C-methyl Groups Research Articles

Anthraquinone‐Sensitized Photooxidation of 5‐Methylcytosine in DNA Leading to Piperidine‐Induced Efficient Strand Cleavage

One-electron photooxidations of 5-methyl-2'-deoxycytidine (d(m)C) and 5-trideuteriomethyl-2'-deoxycytidine ([D(3)]d(m)C) by sensitization with anthraquinone (AQ) derivatives were investigated. Photoirradiation of an aerated aqueous solution containing d(m)C and anthraquinone 2-sulfonate (AQS) afforded 5-formyl-2'-deoxycytidine (d(f)C) and 5-hydroxymethyl-2'-deoxycytidine (d(hm)C) in good yield through an initial one-electron oxidation process. The deuterium isotope effect on the AQS-sensitized photooxidation of d(m)C suggests that the rate-determining step in the photosensitized oxidation of d(m)C involves internal transfer of the C5-hydrogen atom of a d(m)C-tetroxide intermediate to produce d(f)C and d(hm)C. In the case of a 5-methylcytosine ((m)C)-containing duplex DNA with an AQ chromophore that is incorporated into the backbone of the DNA strand so as to be immobilized at a specific position, (m)C underwent efficient direct one-electron oxidation by the photoexcited AQ, which resulted in an exclusive DNA strand cleavage at the target (m)C site upon hot piperidine treatment. In accordance with the suppression of the strand cleavage at 5-trideuterio-methylcytosine observed in a similar AQ photosensitization, it is suggested that deprotonation at the C5-methyl group of an intermediate (m)C radical cation may occur as a key elementary reaction in the photooxidative strand cleavage at the (m)C site. Incorporation of an AQ sensitizer into the interior of a strand of the duplex enhanced the one-electron photooxidation of (m)C, presumably because of an increased intersystem crossing efficiency that may lead to efficient piperidine-induced strand cleavage at an (m)C site in a DNA duplex.

Synthesis of the C19 methyl ether of aspercyclide A via germyl-Stille macrocyclisation and ELISA evaluation of both enantiomers following optical resolution

Aspercyclide A (1) is a biaryl ether containing 11-membered macrocyclic natural product antagonist of the human IgE-FcεRI protein-protein interaction (PPI); a key interaction in the signal transduction pathway for allergic disorders such as asthma. Herein we report a novel approach to the synthesis of the C19 methyl ether of aspercyclide A, employing a Pd(0)-catalysed, fluorous-tagged alkenylgermane/arylbromide macrocyclisation (germyl-Stille reaction) as the key step, and evaluation of both enantiomers of this compound via ELISA following optical resolution by CSP-HPLC. A crystal structure for germyl hydride 27 is also reported.

Free Radical Formation in Novel Carotenoid Metal Ion Complexes of Astaxanthin

The carotenoid astaxanthin forms novel metal ion complexes with Ca(2+), Zn(2+), and Fe(2+). MS and NMR measurements indicate that the two oxygen atoms on the terminal cyclohexene ring of astaxanthin chelate the metal to form 1:1 complexes with Ca(2+) and Zn(2+) at low salt concentrations <0.2 mM. The stability constants of these complexes increased by a factor of 85 upon changing the solvent from acetonitrile to ethanol for Ca(2+) and by a factor of 7 for Zn(2+) as a consequence of acetonitrile being a part of the complex. Optical studies showed that at high concentrations (>0.2 mM) of salt, 2:1 metal/astaxanthin complexes were formed in ethanol. In the presence of Ca(2+) and Zn(2+), salts the lifetime of the radical cation and dication formed electrochemically decreased relative to those formed from the uncomplexed carotenoid. DFT calculations showed that the deprotonation of the radical cation at the carbon C3 position resulted in the lowest energy neutral radical, while proton loss at the C5, C9, or C13 methyl groups was less favorable. Pulsed EPR measurements were carried out on UV-produced radicals of astaxanthin supported on silica-alumina, MCM-41, or Ti-MCM-41. The pulsed EPR measurements detected the radical cation and neutral radicals formed by proton loss at 77 K from the C3, C5, C9, and C13 methyl groups and a radical anion formed by deprotonation of the neutral radical at C3. There was more than an order of magnitude increase in the concentration of radicals on Ti-MCM-41 relative to MCM-41, and the radical cation concentration exceeded that of the neutral radicals.

