Stereocontrolled Synthesis of Dithymidine Phosphorothioates by Use of a Functionalized 5′- Protecting Group Bearing an Imidazole Residue†

Abstract

Diastereoselective formation of internucleotidic phosphorothioate triester bonds was achieved by use of 3-(imidazol-1-ylmethyl)-4′,4″-dimethoxytrityl (IDTr) as a 5′-hydroxyl protecting group in the phosphotriester approach. After removal of all the protecting groups, stereochemistry of the major product was determined as the Spconfiguration by enzymatic digestion.

     

Use of the 2-(4-Pyridyl)Ethyl Protecting Group in the Synthesis of DNA Fragments Via Phosphoramidite Intermediates

Abstract

2-(4-Pyridyl)ethyl is a new protecting group for the internucleotidic bonds in the synthesis of deoxyribooligonucleotides by the phosphoramidite approach. This group is stable to alkali and acid conditions, and can be removed easily by two step procedures under mild conditions. The synthesis of deoxyribo-oligonucleotides by using phosphoramidite units containing 2-(4-pyridyl)ethyl group is also described.     

Synthesis,Biophysical Properties,and Stability Studies of Mixed Backbone Oligonucleotides Containing Novel Non-Ionic Linkages

Abstract

Mixed backbone oligonucleotides (MBOs) (containing ionic and non-ionic internucleotidic linkages) in which the non-ionic segments are either methylphosphotriester (PO-OMe) or primary phosphoramidate (PO-NH₂) linkages have been prepared using the recently described N-pent-4-enoyl (PNT) nucleoside phosphoramidates and H-phosphonates. Biophysical properties and stability studies suggest that these MBOs are novel antisense molecules.     

Use of New Phosphonylating and Condensing Agents in The Synthesis of Oligonucleotides Via The H-Phosphonate Approach

Abstract

Bis (1, 1, 1, 3, 3, 3-hexafluoro-2-propyl) phosphonate was most promising as a phosphonylating agent for the preparation of nucleoside-3′-phosphonate units. 1, 3-Dimethyl-2-chloro-imida-zolinium chloride (UWC) as a coupling agent has successfully been used for the internucleotidic H-phosphonate bonds formation via the H-phosphonate approach on a solid support.     

Use of the 2-(2-Pyridyl)Ethyl Protecting Group in the Synthesis of DNA Fragments via Phosphoramidite Intermediates

Abstract

2-(2-Pyridyl)ethyl is a new protecting group for the internucleotidic linkages in the synthesis of oligodeoxyribo-nucleotides by the phosphoramidite method. This group is stable to alkali and acid, and can be removed by two step procedures under mild conditions. Furthermore, we have found that bis-(diiso-propylamino)chlorophosphine is much more effective for the preparation of bis-(diisopropylamino)alkoxyphosphine than various dichlorophosphines. The synthesis of oligodeoxyribonucleotides by using 2-(2-pyridyl)ethyl-deoxyribonucleoside-3′-0-N,N-diisopropyl-amidite units is also described.     

An Effective Enzyme Interacting with Poly (dT-dA)· Poly (dT-dA): A Dynamic Enhancer-Repressor Action

Abstract

The Green function technique is used to study the open hydrogen bond probability of poly(dT-dA)·poly(dT-dA) when an effective enzyme is attached to the helix. The DNA interstrand hydrogen bond mean motion and probability of fluctuating to an open state depends on the internal vibrational frequency of the enzyme. An enzyme with internal frequency of 80 cm ^?1 reduces hydrogen bond motion and the resulting probability of hydrogen bond fluctuational opening. An enzyme with internal frequency of 72 cm ^?1 increases hydrogen bond motion and the probability of hydrogen bond breaking.     

An Inverse Approach in Oligodeoxyribonucleotide Synthesis

Abstract

We synthesized 3′-O-dimethoxytrityl-5′O-phosphoramidites and 5′-O-succinates which can be used as monomeric building blocks for the built up of oligodeoxyribonucleotides in the alternative 5′-3′ direction. With this inverse strategy oligonucleotide 3′-conjugates as well as 3′-3′ and 5′-5′ internucleotidic linkages can be easily formed.     

