Phosphoramidites of [180] Chiral (Rp)- and (Sp)-Configurated Dimer-Blocks and their Use in Automated Oligonucleotide Synthesis

Abstract

The N,N-diisopropylphosphoramidites 1 and 2 of appropriately protected chiral diastereoisomers of d(T-[P-180]-A) (6a and 6o, resp.) have been synthesized. They were employed in solid-phase synthesis to yield the octamers d(GAGT-(Rp)-[P180]-ACTC) and d(GAGT-(Sp)-[P180]-ACTC).     

Synthesis of Protected 8-Substituted Deoxyribonucleosides and Its Helix Stability in Oligodeoxyribonucleotides Containing the Eco RI Recognition Site

Abstract

Octadeoxyribonucleotides with the sequences d(GGA?ATTCC), d(GGAA?TTCC), and d(GG?AATTCC) have been prepared by solid phase synthesis using the H-phosphonate units containing modified base moieties. These oligomers which have an isosterically altered recognition sequence of the restriction endodeoxyribonuclease Eco RI. The oligomers, with replacement to deoxy-7,8-dihyroadenosine-8-one (dA^OH), 8-methoxydeoxyadenosine (dA_OMe) and 8-methoxydeoxyguanosine (dG^OMe) from deoxyadenosine or deoxyguanosine were used for studying recognition phenomena at the functional group level. From thermodyamic data of these alternating octamers it was shown that the oligomer containing 8-methoxydeoxyadenosine in the center of the recognition sequence destabilizes such duplexes less strongly than the oligomers containing other 8-substituted nucleosides in the 5′-side of the recognition sequences. Further, the hydrolysis by Eco RI of the modified oligomers perfectly resisted compared to d(GGAATTCC).     

The binding of precipitant ions in the tetragonal crystals of hen egg white lysozyme

Abstract

The bonds between lysozyme molecules and precipitant ions in single crystals grown with chlorides of several metals are analysed on the basis of crystal structure data. Crystals of tetragonal hen egg lysozyme (HEWL) were grown with chlorides of several alkali and transition metals (LiCl, NaCl, KCl, NiCl₂ and CuCl₂) as precipitants and the three-dimensional structures were determined at 1.35?Å resolution by X-ray diffraction method. The positions of metal and chloride ions attached to the protein were located, divided into three groups and analysed. Some of them, in accordance with the recently proposed and experimentally confirmed crystal growth model, provide connections in protein dimers and octamers that are precursor clusters in the crystallization lysozyme solution. The first group, including Cu⁺², Ni⁺² and Na⁺¹ cations, binds specifically to the protein molecule. The second group consists of metal and chloride ions bound inside the dimers and octamers. The third group of ions can participate in connections between the octamers that are suggested as building units during the crystal growth. The arrangement of chloride and metal ions associated with lysozyme molecule at all stages of the crystallization solution formation and crystal growth is discussed.

Communicated by Ramaswamy H. Sarma     

NMR Spectroscopic Study of a DNA Duplex with Mercury-Mediated T-T Base Pairs

Recently, we reported that T-T mismatches can specifically recognize Hg ^II (T-Hg ^II -T pair formation). In order to understand the properties of the T-Hg ^II -T pair, we recorded NMR spectra for a DNA duplex, d(CGCG TT GTCC) ? d(GGAC TT CGCG), with two successive T-T mismatches (Hg ^II -binding sites). We assigned ¹ H resonances for mercury-free and di-mercurated duplexes, and performed titration experiments with Hg ^II by using 1D ¹ H NMR spectra. Because of the above mentioned assignments, we could confirm the existence of mono-mercurated species, because individual components gave independent NMR signals in the titration spectra.     

Synthesis and biological evaluation of some thiazole derivatives as new cholinesterase inhibitors

In the present study, some thiazole derivatives were synthesized via the ring closure reaction of 1-[2-(2-oxobenzo[d]thiazol-3(2H)-yl)acetyl]thiosemicarbazide with various phenacyl bromides. The chemical structures of the compounds were elucidated by ¹H NMR, ¹³C NMR and mass spectral data and elemental analyses. Each derivative was evaluated for its ability to inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) using a modification of Ellman’s spectrophotometric method. The compounds were also investigated for their cytotoxic properties using MTT assay. The most potent AChE inhibitor was found as compound 4e (IC₅₀?=?25.5?±?2.12 µg/mL) followed by compounds 4i (IC₅₀?=?38.50?±?2.12 µg/mL), 4c (IC₅₀?=?58.42?±?3.14 µg/mL) and 4g (IC₅₀?=?68?±?2.12 µg/mL) when compared with eserine (IC₅₀?=?0.025?±?0.01 µg/mL). Effective compounds on AChE exhibited weak inhibition on BuChE (IC₅₀ > 80 µg/mL). MTT assay indicated that the cytotoxic dose (IC₅₀?=?71.67?±?7.63 µg/mL) of compound 4e was higher than its effective dose.     

