High thermal stability of 5'-5'-linked alternate hoogsteen triplexes at physiological pH.
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DNA↗
Biomedical subjects
Erik B Pedersen
Publications and source records attributed to Erik B Pedersen.
Synthesis and DNA binding properties of a new benzo[f]imidazo[1,5b]-isoquinoline-butan-1,2-diol derivative.
A benzo[f]imidazo[1,5b]-isoquinoline derivative 4 with a 1,2-butandiol linker was prepared by reaction of a trimethylsilylated 5-naphthylidenehydantoin 3 with a 2,3-dideoxy-D-glycero-pentafuranoside 2 in 22% yield. After deprotection, the resulting compound 5 was converted to a DMT protected phosphoramidite building block 7 for standard DNA synthesis. DNA/DNA, DNA/RNA duplexes with 5 inserted as bulges were destabilized, except when the new amidite was used for the synthesis of a zipping duplex.
DNA↗
Easily denaturing nucleic acids derived from intercalating nucleic acids: thermal stability studies, dual duplex invasion and inhibition of transcription start.
The bulged insertions of (R)-1-O-(pyren-1-ylmethyl)glycerol (monomer P) in two complementary 8mer DNA strands (intercalating nucleic acids) opposite to each other resulted in the formation of an easily denaturing duplex, which had lower thermal stability (21.0 degrees C) than the wild-type double-stranded DNA (dsDNA, 26.0 degrees C), but both modified oligodeoxynucleotides had increased binding affinity toward complementary single-stranded DNA (ssDNA) (41.5 and 39.0 degrees C). Zipping of pyrene moieties in an easily denaturing duplex gave formation of a strong excimer band at 480 nm upon excitation at 343 nm in the steady-state fluorescence spectra. The excimer band disappeared upon addition of a similar short dsDNA, but remained when adding a 128mer dsDNA containing the same sequence. When P was inserted into 2'-OMe-RNA strands, the duplex with zipping P was found to be more stable (42.0 degrees C) than duplexes with the complementary ssDNAs (31.5 and 19.5 degrees C). The excimer band observed in the ds2'-OMe-RNA with zipping P had marginal changes upon addition of both 8 and 128mer dsDNA. Synthesized oligonucleotides were tested in a transcriptional inhibition assay for targeting of the open complex formed by Escherichia coli RNA polymerase with the lac UV-5 promoter using the above mentioned 128mer dsDNA. Inhibition of transcription was observed for 8mer DNAs possessing pyrene intercalators and designed to target both template and non-template DNA strands within the open complex. The observed inhibition was partly a result of unspecific binding of the modified DNAs to the RNA polymerase. Furthermore, the addition of 8mer DNA with three bulged insertions of P designed to be complementary to the template strand at the +36 to +43 position downstream of the transcription start resulted in a specific halt of transcription producing a truncated RNA transcript. This is to our knowledge the first report of an RNA elongation stop mediated by a small DNA sequence possessing intercalators. The insertions of P opposite to each other in ds2'-OMe-RNA showed inhibition efficiency of 96% compared with 25% for unmodified ds2'-OMe-RNA.
DNA↗
Stable and selective formation of hoogsteen-type triplexes and duplexes using twisted intercalating nucleic acids (TINA) prepared via postsynthetic Sonogashira solid-phase coupling reactions.
Bulge insertions of (R)-1-O-[4-(1-pyrenylethynyl)phenylmethyl]glycerol (5) into the middle of homopyrimidine oligodeoxynucleotides (twisted intercalating nucleic acids, TINA) obtained via postsynthetic Sonogashira coupling reaction led to extraordinary high thermal stability of Hoogsteen-type triplexes and duplexes, whereas Watson-Crick-type duplexes of the same nucleotide content were destabilized. Modified oligonucleotides were synthesized using the phosphoramidite of (S)-1-(4,4'-dimethoxytriphenylmethyloxy)-3-(4-iodo-benzyloxy)-propan-2-ol followed by treatment of the oligonucleotide on a CPG-support with the Sonogashira-coupling reaction mixture containing different ethynylaryls. Bulged insertion of the pyrene derivative 5 into oligonucleotides was found to be the best among the tested modifications for binding to the Hoogsteen-type triplexes and duplexes. Thus, at pH 7.2 an oligonucleotide with cytidine content of 36% possessing two bulged insertions of 5 separated by three bases formed a stable triplex (T(m) = 43.0 degrees C), whereas the native oligonucleotide was unable to bind to the target duplex. The corresponding Watson-Crick-type duplex with the same oligonucleotide had T(m) of 38.0 degrees C at pH 7.2, while the T(m) of unmodified dsDNA was 47.0 degrees C. Experiments with mismatched oligonucleotides, luminescent properties, and potential applications of TINA technology is discussed.
