHD1-affected first-degree relatives containing the same contraction, indicating that the epigenetic status of the contracted D4Z4 array is reflective of disease.ConclusionsThe epigenetic status of the distal 4qA D4Z4 repeat correlates with FSHD disease; FSHD-affected subjects have hypomethylation, healthy unaffected subjects have hypermethylation, and nonmanifesting subjects have characteristically intermediate methylation. Thus, analysis of DNA methylation at the distal D4Z4 repeat could be used as a diagnostic indicator of developing clinical FSHD. In addition, the stability of epigenetic repression upstream of DUX4 expression is a key regulator of disease and a viable therapeutic target.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-015-0072-6) contains supplementary material, which is available to authorized users.

Methylation status of HPV16 E2-binding sites classifies subtypes of HPV-associated oropharyngeal cancers.

The human papillomavirus (HPV) E2 protein is a transcriptional repressor of the oncogenes E6/E7 and loss of E2 function is considered a key step in carcinogenesis. Integration of HPV into the host genome may disrupt the E2 gene. Furthermore, methylation of CpG dinucleotides in E2-binding sites (E2BSs) in the HPV upstream regulatory region may interfere with transcriptional repression of E6 and E7 by E2. The authors hypothesized that the CpG methylation status of E2BS identifies subtypes of HPV type 16 (HPV16)-associated oropharyngeal squamous cell cancers (OPSCC) in association with E2 gene integrity and viral integration. Methylation of 10 CpG dinucleotides within the upstream regulatory region, encompassing E2BSs 1, 2, 3, and 4, was quantitatively analyzed by bisulfite pyrosequencing in 57 HPV16-associated OPSCC cases. E2 status was analyzed by gene amplification and quantitative real-time reverse transcriptase-polymerase chain reaction. Viral integration was determined by integration-specific polymerase chain reaction methods. Three subgroups with differential methylation at E2BS3 and E2BS 4 were identified: 1) complete methylation (>80%) associated with the presence of integrated HPV genomes with an intact E2 gene; 2) intermediate methylation levels (20%-80%) with predominantly episomal HPV genomes with intact E2; and 3) no methylation (<20%) with a disrupted E2 gene. Patients with high methylation levels tended to have a worse 5-year overall survival compared with patients with intermediate methylation (hazard ratio, 3.23; 95% confidence interval, 1.13-9.24 [P = .06]). Methylation of E2BS3 and E2BS4 in OPSCC is associated with E2 integrity and viral physical status. It might explain deregulated viral oncogene expression in the presence of E2. The prognostic significance of E2BS methylation for patients with HPV-associated OPSCC needs to be analyzed further.

An Improved and Practical Synthesis of Tranexamic Acid

Tranexamic acid 1, a synthetic antifibrinolytic drug with the treatment being considered highly cost-effective in many countries, has been included in the WHO list of essential medicines. In this paper, we designed the synthesis of 1 via a novel seven-step route from the readily available starting material dimethyl terephthalate, performing with 99.6% purity in 59.2% overall yield. During the process, we successfully developed a direct and efficient method for the preparation of key intermediate methyl 4-(acetamidomethyl)benzoate by one-pot hydrogenation and acylation in acetic anhydride using Ni/Al2O3 as a catalyst. More importantly, it should be a straightforward and practical way to circumvent the usage of toxic reagents (CrO3, Cl2), solvent (CCl4), and expensive catalyst (PtO2), etc., that plagued the previous methodologies.

Diastereoselective Synthesis of (1,3‐Dioxan‐4‐yl)pyrimidine and Purin Nucleoside Analogues

Abstract(1,3‐Dioxan‐4‐yl)‐substituted nucleoside analogues, higher homologues of antiviral and anticancer 1,3‐dioxolanes, were prepared from the key intermediate (4‐acetoxy‐1,3‐dioxan‐2‐yl)methyl benzoate and silylated bases. Glycosylation, carried out under Vorbrüggen conditions in the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf) as a catalyst, afforded the desired compounds with high stereoselectivity and regioselectivity, with only the desired β‐anomeric N‐1 pyrimidine and N‐9 purin nucleosides being obtained. 1H NMR experiments established that the β‐anomers were diequatorial, and this assignment was confirmed by single‐crystal X‐ray diffraction. Despite their structural similarities with natural nucleosides, none of the synthesized nucleosides showed antiviral activity.

