Synthesis of 1'-phenazine-tethered psicofuranosyl oligonucleotides: The thermal stability and fluorescence properties of their duplexes and triplexes (original) (raw)
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Helvetica Chimica Acta, 1999
Dedicated to Prof. Dr. Frank Seela on the occasion of his 60th birthday Dipyrido[3,2-a:2',3'-c]phenazine (dppz) derivatives were conjugated to 9-mer and 18-mer DNA (ODN) at a site without nucleobase, either at the 5'-or 3'-end or at a internucleotide position, via linkers of 7, 12, or 18 atoms lengths. These dppz-linked ODNs were synthesized using novel backbone glycerol phosphoramidites: Glycerol, serving as artificial nucleoside without nucleobase, was modified to amines 10, 23, and 24, which were suitable for the subsequent key reaction with dppz-carboxylic acid 3 (Schemes 2 and 3). The products of these reactions (see 5 ± 7) were then transformed to the standard phosphoramidite derivatives (see 27, 29, and 30) or used for loading on a CPG support (see 28, 31, and 32). The dppz-modified ODNs were subsequently assembled in the usual manner using automated solid-phase DNA synthesis. The 9-mer ODN-dppz conjugates 35 ± 43 were tested for their ability to form stable duplexes with target DNA or RNA strands (D11 (60) or R11 (61)), while the 18-mer ODN-dppz conjugates 48 ± 56 were tested for their ability to form stable triplexes with a DNA target duplex D24´D24 (62) (see Tables 1 and 2). The presence of the conjugated dppz derivative increases the stability of DNA´DNA and DNA´RNA duplexes, typically by a DT m of 7.3 ± 10.98 and 4.5 ± 7.48, respectively, when the dppz is tethered at the 5'-or 3'-terminal (Table 2). The dppz derivatives also stabilize triplexes when attached to the 5'-or 3'-end, with a DT m varying from 3.8 ± 11.18 (Table 3). The insertion of a dppz building block at the center of a 9-mer results in a considerably poorer stability of the corresponding DNA´DNA duplexes (DT m 0.5 to 4.28) and DNA´RNA duplexes (DT m À 1.5 to 0.98), while the replacement of one interior nucleotide by a dppz building unit in the corresponding 8-mer ODN does not reveal the formation of any duplex at all. Different types of modifications in the middle of the 18-mer ODN, in general, do not lead to any triplex formation, except when the dppz derivative is tethered to the ODN through a 12-atom-long linker (Entry 9 in Table 3).
Nucleosides, Nucleotides and Nucleic Acids, 2011
Carboxyalkyl derivative of the intercalating agent imidazo[4,5-b]phenazine was used for the post-synthetic oligonucleotide modification. Model pentadecathymidylate-imidazophenazine conjugate was prepared from 5'-aminoalkyl-modified (dT) 15 using phosphonium coupling reagent BOP in the presence of 1-hydroxybenzotriazole. Spectral-fluorescent properties of the conjugate were studied. The attachment of the dye was found to increase the thermal stability of (dT) 15 duplex with poly(dA) by more than 4°C, probably by intercalation mechanism.
