Synthesis of 4-triazolopyrimidinone nucleotide and its application in synthesis of 5-methylcytosine-containing oligodeoxyribonucleotides.

5'-0-Dimethoxytritylthymidine (2) was phosphorylated and base-modified simultaneously to yield the 4-triazolopyrimidinone nucleotide (3). Coupling between (3) and other common deoxyribonucleotides gave a fully protected nonamer (4). Deblocking under different conditions yielded the nonamer as phosphodiester with concomitant conversion of 4-triazolopyrimidinone to 5-methylcytosine (aqueous ammonia) or thymine (N1,N1,N3,N3-tetramethyl-guanidinium syn-4-nitrobenzaldoximate solution).     

Preparation and photophysical properties of a caged kynurenine

We have prepared l-kyurenine 4-hydroxyphenacyl ester, a caged derivative of L-kynurenine. N(α)-tBOC-L-tryptophan was reacted with 4-hydroxyphenacyl bromide in DMF with K(2)CO(3) as the base to give the N(α)-tBOC 4-hydroxyphenacyl ester. The ester was then treated with O(3) in MeOH at -20°C, followed by trifluoroacetic acid in CH(2)Cl(2), then aqueous HCl to obtain the caged kynurenine as the dihydrochloride salt. The caged kynurenine is stable as a dry solid in the dark at -78°C, but in aqueous solutions in phosphate buffer at pH 7-8 hydrolyzes rapidly (t(1/2) ～5 min). Solutions in Tris at pH 7 are more stable (t(1/2) >30 min), and solutions in 1mM HCl are stable for several hours. As expected, the ester is cleaved in microseconds with laser pulses at 355 nm. The caged kynurenine may be useful for preparation of substrate complexes for crystallography or in biological studies on kynurenine.     

Use of maleimide-thiol coupling chemistry for efficient syntheses of oligonucleotide-enzyme conjugate hybridization probes

Two general methods which exploit the reactivity of sulfhydryl groups toward maleimides are described for the synthesis of oligonucleotide-enzyme conjugates for use as nonradioisotopic hybridization probes. In the first approach, 6-maleimidohexanoic acid succinimido ester was used to couple 5'-thiolated oligonucleotide to calf intestine alkaline phosphatase to provide a 1:1 conjugate in 80-85% yield. The second strategy employed N,N'-1,2-phenylenedimaleimide to cross-link thiolated horseradish peroxidase or beta-galactosidase with a 5'-thiolated oligonucleotide in 58% and 65% yields, respectively. The oligonucleotide-alkaline phosphatase conjugate was able to detect 6 amol of target DNA in 4 h, while the horseradish peroxidase conjugate was found to be 40-fold lower in its sensitivity of detection by using dye precipitation assays.     

An approach to the total synthesis of sinefungin.

Condensation of N6-benzoyl-2',3'-O-isopropylideneadenosine-5'-aldehyde with nitromethane followed by acid catalyzed acetylation and borohydride reduction leads to N6-benzoyl-9-(5,6-dideoxy-2,3-O-isopropylidene-6-nitro-beta-D-ribo-hexofuranosyl)adenine (4). A second nitroaldol condensation between 4 and N-benzyloxycarbonly-L-aspartic acid-beta-semialdehyde alpha-benzyl ester (5) followed by acetylation and borohydride reduction leads to a fully protected 6'-nitro modification of sinefungin and its C6'-epimer (7). Hydrolysis of the acetonide followed by sequential reduction of the benzyl derived protecting groups and the nitro group and debenzoylation leads to a modest yield of a 3:1 mixture of sinefungin (1) and 6'-episinefungin which can only be separated by analytical ion exchange chromatography.     

Ligation with T4 RNA ligase of an oligodeoxyribonucleotide to covalently-linked cross-sectional base-pair analogues of short, normal, and long dimensions.

