An Alternate Process for the Solid-Phase Synthesis and Solid-Phase Purification of Synthetic Nucleic Acid Sequences.

The chemical synthesis of a riboside phosphoramidite has been achieved to provide a 5-O-capture linker and a 2-O-silyl ether protecting group with the intent of enabling an efficient solid-phase purification of synthetic DNA sequences. The riboside phosphoramidite has been incorporated into a DNA sequence while performing the penultimate automated solid-phase synthesis cycle of the sequence. The terminal 5-O-riboside moiety of the resulting DNA sequence is then conjugated to a capture linker to create an anchor for the solid-phase purification of the DNA sequence conjugate. Release of all DNA sequences from the synthesis support is achieved under standard basic conditions to yield a mixture of the desired DNA sequence conjugate along with unconjugated, shorter-than-full-length sequence contaminants. Upon exposure of all DNA sequences to a capture solid support, only the DNA sequence conjugate is chemoselectively captured, thereby allowing the unconjugated shorter-than-full-length DNA sequences to be efficiently washed away from the capture support. After 2-O-cleavage of the silyl ether protecting group from the terminal riboside ethylphosphate triester conjugate, the solid-phase-purified DNA sequence is efficiently released from the capture support through an innovative intramolecular cyclodeesterification of the ethylphosphate triester, prompted by the riboside's rigid cis-diol conformer, to provide a highly pure DNA sequence. Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Preparation of 5-O-(4,4'-dimethoxytrityl)-2-O-tert-butyldimethylsilyl-1,4-anhydro-D-ribitol (3) Basic Protocol 2: Preparation of 5-O-(4,4'-dimethoxytrityl)-2-O-tert-butyldimethylsilyl-3-O-[(N,N-diisopropylamino)ethyloxyphosphinyl]-1,4-anhydro-D-ribitol (6). Basic Protocol 3: Automated synthesis of the chimeric solid-phase-linked DNA sequence 8. Support Protocol: Preparation of 2-cyanoethyl-(5-oxohexyl)-N,N-diisopropylphosphoramidite (9). Basic Protocol 4: Solid-phase purification of the chimeric DNA sequence 10.

Use of Arabinonucleosides for Development and Implementation of a Novel 2'-O-Protecting Group for Efficient Solid-Phase Synthesis and 2'-O-Deprotection of RNA Sequences.

The implementation of protecting groups for 2'-hydroxyl function of ribonucleosides is very demanding in that synthetic RNA sequences must be highly pure to ensure the safety and efficacy of nucleic acid-based drugs for treatment of human diseases. A synthetic approach consisting of a condensation reaction between 2'-O-aminoribonucleosides with ethyl pyruvate has been employed to provide stable 2'-O-imino-2-methyl propanoic acid ethyl esters. Conversion of these esters to fully protected ribonucleoside phosphoramidite monomers has allowed rapid and efficient incorporation of 2'-O-protected ribonucleosides into RNA sequences while minimizing the formation of process-related impurities during solid-phase synthesis. Two chimeric 20-mer RNA sequences have been synthesized and then exposed to a solution of sodium hydroxide to saponify the 2'-O-imino-2-methyl propanoic acid ethyl ester protecting groups to their sodium salts. When subjected to ion-exchange conditions at 65°C and near neutral pH, fully deprotected RNA sequences are isolated without production of alkylating side-products and/or formation of mutagenic nucleobase adducts. © 2022 Wiley Periodicals LLC. This article has been contributed to by US Government employees and their work is in the public domain in the USA. Basic Protocol 1: Synthesis of uridine 2'-O-imino-2-propanoic acid ethyl ester and its fully protected 3'-O-phosphoramidite Basic Protocol 2: Synthesis of N6 -protected adenosine 2'-O-imino-2-propanoic acid ethyl ester and its fully protected 3'-O-phosphoramidite Basic Protocol 3: Synthesis of N4 -protected cytidine 2'-O-imino-2-propanoic acid ethyl ester and its fully protected 3'-O-phosphoramidite Basic Protocol 4: Synthesis of N2 -protected guanosine 2'-O-imino-2-propanoic acid ethyl ester and its fully protected 3'-O-phosphoramidite Basic Protocol 5: Automated solid-phase RNA synthesis and deprotection using 2'-O-imino-2-proponate-protected phosphoramidites.

