Studies of oligoadenylate formation on a poly(U) template

We have studied a variety of condensation reactions involving poly (U) as template and isomeric adenosine dinucleotides as substrates. We find that [3'-5']-linked dinucleotides such as A3pA and pA3pA are better acceptors than the corresponding [2'-5']-linked compounds, while ImpA2pA is a better donor than ImpA3pA. The reaction between A2pA and ImpA3pA, for example, yields only 4% of product while the reaction of A3pA with ImpA2pA yields 86% of product. The more efficient condensation reactions of dimers are about as efficient as the self-condensation of ImpA. They yield a few percent of material in which five or more substrate molecules are linked together. The percentage of the natural [3'-5']-linkage in the product varies greatly, from as little as 1% to as much as 45%.     

Acid-sensitive dissociation between poly(lysine) and histamine-modified poly(glutamate) as a model for drug-releasing from carriers in endosomes.

Histamine was coupled to poly(L-glutamate) (PLG) to give a copolymer, poly(glutamylhistamineglutamate) (PHG), with approx. 40% of carboxyl groups in PLG being modified. Unlike either poly(L-histidine) (PLH) or PLG, PHG precipitated only in buffers with pH between 4 and 5. A complex was formed between PHG and poly(L-lysine) (PLL) at pH 7, but it was rapidly dissociated at pH 5 or lower. When PHG-linked transferrin (Tf-PHG) was used to deliver a PLL-conjugated [3H]methotrexate ([3H]MTX-PLL) in K562 leukemia cell cultures, an intracellular accumulation of the radioactivity was observed. These results suggest that a copolymer with both imidazole and carboxyl groups can be useful in the design of acid-sensitive, carrier-mediated drug delivery systems.     

Synthesis and properties of oligonucleotides containing aminodeoxythymidine units.

Procedures are described for synthesis via solid support methodology of oligonucleotide analogues derived in part from 3'-amino-3'-deoxythymidine or 5'-amino-5'-deoxythymidine. Oligothymidylate decamers terminated with a 3'-amino group or containing a 3'-NHP(O)(O-)O-5' internucleoside link are found to form unusually stable complexes with poly(dA), poly(A), and oligo(dA). For related derivatives of 5'-amino-5'-deoxythymidine enhancement is less or absent, and in the case of multiple substitution destabilization of the heteroduplex may be observed. That the effect of the 3'-amino group is general for oligonucleotide derivatives is indicated by enhanced Tm values for heteroduplex complexes of the mixed-base oligomer, d(TATTCAGTCAT(NH2)), and the methyl phosphonate derivatives, TmTmTmTmTmTmTmTmTmT(NH2) and d(TmAmTmTmCmAmGmTmCmAmT(NH2)).     

Synthesis of amide-linked [(3')CH2CO-NH(5')] nucleoside analogues of small oligonucleotides

We report syntheses of new amide-linked (di-penta)nucleoside analogues of antisense oligonucleotide components. Solution-phase coupling of 3'-(carboxymethyl)-3'-deoxy- and 5'-amino-5'-deoxynucleoside derivatives provides amide dimers. Activated [3'-(carboxymethyl)-3'-deoxy] units with a 5'-azido-5'-deoxy function provide "masked" 5'-amino-5'-deoxy residues for chain extension, and a 5'-O-DMT-protected unit provides the 5'-terminus for attachment to a phosphodiester linkage.     

Nucleosteroids: carbocyclic nucleoside analogs of androst-4-en-17 beta-ol

Steroidal nucleoside analogs were synthesized starting from testosterone. By reduction of the oxime of 17 beta-hydroxy-androst-4-en-3-one (testosterone), a mixture of the two amino epimers of C-3 were obtained. The 3 alpha-amino-androst-4-en-17 beta-ol was crystallized in 73% yield and coupled with 5-amino-4,6-dichloropyrimidine to give 3 alpha-(5'-amino-4'-chloro-pyrimidin-6'-yl)amino-androst-4-en-17 beta-ol. This compound was treated with triethyl orthoformate in acid media to give the corresponding purinyl steroid adduct 3 alpha-(6'-chloro-purin-9'-yl)-androst-4-en-17 beta-ol in 98% yield. This substance, in turn, was converted with good yield into the 6'-thio, 6'-methylamino, and 6'-diethyl aminopurinyl derivatives through nucleophilic reactions at C-6 of the purine nucleus.     

