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Indole was conjugated to deoxyuridine either directly or via ethynyl linkage to synthesize two new DNA monomers. Due to the high reactivity of indole toward electrophilic substitution reactions, unwanted iodination was observed during the oxidation step with iodine oxidizer in automated DNA synthesizer leading to an additional oligonucleotide having extra 126 mass unit beside the desired oligonucleotide. On the other hand, using CSO oxidizer ensured the formation of the wanted oligonucleotide. Oligonucleotide synthesis was confirmed by MALDI-TOF-MS analysis. Polypyrimidine strands containing non-iodinated indole nucleoside were able to form parallel triplexes, antiparallel RNA and DNA duplexes. The thermal denaturation experiments showed higher triplex stabilization for 5-(5-indolylethynyl)-2′-deoxyuridine over 5-(5-indolyl)-2′-deoxyuridine as the triple bond allows twisting to put the indole into a proper position within the triplex encouraging better π-π stacking.

Two new phosphoramidite building blocks for DNA synthesis were synthesized from 1,5- and 2,6-dihydroxyanthraquinones through alkylation with 3-bromo-1-propanol followed by DMT-protection. The novel synthesized 1,5- and 2,6-disubstituted anthraquinone monomers H15 and H26 are incorporated into a G-quadruplex by single and double replacements of TGT and TT loops. Monomers H15 and H26 were found to destabilize G-quadruplex structures for all single replacements of TGT or TT loops. The largest destabilization was observed when H26 linker replaced a TT loop. In contrast, the presence of anthraquinone monomers in two TT loops led to 1–18 °C increase in their thermal stabilities, depending on linker attachment geometry of the monomers. The presence of H15 and H26 linkers replacing two TT loops results in the highest stabilization of the G-quadruplex structure by 18.2 °C. Circular dichroism spectroscopy of all anthraquinone-modified quadruplexes revealed no change of the antiparallel structure when compared with the wild type under potassium buffer conditions. The significantly increased thermostabilities were interpreted by molecular modeling of anthraquinone-modified G-quadruplexes.

Twisted intercalating nucleic acid (TINA) has the ability to stabilize Hoogsteen triplex formation. New oligonucleotides containing 3-(pyren-1-ylethynyl)indole as a DNA linked intercalator backbone have been added to the TINA family and their synthetic route has been devised. The indole ring, under acidic conditions, polarizes the triple bond in the intercalator and this makes hydration of the triple bond possible during the DNA synthesis and an oligonucleotide with 1-(indol-3-yl)-2-(pyren-1-yl)ethanone as the intercalator is formed. Insertion of the unhydrated and hydrated linker systems gave characteristic UV spectra comparable to its monomeric diols due to π-conjugated and isolated pyrene systems, respectively. Thermal denaturation profile at pH 6–7.2 showed excellent stabilization of the triplex upon insertion of 3-(pyren-1-ylethynyl)indole when compared to wild-type triplexes. The investigation includes fluorescence spectroscopic and molecular modeling studies.

Some novel MC-1220 analogs were synthesized by condensation of 4,6-dichloro-N-methylpyrimidin-2-amine derivatives (1a,b and 15) and/or 4-chloro-6-methoxy-N,N,5-trimethylpyrimidin-2-amine (2a) with the sodium salt of 2,6-difluorophenylacetonitrile followed by treatment with aqueous sodium hydroxide in methanol, alkylation, reduction, halogenation, and/or acidic hydrolysis. All synthesized compounds were evaluated for their activity against HIV-1. The most active compound in this study was compound 7, which showed activity against HIV-1 comparable to that of MC-1220. The only difference in structure between compound 7 and MC-1220 is a fluoro atom instead of a CH3 group.

New compounds 5 and 9 using DNA bases e.g. Adenine 1 and Guanine 6 derivatives have been synthesized. The use of simple methods to synthesize compounds 5 and 9 were done using pyrimidine as an alternative DNA base ring. Another design to synthesize new simple pyrimidine rings utilizing thiourea and ethylcyano acetate to afford 6-amino-2-thiouracil was adopted. The reaction of thiouracil 10 with chloro cyano or chloro ester and ketone, resulted in the formation of adduct compounds 18-21 , rather than the formation of compound 17 . All the synthesized compounds were subjected to docking study, in order to gain insights into their binding modes against cyclin-dependent protein kinase 2 (CDK-2) that is involved heavily in cell cycle regulation and receptor protein B-cell lymphoma 2 (BCL-2) which is involved in cell apoptosis. These targets were selected based on their key roles in cancer progression via the regulation of the cell cycle and DNA replication. Molecular-docking analyses showed that compound 14e was the best docked ligand against both targets, as it displayed the lowest binding energy, critical hydrogen bonds and hydrophobic interactions with the targets.