Compounds that are covalent analogues of nucleic acid base pairs of normal, long, and short C1' to C1' dimensions [B. Devadas and N.J. Leonard (1990) J. Am. Chem. Soc., 112, 3125-3135.] have been added to the oligodeoxyribonucleotide d(A)6 with bacteriophage T4 RNA ligase as a prelude to placing them at defined loci within nucleic acid duplexes. Analogue cross sections that represent a normal Watson-Crick base pair as well as a pyrimidine-pyrimidine and a purine-purine apposition were ligated in modest yields (approximately 20%) to the oligonucleotide. Ligation conditions were optimized for each analogue, and the cross section was joined to only a single oligonucleotide in each case. The structures of the ligated products were proved by HPLC, enzymatic degradation, and spectroscopic analyses.     

Study of the structure of the duplex (Phn-NH(CH2)2NH)pd(CCAAACA) .pd(TGTTTGGC) with covalently bound 10-(2-hydroxyethyl)phenazine in an aqueous solution by 2D-1H-NMR spectroscopy]

The spatial structure of duplex (Phn-NH(CH2)2NH)pd(CCAAACA).pd(TGTTTGGC) having a N-(2-oxyethyl)-phenazinium residue covalently linked with the 5'-terminal phosphate of the heptanucleotide was studied by means of one- and two-dimensional 1H-NMR spectroscopy. The resonances of phenazinium protons, ethylenediamine linker protons, as well as, oligonucleotide H5/H6/H8/CH3 base protons and H1',H2'a, H2'b, H3', H4' deoxyribose protons have been assigned by means of 1H-COSY, 1H-NOESY and 1H-13C-COSY. The presence of the phenazine residue in duplex causes an additional imino proton signal of the terminal (G-7).(C-1) base pair, suggesting a higher stability of the duplex (Phn-NH(CH2)2NH)pd(CCAAACA).pd(TGTTTGGC) as compared to the unmodified duplex pd(CCAAACA).pd(TGTTTGGC). Analysis of NOE interactions between protons of the dye and the oligonucleotides show the phenazinium polycyclic system to intercalate between G-7 and C-8 residues of the octanucleotide.     

Preparation and characterization of oligonucleotides containing S-[2-(N7-guanyl)ethyl]glutathione.

S-[2-(N7-Guanyl)ethyl]glutathione is the major adduct derived from modification of DNA with 1,2-dibromoethane in biological systems and is postulated to be a mutagenic lesion [Humphreys, W. G., Kim, D.-H., Cmarik, J. L., Shimada, T., & Guengerich, F. P. (1990) Biochemistry 29, 10342-10350]. Oligonucleotides containing this modified base were prepared by treatment of oligonucleotides with S-(2-chloroethyl)glutathione and purified by chromatography. The self-complementary oligonucleotide d(ATGCAT), when thus modified at the single guanine, appeared to associate with itself as judged by UV measurements, but CD and NMR measurements indicated a lack of hybridization, with a decrease in the melting temperature of greater than 10 degrees C. The same lack of self-association was noted when d(ATGCAT) was modified to contain an N-acetyl-S-[2-(N7-guanyl)ethyl]cysteine methyl ester moiety. The oligomer d-(C1A2T3G4C5C6T7) was modified to contain a single S-[2-(N7-guanyl)ethyl]glutathione moiety at the central position, and UV, CD, and 1H NMR studies indicated that this oligomer hybridized to its normal complement d(A8G9G10C11A12T13G14), although the binding was considerably weakened by adduction (imino proton NMR spectroscopy in the presence of H2O indicated that the hydrogen bond signals seen in the oligomer were all broadened upon modification). All proton resonances were identified using two-dimensional 1H NMR spectroscopy. Adduct formation affected the chemical shifts of the base and 1', 2', and 2" protons of T3 and C5, the 2" proton of C6, and the 8 and 1' protons of C11, while little effect was observed on other protons. No cross-peaks were detected between the glutathione and oligomer moieties in two-dimensional nuclear Overhauser enhanced NMR studies. These results suggest that a rather local structural perturbation occurs in the DNA oligomer upon modification and that the glutathione moiety appears to be relatively unperturbed by its placement in the duplex. When the cytosine in the normal d(AGGCATG) complement to d-(CATGCCT) was changed to each of the other three potential bases at the central position, no hybridization with the oligomer d(CATGCCT) containing S-[2-(N7-guanyl)ethyl]glutathione was detected. We conclude that these N7-guanyl derivatives destabilize hybridization and that bases other than cytosine do not appear to show preferential thermodynamic bonding to these adducts, at least in the sequences examined to date.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synthesis and base pairing properties of 9-deazapurine N7-nucleosides in oligonucleotide duplexes and triplexes

