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Alam M.R.,Girindus America Inc. | Thazhathveetil A.K.,Northwestern University | Li H.,LaserGen | Seidman M.M.,U.S. National Institute on Aging
Methods in Molecular Biology | Year: 2014

Strategies for site-specific modulation of genomic sequences in mammalian cells require two components. One must be capable of recognizing and activating a specific target sequence in vivo, driving that site into an exploitable repair pathway. Information is transferred to the site via participation in the pathway by the second component, a donor nucleic acid, resulting in a permanent change in the target sequence. We have developed biologically active triple helix forming oligonucleotides (TFOs) as site-specific gene targeting reagents. These TFOs, linked to DNA reactive compounds (such as a cross-linking agent), activate pathways that can engage informational donors. We have used the combination of a psoralen-TFO and single strand oligonucleotide donors to generate novel cell lines with directed sequence changes at the target site. Here we describe the synthesis and purification of bioactive psoralen-linked TFOs, their co-introduction into mammalian cells with donor nucleic acids, and the identification of cells with sequence conversion of the target site. We have emphasized details in the synthesis and purification of the oligonucleotides that are essential for preparation of reagents with optimal activity. © 2014 Springer Science+Business Media, LLC.


Gagnon K.T.,Southwestern Medical Center | Pendergraff H.M.,Southwestern Medical Center | Deleavey G.F.,McGill University | Swayze E.E.,Isis Pharmaceuticals | And 10 more authors.
Biochemistry | Year: 2010

Huntington's disease (HD) is a currently incurable neurodegenerative disease caused by the expansion of a CAG trinucleotide repeat within the huntingtin (HTT) gene. Therapeutic approaches include selectively inhibiting the expression of the mutated HTT allele while conserving function of the normal allele. We have evaluated a series of antisense oligonucleotides (ASOs) targeted to the expanded CAG repeat within HTT mRNA for their ability to selectively inhibit expression of mutant HTT protein. Several ASOs incorporating a variety of modifications, including bridged nucleic acids and phosphorothioate internucleotide linkages, exhibited allele-selective silencing in patient-derived fibroblasts. Allele-selective ASOs did not affect the expression of other CAG repeat-containing genes and selectivity was observed in cell lines containing minimal CAG repeat lengths representative of most HD patients. Allele-selective ASOs left HTT mRNA intact and did not support ribonuclease H activity in vitro. We observed cooperative binding of multiple ASO molecules to CAG repeat-containing HTT mRNA transcripts in vitro. These results are consistent with a mechanism involving inhibition at the level of translation. ASOs targeted to the CAG repeat of HTT provide a starting point for the development of oligonucleotide-based therapeutics that can inhibit gene expression with allelic discrimination in patients with HD. © 2010 American Chemical Society.


Patent
Girindus America Inc. and Childrens Hospital Medical Center | Date: 2011-06-01

A method for preparing an enantiomeric chromane, by asymmetrically hydrogenating a chromene compound in the presence of an Ir catalyst having a chiral ligand. The method includes the enantioselective preparation of enantiomeric equol. A preferred Ir catalyst has a chiral phosphineoxazoline ligand. Enantiomeric chromanes of high stereoselective purity can be obtained.


Patent
Girindus America Inc. and Childrens Hospital Medical Center | Date: 2012-09-10

A method for preparing an enantiomeric chromane, by asymmetrically hydrogenating a chromene compound in the presence of an Ir catalyst having a chiral ligand. The method includes the enantioselective preparation of enantiomeric equol. A preferred Ir catalyst has a chiral phosphineoxazoline ligand. Enantiomeric chromanes of high stereoselective purity can be obtained.


Pavlov V.A.,Girindus America Inc. | Laskovics F.M.,Girindus America Inc. | Mazur A.W.,Girindus America Inc.
Phosphorus, Sulfur and Silicon and the Related Elements | Year: 2016

Despite all the advantages of solid-phase oligonucleotide manufacturing, there are significant drawbacks, such as manufacturing capacity and capital investment. The synthesis of oligonucleotides in solution has been proposed as a potential remedy for the economical and scale-up shortcomings of solid-phase manufacturing. We developed a new oligomerization methodology for manufacturing DNA oligos in solution, which alleviates the limitations inherent in the synthesis on solid supports. Since the majority of potential therapeutic DNA oligonucleotides have a thiophosphate moiety, we have created a novel highly efficient sulfurizing reagent for the formation of the P-S bond during elongation of the oligonucleotide. The new protocols for the synthesis and purification of DNA phosphorothioates oligomers have a significant potential for the economical production of oligonucleotides in solution. The method allows the preparation of high-purity oligonucleotides avoiding expensive chromatography during the early stages of oligomerization. The efficient synthesis, consisting of preparing dimer and tetramer building blocks, represents a critical economic advance for large-scale manufacture of up to 16-mer oligonucleotides. © 2016 Taylor & Francis Group, LLC.