The phosphoramidites of DNA monomers of 7-(3-aminopropyn-1-yl)-8-aza-7-deazaadenine (Y) and 7-(3-aminopropyn-1-yl)-8-aza-7-deazaadenine LNA (Z) are synthesized, and the thermal stability at pH 7.2 and 8.2 of anti-parallel triplexes modified with these two monomers is determined. When, the anti-parallel TFO strand was modified with Y with one or two insertions at the end of the TFO strand, the thermal stability was increased 1.2 °C and 3 °C at pH 7.2, respectively, whereas one insertion in the middle of the TFO strand decreased the thermal stability 1.4 °C compared to the wild type oligonucleotide. In order to be sure that the 3-aminopropyn-1-yl chain was contributing to the stability of the triplex, the nucleobase X without the aminopropynyl group was inserted in the same positions. In all cases the thermal stability was lower than the corresponding oligonucleotides carrying the 3-aminopropyn-1-yl chain, especially at the end of the TFO strand. On the other hand, the thermal stability of the anti-parallel triplex was dramatically decreased when the TFO strand was modified with the LNA monomer analog Z in the middle of the TFO strand (ΔTm = -9.1 °C). Also the thermal stability decreased about 6.1 °C when the TFO strand was modified with Z and the Watson-Crick strand with adenine-LNA (AL). The molecular modeling results showed that, in case of nucleobases Y and Z a hydrogen bond (1.69 and 1.72 Å, respectively) was formed between the protonated 3-aminopropyn-1-yl chain and one of the phosphate groups in Watson-Crick strand. Also, it was shown that the nucleobase Y made a good stacking and binding with the other nucleobases in the TFO and Watson-Crick duplex, respectively. In contrast, the nucleobase Z with LNA moiety was forced to twist out of plane of Watson-Crick base pair which is weakening the stacking interactions with the TFO nucleobases and the binding with the duplex part.

In order to gain insight into how to improve thermal stability of i-motifs when used in the context of biomedical and nanotechnological applications, novel anthraquinone-modified i-motifs were synthesized by insertion of 1,8-, 1,4-, 1,5- and 2,6-disubstituted anthraquinone monomers into the TAA loops of a 22mer cytosine-rich human telomeric DNA sequence. The influence of the four anthraquinone linkers on the i-motif thermal stability was investigated at 295 nm and pH 5.5. Anthraquinone monomers modulate the i-motif stability in a position-depending manner and the modulation also depends on the substitution pattern of the anthraquinone. The insertion of anthraquinone was found to stabilize the i-motif structure when replacing any one of the positions of the central TAA loop and the thermal stabilities were typically higher than those previously found for i-motifs containing pyrene-modified uracilyl unlocked nucleic acid monomers or twisted intercalating nucleic acid. The 2,6-disubstituted anthraquinone linker replacing T10 enabled a significant increase of i-motif thermal melting by 8.2 °C. A substantial increase of 5.0 °C in i-motif thermal melting was recorded when both A6 and T16 were modified with a double replacement by the 2,6-isomer into the TAA loops in the outer regions. The largest destabilization is observed for the 1,5-disubstituted anthraquinone linker upon the replacement of A18. CD curves of anthraquinone-modified variants imply no structural changes in all cases under potassium buffer conditions compared with those of the native i-motif. Molecular modeling studies explained the increased thermal stabilities of anthraquinone-modified i-motifs.

