Characterization of an artificial dimer of ribonuclease H using 1H NMR spectroscopy

Summary The protein fusion technique was applied in the synthesis of an artificial dimer of ribonuclease H (305 residues). ¹H NMR spectroscopy was used to analyze the structure of this dimer. Spectral profiles and pK_a values of the histidine residues obtained using ¹H NMR indicate that the dimer retains the secondary and tertiary structures of the intact monomer. Selective spin-lattice relaxation measurements suggest that the two monomeric units in the dimer are in tight contact. Furthermore, the 2D ¹H NMR and paramagnetic relaxation filter results show that the two monomers bind together through interactions between the N- and C-terminal sites of the linked regions.     

Phosphorothioate Analogs OF 2-5A: Activation / Inhibition of Rnase L and Inhibition of HIV-1 Reverse Transcriftase

Abstract

Chemically and enzymatically synthesizeddiastereomeric 2′,5′- phosphorothioate dimer, trimer and tetramer cores and their 5′ - mono- and triphosphates demonstrate marked differences in their ability to bind to and activate RNase L from L929 cell extracts in radiobinding, core-cellulose and rRNA cleavage assays¹,² (Fig. 1). These are the first 2-5A cores that are able to bind to and activate Mase L. The enzymatically synthesized 2′,5′- phosphorothioate dimer and trimer 5′-triphosphates can also bind to and activate mase L¹.     

Oligonucleotides Containing Acyclic Nucleoside Analogues with Carbamate Internucleoside Linkages

Abstract

Synthesis of 2′-deoxy-2′,3′-secothymidine t and its dimer t?t, where the two 2′-deoxy-2′,3′-secothymidine t units are connected via a carbamate, ?=3′-NH-CO-O-5′, internucleoside linkage has been achieved. These building blocks were protected in the 5′-position, converted into their phosphoramidites, or attached onto CPG, and then used for “chimeric oligonucleotide” synthesis.     

Synthesis of Artificially Bent Oligonucleotides by Incorporation of Conformationally Rigid 5′-Cyclouridylic Acid Derivatives

Abstract

This paper describes the design and synthesis of a conformationally rigid dimer building block Umpc3Um having a propylene bridge linked between the uracil 5-position and 5′-phosphate group of pUm. Oligonucleotides incorporating the dimer unit with either the Sp or Rp configuration were synthesized by use of the phosphoramidite approach. The conformational properties of the dimer units and these oligonucleotides were studied in detail.     

Synthesis of a Carboxamide Linked T∗T Dimer with an Acyclic Nucleoside Unit and Its Incorporation in Oligodeoxynucleotides

Abstract

A T?T dimer with ? representing a 2′-OCH₂CH₂NHC(O)-4′ linkage connecting two nucleoside units was prepared by condensation of (S)-1-[2-(2-aminoethoxy)-3-(4,4′-dimethoxytrityloxy)propyl]thymine with 1,2-dideoxy-1-thyminyl-β-D-erythro-pento-furanuronic acid. The T?T dimer was incorporated in oligodeoxynucleotides and investigated for hybridization to DNA.     

PNA-DNA Chimeras Containing 5-Alkynyl-pyrimidine PNA Units. Synthesis,Binding Properties,and Enzymatic Stability

Abstract

Three chimeric dimer synthons (oeg_t^NHT, oeg_up^NHT and oeg_uh^NHT) containing thymine (t), 5-(l-propynyl)-uracil (up) and 5-(1-hexyn-1-yl)-uracil (uh) PNA units with N-(2-hydroxyethyl)glycine (oeg) backbone were synthesized in solution and incorporated into T₂₀ oligonucleotide analogues, using standard P-amidite chemistry. Insertion of dimer blocks led to destabilization of duplexes with dA₂₀ target. The smallest T _m drops were found for chimeras containing oeg_up^NHT dimers. Incorporation of the chimeric synthons into the 3′-end of T₂₀ brought about growing resistance to 3′-exonucleolytic (SV PDE) cleavage in the order of oeg_t^NHT < oeg_up^NHT < oeg_uh^NHT. Due to different endonuclease activities of 3′- and 5′-exonucleases applied, placing of five consecutive dimers at the 5′-terminus resulted in a relatively smaller, but also side-chain dependent, stabilization towards the hydrolysis by 5′-exonuclease (BS PDE). Neither exonucleases (SV and BS PDE) nor an endonuclease (Nuclease P₁) could hydrolyse the unnatural phosphodiester bond linking the 3′-OH of thymidine to the terminal OH of N-(2-hydroxyethyl)glycine PNA backbone.     