An Improved PEG-Linked Solid Support for Minimizing Process-Related Impurities During Solid-Phase Synthesis of DNA and RNA Sequences.

The preparation of controlled pore glass (CPG) supports, functionalized with several hexaethylene glycol spacers, to alleviate the problems associated with the porosity of commercial CPG supports is described in this article. The pore size of CPG restricts the diffusion of reagents to the leader nucleoside embedded in porous supports; this inhibits efficient solid-phase syntheses of DNA and RNA sequences and, by default, the purity of those sequences through formation of a shorter than full-length oligonucleotide. Functionalization of a CPG support with five hexaethylene glycol spacers led to a 42% reduction in process-related impurities contaminating oligonucleotide sequences, compared to that obtained using the commercial long-chain alkylamine (LCAA) CPG support. © 2021 Wiley Periodicals LLC. This article has been contributed to by US Government employees and their work is in the public domain in the USA. Basic Protocol 1: Preparation of the hydroxylated CPG support 3 Basic Protocol 2: Automated preparation of the CPG support 6 Basic Protocol 3: Automated preparation of the poly(hexaethylene glycol)-derived CPG 7 Basic Protocol 4: Automated functionalization of the poly(hexaethylene glycol)-derived CPG support 7 with leader deoxyribo- and ribonucleosides to provide the CPG support 9 Basic Protocol 5: Automated syntheses of DNA and RNA sequences on poly(hexaethylene glycol)-derived CPG support 9 and on a commercial long-chain alkylamine (LCAA) CPG support Support Protocol: Release and deprotection of the DNA and RNA sequences linked to the poly(hexaethylene glycol)-derived CPG support 10 and commercial LCAA-CPG support Basic Protocol 6: Comparative RP-HPLC analyses of crude, fully deprotected DNA or RNA sequences released from the poly(hexaethylene glycol)-derived CPG support 10 and from a commercial LCAA-CPG support.

Innovative 2'-O-Imino-2-propanoate-Protecting Group for Effective Solid-Phase Synthesis and 2'-O-Deprotection of RNA Sequences.

The implementation of protecting groups for the 2'-hydroxyl function of ribonucleosides is still challenging, particularly when RNA sequences must be of the highest purity for therapeutic applications as nucleic acid-based drugs. A 2'-hydroxyl-protecting group should optimally (i) be easy to install; (ii) allow rapid and efficient incorporation of the 2'-O-protected ribonucleosides into RNA sequences to minimize, to the greatest extent possible, the formation of process-related impurities (e.g., shorter than full-length sequences) during solid-phase synthesis; and (iii) be completely cleaved from RNA sequences without the production of alkylating side products and/or formation of mutagenic nucleobase adducts. The reaction of 2'-O-aminoribonucleosides with ethyl pyruvate results in the formation of stable 2'-O-imino-2-methyl propanoic acid ethyl esters and, subsequently, of the fully protected ribonucleoside phosphoramidite monomers, which are required for the solid-phase synthesis of two chimeric RNA sequences (20-mers) containing the four canonical ribonucleosides. Upon treatment of the RNA sequences with a solution of sodium hydroxide, the 2'-O-imino-2-methyl propanoic acid ethyl ester-protecting groups are saponified to their sodium salts, which after ion exchange underwent quantitative intramolecular decarboxylation under neutral conditions at 65 °C to provide fully deprotected RNA sequences in marginally better yields than those obtained from commercial 2'-O-tert-butyldimethylsilyl ribonucleoside phosphoramidites under highly similar conditions.

