Quantification of oligonucleotides by LC-MS/MS: the challenges of quantifying a phosphorothioate oligonucleotide and multiple metabolites.

BACKGROUND: LC-MS/MS allows quantification of therapeutic oligonucleotides in biological fluids at low ng/ml concentrations. Achieving selectivity between metabolites and parent molecules in a single assay is one of the biggest challenges when developing a method. We present a strategy that allows quantification of an 18-mer antisense therapeutic, trabedersen, and six metabolites in human plasma. RESULTS/METHODOLOGY: The method utilizes phenol-chloroform and SPE with UHPLC-MS/MS to independently quantify trabedersen and the 5´n-1, 5´n-2, 5´n-3, 3´n-1, 3´n-2 and 3´n-3 metabolites in a single assay. The qualification data indicate that if the method was validated it would meet regulatory expectations for precision, accuracy and selectivity. CONCLUSION: We show that quantification of an oligonucleotide and multiple metabolites, including isobaric 3´ and 5´ metabolites, is achievable in a single assay through good sample clean-up and careful optimization of the LC-MS/MS parameters. The strategy presented here can be applied elsewhere and may be useful for other oligonucleotides and their metabolites.

Identification of botryticidal proteins with similarity to NBS-LRR proteins in rosemary pepper (Lippia sidoides Cham.) flowers.

Heavy agricultural losses are closely related to attacks by insect-pests and phytopathogens such as bacteria and fungi. Among them, the fungus Botrytis cinerea can cause gray mold in more than 200 different species of plants, and is considered a challenging problem for agribusiness. Fungicides are commonly used to control this pathogen because they are fast-working and easy to apply. However, the continuous use of fungicides may promote the selection of resistant fungi and can also cause profound contamination in ecosystems. Aiming to find alternative strategies to solve these problems, several studies have focused on searching for plant proteins and peptides with antifungal activities (AFPs). With this in mind, this report shows the isolation and characterization of two novels antifungal proteins from flowers of rosemary pepper (Lippia sidoides Cham.) with 10 and 15 kDa. Isolation was performed by using an Octyl-Sepharose hydrophobic column. In vitro bioassays indicated that isolated proteins were able to inhibit B. cinerea development, but were not effective against all bacteria tested. Moreover, N-termini sequences indicate that both proteins showed sequence homology with NBS-LRR R proteins with a lower molecular mass, suggesting possible protein fragmentation. Data reported here could help in the development of biotechnological products for crop protection against phytopathogenic fungi in the near future.

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.

Antisense TRPM-2 oligodeoxynucleotides chemosensitize human androgen-independent PC-3 prostate cancer cells both in vitro and in vivo.

Although numerous chemotherapeutic regimens have been evaluated for patients with hormone-refractory prostate cancer, none has improved survival. Testosterone-repressed prostate message-2 (TRPM-2), which is highly up-regulated after androgen withdrawal and during androgen-independent progression in prostate cancer, has been shown to inhibit apoptosis induced by various kinds of stimuli. The objectives in this study were to test whether antisense (AS) oligodeoxynucleotides (ODNs) targeted against TRPM-2 enhance chemosensitivity in human androgen-independent prostate cancer PC-3 cells both in vitro and in vivo. Initially, the potency of 10 AS ODNs targeting various regions of the TRPM-2 mRNA were evaluated, and the AS ODN targeted to the TRPM-2 translation initiation site (AS ODN#2) was found to be the most potent sequence for inhibiting TRPM-2 expression in PC-3 cells. Despite significant dose-dependent and sequence-specific suppression of TRPM-2 expression, AS ODN#2 had no effect on growth of PC-3 cells both in vitro and in vivo. However, pretreatment of PC-3 cells with AS ODN#2 significantly enhanced chemosensitivity of Taxol (paclitaxel) and mitoxantrone in vitro. Characteristic apoptotic DNA laddering and cleavage of poly(ADP-ribose) polymerase were observed after combined treatment with AS ODN#2 plus paclitaxel or mitoxantrone but not with either agent alone. In vivo administration of AS ODN#2 plus either paclitaxel or mitoxantrone significantly decreased PC-3 tumor volume by 80 or 60%, respectively, compared with mismatch control ODN plus either paclitaxel or mitoxantrone. In addition, terminal deoxynucleotidyl transferase-mediated nick end labeling staining revealed increased apoptotic cells in tumors treated with AS ODN#2 plus paclitaxel or mitoxantrone. These findings confirm that TRPM-2 overexpression confers resistance to cytotoxic chemotherapy in prostate cancer cells and illustrates the potential utility of combined treatment with AS TRPM-2 ODN plus chemotherapeutic agents for patients with hormone-refractory prostate cancer.

