Design, synthesis, and properties of 2',4'-BNA(NC): a bridged nucleic acid analogue

The novel bridged nucleic-acid analogue 2',4'-BNA(NC) (2'-O,4'-C-aminomethylene bridged nucleic acid), containing a six-membered bridged structure with an N-O linkage, was designed and synthesized efficiently, demonstrating a one-pot intramolecular NC bond-forming key reaction to construct a perhydro-1,2-oxazine ring (11 and 12). Three monomers of 2',4'-BNA(NC) (2',4'-BNA(NC)[NH], [NMe], and [NBn]) were synthesized and incorporated into oligonucleotides, and their properties were investigated and compared with those of 2',4'-BNA (LNA)-modified oligonucleotides. Compared to 2',4'-BNA (LNA)-modified oligonucleotides, 2',4'-BNA(NC) congeners were found to possess: (i) equal or higher binding affinity against an RNA complement with excellent single-mismatch discriminating power, (ii) much better RNA selective binding, (iii) stronger and more sequence selective triplex-forming characters, and (iv) immensely higher nuclease resistance, even higher than the S(p)-phosphorthioate analogue. 2',4'-BNA(NC)-modified oligonucleotides with these excellent profiles show great promise for applications in antisense and antigene technologies.     

Formation of modified cytosine residues in the presence of depurinated DNA

[Chemical reaction: See text] Depurination is an important degradation pathway for antisense phosphorothioate oligonucleotides under conditions of thermal stress. We present evidence showing that depurinated oligonucleotides react with cytosine-containing sequences giving products containing a 6-(2-deoxy-beta-D-erythro-pentofuranosyl)-3-(2-oxopropyl)imidazo[1,2-c]pyrimidin-5(6H)-one residue. Further, we demonstrate that the same product is formed upon treatment of 2'-deoxycytidine with 4-oxo-2-pentenal, the latter being an expected byproduct of serial elimination reactions at apurinic sites. In addition to being important for synthetic oligonucleotides, apurinic site formation in cellular DNA is a common occurrence. Because repair of these sites can result in the production of 4-oxo-2-pentenal, it is interesting to speculate whether 6-(2-deoxy-beta-D-erythro-pentofuranosyl)-3-(2-oxopropyl)imidazo[1,2-c]pyrimidin-5(6H)-one residues can form in vivo.     

5' modification of duplex DNA with a ruthenium electron donor-acceptor pair using solid-phase DNA synthesis

Incorporation of metalated nucleosides into DNA through covalent modification is crucial to measurement of thermal electron-transfer rates and the dependence of these rates with structure, distance, and position. Here, we report the first synthesis of an electron donor-acceptor pair of 5' metallonucleosides and their subsequent incorporation into oligonucleotides using solid-phase DNA synthesis techniques. Large-scale syntheses of metal-containing oligonucleotides are achieved using 5' modified phosporamidites containing [Ru(acac)(2)(IMPy)](2+) (acac is acetylacetonato; IMPy is 2'-iminomethylpyridyl-2'-deoxyuridine) (3) and [Ru(bpy)(2)(IMPy)](2+) (bpy is 2,2'-bipyridine; IMPy is 2'-iminomethylpyridyl-2'-deoxyuridine) (4). Duplexes formed with the metal-containing oligonucleotides exhibit thermal stability comparable to the corresponding unmetalated duplexes (T(m) of modified duplex = 49 degrees C vs T(m) of unmodified duplex = 47 degrees C). Electrochemical (3, E(1/2) = -0.04 V vs NHE; 4, E(1/2) = 1.12 V vs NHE), absorption (3, lambda(max) = 568, 369 nm; 4, lambda(max) = 480 nm), and emission (4, lambda(max) = 720 nm, tau = 55 ns, Phi = 1.2 x 10(-)(4)) data for the ruthenium-modified nucleosides and oligonucleotides indicate that incorporation into an oligonucleotide does not perturb the electronic properties of the ruthenium complex or the DNA significantly. In addition, the absence of any change in the emission properties upon metalated duplex formation suggests that the [Ru(bpy)(2)(IMPy)](2+)[Ru(acac)(2)(IMPy)](2+) pair will provide a valuable probe for DNA-mediated electron-transfer studies.     

