Efficient synthesis of antisense phosphorothioate oligonucleotides using a universal solid support

It is demonstrated that solid support containing a novel universal linker could be efficiently used to synthesize both phosphorothioate oligodeoxyribonucleotides and second-generation 2′-O-methoxyethyloligoribonucleotides with high yield and quality as judged by ion-pair-liquid chromatography-electrospray mass spectroscopy, ³¹P NMR and reversed phase HPLC. Analysis of oligonucleotides shows quality being superior to that produced with standard succinyl-linker solid supports, without contamination of materials resulting from linker or support backbone decomposition.     

An efficient two-step synthesis of 3-allylindoles

A two-step synthetic sequence for an efficient synthesis of 3-allylindoles is described.     

An efficient method for solution-phase parallel synthesis of 2-quinoxalinol salen Schiff-base ligands

A solution-phase parallel method for the synthesis of 2-quinoxalinol salen ligands was designed and optimized. The synthesis begins with commercially available 1,5-difluoro-2, 4-dinitrobenzene (DFDNB) and employs a sequence of five straightforward and high-yielding reaction steps. Simple laboratory techniques with low sensitivity to water or air for solution-phase parallel reactions were coupled with convenient workup and purification procedures to give high-purity and yield a small ligand library of 20 compounds. The final step, a Schiff-base condensation of an aldehyde with the diaminoquinoxaline results in a new category of ligands for metal coordination or of potential bioactivity, based on the skeleton 2,2'-(1E,1'E)-(quinoxaline-6,7-diylbis(azan-1-yl-1-ylidene))bis(methan-1-yl-1-ylidene)diphenol. The approach described here is easily adaptable for parallel synthesis of a larger library.     

Highly efficient two-step selective synthesis of 2,6-dimethylnaphthalene

2,6-Dimethylnaphthalene (2,6-DMN), a key raw material for poly(ethylene naphthalate) (PEN), was selectively synthesized via a two-step process in an overall 66% yield from commercially available 4-bromotoluene and 3-methyl-3-buten-1-ol. The ligand-free Heck reaction of the starting materials produced γ-(p-tolyl)-substituted aldehyde that was cyclized with an acid to give 2,6-DMN after in situ oxidation. No other isomers of 2,6-DMN were found.     

A photoactivated precipiton for reagent sequestration in solution-phase synthesis

Precipitons are molecular phase tags for chemical separations. They can be switched from a high-solubility to a low-solubility state to facilitate product, reagent, or catalyst isolation. This paper presents the first photoactivated precipiton and demonstrates that this precipiton is an efficient amine scavenging agent in solution-phase syntheses of amides, ureas, and imines. This approach to amine scavenging offers advantages over solid-phase scavenging methods. The amine is captured in a homogeneous medium, so the capture is much faster than seen with isocyanate resins, and only a small excess of the scavenger is required.     

An efficient two-step total synthesis of the quaterpyridine nemertelline

Regioselective and univocal Suzuki cross-coupling reactions performed on halopyridinyl boronic acids provide a flexible and versatile route to a multigram scale synthesis of 2,2'-dichloro-3,4'-bipyridine 14, which allows couplings with excess pyridin-3-yl boronic acid to give a new and efficient two-step rapid synthesis of nemertelline, the quaterpyridine neurotoxin isolated from a Hoplonemertine sea worm.     

An efficient synthesis of gougerotin and related analogues using solid- and solution-phase methodology

The first solid-phase synthesis of the natural product gougerotin has been accomplished. The synthetic route is versatile and allows for diversification at position C-4 of the heterocycle, C-6' of the sugar ring, and both residues of the peptidic moiety at N-4' in a parallel fashion. [structure: see text]     

Highly efficient two-step synthesis of C-sp3-centered geminal diiodides

[reaction: see text] Trisubstituted gem-diiodoalkenes of functionalized chains are efficiently reduced to the corresponding terminal geminal diiodides in high yields upon treatment with the diazene precursor, diethyl 4-(hydrazinosulfonyl)-benzyl phosphonate.     

Fluorous electrophilic scavengers for solution-phase parallel synthesis

A fluorous isatoic anhydride and isocyanate are synthesized and used as scavengers for amines in solution-phase parallel synthesis of urea, thiourea, and β-hydroxyamine analogs. The resulting fluorous derivatives are readily separated from the reaction mixture by solid-phase extractions (SPE) over FluoroFlash™ cartridges to give products with good purity. The SPE cartridges can be reused.     

A combinatorial method for solution-phase synthesis of labeled bivalent beta-turn mimics

Piperidine-functionalized, 1,4-disubstituted-1,2,3-triazoles of generic structure 1 were conceived as "minimalist" mimics of peptidic beta-turn structures. Key features of these molecules include (i) the possibility of incorporating amino acid side chains corresponding to many of the protein amino acids; (ii) a close correspondence of separations of these side chains to i + 1 to i + 2 residues in turns; (iii) facile adjustment of the side-chain vectors on docking while only influencing two critical degrees of freedom; and (iv) some electrostatic polarity. Fifteen monomers of this type were made via copper-mediated cycloaddition reactions. Solution-phase methodologies were devised to assemble these monomers into bivalent compounds in high purity states (typically >85%) so that they could be used in first-pass biological assays without further purification. The skeleton for forming these bivalent compounds is triazine-based. There is a third site which allowed for introduction of a fluorescent label (library of compounds 2) or an alkyne-functionalized triethylene glycol chain (library of compounds 3) included to promote water-solubility and to allow incorporation of probes via copper-mediated cycloaddition reactions. In the event, two 135-membered libraries were prepared, one consisting of compounds 2 and the other of 3. No protecting groups or coupling agents were required; these attributes of the method were important to allow most of the products to be obtained in over 85% purities. The fluorescein-tagged library of compounds 2 was screened in a fluorescence-activated cell sorting (FACS) assay using cells transfected to overexpress one of the following neurotrophin receptors: TrkA, TrkC, and p75. Preliminary findings indicate four compounds 2gm, 2gn, 2gi, and 2gj bound the TrkA receptor selectively; all of these contain a threonine-lysine turn mimic. Thus, a pharmacological probe for the TrkA receptor has been developed.     