The 9-deazaguanine N7-2'-deoxyribofuranoside (3) as well as the bromo and iodo derivatives 4a,b were synthesized and incorporated in oligonucleotide duplexes and triplexes. Their base pairing properties were investigated and compared with those of the parent purine N7-2'-deoxyribofuanosides.     

Synthesis of 3',5'-cyclic diguanylic acid (cdiGMP) using 1-(4-chlorophenyl)-4-ethoxypiperidin-4-yl as a protecting group for 2'-hydroxy functions of ribonucleosides

We herein report a convenient synthesis of 3',5'-cyclic diguanylic acid via the modified H-phosphonate approach. The 1-(4-chlorophenyl)-4-ethoxypiperidin-4-yl (Cpep) group was used as protecting group for the 2'-hydroxy functions of ribonucleosides. Complete unblocking of the fully protected 3',5'-cyclic diguanylic acid gave cdiGMP as a homogeneous compound in an excellent yield.     

Hydrolysis of 5',5'-tri- or tetraphosphate-mRNA 5'-cap analogs promoted by Cu2+ or Zn2+ metal ions

Kinetics of the hydrolysis of a P(1)-(7-methylguanosinyl-5') P(3)-(guanosinyl-5') triphosphate (m(7)GpppG), P(1)-(7-methylguanosinyl-5') P(4)- (guanosinyl-5') tetraphosphate (m(7)GppppG), diadenosine-5',5'-P(1),P(3)-triphosphate (ApppA), and diadenosine-5',5'-P(1),P(4)-tetraphosphate (AppppA) promoted by Cu(2+) or Zn(2+) has been investigated. Time-dependent products distributions at various metal ion concentrations have been determined by CZE and HPLC-RP. The results show that in acidic conditions, in the presence of metal ion, the predominant hydrolytic reaction is the cleavage of 5',5'-oligophosphate bridge. The 5',5'-oligophosphate bridge of the dinucleotides studied is hydrolyzed by Cu(2+) more efficiently than by Zn(2+). At the catalyst concentration of 2 mM the cleavage of the 5',5'-triphosphate bridge of m(7)GpppG was ～3.6 times faster, and that of the tetraphosphate bridge of m(7)GppppG ～2.3-fold faster in the presence of Cu(2+) compared to the Zn(2+) ion, applied as catalysts. Dependence of the rates of hydrolysis on the catalyst concentration was in some instances not linear, interpreted as evidence for participation of more than one metal ion in the transition complex.     

Endowing RNase H-inactive antisense with catalytic activity: 2-5A-morphants

A convergent synthetic approach was used to conjugate 2',5'-oligoadenylate (2-5A, p5'A2' [p5'A2'](n)()p5'A) to phosphorodiamidate morpholino oligomers (morphants). To provide requisite quantities of 2-5A starting material, commercially and readily available synthons for solid-phase synthesis were adapted for larger scale solution synthesis. Thus, the tetranucleotide 5'-phosphoryladenylyl(2'-->5')adenylyl(2'-->5')adenylyl(2'-->5')adenosine (p5'A2'p5'A2'](2)p5'A2', tetramer 2-5A, 9) was synthesized starting with 2',3'-O-dibenzoyl-N(6),N(6)-dibenzoyl adenosine prepared from commercially available 5'-O-(4-monomethoxytrityl) adenosine. Coupling with N(6)-benzoyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-(tert-butyldimethylsilyl) adenosine-2'-(N,N-diisopropyl-2-cyanoethyl)phosphoramidite, followed by oxidization and deprotection, generated 5'-deprotected dimer 2-5A. Similar procedures lengthened the chain to form protected tetramer 2-5 A. The title product 9 p5'A(2'p5'A)(3) (tetramer 2-5A) was obtained through phosphorylation of the terminal 5'-hydroxy of the protected tetramer and removal of remaining protecting groups using concentrated ammonium hydroxide-ethanol (3:1, v/v) at 55 degrees C and tetrabutylammonium fluoride (TBAF) in THF at room temperature, respectively. The 2-5A-phosphorodiamidate morpholino antisense chimera 11 (2-5A-morphant) was synthesized by covalently linking an aminolinker-functionalized phosphorodiamidate morpholino oligomer with periodate oxidized 2-5A tetramer (p5'A2'[p5'A2'](2)p5'A). The resulting Schiff base was reduced with cyanoborohydride thereby transforming the ribose of the 2'-terminal nucleotide of 2-5A N-substituted morpholine. RNase L assays demonstrated that this novel 2-5A-antisense chimera had significant biological activity, thereby providing another potential tool for RNA ablation.     

