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.

Trans-acting oligodeoxythymidine phosphorothioate triester reagents for transient transfection optimized and facilitated by a high-throughput microbioreactor system.

A rapid and cost-effective transient transfection method for mammalian cells is essential for screening biopharmaceuticals in early stages of development. A library of 25 amphipathic trans-acting oligodeoxythymidine phosphorothioate triester (dTtaPS) transfection reagents, carrying positively charged and lipophilic groups, has been constructed for this purpose. High-throughput screening of the library, using an imaging cytometer and an automated microbioreactor system, has led to the identification of dTtaPS10+ as a potent transfection reagent. This reagent efficiently delivers a plasmid encoding enhanced green fluorescent protein in adherent HeLa cells while exhibiting low cytotoxicity. The microbioreactor system has been particularly useful for assessing the ability of dTtaPS10+ to deliver a plasmid encoding immunoglobulin IgG1 in a fed-batch serum-free suspension CHO cell culture; dTtaPS10+ -mediated transfection resulted in the production of IgG1 in yields comparable to or better than those obtained with commercial lipid-based transfection reagents under similar conditions. The ability of dTtaPS10+ to deliver plasmids is essentially unaffected by the presence of a silicone-based antifoaming reagent, which is commonly used in bioreactor cell cultures. The transfection efficiency of lyophilized dTtaPS10+ -plasmid complexes has been significantly restored upon aqueous reconstitution when compared to that achieved while using commercial transfection reagent complexes under similar conditions. The results of all experiments underscore the potential of dTtaPS10+ for transient transfection of plasmids into adherent cells and fed-batch serum-free suspension CHO cells and rapid screening of reagents in a microbioreactor system.

Structural evaluation of an amyloid fibril model using small-angle x-ray scattering.

Amyloid fibrils are highly structured protein aggregates associated with a wide range of diseases including Alzheimer's and Parkinson's. We report a structural investigation of an amyloid fibril model prepared from a commonly used plasma protein (bovine serum albumin (BSA)) using small-angle x-ray scattering (SAXS) technique. As a reference, the size estimates from SAXS are compared to dynamic light scattering (DLS) data and the presence of amyloid-like fibrils is confirmed using Congo red absorbance assay. Our SAXS results consistently show the structural transformation of BSA from spheroid to rod-like elongated structures during the fibril formation process. We observe the elongation of fibrils over two months with fibril length growing from 35.9 ± 3.0 nm to 51.5 ± 2.1 nm. Structurally metastable fibrils with distinct SAXS profiles have been identified. As proof of concept, we demonstrate the use of such distinct SAXS profiles to detect fibrils in the mixture solutions of two species by estimating their volume fractions. This easily detectable and well-characterized amyloid fibril model from BSA can be readily used as a control or standard reference to further investigate SAXS applications in the detection of structurally diverse amyloid fibrils associated with protein aggregation diseases.

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.

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.

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.

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.

Assessment of the cellular internalization of thermolytic phosphorothioate DNA oligonucleotide prodrugs.

The bioactivity of a CpG-containing phosphorothioate DNA oligonucleotide with thermolytic 2-(N-formyl-N-methylamino)ethyl (fma) thiophosphate groups in mice led us to investigate the parameters affecting the internalization of these thermosensitive DNA prodrugs in various cell lines. Flow cytometry and confocal microscopy analyses indicate that 5'-fluoresceinated fma-phosphorothioate DNA sequences are poorly internalized in Vero, HeLa and GC-2 cells. However, when four fma-thiophosphate groups of a 15-nucleotide long oligothymidylate prodrug are replaced with 3-(N,N-dimethylamino)prop-1-yl thiophosphate functions, internalization of the positively charged prodrug, under physiological conditions, increased fourfold in HeLa and 40-fold in Vero or GC-2 cells. No cytotoxic effects are observed in Vero cells even at an extracellular prodrug concentration of 50 µM over a period of 72 h. Confocal microscopy studies show that internalization of the positively charged oligothymidylate prodrug in Vero cells is time-dependent with early trafficking of the DNA sequence through endosomal vesicles and, eventually, to the nucleus of the cells. Thus, the incorporation of four 3-(N,N-dimethylamino)prop-1-yl thiophosphate groups into thermosentive fma-phosphorothioate DNA prodrugs is an attractive strategy for efficient cellular internalization of these nucleic acid-based drugs for potential therapeutic indications.

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.

Synthesis of an Atom-Specifically 2 H/13 C-Labeled Uridine Ribonucleoside Phosphoramidite.

