Alkyl phosphotriester modified oligodeoxyribonucleotides. VI. NMR and UV spectroscopic studies of ethyl phosphotriester (Et) modified Rp-Rp and Sp-Sp duplexes, (d[GGAA(Et)TTCC])2.

1H NMR chemical shift assignments for the title compounds were made for all but a few H5' and H5" signals using two-dimensional nuclear Overhauser effect (2D-NOE) data, which was also used for the first time to assign absolute configuration at phosphorus. The chemical shifts were, in general, similar to those reported [Broido, M.S., et al. (1985) Eur. J. Biochem. 150, 117-128] for the B-like conformation of the unmodified, parent duplex, [d(GGAATTCC)]2. Differences in chemical shifts for corresponding protons were mostly localized to the AA(Et)TT region, and showed some stereochemical dependence. Unambiguous assignment of the phosphotriester 31P signals was achieved in a novel way using selective insensitive nucleus enhancement by polarization transfer (selective INEPT) NMR. The Rp-Rp duplex melted ca. 11 degrees C lower than either the Sp-Sp or parent duplexes, as evidenced by Tm and variable temperature 1H/31P NMR measurements. The 2D-NOE data for the Rp-Rp duplex suggested possible steric interactions between the ethyl group and the H3' of the flanking A residue. At low ionic strength, the Sp-Sp and parent duplexes had similar stability but at high ionic strength the Sp-Sp duplex was less stable.     

Diastereomers of nonionic oligonucleotide analogs. VI. Isolation of diastereomers of ethyl phosphotriesters of the octanucleotide d(GCCAAACA) by high performance affinity chromatography]

Diastereomers of oligonucleotide ethyl phosphotriesters were separated by high-performance complementary (affinity) chromatography on a column with the immobilized complementary oligonucleotide. The elution buffer contained 0.18 M K2HPO4, pH 7.5, and 30% acetonitrile. The temperature of the separation was a few degrees lower than Tm of corresponding oligonucleotide complexes. The diastereomers separated completely or partially were: d[GCC(Et)AAACA], d[GCCA(Et)AACA], d[GCAA(Et)ACA], d[GCC(Et)A(Et)AACA], d[GCC(Et)AA(Et)ACA], d[GCCA(Et)A(Et)ACA], d[GCC(Et)A(Et)A(Et)ACA].     

Phosphorothioate-modified oligodeoxyribonucleotides. III. NMR and UV spectroscopic studies of the Rp-Rp, Sp-Sp, and Rp-Sp duplexes, [d(GGSAATTCC)]2, derived from diastereomeric O-ethyl phosphorothioates.

2D-NOE and 1H NMR chemical shift data obtained for the title oligonucleotides were compared with similar data previously reported [Broido et al. (1985) Eur. J. Biochem. 150, 117-128] for the unmodified "parent" structure, [d(GGAATTCC)]2. The spectroscopically detectable structural perturbations caused by replacement of phosphate oxygen with sulfur were mostly localized within the GsA moiety, and were greater for the Rp configuration wherein sulfur is oriented into the major groove of the B-helix. UV-derived Tm measurements gave the following order of stability for the duplexes in 0.4 M NaCl: unmodified (33.9 +/- 0.1 degrees C) approximately Sp-Sp (34.1 degrees C) greater than Rp-Rp (31.7 degrees C). The title compounds were prepared by a new and convenient synthetic route which utilized HPLC to separate the diastereomeric O-ethyl phosphorothioate precursors, (Rp)- and (Sp)-d[GG(S,Et)AATTCC], for subsequent de-ethylation by ammonia in water.     

The alternating conformation of analogues of poly[d(AT)].

Synthetic DNAs were prepared containing 6-methyl adenine (m6A) in place of adenine and 5-ethyl uracil (Et5U) or 5-methoxymethyl uracil (Mm5U) in place of thymine. All three modifications destabilized duplex DNAs to varying degrees. The binding of ethidium was studied to analogues of poly[d(AT)]. There was no evidence of cooperative binding and the "neighbour exclusion rule" was obeyed in all cases although the binding constant to poly[d(m6AT)] was approximately 6 fold higher than to poly[d(AT)]. 31P NMR spectra were recorded in increasing concentrations of CsF. Poly[d(AEt5U)] showed two well-resolved signals separated by 0.55 ppm in 1 M CsF compared to 0.32 ppm for poly[d(AT)] under identical conditions. In contrast, poly[d(AMm5U)] and poly[d(m6AT)] showed two signals separated by 0.28 ppm and 0.15 ppm respectively, only when the concentration of CsF was raised to 2 M. The signals for poly[d(AT)] in 2 M CsF were better resolved and were separated by 0.41 ppm. These results suggest that minor modifications to the bases may have conformational effects which could be recognized by DNA-binding proteins.     

