Comparison of the redox chemistry of sulfur- and selenium-containing analogs of uracil.

Selenium is present in proteins in the form of selenocysteine, where this amino acid serves catalytic oxidoreductase functions. The use of selenocysteine in nature is strongly associated with redox catalysis. However, selenium is also found in a 2-selenouridine moiety at the wobble position of tRNAGlu, tRNAGln and tRNALys. It is thought that the modifications of the wobble position of the tRNA improves the selectivity of the codon-anticodon pair as a result of the physico-chemical changes that result from substitution of sulfur and selenium for oxygen. Both selenocysteine and 2-selenouridine have widespread analogs, cysteine and thiouridine, where sulfur is used instead. To examine the role of selenium in 2-selenouridine, we comparatively analyzed the oxidation reactions of sulfur-containing 2-thiouracil-5-carboxylic acid (s2c5Ura) and its selenium analog 2-selenouracil-5-carboxylic acid (se2c5Ura) using 1H-NMR spectroscopy, 77Se-NMR spectroscopy, and liquid chromatography-mass spectrometry. Treatment of s2c5Ura with hydrogen peroxide led to oxidized intermediates, followed by irreversible desulfurization to form uracil-5-carboxylic acid (c5Ura). In contrast, se2c5Ura oxidation resulted in a diselenide intermediate, followed by conversion to the seleninic acid, both of which could be readily reduced by ascorbate and glutathione. Glutathione and ascorbate only minimally prevented desulfurization of s2c5Ura, whereas very little deselenization of se2c5Ura occurred in the presence of the same antioxidants. In addition, se2c5Ura but not s2c5Ura showed glutathione peroxidase activity, further suggesting that oxidation of se2c5Ura is readily reversible, while oxidation of s2c5Ura is not. The results of the study of these model nucleobases suggest that the use of 2-selenouridine is related to resistance to oxidative inactivation that otherwise characterizes 2-thiouridine. As the use of selenocysteine in proteins also confers resistance to oxidation, our findings suggest a common mechanism for the use of selenium in biology.

Crystal structures of (N-methyl-N-phenyl-amino)(N-methyl-N-phenyl-carbamoyl)sulfide and the corresponding disulfane.

The title compounds, (N-methyl-N-phenyl-amino)(N-methyl-N-phenyl-car-bam-oyl)sulfide, C15H16N2OS, (I), and (N-methyl-N-phenyl-amino)-(N-methyl-N-phenyl-carbamo-yl)disulfane, C15H16N2OS2, (II), are stable derivatives of (chloro-carbon-yl)sulfenyl chloride and (chloro-carbon-yl)disulfanyl chloride, respectively. The torsion angle about the S-S bond in (II) is -92.62 (6)°, which is close to the theoretical value of 90°. In the crystal of (II), non-classical inter-molecular C-H⋯O hydrogen bonds form centrosymmetric cyclic dimers [graph set R 2 (2)(10)], while inter-dimer C-H⋯S inter-actions generate chains extending along the b axis.

Crystal structure of bis-(N-methyl-N-phenyl-amino)-tris-ulfane.

The title compound, C14H16N2S3, crystallized with two independent mol-ecules [(1 a ) and (1 b )] in the asymmetric unit. Both mol-ecules display a pseudo-trans conformation. The two consecutive S-S bond lengths of the tris-ulfane unit of mol-ecule (1 a ) are 2.06 (3) and 2.08 (3) Å, and 2.08 (3) and 2.07 (2) Šfor mol-ecule (1 b ). Torsion angles about each of the two S-S bonds are 86.6 (2) and 87.0 (2)° for (1 a ), and -84.6 (2) and -85.9 (2)° for (1 b ). The core atoms, viz. the N-S-S-S-N moiety, of the two mol-ecules superimpose well if one is inverted on the other, but the phenyl groups do not. Thus, the two units are essentially conformational enanti-omers. In mol-ecule (1 a ), the two phenyl rings are inclined to one another by 86.7 (3)°, and in mol-ecule (1 b ), by 81.1 (3)°. In the crystal, mol-ecules are linked via C-H⋯π inter-actions, forming sheets lying parallel to (010).

Bis(N-methyl-N-phenyl-carbamo-yl)disulfane.

The title compound, C(16)H(16)N(2)O(2)S(2), has been synthesized by several different high-yield routes, and has been encountered as a co-product in a number of reaction pathways, ever since it became of inter-est to our research program over 30 years ago. We now confirm the proposed mol-ecular structure in which the mol-ecule exhibits a twofold axis of symmetry through the mid-point of the S-S bond and the two planes defined by the (carbamo-yl)sulfenyl moieties are essentially perpendicular to each other [dihedral angle = 81.55 (14)°].

The use of 2,2'-dithiobis(5-nitropyridine) (DTNP) for deprotection and diselenide formation in protected selenocysteine-containing peptides.