Inhibition of poly(A) polymerase by rifamycin derivatives

The effect of several rifamycin derivatives on poly(A) synthesis in vitro was tested using purified rat liver mitochondrial poly(A) polymerase assayed with an exogenous primer. When used at a concentration of 300 μg/ml, derivatives AF/013, PR/19, AF/AETP, M/88 and AF/ABDP completely inhibited activity corresponding to 50 μg of enzyme protein. Under similar conditions, derivatives DMAO and AF/MO failed to inhibit enzyme activity. Studies with PR/19 showed that the drug interacted directly with the enzyme molecule and did not affect the enzyme-primer complex formation. The inhibition by the drug could be reversed by increasing the substrate (ATP) concentration. It is concluded that some rifamycin derivatives can specifically inhibit template-independent nucleotide chain elongation reactions.     

The 3'-amido and 5'-amido analogues of adenosine 3':5'-monophosphate; interaction with cAMP-specific proteins.

The sensitivity for recognition of adenosine 3:5'-monophosphate (cAMP) by its coordinate proteins towards chemical changes in the six-membered cyclic phosphate ring has been investigated. A comparison of the interaction parameters of the 3' and 5'-amido analogues (I, II) and of unsubstituted cAMP has been made using two different protein kinases and the phosphodiesterase from bovine heart. Binding affinity and the capacity of the amido analogues to stimulate the phosphotransferase activity of the kinases is greatly reeuced relative to cAMP, the 3'-position being more sensitive towards the modification than the 5'-position. The coordinate noncyclic derivatives, 3'-deoxy-3'-amino-5'-AMP (IV) and 5'-deoxy-5'-amino-3'-amp (iii), were also tested. Surprisingly activity towards protein kinases was found to be considerable for the 5'-deoxy-5'-amino-3'-AMP (III), while the 3'-deoxy-3'-amino-5'-AMP (IV) is practically inactive. A possible reason for this is that the noncylic 5'-analogue (III) may be able to assume a cyclic structure maintained by internal salt formation. The phosphodiesterase splits both cyclic amido analogues but with reduced rates compared to that of natural cAMP. Kinetic data obtained from different methods reveal a stronger affinity for the 5'-analogue (I) than the 3'-analogue (II) for the active site, although the reaction rate at saturated substrate concentration is significantly higher with II than with I. The properties of the amido and the noncyclic amino analogues are discussed with available data from chemotaxis of the cellular slime moulds. Furthermore data of the respective methylene cyclic derivatives are used for a more comprehensive comparison. The above is interpreted in terms of the electronic features of the substitutions and of the changes in bond distances or angles upon replacement of O by NH or CH2 in the cyclic phosphate ring (obtained from X-ray work).     

Condensation of activated diguanylates on a poly(C) template

We have studied the metal-ion catalysis of a number of reactions of the isomers of ImpGpG on a poly(C) template. In the absence of a catalytic metal ion, oligomers at least up to (pG)20 are obtained from the ImpGpG isomers in a 1-methylimidazole buffer. The Pb2+ ion improves the yield of longer oligomers and changes substantially the distribution of linkage isomers. The Pb2+ ion greatly improves the yield of longer oligomers obtained from G and ImpGpG on a poly(C) template. The self-condensation of ImpGpG in a 2, 6-lutidine buffer is much less efficient than in a 1-methylimidazole buffer. The Zn2+ greatly increases the yield of products from the [3'-5']-linked dimer, but fails to catalyze the formation of long oligomers from the [2'-5']-linked dimer. The bonds formed in the Zn2+-catalyzed self-condensation of ImpG3pG on poly(C) are mainly [3'-5']-linked.     

Gamma-ray crosslinking of poly(3-hydroxyoctanoate-co-undecenoate)

The gamma-ray crosslinking of films made of poly(3-hydroxyoctanoate) containing undecenoate moieties (up to 33 mole%) were studied. X-ray diffraction, thermal analysis, dynamic mechanical analysis (DMA), solid state nuclear magnetic resonance (NMR) spectroscopy and degree of crosslinking (swelling analysis) as a function of irradiation dose were evaluated for treatments in air or in N(2) atmosphere. After uncrosslinked material was isolated by CHCl(3) extraction, solid state NMR data suggested that only a small percentage of the double bonds took part in the formation of irradiation crosslinks. Crosslinking in N(2) was more efficient than in air and a 20 kGy dose was sufficient for optimal crosslinking. The X-ray diffraction patterns of the polymer films were unaffected by moderate irradiation. The use of sodium hypochlorite to isolate poly(3-hydroxyoctanoate-co-undecenoate) samples resulted in partial chlorination of the double bonds and considerable depolymerization.     

mRNA poly(A) tail, a 3'' enhancer of translational initiation.

