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(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2015/006740 A2 15 January 2015 (15.01.2015) P O P C T (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every A61K 47/48 (2006.01) kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, (21) International Application Number: BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, PCT/US20 14/046425 DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (22) International Filing Date: HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, 11 July 2014 ( 11.07.2014) KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, (25) Filing Language: English OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, (26) Publication Language: English SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, (30) Priority Data: ZW. 61/845,279 11 July 2013 ( 11.07.2013) US (84) Designated States (unless otherwise indicated, for every (71) Applicant: ALNYLAM PHARMACEUTICALS, INC. kind of regional protection available): ARIPO (BW, GH, [US/US]; 300 Third Street, Cambridge, MA 02142 (US). GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, (72) Inventors: MANOHARAN, Muthiah; 300 Third Street, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, Cambridge, MA 02142 (US). NAIR, Jayaprakash, K.; EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, 300 Third Street, Cambridge, MA 02142 (US). KAN- MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, DASAMY, Pachamuthu; 300 Third Street, Cambridge, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, MA 02142 (US). MATSUDA, Shigeo; 300 Third Street, KM, ML, MR, NE, SN, TD, TG). Cambridge, MA 02142 (US). KELIN, Alexander, V.; 300 Third Street, Cambridge, MA 02142 (US). JAYARA- Published: MAN, Muthusamy; 300 Third Street, Cambridge, MA — without international search report and to be republished 02142 (US). RAJEEV, Kallanthottathil, G.; 300 Third upon receipt of that report (Rule 48.2(g)) Street, Cambridge, MA 02142 (US). (74) Agent: LESSLER, Jay, P.; Blank Rome LLP, The Chrysler Building, 405 Lexington Ave., New York, NY 10174-0208 (US). < © ©o © (54) Title: OLIGONUCLEOTIDE-LIGAND CONJUGATES AND PROCESS FOR THEIR PREPARATION (57) Abstract: The present invention relates to ligand conjugates of oligonucleotides (e.g., iRNA agents) and methods for their pre - paration. The ligands are derived primarily from monosaccharides These conjugates are useful for the in vivo delivery of oligonuc- leotides. OLIGONUCLEOTIDE-LIGAND CONJUGATES AND PROCESS FOR THEIR PREPARATION RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 61/845,279, filed July 11, 2013, which is hereby incorporated by reference in its entirety. FIELD OF THE INVENTION The present invention relates to ligand conjugates of oligonucleotides (e.g., iRNA agents) and methods for their preparation. The ligands are derived primarily from monosaccharides. These conjugates are useful for the in vivo delivery of oligonucleotides. BACKGROUND OF THE INVENTION Efficient delivery to cells in vivo requires specific targeting and substantial protection from the extracellular environment, particularly serum proteins. One method of achieving specific targeting is to conjugate a targeting moiety to the iRNA agent. The targeting moiety helps in targeting the iRNA agent to the required target site. One way a targeting moiety can improve delivery is by receptor mediated endocytotic activity. This mechanism of uptake involves the movement of iRNA agent bound to membrane receptors into the interior of an area that is enveloped by the membrane via invagination of the membrane structure or by fusion of the delivery system with the cell membrane. This process is initiated via activation of a cell- surface or membrane receptor following binding of a specific ligand to the receptor. Many receptor-mediated endocytotic systems are known and have been studied, including those that recognize sugars such as galactose, mannose, mannose-6-phosphate, peptides and proteins such as transferrin, asialoglycoprotein, vitamin B12, insulin and epidermal growth factor (EGF). The Asialoglycoprotein receptor (ASGP-R) is a high capacity receptor, which is highly abundant on hepatocytes. The ASGP-R shows a 50-fold higher affinity for N-Acetyl-D-Galactosylamine (GalNAc) than D-Gal. Previous work has shown that multivalency is required to achieve nM affinity, while spacing among sugars is also crucial. Recently, certain carbohydrate conjugates have been shown to be a valuable delivery alternatively to liposomes for siRNA delivery. SUMMARY OF THE INVENTION The present invention relates to ligand conjugates of oligonucleotides or other biologically active agents which have one or more advantageous properties, such as improved delivery of the oligonucleotide or other biologically active agents, lower manufacturing costs or fewer manufacturing issues, or better chemical stability. These conjugates provide effective delivery of oligonucleotides and other biologically active agents. In one embodiment, the present invention relates to a ligand (e.g., carbohydrate) conjugate of an oligonucleotide (e.g., an iRNA agent) or other biologically active agent of the formula I: Formula I wherein the Oligonucleotide is an oligonucleotide, such as an siRNA, microRNA, antimiR, antagomir, microRNA mimic, decoy, immune stimulatory, G-quadruplex, splice altering, ssRNA, antisense, aptamer, stem-loop RNA or DNA or one of the two strands of any double stranded RNA or DNA or double stranded shortemer RNA or DNA (e.g, siRNA); the Biologically Active Agent is any biologically active agent; the Linker is a linking group between the Ligand(s) and the Oligonucleotide or other biologically active agent, where the Linker may be selected from the linking groups in Table 1 or 1A; and the Ligand(s) are sugar-derived, where (i) the Ligand(s) may be attached to the same or different atoms in the Linker, (ii) the conjugate contains from 1 to 12 Ligands (preferably 1 to 5 or 1 to 3 Ligands), and (iii) the Ligand(s) may be selected from (a) the Ligands in Table 2 or 2A, 2 (b) - R -(R )k, where 9 R is absent (in which case k=l) or a spacer (also referred to as a ligand backbone) having two or more sites of attachment for the R groups, R is a targeting monomer selected from Table 3, and k is 1 to 6 (preferably 1 to 5 or 1 to 3), each R3 may be attached to the same or different atoms in R2 ; and (c) the Ligands in Table 4 or 4A. The conjugate includes at least one Linker from Table 1 or 1A or from the examples, one Ligand from Table 2, 2A, 4, or 4A, or one targeting monomer from Table 3 or 3A. For example, the nucleoside linkers described in the examples can be used as the Linker. In one embodiment, the conjugate includes (i) at least one Linker from Table 1 or 1A or from the examples, (ii) one Ligand from Table 2, 2A, 4, or 4A, and (iii) one targeting monomer from Table 3 or 3A. 9 R can be an amino acid-, polypeptide- (e.g., a dipeptide or tripeptide), heteroaryl- (e.g., a triazole), or sugar-containing group. In one preferred embodiment, each R group is attached via 9 an amide, ether, or amino group to R . In one embodiment, R is attached via an amide group. Each entry in Tables 2, 2A, 4, and 4A shows a spacer bound to at least one targeting monomer. The spacers are bound to the targeting monomers either through a heteroatom (which is at a terminus of the spacer), such as a nitrogen atom, or at the anomeric carbon to the sugar group of a targeting monomer (as shown below). The heteroatom attachment point in the structures shown in Tables 2, 2A, 4, and 4A is the first nitrogen atom when walking along the chain from the sugar group (left side of the ligand) to the remainder of the ligand (right side). The spacers for two ligands in Table 2A are shown in the table below (the arrows indicate the points of attachment for the targeting monomers R , and the right side of the spacer is attached to the Linker). Suitable spacers are also shown in Table 5 below. The Oligonucleotide is preferably attached to the Linker through (i) the 3' or 5'-terminal of the oligonucleotide, (ii) one or more sugar moieties of the nucleoside present in the oligonucleotide independent of position, or (iii) one or more base moieties of the nucleoside present independent of position. In one embodiment, the Ligand is conjugated to one of the two strands of a double stranded siRNA via a Linker. In one embodiment, the Ligand(s) target the asialoglycoprotein receptor (ASGPR). In another embodiment, the Ligand(s) target the liver, such as the parenchymal cells of the liver. The Ligand(s) may be an unmodified or modified monosaccharide, disaccharide, trisaccharide, tetrasaccharide, or higher polysaccharide. The oligonucleotide can be attached to the Linker via a cleavable group (e.g., phosphate, phosphorothiate, or amide) or a non-cleavable group (e.g., ether, carbamate, or C-C (e.g., a bond between two carbon atoms or -CH 2-CH2-)). As described herein, the cleavable or non-cleavable group is within the Oligonucleotide of Formula I. In one embodiment, the -Linker- Ligand is not L96 (shown in the examples).
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  • Evolving a New Efficient Mode of Fructose Utilization For

