Cytldlne Cyclic (3′, 5′) Monophosphate: Cyclic Nucleotide With a Non-Characteristic Ribose Ring Conformation

Abstract

Cytidine 3′,-5′-cyclic phosphate (cCMP) occurs in nature and has growth stimulatory activity on L-1210 cells. The initiation of cell growth by cCMP, under conditions where CAMP, cGMP and cUMP delay the onset of proliferation suggests that cCMP may play a regulatory role in the cell metabolism. It has been reported that in 3′,5′-cyclic nucleotides, the phosphate ring fused to the furanose ring resuicts the conformation of the furanose ring to the twist form C(3′) endo C(4′) exo (³T₄), in contrast to the C(2′) endo C(3′) endo (²T₃) and C(3′) endo C(2′) exo (³T₂) twist forms normally found in nucleotides and nucleosides. We have carried out an accurate crystal structure of cCMP and found that the furanose ring in cCMP has the C(3′) endo C(2′) exo conformation (³T₂), with a pseudo rotation amplitude (P) of 44° and phase angle τ_m of 12°. cCMP is in low anti conformation (X_CN = 15.4°) and O(5′) has the fixed g conformation. The phosphate ring is constrained to the chair conformation, as in other cyclic nucleotides. The two exocyclic P-O bond distances are short (1.489, 1.476Å) and the ring angle at N(3) is large (125.2°) suggesting that the molecule in the solid state is a zwitterion with a plus charge on N(3). The crystals are hydrated and highly unstable. The three water molecules are highly disordered in ten locations. The crystals of cCMP 3H₂O are hexagonal, a = 16.294(3), b = c = 11.099(4)Å, space group P6₁, final R value is 0.067 for 1620 reflections 230.     

The use of molecular dynamics simulations to evaluate the DNA sequence-selectivity of G–A cross-linking PBD–duocarmycin dimers

The pyrrolobenzodiazepine (PBD) and duocarmycin families are DNA-interactive agents that covalently bond to guanine (G) and adenine (A) bases, respectively, and that have been joined together to create synthetic dimers capable of cross-linking G–G, A–A, and G–A bases. Three G–A alkylating dimers have been reported in publications to date, with defined DNA-binding sites proposed for two of them. In this study we have used molecular dynamics simulations to elucidate preferred DNA-binding sites for the three published molecular types. For the PBD–CPI dimer UTA-6026 (1), our simulations correctly predicted its favoured binding site (i.e., 5′-C(G)AATTA-3′) as identified by DNA cleavage studies. However, for the PBD–CI molecule (‘Compound 11’, 3), we were unable to reconcile the results of our simulations with the reported preferred cross-linking sequence (5′-ATTTTCC(G)-3′). We found that the molecule is too short to span the five base pairs between the A and G bases as claimed, but should target instead a sequence such as 5′-ATTTC(G)-3′ with two less base pairs between the reacting G and A residues. Our simulation results for this hybrid dimer are also in accord with the very low interstrand cross-linking and in vitro cytotoxicity activities reported for it. Although a preferred cross-linking sequence was not reported for the third hybrid dimer (‘27eS’, 2), our simulations predict that it should span two base pairs between covalently reacting G and A bases (e.g., 5′-GTAT(A)-3′).     

Structures of two Seven-Membered Ring Pyrimidine Nucleoside Derivatives

Abstract

Crystal structure analyses of 1-(2, 3, 5-tri-0-benzoyl-β-D-ribofuranosyl)-tetrahydro-2H-1, 3-diazepine-2, 4(3H)-dione (I) and 1-(2, 3, 5-tri-0-benzoyl-β-D-ribofuranosyl)-6, 7-dihydro-2H-1, 3-diazepine-2(3H)-one (II) show that the major conformational differences between them lie in the aglycone moiety. In I the aglycone is twisted with C(6) and C(7) above, and C(5) below, the plane of the ring. In II the aglycone has a sofa conformation with C(7) above the plane of the ring. The ribose conformation in both compounds is C(1′)-exo-C(2′)-endo (²T₁), the C(4′)-C(5′) orientation is gauche-gauche, and the glycoside linkage is high-anti (X = -104.5(3)° I, -95.2(5)° II, respectively).     