To evaluate the hypothesis that the 3' poly(A) tract of mRNA plays a role in translational initiation, we constructed derivatives of pSP65 which direct the in vitro synthesis of mRNAs with different poly(A) tail lengths and compared, in reticulocyte extracts, the relative efficiencies with which such mRNAs were translated, degraded, recruited into polysomes, and assembled into messenger ribonucleoproteins or intermediates in the translational initiation pathway. Relative to mRNAs which were polyadenylated, we found that nonpolyadenylated [poly(A)-]mRNAs had a reduced translational capacity which was not due to an increase in their decay rates, but was attributable to a reduction in their efficiency of recruitment into polysomes. The defect in poly(A)- mRNAs affected a late step in translational initiation, was distinct from the phenotype associated with cap-deficient mRNAs, and resulted in a reduced ability to form 80S initiation complexes. Moreover, poly(A) added in trans inhibited translation from capped polyadenylated mRNAs but stimulated translation from capped poly(A)- mRNAs. We suggest that the presence of a 3' poly(A) tail may facilitate the binding of an initiation factor or ribosomal subunit at the mRNA 5' end.     

Synthesis and properties of a novel bridged nucleic acid analogue, 5'-amino-3',5'-BNA

An oligonucleotide P3'-->N5' phosphoramidate (5'-amino-DNA) attracts much attention because of its potential for application to DNA sequencing; however, its ability to hybridize with complementary strands is low. To overcome this drawback of the 5-amino-DNA, we have designed and successfully synthesized a novel nucleic acid analogue having a P3'-->N5' phosphoramidate linkage and a constrained sugar moiety, 5'-amino-3'-C,5'-N-methylene bridged nucleic acid (5'-amino-3',5'-BNA). The binding affinity of the 5'-amino-3',5'-BNA towards complementary DNA and RNA strands was investigated by UV melting experiments. The melting temperature (Tm) of the duplex comprising the 5'-amino-3',5'-BNA and its complementary strand was much higher than that of the duplex containing the corresponding 5-amino-DNA.     

Synthesis of thiophenecarboxamides, thieno[3,4-c]pyridin-4(5H)-ones and thieno[3,4-d]pyrimidin-4(3H)-ones and preliminary evaluation as inhibitors of poly(ADP-ribose)polymerase (PARP)

Inhibitors of poly(ADP-ribose)polymerase (PARP) inhibit repair of damaged DNA and thus potentiate radiotherapy and chemotherapy of cancer. Treatment of 3-cyanothiophene with potassium nitrate and concentrated sulphuric acid gave 5-nitrothiophene-3-carboxamide. 4-Nitrothiophene-2-carboxamide and 5-nitrothiophene-2-carboxamide were formed similarly from 2-cyanothiophene. Reduction with tin(II) chloride gave the corresponding aminothiophenecarboxamide salts which were isolated via their N-Cbz derivatives. Lithiation of 3,4-dibromothiophene at -116 degrees C and quenching with alkyl chloroformates gave 4-bromothiophene-3-carboxylates, which were hydrolysed to 4-bromothiophene-3-carboxylic acid. Hurtley reactions with the enolates of pentane-2,4-dione and of 1-phenylbutane-1,3-dione, followed by acyl cleavage, led to 4-(2-oxopropyl)thiophene-3-carboxylic acid and 4-phenacylthiophene-3-carboxylic acid, respectively. Condensation with ammonia in acetic acid gave 6-methyl- and 6-phenylthieno[3,4-c]pyridin-4-ones, which were selectively nitrated at the 1- and 7-positions or were dinitrated. Ethyl 4-acetamido- and 4-benzamido-thiophene-3-carboxylates were cyclised to 2-methyl- and 2-phenyl-thieno[3,4-d][1,3]oxazin-4-ones, respectively. Ring-opening with ammonia and recyclisation led to 2-substituted thieno[3,4-d]pyrimidin-4-ones. The aminothiophenecarboxamides are analogues of 3-aminobenzamide, a selective inhibitor of poly(ADP-ribose)polymerase (PARP); the thienopyridinones and the thienopyrimidinones are analogues of isoquinolin-1-ones and quinazolin-4-ones, respectively, which inhibit this enzyme. In preliminary assays, several thienopyridinones and thienopyrimidinones showed potent inhibitory activity against PARP.     

