Large-scale synthesis of "Cpep" RNA monomers and their application in automated RNA synthesis.

Small interfering RNAs (siRNA) are the latest candidates for oligonucleotide-based therapeutics. Should siRNA be successful in clinical trials, a huge demand for synthetic RNA is anticipated. We believe that 1-(4-chlorophenyl)-4-ethoxypiperidin-4-yl (Cpep) is an ideal 2'-protecting group for large-scale syntheses. Unlike 2'-silyl groups, mild acid hydrolysis instead of fluoride ion is used for the 2'-deprotection. The syntheses of 2'-Cpep protected nucleosides (A, C, G, and U) has been accomplished on a 0.5 Kg scale. The 2'-Cpep monomers were transformed into 3'-O-phosphoramidites for conventional automated solid-phase synthesis. Cost-effective processes for large-scale synthesis of Cpep monomers and initial automated solid-phase synthesis are demonstrated.

Synthesis of 2',3'-dideoxy-2',3'-didehydro nucleosides via a serendipitous route.

This paper describes a "green" synthesis of 2',3'-unsaturated 2',3'-dideoxynucleosides via an electrochemical reaction. Using this approach d4T, d4U, ddA and ddI can be synthesized in high yields.

Unexpected results and recourse in process optimization of nucleoside 3'-O-succinates.

An improved and scalable protocol for the synthesis of 3'-O-succinates of nucleosides has been developed using succinic anhydride. As a result, formation of unwanted dimer has been completely eliminated and use of toxic and smelly reagents have been avoided during synthesis of nucleoside succinates. All succinates were isolated in pure state without silica gel column chromatography.

A case study: oligonucleotide purification from gram to hundred gram scale.

This article describes the purification and scale-up of ISIS 2302, a 20-mer phosphorothioate oligonucleotide by anion-exchange (AX) chromatography. The key operating parameters were optimized at gram scale and further scaled up to hundred gram. SOURCE 30Q, a high efficiency polymeric chromatographic media was used for both the small and large-scale work. High length-based purity and yield were maintained at scale-up. This purification is one of the largest demonstrations of AX purification of phosphorothioate oligonucleotide.

Multiple oligonucleotide synthesis in tandem on solid-phase supports for small and large scale synthesis.

Multiple oligonucleotides linked end-to-end in tandem can be synthesized by adding a nucleoside to the 5'-OH end of a prior sequence. Nucleosides with 3'-succinyl or Q-Linker arms are coupled with HBTU/DMAP. Alternatively, new phosphoramidite reagents with 3'-ester linkages can be used. Hydroxyl or amino supports can also be used as universal starting materials. Treatment with NH4OH cleaves the 3'-ester to yield only 3'-OH groups and no unwanted 3'-phosphorylated products occur.

Large-scale purification of antisense oligonucleotides by high-performance membrane adsorber chromatography.

Very high flux ion-exchange membranes were utilized for a novel purification of antisense oligonucleotides (20-mer). Strong anion-exchange membranes were produced by attaching polymeric ligands onto a microporous cellulosic matrix. The oligonucleotides purified were therapeutic single-stranded phosphorothioates deoxyribonucleotides. Although small-scale membrane devices (15 cm2) had similar resolution to traditional chromatographic columns; their throughputs were superior. Greater than a 1300-fold scale-up produced very similar purity and yields of the phosphorothionate product. Scale-up experiments were conducted with a 2 m2 surface area membrane module. These modules were easily capable of very high throughputs of 0.5 to 2 l/min. High purity and yields were achieved by both step and linear gradient elution.

Purification of antisense oligonucleotides.

Chromatography is an effective tool for obtaining high-purity synthetic oligonucleotides for a variety of end uses, including antisense drug therapy. Reversed-phase and anion-exchange chromatographies are widely used techniques for this application. While selectivity of these techniques can be modified by methods such as ion-pair RP-HPLC or affinity chromatography, these are presently used only at small scales. RP chromatography makes use of terminal hydrophobic-protecting groups to increase retention and selectivity. The main advantages of the RP method are its utility for the purification of a wide variety of modified oligonucleotide structures, its applicability across a range of terminal hydrophobic groups, such as fluorescein, and its ready use from small scale to very large scale with a minimal requirement for process development. AX-HPLC can also give high-purity products at generally higher media capacities. A more extensive method development effort is typically required for the AX-HPLC purification of AO. The AX yield per unit operation can be lower, but the isolated yield of DMT-off desalted oligonucleotide can be equal to or higher than that from RP-HPLC. As additional AO drugs enter and mature in the market, there will be a potential need for ton-scale purification processes. AX provides a way to scale up production on somewhat less expensive equipment with reduced organic solvent requirements.