Synthesis of a New Hydroxyamino Linked Thymidine Dimer via a Radical C-C Bond Formation

Abstract

An efficient synthesis of a thymidine nucleoside dimer [T-3′-β-O-N(CH₃)-CH₂-5′-T] has been accomplished via an intermolecular radical coupling reaction. The novel dimer contains an achiral and neutral backbone linkage which may have potential application in constructing backbone modified antisense oligonucleosides.     

Structure and Conformation of 5′-Chlorocyclocytidine,a Potent Inhibitor of Nucleic Acid Synthesis: X-Ray, 1H and 13C NMR Analyses

Abstract

The structure of the hydrochloride of 5′-chlorocyclocytidine, a potent inhibitor of DNA synthesis, was determined by X-ray crystallography. The nucleoside crystallizes in the orthorhombic space group P2₁2₁2₁ with cell dimensions a = 10.413(4), b = 13.236(5), c = 17.064(6) Å and with two independent molecules in the asymmetric unit (Z = 8). Atomic parameters were refined by full-matrix least squares to a final value of R = 0.053 for 2490 observed reflections. In both molecules the furanose ring has a C4′ endo/04′ exo (⁴ T ₀) pucker. In molecule A the orientation of the -CH₂Cl side chain is gauche. In molecule B the side chain is disordered: in 70% of these molecules the orientation is trans and in 30% it is gauche ⁺. ¹H NMR spectra indicate a conformational equilibrium between C4′ exo/04′ endo (₄ T ⁰) and C4′ endo/C3′ exo (⁴ ₃ T) with a population ratio of 38:62. All three side chain rotamers occur in solution, the trans orientation contributing most. ¹J(C, H) values for C1′ and C2′ are significantly higher than normal and can therefore be used as a diagnostic tool for the assignment of bridgehead carbon atoms in cyclonucleosides.     

An Improved Synthesis of 1-β-D-Arabinofuranosylcytosine 5′-Phosphate-L-1,2-diacylglycerols

Abstract

5′-O-MMTr-cytosine arabinoside was prepared on a large scale from 5′-O-MMTr-cytidine with diphenyl carbonate via 5′-protected cytidine-2′,3′-carbonate-aracytidine-2′,2-anhydro derivative at a 67 % yield. The synthesis of 1,2-L-dipalmitoyl-snglycerol, 1,2-L-distearoyl-sn-glycerol and 1,2-L-dioleoyl-sn-glycerol described here using 9-fluorenylmethoxycarbonyl (FMOC) group for protection of 3-position of glycerol which can be selectively removed by Et₃N treatment on the overall 60–70 % yield based on 1.2,-isopropilidene-sn-glycerol. These glycerols were phosphorylated first with 2-chlorophenyl-phosphoro-bis-triazolide quantitatively¹ in order to avoid acyl migration, then the glycerophosphate intermediates were condensed with 2′,3′,N⁴-trileulinyl-l-β-D-arabinofuranosylcytosine in the presence of 2-mesytilenesulphonyl chloride (MsCl) and 1 -methylimidazole (Melm)-which was used in the coupling of nucleotides²-^? in an 85–95 % yield compared with the low yielding diester method of Ryu³. Deblocking was carried out in two steps with tetrabutylammonium fluoride (TBAF) and hydrazine hydrate, producing target compouns (14a, 14b, 14c) at a 50 % yield.     

1H NMR Structural Analysis of the Interactions of Proflavine with Self-Complementary Deoxytetranucleosides of Different Base Sequence

Abstract

The molecular associations and structures of the complexes between the acridine dye, proflavine, and self-complementary deoxytetraribonucleoside triphosphates 5′-d(GpCpGpC), 5′-d(CpGpCpG), 5′-d(ApCpGpT), 5′-d(ApGpCpT) in aqueous solution have been investigated using one-dimensional and two-dimensional 500 MHz ¹H NMR spectroscopy.     

