Stability of Phosphorothioate Oligonucleotides in Aqueous Ammonia in Presence of Stainless Steel

Abstract

Antisense oligonucleotides as modulators of gene expression represent an exciting new drug technology. Oligodeoxyribonucleotide phosphorothioates are now among the most intensively investigated nuclease-resistant antisense analogs, as evidenced by a number of ongoing clinical trials against several disease targets. Structurally, these differ from natural oligonucleotides by the replacement of one of two nonbridging oxygen atoms by a sulfur atom at each internucleotide linkage. Among factors in the successful development of these complex molecules to support broad clinical trials have been advances made in automation, analysis and purification. The large scale synthesis of oligonucleotide phosphorothioates is presently carried out by initial formation of the internucleotide phosphite linkage using solid-phase phosphoramidite chemistry followed by sulfurization. Efficient synthesis of 20-mer oligophosphorothioates has been achieved on 0.15 mole scale with only 1.75-fold excess of amidite synthons. However, as the scale of synthesis increases to meet future market demands, issues related to fast and efficient synthesis, automation, scalability and product purification are also being investigated. One issue has been the protocol for final product deprotection. Since deprotection involves large quantities of saturated aqueous ammonium hydroxide, one might consider use of stainless steel reactors to withstand resulting vapor pressure at 55°C. A recent report,¹ however, discusses the instability of dimer phosphorothioates in aqueous ammonia in the presence of metal ions. As this is potentially an important issue for phosphorothioate oligonucleotide synthesis, we describe herein our findings regarding deprotection of a 20-mer oligodeoxyribonucleotide phosphorothioate with aqueous ammonia during process development studies.     

Oligodeoxyribonucleotide Phosphorothioates: Substantial Reduction of (N-1)-Mer Content Through the Use of Blockmer Phosphoramidite Synthons

Abstract

Sequence-specific modulation of gene expression for the treatment of diseases has come to reality. Multiple examples of oligodeoxyribonucleotide phosphorothioates, in which one nonbridging oxygen atom of the internucleotide phosphate group of DNA is replaced by a sulfur atom are currently in advanced clinical trials. Recent advances in phosphoramidite coupling chemistry and solid phase synthesis methodology, together with current state of the art large-scale synthesizers, allow complete assembly of a 20-mer deoxyribonucleotide phosphorothioate at 150 mmole scale in just 8 h. Very high average coupling efficiencies (>98.5%) have been achieved at these scales with only 1.75-fold molar amidite excess.     

Sulfurization Efficiency in the Solution Phase Synthesis of Deoxyribonucleoside Phosphorothioates - Comparison of Sulfur Triethylamine with Various Sulfurizing Agents 1

Abstract

Efficient solution-phase synthesis of nucleoside phosphorothioates utilizing phosphoramidite approach is described. Elemental sulfur in combination with triethylamine is the prefered choice for sulfurization of phosphite triester to phosphorothioates.

     

Enzymatic Synthesis of Stereoregular (All Rp) Oligonucleotide Phosphorothioate and Its Properties

Abstract

A protocol has been established for the synthesis of stereoregular (all Rp) oligonucleotide phosphorothioates. A 25-mer oligodeoxynucleotide phosphorothioate has been synthesized and studied for biophysical and biochemical properties.     

Diastereomeric Process Control in the Synthesis of Oligodeoxyribonucleotide Phosphorothioates

Abstract

The emergence of antisense and antigene oligonucleotides as potential sequenceselective inhibitors of gene expression is evidenced by the growing number of ongoing clinicals trials against a variety of diseases. First generation antisense therapeutics utilize a uniformly modified oligodeoxyribonucleotide phosphorothioate where one non-bridging oxygen atom is formally replaced by sulfur, because natural DNA is unstable towards extra- and intracellular enzymes. Phosphoramidite chemistry has been widely used for the synthesis of phosphorothioate oligonucleotides because of its potential for automation, high coupling efficiency, ease of site-specific thioate linkage incorporation, and ready scalability. The large scale solid-supported synthesis of phosphorothioates is presently carried out by initial formation of the internucleotidic phosphite linkage followed by sulfurization of the phosphite triester to phosphorothioate using the Beaucage reagent. The resulting O,O-linked phosphorothioate diester linkage in the oligonucleotide is a chiral functional group. For a typical 20-mer there are 524,288 (2¹⁹) possible diastereoisomers. Separation and individual quantification of this number of diastereomers is currently not feasible. In addition, the best reported methods for stereocontrolled synthesis of phosphorothioate oligomers are not presently useful for drug synthesis; that is, since net 100% enantiomeric excess is not achieved in the coupling step, the oligomeric product still consists of the same mixture of Sp and Rp diastereomers, except that the levels of all but one isomer are reduced to low individual levels. As a result, even a small change in the and Sp phosphorothioate diesters, due to racemization during coupling, indicating that the overall synthetic process is stereo reproducible and under inherent process control.     