Sequence specific hydrogen bond of DNA with denaturants affects its stability: Spectroscopic and simulation studies

BackgroundSeveral different small molecules have been used to target the DNA helix in order to treat the diseases caused by its mutation. Guanidinium(Gdm⁺) and urea based drugs have been used for the diseases related to central nervous system, also as the anti-inflammatory and chemotherapeutic agent. However, the role of Gdm⁺ and urea in the stabilization/destabilization of DNA is not well understood.MethodsSpectroscopic techniques along with molecular dynamics (MD) simulation have been performed on different sequences of DNA in the presence of guanidinium chloride (GdmCl) and urea to decode the binding of denaturants with DNA and the role of hydrogen bond with the different regions of DNA in its stability/destability.Results and conclusionOur study reveals that, Gdm⁺ of GdmCl and urea both intrudes into the groove region of DNA along with the interaction with its phosphate backbone. However, interaction of Gdm⁺ and urea with the nucleobases in the groove region is different. Gdm⁺ forms the intra-strand hydrogen bond with the central region of the both sequences of DNA whereas inter-strand hydrogen bond along with water assisted hydrogen bond takes place in the case of urea. The intra-strand hydrogen bond formation capability of Gdm⁺ with the nucleobases in the minor groove of DNA decreases its groove width which probably causes the stabilization of B-DNA in GdmCl. In contrast, the propensity of the formation of inter-strand hydrogen bond of urea with the nucleobases in the groove region of DNA without affecting the groove width destabilizes B-DNA as compared to GdmCl. This study depicts that the opposite effect of GdmCl and urea on the stability is a general property of B-DNA. However, the extent of stabilization/destabilization of DNA in Gdm⁺ and urea depend on its sequence probably due to the difference in the intra/inter-strand hydrogen bonding with different bases present in both the sequences of DNA.General significanceThe information obtained from this study will be useful for the designing of Gdm⁺ based drug molecule which can target the DNA more specifically and selectively.     

Synthesis of nucleotide oligomer having amino group in the side chain of internucleotidic bond

A convenient synthesis of d(TpT) derivative having amino group in the side chain of internucleotidic bond was achieved in a high yield using dichlorophosphoramidite as a phosphorylating reagent.     

1-Deaza-3′-O-methyladenosine: A Nucleoside with the Syn-conformation in the Solid State and in Solution

Abstract

The 2′-O-methyl (2) and the 3′-O-methyl (3) derivatives of 1-deazaadenosine (1) were prepared. Single crystal X-ray analysis as well as ¹H and ¹³C NMR studies were performed on the 3′-O-methyl-1-deazaadenosine 3. In the solid state, the glycosyl torsion angle (χ = 64.7°) is in the syn-range which is caused by an intramolecular (5′)CH2OH…N(3) hydrogen bond. The ribofuranose moiety adopts a ² E (C-3′-exo; S) conformation and the orientation of the exocyclic C(4′)-C(5′) bond is + sc (γ^(+)g). The conformation in solution was found to be very similar to that in solid state. Whereas the 2′-O-methyl derivative of 1 is a strong inhibitor of adenosine deaminase the 3′-O-methyl derivative is neither inhibitor nor substrate.     

A Molecular Dynamics Computer Simulation Study of Nucleotide Analogues. Comparison of the Hydration Pattern of Dithymidine Phosphate with those of the Dithymidine Methylphosphonate Diastereomers

Abstract

The hydration pattern of thymidyl(3′→5′) thymidine 1 and those of R_p and S_p diastereomers of the corresponding methylphosphonate analogue 2, have been studied using Molecular Dynamics (MD) computer simulation. It was found that the methylphosphonate modification leads to significant changes in the coordination of water molecules around the internucleotidic linkage and these, in turn, affect the hydration pattern of other parts of the molecule. The most notable differences between R_p and S_p diastereomers 2a and 2b were found to occur at the deoxyribose moieties of the nucleosid-5′-yl units.     