Benzyl Compounds↗
Synthesis of 2-(aminocarbonylmethylthio)-1H-imidazoles as novel Capravirine analogues.
Different analogues of Capravirine (AG-1549) or S-1153 were prepared by synthesis of 2-(5-benzyl-4-isopropyl-1-methyl-2,3-dihydro-1H-imidazol-2-ylthio)acetamide (3a-c), ethyl [5-benzyl-1-(ethoxymethyl)-4-ethyl-1H-imidazol-2-ylthio]acetate (10), 2-[5-alkyl-4-substituted 1-(pyridin-4-ylmethyl)-1H-imidazol-2-ylthio]acetamides (12a-f), and 2-[5-benzyl-1-(benzyloxymethyl)-4-isopropyl-1H-imidazol-2-ylthio]acetamides (14a-l) from their corresponding amino acids through a sequence of reactions: Dakin-West reaction, hydrolysis, condensation with thiocyanate derivatives, alkylation with 2-iodoacetamide and ethyl chloroacetate, and coupling with 4-pyridylmethyl chloride, ethoxymethyl chloride and benzyloxymethyl chloride. All the synthesized compounds were screened for their activity against HIV-1 (wild type) and some of them (especially Capravirine like structures) were found active.
Anti-HIV Agents↗
Design, synthesis and ribosome binding of chloramphenicol nucleotide and intercalator conjugates.
Molecular modelling based on X-ray structures of the antibiotic drug chloramphenicol bound in a bacterial ribosome has been used for design of chloramphenicol derivatives. Conjugates of the chloramphenicol amine through appropriate linkers to either a pyrene moiety or to a mono- or dinucleotide moiety were designed to improve binding to ribosomes by providing specific interactions in the peptidyl transferase site or to the P-loop in the ribosome. Specific binding of the conjugates were investigated by footprinting analysis using chemical modifications of accessible nucleotides in ribosomal RNA. The pyrene chloramphenicol conjugate shows enhanced binding to the chloramphenicol binding site compared to the native chloramphenicol, whereas the four nucleotide conjugates could not be shown to bind to the chloramphenicol binding site or to the P-loop.
Binding Sites↗
Synthesis and evaluation of double-prodrugs against HIV. Conjugation of D4T with 6-benzyl-1-(ethoxymethyl)-5-isopropyluracil (MKC-442, emivirine)-type reverse transcriptase inhibitors via the SATE prodrug approach.
This paper reports the synthesis and the antiviral activities of new double-prodrugs against HIV based on the known mixed SATE (S-acyl-2-thioethyl) prodrug approach. The monophosphate of the nucleoside reverse transcriptase inhibitor (NRTI) d4T was masked with one SATE group and one aromatic group through which a nonnucleoside reverse transcriptase inhibitor (NNRTI) was linked. Double-prodrug 1 was a hybrid between d4T monophosphate and the known NNRTI MKC-442, which were linked through a labile p-hydroxybenzoyl protection group in the N-3 position of MKC-442. Double-prodrugs 2 and 3 were conjugates between d4T monophosphate and the new NNRTIs 15 and 19 linked through a stable phenolic linker that was a part of the N-1 substituents of the NNRTIs. The double-prodrugs 1, 2, and 3 all had good activities against wild-type HIV-1, Y181C mutant, and also against a HIV-2 strain that was resistant to NNRTIs.
Cell Line↗
Synthesis of 6-(3,5-dichlorobenzyl) derivatives as isosteric analogues of the HIV drug 6-(3,5-dimethylbenzyl)-1-(ethoxymethyl)-5-isopropyluracil (GCA-186).
The HIV-1 inhibitors described in this paper is closely related to 6-(3,5-dimethylbenzyl)-1-(ethoxy methyl)-5-isopropyluracil (GCA-186) an anti-HIV-1 drug that is highly active against both wild type and mutated HIV-1 strains. The two methyl groups on the 6-benzyl moiety have been shown to improve the binding stability of the drug to the NNRTI-binding site in reverse transcriptase of drug mediated mutant HIV-1 viruses. The methyl groups are replaced with isosteric chloro-atoms to avoid metabolism due to the two methyl groups. However, the isosteric chloro derivatives show tenfold less activity against HIV-1 than their corresponding methyl derivatives. The synthesis and the antiviral activities of the corresponding 1-(allyloxy- and indanyloxy)methyl-6-(3,5-dichlorobenzyl)-5-ethyluracil derivatives are also reported.
Anti-HIV Agents↗
Enhanced inhibition of transcription start by targeting with 2'-OMe pentaribonucleotides comprising locked nucleic acids and intercalating nucleic acids.
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Gene Expression↗
Locked nucleic acids and intercalating nucleic acids in the design of easily denaturing nucleic acids: thermal stability studies.