In Search of a Hoogsteen Base Paired DNA Duplex in Aqueous Solution

Abstract

When the oligodeoxynucleotides d(A)₆ and d(T)₆ are mixed together in a 1:1 ratio (in 100 mM NaCl), the NH signals in the NMR spectrum gave a typical signature of Watson-Crick paired (WC) and Hoogsteen paired (H) AT base pairs. The observation indicates two schemes: Scheme I, WC and H duplexes in slow equilibrium, i.e., WC ? H, Scheme II, the WC helix formed is unstable and that it disproportionates into a triple helix (TR) and free d(A)₆. We show that (i) addition of extra d(A)₆ does not change the helix composition, (ii) addition of a minor-groove specific drug Dst2 (a distamycin analogue) results in an exclusive WC helix- drug duplex, while it does not destabilize triple helix in a 1:2 mixture. In addition we have compared the melting profile, ³¹P NMR spectra, ¹H NMR spectra and the salt dependence of the 1:1 mixture and that of a pure triple helix. All the data from the above experiments overwhelmingly favor Scheme I. However Scheme II cannot be categorically excluded.

Based on 1D/2D NMR studies, we have characterized the structural properties of the Hoogsteen double helix in terms of nucleotide conformations. In addition, we computationally demonstrate that the relative stability of the WC over the H duplexes increases with increasing chain length.     

CONDENSATION OF TRIFORMYLMETHANE WITH GUANOSINE

Guanosine has been reacted with triformylmethane (TFM) in refluxing pyridine. Four different products, 4–7, were isolated by preparative RP-HPLC, and characterized by ¹H and ¹³C NMR and UV spectroscopy and mass spectrometry. One of the products, the cyclic 1:1 adduct 4, is a stable cyclic carbinolamine formed probably by cyclization of the expected aminomethylene derivative 3. Compound 4 then undergoes reversible dehydration to the fully conjugated adduct 5. The appearance of the additional adducts, 6 and 7, suggests that TFM is prone to transformations resulting in the formation of methylenemalonaldehyde (9) and 1,1,3,3-tetraformylpropane (11).     

Glycosylation of Mono- and Bicyclic Dicarbonic Acid Imides

Abstract

Glycosylation of some mono- and bicyclic dicarbonic acid imides was performed via the Friedel-Crafts-catalyzed silyl Hilbert-Johnson reaction. The occurrence of β-N-ribosylation was established by ¹H and ¹³C NMR spectroscopy. The electron distributions in the lactam region of the N-silylated cyclic imides strongly influence the glycosylation. The N-glycosylated cyclic imides are potential agents for glycoalkylation of lysine residues in proteins.     

Cyclic AMP Diphenyl Phosphoric Mixed Anhydride: Synthesis,P NMR Characterization and Reaction with Dimethylamine

Abstract

Phosphorus diastereoisomers, R _p and S _p of p¹-adenosine cyclic 3′, 5′ P² -diphenylpyrophosphate (cyclic AMP diphenylphosphoric mixed anhydride) (1) were prepared from adenosine cyclic 3′, 5′-monophosphate (cyclic AMP) and diphenyl phosphorochloridate and characterized by ³¹p NMR. The synthesis preferentially gave R _p-1. Reaction of 1 with dimethylamine resulted in the formation of a (～ 3:1) mixture of adenosine cyclic 3′,5′-N, N-dimethylphosphoramidate and diphenyl-N, N-dimethylphosphoramidate and occurred with inversion of configuration at cyclic AMP phosphorus.     

Structural and ICAM-1-Docking Properties of a Cyclic Peptide from the I-domain of LFA-1: An inhibitor of ICAM-1/LFA-1-mediated T-cell adhesion