An expedient process for reducing the formation of process-related impurities during solid-phase synthesis of potential nucleic acid-based drugs.

With the intent of mitigating the formation of process-related impurities during solid-phase synthesis of DNA or RNA sequences, a hydroxylated controlled-pore glass support conjugated to three, five or seven hexaethylene glycol spacers was prepared and demonstrated to provide a more efficient and robust synthesis process. Indeed, the use of a support conjugated to five hexaethylene glycol spacers led to a 19% up to 42% reduction of process-related impurities contaminating synthetic nucleic acid sequences, when compared to that obtained from the same DNA/RNA sequences synthesized using a commercial long-chain alkylamine controlled-pore glass support under highly similar conditions.

An Improved Strategy for the Chemical Synthesis of 3',5'-Cyclic Diguanylic Acid.

The physiological functions of c-di-GMP and its involvement in many key processes led to its recognition as a major and ubiquitous bacterial second messenger. Aside from being a bacterial signaling molecule, c-di-GMP is also an immunostimulatory molecule capable of inducing innate and adaptive immune responses through maturation of immune mammalian cells. Given the broad biological functions of c-di-GMP and its potential applications as a nucleic-acid-based drug, the chemical synthesis of c-di-GMP has drawn considerable interest. An improved phosphoramidite approach to the synthesis of c-di-GMP is reported herein. The synthetic approach is based on the use of a 5'-O-formyl protecting group, which can be rapidly and chemoselectively cleaved from a key dinucleotide phosphoramidite intermediate to enable a cyclocondensation reaction leading to a fully protected c-di-GMP product in a yield ∼80%. The native c-di-GMP is isolated, after complete deprotection, in an overall yield of 36% based on the commercial ribonucleoside used as starting material. © 2019 by John Wiley & Sons, Inc.

A High-Throughput Process for the Solid-Phase Purification of Synthetic DNA Sequences.

An efficient process for the purification of synthetic phosphorothioate and native DNA sequences is presented. The process is based on the use of an aminopropylated silica gel support functionalized with aminooxyalkyl functions to enable capture of DNA sequences through an oximation reaction with the keto function of a linker conjugated to the 5'-terminus of DNA sequences. Deoxyribonucleoside phosphoramidites carrying this linker, as a 5'-hydroxyl protecting group, have been synthesized for incorporation into DNA sequences during the last coupling step of a standard solid-phase synthesis protocol executed on a controlled pore glass (CPG) support. Solid-phase capture of the nucleobase- and phosphate-deprotected DNA sequences released from the CPG support is demonstrated to proceed near quantitatively. Shorter than full-length DNA sequences are first washed away from the capture support; the solid-phase purified DNA sequences are then released from this support upon reaction with tetra-n-butylammonium fluoride in dry dimethylsulfoxide (DMSO) and precipitated in tetrahydrofuran (THF). The purity of solid-phase-purified DNA sequences exceeds 98%. The simulated high-throughput and scalability features of the solid-phase purification process are demonstrated without sacrificing purity of the DNA sequences. © 2017 by John Wiley & Sons, Inc.

Solid-Phase Purification of Synthetic DNA Sequences.

Although high-throughput methods for solid-phase synthesis of DNA sequences are currently available for synthetic biology applications and technologies for large-scale production of nucleic acid-based drugs have been exploited for various therapeutic indications, little has been done to develop high-throughput procedures for the purification of synthetic nucleic acid sequences. An efficient process for purification of phosphorothioate and native DNA sequences is described herein. This process consists of functionalizing commercial aminopropylated silica gel with aminooxyalkyl functions to enable capture of DNA sequences carrying a 5'-siloxyl ether linker with a "keto" function through an oximation reaction. Deoxyribonucleoside phosphoramidites functionalized with the 5'-siloxyl ether linker were prepared in yields of 75-83% and incorporated last into the solid-phase assembly of DNA sequences. Capture of nucleobase- and phosphate-deprotected DNA sequences released from the synthesis support is demonstrated to proceed near quantitatively. After shorter than full-length DNA sequences were washed from the capture support, the purified DNA sequences were released from this support upon treatment with tetra-n-butylammonium fluoride in dry DMSO. The purity of released DNA sequences exceeds 98%. The scalability and high-throughput features of the purification process are demonstrated without sacrificing purity of the DNA sequences.