Conformation and dynamics of drug-DNA intercalation.

Molecular dynamics simulations have been undertaken for a B-form dodecanucleotide duplex in solution with and without an intercalated proflavine molecule between the central C.G base pairs. The introduction of this simple intercalator affects both the conformational features and dynamic properties of the oligonucleotide double helix. Changes are seen in the rms atomic fluctuations and anisotropy of phosphate, sugar and base atoms. The backbone conformation is slightly changed on average and more sugars adopt the C3' endo conformation in the simulation of the complex compared with the simulation of the oligonucleotide alone. Both major and minor grooves becomes wider on average with the addition of the intercalating drug. Flanking A.T base pairs on both sides of the intercalation site have undergone an increase in flexibility, with the base pairs, especially at the 5' side, having the N1...N3 hydrogen bonds being broken.     

Oxidative DNA cleavage by free and oligonucleotide-conjugated O-bromobenzoic acid in the presence of copper(II) ions

o-Bromobenzoic acid was found to promote copper-dependent reactive oxygen species formation from molecular oxygen, resulting in DNA base modification and backbone cleavage. The oligonucleotide conjugate bearing 5-(4'-aminopropyl-sulfomoyl)-2-bromobenzoic acid as a reactive group was synthesized and DNA cleavage activity of this oligonucleotide conjugate was tested on a model deoxyoligonucleotide.     

Early steps in the path of nascent ribonucleic acid across the surface of ribonucleic acid polymerase, determined by photoaffinity labeling

The photoaffinity probes beta-(4-azidophenyl) adenosine 5'-diphosphate (N3PhppA) and beta-(4-azidophenyl) adenylyl-(3'--5')-uridine 5'-diphosphate (N3PhppApU) were used to determine the RNA polymerase subunit contacts made by the 5' ends of three nascent RNA chains. Ternary enzyme-poly[d(A-T)].oligonucleotide complexes were prepared in which the nascent oligonucleotide contained a photoaffinity label at the 5' end and a 32P radiolabel only at the 3' end. The length of the RNA was fixed at two, three, or four nucleotides. Photolysis of the ternary complexes was followed by dissociation, polyacrylamide gel electrophoresis, autoradiography, and scintillation counting. With a dinucleotide probe, the enzyme subunits labeled were beta' (71%) and sigma (21%). Photolysis of the ternary complex containing trinucleotide RNA also resulted in labeling of the beta' (64%) and sigma (35%) subunits. With a tetranucleotide, the beta' subunit was very heavily labeled (88%), and a small amount of labeling of the beta (7%) and sigma (4%) subunits was observed. The alpha subunit was not labeled with any of the probes. These results imply that a conformational change, possibly involving dissociation of the sigma subunit, occurs in the enzyme as the ribonucleotide is elongated from a tri- to a tetranucleotide.     

Oligodeoxynucleotide-directed photo-induced cross-linking of HIV proviral DNA via triple-helix formation.