Efficiency Of Coupling in the Synthesis of Deoxyribonucleotide Phosphorothioates Via Phosphotriester Approach Utilizing Various Activating Reagents 1

Abstract

The synthesis of deoxyribonucleotide phosphorothioates via phosphotriester approach utilizing various coupling reagents is described.

     

Use of Phenylacetyl Disulfide (PADS) in the Synthesis of Oligodeoxyribonucleotide Phosphorothioates

Abstract

Investigations into the use of phenylacetyl disulfide (PADS) as an efficient sulfur transfer agent in the solid phase synthesis of oligodeoxyribonucleotide phosphorothioates showed that under suitable solvent conditions, this relatively inexpensive reagent rapidly and efficiently sulfurizes internucleotide phosphite linkages.     

Solution Phase Synthesis of Dithymidine Phosphorothioate by a Phosphotriester Method Using New S-Protecting Groups

Abstract

ABSTRACT

A phosphotriester method for the synthesis of dithymidine phosphorothioates with eight S-protecting groups has been investigated. Three of the S-protecting groups possesed catalytic activity, however side reactions occurred under deprotection. The best S-protecting group was 4-chloro-2-nitrobenzyl which could be removed with a minimum of side reactions (0.3 %). The coupling reagent PyFNOP (11) gave protected dithymidine phosphorothioate in 96% yield after 15 min coupling.     

Ultrafast Cleavage and Deprotection of Oligonucleotides Synthesis and Use of CAc Derivatives

Abstract

We have investigated the use of alkylamines as fast cleavage and deprotection reagents for the solid phase synthesis of oligonucleotides and found methylamine/ammonium hydroxide (or methylamine) as an efficient reagent. The transamination side product formed with the commonly used dC^bz has been eliminated by the use of dC^Ac phosphoramidite. This system has successfully been used in the synthesis of oligonucleotides and oligonucleoside phosphorothioates. DMT dC^Ac hydrogen phosphonate and DMT ribo C^Ac-2′-O Me phosphoramidite also have been prepared and used in the synthesis of oligonucleotides.     

Isolation of Oligodeoxynucleoside Phosphorothioate Diastereomers by the Combination of DEAE Ion-Exchange and Reversed-Phase Chromatography

Abstract

In antisense trials, oligodeoxynucleoside phosphorothioates (S-oligo) stereoisomers arise from the thiophosphate R/S configuration. We have explored effective separation of these isomers by the combination of DEAE ion-exchange (IELC) and reversed-phase (RPLC) liquid chromatography. Twenty-six S-oligos and some of the natural counterparts were examined. In the presence of DMTr at the 5′-end, isomers could be separated well by ELC, while RPLC gave good resolution in the absence of this protecting group. Combination of these two different modes leads to more effective isolation.

     

A Mycobacterial Perspective on Tuberculosis in West Africa: Significant Geographical Variation of M. africanum and Other M. tuberculosis Complex Lineages

BackgroundPhylogenetically distinct Mycobacterium tuberculosis lineages differ in their phenotypes and pathogenicity. Consequently, understanding mycobacterial population structures phylogeographically is essential for design, interpretation and generalizability of clinical trials. Comprehensive efforts are lacking to date to establish the West African mycobacterial population structure on a sub-continental scale, which has diagnostic implications and can inform the design of clinical TB trials.Conclusions/SignificanceBecause of the geographical divide of the mycobacterial populations in West Africa, individual research findings from one country cannot be generalized across the whole region. The unequal geographical family distribution should be considered in placement and design of future clinical trials in West Africa.     

Synthesis and Incorporation of 5″-Amino- and 5′-Mercapto-5′-Deoxy-2′-O-Methyl Nucleosides Into Hammerhead Ribozymes

Abstract

Novel 5′-amino-5′-deoxy-2′-O-methyl uridine, guanosine and adenosine 3′-O-phosphoramidites 5, 11, and 20, as well as protected 5′-mercapto-5′-deoxy-2′-O-methyl uridine 3′-O-phosphoramidite 23 were synthesized from 2′-O-methyl nucleosides. These analogs were incorporated at the 5′-ends of hammerhead ribozymes to evaluate achiral bridging 5′-N- phosphoramidates and 5′-S-phosphorothioates as alternatives for non- bridging phosphorothioates commonly used for end stabilization against nucleases. Oligonucleotide synthesis and deprotection conditions were optimized for better yields of these modified ribozymes.     

