Stereoselective synthesis of P-homochiral oligo(thymidine methanephosphonates).

An approach to the stereoselective synthesis of P-homochiral oligo(thymidine methanephosphonates) is described. Fully protected (Rp)- and (Sp)-diastereomers of MMTrTPMeTAC (3) were prepared in the stereospecific reaction of P-chiral nucleotide component 5'-O-monomethoxytritylthymidine 3'-O-[O-(4-nitrophenyl)methanephosphonate] (1) and 3'-O-acetylthmydine (2) bearing activated 5'-hydroxyl function. Deprotection of the 5'-OH group in 3 and subsequent stepwise reactions of activated 5'-OH oligonucleotide components with (Rp)- or (Sp)- isomers of 1 gave the trinucleotide MMTrTPMeTPMeTAC (4) and, subsequently, the tetranucleotide MMTrTPMeTPMeTPMeTAC (5) possessing all (Rp)- or all (Sp)- configurations at their internucleotide methanephosphonate P-atoms.     

Quadruplex and pentaplex self-assemblies of oligonucleotides containing short runs of 8-aza-7-deaza-2'-deoxyisoguanosine or 2'-deoxyisoguanosine.

Oligonucleotides containing 8-aza-7-deaza-2'-deoxyisoguanosine (4) were investigated regarding their self-assembly in aqueous solution. The aggregation of 4 was compared with that of oligonucleotides containing 2'-deoxyisoguanosine (2b) and 2'-deoxyguanosine (1b). For this purpose the phosphoramidite of 4 was synthesized which was protected by a dibutylaminomethylidene residue at the amino group and a diphenylcarbamoyl residue at the 2-oxo function. Solid-phase synthesis furnished oligonucleotide containing short runs of the nucleoside 4. The self-assembly of the oligonucleotide 5'-d(T(4)4(4)T2) was studied by ion-exchange chromatography. The formation of a pentaplex was observed in the presence of Cs+, while a tetraplex is formed when the counter ion is Na+ or Rb+. The cation selectivity of the oligonucleotide 5'-d(T(4)4(4)T2) was found to be different from the parent 5'-d(T(4)isoG(4)T2) which was forming the tetraplex as well as a pentaplex in aq RbCl solution.     

Enzymes of vitamin B6 degradation. Purification and properties of 5-pyridoxic-acid oxygenase from Arthrobacter sp

5-Pyridoxic-acid oxygenase, a cytoplasmic enzyme formed when Arthrobacter Cr-7 is grown with pyridoxine as a sole source of carbon and nitrogen, was purified about 190-fold to homogeneity from fully induced cells. The enzyme catalyzes Reaction a, (Formula: see text) the essential ring-opening step in the degradation of pyridoxine, and provides a second example of an FAD-dependent oxygenase that adds both two hydrogen and two oxygen atoms to its substrate. 5-Pyridoxic-acid oxygenase has an isoelectric point of 4.6, functions optimally between pH 7 and 8, appears to contain a single subunit of Mr = 51,000 and one FAD (but no iron) per subunit, and is readily resolved by precipitation with ammonium sulfate at pH 3.0. FMN and riboflavin do not replace FAD as coenzyme, but their presence enhances a normally minor side reaction (Reaction b) NAD(P)H + H+ + O2----NAD(P)+ + H2O2 (b) catalyzed by the holoenzyme. Reaction b also is enhanced when the poorly utilized analogues, 3-hydroxy-2-methylpyridine-5-carboxylic acid or NADH, replace 5-pyridoxic acid or NADPH, respectively, as substrates in Reaction a. Each of the enzymes required in two different pathways for degradation of pyridoxine to anabolic intermediates has now been studied. A comparison of these two pathways and their enzymes is provided.     