Cyclic-di-GMP and cyclic-di-AMP activate the NLRP3 inflammasome.

The cyclic dinucleotides 3'-5'diadenylate (c-diAMP) and 3'-5' diguanylate (c-diGMP) are important bacterial second messengers that have recently been shown to stimulate the secretion of type I Interferons (IFN-Is) through the c-diGMP-binding protein MPYS/STING. Here, we show that physiologically relevant levels of cyclic dinucleotides also stimulate a robust secretion of IL-1ß through the NLRP3 inflammasome. Intriguingly, this response is independent of MPYS/STING. Consistent with most NLRP3 inflammasome activators, the response to c-diGMP is dependent on the mobilization of potassium and calcium ions. However, in contrast to other NLRP3 inflammasome activators, this response is not associated with significant changes in mitochondrial potential or the generation of mitochondrial reactive oxygen species. Thus, cyclic dinucleotides activate the NLRP3 inflammasome through a unique pathway that could have evolved to detect pervasive bacterial pathogen-associated molecular patterns associated with intracellular infections.

Biotinylation of a propargylated cyclic (3'-5') diguanylic acid and of its mono-6-thioated analog under "click" conditions.

Commercial N(2)-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-O-(propargyl)guanosine is converted to its 3'-O-levulinyl ester in a yield of 91%. The reaction of commercial N(2)-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-O-tert-butyldimethylsilyl-3'-O-[(2-cyanoethyl)-N,N-diisopropylaminophosphinyl]guanosine with N(2)-isobutyryl-2'-O-propargyl-3'-O-(levulinyl)guanosine provides, after P(III) oxidation, 3'-/5'-deprotection, and purification, the 2'-O-propargylated guanylyl(3'-5')guanosine 2-cyanoethyl phosphate triester in a yield of 88%. Phosphitylation of this dinucleoside phosphate triester with 2-cyanoethyl tetraisopropylphosphordiamidite and 1H-tetrazole, followed by an in situ intramolecular cyclization, gives the propargylated cyclic dinucleoside phosphate triester, which is isolated in a yield of 40% after P(III) oxidation and purification. Complete removal of the nucleobases, phosphates, and 2'-O-tert-butyldimethylsilyl protecting groups leads to the desired propargylated c-di-GMP diester. Cycloaddition of a biotinylated azide with the propargylated c-di-GMP diester under click conditions provides the biotinylated c-di-GMP conjugate in an isolated yield of 62%. Replacement of the 6-oxo function of N(2)-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-3'-O-levulinyl-2'-O-(propargyl)guanosine with a 2-cyanoethylthio group is effected by treatment with 2,4,6-triisopropybenzenesulfonyl chloride and triethylamine to give a 6-(2,4,6-triisopropylbenzenesulfonic acid) ester intermediate. Reaction of this key intermediate with 3-mercaptoproprionitrile and triethylamine, followed by 5'-dedimethoxytritylation, affords the 6-(2-cyanoethylthio)guanosine derivative in a yield of 70%. The 5'-hydroxy function of this derivative is reacted with commercial N(2)-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-O-tert-butyldimethylsilyl-3'-O-[(2-cyanoethyl)-N,N-diisopropylaminophosphinyl]guanosine. The reaction product is then converted to the mono-6-thioated c-di- GMP biotinylated conjugate under conditions highly similar to those described above for the preparation of the biotinylated c-di-GMP conjugate, and isolated in similar yields.