CHEMICAL AND ENZYMATIC SYNTHESIS OF 4′-THIO-β-D-ARABINOFURANOSYLCYTOSINE MONOPHOSPHATE AND TRIPHOSPHATE

N⁴-Acetyl-1-(2, 3-di-O-acetyl-4-thio-β-D-arabinofuranosyl)cytosine (2) was synthesized in three steps from 1-(4-thio-β-D-arabinofuranosyl)cytosine (1). The reaction of this partially blocked 4′-thio-ara-C derivative 2 with 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one gave the 5′-phosphitylate derivative 3, which on reaction with pyrophosphate gave the 5′-nucleosidylcyclotriphosphite 4. Product 4 was then oxidized with iodine/pyridine/water and deblocked with concentrated ammonium hydroxide to provide the desired 4′-thio-ara-C-5′-triphosphate 5. This triphosphate 5 was converted to 4′-thio-ara-C -5′-monophosphate 6 by treatment with snake venom phosphodiesterase I. The details of the synthesis, purification, and characterization of both nucleotides are described.     

Investigation of a Facile Synthetic Method for Phosphorothioate Dimer Synthons In Oligonucleotide Phosphorothioates Synthesis

Abstract

A facile synthetic method of a phosphorothioate dimer block was investigated. Dinucleoside phosphite triester intermediates were obtained in one-pot synthesis by the coupling of a protected nucleoside bearing free 5′-OH and a protected nucleoside bearing free 3′-OH in the presence of phosphorous trichloride (PCl₃) and 1,2,4-triazole. The intermediates were easily sulfurized to afford the desired phosphorothioate dimer blocks in 33-64% overall yields.     

Structural Determination of Silicon-Containing Oligonucleotides by 1H−29Si Long-Range Heteronuclear Multiple Quantum Correlation NMR Spectroscopy

Abstract

The technique of ¹H^?29Si Long-Range Heteronuclear Multiple Quantum Correlation NMR Spectroscopy was used to determine the structure of silicon-containing oligonucleotides. Trimers which contained silicon instead of phosphorus as part of the oligonucleotide link were synthesized through a synthetic route that required minimal hydroxyl protection. The resulting trimer could have one of two possible structures. Through the use of ¹H^?29Si HMQC NMR spectroscopy, it was possible to link the 3′-hydroxymethine proton of one sugar to the 5′-hydroxymethylene proton of an adjacent sugar by correlation to the same silicon atom, thus elucidating the final structure.     

Synthesis of 3′,5′-Dithymidylyl-α-hydroxyphosphonate Dimer Building Blocks for Oligonucleotide Synthesis—A New Pro-oliguncleotide

Abstract

The synthesis of the dimer building blocks 1 and 2 and their introduction into (T)₁₅-oligonucleotides is described. The stability against 3′-exonuclease digestion (SVP) as well as the hybridization properties (T_m values) were examined.     

Synthesis and Enzymatic Studies of Modified Oligonucleotides with Abiological Monomer Units

Abstract

The synthesis and the enzymatic studies of modified oligonucleotides containing a PNA modified PNA-DNA dimer block and a new acyclic racemic serinol nucleoside is described. We show that both, the PNA-DNA dimer block¹ and the modified PNA-spacer (acyclic serinol nucleoside)² can be used as modified templates for the enzymatic generation of single stranded DNA. Degradation studies of the oligonucleotides containing the PNA-DNA dimer block with snake venom phosphodiesterase show that the modified oligonucleotides are stable towards exonucleolytic degradation.     

Synthesis,Biophysical, and Biochemical Properties of PNA-DNA Chimeras

Abstract

Three PNA-DNA chimeric dimer synthons (tT, upT and uhT, see Sch. 1) have been synthesized in solution and used to make T₂₀-analogue chimeras applying standard solid-phase DNA synthesis protocol. Duplex forming ability of chimeras with dA₂₀ and their hydrolyses by 3′- and 5′-exonucleases (snake venom and bovine spleen phosphodiesterase, respectively) have been investigated.     