    Evolving a New Efficient Mode of Fructose Utilization For

    fbioe-09-669093 May 22, 2021 Time: 22:55 # 1 ORIGINAL RESEARCH published: 28 May 2021 doi: 10.3389/fbioe.2021.669093 Evolving a New Efficient Mode of Fructose Utilization for Improved Bioproduction in Corynebacterium glutamicum Irene Krahn1, Daniel Bonder2, Lucía Torregrosa-Barragán2, Dominik Stoppel1, 1 3 1 3,4 Edited by: Jens P. Krause , Natalie Rosenfeldt , Tobias M. Meiswinkel , Gerd M. Seibold , Pablo Ivan Nikel, Volker F. Wendisch1 and Steffen N. Lindner1,2* Novo Nordisk Foundation Center 1 2 for Biosustainability (DTU Biosustain), Chair of Genetics of Prokaryotes, Faculty of Biology and CeBiTec, Bielefeld University, Bielefeld, Germany, Systems 3 Denmark and Synthetic Metabolism, Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany, Institute of Biochemistry, University of Cologne, Cologne, Germany, 4 Department of Biotechnology and Biomedicine, Technical Reviewed by: University of Denmark, Lyngby, Denmark Stephan Noack, Julich-Forschungszentrum, Helmholtz-Verband Deutscher Fructose utilization in Corynebacterium glutamicum starts with its uptake and Forschungszentren (HZ), Germany concomitant phosphorylation via the phosphotransferase system (PTS) to yield Fabien Létisse, UMR 5504 Laboratoire d’Ingénierie intracellular fructose 1-phosphate, which enters glycolysis upon ATP-dependent des Systèmes Biologiques et des phosphorylation to fructose 1,6-bisphosphate by 1-phosphofructokinase. This is known Procédés (LISBP), France to result in a significantly reduced oxidative pentose phosphate pathway (oxPPP) flux *Correspondence: ∼ ∼ Steffen N. Lindner on fructose ( 10%) compared to glucose ( 60%). Consequently, the biosynthesis of [email protected] NADPH demanding products, e.g., L-lysine, by C. glutamicum is largely decreased when fructose is the only carbon source. Previous works reported that fructose Specialty section: This article was submitted to is partially utilized via the glucose-specific PTS presumably generating fructose 6- Synthetic Biology, phosphate.
  • Estimation of Free Methylpentoses in the Presence of Glycosidically Bound Sugar1