New κ-PNN′- and κ-PNN′O-polydentate ligands: Synthesis, coordination and structural studies

The three-step synthesis of new mixed P/N/N′/O-donor ligands C₆H₃(OH){2-NHC(O)CH₂NCHC₆H₄PPh₂}(4-CH₃) 3a·HH and C₆H₄(OH){3-NHC(O)CH₂NCHC₆H₄PPh₂} 3b·HH, by Schiff base condensation of the 1° amines C₆H₃(OH){2-NHC(O)CH₂NH₂}(4-CH₃) 2a or C₆H₄(OH){3-NHC(O)CH₂NH₂} 2b with C₆H₄(CHO)(2-PPh₂) in refluxing EtOH, is described. Reaction of 1 equiv. of 3a·HH or 3b·HH with MCl₂(cod) (M = Pt, Pd; cod = cycloocta-1,5-diene) affords the κ²-PN-chelate complexes MCl₂(3a·HH) (M = Pd 4a; M = Pt 4b) and MCl₂(3b·HH) (M = Pt 4c). The dichlorometal(II) complexes 4d and 4e, bearing instead a pendant 4-phenolic group, were similarly prepared (in >90% yield). Chloro-bridge cleavage of [Pd(μ-Cl)(η³-C₃H₅)]₂ with 3a·HH or 3b·HH gave the monocationic κ²-PN-chelate complexes [Pd(η³-C₃H₅)(3a·HH)]Cl 5a or [Pd(η³-C₃H₅)(3b·HH)]Cl 5b, respectively. Elimination of cod, and single CH₃ protonation, from Pt(CH₃)₂(cod) upon reaction with 1 equiv. of 3a·HH or 3b·HH in C₇H₈ at room temperature afforded the neutral complexes C₆H₃(OH){2-NC(O)CH₂NCHC₆H₄PPh₂Pt(CH₃)}(4-CH₃) 6a and C₆H₄(OH){3-NC(O)CH₂NCHC₆H₄PPh₂Pt(CH₃)} 6b, respectively bearing a monoanionic (3a·H⁻ or 3b·H⁻) κ³-PNN′-tridentate ligand. Amide and phenol deprotonation were readily achieved, using KO^tBu as base, to give high yields of the κ⁴-PNN′O-tetradentate complexes C₆H₃(O){2-NC(O)CH₂NCHC₆H₄PPh₂Pd}(4-CH₃) 7a and C₆H₃(O){2-NC(O)CH₂NCHC₆H₄PPh₂Pt}(4-CH₃) 7b bearing the dianionic ligand 3a²⁻. All new compounds have been characterised by multinuclear NMR, FTIR, mass spectroscopy and microanalysis. Single crystal X-ray studies have been performed on compounds 1b·1.5CH₂Cl₂, 3b·HH·0.5Et₂O, 6b·CHCl₃ and 7b·0.5Et₂O.     

Alteration of the groove width of DNA induced by the multimodal hydrogen bonding of denaturants with DNA bases in its grooves affects their stability

BackgroundDenaturants, namely, urea and guanidinium chloride (GdmCl) affect the stability as well as structure of DNA. Critical assessment of the role of hydrogen bonding of these denaturants with the different regions of DNA is essential in terms of its stability and structural aspect. However, the understanding of the mechanistic aspects of structural change of DNA induced by the denaturants is not yet well understood.MethodsIn this study, various spectroscopic along with molecular dynamics (MD) simulation techniques were employed to understand the role of hydrogen bonding of these denaturants with DNA bases in their stability and structural change.Results and conclusionIt has been found that both, GdmCl and urea intrude into groove region of DNA by striping surrounding water. The hydrogen bonding pattern of Gdm⁺ and urea with DNA bases in its groove region is multimodal and distinctly different from each other. The interaction of GdmCl with DNA is stabilized by electrostatic interaction whereas electrostatic and Lennard-Jones interactions both contribute for urea. Gdm⁺ forms direct hydrogen bond with the bases in the minor groove of DNA whereas direct and water assisted hydrogen bond takes place with urea. The hydrogen bond formed between Gdm⁺ with bases in the groove region of DNA is stronger than urea due to strong electrostatic interaction along with less self-aggregation of Gdm⁺ than urea. The distinct hydrogen bonding capability of Gdm⁺ and urea with DNA bases in its groove region affects its width differently. The interaction of Gdm⁺ decreases the width of the minor and major groove which probably increases the strength of hydrogen bond between the Watson-Crick base pairs of DNA leading to its stability. In contrast, the interaction of urea does not affect much to the width of the grooves except the marginal increase in the minor groove width which probably decreases the strength of hydrogen bond between Watson Crick base pairs leading to the destabilization of DNA.General significanceOur study clearly depicts the role of hydrogen bonding between DNA bases and denaturants in their stability and structural change which can be used further for designing of the guanidinium based drug molecules.     