Intercalating nucleic acids (INA(R)s) with insertions of (R)-1-O-(1-pyrenylmethyl)glycerol were hybridized with locked nucleic acids (LNAs). INA/LNA duplexes were found to be less stable than the corresponding DNA/LNA duplexes when the INA monomer was inserted as a bulge close to the LNA monomers in the opposite strand. This property was used to make "quenched" complements that possess LNA in hairpins and in duplexes and are consequently more accessible for targeting native DNA. The duplex between a fully modified 13-mer LNA sequence and a complementary INA with six pyrene residues inserted after every second base as a bulge was found to be very unstable (Tm=30.1 degrees C) in comparison with the unmodified double-stranded DNA (Tm=48.7 degrees C) and the corresponding duplexes of LNA/DNA (Tm=81.6 degrees C) and INA/DNA (Tm=66.4 degrees C). A thermal melting experiment of a mixture of an LNA hairpin, with five LNA nucleotides in the stem, and its complementary DNA sequence gave a transition with an extremely low increase in optical density (hyperchromicity). When two INA monomers were inserted into the stem of the LNA hairpin, the same experiment resulted in a significant hyperchromicity comparable with the one obtained for the corresponding DNA/DNA duplex.
DNA↗
Hexofuranosyl thymidines inserted into oligodeoxynucleotides via their two exocyclic hydroxy groups. Oligo synthesis and RNase H activity.
Hexofuranosyl nucleosides are considered as conformationally restricted acyclic nucleosides using a furanose ring to link the diol backbone to the nucleobase. The phosphoramidite of 1-(2,3-dideoxy-beta-D-erythro-hexofuranosyl)thymine was synthesized from thymidine with formation of a new stereocentre at C-5' and the nucleoside was used in oligodeoxynucleotide (ODN) synthesis. Binding of mixed sequence ODNs towards complementary DNA and RNA showed decreased affinity compared to the wild-type oligos. Insertion in the middle of poly alphaT sequence led to stabilization of ODN/dA(14) duplexes at low ionic strength, but a decrease was observed in medium and high salt buffers compared to d(alphaT)(14)/dA(14). Both beta and alpha hexofuranosyl thymidines allowed cleavage of complementary mixed-sequence RNA by RNase H to the 3'-site of the modification in ODNs whereas a limited inhibition was detected from the 5'-site.
Base Sequence↗
Facile route for the synthesis of the iminosugar nucleoside (3R,4R)-1-(pyren-1-yl)-4-(hydroxymethyl)pyrrolidin-3-ol.
N-(Pyren-1-yl)-(3R,4S)-4-[(1S,2R)-1,2,3-trihydroxypropyl]pyrrolidin-3-ol (4) was obtained in 36% yield from 3-deoxy-3-C-formyl-1,2:5,6-di-O-isopropylidene-alpha-D-allofuranose (3) by combined hydrolysis and aminoalkylation reactions with 1-aminopyrene in a one-pot reaction. Cleavage reactions of the exocyclic triol chain in 4 with NaIO4 and NaBH4 resulted in iminosugars 7 and 8, which are analogues of the furanose forms of 2-deoxy-D-allose and of 2-deoxy-d-ribose, the latter analogue N-(pyren-1-yl)-(3R,4R)-4-(hydroxymethyl)pyrrolidin-3-ol (8) being formed in 83% yield.
Alkylation↗
Synthesis of 6-arylvinyl analogues of the HIV drugs SJ-3366 and Emivirine.
This paper reports the synthesis and the antiviral activities of a series of 6-arylvinyl substituted analogues of SJ-3366, a highly potent agent against HIV. The objective was to investigate whether substitution of the 6-arylketone with a 6-arylvinyl group could lead to an improved antiviral activity against HIV-1. The most active compounds 1-ethoxymethyl, 1-(2-propynyloxymethyl), and 1-(2-methyl-3-phenylallyloxymethyl) substituted 6-[1-(3,5-dimethylphenyl)vinyl]-5-ethyl-1H-pyrimidine-2,4-dione (5b, 16, and 18) showed activities against HIV-1 wild type in the range of Efavirenz, and moderate activities against Y181C and Y181C+K103N mutant strains were also observed.
Anti-HIV Agents↗
Synthesis and incorporation of an alpha-hexofuranosyl thymidine into oligodeoxynucleotides via its two exocyclic OH-groups.
1-(2,3-Dideoxy-3-amino-alpha-D-arabino-hexofuranosyl)thymine is considered as a conformationally restricted acyclic nucleoside using the furanose ring to link the diol backbone to the nucleobase. The appropriately substituted phosphoramidites were synthesised via 1-(5,6-di-O-acetyl-2,3-dideoxy-3-phthalimido-alpha-D-arabino-hexofuranosyl)thymine and used in oligodeoxynucleotide (ODN) synthesis. However, the binding affinity of the mixed ODNs towards complementary DNA and RNA was decreased compared to the wild-type oligos. The decrease was smaller when the monomer was inserted near the end of the sequence. The insertions into an alpha T sequence or in a beta T sequence gave nearly the same dropping in melting temperature per modification which indicates that the new nucleotide modifications behave both as alpha and beta nucleotides.