Abstract

The purpose of this work was to study the conformation of cyclic peptide 1, cyclo(1,12)- Pen1-Ile2-Thr3-Asp4-Gly5-Glu6-Ala7-Thr8-Asp9-Ser10-Gly11-Cys12-OH, derived from the I-domain of the LFA-1 α-subunit. We found that cyclic peptide 1 can bind to the D1- domain of ICAM-1 and inhibit ICAM-1/LFA-1-mediated homotypic and heterotypic T-cell adhesion. To understand the bioactive conformation and binding requirements for cyclic peptide 1, its solution structure was studied using NMR, CD, and molecular dynamics simulations. Furthermore, possible binding properties between the cyclic peptide and the D1- domain of ICAM-1 were evaluated using docking experiments. This cyclic peptide has a stable βII'-turn at Asp4-Gly5-Glu6-Ala7 and a βI-turn at Pen1-Ile2-Thr3-Asp4; a less stable βV-turn is found at the C-terminal region. The β-turn at Asp4-Gly5-Glu6-Ala7 was also found in the X-ray structure of the I-domain of LFA-1. Our CD studies showed that the peptide binds to calcium/magnesium and forms a 1:1 (peptide:calcium/magnesium) complex with low cation concentrations and multiple types of complexes with higher cation concentrations. Binding to divalent cations causes a conformational change in peptide 1; this is consistent with our previous study that binding of peptide 1 to ICAM-1 was influenced by divalent cations. Docking studies show the interaction between cyclic peptide 1 and the D1- domain of ICAM-1; it indicates that the Ile2-Thr3-Asp4-Gly4-Glu6-Ala7-Thr8 sequence interacts with the F and C strands of the D1-domain. Finally, these studies will help us design a new generation of selective peptides that may bind better to the D1-domain of ICAM-1.     

Microwave-assisted synthesis and bioevaluation of new sulfonamides

In this study, 4-[5-(4-hydroxyphenyl)-3-aryl-4,5-dihydro-1H-pyrazol-1-yl]benzenesulfonamide derivatives (8-14) were synthesized for the first time by microwave irradiation and their chemical structures were confirmed by ¹H NMR, ¹³C NMR and HRMS. Cytotoxic activities and inhibitory effects on carbonic anhydrase I and II isoenzymes of the compounds were investigated. The compounds 9 (PSE?=?4.2), 12 (PSE?=?4.1) and 13 (PSE?=?3.9) with the highest potency selectivity expression (PSE) values in cytotoxicity experiments and the compounds 13 (Ki?=?3.73?±?0.91?nM toward hCA I) and 14 (Ki?=?3.85?±?0.57?nM toward hCA II) with the lowest Ki values in CA inhibition studies can be considered as leader compounds for further studies.     

Synthesis,carbonic anhydrase I and II inhibition studies of the 1,3,5-trisubstituted-pyrazolines

4-(3-(4-Substituted-phenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl) benzenesulfonamides (9–16) were successfully synthesized and their chemical structures were confirmed by ¹H NMR, ¹³C NMR, and HRMS spectra. Carbonic anhydrase I and II inhibitory effects of the compounds were investigated. K_i values of the compounds were in the range of 316.7?±?9.6–533.1?±?187.8?nM towards hCA I and 412.5?±?115.4–624.6?±?168.2?nM towards hCA II isoenzymes. While K_i values of the reference compound Acetazolamide were 278.8?±?44.3?nM and 293.4?±?46.4?nM towards hCA I and hCA II izoenzymes, respectively. Compound 14 with bromine and compound 13 with fluorine substituents can be considered as the leader compounds of the series because of the lowest K_i values in series to make further detailed carbonic anhydrase inhibiton studies.     

Urease inhibitors from Hypericum oblongifolium WALL.

The bioassay-guided fractionation of H. oblongifolium has led to the isolation of potent urease inhibitors 1–3. The structures were elucidated by NMR and mass spectroscopic techniques. Compound 2 showed a potent enzyme inhibition activity (IC₅₀ 20.96?±?0.93), which is comparatively higher than that for the standard thiourea (IC₅₀ 21.01?±?0.51 μM). Compounds 1 and 3 also showed a significant activity, with IC₅₀ 37.95?±?1.93 and 138.43?±?1.23 μM, respectively. The sub crude fractions (F1, F2, F3, and F4) were tested in vitro for their urease inhibition activity. Fractions F2 and F4 showed significant activity with IC₅₀ 140.37?±?1.93 and 167.43?±?3.03 μM, respectively.     

SYNTHESIS OF CYCLIC DINUCLEOTIDES BY AN H-PHOSPHONATE METHOD IN SOLUTION

Abstract

We report synthesis of each of the ten cyclic 2′-deoxyribodinucleotides by a solution-phase H-phosphonate method. The cyclic dimers have been characterized by ³¹P NMR, MS, UV, and enzymatic degradation.     

Common Structural Features of UUCG and UACG Tetraloops in Very Short Hairpins Determined by UV Absorption,Raman, IR and NMR Spectroscopies