Rapid esterification of nucleosides to solid-phase supports for oligonucleotide synthesis using uronium and phosphonium coupling reagents.

Nucleosides can be esterified to solid-phase supports using uronium or phosphonium coupling reagents and a coupling additive, such as 1-hydroxybenzotriazole (HOBT), 7-aza-1-hydroxybenzotriazole (HOAT), N-methylimidazole (NMI), or 4-(dimethylamino)pyridine (DMAP). However, DMAP was far superior to other additives and high nucleoside loadings (up to 60 micromol/g) and rapid coupling reactions (< or = 10 min) were possible. Hydroxyl-derivatized CPG was attached to nucleosides with 3'-succinyl or 3'-hydroquinone-O, O'-diacetic acid (HQDA or Q-Linker) carboxyl groups through a primary ester linkage. Alternatively, supports derivatized with succinic acid or the Q-Linker were attached directly to the 3'-OH group of nucleosides through a secondary ester linkage. Uronium reagents (HATU or HBTU) gave the best results with the HQDA linker arm, while the bromophosphonium (BrOP or PyBrOP) reagents were best with the succinyl linker arm. In all cases, the coupling reactions were much faster than previous methods using carbodiimide coupling reagents. The ease and speed of the reaction make this support derivatization procedure suitable for automated in situ couplings on DNA synthesizers.

Multiple oligodeoxyribonucleotide syntheseson a reusable solid-phase CPG support via the hydroquinone-O,O'-diacetic acid (Q-Linker) linker arm.

A strategy for oligodeoxyribonucleotide synthesis on a reusable CPG solid-phase support, derivatized with hydroxyl groups instead of amino groups, has been developed. Ester linkages, through a base labile hydroquinone- O, O '-diacetic acid ( Q-Linker ) linker arm, were used to couple the first nucleoside to the hydroxyl groups on the support. This coupling was rapidly accomplished (10 min) using O -benzotriazol-1-yl- N, N, N ', N '-tetramethyluronium hexafluorophosphate (HBTU) and 1-hydroxybenzotriazole as the activating reagents. Oligodeoxyribonucleotide synthesis was performed using existing procedures and reagents, except a more labile capping reagent, such as chloro-acetic anhydride, methoxyacetic anhydride or t-butylphenoxyacetic anhydride, was used instead of acetic anhydride. After each oligodeoxyribonucleotide synthesis, the product was cleaved from the support with ammonium hydroxide (3 min) and deprotected as usual. Residual linker arms or capping groups were removed by treatment with ammonium hydroxide/methylamine reagent and the regenerated support was capable of reuse. Up to six different oligodeoxyribonucleotide syntheses or up to 25 cycles of nucleoside derivatization and cleavage were consecutively performed on the reusable support. This method may provide a significant cost advantage over conventional single-use solid supports currently used for the manufacture of antisense oligodeoxyribonucleotides.

Conformational study of DNA-RNA duplexes containing MMI substituted phosphodiester linkages by FTIR spectroscopy.

Six methylene(methylimino) (MMI, Bhat et al. J. Org. Chem., 61, 8186, 1996) linked oligonucleotides a-f (* = MMI linkage; 5'-GCGT*TT*TT*TT*TT*TGCG-3') containing various combinations of 2'-O-methyl and 2'-fluoro substituent were synthesized as a model to study the global conformational change upon hybridization to the complement RNA. Fourier transform infrared (FTIR) spectroscopic technique has been used to study and compare the influence of these modifications on the solution conformation of 2'-modified MMI DNA-RNA duplexes. FTIR analysis of the single-stranded RNA (5'-CGCAAAAAAAAAACGC-3') and the modified oligonucleotides a-f showed that all sugar residues adopted a C3'-endo conformation (North-type). Stable duplexes were formed when oligonucleotides a-f were hybridized to the complement RNA. These duplexes retained the original C3'-endo conformation for all sugar residues, hallmark of an A-form of duplex. We postulate that the observed preorganization of the sugar residues and oligonucleotides containing 2'-modified MMI modifications may play an important role in both improving the recognition of RNA target and enhancing the stability of duplex formation with RNA.