Tetrahedron, 1997
Eleven different planar hydroxy alkylated polyarenes 1-11 with different geometry, bulk and electronic characteristics were synthesised, and used for tethering to the 5'-phosphate of a 9-mer and a 18-met DNA sequences through solid-phase synthesis. The 5'-polyarene-tethered 9-mers 30-40 were tested for their ability to form stable duplexes with four complementary target DNA-strands 25-28 of different length. The 5'-polyarene-tethered 18-mers 44-54 were tested for their ability to form stable triplexes with a 24-mer duplex target 41+42. The T m measurements of duplexes at low salt and pH 7.3 showed that the angular nitro phenanthrene and phenanthrene conjugates 31 and 30 gave the highest duplex stabilisations with targets 25 (ATm 13.8 ° C and 11.8" C) and 26 (ATm 12.3 ° C and 11.9 ° C). With the mismatch sequence 28, only 30 and 31 gave a high ATrn of 11.6 ° C and 10.8 ° C respectively, while lower AT m values were observed for other conjugates (ATm -4.0-5.0 ° C). The T m measurements of triplexes between 43-54 and duplex target 41+42 at low salt and pH 7.3, 6.5 and 6.0 without Mg 2+ showed that the nitro phenanthrene conjugate 45 gave the best triplex stabilisation (ATm 4.1-5.4 ° C). The stabilisation of nitro phcnanthrene conjugate 45 compared to phenanthrene conjugate 44 increased more remarkably when Mg 2÷ was present: 45 (ATm 15" C), 44 (ATm 10 ° C). These results imply that the electron density of the chromophore influences the n-~ stacking interactions between the chromophore and nucleobases, and thereby influencing the duplex and triplex stability. Fluorescence measurements on single strand to double strand transition indicated that the 5'-tethered polyarenes are stacked only on the neighbouring nucleobases of the opposite strand. In case of 5'-9-N-ethylphenazinium conjugate 36, a comparative NMR and fluorescence measurement has unambiguously shown that the tethered phenazinium ion is indeed intercalated between the nucleotides of the opposite target strand 26. Thermodynamic calculations showed the most stable AG°(298K) for 30, 31(+targets 25, 26, 28) and 35, 36(+targets 25, 26) compared to the blank 29 (AAG°(298K)~-10kJ mol-l). The non-palindromic target 27 was shown by Tm measurements to form a stable tertiary structure, which was very little affected by addition of any Y-tethered conjugate, thereby showing the importance of the tertiary structures of an in vivo antisense target and its implication in regard to its bioavailablity to complementary antisense probes. © 1997 Elsevier Science Ltd.
Pharmaceutics, 2021
Oligonucleotides with the sequences 5′-GTG AUPA TGC, 5′-GCA TAUP CAC and 5′-GUPG ATA UPGC, where UP is 2′-O-propargyl uridine, were subjected to post-synthetic Cu(I)-catalyzed azide–alkyne cycloaddition to attach 1,4,7,10-tetraazacyclododecane (cyclen) and two well-known DNA intercalating dyes: thioxanthone and 1,8-naphthalimide. We propose a convenient cyclen protection–deprotection strategy that allows efficient separation of the resulting polyamine–oligonucleotide conjugates from the starting materials by RP-HPLC to obtain high-purity products. In this paper, we present hitherto unknown macrocyclic polyamine–oligonucleotide conjugates and their hybridization properties reflected in the thermal stability of thirty-two DNA duplexes containing combinations of labeled strands, their unmodified complementary strands, and strands with single base pair mismatches. Circular dichroism measurements showed that the B-conformation is retained for all dsDNAs consisting of unmodified and modif...
Nucleic Acids Research, 1994
The 3'-Pzn group tethered to an oligo-DNA stabilizes a DNA -RNA hybrid duplex structure by 13°C compared to the natural counterpart. This report constitutes the first full study of the conformational features of a hybrid DNA -RNA duplex, which has been possible because of the unique stabilization of this rather small duplex by the tethered 3'-Pzn moiety (Tm = 40°C from NMR). In this study, a total of 252 interand intra-strand torsional and distance constraints along with the full NOE relaxation matrix, taking into account the exchange process of imino and amino protons with water, have been used. The 3'-Pznpromoted stabilization of the DNA -RNA hybrid duplex results in detailed local conformational characteristics such as the torsion angles of the backbone and sugar moieties that are close to the features of the other natural DNA -RNA hybrids (i.e. sugars of the RNA strand are 3'-endo, but the sugars of the DNA strand are intermediate between A-and B-forms of DNA, 720 < P < 1800; note however, that the sugars of our DNA strand have a C1,-exo conformation: 1310 < P < 1540). This study suggests that 3'-Pzn-tethered smaller oligo-DNA should serve the same purpose as a larger oligo-DNA as a antisense inhibitor of the viral mRNA. Additionally, these types of tethered oligos have been found to be relatively more resistant to the cellular nuclease. Moreover, they are taken up quite readily through the cellular membrane (14) compared to the natural counterparts.