A series of thalidomide and phthalimide ester analogs were efficiently synthesized from N-chloromethylthalidomide, N-chloromethylphthalimide, and N-(2-bromoethyl)phthalimide derivatives with various biologically important carboxylic acids. The synthesized compounds were purified and characterized by various chromatographic and spectroscopic techniques. The antitumor activity of all the synthesized compounds was screened against human liver and breast cancer cells, which showed that phthalimide ester 6a was the best cytotoxic compound against MCF7 cells, while all of the tested compounds showed a non-cytotoxic effect against HepG2 cells. Compounds 5a, 6a, and 7a possess immunosuppressant effect, while compounds 5c, 5d, 6c, 6d, 7c, and 7d showed an immunostimmulatory effect. Meanwhile, estimation of the binding affinity for all the synthesized compounds toward the vascular endothelial growth factor receptor (VEGFR) showed that compounds 5a, 5b, and 7d were the most potent inhibitors.

Synthesis An allopurinol locked nucleic acid (LNA) monomer was prepared by a novel strategy through C6 deamination of the corresponding 8-aza-7-bromo-7- deazaadenine LNA monomer with aqueous sodium hydroxide. An 8-aza-7-deazaadenine LNA monomer was also synthesized by a modification of the new synthetic pathway. N-Glycosylation at the 8-position was prevented by steric hindrance from the 7-bromo atom in the starting material 8-aza-7-bromo-7-deazaadenine. In the final step of the synthesis, the bromine was removed together with a benzyl protecting group by catalytic reduction with ammonium formate to give the required LNA monomers.

Two-pronged attack: We describe the maturation of a bivalent aptamer by a chemically driven two-step process. From an improved monovalent aptamer subdomain that had been modified by polycyclic aromatic hydrocarbons at individual positions, a mature bivalent variant with superior activities to its progenitor molecule was obtained through domain reassembly.

Free-radical reaction of different carbohydrate educts 2, 5, and 7 with acrylonitrile in the presence of tributyltin hydride and a radical initiator (AIBN) gave the methyl 3-(2-cyanoethyl)-2,3-dideoxypentofuranosides 3a and 6. Similar reaction of 2 with methyl acrylate gave 3-(2-methoxycarbonylethyl>-2,3-dideoxypentofuranose 3b. Nucleoside coupling of 3a with silylated uracil gave an anomeric mixture of β- and α-nucleoside 8 and 9 which were deprotected to give 10 and 11, respectively. Similar reaction of 3b with silylated N4-isobutyrylcytosine gave 12 and 13 which were deprotected to give the final nucleosides 16 and 17, respectively. None of the compounds 10a, 11, 14-17 showed significant activity against HIV. Radikalsynthese von 3-(2-Cyanethyl)- und 3-(2-Methoxycarbonylethyl)-2,3-didesoxy-α-D-erythro-pentofuranosid und ihre Anwendung zur Synthese potentiell antiviraler Nucleoside Die Radikalreaktion der Zuckerderivate 2, 5 und 7 mit Acrylnitril in Gegenwart von Tributylzinnhydrid und dem Radikalstaiter AIBN fuhrte zu den Methyl-3-(2-cyanethyl)-2,3-didesoxypentofuranosiden 3a und 6. Die analoge Reaktion von 2 mit Methylacrylat ergab die 3-(2-Methoxycarbonylethyl)-2,3-didesoxypentofuranose 3b. Die Nucleosidverknupfung von 3a mit silyliertem Urazil lieferte das Anomerengernisch der β- und α-Nucleoside 8 und 9, die zu 10 bzw. 11 entsilyliert wurden. Analog dazu wurden aus 3b und silyliertem N4-lsobutyrylcytosin 12 und 13 und durch Entfernen der Schutzgruppen 16 bzw. 17 erhalten. Keine der Verbindungen 10a, 11, 14-17 zeigte nennenswerte Aktivitat gegen HIV.

This paper describes the synthesis and the antiviral activities of dimeric compounds derived from homo and asymmetric combinations of N-1 propynyloxymethyl analogues 1a,b of MKC-442, an N-1 4-iodobenzyloxymethyl analogue of TNK-651 5, potent contraceptive norgestrel and AZT. They were obtained by Sonogashira reaction, ‘click' chemistry or Pd-catalyzed oxidative coupling. The iodo precursor 5 turned out as a potent compound against wild type and mutated HIV-1 virus. All dimeric compounds showed lower activity against HIV-1 than MKC-442, except the asymmetric dimer of AZT and 1a which showed an activity comparable to MKC-442.

