Facile interstrand migration of the hydrocarbon moiety of a dibenzo[a,l]pyrene 11,12-diol 13,14-epoxide adduct at N2 of deoxyguanosine in a duplex oligonucleotide

When a synthesized deoxyribonucleotide duplex, 5'-CCATCGCTACC-3'.5'-GGTAGCGATGG-3', containing a trans 14R dibenzo[a,l]pyrene (DB[a,l]P) adduct, corresponding to trans opening of the (+)-(11S,12R)-diol (13R,14S)-epoxide by N (2) of the central G residue, was allowed to stand for 2-6 days at ambient temperature in neutral aqueous solution, three new products were observed on denaturing HPLC. One of these corresponded to loss of the DB[a,l]P moiety from the original adducted strand to give an 11-mer with an unmodified central dG. The other two products resulted from a highly unexpected migration of the hydrocarbon moiety to either dG5 or dG7 of the complementary strand, 5'-GGTAG5CG7ATGG-3'. Enzymatic hydrolysis of the two 11-mer migration products followed by CD spectroscopy of the isolated adducted nucleosides indicated that, in both cases, the hydrocarbon moiety had undergone configurational inversion at C14 to give the cis 14S DB[a,l]P dG adduct. MS/MS and partial enzymatic hydrolysis showed that the major 11-mer had the hydrocarbon at dG7. Two 11-mer oligonucleotides were synthesized with a single cis 14S DB[a,l]P dG adduct either at G7 or at G5 and were found to be chromatographically identical to the major and minor migration products, respectively. Although HPLC evidence suggested that a small extent of hydrocarbon migration from the trans 14S DB[a,l]P dG diastereomer also occurred, the very small amount of presumed migration products from this isomer precluded their detailed characterization. This interstrand migration appears unique to DB[a,l]P adducts and has not been observed for their fjord-region benzo[c]phenanthrene or bay-region benzo[a]pyrene analogues.     

GenoMass software: a tool based on electrospray ionization tandem mass spectrometry for characterization and sequencing of oligonucleotide adducts

The analysis of DNA adducts is of importance in understanding DNA damage, and in the last few years mass spectrometry (MS) has emerged as the most comprehensive and versatile tool for routine characterization of modified oligonucleotides. The structural analysis of modified oligonucleotides, although routinely analyzed using mass spectrometry, is followed by a large amount of data, and a significant challenge is to locate the exact position of the adduct by computational spectral interpretation, which still is a bottleneck. In this report, we present an additional feature of the in‐house developed GenoMass software, which determines the exact location of an adduct in modified oligonucleotides by connecting tandem mass spectrometry (MS/MS) to a combinatorial isomer library generated in silico for nucleic acids. The performance of this MS/MS approach using GenoMass software was evaluated by MS/MS data interpretation for an unadducted and its corresponding N‐acetylaminofluorene (AAF) adducted 17‐mer (5'OH‐CCT ACC CCT TCC TTG TA‐3′OH) oligonucleotide. Further computational screening of this AAF adducted 17‐mer oligonucleotide (5′OH‐CCT ACC CCT TCC TTG TA‐3′OH) from a complex oligonucleotide mixture was performed using GenoMass. Finally, GenoMass was also used to identify the positional isomers of the AAF adducted 15‐mer oligonucleotide (5′OH‐ATGAACCGGAGGCCC‐3′OH). GenoMass is a simple, fast, data interpretation software that uses an in silico constructed library to relate the MS/MS sequencing approach to identify the exact location of adduct on oligonucleotides. Copyright © 2012 John Wiley & Sons, Ltd.     

Synthesis of oligonucleotides containing 7-(2-deoxy-β-d-erythro-pentofuranosyl)guanine and 8-amino-2′-deoxyguanosine

The synthesis of oligonucleotides containing 7-(2-deoxy-β-D-erythro-pentofuranosyl)guanine and 8-amino-2′-deoxyguanosine was accomplished. The viable intermediate N²-isobutyryl-7-(2-deoxy-β-D-erythro-pentofuranosyl)guanine ( 6 ) was prepared via a four step deoxygenation procedure from 7-β-D-ribofuranosylguanine ( 1 ). The 5′-hydroxyl group of 6 was protected as 4,4′-dimethoxytrityl ether and then converted to the target phosphoramidite ( 8 ) via conventional phosphitylation procedure. The amino groups of 8-amino-2′-deoxyguanosine ( 9 ) were protected in the form of N-(dimethylainino)methylene functions to give the protected nucleoside 10 , which was subsequently converted to the target phosphoramidite 12 via dimethoxytritylation followed by phosphitylation. The phosphoramidites 8 and 12 were incorporated into a 26-mer and a 31-mer G-rich oligonucleotide using solid-support, phosphoramidite methodology. Analysis of antiparallel triplex formation by the oligonucleotides containing 7-(2-deoxy-β-D-erythro-pentofura-nosyl)guanine in place of 2′-deoxyguanosine showed no enhancement in triple helix formation.     