Characterization of an O-methyltransferase from Streptomyces avermitilis MA-4680

A search of the Streptomyces avermitilis genome reveals that its closest homologs are several O-methyltransferases. Among them, one gene (viz., saomt5) was cloned into the pET-15b expression vector by polymerase chain reaction using sequence-specific oligonucleotide primers. Biochemical characterization with the recombinant protein showed that SaOMT5 was S-adenosyl-L-methionine-dependent Omethyltransferase. Several compounds were tested as substrates of SaOMT5. As a result, SaOMT5 catalyzed Omethylation of flavonoids such as 6,7-dihydroxyflavone, 2',3'-dihydroxyflavone, 3',4'-dihydroxyflavone, quercetin, and 7,8-dihydroxyflavone, and phenolic compounds such as caffeic acid and caffeoyl Co-A. These reaction products were analyzed by TLC, HPLC, LC/MS, and NMR spectroscopy. In addition, SaOMT5 could convert phenolic compounds containing ortho-dihydroxy groups into Omethylated compounds, and 6,7-dihydroxyflavone was known to be the best substrate. SaOMT5 converted 6,7- dihydroxyflavone into 6-hydroxy-7-methoxyflavone and 7-hydroxy-6-methoxyflavone, and caffeic acid into ferulic acid and isoferulic acid, respectively. Moreover, SaOMT5 turned out to be a Mg2+-dependent OMT, and the effect of Mg2+ ion on its activity was five times greater than those of Ca2+, Fe2+, and Cu2+ ions, EDTA, and metal-free medium.     

Attempted stereoselective synthesis of P-chiral analogues of oligodeoxyribonucleotides

Reaction of diastereomeric 5'MMT-nucleoside 3'-O-(4-nitrophenylmethanephosphate) or 3'-O-[O-(4-nitrophenyl)-S-(2-nitrobenzyl)phosphorothioate] with tBuMgCl-activated, 3'-protected nucleoside is stereospecific and leads, after appropriate work-up, to dinucleoside-3',5'-methanephosphonate or dinucleoside-3',5'-phosphorothioate, respectively. This procedure extended to 5'-activated nucleotides, allows to prepare short, stereoregular oligonucleotide analogues bearing at internucleotide positions methanephosphonate or phosphorothioate function of predetermined sense of chirality at P-centers.     

Chemical reactions in double helical nucleic acids. XIII. Directed introduction of acylphosphate internucleotide bonds into the DNA-duplex structure]

DNA duplex, containing an acylphosphate internucleotide bond in a predetermined position of the sugar-phosphate backbone, was synthesized. The synthesis was carried out by condensing on the complementary matrix two heptanucleotides, one of which possessed at the 3'-end a glycine residue, connected with the oligonucleotide by the phosphoramide bond, whereas the 5'-end phosphate group of the other was activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC). The yield of the oligonucleotide with an acylphosphate bond was 24%. The stability and chemical properties of the synthesized compound were studied in comparison with analogous oligonucleotide containing a substituted pyrophosphate internucleotide bond. The former was shown to be an effective acylating agent in the aqueous medium in contrast to the latter which is a phosphorylating agent.     

Wavelength dependence for the photoreactions of DNA-psoralen monoadducts. 1. Photoreversal of monoadducts

