Oligonucleotide Chemistry Research Articles

ChemInform Abstract: A New Protecting Group Strategy for Amino Groups in Oligonucleotide Chemistry: CEOC Group

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New approaches to synthesis of stereospecific sphingomyelin

Enormous progress in the asymmetric synthesis of stereochemically and chemically pure d- erythro-sphingosine and ceramides led to the development of a practical, efficient, easily scaleable process to provide industrial quantities of chiral sphingosine and ceramides. This established a new platform of chiral starting materials which facilitate the synthesis of complex sphingolipids. Utilizing stereochemically homogeneous, fully synthetic ceramides, two efficient synthetic methods were developed for the preparation of ultra pure stereochemically homogeneous sphingomyelins. The first method adapted highly efficient phosphoramidite technology from oligonucleotide chemistry. This method allows selective insertion of a phosphocholine moiety into 3- O-protected ceramide through phosphitylation, followed by choline attachment, phosphite oxidation and deprotection. This route provides stereochemically homogeneous sphingomyelin in 35–79% yield. The second route is based on the reaction of selectively protected ceramides with cyclic chlorophosphate followed by treatment with trimethylamine to give the desired sphingomyelins in 50% yield. Multigram quantities of 14C-labeled N-palmitoyl- d- erythro-sphingomyelin were produced with specific activity >1000 dpm/nmol.

A New Protecting Group Strategy for Amino Groups in Oligonucleotide Chemistry: CEOC Group

A new protecting group, 2-cyanoethyloxycarbonyl, or CEOC, has been developed for amino groups and utilized in synthesizing modified oligonucleotides. (CEOC)-oxy-succinimide reagent has been synthesized to introduce this protecting group. The protecting group is removed by standard oligonucleotide deprotection protocols. Using this approach, oligonucleotides have been synthesized with various types of alkylamine substituents.

Cellular delivery of peptide nucleic acids and inhibition of human telomerase

Human telomerase has an essential RNA component and is an ideal target for developing rules correlating oligonucleotide chemistry with disruption of biological function. Similarly, peptide nucleic acids (PNAs), DNA analogs that bind complementary sequences with high affinity, are outstanding candidates for inducing phenotypic changes through hybridization. We identify PNAs directed to nontemplate regions of the telomerase RNA that can overcome RNA secondary structure and inhibit telomerase by intercepting the RNA component prior to holoenzyme assembly. Relative potencies of inhibition delineate putative structural domains. We describe a novel protocol for introducing PNAs into eukaryotic cells and report efficient inhibition of cellular telomerase by PNAs. PNAs directed to nontemplate regions are a new class of telomerase inhibitor and may contribute to the development of novel antiproliferative agents. The dependence of inhibition by nontemplate-directed PNAs on target sequence suggests that PNAs have great potential for mapping nucleic acid structure and predictably regulating biological processes. Our simple method for introducing PNAs into cells will not only be useful for probing the complex biology surrounding telomere length maintenance but can be broadly applied for controlling gene expression and functional genomics.

Cationic lipids, phosphatidylethanolamine and the intracellular delivery of polymeric, nucleic acid-based drugs (review).