In continuation of our investigation of characteristics and thermodynamic properties of the i-motif 5′-d[(CCCTAA)3CCCT)] upon insertion of intercalating nucleotides into the cytosine-rich oligonucleotide, this article evaluates the stabilities of i-motif oligonucleotides upon insertion of naphthalimide (1H-benzo[de]isoquinoline-1,3(2H)-dione) as the intercalating nucleic acid. The stabilities of i-motif structures with inserted naphthalimide intercalating nucleotides were studied using UV melting temperatures (Tm) and circular dichroism spectra at different pH values and conditions (crowding and non-crowding). This study indicated a positive effect of the naphthalimide intercalating nucleotides on the stabilities of the i-motif structures compared to the wild-type structure which is in contrast to a previous observation for a pyrene-intercalating nucleotide showing a decrease in Tm values.

G-rich anti-parallel DNA triplexes were modified with LNA or α-l-LNA in their Watson-Crick and TFO strands. The triplexes were formed by targeting a pyrimidine strand to a putative hairpin formed by Hoogsteen base pairing in order to use the UV melting method to evaluate the stability of the triplexes. Their thermal stability was reduced when the TFO strand was modified with LNA or α-l-LNA. The same trend was observed when the TFO strand and the purine Watson-Crick strand both were modified with LNA. When all triad components were modified with α-l-LNA and LNA in the middle of the triplex, the thermal melting was increased. When the pyrimidine sequence was modified with a single insertion of LNA or α-l-LNA the ΔTm increased. Moreover, increasing the number of α-l-LNA in the pyrimidine target sequence to six insertions, leads to a high increase in the thermal stability. The conformational S-type structure of α-l-LNA in anti-parallel triplexes is preferable for triplex stability.

A novel series of uracil derivatives with a 3,5-dimethylbenzyl group at the C-6 position were synthesized and evaluated as non-nucleoside HIV-1 reverse transcriptase inhibitors. Purity of the compounds has been confirmed by TLC. Structures of these compounds were established on the basis of elemental analyses and spectral studies. Some of the tested compounds showed moderate to potent activities against wild-type HIV-1, and N-1 alkylated derivatives were highly active.

6H-Indolo[2,3-b]quinoxaline was studied as a covalently bound heteroaromatic intercalator. Six monomers were synthesized and incorporated into DNA oligonucleotides. Through a study of linker length dependence it was concluded that the linker between the oligo and the intercalator must consist of at least five C atoms in order to stabilize a DNA duplex. An intercalator with a 2′-deoxy- d -riboside linker to the oligo could also stabilize a DNA/RNA duplex, while (S)-4-(6-methylindolo[2,3-b]quinoxalin-3-ylmethoxy)-butane-1,2-diol was able to stabilize both DNA/DNA, DNA/RNA and a DNA/LNA duplex. Mismatch studies revealed a huge sensitivity to the C–C mismatch at the 5′-site of the intercalator.

Phosphoramidites of several new twisted intercalating nucleic acid (TINA) monomers and the previously discovered (R)-1-O-[4-(1-pyrenylethynyl)phenylmethyl]glycerol (1) were synthesized and used in DNA synthesis. Stabilization of Hoogsteen-type triplexes was observed in cases of insertion of the novel (R)-1-O-[3-(naphthalen-1-ylethynyl)phenylmethyl]glycerol (2) as a bulge into homopyrimidine oligodeoxynucleotides (ONs), whereas phenylethynyl and 4-(biphenylylethynyl) derivatives of TINAs resulted in destabilization of parallel triplexes relative to the wild-type triplex. It was concluded that TINA monomers should possess at least two fused phenyl rings attached through the triple bond at the 4-position of bulged (R)-1-O-(phenylmethyl)glycerol in homopyrimidine ONs in order to stabilize parallel triplexes. Slight destabilization of DNA/DNA Watson–Crick type duplexes (ΔTm = 1.0–4.5 °C) was detected for 2 inserted as a bulge, while RNA/DNA duplexes and duplexes with other TINA analogues were considerably destabilized (ΔTm > 6.0 °C). In cases of double insertion of 1 opposite to base inversions in dsDNA, the thermal stabilities of the triplexes were higher than that of the wild-type triplex, which is a new solution to overcome the problem of targeting homopurine stretches with single base pair inversions. A DNA three-way junction was considerably stabilized (ΔTm in a range of 10.0–15.5 °C) upon insertion of TINA monomers in the junction point as a bulge. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)

