Reductive Methylation Research Articles

Dimethylsulfoxide as an N‐Methylation Reagent for Amines and Aromatic Nitro Compounds

DMSO a go go: Wang and Xiao have recently reported a simple procedure for the N-methylation of amines, with DMSO and formic acid as the key reagents. The reaction has also been extended to the reductive methylation of aromatic nitro compounds.

Nicotinamide in Friedreich's ataxia: useful or not?

Nicotinamide in Friedreich's ataxia: useful or not?

Reductive methylation and mutation of an anthrax toxin fusion protein modulates its stability and cytotoxicity.

We characterized an anti-cancer fusion protein consisting of anthrax lethal factor (LF) and the catalytic domain of Pseudomonas exotoxin A by (i) mutating the N-terminal amino acids and by (ii) reductive methylation to dimethylate all lysines. Dimethylation of lysines was achieved quantitatively and specifically without affecting binding of the fusion protein to PA or decreasing the enzymatic activity of the catalytic moiety. Ubiquitination in vitro was drastically decreased for both the N-terminally mutated and dimethylated variants, and both appeared to be slightly more stable in the cytosol of treated cells. The dimethylated variant showed greatly reduced neutralization by antibodies to LF. The two described modifications offer unique advantages such as increased cytotoxic activity and diminished antibody recognition, and thus may be applicable to other therapeutic proteins that act in the cytosol of cells.

Dietary uridine supplementation decreases tumor number in Apc(Min/+) mice (829.11)

Colorectal cancer affects 1.23 million people and accounts for more than 600,000 deaths each year worldwide. Deficiency of methyl donors including folate and methionine has been linked to increased colon cancer risk in experimental and epidemiological studies. The Apc(Min/+) mouse model is a powerful tool for studying the genetic and dietary mechanisms which contribute to intestinal cancer phenotypes. A previous study showed that Apc(Min/+) mice also lacking one copy of the Shmt1 gene, which results in perturbed de novo thymidylate (dTMP) synthesis, displayed increased intestinal tumor number that was exacerbated by folate deficiency. dTMP is the only nucleotide that is synthesized at the replication fork as needed for DNA replication and repair. dTMP can either be synthesized de novo from the folate‐dependent reductive methylation of deoxyuridylate (dUMP) to dTMP through a multi‐enzyme complex or can be generated by phosphorylating thymidine to dTMP by the salvage pathway enzyme thymidine kinase (TK1). In this study, we sought to determine whether dietary supplementation of dTMP precursors (thymidine, deoxyuridine or uridine) could modify tumor number in the Apc(Min/+) model. Surprisingly, dietary uridine supplementation reduced intestinal tumor number and tumor burden by 40% (p<0.05) relative to the control diet; neither thymidine nor deoxyuridine affected tumor number.Grant Funding Source: Supported by NIH R37DK58144

Synthesis of 2-(buta-1,3-diynyl)-N,N-dimethylanilines Using Reductive Methylation Step

Synthesis of 2-(buta-1,3-diynyl)-N,N-dimethylanilines based on reductive methylation of ortho-iodoanilines using CH2O–NaBH3CN and coupling with terminal diacetylens was developed. Altered sequence including dimethylation of 2-(buta-1,3-diynyl)anilines was also successfully achieved and gave higher overall yields in the case of anilines without acceptor substituent in the ring.

Methionine synthase reductase A66G polymorphism and leukemia risk: evidence from published studies

Methionine synthase reductase (MTRR) is required for the reductive methylation of cobalamin, which is the functional cofactorial form of methionine synthase (MS) in the remethylation of homocysteine to methionine. The MTRR A66G (rs1801394) polymorphism is found to be associated with decreased enzyme affinity for MTR, the gene that encodes MS, and has been widely investigated for cancer risk, including leukemia. However, the conclusions of epidemiological studies have always been contradictory. To further clarify the association of MTRR A66G polymorphism with the risk of leukemia, this meta-analysis was performed for 2913 cases and 4764 controls. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of associations. Pooled ORs were determined for the co-dominant model (GG vs. AA, AG vs. AA), dominant model (GG + AG vs. AA) and recessive model (GG vs. AA+ AG), respectively. No significant associations were found for all comparisons in the overall pooled analysis. However, the results of stratified analyses revealed that MTRR A66G GG genotype was associated with decreased leukemia risk in the Caucasian population, in children and for acute lymphoblastic leukemia (ALL). In contrast, increased risk was observed in the Asian population and for acute myeloid leukemia (AML). This meta-analysis suggests that MTRR A66G GG is associated with decreased risk of leukemia in a Caucasian population and in children, especially for ALL.