Polymeric, nucleic acid drugs must be protected from endogenous nucleases and delivered to target cell nuclei in order to maximize their activity. Constructs expressing therapeutic genes, antisense oligonucleotides and ribozymes can be delivered into cells by viral vectors, but concerns over safety and clinical utility have led to research into the development of alternative, non-viral delivery systems. Antisense and ribozyme drug development has focused upon modifications to the natural oligonucleotide chemistry which make the molecules resistant to nuclease degradation. These novel oligonucleotides cannot be generated by transgenes and must be administered in similar fashion to conventional drugs. However, oligonucleotides cannot cross membranes by passive diffusion and intracellular delivery for these drugs is very inefficient. Here we review the recent advances in forming lipid-DNA particles designed to mimic viral delivery of DNA. Most evidence now supports the hypothesis that lipid-DNA drugs enter target cells by endocytosis and disrupt the endosomal membrane, releasing nucleic acid into the cytoplasm. The mechanisms of particle formation and endosome disruption are not well understood. Cationic lipids are employed to provide an electrostatic interaction between the lipid carrier and polyanionic nucleic acids, and they are critical for efficient packaging of the drugs into a form suitable for systemic administration. However, their role in endosome disruption and other aspects of successful delivery leading to gene expression or inhibition of mRNA translation are less clear. We discuss the propensity of lipid-nucleic acid particles to undergo lipid mixing and fusion with adjacent membranes, and how phosphatidylethanolamine and other lipids may act as factors capable of disrupting bilayer structure and the endosomal pathway. Finally, we consider the challenges that remain in bringing nucleic acid based drugs into the realm of clinical reality.

Preorganization of DNA: Design Principles for Improving Nucleic Acid Recognition by Synthetic Oligonucleotides.

A. Coverage of This Review This review describes design principles which chemists have applied to the construction of DNA analogues for use in the recognition of specific DNA and RNA sequences. The focus is primarily on molecular strategies which are aimed at increasing binding affinity and specificity, the benchmarks of successful molecular recognition. The field of oligonucleotide chemistry has grown explosively in the last 10 years. For that reason this review is not intended to comprehensively cover the entire field of oligonucleotide chemistry, but will instead focus on this more narrow topic. The reader is invited to visit other recently published reviews for surveys of oligonucleotide synthesis1,2 and of oligonucleotide conjugates, 3 and for in-depth coverage of antisense or antigene therapeutic strategies.4–7

Antisense Strategy: Biological Utility and Prospects in the Treatment of Hematological Malignancies

The use of antisense oligonucleotides for the specific control of cellular genes expression has undergone rapid developments recently. Besides the antisense approach. which usually targets translation initiation or splicing sites. it is also possible to interfere specifically with transcription process through triple helix formation (anti-gene strategy) or through the titration of regulatory proteins (sense and aptanier approaches). Progresses in oligonucleotides chemistry have led to the synthesis of analogs with improved pharmacological properties. while their generation from recombinant vectors in situ has improved oligos delivery to their nuclear or cytoplasmic targets. Hematological malignancies provide an ideal paradigm for the development of antisense therapeutic strategies. Many disease-specific molecular lesions have been identified which provide suitable targets for systemic in vivo administration of oligonucleotides as well as for ex vivo bone marrow purging manipulation. However, oligonucleotides have also been shown to bind to unexpected cellular targets and to induce various unpredictable biological responses as well. In addition, the multi-stage nature of carcinogenesis may indicate that even if successful inhibition of a single gene by oligomer is achieved, it may still be insufficient to induce a major impact on a malignant clone. Thus. much more basic information about both the disease and antisense technology is still required before antisense strategy gains the status of an acceptable therapeutical approach.

Expression monitoring by hybridization to high-density oligonucleotide arrays

The human genome encodes approximately 100,000 different genes, and at least partial sequence information for nearly all will be available soon. Sequence information alone, however, is insufficient for a full understanding of gene function, expression, regulation, and splice-site variation. Because cellular processes are governed by the repertoire of expressed genes, and the levels and timing of expression, it is important to have experimental tools for the direct monitoring of large numbers of mRNAs in parallel. We have developed an approach that is based on hybridization to small, high-density arrays containing tens of thousands of synthetic oligonucleotides. The arrays are designed based on sequence information alone and are synthesized in situ using a combination of photolithography and oligonucleotide chemistry. RNAs present at a frequency of 1:300,000 are unambiguously detected, and detection is quantitative over more than three orders of magnitude. This approach provides a way to use directly the growing body of sequence information for highly parallel experimental investigations. Because of the combinatorial nature of the chemistry and the ability to synthesize small arrays containing hundreds of thousands of specifically chosen oligonucleotides, the method is readily scalable to the simultaneous monitoring of tens of thousands of genes.