Thermolytic 4-methylthio-1-butyl group for phosphate/thiophosphate protection in solid-phase synthesis of DNA oligonucleotides

The thermolabile 4-methylthio-1-butyl phosphate/thiophosphate protecting group for DNA oligonucleotides has been investigated for its potential application to a "heat-driven" process for either oligonucleotide synthesis on diagnostic microarrays or, oppositely, to the large-scale preparation of therapeutic oligonucleotides. The preparation of phosphoramidites 10a-d is straightforward, and the incorporation of these amidites into oligonucleotides via solid-phase techniques proceeds as efficiently as that achieved with 2-cyanoethyl deoxyribonucleoside phosphoramidites. The versatility of the 4-methylthio-1-butyl phosphate/thiophosphate protecting group is exemplified by its facile removal from oligonucleotides upon heating for 30 min at 55 degrees C in an aqueous buffer under neutral conditions or within 2 h at 55 degrees C in concentrated NH(4)OH. The deprotection reaction occurs through an intramolecular cyclodeesterification mechanism leading to the formation of sulfonium salt 18. When mixed with deoxyribonucleosides and N-protected 2'-deoxyribonucleosides or with a model phosphorothioate diester under conditions approximating those of large-scale (>50 mmol) oligonucleotide deprotection reactions, the salt 18 did not significantly alter DNA nucleobases or desulfurize the phosphorothioate diester model to an appreciable extent.     

2′‐Deoxyuridine and 2′‐Deoxyisocytidine as Constituents of DNA with Parallel Chain Orientation: The Stabilization of the iCd⋅Gd Base Pair by the 5‐Methyl Group

Parallel‐stranded oligonucleotides containing 2′‐deoxyuridine ( 2 ) and 2′‐deoxyisocytidine ( 4 ) were synthesized. The phosphoramidite 11 employed in the solid‐phase synthesis carries a (dimethylamino)methylidene residue as amino‐protecting group. This group stabilizes the acid‐labile glycosylic bond of 4 and enables the base‐catalyzed deprotection of oligonucleotides without degrading the nucleoside 4 residues. Oligonucleotide duplexes incorporating the 5‐Me derivatives of 2 (→2′‐deoxythymidine) and 4 (→2′‐deoxy‐5‐methylisocytidine), which are more stable than those containing the unmethylated nucleosides, were also compared. Depending on the nearest‐neighbor environment, Me groups provide an additional stabilization through Me/Me contacts or Me/backbone interactions.     

Synthesis of chimeric oligonucleotides containing phosphodiester, phosphorothioate, and phosphoramidate linkages

[reaction: see text] H-Phosphonate monomers of 2'-O-(2-methoxyethyl) ribonucleosides have been synthesized. Oxidation of oligonucleotide H-phosphonates has been optimized to allow the synthesis of oligonucleotides containing either 2'-deoxy or 2'-O-(2-methoxyethyl) ribonucleoside residues combined with three different phosphate modifications in the backbone, i.e., phosphodiester (PO), phosphorothioate (PS), and phosphoramidate (PN). Phosphodiester linkages were introduced by oxidation with a cocktail of 0.1 M Et(3)N in CCl(4)/Pyr/H(2)O (5:9:1) without affecting phosphorothioate or phosphoramidate linkages. For the synthesis of phosphoramidate-modified oligonucleotides, N(4)-acetyl deoxycytidine-3'-H-phosphonate monomers were used to avoid transamination during the oxidation step.     