Nucleosides, Nucleotides and Nucleic Acids, 2004
Synthetic polycarboxamide minor groove binders (MGB) consisting of N-methylpyrrole (Py), N-methylimidazole (Im), N-methyl-3-hydroxypyrrole (Hp) and balanine (b) show strong and sequence-specific interaction with the DNA minor groove in side-by-side antiparallel or parallel orientation. Two MGB moieties covalently linked to the same terminal phosphate of one DNA strand stabilize DNA duplexes formed by this strand with a complementary one in a sequence-specific manner, similarly to the corresponding mono-conjugated hairpin structures. The series of conjugates with the general formula Oligo-(L-MGB-R) m was synthesized, where ORDER REPRINTS m = 1 or 2, L = linker, R = terminal charged or neutral group, MGB = -(Py) n -, -(Im) n -or -[(Py/Im) n -(CH 2 ) 3 CONH -(Py/Im) n -] and 1 < n < 5. Using thermal denaturation, we studied effects of structural factors such as m and n, linker L length, nature and orientation of the MGB monomers, the group R and the backbone (DNA or RNA), etc. on the stability of the duplexes. Structural factors are more important for linear and hairpin monophosphoroamidates than for parallel bis-phosphoroamidates. No more than two oligocarboxamide strands can be inserted into the duplex minor groove. Attachment of the second sequence-specific parallel ligand [ -L(Py) 4 R] to monophosphoroamidate conjugate CGTTTATT -L(Py) 4 R leads to the increase of the duplex Tm, whereas attachment of [ -L(Im) 4 R] leads to its decrease. The mode of interaction between oligonucleotide duplex and attached ligands could be different (stacking with the terminal A:T pair of the duplex or its insertion into the minor groove) depending on the length and structure of the MGB.
Journal of Fluorescence, 2000
A novel intercalating phenazine derivative (Pzn) was covalently linked to the 3Ј-end of decathymidylate via a ribose residue of the dye. A fluorescence technique was used to study double helix formation by this conjugate with poly(rA) in aqueous solutions of neutral pH, at the presence of 0.1 and 1 M sodium ions. Proportionality between thermally induced changes in the fluorescence intensity of the free conjugate and bound one was revealed, that made it possible to calculate the helix-to-coil transition from fluorescence melting data using a simple equation. The transition curves were found to be in well conformity with those constructed from absorption measurements. It was shown that the attachment of Pzn significantly enhanced the stability of poly(rA) и (dT) 10 duplex due to intercalation of the dye chromophore into the adenine strand. The temperature of halfdissociation was increased by 12ЊC, and the stabilizing increment of standard free energy was 3.2-3.6 kcal/mol at 37ЊC.
Bioorganicheskaia khimiia
The effect of structural factors on the stability of duplexes formed by DNA minor groove binders conjugated with oligonucleotide mono- or diphosphoramidates of the general formula Oligo-MGBm (where Oligo is an oligonucleotide; m = 1 or 2; MGB is -L(Py)2R, L(Py)4R, -L(Im)4R, or -L(Py)4NH(CH2)3CO(Py)4R; Py is a 4-aminopyrrol-2-carboxylic acid residue, L is a gamma-aminobutyric acid or an epsilon-aminocaproic acid residue, R = OEt, NH(CH2)6NEt2, or NH(CH2)6N+Me3) was studied by the method of thermal denaturation. The mode of binder interaction with minor groove depends on the conjugate structure; it may be of the parallel head to head type for bisphosphoramidates and of the antiparallel head to tail type for monophosphoramidates of a hair-pin structure. The effects of the duplexes with parallel orientation (bisphosphoramidates, MGB is L(Py)4R, m = 2) and those of the hairpin structure with the antiparallel orientation (monophosphoramidates, MGB is L(Py)4(CH2)3CO(Py)4R, m = 1) on Tm val...