The 2-cyano-2,2-dimethylethanimine-N-oxymethyl group for the 2'-hydroxyl protection of ribonucleosides in the solid-phase synthesis of RNA sequences.

The reaction of 2-cyano-2-methyl propanal with 2'-O-aminooxymethylribonucleosides leads to stable and yet reversible 2'-O-(2-cyano-2,2-dimethylethanimine-N-oxymethyl)ribonucleosides. Following N-protection of the nucleobases, 5'-dimethoxytritylation and 3'-phosphitylation, the resulting 2'-protected ribonucleoside phosphoramidite monomers are employed in the solid-phase synthesis of three chimeric RNA sequences, each differing in their ratios of purine/pyrimidine. When the activation of phosphoramidite monomers is performed in the presence of 5-benzylthio-1H-tetrazole, coupling efficiencies averaging 99% are obtained within 180 s. Upon completion of the RNA-chain assemblies, removal of the nucleobase and phosphate protecting groups and release of the sequences from the solid support are carried out under standard basic conditions, whereas the cleavage of 2'-O-(2-cyano-2,2-dimethylethanimine-N-oxymethyl) protective groups is effected (without releasing RNA alkylating side-products) by treatment with tetra-n-butylammonium fluoride (0.5 M) in dry DMSO over a period of 24-48 h at 55 °C. Characterization of the fully deprotected RNA sequences by polyacrylamide gel electrophoresis (PAGE), enzymatic hydrolysis, and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry confirmed the identity and quality of these sequences. Thus, the use of 2'-O-aminooxymethylribonucleosides in the design of new 2'-hydroxyl protecting groups is a powerful approach to the development of a straightforward, efficient, and cost-effective method for the chemical synthesis of high-quality RNA sequences in the framework of RNA interference applications.

A novel function of RNAs arising from the long terminal repeat of human endogenous retrovirus 9 in cell cycle arrest.

The human genome contains approximately 50 copies of the replication-defective human endogenous retrovirus 9 (ERV-9) and thousands of copies of its solitary long term repeat (sLTR) element. While some sLTRs are located upstream of critical genes and have enhancer activity, other sLTRs are located within introns and may be transcribed as RNAs. We found that intronic RNAs arising from U3 sLTRs of ERV-9 were expressed as both sense (S) and antisense (AS) transcripts in all human cells tested but that expression levels differed in malignant versus nonmalignant cells. In nonmalignant cells, AS was expressed at higher levels than S and at higher levels than in malignant cells; in malignant cells, AS was expressed at amounts equivalent to those of S RNA. Critically, U3 AS RNA was found to physically bind to key transcription factors for cellular proliferation, including NF-Y, p53, and sp1, indicating that such RNA transcripts may function as decoy targets or traps for NF-Y and thus inhibit the growth of human cancer cells. Indeed, short U3 oligodeoxynucleotides (ODNs) based on these RNA sequences ably inhibited proliferation of cancer cell lines driven by cyclins B1/B2, the gene targets of NF-Y.

2'-Hydroxy protection of ribonucleosides as 2-cyano-2,2-dimethylethanimine-N-oxymethyl ethers in solid-phase synthesis of RNA sequences.