Effect of N-pyridinium positions of quaternized chitosan on transfection efficiency in gene delivery system

Methylated N-pyridylmethyl chitosan chlorides (M-PyMeChCs) with a similar total degree of quaternization (DQT) and molecular weight but different N-pyridinium positions were synthesized by reductive amination and methylation, respectively. The effect of N-pyridinium positions on transfection efficiency and cytotoxicity was investigated in human hepatoma (Huh7) cell lines. The results revealed that M-PyMeChCs are able to form a complete complex formation with DNA since there is an N/P ratio of 5. The particle sizes of M-PyMeChCs/DNA nanopolyplexes were approximately 300nm and indicated a positive charge. The morphology of these nanopolyplexes was found to be in a spherical shape which was investigated by using the transmission electron microscopy (TEM). The M4-PyMeChC/DNA nanopolyplexes showed highest in vitro transfection efficiency in Huh7 cells at N/P ratio of 20 compared to M2-PyMeChC/DNA and M3-PyMeChC/DNA nanopolyplexes. In comparison to M3-PyMeChC/DNA nanopolyplexes, M2-PyMeChC/DNA and M4-PyMeChC/DNA nanopolyplexes showed lower cytotoxicity in Huh7 cells. Our result demonstrated that N-pyridinium positions of M-PyMeChCs are related to transfection efficiency and cytotoxicity.

Structure of fructo-oligosaccharides from leaves and stem of Agave tequilana Weber, var. azul

Fructo-oligosaccharides (FOSs) of a six year old agave plant variety, Agave tequilana, were isolated and fractionated by 2D preparative chromatography (SEC and rpHPLC). Structural analyses of different FOS-fractions were performed by reductive methylation analysis connected to GC/FID identification and NMR-analysis. FOSs from leaves (d.p. 3–8) contain single α-d-Glcp residues as well in terminal as internal position, however (2→1)-linked β-d-Fruf residues only. FOSs from stem, however, contain as well (2→1)- and (2→6)-linked β-d-Fruf residues with branched oligomeric repeating units. These characteristics indicate an enzymatically catalyzed metabolic regulation for the biosynthesis and transformation of fructans in A. tequilana which strongly depends on location and transport activities.

Thymidylate synthase polymorphisms and risks of human orofacial clefts

Underlying mechanisms are unknown by which folic acid use in early pregnancy may reduce risks of orofacial clefts. Thymidylate synthase (TYMS) is a folate-dependent enzyme that catalyzes reductive methylation of deoxyuridylate to thymidylate, thereby playing a central role in DNA synthesis and repair. We investigated two TYMS functional variants (a 28-bp tandem repeat in the promoter enhancer region of the 5'-UTR; and TYMS 1494del6 (rs16430): a 6-bp deletion in the 3'-UTR) for their risk of cleft palate (CP) and of cleft lip with/without CP (CLP). We investigated effect measure modification between these variants and maternal folate intake for cleft risk. This case-control study included deliveries from July 1999 to June 2003 from select areas of California. Case groups included CLP or CP alone. Nonmalformed, liveborn controls were randomly selected. Maternal interviews provided information on vitamin use and dietary folate intake. DNA was derived from newborn bloodspots. Data were available for 304 CLP cases, 123 CP cases, and 581 controls. 1496del6 variants did not appear to influence risk of CP or CLP. Homozygosity for the 28-bp VNTR variant influenced CP risk (odds ratios, OR = 1.8, 95% confidence interval, 1.1-3.1), particularly among Hispanic infants, OR 2.1 (1.0-4.6). Effect measure modification was observed between the 28-bp VNTR and combined folate intake for CP with an OR of 10.0 (1.6-60.9). Although these findings are consistent with biological mechanisms, they were based on relatively small sample sizes and may represent false-positive discoveries. Replication is warranted in other populations.