In contrast to the large number of sidechain protecting groups available for cysteine derivatives in solid phase peptide synthesis, there is a striking paucity of analogous selenocysteine Se-protecting groups in the literature. However, the growing interest in selenocysteine-containing peptides and proteins requires a corresponding increase in availability of synthetic routes into these target molecules. It therefore becomes important to design new sidechain protection strategies for selenocysteine as well as multiple and novel deprotection chemistry for their removal. In this paper, we outline the synthesis of two new Fmoc selenocysteine derivatives [Fmoc-Sec(Meb) and Fmoc-Sec(Bzl)] to accompany the commercially available Fmoc-Sec(Mob) derivative and incorporate them into two model peptides. Sec-deprotection assays were carried out on these peptides using 2,2'-dithiobis(5-nitropyridine) (DTNP) conditions previously described by our group. The deprotective methodology was further evaluated as to its suitability towards mediating concurrent diselenide formation in oxytocin-templated target peptides. Sec(Mob) and Sec(Meb) were found to be extremely labile to the DTNP conditions whether in the presence or absence of thioanisole, whereas Sec(Bzl) was robust to DTNP in the absence of thioanisole but quite labile in its presence. In multiple Sec-containing model peptides, it was shown that bis-Sec(Mob)-containing systems spontaneously cyclize to the diselenide using 1 eq DTNP, whereas bis-Sec(Meb) and Sec(Bzl) models required additional manipulation to induce cyclization.

2,2'-Dithiobis(5-nitropyridine) (DTNP) as an effective and gentle deprotectant for common cysteine protecting groups.

Of all the commercially available amino acid derivatives for solid phase peptide synthesis, none has a greater abundance of side-chain protection diversity than cysteine. The high reactivity of the cysteine thiol necessitates its attenuation during peptide construction. Moreover, the propensity of cysteine residues within a peptide or protein sequence to form disulfide connectivity allows the opportunity for the peptide chemist to install these disulfides iteratively as a post-synthetic manipulation through the judicious placement of orthogonal pairs of cysteine S-protection within the peptide's architecture. It is important to continuously discover new vectors of deprotection for these different blocking protocols in order to achieve the highest degree of orthogonality between the removal of one species in the presence of another. We report here a complete investigation of the scope and limitations of the deprotective potential of 2,2'-dithiobis(5-nitropyridine) (DTNP) on a selection of commercially available Cys S-protecting groups. The gentle conditions of DTNP in a TFA solvent system show a remarkable ability to deprotect some cysteine blocking functionality traditionally removable only by more harsh or forcing conditions. Beyond illustrating the deprotective ability of this reagent cocktail within a cysteine-containing peptide sequence, the utility of this method was further demonstrated through iterative disulfide formation in oxytocin and apamin test peptides. It is shown that this methodology has high potential as a stand-alone cysteine deprotection technique or in further manipulation of disulfide architecture within a more complex cysteine-containing peptide template.

Synthetic routes to, transformations of, and rather surprising stabilities of (N-methyl-N-phenylcarbamoyl)sulfenyl chloride, ((N-methyl-N-phenylcarbamoyl)dithio)carbonyl chloride, and related compounds.

The title compound classes, (carbamoyl)sulfenyl chlorides and ((carbamoyl)dithio)carbonyl chlorides, have been implicated previously as unstable, albeit trappable, intermediates in organosulfur chemistry. The present work reports for each of these functional groups: (i) several routes to prepare it in the N-methylaniline family; (ii) its direct structural characterization by several spectroscopic techniques; (iii) its rather unexpected stability and its ultimate fate when it decomposes; (iv) a series of further chemical transformations that give highly stable derivatives, each in turn subject to thorough characterization. Relevant kinetic and mechanistic experiments were carried out, including some with p-methyl- and 2,6-dimethyl-substituted N-methylanilines. Given that the title compounds can be isolated and are relatively stable, they may find applications in the preparation of thiolyzable and/or photolabile protecting groups for the sulfhydryl function of cysteine and for the development of new protein synthesis and modification reagents.

Chemical footprinting of structural and functional elements of dhfr oribeta during the CHOC 400 cell cycle.

Oribeta, an origin of replication 3' to Chinese hamster dihydrofolate reductase (dhfr) gene, contains several sequence elements that function as components of a chromosomal replicator. Here we have examined sensitivity to KMnO(4) in vitro and in living cells of three regions within dhfr oribeta which contribute to replicator function: the origin of bidirectional DNA replication (OBR) that serves as an initiation site for DNA synthesis, a stably bent DNA region that binds activator protein one (AP-1) and RIP60 in vitro, and an AT-rich region that contains a dA/dT(23) dinucleotide repeat that has properties of a DNA unwinding element. The in vitro patterns of KMnO(4) modification in linear plasmid differed from that in supercoiled plasmid most prominently in the dA/dT(23) repeat, with evidence of palindrome extrusion in supercoiled plasmid. Although palindrome extrusion was not detected in genomic DNA during the cell cycle, the pattern of genomic DNA modification within the dA/dT(23) repeat differed substantially from that of either linear or plasmid DNA in vitro. An AT-rich region that borders the dA/dT repeat was also highly sensitive to modification by KMnO(4) in cells. Within the bent DNA region, the patterns of chemical modification of both the AP-1 and RIP60 sites differed between plasmid and genomic DNA, and minor differences in the in vitro and cellular modification patterns also were observed for the OBR. Nonetheless, there was little evidence of cell cycle-specific modifications in any sequence examined. These studies suggest that sequences within dhfr oribeta adopt specific conformations in cells, with the most prominent changes in the AT-rich region associated with the dA/dT(23) repeat and DNA unwinding.