6-Methyl-5-Azacytidine-Synthesis,Conformational Properties and Biological Activity. A Comparison of Molecular Conformation with 5-Azacytidine

Abstract

The title compound was prepared by the isocyanate procedure and the tri-methylsilyl method. The measurement of ¹H NMR spectrum of 6-methyl-5-azacytidine (1) revealed a preference of γ^t (46%) rotamer around C(5′)-C(4′) bond, a predominance of N conformation of the ribose ring (K_eq 0.33) and a preference of syn conformation around the C-N glycosyl bond. An analogous measurement of 5-azacytidine has shown a preference of γ⁺ (60%) rotamer around the C(5′)-C(4′) bond, a predominance of N conformation of the ribose ring (K_eq 0.41) and a preference of anti conformation around the C-N glycosyl bond. 6-Methyl-5-azacytidine (1) inhibits the growth of bacteria E. coli to the extent of 85% at 4000 μM concentration and the growth of LoVo/L, a human colon carcinoma cell line, to the extent of 30% at 100 μM concentration but did not inhibit L1210 cells at ≤ 100 μM concentration. 6-Methyl-5-azacytidine (1) exhibited no in vitro antiviral activity at ≤ 1 μM concentration.

     

Formation of two centre three electron bond by hydroxyl radical induced reaction of thiocoumarin: evidence from experimental and theoretical studies

Radiation chemical studies of thioesculetin (1), a thioketone derivative of coumarin, were performed by both pulse radiolysis technique and DFT calculations. Hydroxyl (^?OH) radical reaction with 1 resulted transients absorbing at 320, 360 and 500?nm. To identify the nature of the transients, the reaction was studied with specific one-electron oxidant (N₃^?) radical, where 360?nm band was absent. The transient absorption at 500?nm was concentration-dependent. The overall impression for ^?OH radical reaction was that the transient absorbing at 320, 360 and 500?nm was due to sulphur centred monomer radical, hydroxysulfuranyl and dimer radical of 1 respectively. The equilibrium constant between the monomer to dimer radical was 3.75?×?10⁴ M^?1. From the transients’ redox nature, it was observed that 57 and 24% of ^?OH radical yielded to oxidising and reducing products respectively. Further, the product analysis by HPLC suggested that the dimer radical disproportionate to esculetin and thioesculetin. DFT energy calculation for all the possible transients revealed that dimer radical has the lowest energy. The HOMO of 1 and its monomer radical suggested that the electron density was localised on the sulphur atom. The bond length between the two sulphur atoms in dimer radical was 2.88 Å which was less than the van der Waals distance. Bond order between the two sulphur atoms was 0.55, suggesting that the bond was two centre three electron (2c–3e). From TD-DFT calculation, the electronic transition of dimer radical was at 479?nm which was in close agreement with the experimental value. The nature of the electronic transition was σ → σ* from a 2c???3e bond.     

Nucleophilic N1 → N3 Rearrangement of 5′-O-Trityl-O2,3′-Cycloanhydrothymidine

Abstract

5′-O-Trityl-O²,3′-cycloanhydrothymidine (1) heated at 150°C in the presence of O,O-diethyl phosphate or O,O-diethyl phosphorothioate anions undergoes rearrangement into N³-isomer (2); its structure was established by both advanced NMR methods and X-ray crystallographic studies. The most probable mechanism of 1→2 rearrangement relies upon reversibility of glycosidic bond cleavage process.     

Synthesis of Methylenecyclobutyl- and Cyclobutenyl Adenine,Potent Antiviral Carbocyclic Analogues of Oxetanocin A

Abstract

9-Cyclobutyladenines bearing both methylene and hydroxymethyl groups, 3 and 4, were prepared by dehydration of carbocyclic oxetanocin A (1a). Introduction of a double bond into cyclobutane ring was achieved by allylic oxidation of N ⁶-benzoyl-9-[3-methylenecyclobutyl]adenine (12), which after several steps, afforded 9-[3-(hydroxy-methyl)-2-cyclobutenyl)adenine (5).     