The reaction of 2'-O-aminooxymethylribonucleosides with 2-cyano-2-methyl propanal leads to the formation of stable and yet reversible 2'-O-(2-cyano-2,2-dimethylethanimine-N-oxymethyl)ribonucleosides in post-purification yields of 54% to 82%. Phenoxyacetylation of the exocyclic amino functions of these ribonucleosides proceeds in yields of 74% to 89%, and subsequent 5'-O-dimethoxytritylation and 3'-O-phosphitylation of the corresponding N-phenoxyacetylated ribonucleosides provide the fully protected ribonucleoside phosphoramidite monomers in isolated yields of 69% to 88%. These ribonucleoside phosphoramidites are employed in solid-phase synthesis of three chimeric RNA sequences, each differing in purine/pyrimidine content. The stepwise coupling efficiency of the ribonucleoside phosphoramidites (as 0.15 M solutions in acetonitrile) averages 99% over a coupling time of 180 s when 5-benzylthio-1H-tetrazole is used as an activator. Upon completion of RNA chain assembly, removal of the nucleobase- and phosphate-protecting groups and release of sequences from the solid support are carried out under standard basic conditions. Finally, the 2'-O-(2-cyano-2,2-dimethylethanimine-N-oxymethyl) protective groups are cleaved from the RNA sequences by treatment with 0.5 M tetra-n-butylammonium fluoride in dry DMSO for 24 to 48 hr at 55°C without releasing RNA-alkylating side-products. Characterization of the fully deprotected RNA sequences by PAGE, enzymatic hydrolysis, and MALDI-TOF mass spectrometry confirms the identity and high quality of these sequences.

Convenient and efficient approach to the permanent or reversible conjugation of RNA and DNA sequences with functional groups.

The conversion of 3',5'-disilylated 2'-O-(methylthiomethyl)ribonucleosides to 2'-O-(phthalimidooxymethyl)ribonucleosides is achieved in yields of 66% to 94%. Desilylation and dephtalimidation of these ribonucleosides by treatment with NH(4)F in MeOH produce 2'-O-aminooxymethylated ribonucleosides, which are efficient in producing stable and yet reversible 2'-conjugates upon reaction with 1-pyrenecarboxaldehyde. Exposure of 2'-pyrenylated ribonucleosides to 0.5 M tetra-n-butylammonium fluoride (TBAF) in THF or DMSO results in the cleavage of their iminoether functions to give the native ribonucleosides along with an innocuous nitrile side product. Conversely, the reaction of 2'-O-(aminooxymethyl)uridine with 5-cholesten-3-one leads to a permanent uridine 2'-conjugate, which is left unreacted when treated with TBAF. The versatility and uniqueness of 2'-O-(aminooxymethyl)ribonucleosides is demonstrated by the single or double incorporation of a reversible pyrenylated uridine 2'-conjugate into an RNA sequence. Furthermore, the conjugation of 2'-O-(aminooxymethyl)ribonucleosides with various aldehydes, including those generated from their acetals, is also presented. The preparation of 5'-O-(aminooxymethyl)thymidine is also achieved, albeit in modest yields, from the conversion of 5'-O-methylthiomethyl-3'-O-(levulinyl)thymidine to 5'-O-phthalimidooxymethyl-3'-O-(levuliny)lthymidine followed by hydrazinolysis of both 5'-phthalimido and 3'-levulinyl groups. Pyrenylation of the 5'-O-(aminooxymethyl)deoxyribonucleoside also provides a reversible 5'-conjugate that is sensitive to TBAF, thereby further demonstrating the usefulness of 5'-O-(aminooxymethyl)deoxyribonucleosides for permanent or reversible modification of DNA sequences. Curr. Protoc. Nucleic Acid Chem. 50:4.52.1-4.52.36. © 2012 by John Wiley & Sons, Inc.

The overlapping host responses to bacterial cyclic dinucleotides.

Macrophages respond to infection with Legionella pneumophila by the induction of inflammatory mediators, including type I Interferons (IFN-Is). To explore whether the bacterial second messenger cyclic 3'-5' diguanylate (c-diGMP) activates some of these mediators, macrophages were infected with L. pneumophila strains in which the levels of bacterial c-diGMP had been altered. Intriguingly, there was a positive correlation between c-diGMP levels and IFN-I expression. Subsequent studies with synthetic derivatives of c-diGMP, and newly described cyclic 3'-5' diadenylate (c-diAMP), determined that these molecules activate overlapping inflammatory responses in human and murine macrophages. Moreover, UV crosslinking studies determined that both dinucleotides physically associate with a shared set of host proteins. Fractionation of macrophage extracts on a biotin-c-diGMP affinity matrix led to the identification of a set of candidate host binding proteins. These studies suggest that mammalian macrophages can sense and mount a specific inflammatory response to bacterial dinucleotides.