We have studied the wavelength dependence for the photoreversal of a monoadducted psoralen derivative, HMT [4'-(hydroxymethyl)-4,5',8-trimethylpsoralen], in a single-stranded deoxyoligonucleotide (5'-GAAGCTACGAGC-3'). The psoralen was covalently attached to the thymidine residue in the oligonucleotide as either a furan-side monoadduct, which is formed through the cycloaddition between the 4',5' double bond of the psoralen and the 5,6 double bond of the thymidine, or a pyrone-side monoadduct, which is formed through the cycloaddition between the 3,4 double bond of the psoralen and the 5,6 double bond of the thymidine. As a comparison, we have also investigated the wavelength-dependent photoreversal of the isolated thymidine-HMT monoadducts. All photoreversal action spectra correlate with the extinction spectra of the isolated monoadducts. In the case of the pyrone-side monoadduct, two absorption bands contribute to the photoreversal with a quantum yield of 2 X 10(-2) at wavelengths below 250 nm and 7 X 10(-3) at wavelengths from 287 to 314 nm. The incorporation of the monoadduct into the DNA oligomer had little effect upon the photoreversal rate. For the furan-side monoadduct at least three absorption bands contribute to the photoreversal. The quantum yield varied from 5 X 10(-2) at wavelengths below 250 nm to 7 X 10(-4) at wavelengths between 295 and 365 nm. In contrast to the case of the pyrone-side monoadduct, the incorporation of the furan-side monoadduct into the DNA oligomer reduced the photoreversal rate constant at wavelengths above 285 nm.     

Oligonucleotides incorporating 7-(aminoalkynyl)-7-deaza-2'-deoxyguanosines: duplex stability and phosphodiester hydrolysis by exonucleases

Oligonucleotides containing 7-(omega-aminoalkyn-1-yl)-7-deaza-2'-deoxyguanosines (1a-c) were investigated regarding their thermal stability (T(m) values) as well as their phosphodiester hydrolysis catalyzed by exonucleases. Those derivatives are suitable for the labeling of nucleic acid constituents as well as for the postlabeling of DNA. For this, the phosphoramidites 7a,c (obtained from the nucleoside 1a,b), protected by an isobutyryl group at the 2-amino group and a phthaloyl residue at the side-chain amino function, were synthesized. Using compounds 7a,c together with the phosphoramidite of 1c in solid-phase synthesis, a series of self-complementary and non-self-complementary oligonucleotides were prepared and characterized by MALDI-TOF mass spectrometry. A comparison of the T(m) values of the modified oligomers shows that the thermal stability of the duplexes decreases with the length of the nucleobase 7-(omega-aminoalkyn-1-yl) side chain. Exonucleolytic cleavage of oligonucleotide single strands incorporating either the 7-(3-aminopropyn-1-yl)- or the 7-(4-aminobutyn-1-yl)-substituted nucleosides 1a or 1b, respectively, reveals that 3' --> 5' specific snake venom phosphodiesterase liberates 1a 5'-monophosphate but not the methylene-extended 1b 5'-monophosphate. On the contrary, the 5' --> 3' specific bovine spleen exonuclease is able to cleave off single 1a and 1b 3'-monophosphate residues; its action is, however, terminated in the case of oligonucleotides containing two consecutive 1a or 1b nucleotide units.     

Synthesis and properties of oligonucleotides bearing a pendant pyrene group

Two hexathymidine oligonucleotide derivatives bearing a pyrenylbutyl substituent at a designated internucleotide phosphorus were prepared by solid phase syntheses using 5'-O-(di-p-methoxytrityl)thymidyl-3'-4-(1-pyrenyl)butyl phosphorochloridite and 5'-O-(di-p-methoxytrityl)thymidyl-3'2,2,2-trichloro-1,1-dimethylet hyl phosphorochloridite as phosphitylating agents. Spectrophotometric studies showed that these oligomers bind to Poly A, but not to Poly C, Poly G, or Poly U, and that the complexes with Poly A are somewhat more stable than the duplex formed between hexathymidine pentaphosphate and Poly A.     