A combined enzymatic and chemical synthesis of a 2'-O-cyanoethoxymethyl (CEM) protected [1',6-13 C2 , 5-2 H]-uridine phosphoramidite is described herein. This is the first report of an atom-specific nucleobase and ribose labeled 2'-O-CEM protected ribonucleoside phosphoramidite. Importantly, the CEM 2'-OH protecting group permits the efficient solid-phase synthesis of large (>60 nucleotides) RNAs with good yield and purity. The new isotope-labeled phosphoramidite can therefore be applied to nuclear magnetic resonance (NMR) spectroscopy studies. Specifically, the [1',6-13 C2 , 5-2 H]-uridine phosphoramidite can be used to make position-specifically labeled RNAs for NMR analysis without complications from resonance overlap and scalar and dipolar couplings. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of the ribonucleoside 6 Basic Protocol 2: Synthesis of the ribonucleoside phosphoramidite 11.

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.

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%.

Alterations in the HLA-B*57:01 Immunopeptidome by Flucloxacillin and Immunogenicity of Drug-Haptenated Peptides.

Neoantigen formation due to the interaction of drug molecules with human leukocyte antigen (HLA)-peptide complexes can lead to severe hypersensitivity reactions. Flucloxacillin (FLX), a ß-lactam antibiotic for narrow-spectrum gram-positive bacterial infections, has been associated with severe immune-mediated drug-induced liver injury caused by an influx of T-lymphocytes targeting liver cells potentially recognizing drug-haptenated peptides in the context of HLA-B*57:01. To identify immunopeptidome changes that could lead to drug-driven immunogenicity, we used mass spectrometry to characterize the proteome and immunopeptidome of B-lymphoblastoid cells solely expressing HLA-B*57:01 as MHC-I molecules. Selected drug-conjugated peptides identified in these cells were synthesized and tested for their immunogenicity in HLA-B*57:01-transgenic mice. T cell responses were evaluated in vitro by immune assays. The immunopeptidome of FLX-treated cells was more diverse than that of untreated cells, enriched with peptides containing carboxy-terminal tryptophan and FLX-haptenated lysine residues on peptides. Selected FLX-modified peptides with drug on P4 and P6 induced drug-specific CD8+ T cells in vivo. FLX was also found directly linked to the HLA K146 that could interfere with KIR-3DL or peptide interactions. These studies identify a novel effect of antibiotics to alter anchor residue frequencies in HLA-presented peptides which may impact drug-induced inflammation. Covalent FLX-modified lysines on peptides mapped drug-specific immunogenicity primarily at P4 and P6 suggesting these peptide sites as drivers of off-target adverse reactions mediated by FLX. FLX modifications on HLA-B*57:01-exposed lysines may also impact interactions with KIR or TCR and subsequent NK and T cell function.

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.

Solid-phase synthesis of siRNA oligonucleotides.

Since the discovery of RNA interference (RNAi) as a means to silence the expression of specific genes, small interfering RNA (siRNA) oligonucleotides have been recognized as powerful tools for targeting therapeutically important mRNAs and eliciting their destruction. This discovery has created a high demand for synthetic oligoribonucleotides as potential therapeutics and has spurred a renaissance in the development of rapid, efficient methods for solid-phase RNA synthesis. The design and implementation of 2'-hydroxyl protecting groups that provide ribonucleoside phosphoramidites with coupling kinetics and coupling efficiencies comparable to those of deoxyribonucleoside phosphoramidites are key to the production of RNA oligonucleotides in sufficient quantity and purity for pharmaceutical applications. In this context, various siRNAs were chemically modified to identify the biophysical and biochemical parameters necessary for effective and stable RNAi-mediated gene-silencing activities.

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.

Use of thermolytic protective groups to prevent G-tetrad formation in CpG ODN type D: structural studies and immunomodulatory activity in primates.

CpG oligodeoxynucleotides (ODN) show promise as immunoprotective agents and vaccine adjuvants. CpG ODN type D were shown to improve clinical outcome in rhesus macaques challenged with Leishmania major. These ODN have a self-complementary core sequence and a 3' end poly(G) track that favors G-tetrad formation leading to multimerization. Although multimerization appears necessary for localization to early endosomes and signaling via Toll-like receptor 9 (TLR-9), it can result in product polymorphisms, aggregation and precipitation, thereby hampering their clinical applications. This study shows that functionalizing the poly(G) track of D ODN with thermolytic 2-(N-formyl-N-methyl)aminoethyl (fma) phosphate/thiophosphate protecting groups (pro-D ODN) reduces G-tetrad formation in solution, while allowing tetrad formation inside the cell where the potassium concentration is higher. Temperature-dependent cleavage of the fma groups over time further promoted formation of stable G-tetrads. Peripheral blood cells internalized pro-D ODN efficiently, inducing high levels of IFNalpha, IL-6, IFNgamma and IP-10 and triggering dendritic cell maturation. Administration of pro-D35 to macaques challenged with L.major significantly increased the number of antigen-specific IFNgamma-secreting PBMC and reduced the severity of the skin lesions demonstrating immunoprotective activity of pro-D ODN in vivo. This technology fosters the development of more efficient immunotherapeutic oligonucleotide formulations for the treatment of allergies, cancer and infectious diseases.