Progress in antisense therapeutics.

Abstract Antisense technology offers the potential to create compounds specific enough to support highly isotypically selective pharmacology (i.e., pharmacologic agents specific enough to affect only one isotype in a multigene family of related proteins). However, enthusiasm about the potential of the technology has been appropriately tempered by questions about its ability to deliver on its promise. The fundamental issue is: Can oligonucleotides that have acceptable drug properties be created? This review discusses recent progress that helps to define the pharmacokinetic, pharmacologic, and toxicologic properties of phosphorothioate oligonucleotides. Progress in the medicinal chemistry of oligonucleotides that has resulted in a wide range of new chemical classes is also described.

Progress in antisense therapeutics

Antisense technology offers the potential to create compounds specific enough to support highly isotypically selective pharmacology (i.e., pharmacologic agents specific enough to affect only one isotype in a multigene family of related proteins). However, enthusiasm about the potential of the technology has been appropriately tempered by questions about its ability to deliver on its promise. The fundamental issue is: Can oligonucleotides that have acceptable drug properties be created? This review discusses recent progress that helps to define the pharmacokinetic, pharmacologic, and toxicologic properties of phosphorothioate oligonucleotides. Progress in the medicinal chemistry of oligonucleotides that has resulted in a wide range of new chemical classes is also described.

From Oligonucleotides to Phospholipids: Cross-Fertilization Through Complementary Chemistry

With expanded research efforts targeted towards developing gene therapies, oligonucleotide chemistry is enjoying a parallel renaissance. Oligonucleotides and their congeners are utilized as complementary sequence-specific strands in DNA and/or RNA hybridization techniques and, therefore, can also be utilized in vivo to modify the course of various oncological, viral, parasitic, and genetic diseases. This paper illustrates how advances in oligonucleotide chemistry have been applied to the seemingly unrelated area of phospholipid chemistry. A specific example to be addressed is the synthesis of C-14-radiolabeled natural N-palmitoyl D-erythro-sphingomyelin using methods adopted from oligonucleotide chemistry. This and related compounds are valuable analytical tools for the investigation of Niemann-Pick disease, a sphingolipidosis characterized by an inherited sphingomyelinase deficiency. The cross-fertilization of these two chemistries and utilization of the resultant novel approaches in design and synthesis...

Interactions of phosphodiester and phosphorothioate oligonucleotides with intestinal epithelial Caco-2 cells.

Oral bioavailability for antisense oligonucleotides has recently been reported but the mechanistic details are not known. The proposed oral delivery of nucleic acids will, therefore, require an understanding of the membrane binding interactions, cell uptake and transport of oligonucleotides across the human gastro-intestinal epithelium. In this initial study, we report on the cell-surface interactions of oligonucleotides with human intestinal cells. We have used the Caco-2 cell line as an in vitro model of the human intestinal epithelium to investigate the membrane binding interactions of 20-mer phosphodiester (PO) and phosphorothioate (PS) oligonucleotides. The cellular association of both an internally [3H]-labelled and a 5'end [32P]-labelled PS oligonucleotide (3.0% at 0.4 microM extracellular concentration) was similar and was an order of magnitude greater than that of the 5'end [32P]-labelled PO oligonucleotide (0.2%) after 15 minutes incubation in these intestinal cells. The cellular association of PS was highly saturable with association being reduced to 0.9% at 5 microM whereas that of PO was less susceptible to competition (0.2% at 5 microM, 0.1% at 200 microM). Differential temperature-dependence was demonstrated; PS interactions were temperature-independent whereas the cellular association of PO decreased by 75% from 37 degrees C to 17 degrees C. Cell association of oligonucleotides was length and pH-dependent. A decrease in pH from 7.2 to 5.0 resulted in a 2- to 3-fold increase in cell-association for both backbone types. This enhanced association was not due to changes in lipophilicity as the octanol:aqueous buffer distribution coefficients remained constant over this pH range. The ability of NaCl washes to remove surface-bound PS oligonucleotides in a concentration-dependent manner suggests their binding may involve ionic interactions at the cell surface. Cell-surface washing with the proteolytic enzyme, Pronase, removed approximately 50% of the cell-associated oligonucleotide for both backbone types. Binding to surface proteins seems a major pathway for binding and internalization for both oligonucleotide chemistries and appear consistent with receptor (binding protein)-mediated endocytosis. Whether this binding protein-mediated entry of oligonucleotides can result in efficient transepithelial transport, however, requires further study.