[Preparation of 32P-labelled 2-methylthioadenosine di- and triphosphates]. / Poluchenie 32-P-mechenykh di- i trifosfatov 2-metiltioadenozina.

2-Methylthioadenosine 5'-mono- and diphosphates were prepared by chemical synthesis, and enzymatically phosphorylated to 5'-[beta-32P]-diphosphate and 5'-[gamma-32P]-triphosphate, respectively. They were isolated by HPLC and had activity exceeding 1000 Ci/mmol and radiochemical purity higher than 95%.

Quantitation of phosphorothioate oligonucleotides in human blood plasma using a nanoparticle-based method for solid-phase extraction.

Based on the application of cationic polystyrene nanoparticles, a novel method for solid-phase extraction of phosphorothioate oligonucleotides from human plasma has been developed. A high binding affinity, which is required for an effective isolation out of complex mixtures, is mediated by hydrophobic and multiple electrostatic interactions between the oligonucleotides and the nanoparticles. The principle of the method is based on a pH-controlled adsorption/desorption mechanism. Analysis of the extracted samples was performed by capillary gel electrophoresis. Extraction conditions were optimized, providing the isolation of oligonucleotides (> or = 10 nucleotide units) in high yields and purity even at concentrations in the low-nanomolar range (down to 5 nM). The low salt contamination of the samples allows their direct analysis by electrospray mass spectrometry. The combined linearity and accuracy of the assay together with absolute recovery rates in the range of 60-90% indicate that the developed solid-phase extraction method is generally applicable to quantitation of oligonucleotides in human plasma. Further improvement was achieved with an optimized carrier system of 2-fold enlarged particles which reduces the time consumption of the extraction procedure to approximately 30 min.

Inhibition of ADP-induced platelet responses by covalent modification of aggregin, a putative ADP receptor, by 8-(4-bromo-2,3-dioxobutylthio)ADP.

ADP is an important platelet agonist which initiates platelet shape change, aggregation, exposure of fibrinogen receptors, and calcium mobilization. Because of the limitations of previously used affinity analogs and photo-labeling studies as well as controversies surrounding the identity of an ADP receptor on platelets, we have used an affinity label capable of alkylating a putative exofacial receptor on platelets. We now report that 8-(4-bromo-2,3-dioxobutylthio)adenosine-5'-diphosphate (8-BDB-TADP), which is an analog of the natural ligand ADP, blocked ADP-induced platelet shape change, aggregation, exposure of fibrinogen-binding sites, secretion, and calcium mobilization. Following modification by 8-BDB-TADP, the rates of aggregation of platelets induced by thrombin, a calcium ionophore (A23187) or a stimulator of protein kinase C (phorbol myristate acetate) were minimally affected. However, the 8-BDB-TADP-modified platelets exhibited decreased rates of aggregation in response to ADP, as well as collagen and a thromboxane mimetic (U46619), both of which partially require ADP. Autoradiograms of the gels obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized platelets modified by either [beta-32P]8-BDB-TADP, or 8-BDB-TADP and NaB[3H]4 showed the presence of a single radiolabeled protein band at 100 kDa. The intensity of this band was reduced when platelets were preincubated with ADP, ATP, and 8-bromo-ADP prior to labeling by the radioactive 8-BDB-TADP. The results show that 8-BDB-TADP selectively and covalently labeled aggregin (100 kDa), a putative ADP receptor, resulting in a loss of ADP-induced platelet responses.