Sulfurization of dinucleoside phosphite triesters with chiral disulfides

Sixteen chiral analogues of phenylacetyl disulfide (PADS) and 5-methyl-3H-1,2,4-dithiazol-3-one (MEDITH) were used to sulfurize five dithymidine phosphite triesters, each incorporating a β-cyanoethoxy or siloxy group. Each mixture of S(P):R(P) phosphite triester diastereomers was combined with approximately one fourth of an equivalent of each of the sulfurizing reagents, and the R(PS):S(PS) diastereomer ratios of the resulting phosphite sulfides or phosphorothioates were determined by reverse-phase HPLC. Diastereoselectivities and corresponding diastereomeric excess (de) values were calculated by correcting for the starting triester diastereomer ratios. The highest de values for R(PS) and S(PS) phosphorothioates were 14.7% and 7.9%, respectively, both using MEDITH analogues.     

Effects of a trinucleotide ethyl phosphotriester, Gmp(Et)Gmp(Et)U, on mammalian cells in culture

The nonionic 2'-O-methyribooligonucleotide ethyl phosphotriester, Gmp(Et)Gmp(Et)U, is complementary to the...ApCpC...sequence found in the amino acid accepting stem of most tRNAs and the anticodon region of tRNAgly and to the threonine codon of mRNA. Gmp(Et)Gmp(EtU forms hydrogen-bonded complexes with the amino acid accepting stem of tRNApheyeast and unfractionated tRNA Escherichia coli under physiological salt conditions at 37 degrees C as determined by equilibrium dialysis. The extent of phenylalanine aminoacylation of tRNApheE.coli is inhibited 39% by Gmp(Et)Gmp(Et)U at 37 degrees C in solution. The triester is resistant to hydrolysis by serum nucleases and cell lysates. The triester is readily taken up by transformed Syrian hamster fibroblasts growing in monolayer. Within the cell, the triester is deethylated to give the trinucleotide species Gmp(Et)GmpU, GmpGmp(Et)U, and GmpGmpU and is also hydrolyzed to dimeric and monomeric units. Treatment of transformed fibroblasts in monolayer with 25 micronM Gmp(Et)Gmp(Et)U results in a 40% inhibition of cellular protein synthesis with a concurrent slight increase in cellular RNA synthesis during the first 4 h. After 4 h, the rate of cellular protein synthesis begins to recover while RNA synthesis returns to that of the control. Our biochemical studies suggest that inhibition of cellular protein synthesis might be expected if Gmp(Et)Gmp(Et)UGmp(Et)GmpU, GmpGmp(Et)U, and GmpGmpU, which have been taken up by or formed within the cell, physically bind to tRNA and mRNA and inhibit the function of these nucleic acids. The reversible inhibition of protein synthesis may be a consequence of further degradation of the trinucleotide species within the cell as well as to an increase in supply of RNA molecules involved in protein synthesis. The growth of the transformed fibroblasts is inhibited during the first 24 h of incubation with 25 micronM Gmp(Et)Gmp(Et)U after which growth proceeds at a normal rate. In cloning experiments, the number and size of colonies formed by the transformed fibroblasts after 5 days exposure to 25 micronM triester is decreased by 50% relative to untreated controls. The temporary inhibition of cell growth may reflect the transitory inhibition of cellular protein synthesis caused by the triester.     

Complementary addressed modification of nucleic acids with the alkylating derivatives of oligothymidylate ethyl phosphotriesters. Effect of the phosphotriester fragments' configuration

The alkylating derivatives of four individual diastereomers of the oligonucleotide [dTp(Et)]3dTpU and two individual diastereomers of oligonucleotide [dTp(Et)dTp]4 have been synthesized. The reagents with the phosphorus atoms in the enantiomeric p" configuration are shown to be more efficient in reacting with poly(dA) and with nucleic acids in Krebs-2 ascites carcinoma cells compared to those with the phosphorus atoms in the p' configuration.     