Development of Antisense and Antigene Oligonucleotide Analogs

Publisher Summary Many excellent reviews and monographs have been written describing the chemistry and biological activity of various antisense and antigene oligonucleotides. This chapter focuses on two lines of research that are aimed at the development of antisense and antigene oligonucleotide analogs. The first line of research involves the development of nuclease-resistant antisense oligonucleotide analogs, the oligonucleoside methylphosphonates. The second line of research involves the development of nucleoside and oligonucleotide analogs that can be used to recognize sequences in double-stranded DNA. Antisense oligonucleotides are designed to interact with cytoplasmic messenger RNA (mRNA) or with precursor mRNA in the nucleus. Antigene oligonucleotides are designed to bind to double-stranded DNA via the formation of triple-stranded complexes (triplexes). Antisense oligonucleotides depend on Watson–Crick hydrogen-bonding interactions between an oligomer and its target, whereas antigene oligonucleotides make use of Hoogsteen or reversed-Hoogsteen hydrogen-bonding schemes. Antisense and antigene oligonucleotide analogs hold great promise for the design of reagents that can be used to inhibit gene expression selectively at either the mRNA or DNA level.

Direct detection of nucleic acid hybridization on the surface of a charge coupled device.

A method is described for the detection of DNA hybrids formed on a solid support, based upon the pairing of oligonucleotide chemistry and the technologies of electronic microdevice design. Surface matrices have been created in which oligonucleotide probes are covalently linked to a thin SiO2 film. 32P labeled target nucleic acid is then hybridized to this probe matrix under conditions of high stringency. The salient feature of the method is that to achieve the highest possible collection efficiency, the hybridization matrix is placed directly on the surface of a charge coupled device (CCD), which is used to detect 32P decay from hybridized target molecules (1, Eggers, M.D., Hogan, M.E., Reich, R.K., Lamture, J.B., Beattie, K.L., Hollis, M.A., Ehrilich, D.J., Kosicki, B.B., Shumaker, J.M., Varma, R.S., Burke, B.E., Murphy, A., and Rathman, D.D., (1993), Advances in DNA Sequencing Technology, Proc. SPIE, 1891, 13-26). Two implementations of the technology have been employed. The first involves direct attachment of the matrix to the surface of a CCD. The second involves attachment of the matrix to a disposible SiO2 coated chip, which is then placed face to face upon the CCD surface. As can be predicted from this favorable collection geometry and the known characteristics of a CCD, it is found that as measured by the time required to obtain equivalent signal to noise ratios, 32P detection speed by the direct CCD approach is at least 10 fold greater than can be obtained with a commercial gas phase array detector, and at least 100 fold greater than when X-ray film is used for 32P detection. Thus, it is shown that excellent quality hybridization signals can be obtained from a standard hybridization reaction, after only 1 second of CCD data acquisition.

Progress toward oligonucleotide therapeutics: pharmacodynamic properties.