NMR Investigations of the Solution Conformations of Deoxynucleotidyl (3′-5′) Arabinonucleosides

Abstract

The solution conformations of all eight deoxynucleotidyl (3′-5′) arabinonucleosides containing 9-B-D-arabinofuranosyladenine and 1-B-D-arabinonfuranosylcytosine have been analyzed by NMR methods and compared to dinucleoside monophosphates containing the corresponding deoxyriboside units.     

Crystal Structure and Molecular Conformation of 4-Thiouracil-2′-Trifluorothioacetamide -3′, 5′-Diacetyl-β-D-Riboside

Abstract

4-thiouracil-2′-trifluorothioacetamide-3′, 5′-diacetyl-β-D-riboside is one of the modified thiouracil analogs synthesized in our institute. The determination of the crystal and molecular structure of this compound was carried out with a view to study the conformation of the molecule in the solid state as well as to investigate the conformations of the trifluoroacetamide and the acetyl substituents of the ribose and their effects on the conformation of the ribose ring. Crystals of 4-thiouracil-2′-trifluorothioacetamide-3′,5′- diacetyl-β-D-riboside are orthorhombic, space group P2₁ 2₁ 2₁, with cell dimensions a= 15.351 (2), b= 15.535 (1), c= 8.307 (1) Å, V=1981.0 (7) ų, Z=4, D_m= 1.53, D_c=1.527 g/c.c. and μ=30.1cm -¹. The structure was determined using CuKα (λ, =1.5418 Å) at a temperature T of 297K, with 2333 reflections, which were collected on a Enraf-Nonius CAD-4 diffactometer, out of which 2249 (I ≥20) were considered observed. The structure was determined by direct methods using MULTAN and refined by full matrix least squares method to a final reliability factor of 0.054 and a weighted R factor of 0.079. The nucleoside is in the anti conformation [X_CN =51.4 (5)°], the ribose has the unusual C (2′) endo -C (1′) exo (²T₁), and a g+ conformation [ψ=47.5 (4)] across C(4′)-C(5′) bond. The pseudorotation angle P is 152.8 (4) ° and the amplitude of pucker τ_m of 42.7 (3)°. The average C-F bond distance is 1.308 Å. There is no base pairing and the typical base-base hydrogen bonded interactions are not present in this structure. On the other hand, a hydrogen bonded dimer is formed involving C(3′) - H(3′)… O (2) and N(3) -H (N3) … O (Al) hydrogen bonds joining the base, ribose ring and the acetyl group. The trend towards longer exocyclic bonds at the acetyl centers in compounds with strongly electronegative aglycones, is also exhibited in this compound, with C(3′)-O(3′) and C(5′)-0(5′) being much longer than C(1′)-O(4′). The acetyl groups also take part in C-H…O hydrogen bonding with the acetyl oxygen atom OA2.     

Crystal Structure of a Tightly Bound Inhibitor of Adenosine Transport N6-(4-Nitrobenzyl)-β-D-2′-Deoxyadenosine Methanol Solvate,C17H18N6O5.1/2 CH3OH

Abstract

The molecular structure of N⁶-(4-nitrobenzyl)-β-D-2′-deoxyadenosine (I) has been determined by single crystal X-ray diffraction. A potent inhibitor of adenosine permeation in cultured S49 mouse lymphoma cells, I binds tightly (K_D 2.4 nM) to high affinity membrane sites present on the nucleoside transporter elements of these cells. Compound I crystallizes in the trigonal space group P3₂21 with unit cell dimensions a = b = 8.0009(9)Å, c = 49.174(8)Å, and Z = 6. The structure was solved by direct methods and refined by least-squares to a final R = 0.038. The mean plane of the 4-nitrobenzyl group, an important substituent for potent nucleoside transport inhibition in a series of S6-substituted 6-thioinosine derivatives, is inclined at an angle of 120.6° to the plane of the adenine ring. The torsion angles around the methylene carbon atom of this benzyl group are C(6)-N(6)-C(10)-C(11), 96.6° and N(6)-C(10)-C(11)-C(12), 93.6°. The glycosidic torsion angle, X, is 217.1° which corresponds to the common anti nucleoside conformation. The deoxyribose ring, however, has the unusual C(1′)-exo conformation, with C(1′) displaced 0.608Å from the plane of C(2′), C(3′), C(4′) and O(4′). The conformation about the exocyclic C(4′)-C(5′) bond is gauche⁺.     