Pyrrolo-dC oligonucleotides bearing alkynyl side chains with terminal triple bonds: synthesis, base pairing and fluorescent dye conjugates prepared by the azide-alkyne "click" reaction

5-(Octa-1,7-diynyl)-2'-deoxyuridine was converted into the furano-dU derivative 7 by copper-catalyzed cyclization; the pyrolodC-derivative 3 was formed upon ammonolysis. The bicyclic nucleosides 3 and 7 as well as the corresponding non-cyclic precursors 4 and 6 all containing terminal C[triple bond]C bonds were conjugated with the non-fluorescent 3-azido-7-hydroxycoumarin 5 employing the copper(I)-catalyzed Huisgen-Sharpless-Meldal cycloaddition "click reaction". Strongly fluorescent 1H-1,2,3-triazole conjugates (30-33) are formed incorporating two fluorescent reporters-the pyrdC nucleoside and the coumarin moiety. Oligonucleotides incorporating 6-alkynyl and 6-alkyl 7H-pyrrolo[2,3-d]pyrimidin-2(3H)-one nucleosides (3 and 2f) have been prepared by solid-phase synthesis using the phosphoramidite building blocks 10 and 13 ; the pyrrolo-dC oligonucleotides are formed during ammonia treatment. The duplex stability of oligonucleotides containing 3 and related derivatives was studied. Oligonucleotides with terminal triple bonded nucleosides such as 3 are more stabilizing than those lacking a side chain with terminal unsaturation; open-chain derivatives (4) are even more efficient. The click reaction was also performed on oligonucleotides containing the pyrdC-derivative and the fluorescence properties of nucleosides, oligonucleotides and their coumarin conjugates were studied.     

Pentopyranosyl Oligonucleotide Systems,Communication No.12, The β‐D‐Xylopyranosyl‐(4′→2′‐oligonucleotide System

β‐D ‐Xylopyranosyl‐(4′→2′)‐oligonucleotides containing adenine and thymine as nucleobases were synthesized as a part of a systematic study of the pairing properties of pentopyranosyl oligonucleotides. Contrary to earlier expectations based on qualitative conformational criteria, β‐D ‐xylopyranosyl‐(4′→2′)‐oligonucleotides show Watson‐Crick pairing comparable in strength to that shown by pyranosyl‐RNA.     

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.     

Oligonucleotide duplexes containing N8-glycosylated 8-aza-7-deazaguanine and self-assembly of 8-aza-7-deazapurines on the nucleoside and the oligomeric level

The 8-aza-7-deazaguanine N8-(2'-deoxy-beta-D-ribofuranoside) (1) was synthesized, converted into the phosphoramidite 4 and incorporated into oligonucleotides. Nucleoside 1 forms stable base pairs with 2'-deoxy-5-methylisocytidine in DNA with antiparallel chain orientation (aps) and with 2'-deoxycytidine in duplexes with parallel chains (ps). According to the CD spectra self-complementary oligonucleotides d(1-m5isoC)3 and d(1-C), form autonomous DNA-structures. Neither the nucleoside 1 nor the regularly linked 8-aza-7-deaza-2'-deoxyguanosine form G-like tetrads while the regularly linked 8-aza-7-deaza-2'-deoxyisoguanosine gives higher molecular assemblies which are destroyed by bulky 7-bromo substituents. This was verified on monomeric nucleosides by ESI-MS spectrometry and on oligonucleotides by HPLC analysis.     