The HIV proviral genome contains two copies of a 16 bp homopurine.homopyrimidine sequence which overlaps the recognition and cleavage site of the Dra I restriction enzyme. Psoralen was attached to the 16-mer homopyrimidine oligonucleotide, d5'(TTTTCT-TTTCCCCCCT)3', which forms a triple helix with this HIV proviral sequence. Two plasmids, containing part of the HIV proviral DNA, with either one (pLTR) or two (pBT1) copies of the 16-bp homopurine.homopyrimidine sequence and either 4 or 14 Dra I cleavage sites, respectively, were used as substrates for the psoralen-oligonucleotide conjugate. Following UV irradiation the two strands of the DNA targeted sequence were cross-linked at the triplex-duplex junction. The psoralen-oligonucleotide conjugate selectively inhibited Dra I enzymatic cleavage at sites overlapping the two triple helix-forming sequences. A secondary triplex-forming site of 8 contiguous base pairs was observed on the pBT1 plasmid when binding of the 16 base-long oligonucleotide was allowed to take place at high oligonucleotide concentrations. Replacement of a stretch of six cytosines in the 16-mer oligomer by a stretch of six guanines increased binding to the primary sites and abolished binding to the secondary site under physiological conditions. These results demonstrate that oligonucleotides can be designed to selectively recognize and modify specific sequences in HIV proviral DNA.     

Chemical reactions in double-stranded nucleic acids. The nature of the bond formed during chemical ligation using a cis-diol group

Chemical and enzymatic ligation between the 5'-terminal phosphate of one oligonucleotide and the 3'-terminal 2',3'-cis-diol group of the other oligonucleotide on a complementary template was studied. Carbodiimide, imidazolide and N-hydroxybenzotriazole ester methods were used for chemical activation of the phosphate group, and T4 DNA ligase for enzymatic ligation. All the chemical activation methods produced 3',5'- and 2',5'-phosphodiester bonds (40-45 and 55-60%, resp.), whereas enzymatic ligation gave the product only with 3',5'-phosphodiester bond.     

Enhancing the methyl-donor activity of methylcobalamin by covalent attachment of DNA

The preparation of a covalent DNA conjugate of vitamin B12 by means of heterogeneous solid-phase synthesis is reported. The cyano-corrinoid made available, dipotassium Co(beta)-cyanocobalamin-(3'-->2'),(3'-->5')-bis-2'-deoxythymidyl-3'-ate (K(2)-4), was cleanly methylated at the Co center by electrosynthetic means. Aqueous solutions of the resulting organometallic DNA-B12 conjugate K(2)-5 exhibited spectroscopic properties indicative of significant weakening of the axial (Co-N) bond, together with a 25-times higher basicity relative to Co(beta)-methylcobalamin (2). Methyl-transfer equilibria of pH-neutral aqueous solutions of K(2)-5 and cob(I)alamin (K-7) on one side, and of cob(I)alamin-(3'-->2'),(3'-->5')-bis-2'-deoxythymidyl-3'-ate (K(3)-8) and methylcobalamin (2) on the other, were studied at room temperature (Scheme 3). The NMR-derived data provided an equilibrium constant of ca. 0.3. Activation of K(2)-5 for abstraction of its Co-bound Me group by a nucleophile (such as cob(I)alamin) was, thus, indicated.     

DNA structure in which an adenine-cytosine mismatch pair forms an integral part of the double helix

Extensive studies using one- and two-dimensional 1H NMR at 500 MHz revealed that the oligonucleotide d(CGCCGCAGC) in solution at 5 degrees C forms a double helix under conditions of high salt (500 mM in NaCl, 1 mM sodium phosphate), low pH (pH 4.5), and high DNA concentration (4 mM in duplex). The presence of very strong nuclear Overhauser effects (NOEs) from base H8/H6 to sugar H2',H2" and the absence of NOE from base H8/H6 to sugar H3' suggested that the oligomer under these solution conditions forms a right-handed B-DNA double helix. The following lines of experimental evidence were used to conclude that C4 and A7 form an integral part of the duplex: (i) the presence of a NOESY cross-peak involving H8 of A7 and H8 of G8, (ii) the presence of a two-dimensional NOE (NOESY) cross-peak between H6 of C3 and H6 of C4, (iii) base protons belonging to C4 and A7 forming a part of the H8/H6---H1' cross-connectivity route, and (iv) the pattern of H8/H6---H2',H2" NOESY cross-connectivity based upon a B-DNA model requiring that both C4 and A7 form an integral part of the duplex. The possibility of an A-C pair involving H bonds was also examined. Two possible structural models of the duplex at pH 4.5 are proposed: in one model A-C pairing involves two H bonds, and in the other A-C pairing involves a single H bond.     