Conformation and Antiherpes Activity of 3′- and 5′-Azido and Amino Analogs of 5-Methoxymethyl-2′-Deoxyuridine

Abstract

The molecular conformations of 3′- and 5′-azido and amino derivatives of 5-methoxymethyl-2′-deoxyuridine, 1, were investigated by nmr. The glycosidic conformation of 5-methoxymethyl-5′-amino-2′,5′-dideoxy-uridine, 5 had a considerable population of the syn form. The 5′-derivatives show a preference for the S conformation of the furanose ring as in 1. In contrast, the 3′-derivatives show preference for the N conformation. For 5-methoxymethyl-3′-amino-2′,3′-dideoxyuridine, 3, the shift towards the N state is pH dependent. The preferred conformation for the exocyclic (C4′,C5′) side chain is g⁺ for all compounds except 5 which has a strong preference for the t rotamer (79%). Compounds 1, 3 and 5 inhibited growth of HSV-1 by 50% at 2, 18 and 70 μg/ml respectively, whereas 2 and 4 were not active up to 256 μg/ml (highest concentration tested). The compounds were not cytotoxic up to 3,000 μM.     

P1-(Thymidine 5′-)P1-amino-triphosphate: Synthesis,Hydrolysis and Reaction with Cytidine in Alkali1

Abstract

The title compound 1 is prepared from thymidine 5′-phos-phorodiamidate (2) and inorganic pyrophosphate (3) in anhydrous DMF, at 30–32°C. The products of alkaline hydrolysis of 1, at room temperature, are: thymidine 5′-phosphoramidate (4), thymidine 3′-phosphoramidate (8) and thymidine (9) as well as 3 and inorganic trimetaphosphate (10). In 1 N NH₄OH, 1 reacts with cytidine (15) to form cytidylyl-/2^T(3′)-5′/-thymidine (16) and a mixture of cytidine 2′,3′-cyclic phosphate (17) and 9.     

In vitro synthesis of a possible precursor RNA by purified vesicular stomatitis virus.

The RNA products synthesized $\text{in vitro}$ by the virion-associated RNA polymerase of purified vesicular stomatitis virus have previously been shown to contain two distinct 5′-terminal sequences. The mRNA species contain the blocked 5′-terminal G(5′)ppp(5′)A-A-C-A-G sequence and the initiated lead-in RNA segment (approximately 50 bases) contains the unblocked 5′ ppA-C-G sequence. In the present studies, using inosine 5′-triphosphate in place of GTP it is shown that RNA species as large as 14.5S contain an unblocked 5′-ppA-C-(I) sequence indicating that the GTP analogue permits synthesis of a possible precursor of viral mRNA $\text{in vitro}$.     

Nucleotide recognition seouence at the cleavage site of Haemophilus aegyptius II (Hae II) restriction endonuclease

We have determined the nucleotide sequence recognized by the restriction endonuclease Hae II from Haemophilus aegyptius which cleaves the simian virus 40 (SV40) DNA at a single specific site. By using terminal radioactive labeling of the cleavage site at both the 5′ and 3′-ends we have deduced the recognition sequence,

with elements of a two-fold rotational symmetry. The endonuclease produces staggered ends with protruding 3′-terminated single-strands, four nucleotides in length. In plasmid RSF 2124 DNA, which contains multiple Hae II cleavage sites, it was observed that the 5th nucleotide from the 3′ terminus is either a pdA or a pdG, indicating alternating recognition sequences.     

Structure and Conformation of 1-β-D-Ribo Furanosyl Pyridin-2-one-5-Carboxamide: An Anti-Inflammatory Agent