Binding of DNA purine sites to dirhodium compounds probed by mass spectrometry

The adducts formed between the antitumor active compounds [Rh(2)(O(2)CCH(3))(2)(CH(3)CN)(6)](BF(4))(2), Rh(2)(O(2)CCH(3))(4), and Rh(2)(O(2)CCF(3))(4) with DNA oligonucleotides have been assessed by matrix-assisted laser desorption ionization (MALDI) and nanoelectrospray (nanoESI) coupled to time-of-flight mass spectrometry (TOF MS). A series of MALDI studies performed on dipurine (AA, AG, GA, and GG)-containing single-stranded oligonucleotides of different lengths (tetra- to dodecamers) led to the establishment of the relative reactivity cis-[Pt(NH(3))(2)(OH(2))(2)](2+) (activated cisplatin) approximately Rh(2)(O(2)CCF(3))(4) > cis-[Pt(NH(3))(2)Cl(2)] (cisplatin) > [Rh(2)(O(2)CCH(3))(2)(CH(3)CN)(6)](BF(4))(2) > Rh(2)(O(2)CCH(3))(4) approximately Pt(C(6)H(6)O(4))(NH(3))(2) (carboplatin). The relative reactivity of the complexes is associated with the lability of the leaving groups. The general trend is that an increase in the length of the oligonucleotide leads to enhanced reactivity for Rh(2)(O(2)CCH(3))(2)(CH(3)CN)(6)](BF(4))(2) and Rh(2)(O(2)CCH(3))(4) (except for the case of [Rh(2)(O(2)CCH(3))(2)(CH(3)CN)(6)](2+), which reacts faster with the GG octamers than with the dodecamers), whereas the reactivity of Rh(2)(O(2)CCF(3))(4) is independent of the oligonucleotide length. When monitored by ESI, the dodecamers containing GG react faster than the respectiveAA oligonucleotides in reactions with Rh(2)(O(2)CCF(3))(4) and Rh(2)(O(2)CCH(3))(2)(CH(3)CN)(6)](BF(4))(2), whereas AA oligonucleotides react faster with Rh(2)(O(2)CCH(3))(4). The mixed (AG, GA) purine sequences exhibit comparable rates of reactivity with the homopurine (AA, GG) dodecamers in reactions with Rh(2)(O(2)CCH(3))(4). The observation of initial dirhodium-DNA adducts with weak axial (ax) interactions, followed by rearrangement to more stable equatorial (eq) adducts, was achieved by electrospray ionization; the Rh-Rh bond as well as coordinated acetate or acetonitrile ligands remain intact in these dirhodium-DNA adducts. MALDI in-source decay (ISD), collision-induced dissociation (CID) MS-MS, and enzymatic digestion studies followed by MALDI and ESI MS reveal that, in the dirhodium compounds studied, the purine sites of the DNA oligonucleotides interact with the dirhodium core. Ultimately, both MALDI and ESI MS proved to be complementary, valuable tools for probing the identity and stability of dinuclear metal-DNA adducts.     

A theoretical and experimental study of two thiazole orange derivatives with single- and double-stranded oligonucleotides, polydeoxyribonucleotides and DNA

The effect of interaction with DNA and oligonucleotides on the photophysical properties of two thiazole orange (TO) derivatives, with different side chains (-(CH2)3-N+(CH3)3 and -(CH2)6-I)) linked to the nitrogen of the quinoline ring of the thiazole orange, is presented here. The first one called TO-PRO1 is a commercially available dye, whereas the second one called TO-MET has been specially synthesized for further covalent binding to oligonucleotides with the aim of being used for specific in situ detection of biomolecular interactions. Both photophysical measurements and molecular calculations have been done to assess their possible mode of interaction with DNA. When dissolved in buffered aqueous solutions both derivatives exhibit very low fluorescence quantum yields of 8 x 10(-5) and 2 x 10(-4), respectively. However, upon binding to double-stranded DNA, large spectroscopic changes result and the quantum yield of fluorescence is enhanced by four orders of magnitude, reaching values up to phi F = 0.2 and 0.3, respectively, as a result of an intercalation mechanism between DNA base pairs. A modulation of the quantum yield is observed as a function of the base sequence. The two derivatives also bind with single-stranded oligonucleotides, but the fluorescence quantum yield is not so great as that when bound to double-stranded samples. Typical fluorescence quantum yields of 7 x 10(-3) to 3 x 10(-2) are observed when the dyes interact with short oligonucleotides, whereas the fluorescence quantum yield remains below 10(-2) when interacting with single-stranded oligonucleotides. This slight but significant quantum-yield increase is interpreted as a folding of the single strand around the dye, which reduces the internal rotation of the two heterocycles around the central methine bridge that links the two moieties of the dye. From these properties, it is proposed to link monomer covalently to oligonucleotides for the subsequent detection of target sequences within cells.     