Permanent or reversible conjugation of 2'-O- or 5'-O-aminooxymethylated nucleosides with functional groups as a convenient and efficient approach to the modification of RNA and DNA sequences.

2'-O-Aminooxymethyl ribonucleosides are prepared from their 3',5'-disilylated 2'-O-phthalimidooxymethyl derivatives by treatment with NH(4)F in MeOH. The reaction of these novel ribonucleosides with 1-pyrenecarboxaldehyde results in the efficient formation of stable and yet reversible ribonucleoside 2'-conjugates in yields of 69-82%. Indeed, exposure of these conjugates to 0.5 M tetra-n-butylammonium fluoride (TBAF) in THF results in the cleavage of their iminoether functions to give the native ribonucleosides along with the innocuous nitrile side product. Conversely, the reaction of 5-cholesten-3-one or dansyl chloride with 2'-O-aminooxymethyl uridine provides permanent uridine 2'-conjugates, which are left essentially intact upon treatment with TBAF. Alternatively, 5'-O-aminooxymethyl thymidine is prepared by hydrazinolysis of its 3'-O-levulinyl-5'-O-phthalimidooxymethyl precursor. Pyrenylation of 5'-O-aminooxymethyl thymidine and the sensitivity of the 5'-conjugate to TBAF further exemplify the usefulness of this nucleoside for modifying DNA sequences either permanently or reversibly. Although the versatility and uniqueness of 2'-O-aminooxymethyl ribonucleosides in the preparation of modified RNA sequences is demonstrated by the single or double incorporation of a reversible pyrenylated uridine 2'-conjugate into an RNA sequence, the conjugation of 2'-O-aminooxymethyl ribonucleosides with aldehydes, including those generated from their acetals, provides reversible 2'-O-protected ribonucleosides for potential applications in the solid-phase synthesis of native RNA sequences. The synthesis of a chimeric polyuridylic acid is presented as an exemplary model.

Time-dependent thermocontrol of the hydrophilic and lipophilic properties of DNA oligonucleotide prodrugs.

This unit describes the preparation of alkylthioalkylated and formamidoalkylated alcohols, an amidoalkylated alcohol, a hydroxylalkylated phosphoramidate, and their phosphoramidothioate derivatives, all of which have been identified as heat-sensitive thiophosphate-protecting groups in the development of thermolytic immunostimulatory DNA prodrugs. The alcohols are converted to their deoxyribonucleoside phosphoramidite derivatives, which are then used in the preparation of thermosensitive dinucleoside phosphorothioates. The thiophosphate-protecting groups of these dinucleoside phosphorothioates presumably undergo thermolytic cyclodeesterification at elevated temperature under essentially neutral conditions to release the desired phosphorothioate diester function. On the basis of their thermolytic deprotection kinetics, one can identify those thiophosphate-protecting groups that (i) may be useful for thiophosphate protection of CpG motifs of immunostimulatory DNA oligonucleotides (CpG ODNs); (ii) are suitable for protection of phosphodiester functions flanking the CpG motifs; and (iii) offer adequate protection of terminal phosphodiester functions against ubiquitous extracellular and intracellular exonucleases that may be found in biological environments.

Convenient synthesis of a propargylated cyclic (3'-5') diguanylic acid and its "click" conjugation to a biotinylated azide.