Twisted intercalating nucleic acids (TINA) possessing acridine derivatives have been synthesized via the postsynthetic modifications of oligonucleotides possessing insertions of (R)-1-O-(4-iodobenzyl)glycerol (8) or (R)-1-O-(4-ethynylbenzyl)glycerol (9) at the 5'-end or in the middle as a bulge. In the first postsynthetic step, oligonucleotides 8 and 9 on the CPG support were treated with a Sonogashira coupling reaction mixture containing 9-chloro-2-ethynylacridine or 9-chloro-2-iodoacridine, respectively. After the postsynthetic step, treatment of the oligonucleotides with 32% aq ammonia or 50% ethanolic solution of tris(2-aminoethyl)amine led to the substitution of chloride on acridine concurrent with deprotection of the bases and cleavage of the oligonucleotides from CPG. Molecular modeling of the parallel triplex with a bulged insertion of the monomer (R)-3-O-[4-(9-aminoacridin-2-ylethynyl)benzyl]glycerol in the triplex-forming oligonucleotide (TFO) showed that the acridine moiety was stacking between the bases of the duplex, while phenyl was placed between the bases of the TFO. Thermal denaturation studies and fluorescence properties of TINA-acridine oligonucleotide duplexes and triplexes are discussed.

This is the first report describing the design, synthesis, and incorporation of an intercalating linker leading to an efficiently 5′,5′-linked alternate strand Hoogsteen triplex. Using molecular modeling, the 5′,5′ problem was solved in a rather simple way. The two relatively distant 5′-ends have been connected with a molecule that provides rigidity to the structure, while being flexible enough to allow stacking upon all four strands. The synthesis of the core of the designed molecule was conducted by Sonogashira coupling of an appropriately substituted iodobenzene with 1,3-diethynylbenzene to give a conjugated system with three benzene rings interconnected with triple bonds. For the alternate-strand triplex obtained with the intercalating linker, the stability obtained is higher than that of the corresponding homotriplex of equivalent total length and with the same nucleotides. This is deduced from a 5° higher melting temperature of the alternating triplex. The sensitivity towards mismatches in the alternate-strand triplex is in the same range (ΔTm=−13° to −19°) as those observed for homotriplexes.

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.

The synthesis and insertions of ( S )-1- O -(pyren-1-ylmethyl)glycerol into intercalating nucleic acids is described. Insertions of this S -isomer as a bulge lead to reduced binding affinity towards complementary ssDNA compared to intercalating nucleic acids possessing ( R )-1- O -(pyren-1-ylmethyl)glycerol in the same positions. Insertions of both ( R ) or ( S ) 1- O -(pyren-1-ylmethyl)glycerols as bulges into two complementary strands decreased the stability of the complex compared to dsDNA possessing the pyrene pseudo-nucleotide in one of the strands.

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.

The intercalating nucleic acid (INA) presented in this paper is a novel 1-O-(1-pyrenylmethyl)glycerol DNA intercalator that induces high thermal affinity for complementary DNA. The duplex examined contained two INA intercalators, denoted X, inserted directly opposite each other: d(C(1)T(2)C(3)A(4)A(5)C(6)X(7)C(8)A(9)A(10)G(11)C(12)T(13)):d(A(14)G(15)C(16)T(17)-T(18)G(19)X(20)G(21)T(22)T(23)G(24)A(25)G(26)). Unlike most other nucleotide analogues, DNA with INA inserted has a lower affinity for hybridizing to complementary DNA with an INA inserted directly opposite than to complementary unmodified DNA. In this study we used two-dimensional (1)H NMR spectroscopy to determine a high-resolution solution structure of the weak INA-INA duplex. A modified ISPA approach was used to obtain interproton distance bounds from NOESY cross-peak intensities. These distance bounds were used as restraints in molecular dynamics (rMD) calculations. Twenty final structures were generated for the duplex from a B-type DNA starting structure. The root-mean-square deviation (RMSD) of the coordinates for the 20 structures of the complex was 1.95 A. This rather large value, together with broad lines in the area of insertion, reflect the high degree of internal motion in the complex. The determination of the structure revealed that both intercalators were situated in the center of the helix, stacking with each other and the neighboring nucleobases. The intercalation of the INAs caused an unwinding of the helix in the insertion area, creating a ladderlike structure. The structural changes observed upon intercalation were mainly of local character; however, a broadening of the minor groove was found throughout the helix.