CD evidence that the alternating purine-pyrimidine sequence poly[d(A-C).d(G-T)], but not poly[d(A-T).d(A-T)], undergoes an acid-induced transition to a modified secondary conformation.

Circular dichroism and UV absorption data showed that poly[d(A-C).d(G-T)] (at 0.01M Na+ (phosphate), 20 degrees C) underwent two reversible conformational transitions upon lowering of the pH. The first transition was complete at about pH 3.9 and resulted in an acid form of the polymer that was most likely a modified, protonated duplex. The second transition occurred between pH 3.9 and 3.4 and consisted of the denaturation of this protonated duplex to the single strands. UV absorption and CD data also showed that the separated poly[d(A-C)] strand formed two acid-induced self-complexes with pKa values of 6.1 and 4.7 (at 0.01M Na+). However, neither one of these poly[d(A-C)] self-complexes was part of the acid-induced rearrangements of the duplex poly[d(A-C).d(G-T)]. Acid titration of the separated poly[d(G-T)] strand, under similar conditions, did not show the formation of any protonated poly[d(G-T)] self-complexes. In contrast to poly[d(A-C).d(G-T)], poly[d(A-T).d(A-T)] underwent only one acid-induced transition, which consisted of the denaturation of the duplex to the single strands, as the pH was lowered from 7 to 3.     

Analysis of oligo(deoxynucleoside phosphorothioate)s and their diastereomeric composition.

Short oligo(deoxynucleoside phosphorothioate)s were analyzed as a pool of individual diastereomeric species. The composition of such mixtures, determined by means of HPLC, indicates that consecutive couplings in commonly used phosphoramidite chemistry lead to increasing contents of the Rp isomer. Methods of analysis and mathematical basis for diastereomeric composition are discussed. Data presented include all 16 possible combinations of nucleosides in dinucleotide phosphorothioates, as well as examples of trimers and tetramers.     

Synthesis of cyclic oligonucleotides by a modified phosphotriester approach.

Evidence will be presented to show that the allyl group is suitable for the protection of a 3'-terminal phosphodiester function. The latter will be demonstrated by the synthesis, via a phosphotriester approach, of two cyclic tetraribonucleotides [r(AAAA) and r(UAMe2UAMe2)], two cyclic hexadeoxyribonucleotides [d(CGCGCG) and d(TAAAAA)] and a cyclic octadeoxyribonucleotide [d(CGTGCGTG)].     

Stereodifferentiation--the effect of P chirality of oligo(nucleoside phosphorothioates) on the activity of bacterial RNase H.

P stereoregular phosphorothioate analogs of pentadecamer 5'-d(AGATGTTTGAGCTCT)-3' were synthesized by the oxathiaphospholane method. Their diastereomeric purity was assigned by means of enzymatic degradation with nuclease P1 and, independently, with snake venom phosphodiesterase. DNA-RNA hybrids formed by phosphorothioate oligonucleotides (PS-oligos) with the corresponding complementary pentadecaribonucleotide were treated with bacterial RNase H. The DNA-RNA complex containing the PS-oligo of [all-RP] configuration was found to be more susceptible to RNase H-dependent degradation of the pentadecaribonucleotide compared with hybrids containing either the [all-SP] counterpart or the so called 'random mixture of diastereomers' of the pentadeca(nucleoside phosphorothioate). This stereodependence of RNase H action was also observed for a polyribonucleotide (475 nt) hybridized with these phosphorothioate oligonucleotides. The results of melting studies of PS-oligo-RNA hybrids allowed a rationalization of the observed stereodifferentiation in terms of the higher stability of heterodimers formed between oligoribonucleotides and [all-RP]-oligo(nucleoside phosphorothioates), compared with the less stable heterodimers formed with [all-SP]-oligo(nucleoside phosphorothioates) or the random mixture of diastereomers.     

Oligodeoxyribonucleoside methylphosphonates. NMR and UV spectroscopic studies of Rp-Rp and Sp-Sp methylphosphonate (Me) modified duplexes of (d[GGAATTCC])2.