Synthesis of metallointercalator-DNA conjugates on a solid support

Metallointercalator-DNA conjugates were prepared by amide bond formation between active esters on the nonintercalating ligands of transition metal complexes and primary amines presented at the 5' or the 3' termini of oligonucleotides attached to solid supports. The conjugates were liberated from the support by aminolysis and purified by HPLC on C18 or C4 stationary phases, which separates the two diastereomeric forms of the conjugates containing either the Lambda or the Delta enantiomer of the octahedral metal complex. The coupling reaction proceeds with approximately 75% conversion of the amino-terminated oligonucleotide into the conjugate; the isolated yield is approximately 200 nmol for syntheses initiated on DNA-synthesis columns with a loading of 2 micromol. The conjugates were characterized by ultraviolet-visible and circular dichorism absorption spectroscopy, electrospray ionization mass spectrometry, enzymatic digestion, and polyacrylamide gel electrophoresis (PAGE). Oligonucleotides bearing [Rh(phi)(2)(bpy')](3+) (phi = 9, 10-phenanthrene quinone diimine; bpy' = 4-butyric acid-4'-methyl bipyridyl) form 1:1 duplexes with the complementary strand, and the electrophoretic mobility under nondenaturating PAGE of duplexes containing Delta-Rh is notably different from duplexes containing Lambda-Rh. High-resolution PAGE of DNA photocleavage reactions initiated by irradiation of the tethered Rh complexes reveal intercalation of the complex only near the tethered end of the duplex. Analogous DNA-binding properties were observed with [Rh(phi)(2)(bpy')](3+) tethered to the 3' terminus. By combining the 3' and 5' modification strategies, a mixed-metal DNA conjugate containing both [Os(phen)(bpy')(Me(2)-dppz)](2+) (Me(2)-dppz = 7, 8-dimethyldipyridophenazine) on the 3' terminus and [Rh(phi)(2)(bpy')](3+) on the 5' terminus was prepared and isolated. Taken together, these strategies for preparing metallointercalator-DNA conjugates offer a useful approach to generate chemical assemblies to probe long-range DNA-mediated charge transfer where the redox initiator is confined to and intercalated in a well-defined binding site.     

Sequence-specific protection of plasmid DNA from restriction endonuclease hydrolysis by pyrrole-imidazole-cyclopropapyrroloindole conjugates

The pyrrole-imidazole (Py-Im) triamide-cyclopropa pyrroloindole (CPI) conjugates ImPyImLDu86 (7) and ImImPyLDu86 (14) were synthesized and their alkylating activities and inhibitory effects on DNA hydrolysis by restriction endonucleases were examined. Sequencing gel analysis demonstrated that conjugates 7 and 14 specifically alkylated DNA at 5'-CGCGCG-3' and 5'-PyGGCCPu-3', respectively. Agarose gel electrophoresis indicated that incubation of a supercoiled plasmid, pSPORT I (4109 bp), with conjugate 7 effectively inhibited its hydrolysis by BssHII (5'-G_CGCGC-3'), whereas conjugate 14 had no effect on this hydrolysis. These results suggest that conjugate 7 sequence-specifically inhibits the hydrolysis of DNA by BssHII. Sequence-specific alkylation by the Py-Im triamide-CPI conjugates was further confirmed by inhibition of the Eco52I (5'-C_GGCCG-3') hydrolysis of conjugate 14-treated pQBI PGK (5387 bp). In clear contrast, hydrolysis of pQB1 PGK by DraI (3'-TTT_AAA-3') was not inhibited by 5 micro M conjugate 14. That ImImPy did not inhibit the hydrolysis of pQB1 PGK indicates that covalent bond formation is necessary for inhibition. A similar experiment, using linear pQBI PGK, achieved the same extent of protection of the DNA with approximately half the concentration of conjugate 14 as was required to protect supercoiled DNA from hydrolysis.