Efforts Toward Synthesis of Oligonucleotides for Commercialization

Abstract

Recent progress on the development of oligonucleotide phosphorothioates to meet market demands is reported.     

Chemoselective Synthesis of Dithymidine Phosphorothioate in Solution Using O-Protected Thiophosphate Monomers

Abstract

Diastereomerically pure O-protected thymine monothioate nucleotide (I) is efficiently coupled to protected thymidine (II) in a chemoselective, but not stereoselective manner, to give dithymidine phosphorothioates (III).     

Synthesis of the tBuSATE Pronucleotide of AZT by Two Different Synthetic Approaches

Abstract

A large scale synthesis of the tBuSATE pronucleotide of AZT was required for in vivo studies. A comparative synthesis of this derivative by phosphoramidite and monophosphate approaches is reported.     

Incorporation of terminal phosphorothioates into oligonucleotides.

Considerable effort has been directed towards studying the structure and function of oligonucleotides and several approaches rely on the attachment of reporter groups to oligonucleotides. We report here the introduction of 3'- and 5'-terminal phosphorothioates into heptameric oligonucleotides and their post-synthetic modification with several reporter groups. The synthesis of terminal phosphorothioates is based on the coupling of a ribonucleoside phosphoramidite at the first or last nucleotide, respectively, which, after sulphurization, is removed by sequential oxidation of the vicinal hydroxyl groups and then beta-elimination. Product formation is of the order of 95%. The ratio of phosphorothioate- versus phosphate-terminated oligodeoxynucleotides as analysed by electrophoresis on a Hg2+gel is in general 85/15. Examples for the reactivity of the terminal phosphorothioates for conjugation with cholesterol, bimane and for sulphydryl exchange are described.     

Synthesis and Biological Activity of O,O-Dialkyl S-(5-Aryl-l,3,4-oxadiazol-2(3H)-on-3-yl)methyl Phosphorothioates and Phosphorodithioates

Some phosphorus derivatives of oxadiazoles were synthesized to seek insecticidal lead compounds. The l,3,4-oxadiazol-2-ones were converted via the N-methylol derivatives to the corresponding N-chloromethyl derivatives. From these derivatives a variety of O,O-dimethyl phosphorodithioates 4, O,O-dimethyl phosphorothioates 5 and O,O-di-i-propyl phosphorothioates 6 were prepared.

These phosphorus derivatives were examined for insecticidal activity towards houseflies and for anti-acetylcholinesterase (anti-AChE) activity using the housefly heads as an enzyme source. Most of the compounds 4 and 5 showed contact toxicity as high as the analogous methidathion insecticides, which appeared to correlate with the strong anti-AChE activity. On the other hand, all the compounds 6 showed a high activity in AChE inhibition but only a poor insecticidal activity.     

Continual improvement of nasopharyngeal carcinoma segmentation with less labeling effort

PurposeConvolutional neural networks (CNNs) offer a promising approach to automated segmentation. However, labeling contours on a large scale is laborious. Here we propose a method to improve segmentation continually with less labeling effort.MethodsThe cohort included 600 patients with nasopharyngeal carcinoma. The proposed method was comprised of four steps. First, an initial CNN model was trained from scratch to perform segmentation of the clinical target volume. Second, a binary classifier was trained using a secondary CNN to identify samples for which the initial model gave a dice similarity coefficient (DSC) < 0.85. Third, the classifier was used to select such samples from the new coming data. Forth, the final model was fine-tuned from the initial model, using only selected samples.ResultsThe classifier can detect poor segmentation of the model with an accuracy of 92%. The proposed segmentation method improved the DSC from 0.82 to 0.86 while reducing the labeling effort by 45%.ConclusionsThe proposed method reduces the amount of labeled training data and improves segmentation by continually acquiring, fine-tuning, and transferring knowledge over long time spans.     

Conjugated Antisense Oligonucleotides for Inhibition of Human Cytomegalovirus In Vitro

Abstract

Several thiono triester containing oligonucleotide phosphorothioates linked with a lipophilic group have been synthesized. Some of these modified antisense oligonucleotides show potent anti-HCMV activity as well as improved cellular association and nuclease resistance.     

Large Scale Synthesis of the Cap Part in Messenger RNA Using a New Type of Bifunctional Phosphorylating Reagent

Abstract

Bifunctional phosphorylating reagents 1 and 2 were employed for the synthesis of the cap part, m⁷G⁷ pppG, from guanosine 5′-phosphates on a large scale without any protecting groups.