Conformational studies of antisense DNA by PFG NMR.

Pulsed field gradient diffusion constant measurements were used to resolve the ambiguity in determining the conformational states of single-stranded DNA dodecanucleotides (d1s, d4s and d5s). For d1s and d5s, because of the spectral symmetry conventional NMR analyses cannot differentiate whether they are hairpins or homo-duplexes. However, the diffusion constants of these sequences at 300 K are 1.4 times greater than those of the comparison complementary duplexes. This result agrees well with what is expected for Dhairpin/Dduplex based on classic liquid-phase translational diffusion models and the Einstein-Stokes equation, confirming that d1s and d5s form hairpins. d4s did not show a structured spectral pattern, but its diffusion constant measurement suggests that this sequence may not be a random coil. The DNA sequences studied contain chemically modified backbone linkages and are potential antisense agents for gene regulation. The knowledge of their diffusion constants, in combination with conventional NMR analysis and other biophysical spectroscopic measurements, provides new insights into the relationships of chemical structure and conformational preference of antisense oligonucleotides and their analogs.

Antisense oligodeoxynucleotides: synthesis, biophysical and biological evaluation of oligodeoxynucleotides containing modified pyrimidines.

6-Azathymidine, 6-aza-2'-deoxycytidine, 6-methyl-2'-deoxyuridine, and 5,6-dimethyl-2'-deoxyuridine nucleosides have been converted to phosphoramidite synthons and incorporated into oligodeoxynucleotides (ODNs). ODNs containing from 1 to 5 of these modified pyrimidines were compared with known 2'-deoxyuridine, 5-iodo-2'-deoxyuridine, 5-bromo-2'-deoxyuridine, 5-fluoro-2'-deoxyuridine, 5-bromo-2'-deoxycytidine, and 5-methyl-2'-deoxycytidine nucleoside modifications. Stability in 10% heat inactivated fetal calf serum, binding affinities to RNA and DNA complements, and ability to support RNase H degradation of targeted RNA in DNA-RNA heteroduplexes were measured to determine structure-activity relationships. 6-Azathymidine capped ODNs show an enhanced stability in serum (7- to 12-fold increase over unmodified ODN) while maintaining hybridization properties similar to the unmodified ODNs. A 22-mer ODN having its eight thymine bases replaced by eight 6-azathymines or 5-bromouracils hybridized to a target RNA and did not inhibit RNase H mediated degradation.

Novel strategies towards construction of non-ionic and achiral backbones in antisense oligonucleotides.

A convenient synthesis of a thymidine (T) nucleoside dimer (T-3'-CH2-O-NCH3-CH2-4'-T) 12 has been accomplished via a nucleoside coupling reaction. An alternative synthesis of 3'-deoxy-3'-C-hydroxymethyl-thymidine is described. The new dimer and methodology is useful for the development of backbone-modified antisense oligonucleosides.

Synthesis and antiviral activity of certain guanosine analogues in the thiazolo[4,5-d]pyrimidine ring system.

Several sugar-modified nucleoside derivatives of the purine analogue 5-amino-3-beta-D-ribofuranosylthiazolo[4,5-d]pyrimidine-2,7-dione (1) were synthesized. Phosphorylation of 1 using POCl3 resulted in 5'-monophosphate 2, which was subsequently converted to 3',5'-cyclic phosphate 3, by reported methods. 5'-Sulfamoyl derivative 4 was synthesized by treatment of the 2,3-O-isopropylidene derivative of 1 with chlorosulfonamide followed by acid deprotection. Compounds 5-7, the 5'-deoxy, the tri-O-acetyl, and the 2'-deoxy derivatives of 1, respectively, were synthesized by glycosylation of 5-aminothiazolo[4,5-d]pyrimidine-2,7-dione, the aglycon of 1, with the appropriate sugar moieties, utilizing the Vorbruggen procedure. Oxidative cleavage of the C2'-C3' bond in 1 followed by reduction with sodium borohydride led to "seco" analogue 8. Nucleosides 2-8 were evaluated for antiviral activity in vivo against the Semliki Forest virus. The activity of compounds 2, 5, and 7 were similar to that of 1. Cyclic phosphate 3 was toxic at the high dose and weakly active at the lower dose. Compounds 4, 6, and 8 were inactive in this system.

