Synlett 2010(19): 2959-2963  
DOI: 10.1055/s-0030-1259022
LETTER
© Georg Thieme Verlag Stuttgart ˙ New York

Modified GSMP Synthesis Greatly Improves the Disulfide Crosslink of T7 Run-Off siRNAs with Cell Penetrating Peptides

Katja Schmitza,b, Frank Hahna,c, Ute Schepers*a,d,e
a Kekulé-Institut für Organische Chemie und Biochemie, Rheinische Friedrich-Wilhelms Universität Bonn, Gerhard-Domagk-Str.1, 53121 Bonn, Germany
b Institute of Organic Chemistry, Karlsruhe Institute of Technology (KIT), Fritz-Haber-Weg 6, 76131 Karlsruhe, Germany
c Department of Biochemistry, University of Cambridge, Downing Site, Cambridge CB2 1QW, U.K.
d Institute of Applied Biosciences, Karlsruhe Institute of Technology (KIT), Herzstr. 16, 76187 Karlsruhe, Germany
e Institute of Toxicology and Genetics, Karlsruhe Institute of Technology (KIT), Hermann-von-Helmholtz Platz 1,PO Box 3640, 76021 Karlsruhe, Germany
Fax: +49(7247)823354; e-Mail: [email protected];
Further Information

Publication History

Received 31 July 2010
Publication Date:
03 November 2010 (online)

Abstract

Preventing intramolecular cyclization greatly improves 5′-deoxy-5′-thioguanosine monophosphorothioate (GSMP) synthesis and its application as a potent initiator nucleotide for T7 run-off transcription of noncoding RNAs. GSMP was efficiently incorporated into the 5′-end of siRNA sense strands and the resulting thiol-modified siRNA was crosslinked with a free cysteine of cell penetrating peptide Penetratin as monitored by mass spectrometry. Cellular uptake and the knockdown potential of the peptide-coupled siRNA (pepsiRNA) were evaluated in primary cells.

    References and Notes

  • 1 Brantl S. Biochim. Biophys. Acta  2002,  1575:  15 
  • 2 Famulok M. Curr. Opin. Struct. Biol.  1999,  9:  324 
  • 3a Arenz C. Schepers U. Naturwissenschaften  2003,  90:  345 
  • 3b Aagaard L. Rossi JJ. Adv. Drug Deliv. Rev.  2007,  59:  75 
  • 4a Zhang BL. Cui ZY. Sun LL. Org. Lett.  2001,  3:  275 
  • 4b Igloi GL. Anal. Biochem.  1996,  233:  124 
  • 4c Hausch F. Jaschke A. Bioconjugate Chem.  1997,  8:  885 
  • 5a Davidson TJ. Harel S. Arboleda VA. Prunell GF. Shelanski ML. Greene LA. Troy CM. J. Neurosci.  2004,  24:  10040 
  • 5b Muratovska A. Eccles MR. FEBS Lett.  2004,  558:  63 
  • 6 Fischer PM. Krausz E. Lane DP. Bioconjugate Chem.  2001,  12:  825 
  • 7a Seelig B. Jäschke A. Tetrahedron Lett.  1997,  38:  7729 
  • 7b Seelig B. Jäschke A. Bioconjugate Chem.  1999,  10:  371 
  • 8 Lorsch JR. Szostak JW. Biochemistry  1994,  33:  973 
  • 9a Sengle G. Jenne A. Arora PS. Seelig B. Nowick JS. Jäschke A. Famulok M. Bioorg. Med. Chem.  2000,  8:  1317 
  • 9b Goody RS. Walker RT. J. Org. Chem.  1971,  36:  727 
  • 10 Dean DK. Synth. Commun.  2002,  32:  1517 
  • 11 Verheyden JP. Wagner D. Moffatt JG. J. Org. Chem.  1970,  35:  2319 
  • 12 Dimitrijevich SD. Verheyden JPH. Moffat JG.
    J. Org. Chem.  1978,  44:  400 
  • 13 Natarajan A. Moerke N. Fan YH. Chen H. Christ WJ. Wagner G. Halperin JA. Bioorg. Med. Chem. Lett.  2004,  14:  2657 
  • 15 Zhang L. Sun LL. Cui ZY. Gottlieb RL. Zhang BL. Bioconjugate Chem.  2001,  12:  939 
  • 16a Hardouin N. Nagy A. Genesis  2000,  26:  245 
  • 16b Hadjantonakis AK. Gertsenstein M. Ikwa M. Okabe M. Nagy A. Mech. Dev.  1998,  76:  79 
  • 17 Sprong H. Degroote S. Claessens T. van Drunen J. Oorschot V. Westerink BH. Hirabayashi Y. Klumperman J. van der Sluijs P. van Meer G. J. Cell Biol.  2001,  155:  369 
14