The meta-epigenomic structure of purified human stem cell populations is defined at cis-regulatory sequences

The mechanism and significance of epigenetic variability in the same cell type between healthy individuals are not clear. Here, we purify human CD34+ hematopoietic stem and progenitor cells (HSPCs) from different individuals and find that there is increased variability of DNA methylation at loci with properties of promoters and enhancers. The variability is especially enriched at candidate enhancers near genes transitioning between silent and expressed states, and encoding proteins with leukocyte differentiation properties. Our findings of increased variability at loci with intermediate DNA methylation values, at candidate “poised” enhancers, and at genes involved in HSPC lineage commitment suggest that CD34+ cell subtype heterogeneity between individuals is a major mechanism for the variability observed. Epigenomic studies performed on cell populations, even when purified, are testing collections of epigenomes, or meta-epigenomes. Our findings show that meta-epigenomic approaches to data analysis can provide insights into cell subpopulation structure.

Setdb1 is required for germline development and silencing of H3K9me3-marked endogenous retroviruses in primordial germ cells.

Transcription of endogenous retroviruses (ERVs) is inhibited by de novo DNA methylation during gametogenesis, a process initiated after birth in oocytes and at approximately embryonic day 15.5 (E15.5) in prospermatogonia. Earlier in germline development, the genome, including most retrotransposons, is progressively demethylated. Young ERVK and ERV1 elements, however, retain intermediate methylation levels. As DNA methylation reaches a low point in E13.5 primordial germ cells (PGCs) of both sexes, we determined whether retrotransposons are marked by H3K9me3 and H3K27me3 using a recently developed low-input ChIP-seq (chromatin immunoprecipitation [ChIP] combined with deep sequencing) method. Although these repressive histone modifications are found predominantly on distinct genomic regions in E13.5 PGCs, they concurrently mark partially methylated long terminal repeats (LTRs) and LINE1 elements. Germline-specific conditional knockout of the H3K9 methyltransferase SETDB1 yields a decrease of both marks and DNA methylation at H3K9me3-enriched retrotransposon families. Strikingly, Setdb1 knockout E13.5 PGCs show concomitant derepression of many marked ERVs, including intracisternal A particle (IAP), ETn, and ERVK10C elements, and ERV-proximal genes, a subset in a sex-dependent manner. Furthermore, Setdb1 deficiency is associated with a reduced number of male E13.5 PGCs and postnatal hypogonadism in both sexes. Taken together, these observations reveal that SETDB1 is an essential guardian against proviral expression prior to the onset of de novo DNA methylation in the germline.

Solid–liquid equilibria and the physical properties of binary systems of diphenyl carbonate, dimethyl carbonate, methyl phenyl carbonate, anisole, methanol and phenol

Non-phosgene diphenyl carbonate (DPC) can be synthesized via many routes. A preferable green route of synthesis consists of a two-step reaction from dimethyl carbonate (DMC) and phenol because the direct synthesis of DPC is limited due to the low equilibrium constants of the forward reaction. In this synthesis process, the reaction products with intermediates should be separated from each other for higher yields of DPC and to recycle the reactants. However, relatively few studies have examined the separation design properties, particularly, the phase equilibria and the physical properties for systems containing the reaction intermediate methyl phenyl carbonate (MPC). In the present work, we analytically determined the binary solid–liquid equilibria (SLE) for the {DMC (1)+DPC (2)}, {DPC (1)+MPC (2)}, {phenol (1)+MPC (2)} and {methanol (1)+DPC (2)} systems. The former three systems showed a simple eutectic point, while the {methanol (1)+DPC (2)} system did not. The determined SLE data correlated well with a non-random two-liquid (NRTL) activity coefficient model. In addition, the temperature dependence of the physical properties of the reaction products densities, refractive indices and kinematic viscosities were reported. The experimental properties were correlated well with the Daubert and Danner (DIPPR) equation for density, the linear equation for refractive index, and the Goletz and Tassion equation for kinematic viscosity, respectively.

