Formacetal Linkage Research Articles

ChemInform Abstract: Advances in Chemical Synthesis of Structurally Modified Bioactive RNAs

Methods for the chemical synthesis of RNA have been available for almost half century, and presently, RNA could be chemically synthesized by automated synthesizers, using protected ribonucleosides preactivated as phosphoramidites, which has already been covered by many reviews. In addition to advancement on synthetic methods, a variety of modifications have also been made on the synthesized oligonucleotides, and previous reviews on the general synthesis of RNAs have not covered this area. In this tutorial review, three types of modifications have been summarized standing at the viewpoint of medicinal chemistry: (1) modifications on nucleobase, comprising substituent introduction and replacement with pseudobase; (2) modifications on ribose, consisting of modifications on the 2’, 3’ or 5’-position, alternation of configuration, and conformational restriction on ribose; (3) modifications on internucleoside linkages, including amide, formacetal, sulfide, sulfone, ether, phosphorothiolate and phosphorothioate linkages. Synthetic methods achieving these modifications along with the functions or values of these modifications have also been discussed and commented on.

ChemInform Abstract: In vitro and in vivo Activities of Oligodeoxynucleotide-Based Thrombin Inhibitors Containing Neutral Formacetal Linkages.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Interplay of Structure, Hydration and Thermal Stability in Formacetal Modified Oligonucleotides: RNA May Tolerate Nonionic Modifications Better than DNA

DNA and RNA oligonucleotides having formacetal internucleoside linkages between uridine and adenosine nucleosides have been prepared and studied using UV thermal melting, osmotic stress, and X-ray crystallography. Formacetal modifications have remarkably different effects on double helical RNA and DNA-the formacetal stabilizes the RNA helix by +0.7 degrees C but destabilizes the DNA helix by -1.6 degrees C per modification. The apparently hydrophobic formacetal has little effect on hydration of RNA but decreases the hydration of DNA, which suggests that at least part of the difference in thermal stability may be related to differences in hydration. A crystal structure of modified DNA shows that two isolated formacetal linkages fit almost perfectly in an A-type helix (decamer). Taken together, the data suggest that RNA may tolerate nonionic backbone modifications better than DNA. Overall, formacetal appears to be an excellent mimic of phosphate linkage in RNA and an interesting modification for potential applications in fundamental studies and RNA-based gene control strategies, such as RNA interference.

Nucleoside–boron cluster conjugates – Beyond pyrimidine nucleosides and carboranes

Abstract General methods for the synthesis of new purine and pyrimidine nucleosides modified with borane clusters and metallacarborane complexes are presented. They include: (1) attachment of carborane modification at 2′ position of nucleoside via formacetal linkage formation, (2) tethering of the metallacarborane group at nucleobase part of the nucleoside via dioxane ring opening in oxonium metallacarborane, carborane or dodecaborate derivatives, and (3) ‘‘click” chemistry approach based on Huisgen 1,3-dipolar cycloaddition. Proposed methodologies extend the range of nucleoside–boron cluster conjugates available and open new areas for their applications.

Cover Picture: Oligoribonucleotide Analogues Containing a Mixed Backbone of Phosphodiester and Formacetal Internucleoside Linkages, Together with Vicinal 2′‐O‐Methyl Groups (ChemBioChem 5/2007)

The cover picture shows the chemical structure and a 3D representation (unoptimized Chem3D model) of RNA with hydrophobic chemical modifications. Replacement of selected phosphodiesters with formacetal linkages (blue) and methylation of the adjacent 2′‐hydroxyl groups (red) have independent and additive positive effects on the thermal stability of the RNA helices. Biophysical studies suggest that several complementary factors—reduction of electrostatic repulsion, solvation changes, conformational preorganization, hydrophobic interactions, etc.—interact to produce the observed stabilization. Modifications of RNA's sugar–phosphate backbone can be used to tune the thermal and enzymatic stability, hydrophobicity and electrostatic properties of RNA molecules. Chemically modified RNA has potential applications in gene‐control technologies, such as antisense and RNA interference. Further details can be found in the article by E. Rozners, et al. on p. 537 ff.