Gram-scale purification of phosphorothioate oligonucleotides using ion-exchange displacement chromatography.

The purification of oligonucleotides by ion-exchange displacement chromatography is demonstrated on the gram-scale. Using a 50 mmD x 100 mmL (203 ml) column operated in the displacement mode, 1.2 g of a 24mer phosphorothioate oligonucleotide was purified. Product yield for this separation was 70% (780 mg) at a purity of 96.4% and the mass balance recovery of all oligonucleotide was 97.5%. The displacement purification of four additional phosphorothioate oligonucleotides ranging in length from 18 to 25 bases is also demonstrated on the semi-preparative (10-50 mg) scale. All of these oligonucleotides were purified using similar displacement conditions and typical results were 60% yield at 96% purity. The displacement portion of these separations required <15 min and total cycle time including equilibration, feed loading and regeneration can be performed in under 30 min. These results seem to indicate that displacement chromatography may be amenable to generalizations in separation protocol that would greatly reduce the effort required to obtain an optimized purification scheme for moderately long oligonucleotides.

HPLC analysis of synthetic oligonucleotides using spectral contrast techniques.

The need for high-purity oligodeoxyribonucleotides for various applications has resulted in the development of novel synthesis, purification and analytical techniques. A diversity of methods, including polyacrylamide slab gel electrophoresis, capillary gel electrophoresis, as well as HPLC, have been successfully used to analyze material throughout the purification process. This study demonstrates how the application of spectral comparison techniques to synthetic products resolved by anion-exchange HPLC can distinguish deletion fragments (i.e., "N-1" sequences) from full-length products. Such analysis techniques can also differentiate partial from fully phosphorothioated DNA sequences. In combination, HPLC separation and spectral analysis technology provide information not obtainable with any other single analytical method.

Analysis of oligo(deoxynucleoside phosphorothioate)s and their diastereomeric composition.

Short oligo(deoxynucleoside phosphorothioate)s were analyzed as a pool of individual diastereomeric species. The composition of such mixtures, determined by means of HPLC, indicates that consecutive couplings in commonly used phosphoramidite chemistry lead to increasing contents of the Rp isomer. Methods of analysis and mathematical basis for diastereomeric composition are discussed. Data presented include all 16 possible combinations of nucleosides in dinucleotide phosphorothioates, as well as examples of trimers and tetramers.

Solution-phase synthesis of phosphorothioate oligodeoxynucleosides by the phosphotriester method.

A "phosphorothioate triester method" was investigated for the solution-phase synthesis of phosphorothioate oligonucleosides. Using fully protected 3'-phosphorothiolate thymidine bearing O-cyanoethyl and S-2,4-dichlorobenzyl groups as phosphorothioate protecting groups, decathymidine nonaphosphorothioate was efficiently assembled through a blockwise procedure. Two side reactions occurred during the deprotection steps: breakage of internucleoside linkages (1.8% per linkage) and formation of phosphate diester linkages (0.9%). Substitution of the dichlorobenzyl group by the more labile 4-nitrobenzyl S-protecting group reduced the extent of internucleoside bond breakage by one-half.

Separation and characterization of diastereoisomers of antisense oligodeoxyribonucleoside phosphorothioates.