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.

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.

Fluorescence and hybridization specificity is reported for intercalating nucleic acids (INAs), which are here oligodeoxynucleotides (ODNs) synthesized with insertions using (S)-1-[bis(4-methoxyphenyl)(phenyl)methoxy]-3-[(pyren-1-yl)methoxy]propan-2-ol phosphoramidites. It is shown that an INA with two insertions placed as next-nearest neighbors can be used for discrimination between nucleic acids and their single-point mutants. Quenching of an excimer band at 480 nm is observed upon hybridizing to a complementary sequence, whereas the excimer band is present when the nucleobase pair between the two pyrene moieties is mismatched. It is the first example of a solution based on fluorescence detection of single-point mutants that uses excimer formation and does not rely on stringent hybridization conditions. Furthermore, it is shown that INAs with pyrene insertions retain their sequence specificity in thermal melting.

alpha-Aminoketone hydrochlorides 2a-d were synthesized by Dakin-West reaction from L-phenylalanine and L-cyclohexylalanine followed by hydrolysis in acidic medium. Treatment of 2a-d with aqueous potassium thiocyanate afforded 1, 3-imidazole-2-thiones 3a-d which were alkylated with methyl iodide to give 2-methylsulfanyl-1H-imidazoles 4a-d with 4-benzyl/4-cyclohexylmethyl and 5-ethyl/5-isopropyl substituents. Coupling of 4a-d with ethoxymethyl chloride or benzyloxymethyl chloride furnished N-1 5a-d and N-3 6a-h alkylated products. The synthesised compounds were tested for their activity against HIV-1. The most active compounds have a cyclohexylmethyl group in the 5-position of 6 and showed an activity against HIV-1 comparable to the activity of Nevirapine.

Thymidine dimers in which the natural phosphodiester linkage has been replaced by a 2,5-disubstituted tetrazole ring are synthesized and incorporated into oligodeoxynucleotides (ODNs). The synthesis is accomplished by two strategies based on an alkylation of 5′-O-trityl-on and 5′-O-trityl-off 3′-deoxy-3′-(1H-tetrazol-5-yl)thymidines with 5′-iodo-5′-deoxythymidine in the presence of Et3N, and the formation of only 2-substituted tetrazol-5-yl linkages is observed in 89 and 46% yields, respectively. The nucleoside dimer formed is reacted with 4,4′-dimethoxytrityl chloride (DMTCl), followed by treatment with 2-cyanoethyl tetraisopropylphosphordiamidite in the presence of N,N-diisopropylammonium tetrazolide, to afford the 5′-O-DMT-protected dinucleoside phosphoramidite that is used for incorporation into ODNs on an automated DNA synthesizer. The modified ODNs with one and up to five tetrazole internucleosidic linkages are obtained in good yields. The thermal stability of DNA/DNA and DNA/RNA duplexes is studied by UV experiments and reported also.

Novel analogues of MKC442 (6-benzyl-1-(ethoxymethyl)-5-isopropylpyrimidine- 2,4(1H,3H)-dione) were synthesized by reaction of 6-[(3,5-dimethylphenyl) fluoromethyl]-5-ethyluracil (5) with ethoxymethyl chloride and formaldehyde acetals. The Sonogashira reaction was carried out on the N1-(p-iodobenzyl)oxy] methyl derivative of compound 5 using propagyl alcohol to afford compound 12 (YML220). The latter compound was selected for further studies since it showed the most potent and selective activity in vitro against wild-type HIV-1 and non-nucleoside reverse transcriptase inhibitor-, nucleoside reverse transcriptase inhibitor-, and protease inhibitor-resistant mutants and a wide range of HIV-1 clinical isolates. 12 also showed microbicidal activity in long-term assays with heavily infected MT-4 cells.

Deprotected compounds 1 and 9 were allowed to react with 4,4'-dimethoxytrityl chloride in pyridine to give 5'-O-DMT nucleosides 2 and 10. The 3'-phosphoramidites 4 and 11 were incorporated into oligodeoxynucleosides (ODNs). The hybridization properties of the modified ODNs with their complementary DNA strands were studied. Interesting results were obtained when 11 was inserted as a bulged nucleoside into TWAs, duplexes, and triplexes.