Nucleotides. LXXIV Synthesis of a-D-Arabino-oligonucleotides

The 5 α-D-arabinofuranosylnucleosides α-araU (15), α-araT (18), α-araC (22), α-araA (25), and α-araG (28) have been synthesized by the modified silyl-method. The amino groups at the nucleobases and the 2′-hydroxy group at the sugar moiety were protected by the 2-(4-nitro-phenyl) ethoxycarbonyl (npeoc) group (37-40) and the amide function in α-araG was additionally blocked by the 2-(4-nitrophenyl)ethyl group (63) to improve solubility in organic solvents. Mono-and dimethoxytritylation of the 5′-OH group was performed in the usual manner to give 41-48, 64, and 65 in high yields and further substitution of the 3′-OH group led to the monomeric building blocks 66-75 as well as the 3′-O-succinoyl derivatives 76-85 functioning as starting units in solid-support oligonucleotide synthesis. A large number of oligo-α-arabinonucleotides have been prepared on modified CPG-material applying the npeoc/npe strategy as a very efficient synthetic tool for highly purified, homogenous oligomers. Hybridizations between α-arabinonucleotide strands revealed in analogy to earlier findings an antiparallel orientation whereas the combination of an oligo-α-D-arabinonucleotide with a complementary oligo-2′-deoxy-β-D-ribofuranosylnucleotide showed base-pairing only if a parallel polarity was present. The advantages in oligo-α-arabinonucleotide synthesis were furthermore demonstrated by the synthesis of the tα-ANA ^his a structural analog of the natural tRNA ^his of the phage T5.     

A One Step Synthesis of O6-Methyl-2′-deoxyguanosine

Abstract

A single step chemical synthesis of N⁷-methyl-2′-deoxyguanosine (m⁷dG), N¹-methyl-2′-deoxyguanosine (m¹dG) and O⁶-methyl-2′-deoxyguanosine (m⁶dG) is described. The products were separated on the silical gel plates and characterized by nuclear magnetic resonance and mass spectrometry.     

Improved Targeting of the Flanks of a DNA Stem Using α-Oligodeoxynucleotides.-The Enhanced Effect of an Intercalator

Abstract

2′-Deoxy-5′-0-(4,4′-dimethoxytrityl)-5-methyl-N ⁴-(1-pyrenylmethyl)-α-cytidine (5) was prepared by reaction of 1-pyrenylmethylamine with an appropriate protected 4-(l,2,4-triazolyl)-α-thymidine derivative 3 which was synthesized from 5-O-DMT protected α-thymidine 1. Aminolysis of 3 afforded 3′-O-acetyl-2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-5-methyl-α-cytidine (8). Benzoylation of 8 and removal of acetyl afforded N ⁴-benzoyl-2-deoxy-5–0-(4,4′-dimethoxytrityl)-5-methyl-α-cytidine (10). The amidites of compounds 5and 10 were prepared and used in α-oligonucleotide synthesis. DNA three-way junction (TWJ) is stabilized when an α-ODN is used for targeting the dangling flanks of the stem in a DNA hairpin. Further stabilization of the TWJ is observed when 5 is inserted into the α-ODN at the junction region.

     

15N-Labeled Oligodeoxynucleotides -Useful Probes for 1H-NMR Investigations

Abstract

The amino protons of ¹⁵N-labeled deoxyoligonucleotides were studied as possible structural probes for NMR investigations of the interaction between DNA and regulatory proteins. To apply this strategy, 6-¹⁵NH₂-2′ -deoxyadenoslne, 4-¹⁵NH -2′-deoxycytidine and 2-¹⁵NH -2′-deoxyguanosine were chemically synthesized. The labeled nucleosides were introduced into distinct positions of oligodeoxy-nucleotides by large-scale DNA synthesis. The behaviour of the ¹⁵N-coupled cytidine amino protons in a 18 base pair (bp) lac operator sequence were investigated using H-¹⁵N INDOR spectroscopy .