1H NMR chemical shift assignments for the title compounds were made for most of the 1H signals using two-dimensional nuclear Overhauser effect (2D-NOE) data, which were also used to establish the absolute configuration at the modified phosphorus. The chemical shifts were similar to those reported [Broido, M.S., et al. (1985) Eur. J. Biochem. 150, 117-128] for the unmodified, parent, B-type duplex [d(GGAATTCC)]2. Differences in chemical shifts were mostly localized to the nucleotides on the 5'- and 3'-sides of the modified phosphorus. The Rp-Rp isomers exhibited UV-derived Tm values similar to that of the parent duplex. On the other hand, the Sp-Sp isomers generally exhibited lower Tm values which correlated with P-CH3--H3' (n-1 nucleotide) cross peak intensities and 31P spectral parameters. The combined data argue for increased steric interactions with the Sp-P-Me methyl group as the modification position is moved toward the center of the oligomer. All of the Tm results can be explained in terms of three factors which result from replacement of a phosphate by a methylphosphonate group: reduction of oligomer charge; electronic and other substituent effects; steric interactions.     

Isoguanine quartets formed by d(T4isoG4T4): tetraplex identification and stability.

The self-aggregation of the oligonucleotide d(T4isoG4T4) (1) is investigated. Based on ion exchange HPLC experiments and CD spectroscopy, a tetrameric structure is identified. This structure was formed in the presence of sodium ions and shows almost the same chromatographic mobility on ion exchange HPLC as d(T4G4T4) (2). The ratio of aggregate versus monomer is temperature dependent and the tetraplex of [d(T4isoG4T4)]4 is more stable than that of [d(T4G4T4)]4. A mixture of d(T4isoG4T4) and d(T4G4T4) forms mixed tetraplexes containing strands of d(T4isoG4T4) and d(T4G4T4).     

Circular dichroism spectra of DNA quadruplexes [d(G(5)T(5))](4) as formed with G(4) and T(4) tetrads and [d(G(5)T(5)). d(A(5)C(5))]2 as formed with Watson-Crick-like (G-C)(2) and (T-A)(2) tetrads

We utilize electrophoresis and find that a thermally treated equimolar mixture of the oligonucleotide d(G(5)T(5)) and its complementary oligonucleotide d(A(5)C(5)) exhibits either two bands or a single band in one lane, depending on the conditions of the incubation solutions. The thermally treated d(G(5)T(5)) solution loaded in a different lane exhibits a single band of the parallel quadruplex [d(G(5)T(5))](4), which is composed of homocyclic hydrogen-bonded G(4) and T(4) tetrads previously proposed. For the thermally treated equimolar mixture of d(G(5)T(5)) and d(A(5)C(5)), the fast band is assigned to a Watson-Crick d(G(5)T(5)). d(A(5)C(5)) duplex, so that the slow band with the same low mobility as that of [d(G(5)T(5))](4) may be assigned to either [d(G(5)T(5))](4) itself or a [d(G(5)T(5)). d(A(5)C(5))](2) quadruplex. If the latter compound is true, this may be the antiparallel quadruplex composed of the heterocyclic hydrogen-bonded G-C-G-C and T-A-T-A tetrads proposed previously. After removing these three bands for the duplex and two kinds of hypothetical quadruplexes, we electrophoretically elute the corresponding compounds in the same electrophoresis buffer using an electroeluter. The eluted compounds are ascertained to be stable by electrophoresis. The circular dichroism (CD) and UV absorption spectra measured for the three isolated compounds are found to be clearly different. For the electrophoretic elution of the hypothetical [d(G(5)T(5))](4) quadruplex, the result of the molecularity of n = 4 obtained from the CD melting curve analysis provides further support for the formation of the parallel [d(G(5)T(5))](4) quadruplex already proposed. For the thermally treated equimolar mixture of d(G(5)T(5)) and d(C(5)A(5)), the fast band with a molecularity of n = 2 corresponds to the Watson-Crick duplex, d(G(5)T(5)). d(A(5)C(5)). The slow band with a molecularity of n = 4 indicates the antiparallel quadruplex [d(G(5)T(5)). d(A(5)C(5))](2), whose observed CD and UV spectra are different from those of [d(G(5)T(5))](4). By electrophoresis, after reannealing the eluted compound [d(G(5)T(5)). d(A(5)C(5))](2), a distinct photograph showing the band splitting of this quadruplex band into the lower duplex and upper quadruplex bands is not possible; but by a transilluminator, we occasionally observe this band splitting with the naked eye. The linear response polarizability tensor calculations for the thus determined structures of the [d(G(5)T(5))](4) quadruplex, the McGavin-like [d(G(5)T(5)). d(A(5)C(5))](2) quadruplex, and the Watson-Crick d(G(5)T(5)). d(A(5)C(5)) duplex are found to qualitatively predict the observed CD and UV spectra.     