Stereoselective VO(acac) 2 catalyzed epoxidation of acyclic homoallylic diols. Complementary preparation of C2- syn-3,4-epoxy alcohols

A substrate-controlled stereoselective epoxidation of free and monoprotected homoallylic diols was developed. This second-generation approach is based on the incorporation of a primary hydroxy directing group at the C2 methyl carbon, which changes the nature of the vanadium ester intermediate providing a new diastereoselectivity manifold for the preparation of 3,4-epoxy alcohols. This modification favored the formation of the challenging C2- syn epoxy alcohol product not previously available using the standard homoallylic alcohol substrates. These new epoxy alcohol diastereomers expand the scope and generality for the utilization of 3,4-epoxy alcohols as precursors for stereoselective polypropionate synthesis.

Synthesis, crystal structure and spectroscopic studies of copper(II) complex of C-meso-1,5,8,12-tetramethyl-1,4,8,11-tetraazacyclotetradecane

Copper(II) complex coordinated with cyclam-type macrocyclic tetramine, C-meso-1,5,8,12-tetramethyl-1,4,8,11-tetraazacyclotetradecane (2Cdmc) has been synthesized and characterized by single crystal X-ray crystallographic analysis and UV–Vis absorption spectra. Solved molecular structure of [Cu(2Cdmc)(H2O)]Cl2·2H2O (1) revealed that macrocyclic ligand 2Cdmc was in the most stable trans-III structure, but the C-methyl groups of 2Cdmc adopted energetically unfavorable axial configuration (C-axial). This complex 1 is the second example of complex of cyclam-type tetramine with only axially oriented C-methyl groups. This complex adopted five-coordinated square-pyramidal geometry with one water molecule occupying one of the two apical sites. Network of hydrogen bonds involving counter anions (Cl−), coordinating and non-coordinating water molecules, and N–H of 2Cdmc was present. UV–Vis spectra in various solutions also indicated the formation of five-, or six-coordinated complex in methanol and DMSO, but four-coordinated complex in water.

CYP99A3: functional identification of a diterpene oxidase from the momilactone biosynthetic gene cluster in rice

Rice (Oryza sativa) produces momilactone diterpenoids as both phytoalexins and allelochemicals. Strikingly, the rice genome contains a biosynthetic gene cluster for momilactone production, located on rice chromosome 4, which contains two cytochrome P450 (CYP) mono-oxygenases, CYP99A2 and CYP99A3, with undefined roles; although it has been previously shown that RNA interference double knock-down of this pair of closely related CYPs reduced momilactone accumulation. Here we attempted biochemical characterization of CYP99A2 and CYP99A3, which was ultimately achieved by complete gene recoding, enabling functional recombinant expression in bacteria. With these synthetic gene constructs it was possible to demonstrate that while CYP99A2 does not exhibit significant activity with diterpene substrates, CYP99A3 catalyzes consecutive oxidations of the C19 methyl group of the momilactone precursor syn-pimara-7,15-diene to form, sequentially, syn-pimaradien-19-ol, syn-pimaradien-19-al, and syn-pimaradien-19-oic acid. These are presumably intermediates in momilactone biosynthesis, as a C19 carboxylic acid moiety is required for formation of the core 19,6-γ-lactone ring structure. We further were able to detect syn-pimaradien-19-oic acid in rice plants, which indicates physiological relevance for the observed activity of CYP99A3. In addition, we found that CYP99A3 also oxidized syn-stemod-13(17)-ene at C19 to produce, sequentially, syn-stemoden-19-ol, syn-stemoden-19-al, and syn-stemoden-19-oic acid, albeit with lower catalytic efficiency than with syn-pimaradiene. Although the CYP99A3 syn-stemodene-derived products were not detected in planta, these results nevertheless provide a hint at the currently unknown metabolic fate of this diterpene in rice. Regardless of any wider role, our results strongly indicate that CYP99A3 acts as a multifunctional diterpene oxidase in momilactone biosynthesis.

ChemInform Abstract: Synthesis of a Chiral Polysubstituted cis-Fused Decalin Containing an Angular C-Methyl Group by an Intramolecular Diels-Alder Reaction.