Enhanced Detection of Ubiquitin Isopeptides Using Reductive Methylation

Identification of ubiquitination (Ub) sites is of great interest due to the critical roles that the modification plays in cellular regulation. Current methods using mass spectrometry rely upon tryptic isopeptide diglycine tag generation followed by database searching. We present a novel approach to ubiquitin detection based upon the dimethyl labeling of isopeptide N-termini glycines. Ubiquitinated proteins were digested with trypsin and the resulting peptide mixture was derivatized using formaldehyde-D2 solution and sodium cyanoborohydride. The dimethylated peptide mixtures were next separated by liquid chromatography and analyzed on a quadrupole-TOF based mass spectrometer. Diagnostic b2' and a1' ions released from the isopeptide N-terminus upon collision-induced dissociation (CID) were used to spectrally improve the identification of ubiquitinated isopeptides. Proof of principle was established by application to a ubiquitinated protein tryptic digest spiked into a six-protein mix digest background. Extracted ion chromatograms of the a1' and b2' diagnostic product ions from the diglycine tag resulted in a significant reduction in signal complexity and demonstrated a selectivity towards the identification of diglycine branched isopeptides. The method was further shown to be capable of identifying diglycine isopeptides resulting from in-gel tryptic digests of ubiquitin enriched material from a His-Ub transfected cell line. We envisage that these ions may be utilized in global ubiquitination studies with post-acquisition MS/MS (or MSe) data interrogation on high resolution hybrid mass spectrometers. ᅟ

Two optimized synthetic pathways toward a chiral precursor of Mivacurium chloride and other skeletal muscle relaxants

A chiral precursor of Mivacurium chloride, (R)-5′-methoxylaudanosine, was prepared using two different methods. The chiral resolution of racemic 5′-methoxylaudanosine, typically used in industry, was carried out in parallel with a procedure consisting of asymmetric transfer hydrogenation (ATH) and reductive methylation. A novel one-pot synthetic step was developed for the synthesis of racemic 5′-methoxylaudanosine. In both routes, the enantioselectivity was high but further purification was necessary to reach the level of a pharmaceutical standard. The individual synthetic steps reported herein can also be used for the synthesis of analogous bistetrahydroisoquinoline-based skeletal muscle relaxants.

Self sensitized photooxidation of tetrathienyltetrathiafulvalenes: synthesis of thienyl substituted 1,2,5,8-tetrathiecine-6,7-dione, a new heterocyclic system

Irradiation of tetrathienyltetrathiafulvalenes in an oxygen saturated solution gives tetrathienyl substituted 1,2,5,8-tetrathiecine-6,7-diones, via singlet oxygen generation followed by cycloaddition onto the TTF double bond and a final rearrangement process. The thermal and photochemical behaviour and reductive methylation of this new class of macro-heterocyclic system have also been investigated.

Metal-template Condensation of 1,3-Diamino-2-propanol and Formaldehyde: Synthesis, Crystal Structure, and Magnetic Properties of a New Dinuclear Copper(II) Complex

Department of Chemistry, Kongju National University, Kongju 314-701, KoreaReceived August 25, 2012, Accepted September 17, 2012Key Words : Dinuclear copper(II) complex, Reductive methylation, Crystal structure, Magnetic properties,Template condensationPolynuclear transition metal complexes with relativelystrong intramolecular metal-metal interactions have receivedconsiderable attention because of their interesting chemicalproperties and/or potential applications in various fields,such as supramolecular chemistry, catalysts for organicreactions, and materials chemistry.

A mass spectrometry-based plasma protein panel targeting the tumor microenvironment in patients with breast cancer

Proteins secreted or shed by cancerous cells are seen as a rich source of biomarkers and novel therapeutic targets. Recently, the importance of the tumor microenvironment, which comprises the surrounding non-tumor cells, has received increased attention for its role in tumor progression. We developed a targeted proteomics assay to monitor a panel of plasma proteins postulated to be present in the tumor microenvironment. The plasma of 76 breast cancer patients was depleted of abundant circulating proteins, enzymatically digested and labeled by reductive methylation. The labeled digests were analyzed by tandem mass spectrometry using a multiple reaction monitoring acquisition method. The protein targets were correlated with the tumor characteristics, the extent of the disease and the clinical staging of the patients. Linear discriminant analysis revealed that infiltrating ductal and invasive mammary breast carcinomas could be grouped based on distinctive peptide levels of fibronectin, clusterin, gelsolin and α-1-microglobulin/Inter-α-trypsin inhibitor light chain precursor (AMBP). These proteins have been previously associated with breast cancer at the tissue level, however, this is the first study to measure plasma levels of these proteins and correlate these levels with clinical features. Significant variability was seen between unique peptides belonging to the same protein. This article is part of a Special Issue entitled: From protein structures to clinical applications.