A PELDOR-based nanometer distance ruler for oligonucleotides

A pulsed electron paramagnetic resonance (EPR) spectroscopic ruler for oligonucleotides was developed using a series of duplex DNAs. The spin-labeling is accomplished during solid-phase synthesis of the oligonucleotides utilizing a palladium-catalyzed cross-coupling reaction between 5-iodo-2'-deoxyuridine and the rigid spin-label 2,2,5,5-tetramethyl-pyrrolin-1-yloxyl-3-acetylene (TPA). 4-Pulse electron double resonance (PELDOR) was then used to measure the intramolecular spin-spin distances via the dipolar coupling, yielding spin-spin distances of 19.2, 23.3, 34.7, 44.8, and 52.5 A. Employing a full-atom force field with explicit water, molecular dynamic (MD) simulations on the same spin-labeled oligonucleotides in their duplex B-form gave spin-spin distances of 19.6, 21.4, 33.0, 43.3, and 52.5 A, respectively, in very good agreement with the measured distances. This shows that the oligonucleotides adopt a B-form duplex structure also in frozen aqueous buffer solution. It also demonstrates that the combined use of site-directed spin-labeling, PELDOR experiments, and MD simulations can yield a microscopic picture about the overall structure of oligonucleotides. The technique is also applicable to more complex systems, like ribozymes or DNA/RNA-protein complexes, which are difficult to access by NMR or X-ray crystallography.     

Synthesis and Properties of Oligonucleotides Containing 2,4‐Dihydroxycyclohexyl Nucleosides

Cyclohexyl nucleosides with an adenine and uracil base have been synthesized from 2‐azidocyclohexane‐1,5‐diol. The obtained racemic nucleosides were resolved using (R)‐O‐methylmandelic acid. Short oligonucleotides were synthesized using phorphoramidite chemistry. However, these oligonucleotides do not show self‐hybridization, and duplexes are less stable than those of ribopyranosyl‐(4′ → 2′)‐oligonucleotides.     

Nucleotides,Part LXIX,Synthesis of Phosphoramidite Building Blocks of Isoxanthopterin N8‐(2′‐Deoxy‐β‐D‐ribonucleosides): New Fluorescence Markers for Oligonucleotide Synthesis

The chemical synthesis of isoxanthopterin and 6‐phenylisoxanthopterin N⁸‐(2′‐deoxy‐β‐D ‐ribofuranosyl nucleosides) is described as well as their conversion into suitably protected 3′‐phosphoramidite building blocks to be used as marker molecules for DNA synthesis. Applying the npe/npeoc (=2‐(4‐nitrophenyl)ethyl/[2‐(4‐nitrophenyl)ethoxy]carbonyl) strategy, we used the new building blocks in the preparation of oligonucleotides by an automated solid‐support approach. The hybridization properties of a series of labelled oligomers were studied by UV‐melting techniques. It was found that the newly synthesized markers only slightly interfered with the abilities of the labelled oligomers to form stable duplexes with complementary oligonucleotides.     

Novel interstrand communication systems within DNA duplexes based on 1-, 2- and 4-(phenylethynyl)pyrenes attached to 2'-amino-LNA: high-affinity hybridization and fluorescence sensing

Functionalisation of 2'-amino-LNA oligonucleotides with 1-, 2- and 4-(phenylethynyl)pyrene fluorophores via a carbonyl linker (PEPyc) resulted in efficient interstrand communication systems in nucleic acid duplexes, providing effective tools for stabilization of nanostructures and fluorescence monitoring of DNA self-assembly.     

Efficient conjugation of peptides to oligonucleotides by "native ligation"

