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.

     

Quantum Molecular Modeling of the Interaction Between Guanine and Alkylating Agents - 1 - Sulfur Mustard

Abstract

Interaction between Guanine and the episulfonium form of Sulfur mustard (HD) was studied using the ab initio LCAO-MO method at the HF/6–31G level. The alkylation mechanism on guanine-N7 was analyzed by using a supermolecular modeling. Our stereostructural results associated with the molecular electrostatic potentials and HOMO-LUMO properties, show that in vacuum the alkylation of the N7 of guanine by HD in the agressive episulfonium form is a direct process without transition state and of which the pathway is determined.     

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.     

Polysulfide Reagent in Solid-Phase Synthesis of Phosphorothioate Oligonucleotides: Greater than 99.8% Sulfurization Efficiency

A solution of sulfur (0.1 M) and sodium sulfide (0.01 M) in 3-picoline, referred to as polysulfide reagent, rapidly converts trialkyl and triaryl phosphite triesters to the corresponding phosphorothioate derivatives. Greater than 99.8% average stepwise sulfurization efficiency is obtained in the solid-phase synthesis of DNA and RNA phosphorothioate oligonucleotides via the phosphoramidite approach.     

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.     

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

Abstract

Oligodeoxyribonucleotide phosphorothioates, where one of the nonbridging oxygen of the internucleotide phosphate is formally replaced by a sulfur atom, are the first class to undergo human clinical trials. Ongoing phosphorothioate clinical trials against several disease targets has necessitated manufacture of very large quantities of oligonucleotide active pharmaceutical ingredient (API). Clinical trial and future market demands have stimulated effort towards developing cost efficient large scale synthesis of these complex bio-molecules. This effort has culminated in the routine synthesis of 20-mer oligodeoxyribonucleotide phosphorothioates at 150 mmole scale using only 1.75-fold molar excess of amidites in less than 8 h total synthesis time.     

Efficiency of New Dose Escalation Designs in Dose-Finding Phase I Trials of Molecularly Targeted Agents

Background

Statistical simulations have consistently demonstrated that new dose-escalation designs such as accelerated titration design (ATD) and continual reassessment method (CRM)-type designs outperform the standard “3+3” design in phase I cancer clinical trials.

Methods

We evaluated the actual efficiency of different dose escalation methods employed in first-in-human phase I clinical trials of targeted agents administered as single agents published over the last decade.

Results

Forty-nine per cent of the 84 retrieved trials used the standard “3+3” design. Newer designs used included ATD in 42%, modified CRM [mCRM] in 7%, and pharmacologically guided dose escalation in 1%. The median numbers of dose levels explored in trials using “3+3”, ATD and mCRM designs were 6, 8 and 10, respectively. More strikingly, the mean MTD to starting dose ratio appeared to be at least twice as high for trials using mCRM or ATD designs as for trials using a standard “3+3” design. Despite this, the mean number of patients exposed to a dose below the MTD was similar in trials using “3+3”, ATD and mCRM designs.

Conclusion

Our results support a more extensive implementation of innovative dose escalation designs such as mCRM and ATD in phase I cancer clinical trials of molecularly targeted agents.     

Synthesis of 9-[1-(1-Hydroxyethyl)-3-(Phosphonomethoxy)Propyl] Adenine and Prodrug as Possible Antiviral Agents

The appropriately protected C-1′-hydroxyethyl-3-hydroxypropyl-N⁹-adenine nucleoside was prepared from 1-pivaloyloxy-5-tert-butyldiphenylsilyloxy-3-pentanol and adenine through the Mitsunobu reaction. One of the terminal hydroxyls was converted to the phosphonomethoxy derivative and the prodrug.     

Solid Phase Synthesis and Chromatographic Characteristics of Nucleophilic Agents Conjugated with Oligonucleotides Containing 5'-Terminal Carboxyl Group

Conjugates of amines or short peptides with oligonucleotides containing 5'-terminal carboxyl group were prepared by solid phase chemical synthesis. A correlation between the physicochemical parameters and retention times of the synthesized conjugates was established by ion-pair reversed-phase HPLC.     