ChemInform Abstract: Synthesis of a Chiral Polysubstituted cis-Fused Decalin Containing an Angular C-Methyl Group by an Intramolecular Diels-Alder Reaction.

Error-free replicative bypass of thymine glycol by the combined action of DNA polymerases κ and ζ in human cells

Thymine glycol (Tg) is the most common DNA lesion of thymine induced by interaction with reactive oxygen species. Because of the addition of hydroxyl groups at C5 and C6 in a Tg lesion, the damaged base loses its aromatic character and becomes nonplanar; consequently, the C5 methyl group protrudes in an axial direction and that prevents the stacking of the 5' base above the Tg lesion. Because Tg presents a severe block to continued synthesis by replicative DNA polymerases, we determine here how human cells manage to replicate through this lesion. Using a duplex plasmid system where bidirectional replication ensues from an origin of replication, we show that translesion synthesis (TLS) makes a prominent contribution to Tg bypass and that it occurs in a predominantly error-free fashion. Also, we provide evidence that Pol kappa and Pol zeta function together in promoting error-free replication through the lesion, and based on structural and biochemical information, we propose a role for Pol kappa at the insertion step and of Pol zeta at the extension step of Tg bypass. We discuss the implications of these observations and suggest that human cells have adapted the TLS machinery to function in a much more error-free fashion than could have been predicted from the intrinsic catalytic efficiencies and fidelities of TLS polymerases.

Probing Ligand-binding Pockets of the Mevalonate Pathway Enzymes from Streptococcus pneumoniae

Diphosphomevalonate (Mev.pp) is the founding member of a new class of potential antibiotics targeting the Streptococcus pneumoniae mevalonate (Mev) pathway. We have synthesized a series of Mev.pp analogues designed to simultaneously block two steps in this pathway, through allosteric inhibition of mevalonate kinase (MK) and, for five of the analogues, by mechanism-based inactivation of diphosphomevalonate decarboxylase (DPM-DC). The analogue series expands the C(3)-methyl group of Mev.pp with hydrocarbons of varying size, shape, and chemical and physical properties. Previously, we established the feasibility of a prodrug strategy in which unphosphorylated Mev analogues could be enzymatically converted to the active Mev.pp forms by the endogenous MK and phosphomevalonate kinase. We now report the kinetic parameters for the turnover of non-, mono-, and diphosphorylated analogues as substrates and inhibitors of the three mevalonate pathway enzymes. The inhibition of MK by Mev.pp analogues revealed that the allosteric site is selective for compact, electron-rich C(3)-subsitutents. The lack of reactivity of analogues with DPM-DC provided evidence, counter to the existing model, for a decarboxylation transition state that is concerted rather than dissociative. The Mev pathway is composed of three structurally and functionally conserved enzymes that catalyze consecutive steps in a metabolic pathway. The current work reveals that these enzymes exhibit significant differences in specificity toward R-group substitution at C(3) and that these patterns are explained well by changes in the volume of the C(3) R-group-binding pockets of the enzymes.

Methylation-independent DNA Binding Modulates Specificity of Repressor of Silencing 1 (ROS1) and Facilitates Demethylation in Long Substrates

DNA cytosine methylation is an epigenetic mark that promotes gene silencing and performs critical roles during reproduction and development in both plants and animals. The genomic distribution of DNA methylation is the dynamic outcome of opposing methylation and demethylation processes. In plants, active demethylation occurs through a base excision repair pathway initiated by 5-methycytosine (5-meC) DNA glycosylases of the REPRESSOR OF SILENCING 1 (ROS1)/DEMETER (DME) family. To gain insight into the mechanism by which Arabidopsis ROS1 recognizes and excises 5-meC, we have identified those protein regions that are required for efficient DNA binding and catalysis. We have found that a short N-terminal lysine-rich domain conserved in members of the ROS1/DME family mediates strong methylation-independent binding of ROS1 to DNA and is required for efficient activity on 5-meC.G, but not for T.G processing. Removal of this domain does not significantly affect 5-meC excision from short molecules, but strongly decreases ROS1 activity on long DNA substrates. This region is not required for product binding and is not involved in the distributive behavior of the enzyme on substrates containing multiple 5-meC residues. Altogether, our results suggest that methylation-independent DNA binding allows ROS1 to perform a highly redundant search for efficient excision of a nondamaged, correctly paired base such as 5-meC in long stretches of DNA. These findings may have implications for understanding the evolution of structure and target specificity in DNA glycosylases.