Thermostability of double-stranded deoxyribonucleic acids: effects of covalent additions of a psoralen

We have carried out a thermodynamic study on the effects of covalent additions of the psoralen derivative HMT, 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen, on the stability of double-stranded deoxyoligonucleotides. This was done with two systems. The first was a double-stranded DNA formed by two non-self-complementary oligonucleotides, 5'-GAAGCTACGAGC-3' and 5'-GCTCGTAGCTTC-3', where we site specifically placed an HMT molecule on the thymidine residue in oligonucleotide 5'-GAAGCTACGAGC-3' as either a furan-side monoadduct or a pyrone-side monoadduct. The second was a double-stranded DNA formed by a self-complementary oligonucleotide, 5'-GGGTACCC-3', where we placed an HMT molecule on the thymidine residue of each strand as a furan-side monoadduct or cross-linked the two strands with an HMT molecule linked to the two thymidines. We found that HMT cross-linking of the two strands stabilizes the double helix formed by 5'-GGGTACCC-3', as one might expect. Less predictable results were that the monoaddition of a psoralen stabilizes the double helix formed by the two non-self-complementary oligonucleotides by as much as 1.3 kcal/mol as a furan-side monoadduct and 0.7 kcal/mol as a pyrone-side monoadduct at 25 degrees C in 50 mM NaCl. In contrast, the monoaddition of a psoralen on each of the two thymidines in the double helix formed by 5'-GGGTACCC-3' destabilizes the helix by 1.8 kcal/mol at 25 degrees C in 1 M NaCl. This destabilization arises from an unfavorable enthalpy change (8.6 kcal/mol) and a favorable entropy change (23 cal/K X mol) due to the two HMT molecules at the centers of each strand.(ABSTRACT TRUNCATED AT 250 WORDS)     

Trichloroacetaldehyde modified oligonucleotides

Some commercial batches of dichloroacetic acid (DCA) contain traces of chloral (trichloroacetaldehyde). Using such DCA to effect detritylation during solid-phase oligonucleotide synthesis results in the formation of a family of process impurities in which the atoms of chloral (Cl3CCHO) are incorporated between the 5'-oxygen and phosphorus atoms of an internucleotide linkage. The structure was elucidated by HPLC with UV and MS detection, digestion of the oligonucleotide, synthesis of model compounds, and 1H and 31P NMR spectroscopy. By understanding the chemistry behind its formation, we are now able to limit levels of this impurity in synthetic oligonucleotides by limiting chloral in DCA.     

Oligonucleotide lipoplexes: the influence of oligonucleotide composition on complexation

Despite extensive investigations into oligonucleotide lipoplexes, virtually no work has addressed whether the physicochemical properties of these assemblies vary as a function of the constituent oligonucleotide (ODN) sequence and/or composition. The present study was aimed at answering this question. To this end, we complexed N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP) liposomes, in dispersion, with either 18-mer phosphorothiote homo-oligonucleotides composed of either adenine, thymidine or cytosine; or one of three structurally related 18-mer phosphorothioate oligonucleotides (S-ODNs) (G3139, its reverse sequence and its two-base mismatch). After ODN addition to vesicles at different mole ratios, changes in pH and electrical surface potential at the lipid–water interface were analyzed by using the fluorophore heptadecyl-7-hydroxycoumarin while particle size distributions were analyzed by static-light scattering. The results indicate that each homo-oligonucleotide does indeed exhibit different complexation behavior. In particular, the maximal level of DOTAP neutralization by the polyadenine S-ODN is much lower than that for the two other homo-oligonucleotides and hence its lipoplex is much more positively charged. Much smaller electrostatic differences are also apparent between lipoplexes formed from each of the G3139-related ODNs. This paper identifies nucleotide base selection and sequence as a variable that can affect the physicochemical properties of oligonucleotide lipoplexes and hence probably their transfection competency.     

Nucleoterpenes of Thymidine and 2′‐Deoxyinosine: Synthons for a Biomimetic Lipophilization of Oligonucleotides

2′‐Deoxyinosine ( 1 ) and thymidine ( 7 ) were N‐alkylated with geranyl and farnesyl moieties. These hydrophobic derivatives, 3a and 3b , and 9a and 9b , respectively, represent the first synthetic biomimetic nucleoterpenes and were subsequently 5′‐protected and converted into the corresponding 3′‐O‐phosphoramidites, 5a and 5b and 11a and 11b , respectively. The latter were used to prepare a series of lipophilized oligonucleotide dodecamers, a part of which were additionally labelled with indocarbocyanine fluorescent dyes (Cy3 or Cy5), 18 – 23 . The insertion of the lipooligonucleotides into, as well as duplex formation at artificial lipid bilayers was studied by single‐molecule fluorescence spectroscopy and fluorescence microscopy.     