Abstract

Thermodynamic and structural properties of two UNCG tetraloops in very short hairpin octamers, 5′-r (GCUUCGGC)-3′ and 5′-r (GCUACGGC)-3′. have been studied by means of various physical techniques. Melting profiles of both octamers, obtained from UV absorption spectra taken as a function of temperature, are consistent with a monophasic, progressive and completely reversible order-to-disorder transition and confirm their unusual structural stability (T_m>51^° C). The ¹H, ¹³C and ³¹P NMR chemical shifts and coupling constants of the UACG loop nucleotides are comparable with those reported previously for UUCG loops, i.e. 2′-endo/anti conformation of the second and third nucleotide of the loop as well as the syn orientation of the ultimate guanine base and the A-type double helical conformation of the hairpin stem. Simulation of quantitative NOESY volumes shows that the UACG octamer adopts a very rigid compact structure which is well represented by an average order parameter of 0.9. Three base-pairs and four additional strong hydrogen bonds are undoubtedly responsible for such limited flexibility. Raman and infrared spectra as a function of temperature reflect the order-to-disorder transition, as well. Vibrational conformational markers in low temperature spectra of both octamers indicate the hairpin structure as the major conformer in aqueous phase. These spectra further support the structural features of most of the nucleotides involved in the tetraloops and clearly demonstrate the structural similarities of the phosphodiester backbone in both hairpins. Consequently, on the basis of all present results, one can deduce that the conformational features of the UUCG and UACG tetraloops seem to be inherent to the UNCG type tetraloops, regardless of either the nature of the tetraloop second base or the stem length.     

Cell-free Expressed Bacteriorhodopsin in Different Soluble Membrane Mimetics: Biophysical Properties and NMR Accessibility

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Highlights? Bacteriorhodopsin (bR) is NMR accessible in nanodiscs and amphipols ? Nonconventional surfactants offer increased stability for cell-free expressed bR ? The bR-membrane interface is highly adaptable in the selected membrane mimetics ? Applications and limitations of the environments for NMR studies are discussed     

Structural Characterization of Cyclic ADP-Ribose by NMR Spectroscopy

Abstract

Structure of cyclic adenosine diphosphoribose (cADPR) was reinvestigated by using ¹H, ¹³C, and ³¹P NMR spectroscopy. The ¹H-¹H coupling constants and NOE data suggested that the adenosine and ribose moieties have a predominant C2′-endo conformation and an unusual flat conformation, respectively.     

Influence of uracil defect on DNA structure: 1H NMR investigation at 500 MHz.

The local structure of two self complementary oligonucleotides d(GTAC-GTAC) and d(GTACGUAC) which differ only by the presence of uracil, not a normal component of DNA, have been investigated by 1H NMR at 500 MHz. The two octamers exhibit the same thermodynamical constants (t 1/2, delta H), their exchangeable protons broaden and disappear at the same temperature. The T-U substitution did not induce any significant changes on non exchangeable protons resonances from 2-D COSY and 2-D NOESY experiments. So the two octamers exhibit the same global structure. The only variation was detected by 1D NOE measurements: the base orientations around the N glycosidic bonds (chi angles) are different.     

Synthesis and Physical Characterization of Bis 3′→5′ Cyclic Dinucleotides (NpNp): RNA Polymerase Inhibitors

Abstract

A defined chemical synthesis of the cyclic dinucleotides, ApAp, ApUp, and UpUp has been devised, based on modern phosphotriester methods. These cyclic dinucleotides have been shown to inhibit RNA polymerase. The ¹H NMR spectra of the protected dimers show a preference for the ²E form, while the spectra of the unprotected dimers show the ³E form to be favored. The circular dichroism spectra show a negative long wavelength transition; however, ApUp does not show the short wavelength maximum present in the spectra of APAP and UpUp.     

Spectroscopic investigations on DNA binding profile of two new naphthyridine carboxamides and their application as turn-on fluorescent DNA staining probes

Abstract

Two new 10-methoxydibenzo[b,h][1,6]naphthyridine-2-carboxamide derivatives (R1 and R2) have been synthesized and characterized using different spectral techniques. The binding of these probes with DNA was investigated using spectral (Electronic, fluorescence, ¹H NMR and circular dichroism) and molecular docking studies. These probes exhibited a strong fluorescence around 440?nm upon excitation around 380?nm. Electronic and competitive fluorescence titration studies, in HEPES [(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)] buffer/dimethyl sulfoxide (pH 7.4) medium, suggest that these probes bind strongly to DNA, which is substantiated by ¹H NMR study. The binding constants are calculated to be 5.3?×?10⁷ and 6.8?×?10⁶ M^?1 for R1 and R2, respectively. From the results of spectral studies, it is proposed that the mechanism of binding of these probes with DNA is through minor groove binding mode, which is further confirmed by circular dichroism and molecular docking studies. Initial cell viability screening using MTT (3-[4,5-methylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay shows that normal Vero cells are viable towards these probes at nano molar concentration, which is the concentration range employed in the present study for DNA staining (IC₅₀ in the order of 0.023?mM). The enhancement in fluorescence intensity of these probes upon binding with DNA enables the staining of DNA in agarose gel in gel electrophoresis experiment. The sensitivity of these probes is comparable with that of ethidium bromide and DNA amounts as low as 4 nano gram are detectable.

Communicated by Ramaswamy H. Sarma