ChemInform Abstract: Total Synthesis of (.+‐.)‐Aspercyclide A and Its C19 Methyl Ether.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Total synthesis and evaluation of a key series of C5-substituted vinblastine derivatives.

A remarkably concise seven- to eight-step total synthesis of a systematic series of key vinblastine derivatives is detailed and used to characterize the importance and probe the role of the C5 ethyl substituent (R = H, Me, Pr, CH=CH(2), C[triple bond]CH, CH(2)OH, and CHO vs Et). The analogues, which bear deep-seated structural changes accessible only by total synthesis, were prepared using a powerful intramolecular [4 + 2]/[3 + 2] cycloaddition cascade of 1,3,4-oxadiazoles ideally suited for use in the assemblage of the vindoline-derived lower subunit followed by their incorporation into the vinblastine analogues through the use of a single-step biomimetic coupling with catharanthine. The evaluation of the series revealed that the tubulin binding site surrounding this C5 substituent is exquisitely sensitive to the presence (Et > H, 10-fold), size (Me < or = Et > Pr, 10-fold), shape (Et > CH=CH(2) and C[triple bond]CH, > 4-fold), and polarity (Et > CHO > CH(2)OH, >10-20-fold) of this substituent and that on selected occasions only a C5 methyl group may provide analogues that approach the activity observed with the naturally occurring C5 ethyl group.

Total Synthesis of (±)-Aplykurodinone-1: Traceless Stereochemical Guidance

The total synthesis of the highly degraded steroidal natural product, aplykurodinone-1 (1), has been accomplished. Key features include a one-flask hydrolysis/retro-aldol/iodolactonization sequence to excise the C(8) hydroxymethylene functionality with retention of stereochemistry and the stereoselective installation of the C(13) methyl group through hydrogenation with homogeneous catalyst.

A Simple Method for Enzymatic Synthesis of Unlabeled and Radiolabeled Hydroxycinnamate-CoA

Hydroxycinnamate coenzyme A (CoA) thioesters are substrates for biosynthesis of lignin and hydroxycinna- mate esters of polysaccharides and other polymers. Hence, a supply of these substrates is essential for investigation of cell wall biosynthesis. In this study, three recombinant enzymes, caffeic acid 3-O-methyltransferase, 4-coumarate- CoA ligase 1, and 4-coumarate-CoA ligase 5, were cloned from wheat, tobacco, and Arabidopsis, respectively, and were used to synthesize 14 C-feruloyl-CoA, caffeoyl-CoA, p-coumaroyl-CoA, feruloyl-CoA, and sinapoyl-CoA. The corresponding hydroxycinnamoyl-CoA thioesters were high-performance liquid chromatography purified, the only extraction/purification step necessary, with total yields between 88-95%. Radiolabeled 14 C-feruloyl-CoA was gener- ated from caffeic acid and S-adenosyl- 14 C-methionine under the combined action of caffeic acid 3-O-methyltransferase and 4-coumarate-CoA ligase 1. About 70% of 14 C-methyl groups from S-adenosyl methionine were incorporated into the final product. The methods presented are simple, fast, and efficient for the preparation of the hydroxycinnamate thioesters.

A Synthetic 18-Norsteroid Distinguishes between Two Neuroactive Steroid Binding Sites on GABAA Receptors