Diastereoisomers of oligodeoxyribonucleoside phosphorothioates (OPT) up to a tetramer were effectively separated with reversed-phase high-performance liquid chromatography (reversed-phase HPLC) under optimized conditions. The diastereoisomers of OPT resulted in different retention times on the reversed-phase HPLC. From the results, we found that there were certain rules in the elution order of the diastereoisomers. The configurational sequence of the diastereoisomers was determined by digestion with nuclease P1, snake venom phosphodiesterase, and alkaline phosphatase. The diastereoisomers were studied by CD spectroscopy with respect to their conformation in aqueous media. We found that a large variation in the conformation of diastereoisomers exists. Results suggest that much attention should be paid to the diastereoisomerism in antisense molecules having chiral internucleotide linkages such as OPT.

Preparation of oligodeoxyribonucleoside phosphorodithioates by a triester method.

A method to prepare thymidine phosphorodithioate dimers (ref. 1) has been extended to allow the preparation of oligo-2'-deoxyribonucleotide phosphorodithioates containing all four bases. The method is suitable for large-scale synthesis and gives phosphorodithioates without phosphorothioate impurities (31P nmr, detection limit 0.5 to 1%). Oligonucleotides up to octamers which contain -0-(PS2-)-0- linkages at all positions have been prepared by block synthesis in solution. The phosphorodithioate linkage is introduced by the reaction of a 5'-O, N-protected nucleoside (or oligonucleotide) with a dithiophosphorylating agent RSP(S)(ODhbt)2, R = 2,4-dichlorobenzyl, Dhbt = 3,4-dihydro-4-oxo-benzotriazin-3-yl, followed by coupling of the product to a 3'-O,N-protected nucleoside (or oligonucleotide). This method gives pure protected oligodeoxyribonucleoside phosphorodithioates, and phosphorothioate linkages are only introduced if contact with conc. aqueous ammonia during or after deblocking is unduly prolonged.

Large-scale synthesis, purification, and analysis of oligodeoxynucleotide phosphorothioates.

Synthesis of oligonucleotides has been carried out on 1-, 2-, and 5-mmol scales using an appropriately modified automated, commercially available DNA synthesizer. The reaction cycles were optimized to obtain efficient coupling (> or = 97%). The synthesized oligonucleotide was purified by preparative reversed-phase liquid chromatography, followed by detritylation and desalting to obtain the oligonucleotides in the Na+ form. The purified oligonucleotides were characterized by 31P NMR, mass spectrometry, capillary gel electrophoresis, and ion-exchange high-performance liquid chromatography. By using these protocols, a 25-mer oligodeoxyribonucleotide can be synthesized on a 1-, 2-, or 5-mmole scale to obtain approximately 2.4, 4.8, or 12 g of purified product.

Ion-exchange high-performance liquid chromatography analysis of oligodeoxyribonucleotide phosphorothioates.

Oligodeoxynucleotide-containing phosphorothioate backbones have been used to regulate viral as well as cellular gene expression. The studies carried out in tissue culture have shown promising results on the use of oligonucleotide phosphorothioates as antiviral agents and, at present, study is underway to develop these oligonucleotide analogues as chemotherapeutic agents. To analyze and purify oligonucleotide analogues, high-performance liquid chromatography using weak anion exchange column has been described. The separation of oligonucleotide phosphorothioate is found to be length dependent.

Solid phase synthesis of oligodeoxyribonucleoside phosphorodithioates from thiophosphoramidites.

Oligonucleoside phosphorodithioates 1 are modified DNA sequences with potential use as antisense oligonucleotides. The preparation of up to 20-mers containing all four bases by solid phase synthesis is described, with details on the preparation of the four monomer units (protected nucleoside thiophosphoramidites 2), the conditions used for the assembly of the strands with up to 19 phosphorodithioate linkages, and the purification and characterisation of the products. Full-length homogeneity of HPLC-purified all-phosphorodithioate products is demonstrated by PAGE, but 31P NMR discloses the presence of phosphorothioate impurities (typically 8-9%), the origin of which is discussed. Oligonucleoside phosphorodithioates are freely soluble in water at neutral or basic pH, and are very stable towards oxidation, hydrolysis, and nuclease cleavage. Their ability to hybridize to complementary DNA has been studied by UV melting point (Tm) measurements. The observed depression of Tm, 0.5-2 degrees C per phosphorodithioate linkage, is higher that the 0.4-0.6 degrees C found for phosphorothioates.

