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Preparation of 5′‐Silyl‐2′‐Orthoester Ribonucleosides for Use in Oligoribonucleotide Synthesis

Stephen A. Scaringe¹, David Kitchen¹, Robert J. Kaiser¹, William S. Marshall¹

¹Dharmacon Inc., Lafayette, Colorado

Publication Name:

Current Protocols in Nucleic Acid Chemistry

Unit Number:

Unit 2.10

DOI:

10.1002/0471142700.nc0210s16

Online Posting Date:

May, 2004

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Abstract

The recent discovery that small interfering RNAs (siRNAs) induce gene suppression in mammalian cells has sparked tremendous interest in using siRNA-based assays and high-throughput screens to study gene function. As a result, research programs at leading academic and commercial institutions have become a substantial and rapidly growing market for synthetic RNA. Important considerations in synthesizing RNA for biological gene function studies are sequence integrity, purity, scalability, and resistance to nucleases; ease of chemical modification, deprotection, and handling; and cost. Of the well-established RNA synthesis methods, 2¢-ACE chemistry is the only one that meets all of these criteria. 2¢-ACE technology employs a unique class of silyl ethers to protect the 5¢-hydroxyl, in combination with an acid-labile orthoester protecting group on the 2¢-hydroxyl (2¢-ACE). 2¢-ACE-protected phosphoramidite monomers are joined using standard solid-phase technology to achieve RNA synthesis at efficiencies rivaling those for DNA. This unit describes the synthesis of standard 5¢-silyl-2¢-ACE-protected phosphoramidites.

Keywords: RNA synthesis; silyl protection; orthoester; ribonucleoside; phosphoramidite

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Unit Introduction
Basic Protocol: Synthesis of 2¢-ACE-Protected Uridine Ribonucleoside Phosphoramidite
Alternate Protocol 1: Synthesis of 2¢-ACE-Protected Adenosine Ribonucleoside Phosphoramidite
Alternate Protocol 2: Synthesis of 2¢-ACE-Protected Guanosine Ribonucleoside Phosphoramidite
Alternate Protocol 3: Synthesis of 2¢-ACE-Protected Cytidine Ribonucleoside Phosphoramidite
Commentary
Literature Cited
Figures

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Materials

Basic Protocol: Synthesis of 2¢-ACE-Protected Uridine Ribonucleoside Phosphoramidite

Materials

Uridine (S.1; 244 g/mol; Monomer Sciences)
Pyridine
1,3-Dichloro-1,1,3,3-tetraisopropyldisiloxane (TIPS-Cl₂; 315 g/mol; d = 0.986 g/mL; Monomer Sciences)
Toluene
Dichloromethane
5% and 8% (w/v) aqueous sodium bicarbonate (NaHCO₃)
Saturated aqueous sodium chloride (NaCl)
Anhydrous sodium sulfate (Na₂SO₄)
Silica gel 60 (for column chromatography)
Hexane
Ethyl acetate (EtOAc)
Silica gel thin-layer chromatography (TLC) plates
Methanol (MeOH)
Acetonitrile (MeCN)
Tris(2-acetoxyethyl) orthoformate (322 g/mol; Dharmacon Inc.)
Pyridinium p-toluenesulfonate (251 g/mol)
Triethylamine (TEA)
4-tert-Butyldimethylsiloxy-3-penten-2-one (238 mL/mol; Silar)
N,N,N¢,N¢-Tetramethylethylene diamine (TEMED; 150 mL/mol)
48% (w/v) aqueous hydrofluoric acid (HF; 32 mL/mol)
Diisopropylamine (140 mL/mol)
Benzhydroxy-bis(trimethylsilyloxy)chlorosilane (BzH-Cl; 425 g/mol; Dharmacon Inc.)
Acetone
Bis(N,N-diisopropylamino)methoxyphosphine (262 g/mol; Monomer Sciences)
0.45 M 1H-tetrazole in MeCN (AIC)
Ethanol (EtOH)

Rotary evaporator
1-L separatory funnel
Glass-fritted Buchner funnel (coarse porosity) and side-arm Erlenmeyer flask
High-vacuum pump
Water aspirator
50 × 600–mm chromatography column

Additional reagents and equipment for flash chromatography (appendix 3E) and TLC (appendix 3D)

NOTE: For solvent evaporation on a rotary evaporator, the required vacuum source will depend on the boiling point of the solvent involved. For dichloromethane and hexane, a water aspirator or diaphragm pump will suffice. For all other solvents, a high-vacuum oil pump is needed.