Direct Regioselective Enzymatic Acylation of Nucleosides: Building-Blocks for the Solution Phase Synthesis of Oligonucleotides

Abstract

The synthesis of 3′- and 5′-O-levulinyl nucleosidic monomers through enzymatic acylation with acetonoxime levulinate is demonstrated. The acylation process takes place in one-step and use of expensive reagents, such as DMTrCl is avoided. The regioselectivity of the procedure makes it very convenient for acylated monomers required for solution phase synthesis of oligonucleotides.     

A Comparison of Various Selective Isolation Media for their Efficiency in the Diagnosis and Enumeration of Soft Rot Coliform Bacteria

The efficiency of various selective isolation media for diagnosis and enumeration of Erwinia carotovora var. atroseptica is described. In both pure culture and mixed population studies the medium of choice is that of Cuppels and Kelman's modified by the addition of manganous sulphate. Difficulties in the preparation of this pectate medium have been overcome by including an antifoam agent.     

Synthesis and Evaluation of Disubstituted N1- and N3-Imidazolidin-2-ones Acting as Potential Immunosuppressive Agents

New N1-mono and N1, N2-disubstituted imidazolidin-2-one with a significant immunosuppressive activity have been discovered. Among the 17 synthesized and tested compounds, five of them showed maximal inhibition of proliferation of concanavallin A (Con A)- stimulated splenocytes at 90?μM, identical to that obtained with cyclosporin A (CsA) at 5?μM, an optimal concentration.     

Relating Quinone Profile to the Aerobic Biodegradation in Thermophilic Composting Processes of Cattle Manure with Various Bulking Agents

Summary Cattle manure was composted aerobically with various bulking agents (rice straw, vermiculite, sawdust or waste paper) at a constant incubation temperature of 60 °C. Increased quinone content (IQC) was used to assess microbial biomass in the composted material. IQC was proportional to mass reduction (MR) (R = 0.812) and cumulative O₂ consumption (COC) (R = 0.810) irrespective of the bulking agent used, indicating that the yield of quinone was constant. Quinone yields were 0.44 ± 0.03 μmol quinone/g MR and 0.34 ± 0.02 μmol quinone/g COC. The material that was decomposed by microorganisms was considered to be mainly cattle manure. Bulking agents were not degraded within the 14 day trial period and did not affect microbial succession because composting runs with various bulking agents exhibited similar quinone yields.     

Cell Wall Synthesis in Staphylococcus aureus in the Presence of Protein Synthesis Inhibitory Agents

Electron-microscopic and biochemical studies on morphological changes in Staphylococcus aureus following exposure to protein synthesis inhibitory agents such as lincomycin (LCM), clindamycin (CLM), erythromycin (EM), and spiramycin (SP) are presented in this paper. It was demonstrated that bacterial cell walls became extremely thickened usually with the formation of multilayers, when exposed to each of the above-mentioned antibiotics. Furthermore, electron density of the cytoplasm was higher in those cells exposed to drugs than in intact control cells. Incorporations of ¹⁴C-labeled l-lysine into the cell-wall fraction and the protein fraction were measured for biochemical elucidation of these phenomena. Labeled lysine was selectively incorporated into the cell-wall fraction when the test organism was exposed to the respective antibiotics. Uptake at 15 min after exposure was about twice as large as that of intact control cells. SP and CLM inhibited protein synthesis while they stimulated cell-wall synthesis. The evidences for thickening of, and formation of multilayers in the bacterial cell walls following exposure to drugs were closely related to the stimulating action of these antibiotics on the cell-wall synthesizing system. Morphology of resistant clinical isolates following such antibiotic exposure was also investigated using two staphylococcal strains, one resistant to EM alone and the other completely cross-resistant to all the macrolides.     

Solution Phase Synthesis of Dithymidine Phosphorodithioate Using New S-Protecting Groups in Combination with a Chemoselective Coupling Reagent (PyNOP)

Abstract

ABSTRACT

A method for the synthesis of O-thymidin-3′-yl S-alkyl dithiophosphate monomers 1 with different S-protecting groups has been developed. These have been used for solution phase synthesis of dithymidine phosphorodithioate by a new phosphotriester method. Coupling reactions are fast (15 min.) and the products are free from phosphorothioate contaminations.     