Monoclonal antibodies specific for poly(dG) X poly(dC) and poly(dG) X poly(dm5C)

Most duplex DNAs that are in the "B" conformation are not immunogenic. One important exception is poly(dG) X poly(dC), which produces a good immune response even though, by many criteria, it adopts a conventional right-handed helix. In order to investigate what features are being recognized, monoclonal antibodies were prepared against poly(dG) X poly(dC) and the related polymer poly(dG) X poly(dm5C). Jel 72, which is an immunoglobulin G, binds only to poly(dG) X poly(dC), while Jel 68, which is an immunoglobulin M, binds approximately 10-fold more strongly to poly(dG) X poly(dm5C) than to poly(dG) X poly(dC). For both antibodies, no significant interaction could be detected with any other synthetic DNA duplexes including poly[d(Gm5C)] X poly[d(Gm5C)] in both the "B" and "Z" forms, poly[d(Tm5Cm5C)] X poly[d(GGA)], and poly[d(TCC)] X poly[d(GGA)], poly(dI) X poly(dC), or poly(dI) X poly(dm5C). The binding to poly(dG) X poly(dC) was inhibited by ethidium and by disruption of the DNA duplex, confirming that the antibodies were not recognizing single-stranded or multistranded structures. Furthermore, Jel 68 binds significantly to phage XP-12 DNA, which contains only m5C residues and will precipitate this DNA in the absence of a second antibody. The results suggest that (dG)n X (dm5C)n sequences in natural DNA exist in recognizably distinct conformations.     

Oligomerization of (guanosine 5′-phosphor)-2-methylimidazolide on poly(C): An RNA polymerase model

(Guanosine 5′-phosphor)-2-methylimidazolide (2-MeImpG), unlike guanosine 5′-phosphorimidazolide (ImpG), undergoes an efficient, buffer-independent, template-directed oligomerization in the presence of poly(C) at pH values above 7.6. The reaction occurs in a Watson-Crick double helix and yields predominantly 3′-5′-linked oligomers up to the 50-mer in above 80% yield. Synthesis proceeds in the 5′ → 3′ direction and has high fidelity in the sense that nucleotides other than G are not incorporated significantly into oligomers. Under some conditions, oligomers corresponding to approximately one and two turns of the helix are obtained in higher yield than somewhat longer or somewhat shorter oligomers.In the protonated triple-helical structure formed below pH 7, the efficiency of the oligomerization is much lower. Oligomers up to about the 10-mer are obtained. The most abundant products are “capped” at the 5′ terminus with a GppG pyrophosphate group.     

Coordination property of poly(1-vinyl-2-methylimidazole)-heme complexes

Poly(1-vinyl-2-methylimidazole) and poly(1-vinyl-2-methylimidazole-co-1-vinylpyrrolidone) predominantly form five-coordinate heme complexes in aqueous solution. The apparent formation constants (K) of the heme complexes were estimated spectroscopically. The K values of the polymer-heme complexes were about 10² to 10³ times those of the corresponding monomeric ones. These large K values were canceled by adding poly(1-vinylpyrrolidone), alcohol, or dimethylformamide. The viscometric measurement of the polymer-heme solution showed that the polymer complex took a compact shape. These results indicate a hydrophobic interaction of heme with the polymer-ligand. Poly(1-vinyl-2-methylimidazole) could form the five-coordinate heme complex, even in the presence of a large amount of imidazole.     

Interactions of 2-methyladenines and poly(m2A) with poly(br5U)

Mixing curve experiments and melting curve analyses have shown that poly(m2A) forms complexes with poly(br5U) with stoichiometries of either 1:1 or 1:2 in high ionic strengths. CD spectra of poly(m2A).poly(br5U) and poly(m2A).2 poly(br5U) both resemble quite well to those of poly(A). poly(br5U) and poly(A).2poly(br5U), respectively. This suggests that the corresponding complexes are closely related in the structural details. Significant similarities of the CD spectra were observed for poly(m2A).2poly(br5U) and complexes between 2,9-dimethyladenine or 2-methyladenosine and poly(br5U) in the presence of spermine, indicating also the 1:2 stoichiometry. Thus, a methyl group at the position 2 of adenine ring is not necessarily hindering a formation of the Watson-Crick type base pairings.     