Purification and characterization of five different alpha subunits of guanine-nucleotide-binding proteins in bovine brain membranes. Their physiological properties concerning the activities of adenylate cyclase and atrial muscarinic K+ channels.

We have purified five different alpha subunits of guanine-nucleotide-binding proteins (G proteins) from bovine brain membranes as active forms bound to guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]). All the purified alpha subunits were interacted with beta gamma subunits and served as a substrate for pertussin-catalyzed ADP-ribosylation. Based on the findings of immunoblot analyses using specific antibodies raised against various alpha subunits of G proteins, three of them were identified as alpha i-1, alpha i-2 and alpha i-3, and the other two were classified into alpha o type. One of the alpha o-type proteins was the most abundant in the brain membranes (termed alpha o), and the other (alpha o2) appeared to differ from alpha o in its proteolytic digestion data. The physiological properties of these purified GTP[gamma S]-bound alpha subunits towards adenylate cyclase and atrial muscarinic K+ channels were studied. The nucleotide-bound forms of alpha i-1, alpha i-2, alpha i-3 and alpha o2 inhibited the adenylate cyclase activity of S49 cyc- membranes which had been reconstituted with GTP[gamma S]-treated Gs; this inhibition appeared to be mainly competitive with the activated Gs, alpha i-1 having the most potent inhibitory activity among them. GTP[gamma S]-bound alpha o, however, could not inhibit the Gs-stimulated activity at all. On the other hand, all the GTP[gamma S]-bound alpha subunits activated atrial muscarinic K+ channels, accompanied by a lag time, at picomolar concentrations. The beta gamma subunits resolved from G proteins also activated the K+ channels without a lag time at nanomolar concentration. The maximum activation by the beta gamma subunits appeared to be more potent than that by any of the alpha subunits. These results suggest that alpha and beta gamma subunits might activate the K+ channels by mechanisms different from each other.

The quantitative determination of metabolites of 6-mercaptopurine in biological materials. VII. Chemical synthesis by phosphorylation of 6-thioguanosine 5'-monophosphate, 5'-diphosphate and 5'-triphosphate, and their purification and identification by reversed-phase/ion-pair high-performance liquid chromatography and by various enzymatic assays.

A fast and reliable two-step method has been established for the chemical synthesis of 6-thioguanosine 5'-monophosphate, 6-thioguanosine 5'-diphosphate and 6-thioguanosine 5'-triphosphate starting from the ribonucleoside. In the first step, 6-thioguanosine dissolved in triethyl phosphate, at high yield reacts with phosphorus oxide trichloride to 6-thioguanosine 5'-monophosphate which is purified by anion-exchange chromatography on DEAE-Sephadex using a step gradient of hydrochloric acid. In the second step, 6-thioguanosine 5'-monophosphate dissolved in water, reacts with phosphoric acid in the presence of pyridine/dicyclohexyl carbodiimide and is converted to 6-thioguanosine 5'-diphosphate and 6-thioguanosine 5'-triphosphate which are separated from each other and from the 6-thioguanosine 5'-monophosphate by anion-exchange chromatography on DEAE-Sephadex using a gradient of ammonium bicarbonate. Material from each step of the preparation procedure is separated by reversed-phase HPLC chromatography and analyzed for its free ribonucleoside content, 5'-monophosphate, 5'-diphosphate, 5'-triphosphate and small amounts of unidentified phosphorylated compounds. The purity of the final preparations and the identity of each 6-thioguanosine 5'-phosphate are proven by highly specific enzymatic peak-shifting/HPLC analyses using alkaline phosphatase, 5'-nucleotidase, pyruvate kinase, nucleoside diphosphate kinase and combined hexokinase/glucose 6-phosphate dehydrogenase.