Identification of a novel thiopurine S-methyltransferase allele (TPMT*37)

Thiopurine S-methyltransferase (TPMT) is a key enzyme in the methylation of the thiopurine drugs azathioprine and 6-mercaptopurine. TPMT is subject to genetic polymorphism that results in a trimodal distribution of enzyme activity. All poor methylators (PMs) and 30-60% of intermediate methylators develop potentially life-threatening myelosuppression on standard doses of azathioprine and 6-mercaptopurine because of excess production of the thioguanine nucleotides (6-TGNs). Over 95% of PMs are explained by the alleles TPMT*2 and TPMT*3, whereas one in 20 intermediate methylators are heterozygous for a novel PM allele. In this brief report, we describe the identification of a novel allele (TPMT*37) in a Caucasian male who had a red blood cell TPMT activity of 8.9 U/ml (reference range: 9.3-17.6 U/ml). TPMT*37 introduces a premature stop codon at position 216, resulting in loss of the last 29 amino acid residues from the C terminal of the TPMT protein.

Methylation epigenotypes and genetic features in colorectal laterally spreading tumors

Aberrant DNA methylation plays an important role in genesis of colorectal cancer (CRC). Previously, we identified Group 1 and Group 2 methylation markers through genome-wide DNA methylation analysis, and classified CRC and protruded adenoma into three distinct clusters: high-, intermediate- and low-methylation epigenotypes. High-methylation epigenotype strongly correlated with BRAF mutations and these aberrations were involved in the serrated pathway, whereas intermediate-methylation epigenotype strongly correlated with KRAS mutations. Here, we investigated laterally spreading tumors (LSTs), which are flat, early CRC lesions, through quantitative methylation analysis of six Group 1 and 14 Group 2 methylation markers using pyrosequencing. Gene mutations in BRAF, KRAS and PIK3CA, and immunostaining of TP53 and CTNNB1 as well as other clinicopathological factors were also evaluated. By hierarchical clustering using methylation information, LSTs were classified into two subtypes; intermediate-methylation epigenotype correlating with KRAS mutations (p = 9 × 10(-4)) and a granular morphology (LST-G) (p = 1 × 10(-7)), and low-methylation epigenotype correlating with CTNNB1 activation (p = 0.002) and a nongranular morphology (LST-NG) (p = 1 × 10(-7)). Group 1 marker methylation and BRAF mutations were barely detected, suggesting that high-methylation epigenotype was unlikely to be involved in LST development. TP53 mutations correlated significantly with malignant transformation, regardless of epigenotype or morphology type. Together, this may suggest that two molecular pathways, intermediate methylation associated with KRAS mutations and LST-G morphology, and low methylation associated with CTNNB1 activation and LST-NG morphology, might be involved in LST development, and that involvement of TP53 mutations could be important in both subtypes in the development from adenoma to cancer.

Epigenetic profiling of prostate cancer tissue to identify men with high-risk disease.