Oligoribonucleotide Analogues Containing a Mixed Backbone of Phosphodiester and Formacetal Internucleoside Linkages, Together with Vicinal 2′‐O‐Methyl Groups

Oligoribonucleotides containing formacetal internucleoside linkages have been prepared and studied by UV melting experiments. In RNA duplexes, the formacetal substitution is stabilizing (Deltat(m)=0 to +0.9 degrees C per modification) at physiological salt concentrations (0.1 M) but destabilizing (Deltat(m)=-0.4 to -0.8 degrees C per modification) at high salt concentrations (1 M); this suggests that reduction of electrostatic repulsion contributes substantially to the stabilization. The presence of 2'-O-Me substituents increases the stabilities of the duplexes (Deltat(m)=+0.5 to +1.1 degrees C per modification). The positive effects of formacetals and 2'-O-Me groups were independent and additive. (1)H NMR studies on monomeric model compounds containing 3'-(ethyl phosphate) or 3'-O-ethoxymethyl groups showed that the formacetal and 2'-O-Me substitutions shift the conformational equilibria of the ribose residues towards the North conformers by 5 to 12 %. Although the preference for the North conformers qualitatively correlates with increased duplex stabilities, changes in thermodynamic parameters (DeltaH degrees and TDeltaS degrees ) for formation of oligonucleotide duplexes and differences in dependence on concentrations of sodium acetate, sodium chloride and sodium perchlorate suggest that solvation effects are also important for the duplex stabilities. Overall the formacetal linkages fit well in A-type RNA duplexes, making them potentially interesting modifications for RNA-based gene-control strategies (e.g., antisense and RNA interference).

ChemInform Abstract: A New Method for Formacetal Linkage Formation: Protection of Alcohols, Phenols and Carboxylic Acids.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

A new method for formacetal linkage formation: protection of alcohols, phenols and carboxylic acids

A new formacetal linkage (ROCH 2OR′) forming reaction was developed, which exploited a combination of sulfide (R′OCH 2SR″) and CuBr 2-Bu 4NBr. This reaction proceeded to give high yields under neutral conditions and was applied to the protection of alcohols, phenols and carboxylic acids by several types of α-oxymethyl groups.

Efficient chemical synthesis of a pyrimidine (6–4) pyrimidone photoproduct analog and its properties

We synthesized an analog of a pyrimidine (6–4) pyrimidone photoproduct with a formacetal linkage instead of the phosphodiester in the (6–4) photoproduct, and investigated the interaction between a monoclonal antibody specific to the natural (6–4) photoproduct and a DNA containing the photoproduct analog.

Chemical synthesis and properties of (6-4) photoproduct and its analogs.

We report the preparation of a deoxyribooligonucleotide containing a new thymine (6-4) photoproduct analog. The (6-4) photoproduct is one of the major forms of DNA lesions, and leads to mutation in DNA. An antibody (64M5) that binds the (6-4) photoproduct has been described. To investigate the interaction of the photoproduct with the 64M5 antibody, we prepared a (6-4) photoproduct analog in which the two thymines were connected with a formacetal linkage. With UV-irradiation, the thymine dimer with the formacetal linkage reacted to the (6-4) photoproduct faster than the phosphodiesterified dimer, and the yields of the analog was higher than those of the natural thymine dimer. The 64M5 antibody exhibited sufficient binding to a tetranucleotide containing the (6-4) photoproduct analog with a formacetal linkage, although the association constant was slightly lower than that for the natural lesion. This (6-4) photoproduct analog may be useful for investigation of other proteins that recognize the (6-4) photoproduct.

In vitro and in vivo activities of oligodeoxynucleotide-based thrombin inhibitors containing neutral formacetal linkages.

A series of 15-mer oligodeoxynucleotide analogues were synthesized, and their thrombin inhibitory activities in vitro and in vivo were evaluated. These oligodeoxynucleotide analogues share the same sequence (GGTTGGTGTGGTTGG) but have one or more phosphodiester linkages replaced by a neutral formacetal group. The results obtained from monosubstitutions show that no single phosphodiester group is critical for the thrombin inhibitory activity, suggesting that the interaction between the oligodeoxynucleotide and thrombin is based on a multiple-site charge-charge interaction. Analysis of the effects of different phosphodiester replacements indicates that the backside and left side of the chairlike structure formed by the molecule may be involved in binding with thrombin, presumably by having direct contacts with the anion-binding exosite of the enzyme. For the oligodeoxynucleotides containing two noncontiguous formacetal groups, the effect of the disubstitution is the sum of the effects obtained from the corresponding two monosubstitutions. Infusion of an oligodeoxynucleotide containing four formacetal groups into monkeys showed an increased in vivo anticoagulant effect and an extended in vivo half-life compared to the unmodified oligodeoxynucleotide.

Synthesis and hybridization properties of an oligonucleotide analog containing a glucose-derived conformation-restricted ribose moiety and 2′, 5′ formacetal linkages

A pyrimidine trinucleoside containing a conformationally restricted ribose moiety and two 2′, 5′ formacetal internucleoside linkages was synthesized. The bicyclic ribose moiety was identified by molecular modeling as a candidate to preorder a nucleoside to resemble the bound duplex conformation. The thymine nucleoside analog was simply synthesized from glucose. The trimer was incorporated at the 3′ end of a larger unmodified ON. The hybridization affinity of this chimeric ON analog to a complementary RNA was found to be somewhat inferior relative to a non conformationally restricted control.