Cytoplasmic pH in the action of epidermal growth factor (EGF) in cultured porcine thyroid cells

We demonstrate measurement of cytoplasmic pH (pHi), using 2',7'-bis(2-carboxyethyl)-5 (and 6-) carboxyfluorescein (BCECF), and internalized fluorescent pHi indicator, in thyroid cells. Using cultured porcine thyroid cells, we studied the effects of epidermal growth factor (EGF) on pHi and [3H] thymidine incorporation; 10 nM EGF alkalinizes thyroid cells and stimulates thymidine incorporation. The results indicate that Na+/H+ exchange or cell alkalinization may function as a transmembrane signal transducer in the action of EGF in the thyroid cells.     

Stabilization of triple-stranded oligonucleotide complexes: use of probes containing alternating phosphodiester and stereo-uniform cationic phosphoramidate linkages.

Pyrimidine oligonucleotides containing alternating anionic and stereo-uniform cationic N-(dimethylamino-propyl)phosphoramidate linkages [e.g. d(T+T-)7T, d(T+T-)2(T+C-)5T and (U'+U'-)7dT, where U' is 2'-O-methyluridine)] are shown to bind to complementary double-stranded DNA segments in 0.1 M NaCl at pH 7 to form triple-stranded complexes with the pyrimidine.purine.pyrimidine motif. For each of the sequences investigated, one stereoisomer bound with higher affinity, and the other stereoisomer with lower affinity, than the corresponding all-phosphodiester oligonucleotide. The stereoisomer of d(T+T-)7T that interacted weakly with a dT.dA target in 0.1 M NaCl formed a novel dA.dA.dT triple-stranded complex with poly(dA) or d(Al5C4A15) in 1 M NaCl; in contrast, the stereoisomer that bound strongly to the dT.dA target failed to form a dA.dA.dT triple-stranded complex.     

Specific modification of nucleic acids inside the cell with alkylating derivatives of oligonucleotides ethylated at internucleotide phosphates

The alkylating derivatives of (C6H5NH)2P[dTp(Et)]4U and [dTp(Et)]9 U with completely esterified internucleotide phosphates bearing reactive 2,3 -O-4(N-2-chloroethyl N-methylamino)-benazylidene moiety attached to 3 -end cis-diol group were prepared. These alkylating derivatives of non-ionisable oligonucleotide analogs were demonstrated to penetrate efficiently into Krebs ascites tumor cells and to alkylate nucleic acids inside the cells with a strong preference towards complementary poly(A)-fragments of mRNA.     

Synthesis and application of a new phosphorylating agent: S-(N-monomethoxytritylaminoethyl)-O-(o-chlorophenyl)phosphorothi oate

A new phosphorylating agent, S-(N-monomethoxytritylaminoethyl)-O-(O-chlorophenyl) phosphorothioate, was prepared and reacted with a 5'-hydroxyl group of an oligonucleotide using 1-mesitylene-sulfonyl-3-nitrotriazole (MSNT) as a condensing agent. After base labile protecting groups were removed, the partially deprotected oligonucleotide was separated on a reversed phase column and converted to the oligonucleotide with an aminoethyl or a phosphoryl group at the 5'-end by treatment with 80% acetic acid or iodine-water, respectively. The syntheses of ppT, pppT, A5'pp5'T and A5'ppp5'T were also performed by treatment of 5'-O-(N-monomethoxytritylaminoethylthiophosphoryl) thymidine with tri-n-octylammonium salt of phosphoric acid, pyrophosphoric acid, pA and ppA, respectively.