Carboxyl Proteinase from the Wood Deteriorating Basidiomycete Pycnoporus coccineus :Substrate Specificity with Oxidized Insulin Peptide B1 ~ B16 and B15 ~ B24, Angiotensin and Proangiotensin

The specificity of highly purified carboxyl proteinase from Pycnoporus coccineus (formerly designated Trametes sanguined) was investigated with oligopeptides at pH 2.7. Hydrolysis of oxidized insulin peptide Bl ~ B16 was observed at two peptide bonds (His¹⁰-Leu¹¹ and Ala¹⁴-Leu¹⁵) during 3-hr incubation. The enzyme did not hydrolyze oxidized insulin peptide B15 ~ B24. Hydrolysis of angiotensin (formerly designated angiotensin II) was observed at the Tyr⁴-Ile⁵ bond. Hydrolysis of proangiotensin (formerly designated angiotensin I) was also at the Tyr⁴-Ile⁵ bond. In conclusion, peptide bonds which have a hydrophobic amino acid in the P′₁ position (as defined by Schechter and Berger, Biochem. Biophys. Res. Commun., 27, 157 (1967)) are preferentially cleaved by the trypsinogen activating carboxyl proteinase of Pycnoporus coccineus.     

Aflatoxin B1 and DNA Adducts. Proposed Model for Surface Noncovalent and Covalent Complexes with N(7) of Guanine. II.

Abstract

An alternate model for surface noncovalent and surface covalent binding of aflatoxin B₁ to N(7) of guanine in DNA is proposed. This model considers the out-of-plane motions of C(8) of aflatoxin B₁ in those interactions.

The covalent intercalated fit of aflatoxin B₁ into DNA arises from steric adjustments made by DNA at the covalent intercalation site as well as local strain in the bond angles about N(7) of guanine and C(8) of aflatoxin B₁. The bond angle about N(7) deviates modestly from the sp² value toward the sp³ value.

This study suggests that the surface covalent aflatoxin B₁ -DNA complex serves only a minor role in aflatoxin's precarcinogenic interaction with DNA and is a likely correctable error.     

Conformational Studies of Two Bradykinin Antagonists by Using Two-dimensional NMR Techniques and Molecular Dynamics Simulations

Abstract

The solution conformations of two potent antagonists of bradykinin (Arg¹-Pro²-Pro³-Gly⁴- Phe⁵-Ser⁶-Pro⁷-Phe⁸-Arg⁹), [Aca-¹, DArg⁰, Hyp³, Thi⁵, DPhe⁷,(N-Bzl)Gly⁸]BK (1) and [Aaa- ¹, DArg⁰, Hyp³, Thi⁵,(2-DNal)⁷, Thi⁸]BK (2), were studied by using 2D NMR spectroscopy in DMSO-dg and molecular dynamics simulations. The NMR spectra of peptide 1 reveals the existence of at least two isomers arising from isomerization across the DPhe⁷-(N-Bzl)Gly⁸peptide bond. The more populated isomer possesses the cis peptide bond at this position. The ratio of cis/trans isomers amounted to 7:3. With both antagonists, the NMR data indicate a β-turn structure for the Hyp³-Gly⁴ residues. In addition, for peptide 2, position 2,3 is likely to be occupied by turn-like structures. The cis peptide bond between DPhe⁷ and (N- Bzl)Gly⁸ in analogue 1 suggests type VI β-turn at position 7,8. The molecular dynamics runs were performed on both peptides in DMSO solution. The results indicate that the structure of peptide 1 is characterized by type VIb β-turn comprising residues Ser-Arg⁹ and the βI or βII-turn involving the Pro²-Thi⁵ fragment, whereas peptide 2 shows the tendency towards the formation of type I β-turn at position 2,3. The structures of both antagonists are stabilized by a salt bridge between the guanidine moiety of Arg¹ and the carboxyl group of Arg⁹. Moreover, the side chain of DArg⁰ is apart of the rest of molecule and is not involved in structural elements except for a few calculated structures.