Unusual conformation of a 3?-thioformacetal linkage in a DNA duplex

The DNA.DNA duplex d(CGCGTTSCH2OTTGCGC).d(GCGCAAAACGCG) (designated duplex III) containing a 3'-thioformacetal (3'-TFMA) linkage in the center of the sequence was characterized in detail by two- and three-dimensional homonuclear NMR spectroscopy. The NMR results were analyzed and compared with those of two duplexes of the same sequence: One is an unmodified reference sequence and the other contains a formacetal (OCH2O) linkage at the central T--T step (designated duplex I and duplex II, respectively). In general, the NMR spectra of duplex III closely resemble those of the analogous duplexes I and II, suggesting an overall B-type structure adopted by the 3'-TFMA-modified duplex III. Nonetheless, the detection of several distinct spectral features originating from the protons at the T6(3'-SCH2O)T7 modification site is indicative of a local conformation that is clearly different from the corresponding region in duplexes I and II. The 3'-thioformacetal linker, in contrast to the formacetal (FMA) linkage, cannot be accommodated in a conformation usually found in natural nucleic acid duplexes. As a consequence, the 3'-TFMA-modified T6 sugar adopts an O4'-endo form (an intermediate structure between the usual C2'-endo and C3'-endo forms). This change is accompanied by a change in the epsilon (C4'-C3'-S3'-CH2) dihedral angle and by subsequent adjustments of other torsion angles along the backbone. Notably, this conformational readjustment at the T6-T7 backbone linkage is localized; its collective result has negligible effect on base-base stacking of the T6 and T7 residues. A close examination of the COSY data in all three duplexes reveals a subtle variation in sugar geometry, with more S-type character adopted by the modified duplexes II and III. The results of this study illustrate that, although the difference between FMA and 3'-TFMA linkages is merely in the substitution of the T6(O3') in the former by a sulfur atom in the latter, the stereoelectronic difference in a single atom can induce significant local structural distortion in an otherwise well-structured oligonucleotide duplex.

Probing structural factors stabilizing antisense oligonucleotide duplexes: NMR studies of a DNA.DNA duplex containing a formacetal linkage.

The duplex formed by annealing the formacetal backbone modified dodecamer d-(CGCGTTOCH2OTTGCGC) to its complementary strand, d(GCGCAAAACGCG) (duplex I), has been studied by NMR techniques and analyzed with reference to its unmodified counterpart (duplex II). Comparison of parameters such as 2D cross-peak intensities, coupling constants, and spectral patterns indicates that structural perturbations caused by the incorporation of the formacetal linkage are minimal and localized to the central T4.A4 block. Duplex I adopts a B-type helical conformation with regular Watson-Crick base pairing and normal minor groove width. The methylene group is accommodated along the phosphate backbone in a conformation similar to that of the PO2 group found in the B-form DNA family. The central T6-T7 base pairs of duplex I melt simultaneously with the duplex, indicating a cooperative transition to single strands. Although the formacetal linkage affects global melting, as evidenced by a 3 degree C reduction in Tm for duplex I with respect to duplex II, the present study indicates that this is not the result of localized premelting at the formacetal site of duplex I but rather reflects the subtle interplay of several structural and energy factors which need to be further explored.

Hybridization Properties of a DeoxyoIigonucleotide Containing Four Formacetal Linkages

Abstract The deoxyoiigonucleotide “TCTCTCTCTCT” bearing four formacetal linkages has been synthesized. The hybridization properties of this compound with complementary RNA and single stranded DNA has been compared to control sequences bearing phosphodiester, phosphoramidate and methylphosphonate linkages. The formacetal oligomer hybridizes to RNA similarly to a diester and significantly better than the other neutral linkages. Recently we reported the synthesis of a deoxyoligonucleotide bearing two formacetal linkages at the three prime end (1). This oligomer was characterized for binding to a complementary RNA sequence. We now report the synthesis of the sequence 5′ TCTC∗TC∗TC∗TCTTT where C is 5 methyl deoxycytidine and ∗ is the formacetal linkage. Controls were prepared in which the modified linkage position ∗ was the normal phosphodiester, methylphosphonate and methoxyethyl phosphoramidate. These four sequences which differ only in the backbone linkage at the starred positions were compared for their ab...

Deoxyoligonucleotide analogs based on formacetal linkages

This report describes the synthesis of a trimer of thymidine bearing formacetal linkages, its incorporation into a longer deoxyoligonucleotide and characterization of the hybridization properties with a complementary RNA sequence.