Straightforward Synthesis of 6-Thiodeoxyguanosine and Its Incorporation into Oligodeoxynucleotides

Abstract

A convenient synthesis of 6-thiodeoxyguanosine from deoxyguanosine is described. Simple methods for incorporating 6-thiodeoxyguanosine into oligodeoxynucleotides are also given.     

Straightforward Synthesis of 6-Thiodeoxyguanosine and Its Incorporation into Oligodeoxynucleotides

Abstract

A convenient synthesis of 6-thiodeoxyguanosine from deoxyguanosine is described. Simple methods for incorporating 6-thiodeoxyguanosine into oligodeoxynucleotides are also given.     

Synthesis of Oligodeoxynucleotides Containing 5-Bromouracil and N6-Methyladenine

Abstract

Oligodeoxynucleotides containing uracil, 5-bromouracil and N6-methyladenine were prepared by phosphotriester or amidite approaches and by a hybrid phosphotriester-phosphoramidite method.     

Preparation of Oligodeoxynucleotides Containing 6-Methylthiopurine Residues by Chemical Synthesis or Specific Methylation

Abstract

Two methods (chemical synthesis and specific methylation) are described for the preparation of oligodeoxynucleotides containing 6-methylthiopurine residues. 6-Methylthiopurine phosphoramidite (6) is prepared and incorporated into oligomers. Methylation with methyl iodide of 6-thiopurine (or 6-thioguanine) in oligonucleotides also leads to exclusive production of 6-methylthiopurine (or 6-methylthioguanine) oligomers.     

Extensive Natural Epigenetic Variation at a De Novo Originated Gene

Epigenetic variation, such as heritable changes of DNA methylation, can affect gene expression and thus phenotypes, but examples of natural epimutations are few and little is known about their stability and frequency in nature. Here, we report that the gene Qua-Quine Starch (QQS) of Arabidopsis thaliana, which is involved in starch metabolism and that originated de novo recently, is subject to frequent epigenetic variation in nature. Specifically, we show that expression of this gene varies considerably among natural accessions as well as within populations directly sampled from the wild, and we demonstrate that this variation correlates negatively with the DNA methylation level of repeated sequences located within the 5′end of the gene. Furthermore, we provide extensive evidence that DNA methylation and expression variants can be inherited for several generations and are not linked to DNA sequence changes. Taken together, these observations provide a first indication that de novo originated genes might be particularly prone to epigenetic variation in their initial stages of formation.     

Oligodeoxynucleotides, containing 6-O-alkylated guanine segments recognized by HaeIII restriction endonuclease]

A series of self-complementary decadeoxynucleotides containing a native or modified HaeIII site GGCC (with one or both guanine residues 6-O-cetylated) have been synthesized by the phosphotriester approach. The nonmodified decanucleotide is normally digested with snake venom phosphodiesterase as well as with HaeIII and BspI restriction endonucleases, whereas the bulky 6-O-alkyl substituent strongly inhibits the VPDE hydrolysis and completely prevents digestion with the endonucleases.     

反义脱氧寡核苷酸的抗SRS病毒活性

本文研究了合成的反义脱氧寡核苷酸片段,包括修饰的和非修饰的,同聚的和异聚的脱氧寡核苷酸对SRS病毒感染细胞的增殖,细胞群落形成,xc融合细胞产生和逆转录酶活性的抑制作用。结果表明上述寡聚物对病毒都有抑制活性。小鼠急性毒性试验在高剂量下未见毒性。各种寡聚物的作用机制不同。     

O6-Protection and Other Transformations at Guanosine and Inosine Lactam Sites with Application of Related Pyridinium Salts

Abstract

Nucleophilic displacement reactions of guanosine- and inosine-derived pyridinium salts will be discussed in a view of their preparative applications in nucleoside and oligonucleotide chemistry.     

Melting of Oligodeoxynucleotides with Various Structures

Abstract

Effects of DNA fragments end structures on their melting profiles were studied experimentally and theoretically. We examined melting of hairpins and dumbbells obtained from 62- bp-long linear DNA duplex which is a perfect palindromic sequence. To fit theoretical melting profile to experimental ones additional theoretical parameters were incorporated into the standard statistical mechanical helix-coil transition theory. From comparison theoretical and experimental melting profiles theoretical parameters connected with end- structure effects were evaluated. Analysis revealed the stabilization effect of the hairpin loops and helix ends with respect to DNA duplex melting. Both type of ends make melting these oligodeoxynucleotides more cooperative than predicted by the standard helix-coil transition theory. At low ionic strength ([Na⁺] < 0.04 M) this effect becomes so pronounced that melting of the DNA duplexes 30–40 bp-long conforms to the two state model.

From the analysis experimental data obtained for dumbbell structures loop-weighting factor for single-stranded loop consisting of 132 nucleotides was determined. This parameter decreases 10 times with the ionic strength decreasing by an order of magnitude from 0.2 to 0.02 M Na⁺.     