Practical enantioresolution of alcohols with 2-methoxy-2-(1-naphthyl)propionic acid and determination of their absolute configurations by the (1)H NMR anisotropy method.

The enantioresolution of racemic alcohols as esters of 2-methoxy-2-(1-naphthyl)propionic acid (MalphaNP acid 1) and the determination of their absolute configurations on the basis of (1)H NMR anisotropy effect are described. The enantiopure MalphaNP acid (S)-(+)-1 was allowed to react with racemic 2-alkanols and 1-octyn-3-ol, yielding diastereomeric mixtures of esters, which were easily separated by HPLC on silica gel. To determine the absolute configurations of the first-eluted diastereomeric esters by the (1)H NMR anisotropy method, the general scheme was proposed. Separated esters were reduced with LiAlH(4) or hydrolyzed with KOH/EtOH to recover enantiopure alcohols.     

alpha-DNA-V. Parallel annealing, handedness and conformation of the duplex of the unnatural alpha-hexadeoxyribonucleotide alpha-[d(CpApTpGpCpG)] with its beta-complement beta-[d(GpTpApCpGpC)] deduced from high field 1H-NMR.

The beta-complementary hexamer, beta-d[GTACGC], to the alpha-sequence, alpha-d[CATGCG], was synthesized by the phosphotriester method. The non-exchangeable proton assignments were obtained using 1D- and 2D-NMR techniques, including NOE, COSY and NOESY. The beta-strand exists as a random coil at 21 degrees C; however, at 4 degrees C, it forms an antiparallel self-recognition duplex annealing at positions 1-4. The beta-strand was annealed to the alpha-strand, and confirmation of complete annealing was obtained by detection and assignment of the six base pair imino protons in H2O/D2O solution at 21 degrees C. 1D-NOE experiments of the alpha, beta duplex d[alpha-(CATGCG) X beta-(GTACGC)] reveal that (i) it exists in aqueous solution in a conformation that belongs to the B family, (ii) it is 70 +/- 10% right-handed, (iii) the sugar-base orientations of the beta-strand are anti, and the deoxyribose units exist predominantly in the 2'-endo-3'-exo conformation. NOE measurements of the imino proton signals in the alpha, beta duplex reveal that the duplex exhibits parallel polarity.     

Substrate specificity and stereospecificity of calf spleen phosphodiesterase towards deoxyribonucleosidyl 3'-(4-nitrophenyl phosphates) and phosphorothioates

Phosphodiesterase from calf spleen exhibits nucleotidyltransferase activity when incubated with either the (PR) or the (PS) diastereomer of thymidyl 3'-(4-nitrophenyl phosphorothioate). Thymidylyl(3'-5')thymidyl phosphorothioate 3'-(4-nitrophenyl phosphorothioate) was identified as the main product of the enzyme-catalyzed reaction and the absolute configuration at the internucleotide phosphorus atom of the product was determined. The nucleotidyltransferase reaction is shown to proceed with retention of configuration at phosphorus, implying involvement of a double displacement mechanism with the formation of a nucleotidylated enzyme intermediate. To study the substrate specificity of spleen phosphodiesterase a series of deoxyribonucleosidyl 3'-(4-nitrophenyl phosphates) and phosphorothioates were synthesized, and Km and V parameters for each substrate were measured. The results obtained show virtually no specificity for substrates with different nucleosidyl moieties, while about a 20 - 30-fold drop in V and a slight increase in Km values is observed for phosphorothioate analogues as compared with corresponding phosphates. The enzyme showed no significant stereoselectivity towards phosphorothioates of opposite configurations at phosphorus.     