Chromomycin A3 binds to left-handed poly(dG-m5dC)

The interaction of chromomycin A3 (an antitumor antibiotic) with right-handed and left-handed polynucleotides has been studied by absorbance, fluorescence, circular dichroism, 31P-NMR and 1H-NMR techniques. Binding to either the B form of poly(dG-dC) or the Z form of poly(dG-m5dC) shifts the absorbance maximum to higher wavelength and enhances the fluorescence of the drug. Circular dichroic spectra of solutions containing various concentrations of chromomycin A3 and fixed concentrations of either B or Z polynucleotides show well defined isoelliptic points at similar wavelengths. At the isoelliptic point, the drug complex with B DNA exhibits positive ellipticity while with Z DNA it exhibits negative ellipticity. 31P-NMR spectra of the chromomycin A3 complex with the Z form of poly(dG-m5dC) demonstrate that the Z conformation is retained in the drug complex up to one molecule drug/four base pairs. At Mg2+ concentrations lower than that necessary to stabilize the left-handed conformation of poly(dG-m5dC) alone, 31P analysis shows that chromomycin A3 can bind simultaneously to both the B and Z conformations of poly(dG-m5dC), with no effect on the B-Z equilibrium. These data demonstrate that chromomycin A3 binds to left-handed poly(dG-m5dC) with retention of the left-handed conformation up to saturating drug concentrations.     

Hexammineruthenium (III) chloride: a highly efficient promoter of the B-DNA to Z-DNA transition of poly-(dG-m5dC).poly(dG-m5dC) and poly(dG-dC).poly(dG-dC)

The effects of Ru(NH3)(3+)6 on the conformation of poly(dG-m5dC).poly(dG-m5dC) and poly(dG-dC).poly(dG-dC) were studied by circular dichroism (CD) spectroscopy. Ru(NH3)(3+)6 at very low concentrations provokes the Z-DNA conformation in both polynucleotides. In the presence of 50 mM NaCl, the concentration of Ru(NH3)(3+)6 at the midpoint of B to Z transition of poly(dG-m5dC).poly(dG-m5dC) is 4 microM compared to 5 microM for Co(NH3)(3+)6. The half-lives of B to Z transition of poly(dG-m5dC).poly(dG-m5dC) in the presence of 10 microM Ru(NH3)(3+)6 and Co(NHG3)(3+)6 are at 23 and 30 min, respectively. The concentration of Ru(NH3)(3+)6 at the midpoint of B to Z transition of poly(dG-dC).poly(dG-dC) is 50 microM. These results demonstrate that Ru(NH3)(3+)6 is a highly efficient trivalent cation for the induction of B to Z transition in poly(dG-m5dC).poly(dG-m5dC) and poly(dG-dC).poly(dG-dC). In contrast, Ru(NH3)(3+)6 has no significant effect on the conformation of calf thymus DNA, poly(dA-dT).poly(dA-dT) and poly(dA-dC).poly(dG-dT).     

Helix-helix transitions in DNA: fibre X-ray study of the particular cases poly(dG-dC) · poly(dG-dC) and poly(dA) · 2poly(dT)

The helix-helix transitions which occur in poly(dG-dC) · poly(dG-dC) and in poly (dG-m⁵dC) · poly(dG-m⁵dC) are commonly assumed to be changes between the right-handed A- or B-DNA double helices and the left-handed Z-DNA structure. The mechanisms for such transconformations are highly improbable, especially when they are supposed to be active in long polynucleotide chains organised in semicrystalline fibres. The present alternative possibility assumes that rather than the Z-DNA it is a right-handed double helix (S-DNA) which actually takes part in these form transitions. Two molecular models of this S form, in good agreement with X-ray measurements, are proposed. They present alternating C(2′)-endo and C(3′)-endo sugar puckering like the “alternating B-DNA” put forward some years ago. Dihedral angles, sets of atomic coordinates and stereo views of the two S-DNA structures are given, together with curves of calculated diffracted intensities. Furthermore, we question the possibility of obtaining semicrystalline fibres with triple helices of poly(dA) · 2poly(dT) in a way which renders X-ray diffraction efficient. It is suggested that, up to now, only double helices of poly(dA) · poly(dT) can actually be observed by fibre X-ray diffraction measurements. Received: 30 March 1999 / Revised version: 30 June 1999 / Accepted: 30 June 1999     

Coordination of poly(1-vinylimidazole) by Hemin

It is shown that when hemin (Fe) coordinates imidazole groups (L) attached to a polymer such as poly(1-vinylimidazole) (PVI), then the experimentally observed equilibrium will correspond to the apparent equilibrium constant K_x = [FeL_x]/[Fe][L] and the plot of log ([FeL_x]/[Fe]) against log [L] will give a slope (n) of unity both when x = 1 and, provided both imidazole groups are attached to the same polymer molecule, when x = 2. In agreement with this, hemin reacts with PVI in dimethylsulfoxide at 25°C to give a single product that has a spectrum identical to that of the bisimidazole adduct and the equilibrium corresponds to n = 1.