Microbiological Transformations of Δ6a,10a-Tetrahydrocannabinol

A screening program was conducted to find microorganisms that catalyze transformation reactions with cannabinoids. Three hundred fifty-eight cultures, consisting of 97 bacteria, 175 actinomycetes, and 86 molds, were incubated in media containing 0.5 mg of Δ^6a,10a-tetrahydrocannabinol (Δ^6a,10a-THC) per ml. After 120 h of cultivation, ethyl acetate extracts of the cultures were examined by thin-layer chromatography (TLC) for transformation products. About 18% of the cultures modified Δ^6a,10a-THC. The ability to modify the substrate did not predominate among any particular group of microorganisms. After purification, the products from three cultures were analyzed by high-resolution mass spectrometry, 100-mHz proton magnetic resonance spectrometry, ultraviolet spectrometry, and infrared spectrometry. These spectral data indicated that a Mycobacterium sp. oxidized Δ^6a,10a-THC to cannabinol and a diastereomeric pair of 6a-hydroxy-Δ^10,10a-THC isomers; a Streptomyces sp. and a Bacillus sp. oxidized Δ^6a,10a-THC to 7-keto-Δ^6a,10a-THC and 4′-hydroxy-Δ^6a,10a-THC, respectively. The occurrence of these products and the presence of others that have not yet been isolated or identified indicate that microbial transformation may be a useful tool for the preparation of new cannabinoids that have desirable pharmacological properties.     

Postsynthetic Conjugation of RNA to Carboxylate and Dicarboxylate Molecules

Carboxylates and dicarboxylates are important phosphate mimics. Herein, we present a simple synthetic route for the preparation of RNA carboxylate/dicarboxylate conjugates, starting from suitably protected NH₂- and COOH-containing molecules that are coupled to the RNA on the solid support. The key point in our method was the use of trimethylsilylethanol (TMSE-OH) protecting group, which is removed simultaneously with the silyl protecting group on the 2′-OH of the RNA ribose (e.g. t-Butyldimethylsilyl) during the final RNA cleavage/deprotection steps. The usefulness of this method was demonstrated by preparing different RNA-phosphate mimics oligos.     

Synthesis and Chemical Transformations of N-Hydroxy- and N-Hydroxyalkylcycloimides of Chlorin p6

Two series of chlorin p₆ 13,15-cycloimides that differ in their substituents at the nitrogen atom of the additional six-membered ring were synthesized. The compounds of the first series have a hydroxy, alkoxy, or acyloxy group at the 13,15-cycloimide nitrogen and those of the second series, residues of aliphatic alcohols. The cycloimides synthesized are satisfactorily stable and display an intensive light absorption maximum at 710–718 nm. Treatment of the cycloimides with sodium periodate in the presence of osmium tetroxide and with the Vilsmeier reagent resulted in the formation of 3-formyl- and 3-(2-formylvinyl)derivatives, respectively. The conversion of vinyl into formyl group or 2-formylvinyl group leads to an additional bathochromic shift of the long-wave maximum by 30 nm on average. An extra hydroxy group was introduced at position 18 of the macrocycle to increase the cycloimide hydrophilicity.     

N-Methyl-D-Aspartate Recognition Site Ligands Modulate Activity at the Coupled Glycine Recognition Site

In synaptic plasma membranes from rat forebrain, the potencies of glycine recognition site agonists and antagonists for modulating [3H]1-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) binding and for displacing strychnine-insensitive [3H]glycine binding are altered in the presence of N-methyl-D-aspartate (NMDA) recognition site ligands. The NMDA competitive antagonist, cis-4-phosphonomethyl-2-piperidine carboxylate (CGS 19755), reduces [3H]glycine binding, and the reduction can be fully reversed by the NMDA recognition site agonist, L-glutamate. Scatchard analysis of [3H]glycine binding shows that in the presence of CGS 19755 there is no change in Bmax (8.81 vs. 8.79 pmol/mg of protein), but rather a decrease in the affinity of glycine (KD of 0.202 microM vs. 0.129 microM). Similar decreases in affinity are observed for the glycine site agonists, D-serine and 1-aminocyclopropane-1-carboxylate, in the presence of CGS 19755. In contrast, the affinity of glycine antagonists, 1-hydroxy-3-amino-2-pyrrolidone and 1-aminocyclobutane-1-carboxylate, at this [3H]glycine recognition site increases in the presence of CGS 19755. The functional consequence of this change in affinity was addressed using the modulation of [3H]TCP binding. In the presence of L-glutamate, the potency of glycine agonists for the stimulation of [3H]TCP binding increases, whereas the potency of glycine antagonists decreases. These data are consistent with NMDA recognition site ligands, through their interactions at the NMDA recognition site, modulating activity at the associated glycine recognition site.     

Structural Transformations of Ice at High Pressures Via Molecular Dynamics Simulations

Abstract

A simple classical model is used for the study of the structural transformations of ice under high pressures, such as ice VIII to VII and X, via classical molecular dynamics (MD) simulation. In the present MD simulation, pair potentials of a simple form between pair of atoms and a thee-body potential representing the H-O-H angle dependence, originally developed by Kawamura et al., were used. Starting with a stable ice VIII at low pressure and low temperature, we have carried out two different MD runs, one with increasing pressure keeping the temperature constant (simulation I) and the other with increasing temperature under constant pressure (simulation II). From these MD simulations we have obtained the structural transformations from ice VIII to VII for both simulations; the former was finally transformed into ice X for the simulation I. The present results are compatible with recent experiments on high pressure ices.     

After Authenticity at an American Heritage Site

This essay explores what happens to a heritage site "after authenticity." The site is Colonial Williamsburg, one of the most ambitious reconstruction projects ever undertaken and a place intended to be experienced as an objective correlate of an American national "identity." Because it makes such claims, throughout its history the site has been subject to critiques by those wishing to undermine its authority to speak as the voice of an all-encompassing America. Moreover, in the past 20 years, professional historians at Colonial Williamsburg have become articulate on-site critics of the epistemology of authenticity as they promulgate a historiography currently popular in history museums and in the academy.