Nikcevic I.,University of Cincinnati | Wyrzykiewicz T.K.,Girindus America Inc. | Limbach P.A.,University of Cincinnati
International Journal of Mass Spectrometry | Year: 2011

Oligonucleotide phosphorothioatediesters (phosphorothioate oligonucleotides), in which one of the non-bridging oxygen atoms at each phosphorus center is replaced by a sulfur atom, are now one of the most popular oligonucleotide modifications due to their ease of chemical synthesis and advantageous pharmacokinetic properties. Despite significant progress in the solid-phase oligomerization chemistry used in the manufacturing of these oligonucleotides, multiple classes of low-level impurities always accompany synthetic oligonucleotides. Liquid chromatography-mass spectrometry has emerged as a powerful technique for the identification of these synthesis impurities. However, impurity profiling, where the entire complement of low-level synthetic impurities is identified in a single analysis, is more challenging. Here we present an LC-MS method based the use of high resolution-mass spectrometry, specifically Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS or FTMS). The optimal LC-FTMS conditions, including the stationary phase and mobile phases for the separation and identification of phosphorothioate oligonucleotides, were found. The characteristics of FTMS enable charge state determination from single m/z values of low-level impurities. Charge state information then enables more accurate modeling of the detected isotopic distribution for identification of the chemical composition of the detected impurity. Using this approach, a number of phosphorothioate impurities can be detected by LC-FTMS including failure sequences carrying 3′-terminal phosphate monoester and 3′-terminal phosphorothioate monoester, incomplete backbone sulfurization and desulfurization products, high molecular weight impurities, and chloral, isobutyryl, and N3 (2-cyanoethyl) adducts of the full-length product. When compared with low resolution LC-MS, ∼60% more impurities can be identified when charge state and isotopic distribution information is available and used for impurity profiling. © 2010 Elsevier B.V. All rights reserved.


Patent
Girindus America Inc. | Date: 2012-07-24

Described herein are novel solid-supported sulfurization reagents having the general structure: (I) wherein (P) is a polymer; X is a linker; R_(1 )is an alkyl group, a cycloalkyl group, an aryl group, or a heterocycle; and R_(2 )is an alkyl group, an aryl group, a methyleneacyloxy group having the formula CH_(2)OC(O)R_(7), a methylene carbonate group having the formula CH_(2)OC(O)OR_(8), or a methylene carbamate group having the formula CH_(2)OC(O)NR_(9)R_(10), wherein R_(7 )is a C_(1 )to C_(20 )hydrocarbon residue, R_(8 )is any alkyl, cycloalkyl, aryl, or heteroaryl, and R_(9 )and R^(10 )are independently hydrogen, alkyl, cycloalkyl, aryl, or heteroaryl. Other embodiments include solid-supported sulfurization reagents having the structure of Formula I, wherein (P) is a polystyrene-based solid support and X is an aromatic linker. Also described herein are methods for synthesizing the solid-supported sulfurization reagents and their use during the synthesis of oligonucleotides.


Patent
Girindus America Inc. | Date: 2011-06-22

An oligonucleotide which comprises at least one internucleotide linkage comprising a PSR bond and at least two nucleosides, wherein R corresponds to the formula (I) wherein A is a geminally substituted alkylene group, preferably CH_(2), X and Y are independently selected from S and O, and R_(0 )is selected from the group consisting of optionally substituted carbon bonded organic residue, such as in particular optionally substituted alkyl or aryl, SRx, ORx and NRxRy wherein Rx and/or Ry are selected from H and organic residues and at least Rx is a substituent other than H. Another object of the invention is a sulfurizing agent useful for oligonucleotide manufacture and the manufacture thereof. Other nucleotides described comprise at least one internucleotide linkage comprising a PSR bond, at least one internucleotide linkage comprising a PSR bond and at least three nucleosides wherein R is an organic residue other than the group R, preferably selected from a group consisting of an aryl group and a heteroaryl group which is bonded to the S-atom through an annular carbon atom.


Patent
Girindus America Inc. | Date: 2011-06-30

A method for synthesizing an oligonucleotide which comprises using a sulfurizing agent of general formula (I) for sulfurizing at least one phosphorus internucleotide linkage of a precursor of the oligonucleotide, wherein R is an aryl group or a heteroaryl group, which is bonded to the S-atom through an annular carbon atom; and R_(1 )and R_(2 )are independently organic residues, preferably a C1-C20 hydrocarbon residue. The method may further comprise purifying the oligonucleotide. Also included is a process for the synthesis of the sulfurizing agent.


PubMed | Girindus America Inc.
Type: | Journal: Methods in molecular biology (Clifton, N.J.) | Year: 2014

Strategies for site-specific modulation of genomic sequences in mammalian cells require two components. One must be capable of recognizing and activating a specific target sequence in vivo, driving that site into an exploitable repair pathway. Information is transferred to the site via participation in the pathway by the second component, a donor nucleic acid, resulting in a permanent change in the target sequence. We have developed biologically active triple helix forming oligonucleotides (TFOs) as site-specific gene targeting reagents. These TFOs, linked to DNA reactive compounds (such as a cross-linking agent), activate pathways that can engage informational donors. We have used the combination of a psoralen-TFO and single strand oligonucleotide donors to generate novel cell lines with directed sequence changes at the target site. Here we describe the synthesis and purification of bioactive psoralen-linked TFOs, their co-introduction into mammalian cells with donor nucleic acids, and the identification of cells with sequence conversion of the target site. We have emphasized details in the synthesis and purification of the oligonucleotides that are essential for preparation of reagents with optimal activity.

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