NOTE: The efficiency of reactions as well as the results of column chromatography at each step are assessed by TLC on silica gel plates. It is suggested that the TLC plates be spotted in three horizontal locations at the origin of the plate as follows: (1) starting material alone, (2) co-spot of starting material plus crude reaction mixture or product fraction, and (3) crude reaction mixture or product fraction alone. In this way, the conversion of starting material to product, or the purity of the product fraction, can be conveniently monitored.

Alternate Protocol 1: Synthesis of 2¢-ACE-Protected Adenosine Ribonucleoside Phosphoramidite

Additional Materials (also see Basic Protocol)

N⁶-Benzoyladenosine (337 g/mol; Monomer Sciences)

Alternate Protocol 2: Synthesis of 2¢-ACE-Protected Guanosine Ribonucleoside Phosphoramidite

Additional Materials (also see Basic Protocol)

N²-Isobutyrylguanosine (353 g/mol; Monomer Sciences)

Alternate Protocol 3: Synthesis of 2¢-ACE-Protected Cytidine Ribonucleoside Phosphoramidite

Additional Materials (also see Basic Protocol)

N⁴-Acetylcytidine (285 g/mol; Monomer Sciences)

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Figures

Figure 2.10.1

Protected ribonucleoside phosphoramidites for 5¢-O-silyl-2¢-O-orthoester RNA synthesis.

View Image
Figure 2.10.2

3¢,5¢-Protection of the nucleoside. Synthesis of 3¢,5¢-O-(tetraisopropyldisiloxane-1,3-diyl)uridine from uridine. TIPS-Cl₂, 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane.

View Image
Figure 2.10.3

2¢-ACE protection. Synthesis of 3¢,5¢-O-(tetraisopropyldisiloxane-1,3-diyl)-2¢-O-bis(acetoxyethyloxy)methyl uridine from 3¢,5¢-O-(tetraisopropyldisiloxane-1,3-diyl)uridine.

View Image
Figure 2.10.4

Removal of 3¢,5¢-protection. Synthesis of 2¢-O-bis(acetoxyethyloxy)methyl uridine from 3¢,5¢-O-(tetraisopropyldisiloxane-1,3-diyl)-2¢-O-bis(acetoxyethyloxy)methyl uridine. TEMED, N,N,N¢,N¢-tetramethylethylenediamine.

View Image
Figure 2.10.5

5¢-Silylation. Synthesis of 5¢-O-benzhydroxy-bis(trimethylsilyloxy)silyl-2¢-O-bis(acetoxyethyloxy)methyl uridine from 2¢-O-bis(acetoxyethyloxy)methyl uridine. BzH-Cl, benzhydroxy-bis(tri-methylsilyloxy)silyl chlorosilane; DIPA, N,N-diisopropylamine.

View Image
Figure 2.10.6

Preparation of the phosphoramidite monomer for oligoribonucleotide synthesis. Synthesis of 5¢-O-benzhydroxy-bis(trimethylsilyloxy)silyl-2¢-O-bis(acetoxyethyloxy)methyl-3¢-O-(N,N-diisopropylamino)methoxyphosphinyl uridine from 5¢-O-benzhydroxy-bis(trimethylsilyloxy)silyl-2¢-O-bis(acetoxyethyloxy)methyl uridine. i: bis(N,N-diisopropylamino)methoxyphosphine; 1H-tetrazole, CH₂Cl₂, 12 hr.

View Image

Literature Cited

Literature Cited
	Caruthers, M.H. 1985. Gene synthesis machines: DNA chemistry and its uses. Science 230:281-285.
	Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. 2001. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494-498.
	Matteucci, M.D. and Caruthers, M.H. 1981. Synthesis of deoxyoligonucleotides on a polymer support. J. Am. Chem. Soc. 103:3185-3191.
	Scaringe, S.A., Wincott, F.E., and Caruthers, M.H. 1998. Novel RNA synthesis method using 5¢-silyl-2¢-orthoester protecting groups. J. Am. Chem. Soc. 120:11820-11821.
	Usman, N.O., Ogilvie, K.K., Jiang, M.Y., and Cedergren, R.J. 1987. The automated chemical synthesis of long oligoribonucleotides using 2¢-O-silylated ribonucleoside 3¢-O-phosphoramidites on a controlled-pore glass support: Synthesis of a 43-nucleotide sequence similar to the 3¢-half molecule of an Escherichia coli formylmethionine tRNA. J. Am. Chem. Soc. 109:7845-7854.
	Wu, X. and Pitsch, S. 1998. Synthesis and pairing properties of oligoribonucleotide analogues containing a metal-binding site attached to -d-allofuranosyl cytosine. Nucl. Acids Res. 26:4315-4323.