197 Background: Prostate cancer (PCa) prognosis and aggressiveness are guided by Gleason Scores (GS). Moreover, biopsied prostate tissue might not be representative of the actual disease. Epigenetics could provide a quantitative method for confirming or upgrading disease status obtained by GS, providing additional information with regard to aggressiveness. Methods: Eighty-four prostatectomy samples from PCa patients with GS6 (n=6), GS3+4 (n=41), GS4+3 (n=26) and GS greater than or equal to 8 (n=11) were epigenetically profiled, for GSTP1, APC, RASSF1, RARB, and GAL3 methylation, using methylation-specific PCR. Unsupervised hierarchical clustering, based on Ward’s method, was performed to discover an association between DNA-methylation patterns and GS. Methylation ratios were scaled across all samples by calculating, per gene, a Z-score. Results: Hierarchical clustering analysis resulted in three distinct clusters. One cluster with a relative, general depletion of methylation, a second cluster with high methylation levels for most genes, particularly for RARB, and a third cluster exhibiting intermediate methylation signals and lower, but distinct, RARB methylation. Kendall’s rank correlation tau indicated a significant positive correlation between GS and methylation levels of all five genes, with tau ranging from 0.010 to 0.376 (all p values&lt;0.05). Methylation levels of all five genes were significantly lower in the samples with GS less than or equal to 3+4 versus those with GS greater than or equal to 4+3 (all p values&lt;0.05). Clustering showed that 100% of the GS6 and 80% of the GS3+4 patient’ samples were associated with low methylation signals. In contrast, 81% and 82% of the GS4+3, and GS greater than or equal to 8 samples respectively, fell into the category with intermediate to high methylation levels. Conclusions: Methylation levels of all genes correlated with GS and were significantly lower in patient' samples with GS6 and GS7 (3+4) compared to GS7 (4+3) and GS greater than or equal to 8. This study shows that epigenetic profiling of selected genes has prognostic value and may help to identify patients with aggressive PCa.

An environmentally benign approach to the synthesis of thymine via hydroformylation of methyl acrylate

A green and practical method for the synthesis of thymine was established by using methyl acrylate under hydroformylation conditions to give the important intermediate methyl α-formylpropionate. The incorporation of this protocol into the synthesis of thymine resulted in substantial reductions in waste emissions and production costs. .

A Facile Convergent Synthetic Procedure for 3-Deoxydapagliflozin as SGLT2 Inhibitor

3-Deoxydapagliflzoin (1) is a highly selective, potent sodium-dependent glucose transporter 2 (SGLT2) inhibitor discovered earlier. A facile convergent synthetic procedure for 3-deoxydapagliflzoin (1) was developed in 13 steps and in 38% overall yield starting from inexpensive methyl α-D-glucopyranoside 2. Regioselective protection of 2-OH and 3-OH in the key intermediate methyl 4,6-O-benzylidene-α-D-glucopyranoside 3 was systematically studied; the regioisomers thus obtained were distinguished from each other with nuclear overhauser effect (NOE) and their ratios and relative polarities were rationally explained. The strategy for deoxygenation of the sterically hindered 3-OH in another key intermediate 4,6-O-benzylidene-2-O-t-butyldiphenylsilyl-α-D-glucopyranoside (4) was also systematically studied. The facile synthetic route is characterized by convergence characteristics and inexpensive starting material, also representing an alternative general synthetic approach to phenyl 3-deoxy-C-glucosides.

ZAP70 Methylation Is An Independent Prognostic Biomarker For Front Line Therapy Of Chronic Lymphocytic Leukemia : Results From The UK LRF CLL4 Trial

