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Protection of 2′‐Hydroxy Functions of Ribonucleosides
Publication Name:
Current Protocols in Nucleic Acid Chemistry
Unit Number:
Unit 2.2
DOI:
10.1002/0471142700.nc0202s00
Online Posting Date:
May, 2001 Abstract
The main purpose of this article is to discuss 2¢-protection in the context of effective oligoribonucleotide synthesis. Emphasis is placed on the 2¢-protecting groups of choice in the synthesis of oligo-and polyribonucleotides, and the requirements that a protective group must satisfy to become the 2¢-hydroxyl-protecting group of choice. Finally, the unit discusses the issue of 2¢-O-acyl and 2¢-O-silyl group migration to the 3¢-hydroxy function of ribonucleosides during protection, along with the consequences of the conditions used for their removal on the stability of internucleotide linkages.
Figures
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Figure 2.2.1Scheme showing protected 2¢-hydroxy functions. B and B¢ are bases. -
Figure 2.2.2Scheme showing cleavage of interribonucleotide linkages under (A) basic and (B) acidic conditions. Although only shown in panel A, the hydrolysis of S.3 can yield either the 2¢-phosphate (S.5) or the 3¢-phosphate (S.6) under either basic or acidic conditions. -
Figure 2.2.3Several protecting groups for 5¢-terminal hydroxy functions. -
Figure 2.2.4Several protecting groups for base residues (A: S.16 and S.17; C: S.18; G: S.19 to S.22; U: S.23 to S.25). S.26 and S.27 are used in oximate treatment for the removal of aryl (Ar) groups. -
Figure 2.2.5Several protecting groups for internucleotide linkages. Ar is phenyl or another aryl group. -
Figure 2.2.6Scheme showing the preparation of uridylyl-(3¢5¢)-uridine from 2¢-O-benzyl (Bn)-uridine. -
Figure 2.2.7The 2-nitrobenzyl (S.34), 4-methoxybenzyl (S.35a), and 3,4-dimethoxybenzyl (S.35b) protecting groups. -
Figure 2.2.8Scheme showing introduction of the tert-butyldimethylsilyl (TBDMS) protecting group. -
Figure 2.2.9Scheme showing the interconversion of the 2¢-O- (S.39) and 3¢-O- (S.40) TBDMS adenosine derivatives. -
Figure 2.2.10Scheme showing conversion of 2¢-O-TBDMS-5¢-O-DMTr-ribonucleoside into its corresponding 3¢-phosphoramidite (S.41) and the structure of the possibly contaminating isomeric 2¢-phosphoramidite (S.42). Reagents (i): NCCH2 CH2 OPN(i-Pr)2 Cl, base. -
Figure 2.2.11Phosphoramidite activators 5-ethylthio-1H-tetrazole (S.43b) and 1H-tetrazole (S.43a), and the unblocking reagent triethylamine trihydrofluoride (S.44). -
Figure 2.2.122¢-O-TBDMS-ribonucleoside 3¢-H-phosphonate. -
Figure 2.2.13Scheme showing preparation of 2¢-O-Thp derivatives of uridine and adenosine. Reagents: (i) 3,4-dihydro-2H-pyran (S.48), toluene-4-sulfonic acid (TsOH), dioxane; (ii) NaOMe, MeOH. -
Figure 2.2.14Scheme showing conversion of 2¢-O-Thp-UpU into unprotected uridylyl-(3¢5¢)-uridine (S.50) and the structure of its (2¢5¢)-isomer (S.51). -
Figure 2.2.15Scheme showing preparation of 2¢-O-Mthp ribonucleoside derivatives (S.54) via 3¢,5¢-di-O-acyl-ribonucleosides (S.53) or 3¢,5¢-O-(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl) derivatives (S.56). Reagents: (i) 4-methoxy-5,6-dihydro-2H-pyran (S.55), toluene-4-sulfonic acid (TsOH), dioxane; (ii) NH3 , MeOH; (iii) (i-Pr)2 Si(Cl)OSi(Cl)(i-Pr)2 , imidazole, MeCN; (iv) Et4 NF, MeCN. -
Figure 2.2.16Scheme illustrating the removal of the Mthp protecting group. -
Figure 2.2.17Tetrahydrofuran-2-yl (S.59) and 9-(4-methoxyphenyl)xanthen-9-yl (S.60) protecting groups. -
Figure 2.2.18Scheme showing removal of levulinyl (top) and Fmoc (bottom) protecting groups. Reagents: (i) N2 H4 ×H2 O, C5 H5 N, AcOH; (ii) 0.1 M 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), MeCN. -
Figure 2.2.19Acetal protecting groups labile to acidic hydrolysis. -
Figure 2.2.20(A) Scheme showing preparation of Ctmp (S.71a) and Fpmp (S.71a) ribonucleoside derivatives. (B) Preparation of the 1-aryl-4-methoxy-1,2,5,6-tetrahydropyridines (S.70) required in (A). Reagents (i) S.70, CF3 CO2 H, CH2 Cl2 ; (ii) Et4 NF, MeCN; (iii) ethylene, AlCl3 , CH2 Cl2 ; (iv) toluene-4-sulfonic acid monohydrate (TsOH×H2 O), MeOH, and reflux followed by (MeO)3 CH; (v) (i-Pr)2 NEt, Et2 OBF3 , CH2 Cl2 , 0°C. -
Figure 2.2.21Dependence of half times (t1/2 ) on pH for hydrolysis of 2¢-O-Ctmp-uridine (S.71a) and 2¢-O-Fpmp-uridine (S.71b) at 30°C. -
Figure 2.2.22Ctmp-protected phosphoramidite (S.76) and H-phosphonate (S.77). -
Figure 2.2.235¢-O-Px-2¢-O-Fpmp phosphoramidite (S.78), 5¢-O-DMTr-2¢-O-Fpmp phosphoramidite (S.79), and 5¢-O-DMTr-2¢-O-methyl-ribonucleoside phosphoramidite (S.80). -
Figure 2.2.24Additional acetal protecting groups: (2-nitrobenzyloxy)methyl (S.81a), (4-nitrobenzyloxy)methyl (S.81b), (2,6-dimethoxycarbonyl)phenoxymethyl (S.82a), and (2,6-dicarboxy)phen- oxymethyl (S.82b). -
Figure 2.2.25Scheme showing interconversion of isomeric 2¢- and 3¢-O-acyl-ribonucleoside derivatives. -
Figure 2.2.26Scheme showing preparation of guanylyl-(3¢5¢)-uridine and cytidylyl-(3¢5¢)-uridine using a 2¢-O-acyl protecting group. Reagents: (i) mesitylene-2-sulfonyl chloride, C5 H5 N; (ii) MeNH2 , EtOH, or NH3 , MeOH. -
Figure 2.2.272¢-O-Benzoyl-protected 3¢-phosphoramidite (S.88), 2¢-O-(2-chlorobenzoyl)-protected 3¢-H-phosphonate (S.89), and the isomeric 2¢-H-phosphonate (S.90). -
Figure 2.2.28Structures relating to a discussion of the relative merits of the TBDMS and Fpmp protecting groups. -
Figure 2.2.29Unblocking of 2¢-O-Fpmp-protected oligoribonucleotides under mild conditions of acidic hydrolysis. -
Figure 2.2.30Substituted silyl and 1-aryl-4-alkoxypiperidin-4-yl protecting groups.
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