Arabinonucleosides↗
Synthesis of furoannelated analogues of Emivirine (MKC-442).
Furoannelated analogues 1-alkoxymethyl-7-phenyl-5, 7-dihydro-1H-furo[3, 4-d]-pyrimidine-2, 4-diones of Emivirine (3a, b) were synthesized from the primary alcohols 1-alkoxymethyl-6-benzyl-5-hydroxymethyl-1H-pyrimidine-2, 4-dione (5a, b) using a radical ring closure reaction with Pb(OAc)(4). These analogues are conformationally restricted in order to fix the aromatic substituent of Emivirine in nearly the same position as it is in the case when the complex of Emivirine is bound to HIV-1 RT. However, the anti HIV-1 activities of 3a, b were considerably lower than those of the lead Emivirine.
Cell Line↗
Intercalating nucleic acids: the influence of linker length and intercalator type on their duplex stabilities.
Six new examples of intercalating nucleic acids were synthesized in order to evaluate the dependence of the length of the linker between oligo and intercalator on the thermal stability of their corresponding duplexes and triplexes.
Hot Temperature↗
Synthesis and evaluation of new potential HIV-1 non-nucleoside reverse transcriptase inhibitors. New analogues of MKC-442 containing Michael acceptors in the C-6 position.
Analogues of MKC-442 capable of undergoing Michael addition reactions were synthesised in order to investigate the activity against the HIV-1 mutant (Y181C). An improved activity was postulated on the basis of a possible covalent binding to the mercapto group of Cys181. Lithiation of the C-6 position of 1-ethoxymethyl-5-ethyl-1H-pyrimidine-2,4-dione (5) was followed by reaction with alpha,beta-unsaturated aldehydes and oxidation of the alcohols formed to give the alkenoyl analogues 1a-3a. Analogues 1b-3b containing an allyloxymethyl group in the N-1 position instead of the ethoxymethyl group could not be synthesised due to isomerisation of the allylic group during the metallation reaction. The NMR data for compounds 1a-3a showed a hindered rotation, which was more pronounced for the 6-cyclohexenylcarbonyl derivative 3a than for the propenyl derivatives 1a and 2a. Moderate activity against wild type HIV-1 was observed for the alcohol 8 and the ketones 2a-3a. However, no activity was observed against the Y181C mutant.
Anti-HIV Agents↗
Multiple pathways in the synthesis of new annelated analogues of 6-benzyl-1-(ethoxymethyl)-5-isopropyluracil (emivirine).
Condensation of 3-(3,5-dimethylphenyl)-2-oxocyclopentanecarboxamide (11) with oxalyl chloride and condensation of ethyl 2-benzylamino-5-methyl-3-phenylcyclopent-1-enecarboxylate (17a) with trimethylsilyl isothiocyanate gave 7-(3,5-dimethylphenyl)-6,7-dihydro-5H-cyclopenta[e][1,3]oxazine-2,4-dione (12) and 1-benzyl-5-methyl-7-phenyl-2-thioxo-1,2,3,5,6,7- hexahydrocyclopentapyrimidin-4-one (18a), respectively. Acid catalyzed ring-closure of 6-(4-methyl-1-phenylpent-3-enyl)-2-thioxo-2,3-dihydro-1H-pyrimidin-4-one (26) and radical mediated ring-closure of 1,3-bis(benzyloxymethyl)-5-bromo-6-(1-phenylbut-3-enyl)-1H-pyrimidine-2,4- dione (32a) gave 5,5-dimethyl-8-phenyl-5,6,7,8-tetrahydro-1H-quinazoline-2,4- dione (28) and 1,3-bis(benzyloxymethyl)-5-methyl-7-phenyl-1,5,6,7- tetrahydrocyclopentapyrimidine-2,4-dione (33), respectively. Annelated emivirine analogues 7-(3,5-dimethylphenyl)-1- ethoxymethyl-1,5,6,7-tetrahydrocyclopentapyrimidine-2,4-dione (4), 1-ethoxymethyl-5,5-dimethyl-8-phenyl-5,6,7,8-tetrahydro-1H-quinazoline- 2,4-dione (5) and 1-ethoxymethyl-5-methyl-7-phenyl-1,5,6,7- tetrahydrocyclopentapyrimidine-2,4-dione (6) were obtained in few steps from 12, 28 and 18a/33, respectively. These new analogues can be considered as conformationally locaTed analogues of emivirine. However, the compounds 4 6 showed lower activities against HIV-1 than emivirine and it is concluded that the locked conformation disfavours activity against HIV-1.
Anti-HIV Agents↗