Structure of the alpha-form of poly[d(A)].poly[d(T)] and related polynucleotide duplexes

The alpha-form of poly[d(A)].poly[d(T)], observed in fibers at high (greater than 80%) relative humidity, is a 10-fold double-helical structure of pitch 3.2 nm. This new X-ray analysis shows that the two strands of the double helix are of the same kind conformationally and both B-like in containing C-2'-endo-puckered deoxyribose rings. Nevertheless, the two strands are different enough for the overall morphology of the duplex to resemble that of the heteromerous model for the drier (beta) form of poly[d(A)].poly[d(T)] in which one strand has C-2'-endo rings and the other C-3'-endo. Since the orientations of the bases in poly[d(A)].poly[d(T)] are persistently different from those of classical B-DNA it is likely that there will be local bending (about 10 degrees) at the junctions between general sequence tracts and the oligo[d(A)].oligo[d(T)] tracts that occur in some native DNAs. The conclusions about the structure of alpha-poly[d(A)].poly[d(T)] are reinforced by independent analyses of similar X-ray diffraction patterns from poly[d(A)].poly[d(U)] and poly[d(A-I)].poly[d(C-T)].     

Synthesis, complete 1H assignments and conformations of the self-complementary hexadeoxyribonucleotide [d(CpGpApTpCpG)]2 and its fragments by high field NMR.

The two deoxyribonucleotides [d(CpGpApTpCpG)]2 and [d(CpGpCpG)]2 were synthesized by the phosphotriester method. Their duplex form under the conditions of the 1H-nmr experiments was proven by end 32P labeling with T4 polynucleotide kinase followed by butt end joining employing the absolute specificity of T4 ligase for double stranded DNA and analysis using gel electrophoresis and autoradiography. Complete nmr assignment of the 1H chemical shifts and coupling constants was achieved. The assignments were secured using sequential decoupling, NOE difference measurements, and two-dimensional COSY and SECSY experiments. Spectrum simulation confirmed the experimental values of chemical shifts and coupling constants. The techniques for the assignment outlined together with 31P and 2-D heteronuclear shift correlation permit an approach to a systematic analysis of more complex single-strand and duplex oligodeoxyribonucleotides.     

Raman spectra of single crystals of r(GCG)d(CGC) and d(CCCCGGGG) as models for A DNA, their structure transitions in aqueous solution, and comparison with double-helical poly(dG).poly(dC)

The self-complementary oligonucleotides [r(CGC)d(CGC)]2 and [d(CCCCGGGG)]2 in single-crystal and solution forms have been investigated by Raman spectroscopy. Comparison of the Raman spectra with results of single-crystal X-ray diffraction and with data from polynucleotides permits the identification of a number of Raman frequencies diagnostic of the A-helix structure for GC sequences. The guanine ring frequency characteristic of C3'-endo pucker and anti base orientation is assigned at 668 +/- 2 cm-1 for both dG and rG residues of the DNA/RNA hybrid [r(GCG)d(CGC)]2. The A-helix backbone of crystalline [r(GCG)d(CGC)]2 is altered slightly in the aqueous structure, consistent with the conversion of at least two residues to the C2'-endo/anti conformation. For crystalline [d(CCCCGGGG)]2, the Raman and X-ray data indicate nucleosides of alternating 2'-endo-3'-endo pucker sandwiched between terminal and penultimate pairs of C3'-endo pucker. The A-A-B-A-B-A-A-A backbone of the crystalline octamer is converted completely to a B-DNA fragment in aqueous solution with Raman markers characteristic of C2'-endo/anti-G (682 +/- 2) and the B backbone (826 +/- 2 cm-1). In the case of poly(dG).poly(dC), considerable structural variability is detected. A 4% solution of the duplex is largely A DNA, but a 2% solution is predominantly B DNA. On the other hand, an oriented fiber drawn at 75% relative humidity reveals Raman markers characteristic of both A DNA and a modified B DNA, not unlike the [d-(CCCCGGGG)]2 crystal. A comparison of Raman and CD spectra of the aqueous [d(CCCCGGGG)]2 and poly(dG).poly(dC) structures suggests the need for caution in the interpretation of CD data from G clusters in DNA.