The pyrimidine nucleoside, 1-β-D-ribofuranosyl pyridine-2-one-5-carboxamide, is an anti inflammatory agent used in the treatment of adjuvant-induced arthritis. It is the 2-one isomer of 1-β-D-ribofuranosyl pyridine-4-one 5-carboxamide, an unusual nucleoside isolated from the urine of patients with chronic myelogenic leukemia and an important cancer marker. Crystals of 1-β-D-ribofuranosyl pyridine-2-one-5-carboxamide are monoclinic, space group C2, with the cell dimensions a = 31.7920(13), b = 4.6872 (3), c = 16.1838(11), β = 93.071(3)°, V = 2408.2(2) ų, D_calc = 1.496 mg/m³ and Z = 8 (two molecules in the asymmetric unit). The structure was obtained by the application of direct methods to diffractometric data and refined to a final R value of 0.050 for 1669 reflections with I ≥ 3σ. The nucleoside exhibits an anti conformation across the glycosidic bond (χ_CN = ?15.5°, ?18.9°), a C3 ′- endo C2 ′ -exo [³ ₂T] ribose pucker and g⁺ across the C(4 ′)-C(5 ′) exocyclic bond. The amino group of the carboxamide group is distal from the 2-one and lacks the intramolecular hydrogen bonding found in the related 2-one molecule. Nuclear magnetic resonance studies shows also an anti conformation across the glycosidic bond but the solution conformation of the furanose ring is not the same as that found in the solid state.     

Interaction of wheat protein synthesis elongation factor 1 with GTP analogs

Wheat protein synthesis elongation factor 1 was tested for binding to GTP analogs, including structures resembling “caps” that are present at the 5′-termini of most eukaryotic mRNAs. The interaction was assayed by determining the capacity of the analogs to inhibit the binding of [³H]GTP to elongation factor 1. Significant interaction of elongation factor 1 with G(5′)ppp(5′)G, G(5′)pppp(5′)G, and G(5′)ppp(5′)A was observed. Methylation of a ribose 2′-hydroxyl had very little effect, but methylation of the 7 position of guanosine greatly diminished the affinity of elongation factor 1 for these compounds. m⁷G(5′)ppp(5′)Cm, m⁷G(5′)ppp(5′)Um, and m⁷G(5′)ppp(5′)Am gave no detectable binding with EF1.     

Effect of N-terminal deletion of signal peptide on the secretion of human granulocyte-colony stimulating factor in mammalian cells

Summary The nine N-terminal amino acids of signal peptide differ between human and mouse granulocyte-colony stimulating factors (G-CSF). To study the function of this non-conserved sequence, cDNA lacking the sequence has been constructedin vitro. The deleted signal peptide promoted the secretion of hG-CSF in CHO cells.     

Correlation between the backbone and side chain conformations in 5′-nucleotides. The concept of a ‘rigid’ nucleotide conformation

Conformational energies of the 5′-adenosine monophosphate have been computed as a function of χ and ψ, of the torsion angles about the side-chain glycosyl C(1′)–N(9) and of the main-chain exocyclic C(4′)–C(5′) bonds by considering nonbonded, torsion, and electrostatic interactions. The two primary modes of sugar puckering, namely, C(2′)-endo and C(3′)-endo have been considered. The results indicate that there is a striking correlation between the conformations about the side-chain glyocsyl bond and the backbone C(4′)–C(5′) bond of the nucleotide unit. It is found that the anti and the Gauche–Gauche (gg), conformations about the glycosyl and the C(4′)–C(5′) bonds, respectively, are energetically the most favored conformations for 5′-adenine nucleotide irrespective of whether the puckering of the ribose is C(2′)-endo or C(3′)-endo. Calculations have also shown that the other common 5′-pyrimidine nucleotides will show similar preferences for the glycosyl and C(4′)–C(5′) bond conformations. These results are in remarkable agreement with the concept of the “rigid” nucleotide unit that has been developed from available data on mononucleotides and dinucleoside monophosphates. It is found that the conformational ‘rigidity’ in 5′-nucleotides compared with that of nucleosides is a consequence of, predominantly, the coulombic interactions between the negatively charged phosphate group and the base. The above result permits one to consider polynucleotide conformations in terms of a “rigid” C(2′)-endo or C(3′)-endo nucleotide unit with the major conformational changes being brought about by rotations about the P–O bonds linking the internucleotide phosphorus atom. IT is predicted that the anti and the gg conformations about the glycosyl and the C(4′)–C(5′) bonds would be strongly preferred in the mononucleotide components of different purine and pyrimidine coenzymes and also in the nucleotide phosphates like adenodine di- and triphosphates.     