In the absence of GABA, neuroactive steroids that enhance GABA-mediated currents modulate binding of [35S]t-butylbicyclophosphorothionate in a biphasic manner, with enhancement of binding at low concentrations (site NS1) and inhibition at higher concentrations (site NS2). In the current study, compound (3alpha,5beta,17beta)-3-hydroxy-18-norandrostane-17-carbonitrile (3alpha5beta-18-norACN), an 18-norsteroid, is shown to be a full agonist at site NS1 and a weak partial agonist at site NS2 in both rat brain membranes and heterologously expressed GABAA receptors. 3alpha5beta-18-norACN also inhibits the action of a full neurosteroid agonist, (3alpha,5alpha,17beta)-3-hydroxy-17-carbonitrile (3alpha5alphaACN), at site NS2. Structure-activity studies demonstrate that absence of the C18 methyl group and the 5beta-reduced configuration both contribute to the weak agonist effect at the NS2 site. Electrophysiological studies using heterologously expressed GABAA receptors show that 3alpha5beta-18-norACN potently and efficaciously potentiates the GABA currents elicited by low concentrations of GABA but that it has low efficacy as a direct activator of GABAA receptors. 3alpha5beta-18-norACN also inhibits direct activation of GABAA receptors by 3alpha5alphaACN. 3alpha5beta-18-norACN also produces loss of righting reflex in tadpoles and mice, indicating that action at NS1 is sufficient to mediate the sedative effects of neurosteroids. These data provide insight into the pharmacophore required for neurosteroid efficacy at the NS2 site and may prove useful in the development of selective agonists and antagonists for neurosteroid sites on the GABAA receptor.

Dynamics of Retinal Studied by 2H NMR Relaxation Underlie Multiscale Conformational Changes in Rhodopsin Activation

The activation mechanism of the G protein-coupled receptor rhodopsin has been investigated through solid-state 2H NMR relaxation studies. Rhodopsin was regenerated with retinal 2H-labeled at the C5-, C9-, and C13-methyl groups and was recombined into POPC (dark state and Meta I) or POPC:DOPE (Meta II) [1-4]. The 2H NMR relaxation rates of Zeeman order (R1Z) or quadrupolar order (R1Q) were measured from −30 to −150°C in the dark, Meta I, and Meta II states. Relaxation data were interpreted in terms of axial rotation and off-axial motion of the methyl groups and revealed interactions between the retinal cofactor and the rhodopsin binding pocket. Relaxation rates show considerable mobility of the retinal ligand in the dark state despite the complete absence of basal activity. 2H NMR data indicate that the retinal C9-methyl group does not change its orientation upon photoisomerization. Rather it acts as a hinge for light-induced rotation of the C13-methyl toward the second extracellular loop E2. The C13-methyl rotation leads to a displacement of the E2 loop that is observed already in the Meta I state. Short T1Z relaxation times for the C5-methyl indicate that the β-ionone ring adopts a twisted 6-s-cis conformation, and remains in its hydrophobic binding pocket up to the Meta II state. An activation mechanism is proposed whereby the photonic energy is channeled by 11-cis to trans isomerization of retinal against the E2 loop by the C9- and C13-methyl groups, and toward helices H5 and H3 by the β-ionone ring and C5-methyl group.[1] G.F.J. Salgado et al. (2004) Biochemistry43, 12819. [2] G.F.J. Salgado et al. (2006) JACS128, 11067. [3] A.V. Struts et al. (2007) JMB372, 50. [4] M.F. Brown et al. (2009) Photochem. Photobiol.85, 442.

Total synthesis of (±)-aspercyclide A and its C19 methyl ether

The total syntheses of (+/-)-aspercyclide A (1) and its C19 methyl ether (15a) featuring Heck-Mizoroki macrocyclisation to form the 11-membered (E)-styrenyl biaryl ether lactone core are described.

Candida albicans sphingolipid C9-methyltransferase is involved in hyphal elongation

C9-methylated glucosylceramide is a fungus-specific sphingolipid. This lipid is a major membrane component in the cell and is thought to play important roles in the growth and virulence of several fungal species. To investigate the importance of the methyl branch of the long-chain base in glucosylceramides in pathogenic fungi, we identified and characterized a sphingolipid C9-methyltransferase gene (MTS1, C9-MethylTransferase for Sphingolipid 1) in the pathogenic yeast Candida albicans. The mts1 disruptant lacked (E,E)-9-methylsphinga-4,8-dienine in its glucosylceramides and contained (E)-sphing-4-enine and (E,E)-sphinga-4,8-dienine. Reintroducing the MTS1 gene into the mts1 disruptant restored the synthesis of (E,E)-9-methylsphinga-4,8-dienine in the glucosylceramides. We also created a disruptant of the HSX11 gene, encoding glucosylceramide synthase, which catalyses the final step of glucosylceramide synthesis, in C. albicans and compared this mutant with the mts1 disruptant. The C. albicans mts1 and hsx11 disruptants both had a decreased hyphal growth rate compared to the wild-type strain. The hsx11 disruptant showed increased susceptibility to SDS and fluconazole, similar to a previously reported sld1 disruptant that contained only (E)-sphing-4-enine in its glucosylceramides, suggesting that these strains have defects in their cell membrane structures. In contrast, the mts1 disruptant grew similarly to wild-type in medium containing SDS or fluconazole. These results suggest that the C9-methyl group of a long-chain base in glucosylceramides plays an important role in the hyphal elongation of C. albicans independent of lipid membrane disruption.