New S-DABOs with a long alkylating S-alkyl substituent showing antiretroviral activity against HIV-1 in the micromolar range were prepared from 5,6-disubstituted 4-oxo-2-thiopyrimidines and 1,7-dibromo-3,5-dioxaheptane. The analogues with an ethyl group in position 5 also showed activity in the micromolar range against a Tyr/8/Cys mutant strain of HIV-1. The S-DABO analogues showing activity against the HIV-1 RT mutant strain were transformed to the N-3 and N-1 ring closed 7-oxo-pyrimidino-1,3,5-oxathiazepines which surprisingly all showed activity against HIV-1 in the micromolar range, as well as against a Tyr/8/Cys mutant strain of HIV-1. Some analogues of S-DABO with a thien-2-ylmethyl residue in position 6 were synthesized and tested against HIV-1 wild type, but they showed less or comparable activities to those of the corresponding 6-benzyl analogues.

The synthesis of two new phosphoramidite building blocks for the incorporation of 5-(pyren-1-yl)uracilyl unlocked nucleic acid (UNA) monomers into oligonucleotides has been developed. Monomers containing a pyrene-modified nucleobase component were found to destabilize an i-motif structure at pH 5.2, both under molecular crowding and noncrowding conditions. The presence of the pyrene-modified UNA monomers in DNA strands led to decreases in the thermal stabilities of DNA*/DNA and DNA*/RNA duplexes, but these duplexes' thermal stabilities were better than those of duplexes containing unmodified UNA monomers. Pyrene-modified UNA monomers incorporated in bulges were able to stabilize DNA*/DNA duplexes due to intercalation of the pyrene moiety into the duplexes. Steady-state fluorescence emission studies of oligonucleotides containing pyrene-modified UNA monomers revealed decreases in fluorescence intensities upon hybridization to DNA or RNA. Efficient quenching of fluorescence of pyrene-modified UNA monomers was observed after formation of i-motif structures at pH 5.2. The stabilizing/destabilizing effect of pyrene-modified nucleic acids might be useful for designing antisense oligonucleotides and hybridization probes.

The thiazole orange dye 1,1'-(4,4,8,8-tetramethyl-4,8-diazaundecamethylene)-bis[4-[3-methy l-2, 3-dihydro(benzo-1,3-thiazole)-2-methylidene]]quinolinium tetraiodide (TOTO) binds to double-stranded DNA (dsDNA) in a sequence selective bisintercalation. Each chromophore is sandwiched between two base pairs in a (5'-CpT-3'):(5'-ApG-3') site, and the linker spans over two base pairs in the minor groove. The binding of analogs of TOTO in which the linker has been modified is examined. The aim of the study is to utilize the sequence selectivity of the TOTO chromophores to enhance and/or alter the overall selectivity of the binding. One- and two-dimensional 1H-NMR investigations of complexes between TOTO analogs and various dsDNA oligonucleotides are reported. The following analogs were synthesized and used: 1,1'-(4,4,8,8-tetramethyl-4,8-diazadodecamethylene) -bis[4-[3-methyl-2,3-dihydro- (benzo-1,3-thiazole)-2-methylidene]]quinolinium tetraiodide (TOTO10), 1,1'-(5,5,9,9-tetramethyl-5,9-diazatridecamethylene)-bis[4-[3-meth yl-2, 3-dihydro(benzo-1,3-thiazole)-2-methylidene]]quinolinium tetraiodide (TOTO11), and 1,1'-(6,6,10,10-tetramethyl-6,10-diazapentadecamethylene)-bis[4-[3 -methyl-2, 3-dihydro(benzo-1,3-thiazole)-2-methylidene]]quinolinium tetraiodide (TOTO13). The results show that with a longer linker the dyes can bisintercalate into two (5'-CpT-3'):(5'-ApG-3') sites separated by one or two base pairs. Bisintercalation in two such "isolated" binding sites yields non-nearest-neighbor bisintercalation in which the linker spans over more than two base pairs. The investigations also showed that an exact length of the linker is not crucial for the site selectivity since TOTO, TOTO10, and TOTO11 are almost equally suitable in binding selectively to the (5'CTAG-3')2 sequence. Fluorescence measurements show that TOTO10, TOTO11, and TOTO13 have higher fluorescence quantum yields than TOTO when bound to d(CGCTAGCG)2. This indicates that the length of the linker in TOTO may not be the optimum one in terms of using the dye as a fluorescence marker.