A new strategy has been developed for conjugation of peptides to oligonucleotides. The method is based on the "native ligation" of an N-terminal thioester-functionalized peptide to a 5'-cysteinyl oligonucleotide. Two new reagents were synthesized for use in solid-phase peptide and oligonucleotide synthesis, respectively. Pentafluorophenyl S-benzylthiosuccinate was used in the final coupling step in standard Fmoc-based solid-phase peptide assembly. Deprotection with trifluoracetic acid generated in solution peptides substituted with an N-terminal S-benzylthiosuccinyl moiety. O-trans-4-(N-alpha-Fmoc-S-tert-butylsulfenyl-L-cysteinyl)aminoc yclohe xyl O-2-cyanoethyl-N,N-diisopropylphosphoramidite was used in the final coupling step in standard phosphoramidite solid-phase oligonucleotide assembly. Deprotection with aqueous ammonia solution generated in solution 5'-S-tert-butylsulfenyl-L-cysteinyl functionalized oligonucleotides. Functionalized peptides and oligonucleotides were used without purification in native ligation conjugation reactions in aqueous/organic solution using tris-(2-carboxyethyl)phosphine to remove the tert-butylsulfenyl group in situ and thiophenol as a conjugation enhancer. A range of peptide-oligonucleotide conjugates were prepared by this route and purified by reversed-phase HPLC.     

Template-free segmental synthesis of oligonucleotides containing nonnative linkages

Protected oligonucleotides containing 3'-alkyl carboxylic acids or 3'-alkylamines were obtained from photolabile solid-phase synthesis supports (1 and 4). Protected oligonucleotides containing 5'-alkylamines and 3'-hydroxyl groups were obtained using a photolabile solid-phase synthesis support (2) and a commercially available phosphoramidite reagent (3). Depending upon the source of alkylamine-containing oligonucleotide, the segments were coupled under mild conditions to form products containing either 5'-3' or 3'-3' linkages in good yield and high purity. Oligonucleotides as long as 40 nucleotides were prepared, and coupling yields of protected biopolymer segments were independent of length over the range examined. This method is particularly well suited for the convergent synthesis of oligonucleotides containing nonnative linkages and should be useful for the rapid assembly of modified biopolymers that are useful in biochemical studies.     

Synthesis and properties of a bridged nucleic acid with a perhydro-1,2-oxazin-3-one ring

A novel derivative of 2',4'-bridged nucleic acid, named hydroxamate-bridged nucleic acid (HxNA), containing a six-membered perhydro-1,2-oxazin-3-one ring, was designed and synthesized. The introduction of a carbonyl function along with an N-O linkage in the six-membered bridged structure is the unique structural feature of the novel 2',4'-bridged nucleic acid analogue. The design was carried out to restrict the flexibility of the sugar moiety through the trigonal planarity of carbonyl function, which would improve the properties of the modification. The synthesized monomer was incorporated into oligonucleotides, and their properties were examined. The HxNA-modified oligonucleotides exhibited selectively high affinity toward complementary ssRNA. Furthermore, the nuclease resistance of the HxNA-modified oligonucleotide was found to be higher than that of the corresponding natural and 2',4'-BNA/LNA-modified oligonucleotides. Interestingly, exposure of HxNA modified oligonucleotide to 3'-exonuclease resulted in gradual opening of the bridge, which stopped further digestion. Moreover, ring-opening of only one modification at the 3'-end of the oligonucleotides was observed, even if two or three HxNA modifications were present in the sequence. The results demonstrate the strong potential of the HxNA modification as a switch for the generation of highly nuclease-resistant RNA selective oligonucleotide in situ, which could have potential applications in antisense technology.     

Analysis of single nucleotide polymorphism sites in exon 4 of the p53 gene using high-performance liquid chromatography electrospray ionization mass spectrometry tandem mass spectrometry

Three groups of four oligonucleotides with special single nucleotide polymorphisms (SNP) sites in exon 4 of the p53 gene were analyzed with ion-pair reversed-phase high-performance liquid chromatography electrospray ionization mass spectrometry tandem mass spectrometry. The retention order of four oligonucleotides with SNPs was C < G < A < T, regardless of whether the polymorphisms were at the 3' end, the 5' end, or the middle of the oligonucleotides. The charge state of the molecular ion affects the MS/MS spectra of the oligonucleotides. SNPs at the 3' end can be easily identified from the fragmentation pattern of the 2- charge state, but not from the 3- charge state, especially from the w1 fragment. The single base may be taken as the symbol of the 5' end SNP site derived from [M3H]2, but not from the [M3H]2 charge state. The oligonucleotides with SNPs in the middle were also determined from the [M2H]2 precursor ion.