Inhibition of pyrimidine metabolism in myeloid leukemia cells by triazole and pyrazole nucleosides.

Two triazole nucleosides, 1 (3-beta-D-ribofuranosyl-1,2,4-triazole-5-carboxamide) and 2 (2-beta-D-ribofuranosyl-1,2,3-triazole-4,5-dicarboxamide), and a pyrazole nucleoside, 3 (1-beta-D-ribofuranosylpyrazole-3,4-dicarboxamide), were found to inhibit pyrimidine nucleotide biosynthesis in the human myeloid leukemia cell line, K562. Cells treated with these inhibitors released orotate in quantities of 8-35 nmol/10(5) cells/day. Treatment with these compounds caused the K562 cells to accumulate in the S phase of the cell cycle and induced the cells to synthesize hemoglobin.

Growth inhibition and induction of cellular differentiation of human myeloid leukemia cells in culture by carbamoyl congeners of ribavirin.

A series of 1,2,3-triazole (2), pyrazole (3 and 5), and pyrrole (4) ribonucleosides with two adjacent carbamoyl groups have been synthesized and evaluated for cell growth inhibition and induction of cellular differentiation of HL-60 cells in culture. Glycosylation of the TMS derivatives of dimethyl 1,2,3-triazole-4,5-dicarboxylate (6) and diethyl pyrazole-3,4-dicarboxylate (7) with 1-O-acetyl-2,3,5-tri-O-benzoyl-D- ribofuranose (8) in the presence of TMS triflate gave predominantly the beta-nucleosides 9 and 14, respectively. Ammonolysis of 9 and 14 furnished 2-beta-D-ribofuranosyl-1,2,3-triazole-4,5-dicarboxamide (2) and 1-beta-D-ribofuranosylpyrazole-3,4-dicarboxamide (3), respectively. Stereoselective ring annulation of 1-deoxy-1-hydrazinyl-2,3-O-isopropylidene-D- ribose (16) with tetracyanoethylene (15) gave 5-amino-1-(2,3-O-isopropylidene-beta-D-ribofuranosyl)pyrazole-3,4- dicarbonitrile (17). Deisopropylidenation of 17, followed by oxidative hydrolysis of the reaction product (18), gave the 5-amino derivative of 3 (5). Stereospecific glycosylation of the sodium salt of preformed diethyl pyrrole-3,4-dicarboxylate (22) with 1-chloro-2,3-O-isopropylidene-5-O-(tert-butyldimethylsilyl)-alpha-D- ribofuranose (23) was accomplished to furnish blocked nucleoside 24, which on ammonolysis and deisopropylidenation gave 1-beta-D-ribofuranosylpyrrole-3,4-dicarboxamide (4). The structures of 2 and 3 were assigned by single-crystal X-ray diffraction studies, which showed extensive inter- and intramolecular hydrogen bonding. Nucleosides 2-5 are devoid of significant cytotoxic properties against L1210 and WI-L2 leukemia cells in culture. However, these compounds were found to be inducers of cellular differentiation of HL-60 cells in the range of 30-60 microM and were comparable to ribavirin in this regard.

Low molecular weight human T-cell response immunopotentiator: alpha-2'-deoxy-3-deazaguanosine.