    Estimation of Free Methylpentoses in the Presence of Glycosidically Bound Sugar1

    ANALYTICAL BIOCHEMISTRY 14, 278-289 (1966) Estimation of Free Methylpentoses in the Presence of Glycosidically Bound Sugar1 A. K. BHATTACHARYYA AND D. AMINOFF From the Simpson Memorial Institute, The University of Michigan, Ann Arbor, Michigan Received August 3, 1965 In the course of our investigations on the immunochemistry and bio- synthesis of the human blood group substances, it became necessary to determine the amount of free fucose in the presence of the glycosidically bound sugar. Dische and Shettles (1) have described a specific spectro- photometric procedure for the determination of methylpentoses. This involved heating with concentrated sulfuric acid and the development of a specific color with cysteine hydrochloride. Modifications of the method have been described (2, 3) but in each case a strong acid is employed in the reagent. The heating with the acidic reagents releases methyl- pentose and converts it to the appropriate chromogen. Thus, these methods do not distinguish between free and bound methylpentoses. In- direct methods were therefore used to determine the rate of release of methylpentose under varying conditions of hydrolysis. One such method involved chromatographic separation of the sugars in the hydrolysis products followed by quantitative determination of the area correspond- ing to free fucose either by the cysteine-H&SO4 method (1) or by the Somogyi-Nelson procedure (4). Recovery of methylpentoses by these techniques was invariably low. This paper describes a procedure which involves the oxidation of methylpentoses with periodate. Only the free methylpentose will release acetaldehyde on oxidation with periodate and thus can be determined in the presence of the glycosidically bound compound.
  • Selective Fermentation of Potential Prebiotic Lactose-Derived Oligosaccharides By

    Selective Fermentation of Potential Prebiotic Lactose-Derived Oligosaccharides By

    1 Selective fermentation of potential prebiotic lactose-derived oligosaccharides by 2 probiotic bacteria 3 4 Tomás García-Cayuela, Marina Díez-Municio, Miguel Herrero, M. Carmen Martínez- 5 Cuesta, Carmen Peláez, Teresa Requena*, F. Javier Moreno 6 7 Instituto de Investigación en Ciencias de la Alimentación, CIAL (CSIC-UAM), CEI 8 (UAM+CSIC), Nicolás Cabrera 9, 28049 Madrid, Spain. 9 10 * Corresponding author: Tel.: +34 91 0017900; E-mail address: [email protected] 11 1 12 Abstract 13 The growth of potential probiotic strains from the genera Lactobacillus, 14 Bifidobacterium and Streptococcus was evaluated with the novel lactose-derived 15 trisaccharides 4’-galactosyl-kojibiose and lactulosucrose and the potential prebiotics 16 lactosucrose and kojibiose. The novel oligosaccharides were synthesized from 17 equimolar sucrose:lactose and sucrose:lactulose mixtures, respectively, by the use of a 18 Leuconostoc mesenteroides dextransucrase and purified by liquid chromatography. The 19 growth of the strains using the purified carbohydrates as the sole carbon source was 20 evaluated by recording the culture optical density and calculating maximum growth 21 rates and lag phase parameters. The results revealed an apparent bifidogenic effect of 22 lactulosucrose, being also a moderate substrate for streptococci and poorlybut badly 23 utilized by lactobacilli. In addition, 4’-galactosyl-kojibiose was selectively fermented by 24 Bifidobacterium breve, which was also the only tested bifidobacterial species able to 25 ferment kojibiose. The described fermentation properties of the specific probiotic strains 26 on the lactose-derived oligosaccharides would enable the design of prebiotics with a 27 high degree of selectivity. 28 29 Keywords: Prebiotic; Lactose-derived oligosaccharides; Probiotic; Bifidobacterium; 30 Lactobacillus; Streptococcus 31 2 32 1.