Folate binding site of flavin-dependent thymidylate synthase

The DNA nucleotide thymidylate is synthesized by the enzyme thymidylate synthase, which catalyzes the reductive methylation of deoxyuridylate using the cofactor methylene-tetrahydrofolate (CH(2)H(4)folate). Most organisms, including humans, rely on the thyA- or TYMS-encoded classic thymidylate synthase, whereas, certain microorganisms, including all Rickettsia and other pathogens, use an alternative thyX-encoded flavin-dependent thymidylate synthase (FDTS). Although several crystal structures of FDTSs have been reported, the absence of a structure with folates limits understanding of the molecular mechanism and the scope of drug design for these enzymes. Here we present X-ray crystal structures of FDTS with several folate derivatives, which together with mutagenesis, kinetic analysis, and computer modeling shed light on the cofactor binding and function. The unique structural data will likely facilitate further elucidation of FDTSs' mechanism and the design of structure-based inhibitors as potential leads to new antimicrobial drugs.

Lysine methylation strategies for characterizing protein conformations by NMR

In the presence of formaldehyde and a mild reducing agent, reductive methylation is known to achieve near complete dimethylation of protein amino groups under non-denaturing conditions, in aqueous media. Amino methylation of proteins is employed in mass spectrometric, crystallographic, and NMR studies. Where biosynthetic labeling is prohibitive, amino (13)C-methylation provides an attractive option for monitoring folding, kinetics, protein-protein and protein-DNA interactions by NMR. Here, we demonstrate two improvements over traditional (13)C-reductive methylation schemes: (1) By judicious choice of stoichiometry and pH, ε-aminos can be preferentially monomethylated. Monomethyl tags are less perturbing and generally exhibit improved resolution over dimethyllysines, and (2) By use of deuterated reducing agents and (13)C-formaldehyde, amino groups can be labeled with (13)CH(2)D tags. Use of deutero-(13)C-formaldehyde affords either (13)CHD(2), or (13)CD(3) probes depending on choice of reducing agent. Making use of (13)C-(2)H scalar couplings, we demonstrate a filtering scheme that eliminates natural abundance (13)C signal.

Crystal structure of the glycosyltransferase SnogD from the biosynthetic pathway of nogalamycin in Streptomyces nogalater

The glycosyltransferase SnogD from Streptomyces nogalater transfers a nogalamine moiety to the metabolic intermediate 3',4'-demethoxynogalose-1-hydroxynogalamycinone during the final steps of biosynthesis of the aromatic polyketide nogalamycin. The crystal structure of recombinant SnogD, as an apo-enzyme and with a bound nucleotide, 2-deoxyuridine-5'-diphosphate, was determined to 2.6 Å resolution. Reductive methylation of SnogD was crucial for reproducible preparation of diffraction quality crystals due to creation of an additional intermolecular salt bridge between methylated lysine residue Lys384 and Glu374* from an adjacent molecule in the crystal lattice. SnogD is a dimer both in solution and in the crystal, and the enzyme subunit displays a fold characteristic of the GT-B family of glycosyltransferases. Binding of the nucleotide is associated with rearrangement of two active-site loops. Site-directed mutagenesis shows that two active-site histidine residues, His25 and His301, are critical for the glycosyltransferase activities of SnogD both in vivo and in vitro. The crystal structures and the functional data are consistent with a role for His301 in binding of the diphosphate group of the sugar donor substrate, and a function of His25 as a catalytic base in the glycosyl transfer reaction.