N , N -Dimethylaminomethylene-2′,3′- O , O -isopro-pylideneguanosine (8): 2′,3′-O,O-Isopropylideneguanosine (2; 5.75 g, 17.8 mmol) was resuspended in DMF (60 mL) and N,N-dimethylformamide dimethyl acetal (8.91 mL) was added under argon to yield an orange-brown solution. The reaction mixture was stirred at 50 ˚C for 4 h. The solvent was removed under reduced pressure and at elevated temperatures (ca. 55 ˚C), the white residue was then removed by filtration. The filtrate was dried under reduced pressure, redissolved in MeOH (25 mL) yielding a fluorescent green solution and precipitated with 50 mL of EtOAc. After storage overnight at 4 ˚C, the residue was removed by filtration and the combined filtrates were thoroughly washed with EtOAc and dried under reduced pressure. The product was obtained as a white powder in 80% yield (5.38 g, 14.2 mmol). All steps were carried out under protection from light; mp >200 ˚C; R f 0.47 (CHCl3-MeOH, 8:1), 0.08 (CHCl3-MeOH, 19:1). ¹H NMR (400 MHz, DMSO-d 6): δ = 11.32 (br s, 1 H, NH), 8.56 (s, 1 H, H-imine), 8.00 (s, 1 H, H-8), 6.03 (d, J = 3.0 Hz, 1 H, H-1′), 5.26 (dd, J = 3.0, 6.3 Hz, 1 H, H-2′), 5.03 (dd, J = 5.4, 5.4 Hz, 1 H, 5′-OH), 4.95 (dd, J = 2.9, 6.3 Hz, 1 H, H-3′), 4.13 (ddd, J = 2.9, 4.9, 4.9 Hz, 1 H, H-4′), 3.59-3.47 (m, 2 H, H-5′), 3.15 (s, 3 H, NMe), 3.03 (s, 3 H, NMe), 1.53 (s, 3 H, Me), 1.33 (s, 3 H, Me). ¹³C NMR (101 MHz, DMSO-d 6): δ = 158.3 (C-imine), 157.6, 157.5 (C-4, C-6), 149.6 (C2), 136.0 (C-8), 119.9 (C-5), 113.2 (C-quat), 88.6, 86.4, 83.6, 81.2 (C-1′ to C-4′), 61.5 (C-5′), 40.8 (NMe), 34.8 (NMe), 27.2 (Me), 25.3 (Me). MS (EI; 70 eV): m/z (%) = 378 (89%) [M]+, 363 (6%) [M - CH]+, 348 (6%) [M - 2 Me]+, 333 (2%) [M - NH(Me)2]+, 206 (100%) [M - DAMG]+, 191 (26%) [DAMG - Me]+, 176 (3%) [DAMG - 2 Me]+, 150 (6%) [guanine]+. HRMS: m/z calcd for C16H22N6O5: 378.1652; found: 378.1654 (±0.0095); (DAMG = 2-N′,N-dimethylaminomethylene guanine).
N , N -Dimethylaminomethylene-2′,3′- O , O -isopro-pylidene-5′-deoxy-5′-iodoguanosine (9): Ground 8 (2.20 g, 5.81 mmol) was resuspended in anhyd THF (110 mL) under argon and cooled to -70 ˚C by a mixture of acetone and dry ice. Methyltriphenoxyphosphonium iodide (3.94 g, 8.71 mmol; 1.5 equiv) was added. Due to the light sensitivity of the reactant and the product all subsequent steps were carried out under exclusion of light. After 30 min of stirring the reaction mixture was allowed to warm to r.t. and stirred for another 4 h. The reaction was stopped by the addition of MeOH (5 mL) and the solvent was removed under reduced pressure. The dark red residue was dissolved in MeOH-CHCl3 (1:4; 2.5 mL) and CHCl3 (7 mL) was added. The solution of the crude product was subjected to chromatography on silica gel (Merck 0.40-0.65 µm; eluent: CHCl3-MeOH, 9:1). After removal of the solvent, 9 was obtained as an orange solid in 99% yield (2.81 g, 5.75 mmol); mp 95-97 ˚C; R f 0.74 (CHCl3-MeOH, 5:1), 0.22 (CHCl3-MeOH, 19:1). ¹H NMR (400 MHz, DMSO-d 6): δ = 11.39 (br s, 1 H, NH), 8.58 (s, 1 H, H-imine), 8.01 (s, 1 H, H-8), 6.15 (d, J = 2.1 Hz, 1 H, H-1′), 5.42 (dd, J = 2.1, 6.3 Hz, 1 H, H-2′), 5.02 (dd, J = 2.8, 6.3 Hz, 1 H, H-3′), 4.28 (ddd, J = 2.8, 5.7, 9.0 Hz, 1 H, H-4′), 3.49 (dd, J = 9.0, 9.9 Hz, 1 H, H-5′), 3.30 (dd, J = 5.7, 9.9 Hz, 1 H, H-5′), 3.19 (s, 3 H, NMe), 3.04 (s, 3 H, NMe), 1.53 (s, 3 H, Me), 1.35 (s, 3 H, Me). ¹³C NMR (101 MHz, DMSO-d 6): δ = 158.2 (C-imine), 157.7, 157.5 (C-4, C-6), 149.2 (C2), 137.9 (C-8), 120.2 (C-5), 113.5 (C-quart), 89.4, 86.4, 84.0, 83.9 (C-1′ to C-4′), 41.1 (NMe), 34.9 (NMe), 27.2 (Me), 25.3 (Me), 6.7 (C-5′). MS (EI; 70 eV): m/z = 488 (13%) [M]+, 361 (7%)
[M - I]+, 360 (35%) [M - HI]+, 345 (1%) [M - HI, Me]+, 206 (3%) [DAMG]+, 188 (2%) [DAMG - H2O]+, 176 (26%) [DAMG - 2 Me]+, 149 (12%) [guanine - H]+, 128 (100%) [HI]. HRMS: m/z calcd for C16H21N6O4I: 488.0669; found: 488.06631; (DAMG = 2-N′,N-dimethylaminomethylene guanine).