Synthesis of protected peptides with the sequence 8-13-1-3 and 4-13-1-3 of mycobacillin and their analogs

We have synthesized both a protected nonapeptide of the mycobacillin 8-13-1-3 amino acid sequence and a protected tridecapeptide of the 4-13-1-3 sequence, which are a fragment and a open chain analog of this antibiotic, respectively. Some of their analogs with a reversed configuration of the amino acids at fixed positions have also been synthesized. The nonapeptides were obtained by coupling partially protected mycobacillin fragments with the sequence 8-10 and 11-13-1-3 while the tridecapeptides were synthesized by coupling partially protected fragments 4-7 and 8-13-1-3. Configuration analogs of these fragments were also used. The coupling methods applied were DCCI/HONSu or DCCI/HOBt. The purification of the synthesized peptides was achieved by means of recrystallization or column chromatography on silica gel. They were characterized mainly by m.p., degree of optical rotation, elemental and amino acid analysis.     

Synthesis of Potential Purinoceptor Antagonists: Application of P1-tBu Phosphazene Base for Alkylation of Adenine in Solution and on Solid Phase

Alkylation of adenine in solution and on solid phase was accelerated by phosphazene base P1-tBu compared to mineral bases. The reactions in solution afforded regioselectively the appropriate N9-alkylated adenines with high preparative yields while the reaction with polystyrene resin-bound N-bromoacetylated peptides gave three regioisomers (alkylated at the N9, N7, and N3 position of adenine) in a 4:2:1 molar ratio. Ten novel nonphosphate nucleotide analogues were tested in an ADP-induced platelet aggregation assay.     

Distribution of Protein Phosphatase Inhibitor-1 in Brain and Peripheral Tissues of Various Species: Comparison with DARPP-32

The distribution of inhibitor-1, a cyclic AMP-regulated inhibitor of protein phosphatase-1, was analyzed in various brain regions and peripheral tissues of various species by immunolabeling of sodium dodecyl sulfate-poly-acrylamide gel transfers using specific antibodies. The distribution of inhibitor-1 was directly compared to that of DARPP-32, a structurally related cyclic AMP-regulated inhibitor of protein phosphatase-1. In rat CNS, a single immunoreactive protein of M(r) 30,000, identified as inhibitor-1, was widely distributed. In contrast, DARPP-32 was highly concentrated in the basal ganglia. Inhibitor-1 was detected in brain tissue from frog (M(r) 27,000), turtle (M(r) 29,000/33,000), canary (M(r) 26,000), pigeon (M(r) 28,000), mouse (M(r) 30,500), rabbit (M(r) 26,500), cow (M(r) 27,000), and monkey (M(r) 27,500), but not from goldfish. Inhibitor-1 was detected at various levels in most peripheral tissues of the species studied; however, it was not detectable in certain tissues of particular species (e.g., rat and cow liver). DARPP-32 was detected in brain tissue of all the species tested except frog and goldfish, but was not detectable in most peripheral tissues. Both inhibitor-1 and DARPP-32 were concentrated in the cytosol and synaptosomal cytosol of rat striatum. The developmental expressions of inhibitor-1 and DARPP-32 in rat striatum differed: the level of inhibitor-1 peaked in the first postnatal week and then declined by the third postnatal week, whereas the level of DARPP-32 increased to a peak level by the third postnatal week and remained elevated thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of 1-(omega-aminoalkyl)naphthoindolediones with antiproliferative properties

The preparation and cytotoxic properties of 4,11-dihydroxynaphtho[2,3-f]indole-5,10-dione derivatives carrying N-aminoalkyl substituents are described. The N-aminobutylnaphthoindolediones obtained were studied in National Cancer Institute Screening Program and demonstrated high antiproliferative activity against 60 human cancer cell lines. All N-(4-aminobutyl) derivatives have higher potency than adriamycin or mitoxantrone against adriamycin selected multidrug resistant breast cancer cell line NCI/ADR.