Design,synthesis, and DNA binding characteristics of a group of orthogonally positioned diamino,N-formamido,pyrrole- and imidazole-containing polyamides

Orthogonally positioned diamino/dicationic polyamides (PAs) have good water solubility and enhanced binding affinity, whilst retaining DNA minor groove and sequence specificity compared to their monoamino/monocationic counterparts. The synthesis and DNA binding properties of the following diamino PAs: f-IPI (3a), f-IPP (4), f-PIP (5), and f-PPP (6) are described. P denotes the site where a 1-propylamino group is attached to the N1-position of the heterocycle. Binding of the diamino PAs to DNA was assessed by DNase I footprinting, thermal denaturation, circular dichroism titration, biosensor surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC) studies. According to SPR studies, f-IPI (3a) bound more strongly (K_eq = 2.4 × 10⁸ M^?1) and with comparable sequence selectivity to its cognate sequence 5′-ACGCGT-3′ when compared to its monoamino analog f-IPI (1). The binding of f-IPI (3a) to 5′-ACGCGT-3′ via the stacked dimer motif was balanced between enthalpy and entropy, and that was quite different from the enthalpy-driven binding of its monoamino parent f-IPI (1). f-IPP (4) also bound more strongly to its cognate sequence 5′-ATGCAT-3′ (K_eq = 7.4 × 10⁶ M^?1) via the side-by-side stacked motif than its monoamino analog f-IPP (2a). Although f-PPP (6) bound via a 1:1 motif, it bound strongly to its cognate sequence 5′-AAATTT-3′ (K_eq = 4.8 × 10⁷ M^?1), 15-times higher than the binding of its monoamino analog f-PPP (2c), albeit f-PPP bound via the stacked motif. Finally, f-PIP (5) bound to its target sequence 5′-ATCGAT-3′ as a stacked dimer and it has the lowest affinity among the diamino PAs tested (K_eq <1 × 10⁵ M^?1). This was about two times lower in affinity than the binding of its monoamino analog f-PIP (2b). The results further demonstrated that the ‘core rules’ of DNA recognition by monoamino PAs also apply to their diamino analogs. Specifically, PAs that contain a stacked IP core structure bind most strongly (highest binding constants) to their cognate GC doublet, followed by the binding of PAs with a stacked PP structure to two degenerate AT base pairs, and finally the binding of PAs with a PI core to their cognate CG doublet.     

Dynamic Bis-Intercalation of a Homodimeric Thiazole Orange Dye in DNA: Evidence of Intercalator Creeping

Abstract

We have used one and two dimensional exchange ¹H NMR spectroscopy to characterize the dynamics of the binding of a homodimeric thiazole orange dye, 1,1′-(4,4,8,8-tetramethyl-4,8-diaza-undecamethylene)-bis-4-(3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)-quinolinium tetraiodide (TOTO), to double stranded DNA (dsDNA). The double stranded oligonucleotides used were d-(CGCTAGCG)₂ ( 1 ) and d(CGCTAGCTAGCG)₂ ( 2 ). TOTO binds preferentially to the (5′-CTAG-3′)₂ sites and forms mixtures of 1:1 and 1:2 dsDNA-TOTO complexes with 2 in ratios dependent on the relative amount of TOTO and the oligonucleotide in the sample. The dynamic exchange between preferential binding sites in the case of a 2:1 1 -TOTO mixture is an intermolecular exchange process between two binding sites on different oligonucleotides. In the case of the 1:1 2 -TOTO complex an intramolecular exchange process occur between two different binding sites on the same strand. Both processes were studied. The results demonstrate the ability of TOTO to migrate along a dsDNA strand in an intramolecular exchange process. The migration process (“creeping”) along the DNA strand is 6 times faster than the rate of intermolecular exchange between sites in two different oligonucleotides.     

Sequence of tRNAIAUIle from brewer's yeast

The nucleotide sequence of tRNA^Ile from brewer's yeast Sacharomyces cerevisiae was determined. Its primary structure is pG-G-U-C-U-C-U-U-m¹G-m²G- C-C-C-A-G-D-D-G-G-D-D-A-A-G-G-C-A-C-C-G-U-G-C-U-I-A-U-t⁶A-A-C-G-C-G-G-G-G-A- D-m⁵C-A-G-C-G-G-T-ψ-C-G-m¹A-U-C-C-C-G-C-U-A-G-A-G-A-C-C-A-C-C-A. Its anticodon is I-A-U. It should therefore recognize the three isoleucine codons and is for this reason probably the only isoacceptor tRNA for isoleucine in brewer's yeast. It presents a large homology with its counterpart from Torulopsis utilis (87%).