Simple and Efficient Synthesis of (S)-Dapoxetine

A refinement in the synthetic strategy for (S)-dapoxetine 1 is described. The key features of synthetic strategy include (a) a Sharpless asymmetric epoxidation reaction and regioselective reductive ring opening of a 2,3-epoxy alcohol to elaborate the hydroxy-bearing stereogenic center at benzylic position; (b) regioselective functionalization of 1-naphthol and amine functionality through Mitsunobu procedures; and (c) Eschweiler–Clarke reductive methylation condition to access the target molecule.

Abstract 3892: Anti-cooperative allosteric inhibitors of thymidylate synthase as novel antineoplastic agents

Abstract Thymidylate synthase (TS) inhibitors are a class of antineoplastic agents widely used for the treatment of gastrointestinal, breast, ovarian and other cancers. TS catalyzes reductive methylation of dUMP to dTMP and serves as the sole de novo source of thymidylate inside the cell. While TS is essential for DNA biosynthesis and normal cell proliferation, TS overexpression has been associated with resistant tumor phenotype and poor prognosis. Although TS inhibitor 5-flourouracil (5-FU) continues to be one of the most frequently used chemo-therapeutic agents, 5-FU and its metabolites can cause significant liver, cardiac and bone marrow toxicity and are associated with development of drug resistance. In this study, we developed a new strategy that targets TS subunit cooperativity by freezing isoform interconversions and structural dynamics of the enzyme. In TS catalytic mechanism, conserted motion of amino acid residues 181-197 brings the catalytic Cys195 in and out of the active site. These small scale motions that translate across the dimer interface upon substrate binding and product release are integral to TS enzymatic function and can be taken advantage of in structure-based design of its inhibitors. For this purpose, we selected TS crystal structure with high asymmetry between the subunits (PDBID:1HVY) and used it as a template for molecular docking. The structure was prepared by removing heteroatoms and liganded molecules, followed by energy minimization in UCSF Chimera (1.5.2) and testing resulting geometries in Coot (0.6.2). We identified 5 potential docking sites and selected the site at the dimer interface specific to inactive conformation of TS. National Cancer Institute (NCI) small molecule library of 139,735 compounds was extracted from ZINC and pruned for redundant structures and molecules with molecular weight less than 200 Da. Resulting compound library was docked (DOCK6.4) into the selected site in 1000 orientations with flexible bond parameters enabled. The top 1000 hits were re-scored using AMBER and visualized in PyMOL to check for consistency and geometric fit. 837 out of 1000 molecules were further selected and re-ranked based on predicted physicochemical characteristics (cLogP, pKa, solubility, etc.). The twenty-seven highest ranked compounds were requested from the NCI and tested for their effects on viability in HeLa, K562 and A549 human cancer cell lines. Five out of 27 compounds reduced viability by more than 50% at a concentration of 10uM; one of the compounds was effective below 1uM. In conclusion, by using this new approach that targets TS subunit cooperativity, we identified small molecules with potential anti-cancer activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3892. doi:1538-7445.AM2012-3892

Structural investigations of ClC‐ec1, a large integral membrane protein, using solution‐state NMR and nanodisc technology

CLC antiporters mediate Cl−/H+ exchange across cell membranes in organisms ranging from E. coli to humans via coupling of protein conformational changes to ion binding, unbinding and translocation. X‐ray crystallographic structures have provided essential molecular pictures, but details of conformational changes remain elusive. We are using solution state NMR to probe conformational change in ClC‐ec1, a CLC homolog of known structure. 13C labeling of lysine residues is achieved by post translational reductive methylation. 1H‐13C HSQC spectra reveal reversible, substrate‐dependent spectral changes that may reflect conformational changes. To address the concern that the detergent‐solubilized protein is not in a native environment, we are also using nanodiscs, which consist of two amphipathic helices wrapped around a bilayer patch of lipids surrounding the membrane‐embedded protein. The monomeric, 13C‐methylated mutant form of ClC‐ec1 was incorporated into discs and tested for homogeneity and monodispersity. The 1H‐13C HSQC spectra show substrate‐dependent spectral changes in this system, although further experiments will be needed to determine if these represent functionally‐relevant conformational changes. These studies demonstrate the potential for using solution‐state NMR and nanodisc technology to study ClC‐ec1 in a native lipid environment.Supported by: National Science Foundation