Hydrogen bonding between the 17β‐substituent of a neurosteroid and the GABAA receptor is not obligatory for channel potentiation

Potentiating neurosteroids are some of the most efficacious modulators of the mammalian GABA(A) receptor. One of the crucial interactions may be between the C20 ketone group (D-ring substituent at C17) of the neurosteroid, and the N407 and Y410 residues in the M4 domain of the receptor. In this study, we examined the contribution of hydrogen bonding between 17beta-substituents on the steroid D-ring and the GABA(A) receptor to potentiation by neurosteroids. Whole-cell and single-channel recordings were made from HEK 293 cells transiently expressing wild-type and mutant alpha1beta2gamma2L GABA(A) receptors. A steroid with a 17beta-carbonitrile group (3alpha5alpha18nor17betaCN) was a potent and efficacious potentiator of the GABA(A) receptor. Potentiation was also shown by a cyclosteroid in which C21 and the C18 methyl group of (3alpha,5alpha)-3-hydroxypregnan-20-one are connected within a six-membered ring containing a double bond as a hydrogen bond acceptor (3alpha5alphaCDNC12), a steroid containing a 17beta-ethyl group on the D-ring (3alpha5alpha17betaEt) and a steroid lacking a 17beta-substituent on the D-ring (3alpha5alpha17H). Single-channel kinetic analysis indicates that the kinetic mechanism of action is the same for the neurosteroid 3alpha5alphaP, 3alpha5alpha18nor17betaCN, 3alpha5alphaCDNC12, 3alpha5alpha17betaEt and 3alpha5alpha17H. Interestingly, 3alpha5alpha17betaEt, at up to 3 microM, was incapable of potentiating the alpha1N407A/Y410F double mutant receptor. Hydrogen bonding between the steroid 17beta-substituent and the GABA(A) receptor is not a critical requirement for channel potentiation. The alpha1N407/Y410 residues are important for neurosteroid potentiation for reasons other than hydrogen bonding between steroid and receptor.

6-s-cis Conformation and Polar Binding Pocket of the Retinal Chromophore in the Photoactivated State of Rhodopsin

The visual pigment rhodopsin is unique among the G protein-coupled receptors in having an 11-cis retinal chromophore covalently bound to the protein through a protonated Schiff base linkage. The chromophore locks the visual receptor in an inactive conformation through specific steric and electrostatic interactions. This efficient inverse agonist is rapidly converted to an agonist, the unprotonated Schiff base of all-trans retinal, upon light activation. Here, we use magic angle spinning NMR spectroscopy to obtain the (13)C chemical shifts (C5-C20) of the all-trans retinylidene chromophore and the (15)N chemical shift of the Schiff base nitrogen in the active metarhodopsin II intermediate. The retinal chemical shifts are sensitive to the conformation of the chromophore and its molecular interactions within the protein-binding site. Comparison of the retinal chemical shifts in metarhodopsin II with those of retinal model compounds reveals that the Schiff base environment is polar. In particular, the (13)C15 and (15)Nepsilon chemical shifts indicate that the C horizontal lineN bond is highly polarized in a manner that would facilitate Schiff base hydrolysis. We show that a strong perturbation of the retinal (13)C12 chemical shift observed in rhodopsin is reduced in wild-type metarhodopsin II and in the E181Q mutant of rhodopsin. On the basis of the T(1) relaxation time of the retinal (13)C18 methyl group and the conjugated retinal (13)C5 and (13)C8 chemical shifts, we have determined that the conformation of the retinal C6-C7 single bond connecting the beta-ionone ring and the retinylidene chain is 6-s-cis in both the inactive and the active states of rhodopsin. These results are discussed within the general framework of ligand-activated G protein-coupled receptors.