Pyrazolo[3,4-d][1,2,3]triazine DNA: synthesis and base pairing of 7-deaza-2,8-diaza-2'-deoxyadenosine

7-deaza-2,8-diaza-2'-deoxyadenosine (4) was synthesized from 8-aza-7-deaza-2'-deoxyadenosine (1) via the 1,N(6)-etheno derivative 5. Ring opening with sodium hydroxide followed by ring closure in the presence of sodium nitrite formed the tricyclic intermediate 5 from which the transiently introduced "etheno" moiety was removed with NBS. Compound 4 was converted to the phosphoramidite 11, which was employed in solid-phase oligonucleotide synthesis. Base pairing studies on 4, incorporated in a 12-mer duplex, showed that this adenine nucleoside analogue forms a strong base pair with dG but not with dT. This novel base pair is as stable as that of the canonical dA-dT pair. As a result of the absence of nitrogen-7 compound 4 is expected to form a face to face base pair with dG.     

DNA microarray synthesis by using PDMS molecular stamp (II)

Based on the standard phosphoramidites chemistry protocol, two oligonucleotides synthetic routes were studied by contact stamping reactants to a modified glass slide. Route A was a contact coupling reaction, in which a nucleoside monomer was transferred and coupled to reactive groups (OH) on a substrate by spreading the nucleoside activated with tetrazole on a polydimethylsiloxane (PDMS) stamp. Route B was a contact detritylation, in which one nucleoside was fixed on the desired synthesis regions where dimethoxytrityl (DMT) protecting groups on the 5’-hydroxyl of the support-bound nucleoside were removed by stamping trichloroacetic acid (TCA) distributed on features on a PDMS stamp. Experiments showed that the synthetic yield and the reaction speed of route A were higher than those of route B. It was shown that 20 mer oligonucleotide arrays immobilized on the glass slide were successfully synthesized using the PDMS stamps, and the coupling efficiency showed no difference between the PDMS stamping and the conventional synthesis methods.     

DNA microarray synthesis by using PDMS molecular stamp (II) Oligonucleotide on-chip synthesis using PDMS stamp

Based on the standard phosphoramidites chemistry protocol, two oligonucleotides synthetic routes were studied by contact stamping reactants to a modified glass slide. Route A was a contact coupling reaction, in which a nucleoside monomer was transferred and coupled to reactive groups (OH) on a substrate by spreading the nucleoside activated with tetrazole on a polydimethylsiloxane (PDMS) stamp. Route B was a contact detritylation, in which one nucleoside was fixed on the desired synthesis regions where dimethoxytrityl (DMT) protecting groups on the 5’-hydroxyl of the support-bound nucleoside were removed by stamping trichloroacetic acid (TCA) distributed on features on a PDMS stamp. Experiments showed that the synthetic yield and the reaction speed of route A were higher than those of route B. It was shown that 20 mer oligonucleotide arrays immobilized on the glass slide were successfully synthesized using the PDMS stamps, and the coupling efficiency showed no difference between the PDMS stamping and the conventional synthesis methods.     

Base excision repair synthesis of DNA containing 8-oxoguanine in Escherichia coli

8-oxo-7,8-dihydroguanine (8-oxo-G) in DNA is a mutagenic adduct formed by reactive oxygen species. In Escherichia coli, 2,6-dihydroxy-5N-formamidopyrimidine (Fapy)-DNA glycosylase (Fpg) removes this mutagenic adduct from DNA. In this report, we demonstrate base excision repair (BER) synthesis of DNA containing 8-oxo-G with Fpg in vitro. Fpg cut the oligonucleotide at the site of 8-oxo-G, producing one nucleotide gap with 3' and 5' phosphate termini. Next, 3' phosphatase(s) in the supernatant obtained by precipitating a crude extract of E. coli with 40% ammonium sulfate, removed the 3' phosphate group at the gap, thus exposing the 3' hydroxyl group to prime DNA synthesis. DNA polymerase and DNA ligase then completed the repair. These results indicate the biological significance of the glycosylase and apurinic/ apyrimidinic (AP) lyase activities of Fpg, in concert with 3' phosphatase(s) to create an appropriately gapped substrate for efficient BER synthesis of DNA containing 8-oxo-G.     