In CLL, over-expression of ZAP70 is well established as a prognostic biomarker for unfavourable disease course. However, technical challenges limit the application of flow cytometry for ZAP70 detection and measurement of ZAP70 mRNA expression is complicated by the need for T cell depletion. Having identified a correlation between DNA methylation of a 5' regulatory region of ZAP70 and expression of the gene, we and others have proposed the measurement of ZAP70 methylation as an alternative biomarker. Of note, recent work by Claus et al showed ZAP70 methylation to be prognostic for progression free survival (PFS) after therapy in two small, phase II, trial cohorts, treated with regimes incorporating Rituximab. However, as no comprehensive assessment of the prognostic value of ZAP70 methylation has been reported, we analysed ZAP70 methylation in 416 samples from patients entered into the phase III UK LRF CLL4 trial (comparing Chlorambucil(C) with Fludarabine alone (F) or in combination with Cyclophosphamide(FC)).The methylation of a single CpG site 334bp downstream of the transcription start site (C334) was interrogated in peripheral blood mononuclear cell DNA samples, using a quantitative bisulfite pyrosequencing assay. All samples were bisulfite converted, amplified and pyrosequenced in triplicate with a 6 point standard curve in each run. Standard curves for each of the 20 runs were linear (r2 > 0.98) and sensitivity of pyrosequencing was ±5%.C334 methylation ranged from 0-100% and showed a significant inverse correlation with percentage of ZAP70 positive cells, IGHV identity to germline and percentage of CD38 positive cells (all p≤0.001). Methylation levels showed a trimodal, W-shaped, distribution with major modes at 5% and 95% and a minor mode at 50%. Based on this distribution, samples were classified as having Low (C334L<33.3%), Intermediate (C334Int 33.3-66.6%) or High (C334H >66.6%) methylation (Figure 1). [Display omitted] In univariate Kaplan Meier (KM) analysis, no significant difference was seen between C334L and C334Int groups in terms of PFS or OS (median PFS 1.68 versus 1.76 years, p=0.36 and median OS 4.71 years versus 5.41 years, p=0.18). However, C334H cases had a significantly longer PFS and OS than either group (median PFS=2.86 years, median OS=8.60 years, all p<0.001). C334H cases were significantly enriched with good risk features such as the presence of a 13q deletion, absence of 11q deletion, absence of NOTCH1 mutation, ZAP70 negativity, CD38 negativity and IGHV mutated status (all p<0.02). However, C334H status retained significant prognostic value for PFS and OS in KM analysis when cases were stratified by each of these good risk features. Furthermore, in multivariate Cox regression analysis, C334H cases showed a significantly reduced risk of progression compared to all other cases (HR=0.67 (95%CI 0.48-0.95), p=0.023), independent of treatment received, IGHV status and 11q and 17p deletion status (Table 1).By identifying a group of patients with hypermethylation of ZAP70, who display a superior outcome with first line chemotherapy, our results confirm the prognostic value of ZAP70 methylation and show it to be superior to ZAP70 expression as measured by flow cytometry. The independent prognostic value of this, compared with established biomarkers, together with the ease and low cost of the assay, warrant its validation in other large trial cohorts. Disclosures:No relevant conflicts of interest to declare.

Identification of an intermediate methyl carrier in the radical S-adenosylmethionine methylthiotransferases RimO and MiaB.

RimO and MiaB are radical S-adenosylmethionine (SAM) enzymes that catalyze the attachment of methylthio (-SCH3) groups to macromolecular substrates. RimO attaches a methylthio group at C3 of aspartate 89 of protein S12, a component of the 30S subunit of the bacterial ribosome. MiaB attaches a methylthio group at C2 of N(6)-(isopentenyl)adenosine, found at nucleotide 37 in several prokaryotic tRNAs. These two enzymes are prototypical members of a subclass of radical SAM enzymes called methylthiotransferases (MTTases). It had been assumed that the sequence of steps in MTTase reactions involves initial sulfur insertion into the organic substrate followed by capping of the inserted sulfur atom with a SAM-derived methyl group. In this work, however, we show that both RimO and MiaB from Thermotoga maritima catalyze methyl transfer from SAM to an acid/base labile acceptor on the protein in the absence of their respective macromolecular substrates. Consistent with the assignment of the acceptor as an iron-sulfur cluster, denaturation of the SAM-treated protein with acid results in production of methanethiol. When RimO or MiaB is first incubated with SAM in the absence of substrate and reductant and then incubated with excess S-adenosyl-l-[methyl-d3]methionine in the presence of substrate and reductant, production of the unlabeled product precedes production of the deuterated product, showing that the methylated species is chemically and kinetically competent to be an intermediate.