Gerald Zon

Assignment of the 31P and 1H resonances in oligonucleotides by two-dimensional NMR spectroscopy

The use of new 1H‐detected heteronuclear 1H‐31P shift correlation experiments is demonstrated for oligonucleotides of 12 and 40 base pairs. The methods give unambiguous assignments of the 31P resonances and also permit identification of the C4′ and C5′ sugar protons. Use of the new methods enables one to make sequence‐specific resonance assignments without reference to a known or assumed conformation of the DNA fragment.

Reversed-phase high-performance liquid chromatographic separation of diastereomeric phosphorothioate analogues of oligodeoxyribonucleotides and other backbone-modified congeners of DNA.

Diastereomeric phosphorothioate analogues of oligodeoxyribonucleotides, which were synthesized by an automated, solid-phase, phosphoramidite-coupling method, were conveniently separated by reversed-phase high-performance liquid chromatography on a muBondapak C18 column with a gradient of acetonitrile in triethylammonium acetate buffer. These synthetic and chromatographic methods were also used to obtain diastereomerically pure bis-phosphorothioate, alkanephosphonate, and O-alkyl phosphotriester analogues of DNA for exploratory studies of stereochemistry and phosphorolytic enzymes.

Stereochemical studies of the formation of chiral internucleotide linkages by phosphoramidite coupling in the synthesis of oligodeoxyribonucleotides

HPLC-fractionated (Rp)- and (Sp)-phosphoramidites underwent tetrazole-catalyzed coupling to support-bound nucleosides to give intermediary phosphates with epimerization at phosphorus, as shown by conversion to epimerized mixtures of dinucleoside phosphorothioates.

Ultrasonic irradiation of bacterial polysaccharides. Characterization of the depolymerized products and some applications of the process

Ultrasonic irradiation (u.i.) has been used to depolymerize biopolymers including DNA, dextran, and the Vi capsular polysaccharide from Citrobacter freundii. Representative bacterial polysaccharides were subjected to u.i. and the effect of this energy upon their molecular weight and chemical structure was characterized. U.i. depolymerized a neutral polysaccharide (dextran) and acidic polysaccharides containing either a phosphoric diester linkage [Haemophilus influenzae type b (Hib) and pneumococcus types 6A and 6B] or a uronic acid moiety (pneumococcus type 9N). Prolonged u.i. depolymerized all the polysaccharides to a finite and similar molecular mass (approximately 50 000 daltons). The rate of depolymerization induced by u.i. depended on the viscosity of the solvent and the concentration of the polysaccharide. 13C-N.m.r. data of the native Hib polysaccharide and its depolymerized products indicated that u.i. did not alter the chemical structure of the repeating units. Determination of the monophosphate terminal residues by 31P-n.m.r. spectroscopy and of the reducing end groups by the Park-Johnson reaction indicated that both the phosphoric diester and the glycosidic linkages were cleaved. The Vi polysaccharide, prepared as an investigational vaccine, could not be analyzed for its chemical structure by 13C-n.m.r. spectroscopy owing to its high viscosity but depolymerization by u.i. permitted this analysis. The finite molecular weight of the products observed after prolonged u.i. is best explained by the postulation that the mechanical torque necessary to rupture the linkages is dependent upon the length of the polysaccharide. The method of u.i. for depolymerization is useful for the preparation of homogeneous, low-molecular-weight polysaccharides without alteration of the chemical structure of the repeating units.

A new approach for assigning phosphorus-31 NMR signals and correlating adjacent nucleotide deoxyribose moieties via proton-detected multiple-quantum NMR. Application to the adduct of d(TGGT) with the anticancer agent (ethylenediamine)dichloroplatinum.

Compared to ‘H, I3C, and I5N NMR spectra, useful 31P spectra of DNA and nucleosomes under physiological conditions can be obtained more readily.’ Studies of oligodeoxyribonucleotides, where even small 31P shifts can be observed and interpreted* and spectra of other nuclei can be observed easily, can provide greater insight into 31P spectral changes on drug, carcinogen, or protein binding to DNA. Unfortunately, full utilization of 31P NMR spectroscopy is hampered by difficulties in signal assignment.* The most reliable method to date (170/’80 labeling) is limited to synthetic materials3 We report a new approach for the assignment of the 31P NMR resonances in oligodeoxyribonucleotides via ‘H-detected heteronuclear multiple-quantum coherence (HMQC) two-dimensional correlation spectroscopy” combined with 2D-NOE methods. This approach exploits the method reported for sensitivity enhancement of heteronuclei in numerous system^;^ however, we illustrate the advantage of this experiment for information content involving abundant nuclei. The HMQC experiment selects only those ‘H nuclei that are spin-coupled to (e.g., deoxyribose H3/,H{,Hr) and correlates them with their respective 31P signals. The H3’,HS’,H5’’ signals can be assigned via ’H 2D-NOE methods. Due to the narrow shift range of 3’P in these systems, it is essential to record the spectrum as a direct correlation of ‘H and 31P chemical shifts by removal of the ‘H resonance offset contribution to the F1 dimension.48*e This has been accomplished via the pulse sequencek