Quantitative parameters of cooperative interactions of oligonucleotides within tandem complexes

Quantitative parameters of cooperative interactions of deoxyribooligonucleotides within perfect complementary complexes with a nick in one strand and imperfect complexes containing one mismatched base pair in the nick were obtained. One complementary strand was represented by 22-mer oligonucleotides, while the other, by two short 8-mer oligonucleotides forming a tandem complex with the central part of the 22-mer. In the tandem complexes, the 8-mers form contacts of the following types: 5"-Py*pPy-3", 5"-Pu*pPy-3", and 5"-Pu*pPu-3", where p is phosphate, Py and Pu are pyrimidine and purine nucleosides, respectively, and * stands for a nick. In each incompletely complementary complex, the mismatched base pair in the nick is formed by the 3"-end nucleoside of the 8-mer oligonucleotide and by the nucleoside located in the middle of the 22-mer oligonucleotide. The alkylating 4-[N-(2-chloroethyl)-N-methylamino]phenyl}methylamino group (RCl) is linked through the 5"-end phosphate of the 8-mers (reagents) close to 3"-ends of the 22-mers. The dependences of the limit extents of alkylation of 22-mers (targets) at zero and saturating concentrations of the neighbor oligonucleotides (effectors) on the initial concentration of RCl-derivatives of oligonucleotides (reagents) were used to calculate the association constants K _X of the reagent with the target. The ratio of these constants was used to determine the parameters of contact cooperativity , which characterize the interactions at the junction of two oligonucleotides within the tandem complexes.     

Oligomeric building block approach to the synthesis of diastereomerically pure pentathymidine 3',5'-methanephosphonates

[formula: see text] A method for a large-scale synthesis of stereodefined oligo(nucleoside 3',5'-methanephosphonates) has been developed, based on transient 3'-O protection, which allows for the conversion of the protecting chirally defined methanephosphonanilidate group, located at the 3' end of a stereoregular oligomer, into diastereomerically pure "oligomeric building blocks" for stereospecific coupling with the 5'-OH group of another oligonucleotide.     

Structural characterization of carcinogen-modified oligodeoxynucleotide adducts using matrix-assisted laser desorption/ionization mass spectrometry

The aim of this study was to determine the chemical structure of in vitro 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-modified oligodeoxynucleotides (ODNs) by exonuclease digestion and matrix-assisted laser desorption/ionization mass spectrometry. A single-stranded 11-mer ODN, 5'-d(CCATCGCTACC), was reacted with N-acetoxy-PhIP, resulting in the formation of one major and eight minor PhIP-ODN adducts. A 10 min treatment of the major and one minor PhIP-ODN adduct with a 3'-exonuclease, bovine intestinal mucosa phosphodiesterase (BIMP), and a 5'-exonuclease, bovine spleen phosphodiesterase, results in inhibition of the primary exonuclease activity at deoxyguanosine (dG) producing 5'-d(CCATCG(PhIP)) and 5'-d(G(PhIP)CTACC) product ions, respectively. Post-source decay (PSD) of these enzymatic end products identifies dG as the sole modification site in two 11-mer ODN-PhIP adducts. PSD of the minor PhIP-ODN adduct digestion end product, 5'-d(CCATCG(PhIP)), also reveals that the PhIP adducted guanine moiety is in an oxidized form. Prolonged treatment of the PhIP-ODN adducts at 37 degrees C with BIMP induces a non-specific, or endonuclease, enzymatic activity culminating in the formation of deoxyguanosine 5'-monophosphate-PhIP (5'-dGMP-PhIP). The PSD fragmentation pattern of the 5'-dGMP-PhIP [M + H](+) ion of the major adduct confirms PhIP binds to the C-8 position of dG. For the minor adduct, PSD results suggest that PhIP binds to the C-8 position of an oxidized guanine, supporting the hypothesis that this adduct arises from oxidative degradation, resulting in a spirobisguanidino structure.     