Although many issues remain to be resolved, recent progress suggests that oligonucleotides may have therapeutic potential. The first therapeutic oligonucleotide, ISIS 2105, an oligonucleotide designed to treat genital warts, has completed Phase I clinical trials and is undergoing Phase II testing. Conceptually, oligonucleotides may interact with nucleic acid or nonnucleic acid receptors; however, rational design of oligonucleotide drugs to interact with nucleic acid receptors is significantly simpler because the factors that determine affinity and selectivity are better understood. Recent progress demonstrates that this information can be coupled to rapid advances in the medicinal chemistry of oligonucleotides to create new generations of oligonucleotide drugs with dramatically improved properties. The purposes of this review are to place oligonucleotide therapeutics in the context of modern molecular drug discovery and to summarize progress in understanding the pharmacodynamic properties of oligonucleotides designed to bind to nucleic acid receptors. The pharmacokinetic and toxicologic properties of oligonucleotides have been reviewed recently (1-4). The activities of oligonucleotides that bind to nonnucleic acid receptors will be considered in future reviews.

Dipentafluorophenyl carbonate--a reagent for the synthesis of oligonucleotides and their conjugates.

Dipentafluorophenyl carbonate has been successfully used as condensing agent for the internucleotide bond formation in the synthesis of oligonucleotides via H-phosphonate approach. The mechanism of a nucleotide component activation with this reagent has been investigated with the help of 31P NMR spectroscopy. It was shown that preactivation of deoxynucleoside H-phosphonate with dipentafluorophenyl carbonate has no influence on the efficiency of the synthesis. This reagent is highly reactive, nonhygroscopic and stable on storage at room temperature. The effectiveness of dipentafluorophenyl carbonate in the oligonucleotide chemistry has been demonstrated in the solid-phase synthesis of 10-50-mers on 0.2, 1 and 10 mumol scales. The use of this reagent for the derivatisation of polymer supports as well as for the synthesis of oligonucleotide conjugates with polyethylene glycol and a lipid is described.

Oligonucleotide therapy.

Rapid progress in oligonucleotide therapeutics has continued over the past year as major programs established in the past four years have grown and begun to be productive. Important advances were reported in the medicinal chemistry of oligonucleotides and in understanding their pharmacodynamic properties. Significant progress was made in understanding the pharmacokinetic and toxicologic properties of first generation analogs, particularly phosphorothioates and one oligonucleotide, ISIS 2105, entered clinical trials. Additionally, combinatorial approaches designed to identify oligonucleotides that may bind to a variety of targets were reported.

HPLC and Synthesis Strategy in Nucleoside and (Oligo)Nucleotide Chemistry

Abstract A judicious use of HPLC allows to simplify the synthetic approach of an (oligo)nucleotide. As an example, is reported a preparation of an antiviral (3′-5′)dinucleotide with simultaneous isolation of its (3′-3′) isomer.

A synthesis of phospholipids using a phosphite-triester approach and uniformly cleavable β-halogenated protecting groups

A new method for the synthesis of phospholipids is described. It is characterized by the use of a phosphite-amide-triester strategy, adopted from oligonucleotide chemistry, and by the use of protective groups which are cleavable by reductive fragmentation. Phosphorous acid-1,1,1-trichloro-2-methyl-2-propylester- (TCB) chloride-dimethylamide is used as the phosphorylating agent. Thus TCB is used for the protection of phosphodiesters. Nitrogenfunctions are protected by the 1,1,1-trichloro-2-methyl-2-propyl-oxycarbonyl group (TCBOC) and 2-bromoethanole is used for the protection of carboxyl groups. The versatility of this method is demonstrated by the syntheses of 1,2-dimiristoyl- sn-glycero-3-phosphatidyl-ethanolamine (DMPE) and 1,2-dimyristoyl- sn-glycero-3-phosphatidyl-serine (DMPS).

ChemInform Abstract: Novel Approach to the Preparation of Deoxynucleoside‐3′‐monophosphates.

AbstractThe p‐chlorophenyl group, widely used as protecting group in the oligonucleotide chemistry, can be reductively cleaved from nucleotide triesters such as (I) (→ (II), after complete deblocking) or oligonucleotides.