Selective covalent conjugation of phosphorothioate DNA oligonucleotides with streptavidin

Protein-DNA conjugates have found numerous applications in the field of diagnostics and nanobiotechnology, however, their intrinsic susceptibility to DNA degradation by nucleases represents a major obstacle for many applications. We here report the selective covalent conjugation of the protein streptavidin (STV) with phosphorothioate oligonucleotides (psDNA) containing a terminal alkylthiolgroup as the chemically addressable linking unit, using a heterobifunctional NHS-/maleimide crosslinker. The psDNA-STV conjugates were synthesized in about 10% isolated yields. We demonstrate that the terminal alkylthiol group selectively reacts with the maleimide while the backbone sulfur atoms are not engaged in chemical conjugation. The novel psDNA-STV conjugates retain their binding capabilities for both biotinylated macromolecules and the complementary nucleic acid. Moreover, the psDNA-STV conjugate retained its binding capacity for complementary oligomers even after a nuclease digestion step, which effectively degrades deoxyribonucleotide oligomers and thus the binding capability of regular DNA-STV conjugates. The psDNA-STV therefore hold particular promise for applications e.g. in proteome research and novel biosensing devices, where interfering endogenous nucleic acids need to be removed from analytes by nuclease digestion.     

Plate-to-plate fluorous solid-phase extraction for solution-phase parallel synthesis

A commercially available Argonaut VacMaster-96 plate-to-plate solid-phase extraction (SPE) station equipped with 24 FluoroFlash cartridges is employed for parallel purification of fluorous reaction mixtures. Each cartridge charged with 3 g of fluorous silica gel has the capability to produce up to 100 mg of purified small molecules. The 24-well receiving plate has a standard footprint that can be directly concentrated in a Genevac vacuum centrifuge. Important issues such as sample loading, product cross-contamination, cartridge reuse, and reproducibility are investigated. The SPE system has been demonstrated in the purification of three small libraries that were produced involving amine scavenging reactions with fluorous isatoic anhydride, amide coupling reactions with 2-chloro-4,6-bis[(perfluorohexyl)propyloxy]-1,3,5-triazine (fluorous CDMT), and amide coupling reactions with a newly developed fluorous Mukaiyama condensation reagent.     

Toward solution-phase automated iterative synthesis: fluorous-tag assisted solution-phase synthesis of linear and branched mannose oligomers

We report herein the first synthesis of linear and branched mannose oligosaccharides using fluorous-tag assistance with reagents and FSPE protocols that are amenable to automation. The particular fluorous linker proved to maintain solubility of the growing oligosaccharide chain such that identical reaction solvent conditions and purification protocols could be used between glycosylation and deprotection reactions, thereby rendering the procedures amenable to automation.     

A solution-phase combinatorial synthesis of selective dopamine D4 ligands

Utilizing combinatorial synthesis and a preparative LC-MS automated chromatography system we have prepared and purified a library of 4-[2-(1,2,4-oxadiazolyl)]piperidines that were designed to be novel and selective dopamine D4 ligands. In one round of synthesis we identified N-4-chlorobenzyl-4-[2-(3-(2-thienyl)-1,2, 4-oxadiazolyl)]piperidine with a Kd of 5 nM for the human D4 receptor.     

A base-stable dithiomethyl linker for solid-phase synthesis of oligonucleotides

A novel linkage, useful for the synthesis of oligonucleotides is described. The linking function is compatible with all conditions used for oligonucleotide synthesis, orthogonal to all other protecting groups, but regenerates 3′-OH rapidly upon mild reduction under aqueous conditions. This method is employed in the removal of depurinated fragments during the synthesis of oligonucleotides.     

A ‘click chemistry’ approach to the efficient synthesis of modified nucleosides and oligonucleotides for PET imaging

Different thymidine derivatives bearing either an iodoaryl moiety at the 5′ position or a dialkylsilyl group at the 3′ position have been efficiently synthesized as precursors for carbon-11 or fluorine-18 labeling, respectively. Furthermore, iodoarylated thymidine derivatives have been incorporated into oligonucleotides giving an original way to label them with carbon-11.     

A two-step practical synthesis of dehydroalanine derivatives

A two-step practical synthesis of dehydroalanine derivatives from commercially available starting materials is reported. The approach comprises an Ugi four-component reaction using 2-benzoylacetaldehyde, followed by an elimination process of the benzoate group. This protocol provided access to several dehydroalanine derivatives modified with diverse functional groups, which might be useful for further transformations in the construction of more complex molecules.     

Quantitative capillary gel electrophoresis assay of phosphorothioate oligonucleotides in pharmaceutical formulations

Quantitative capillary gel electrophoresis (QCGE) has been developed for the accurate quantitation of a 21-mer phosphorothioate oligonucleotide, ISIS 2922, and its degradation products in an intravitreal formulation. The electrokinetic mode of injection employed by CGE necessitates formulation of the external reference standard in a sample matrix similar to that of the drug product and the use of an internal standard for improved accuracy and precision. The analytical method detailed in this paper has demonstrated the necessary accuracy, precision, linearity, range, selectivity and ruggedness for use in routine drug product analysis and stability monitoring of phosphorothioate oligonucleotides.