Synthesis of backbone-modified DNA analogues for biological applications

The phosphite triester method was adapted for automated synthesis of phosphate (“backbone”)-modified analogues of DNA. The use of phosphoramidite reagents to achieve substitution of the nonequivalent (diastereotopic) oxygens of an internucleotide phosphate linkage is nonselective, and leads to nonequivalent (diastereomeric) strands of DNA with Rp and Sp absolute configurations at the chiral, modified phosphorus position. Indications of the scope and limitations of the use of reversed-phase HPLC to separate these diastereomers have been obtained through studies of backbone-modified DNA analogues having phosphorothioate (P-S), phosphotriester (P-OR), and alkanephosphonate (P-R) linkages. Incorporation of these modifications in the self-complementary octanucleotide d(GGAATTCC) led to separable diastereomers, which form nonequivalent Rp · Rp and Sp · Sp duplexes. A combination of chemical and nuclear Overhauser effect NMR spectroscopic methods was developed to assign unambiguously, for the first time, the absolute configurations at phosphorus in these prototypal cases. The effects of backbone ethylation on DNA structure and dynamics were evaluated by NMR methods. Modified duplexes were used to probe for proposed phosphate contacts for EcoRI endonuclease, and to define, in concert with base-modified analogues, a recognition site for monoclonal anti-native DNA autoantibody.

Hot Start PCR with heat-activatable primers: a novel approach for improved PCR performance

The polymerase chain reaction (PCR) is widely used for applications which require a high level of specificity and reliability, such as genetic testing, clinical diagnostics, blood screening, forensics and biodefense. Great improvements to PCR performance have been achieved by the use of Hot Start activation strategies that aim to prevent DNA polymerase extension until more stringent, higher temperatures are reached. Herein we present a novel Hot Start activation approach in PCR where primers contain one or two thermolabile, 4-oxo-1-pentyl (OXP) phosphotriester (PTE) modification groups at 3′-terminal and 3′-penultimate internucleotide linkages. Studies demonstrated that the presence of one or more OXP PTE modifications impaired DNA polymerase primer extension at the lower temperatures that exist prior to PCR amplification. Furthermore, incubation of the OXP-modified primers at elevated temperatures was found to produce the corresponding unmodified phosphodiester (PDE) primer, which was then a suitable DNA polymerase substrate. The OXP-modified primers were tested in conventional PCR with endpoint detection, in one-step reverse transcription (RT)–PCR and in real-time PCR with SYBR Green I dye and Taqman® probe detection. When OXP-modified primers were used as substitutes for unmodified PDE primers in PCR, significant improvement was observed in the specificity and efficiency of nucleic acid target amplification.

Synthesis and absolute configuration of P-chiral O-isopropyl oligonucleotide triesters

Abstract New O -isopropylphosphomorpholidite reagents provided the title compounds as mixtures of P-chiral diastereomers, which were separated by HPLC for enzymatic digestion studies and assignment of configuration at phosphorus by chemical correlation with known phosphorothioates.

Synthesis, separation, and stereochemistry of diastereomeric oligodeoxyribonucleotides having a 5′-terminal internucleotide phosphorothioate linkage

Abstract An automated, solid-phase, phosphoramidite-coupling method provided the title compounds, up to a hexadecamer, for HPLC isolation of diastereomerically pure samples, and comparative digestions with snake venom phosphodiesterase and nuclease P1.

Antisense phosphorothioate oligodeoxynucleotides: introductory concepts and possible molecular mechanisms of toxicity

Over the past 5 years or so, much attention has been given to the possible use of synthetic antisense oligonucleotide analogs as a new class of therapeutic agents that function by sequence-specific inhibition of genetic expression. The basic design concepts which underline this novel approach to drug discovery are briefly described herein, together with some of the chemical, biochemical, and pharmacological aspects of phosphorothioate oligodeoxynucleotides that are first-generation antisense compounds now under clinical investigation. Possible molecular mechanisms of toxicity for this class, and other structural types of antisense compounds are discussed with the hope of stimulating interest in future toxicological studies in this emerging area of drug development.