The ribonucleoside building block, N²-isobutyryl-2'-O-propargyl-3'-O-levulinyl guanosine, was prepared from commercial N²-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-O-propargyl guanosine in a yield of 91%. The propargylated guanylyl(3'-5')guanosine phosphotriester was synthesized from the reaction of N²-isobutyryl-2'-O-propargyl-3'-O-levulinyl guanosine with N²-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-O-tert-butyldimethylsilyl-3'-O-[(2-cyanoethyl)-N,N-diisopropylaminophosphinyl] guanosine and isolated in a yield of 88% after P(III) oxidation, 3'-/5'-deprotection, and purification. The propargylated guanylyl(3'-5')guanosine phosphotriester was phosphitylated using 2-cyanoethyl tetraisopropylphosphordiamidite and 1H-tetrazole and was followed by an in situ intramolecular cyclization to give a propargylated c-di-GMP triester, which was isolated in a yield of 40% after P(III) oxidation and purification. Complete N-deacylation of the guanine bases and removal of the 2-cyanoethyl phosphate protecting groups from the propargylated c-di-GMP triester were performed by treatment with aqueous ammonia at ambient temperature. The final 2'-desilylation reaction was effected by exposure to triethylammonium trihydrofluoride affording the desired propargylated c-di-GMP diester, the purity of which exceeded 95%. Biotinylation of the propargylated c-di-GMP diester was easily accomplished through its cycloaddition reaction with a biotinylated azide derivative under click conditions to produce the biotinylated c-di-GMP conjugate of interest in an isolated yield of 62%.

Release of DNA oligonucleotides and their conjugates from controlled-pore glass under thermolytic conditions.

The sequential functionalization of long-chain alkylamine controlled-pore glass (CPG) with a 3-hydroxypropyl-(2-cyanoethyl)thiophosphoryl linker and a dinucleoside phosphorotetrazolide leads to a uniquely engineered support for solid-phase synthesis. Unlike conventional succinylated-CPG supports, this support is designed to allow oligonucleotide deprotection and elimination of deprotection side-products to proceed without release of the oligonucleotide. When needed, the DNA oligonucleotide can be thermolytically released in 2 hr under essentially neutral conditions. The modified CPG support has been successfully employed in the synthesis of both native and fully phosphorothioated DNA 20-mers. On the basis of reversed-phase HPLC and electrophoretic analyses, the purity of the released oligonucleotides is comparable to that of identical oligonucleotides synthesized from succinylated-CPG supports, in terms of both shorter-than-full-length oligonucleotide contaminants and overall yields. The detailed preparation of DNA oligonucleotides conjugated with exemplary reporter or functional groups, either at the 3'-terminus or at both 3'- and 5'-termini, is also described.

Thermolytic release of covalently linked DNA oligonucleotides and their conjugates from controlled-pore glass at near neutral pH.

The functionalization of long chain alkylamine controlled-pore glass (CPG) with a 3-hydroxypropyl-(2-cyanoethyl)thiophosphoryl linker and its conversion to the support 7 has led to the synthesis of DNA oligonucleotides and their 3'- or (3',5')-conjugates. Indeed, CPG support 7 has been successfully employed in the synthesis of both native and fully phosphorothioated DNA 20-mers. Unlike conventional succinylated CPG supports, this distinctively functionalized support allows oligonucleotide deprotection and removal of the deprotection side products to proceed without releasing the oligonucleotide into the aqueous milieu. When freed from deprotection side products, the DNA oligonucleotide is thermolytically released from the support within 2 h under nearly neutral conditions (pH 7.2, 90 degrees C). The quality of these oligonucleotides is comparable to that of identical oligonucleotides synthesized from succinylated CPG supports in terms of shorter than full length oligonucleotide contaminants and overall yields. The versatility of the thermolytic CPG support 7 is further demonstrated by the synthesis of a DNA oligonucleotide (20-mer) and its conjugation with an azido and alkynyl groups at both 5'-and 3'-termini, respectively. The functionality of the (3',5')-heteroconjugated oligonucleotide 18 is verified by its circularization to the DNA oligonucleotide 19 under "click" chemistry conditions.