We have examined the immunological activity of a unique alpha-nucleoside analog of 2'-deoxyguanosine in which the pyrimidine ring nitrogen in the 3 position is replaced by CH [6-amino-1, 5-dihydro-1-(2-deoxy-alpha-D-erythro-pentofuranosyl)imidazo[4,4-c] pyridin-4-one, alpha-d3DGuo, 1] and its structural analogs. The alpha-d3DGuo is not mitogenic to human PBL. It displayed consistently, however, a potent immunoenhancing activity on PHA-induced human lymphocyte proliferation at concentrations ranging from 0.0125 mM to 0.4 mM in a dose dependent manner. These findings thus suggest that mitogenicity is not a pre-requisite for the immunoenhancing effect. The maximal potentiating effect of alpha-d3DGuo is usually exerted at the bottom range of the dose response to PHA. The magnitude of increase is about the same as that mediated by rIL-2. Similarly, Con A mediated lymphocyte proliferation is markedly enhanced by alpha-d3DGuo. When added during allogeneic MLR, alpha-d3DGuo also augmented the proliferation of alloreactive T-cells and the magnitude of response was similar to that induced by rIL-2. The alpha-d3DGuo induced increase in allogeneic response was dependent on concentrations of both alpha-3dDGuo and alloantigens as noted with T-mitogen induced proliferative responses. The cytotoxic activity of lymphocytes induced in allogeneic mixed cultures was also augmented by alpha-d3DGuo. It showed, however, no potentiating effect on B-lymphocytes proliferation stimulated either with SAC or PWM. The alpha-d3DGuo is also able to restore, at least partially, the depressed proliferative responses of T-cells to both PHA and Con A.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural mimicry of adenosine by the antitumor agents 4-methoxy- and 4-amino-8-(beta-D-ribofuranosylamino)pyrimido[5,4-d]pyrimidine as viewed by a molecular modeling method.

A rationale for the antitumor activity of 4-methoxy- and 4-amino-8-(beta-D-ribofuranosylamino)pyrimido-[5,4-d]pyrimidine (beta-MRPP and beta-ARPP, respectively) was studied by a molecular modeling method. Although these nucleoside analogues are structurally different from adenosine, they act as substrates for adenosine kinase. The molecular modeling method, which considered the three-dimensional structure and atom-based physicochemical properties of the nucleosides to quantify the molecular similarities, showed that certain low-energy conformations of the beta anomers of a series of nucleosides including beta-MRPP, beta-ARPP, and their 4-hydroxy, 4-amino-6-chloro, 4-methylthio-2,6-dichloro, 4,6-diamino, 4-dimethylamino, 4-methylamino, and 4-hydroxy-2,6-dichloro analogues have remarkable structural similarity to adenosine. The method also suggested that the selection of the reference compound adenosine in the structural comparison is of primary importance to gain insight into the observed antitumor activity. The success of the present method led to AM1 (Austin model 1) molecular orbital calculations and experimental studies indicating that the antitumor activity of the alpha anomer of ARPP is probably due to equilibration to the beta anomer. The AM1 calculation of the protonation energy of N5 of pyrimido[5,4-d]pyrimidines, which occupies the same position in space as the N1 of adenosine, gave a direct correlation between the basicity of the nitrogen with a lone pair of electrons and the observed antitumor activity.

Inhibition of phosphoribosylpyrophosphate synthetase by 4-methoxy-(MRPP) and 4-amino-8-(D-ribofuranosylamino) pyrimido[5,4-d]pyrimidine (ARPP).

The basis for the antitumor activities of the exocyclic amino nucleosides 4-amino-(ARPP) and 4-methoxy-8-(D-ribofuranosylamino)pyrimido[5,4-d]pyrimidine (MRPP) was investigated. The primary target of these nucleosides appeared to be 5-phospho-alpha-D-ribofuranose-1-pyrophosphate (PRPP) synthetase. MRPP-5'-monophosphate was a competitive inhibitor (Ki = 40 microM) of the activation of this enzyme by the cofactor inorganic phosphate (K alpha = 2.2 mM). Consequently, ARPP and MRPP treatment of WI-L2 cultures rapidly inhibited both de novo pyrimidine and purine synthesis as well as the nucleotide salvage reactions dependent on PRPP, ARPP or MRPP treatment completely prevented [14C]bicarbonate incorporation into acid-soluble pyrimidine and purine nucleotides. The rate of salvage of [8-14C]hypoxanthine to form IMP was decreased by 85%. Treatment of cells with these agents caused a 50% reduction in the steady-state level of PRPP. When the capacity of the treated cells for sustained synthesis of PRPP was examined by adenine incorporation, the rate of adenine uptake was inhibited by greater than 50%. In vivo treatment of BDF1 mice with a single dose of ARPP (173 mg/kg) or MRPP (62 mg/kg) extended the mean life span of the mice, which had been inoculated intraperitoneally 1 day earlier with 1 x 10(6) L1210 murine leukemia cells, by 62 and 82% respectively. These studies indicate that MRPP and ARPP inhibit PRPP synthetase, and that PRPP synthetase may be a viable target in the development of certain antitumor agents.