Synthesis of regioisomeric analogues of crisamicin A

The synthesis of bis-furonaphthopyrans 12a and 12b, regioisomeric analogues of the dimeric pyranonaphthoquinone antibiotic crisamicin A 1 is described. The key intermediate 16 was prepared via a one-pot in situ Suzuki-Miyaura homocoupling of naphthyl triflate 23 using bis(pinacolato)diboron. Oxidation of binaphthyl 16 to bis-naphthoquinone 14 was then effected with silver(II) oxide and nitric acid. Efficient double furofuran annulation of bis-naphthoquinone 14 with 2-trimethylsilyloxyfuran 8 afforded bis-furonaphthofuran adducts 13a and 13b as an inseparable 1:1 mixture of diastereomers. Oxidative rearrangement of this mixture of bis-furonaphthofuran adducts 13a and 13b using silver(II) oxide and nitric acid afforded unstable bis-furonaphthopyrans 12a and 12b also as a 1:1 mixture of diastereomers. Addition of 2-trimethylsilyloxyfuran 8 to naphthoquinone 25 afforded adduct 26 that underwent oxidative rearrangement to furonaphthopyran 27, however attempts to effect Suzuki-Miyaura homocoupling of triflates 26 and 27 to their respective dimers 13 and 12, was unsuccessful.     

An efficient synthesis of cyclic IDP- and cyclic 8-bromo-IDP-carbocyclic-riboses using a modified Hata condensation method to form an intramolecular pyrophosphate linkage as a key step. An entry to a general method for the chemical synthesis of cyclic ADP-ribose analogues

An efficient synthesis of cyclic IDP-carbocyclic-ribose (3) and its 8-bromo derivative 6, as stable mimics of cyclic ADP-ribose, was achieved, and a condensation reaction with phenylthiophosphate-type substrate 15 or 16 to form an intramolecular pyrophosphate linkage was a key step. N-1-Carbocyclic-ribosylinosine derivative 28 and the corresponding 8-bromo congener 24 were prepared via condensation between N-1-(2,4-dinitrophenyl)inosine derivative 17 and a known optically active carbocyclic amine 18. Compounds 24 and 28 were then converted to the corresponding 5"-phosphoryl-5'-phenylthiophosphate derivatives 15 and 16, respectively, which were substrates for the condensation reaction to form an intramolecular pyrophosphate linkage. Treatment of 8-bromo substrate 15 with I2 or AgNO3 in the presence of molecular sieves 3A (MS 3A) in pyridine at room temperature gave the desired cyclic product 12 quantitatively, while the yield was quite low without MS. The similar reaction of 8-unsubstituted substrate 16 gave the corresponding cyclized product 32 in 81% yield. Acidic treatment of these cyclic pyrophosphates 12 and 32 readily gave the targets 6 and 3, respectively. This result suggests that the construction of N-1-substituted hypoxanthine nucleoside structures from N-1-(2,4-dinitrophenyl)inosine derivatives and the intramolecular condensation by activation of the phenylthiophosphate group with I2 or AgNO3/MS 3A combine to provide a very efficient route for the synthesis of analogues of cyclic ADP-ribose such as 3 and 6. Thus, this may be an entry to a general method for synthesizing biologically important cyclic nucleotides of this type.     

A New and Efficient Method for Synthesis of 5′‐Conjugates of Oligonucleotides through Amide‐Bond Formation on Solid Phase

An efficient method for synthesis of oligonucleotide 5′‐conjugates through amide‐bond formation on solid phase is described. Protected oligonucleotides containing a 5′‐carboxylic acid function were obtained by use of a novel non‐nucleosidic phosphoramidite building block, where the carboxylic acid moiety was protected by a 2‐chlorotrityl group. The protecting group is stable to the phosphoramidite coupling conditions used in solid‐phase oligonucleotide assembly, but is easily deprotected by mild acidic treatment. The protecting group may be removed also by ammonolysis. 5′‐Carboxylate‐modified oligonucleotides were efficiently conjugated on solid support under normal peptide‐coupling conditions to various amines or to the N‐termini of small peptides to yield products of high purity. The method is well‐suited in principle for the synthesis of peptide‐oligonucleotide conjugates containing an amide linkage between the 5′‐end of an oligonucleotide and the N‐terminus of a peptide.     

pH-Independent triplex formation: hairpin DNA containing isoguanine or 9-deaza-9-propynylguanine in place of protonated cytosine