5′- O -(4,4′-dimethoxytrityl)-5-methyl- N 4 -(1-pyrenylmethyl)cytidine( 5 ) was prepared by reaction of 1-pyrenylmethylamine with an appropriate protected 4-(1,2,4-triazolyl)thymidine derivative which was synthesized from 5- O -DMT protected thymidine by acetylation with acetic anhydride and subsequent reaction with triethylamine, 1,2,4-triazole and POCl 3 . Stabilities are reported for DNA duplexes, three-way junctions and triplexes when 5 is inserted. Most interestingly, the three-way junctions are stabilized when 5 is used for insertion into the junction region. This break-through for recognizing the foot of a stem could have far-reaching importance because new targets for antisense oligos is now rendered possible on intact secondary structures.

A UBr∗UBr dimer was synthesized by connecting the appropiate nucleoside monomers through a 5-atom carboxamide linkage. The dimer was incorporated in oligodeoxynucleotides and investigated for hybridization properties toward single and double stranded DNA.

A T∗T dimer with ∗ representing a 2′-OCH2CH2NHC(O)-4′ linkage connecting two nucleoside units was prepared by condensation of (S)-1-[2-(2-aminoethoxy)-3-(4,4′-dimethoxytrityloxy)propyl]thymine with 1,2-dideoxy-1-thyminyl-β-D-erythro-pento-furanuronic acid. The T∗T dimer was incorporated in oligodeoxynucleotides and investigated for hybridization to DNA.

Two carboxamide linked dimers were synthesized by condensation of the appropiate a nucleosides with a thymidine-5′-carboxylic acid and incorporated in oligodeoxynucleotides. The oligodeoxynucleotides were investigated for hybridization to single and double stranded DNA.

Oligodeoxynucleotides modified with carboxamide linked dimeric nuclcotides and an acyclic nucleoside were prepared and investigated for their hybridization properties toward DNA.

1-(2-Deoxy-3,5-bis-O-(4-methylbenzoyl)-D-erythro-pentofuranosyl)-5-formyluracil (4) was synthesized from 5-formyluracil and an appropriate methyl glycoside and condensed with 2-thiohydantoin (5a) and its corresponding 3-phenyl derivative5b to give 5-[1-(2-deoxy-3,5-bis-O-(4-methylbenzoyl)-D-erythro-pentofuranosyl)uracil-5-ylmethylene]-2-thiohydantoins7a and7b, respectively, in 65–70% yield. They were deprotected with sodium methoxide in methanol to give both anomers of the free nucleosides. In a different route 5-formyluracil (1) was condensed with5b and subsequently with an appropriate methyl glycoside to give7b.

3′-Amino and 5′-amino derivatives of hydantoin 2′-deoxynucleosides have been prepared from the corresponding 3′-phthalimido and 5′-azido nucleosides, respectively, which in turn were prepared by condensation of appropriate sugars with 5-benzylidenehydantoin. The amino nucleosides were tested for their potential activity against HIV and HSV.

Coupling of 6-benzyl-5-hydroxymethyluracil (1) with formaldehyde acetals followed by fluorination using (diethylamino)sulfur trifluoride (DAST) afforded 1-alkenyloxymethyl and 1-propargyloxymethyl 5-fluoromethyl-6-benzyluracils 3a-c. 6-(3,5-Dimethylbenzyl)-5-ethyl-1-[(2-fluoroethoxy)methyl]pyrimidine-2,4(1H,3H)-dione (6) was synthesized by fluorination of the corresponding hydroxy derivative 5. Sonogoshira reaction was performed on 6-(3,5-dimethylbenzyl)-5-ethyl-1-(4-iodobenzyl)uracil (7) with propargyl alcohol to afford 8 which was fluorinated to give the fluoro propargyl derivative 9. Compound 7 was synthesized by N1-alkylation of the corresponding uracil. Significant activity was found against HIV-1 except for compounds with 5-hydroxymethyl and 5-fluoromethyl substituents.