Triplex-forming oligonucleotides (TFOs) containing 2'-deoxyisoguanosine (2), 7-bromo-7-deaza-2'-deoxyisoguanosine (2) as well as the propynylated 9-deazaguanine N7-(2'-deoxyribonucleoside) were prepared. For this the phosphoramidites 9a, b of the nucleoside 1 and, the phosphoramidites 19, 20 of compound 3b were synthesized. They were employed in solid-phase oligonucleotide synthesis to yield the protected 31-mer oligonucleotides. The deblocking of the allyl-protected oligonucleotides containing 1 was carried out by Pd(0)[PPh3]4-PPh3 followed by 25% aq. NH3. Formation of the 31-mer single-stranded intramolecular triplexes was studied by UV-melting curve analysis. In the single-stranded 31-mer oligonucleotides the protonated dC in the dCH(+)-dG-dC base triad was replaced by 2'-deoxyisoguanosine (1), 7-bromo-7-deaza-2'-deoxyisoguanosine (2) and, 9-deaza-9-propynylguanine N7-(2'-deoxyribonucleoside) (3b). The replacement of protonated dC by compounds 1 and 3b resulted in intramolecular triplexes which are formed pH-independently and are stable under neutral conditions. These triplexes contain "purine" nucleosides in the third pyrimidine rich strand of the oligonucleotide hairpin.     

Asymmetric synthesis of cyclohexene nucleoside analogues

The asymmetric synthesis of novel cyclohexene nucleoside analogues 12 and 15 is described. An enantiospecific Diels-Alder reaction between (E,E)-diene 2 and (+)-5-(d-mentyloxy)-2(5H)-furanone 3 provided the cycloadduct isomer 4. Three additional steps yielded amine 8 allowing the constructions of the thymine and adenine moieties to afford intermediates 11 and 14, respectively. Amination or cyclization and removal of the protecting groups occurred in one step in the presence of ammonia, giving the target six-membered ring nucleosides.     

Total synthesis of (+/-)-symbioimine

The synthesis of (+/-)-symbioimine (1) has been completed in only 12 linear steps in 8% overall yield. The key step is the treatment of 13b with BF3.Et2O to generate N-carboalkoxydihydropyridinium cation 14b, which undergoes a novel stereospecific intramolecular Diels-Alder reaction to give adduct 16b in 42% yield. Cleavage of the N-Troc group of 16b afforded imine 24b stereospecifically. Cleavage of the TBDMS ethers and sulfation provided (+/-)-symbioimine (1). [reaction: see text].     

A practical synthesis of (+)-biotin from L-cysteine

Alpha-amino aldehyde 4, which is readily derived from L-cysteine through cyclization and elaboration of the carboxy group, was subjected to the Strecker reaction, which, via sodium bisulfite adduct 16, afforded alpha-amino nitrile 5 with high diastereoselectivity (syn/anti=11:1) and in high yield. Amide 6, derived from 5, was converted to thiolactone 8, a key intermediate in the synthesis of (+)-biotin (1), by a novel S,N-carbonyl migration and cyclization reaction. The Fukuyama coupling reaction of 8 with the zinc reagent 21, which has an ester group, in the presence of a heterogeneous Pd/C catalyst allowed the efficient installation of the 4-carboxybutyl chain to provide 9. Compound 9 was hydrogenated and the protecting groups removed to furnish 1 in 10 steps and in 34 % overall yield from L-cysteine.     

A conformationally preorganized universal solid support for efficient oligonucleotide synthesis

A novel, conformationally preorganized nonnucleosidic universal solid support for oligonucleotide synthesis was developed. The solid support featured two chemically equivalent hydroxy groups locked in syn-periplanar orientation and orthogonally protected with 4,4'-dimethoxytrityl and acetyl groups. The solid support was extensively tested in the preparation of oligonucleotides and their phosphorothioate analogues containing 2'-deoxy, 2'-O-methyl, and 2'-O-methoxyethylnucleoside residues at the 3'-terminus. Upon completion of oligonucleotide chain assembly, the support-bound oligonucleotide material was treated with concentrated ammonium hydroxide, which removed the O-acetyl protection. The deprotected hydroxy group then effected the transesterification of a phosphate linkage between the solid support and the 3'-terminal nucleoside residue to result in a facile release of the oligonucleotide to solution. The kinetics of the release process was studied in a continuous flow of concentrated aqueous ammonium hydroxide at a temperature of 300.15 K. Optimal conditions for the release of oligonucleotides depending on the chemistry of the backbone and 3'-terminal nucleoside residue were formulated.     

Infrared photodissociation of non-covalent adducts of electrosprayed nucleotide ions

Efficient dissociation of gaseous non-covalent adducts of multiply charged DNA anions can be effected by infrared irradiation to yield minimal dissociation of the DNA molecular ions-far less dissociation than by collisional activation. Examples include removal of adducted impurities from 100-mer DNA anions and removal of all 14 adducted molecules of 1,4-diaminobutane from M^15? to M^17? of a 50-mer DNA.