Biotinylation of a Propargylated Cyclic (3′‐5′) Diguanylic Acid and of Its Mono‐6‐Thioated Analog Under “Click” Conditions
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Abstract
Commercial N 2 ?isobutyryl?5??O ?(4,4??dimethoxytrityl)?2??O ?(propargyl)guanosine is converted to its 3??O ?levulinyl ester in a yield of 91%. The reaction of commercial N 2 ?isobutyryl?5??O ?(4,4??dimethoxytrityl)?2??O ?tert ?butyldimethylsilyl?3??O ?[(2?cyanoethyl)?N ,N ?diisopropylaminophosphinyl]guanosine with N 2 ?isobutyryl?2??O ?propargyl?3??O ?(levulinyl)guanosine provides, after P (III) oxidation, 3??/5??deprotection, and purification, the 2??O ?propargylated guanylyl(3??5?)guanosine 2?cyanoethyl phosphate triester in a yield of 88%. Phosphitylation of this dinucleoside phosphate triester with 2?cyanoethyl tetraisopropylphosphordiamidite and 1H ?tetrazole, followed by an in situ intramolecular cyclization, gives the propargylated cyclic dinucleoside phosphate triester, which is isolated in a yield of 40% after P (III) oxidation and purification. Complete removal of the nucleobases, phosphates, and 2??O ?tert ?butyldimethylsilyl protecting groups leads to the desired propargylated c?di?GMP diester. Cycloaddition of a biotinylated azide with the propargylated c?di?GMP diester under click conditions provides the biotinylated c?di?GMP conjugate in an isolated yield of 62%. Replacement of the 6?oxo function of N 2 ?isobutyryl?5??O ?(4,4??dimethoxytrityl)?3??O ?levulinyl?2??O ?(propargyl)guanosine with a 2?cyanoethylthio group is effected by treatment with 2,4,6?triisopropybenzenesulfonyl chloride and triethylamine to give a 6?(2,4,6?triisopropylbenzenesulfonic acid) ester intermediate. Reaction of this key intermediate with 3?mercaptoproprionitrile and triethylamine, followed by 5??dedimethoxytritylation, affords the 6?(2?cyanoethylthio)guanosine derivative in a yield of 70%. The 5??hydroxy function of this derivative is reacted with commercial N 2 ?isobutyryl?5??O ?(4,4??dimethoxytrityl)?2??O ?tert ?butyldimethylsilyl?3??O ?[(2?cyanoethyl)?N ,N ?diisopropylaminophosphinyl]guanosine. The reaction product is then converted to the mono?6?thioated c?di? GMP biotinylated conjugate under conditions highly similar to those described above for the preparation of the biotinylated c?di?GMP conjugate, and isolated in similar yields. Curr. Protoc. Nucleic Acid Chem. 52:14.9.1?14.9.20. © 2013 by John Wiley & Sons, Inc.
Keywords: propargylated c?di?GMP; biotinylated azide; click conjugation reaction; biotinylated c?di?GMP conjugate; 6?(2?cyanoethylthio)guanosine; c?di?GMP mono?6?thioated analog
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PDF or HTML at Wiley Online LibraryTable of Contents
- Introduction
- Basic Protocol 1: Synthesis, Purification, and Characterization of the Mono‐6‐Thioated Analog of 2′‐O‐Propargylated Cyclic‐DI‐GMP
- Commentary
- Literature Cited
- Figures
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Basic Protocol 1:
Materials
Basic Protocol 2:
Materials
Basic Protocol 3: Synthesis, Purification, and Characterization of the Mono‐6‐Thioated Analog of 2′‐O‐Propargylated Cyclic‐DI‐GMP
Materials
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Figure 14.9.1 Synthesis of the propargylated dinucleoside phosphate triester 5 . Abbreviations: DMTr, 4,4′‐dimethoxytrityl; Lev, levulinyl; TBDMS, tert ‐butyldimethylsilyl; GiBu , 2‐( N ‐isobutyryl)guanin‐9‐yl. Adapted with permission from Grajkowski et al. (). Copyright 2010 American Chemical Society. View Image -
Figure 14.9.2 Synthesis of the propargylated c‐di‐GMP 9 from the propargylated dinucleoside phosphate triester 5 . Abbreviations: GiBu , 2‐( N ‐isobutyryl)guanin‐9‐yl; TBDMS, tert ‐butyldimethylsilyl; Gua, guanin‐9‐yl. Adapted with permission from Grajkowski et al. (). Copyright 2010 American Chemical Society. View Image -
Figure 14.9.3 Click synthesis of the biotinylated c‐di‐GMP conjugate 12 from the biotinylated azide 11 . Abbreviations: TBTA, tris ‐(benzyltriazolylmethyl)amine; Gua, guanin‐9‐yl. Adapted with permission from Grajkowski et al. (). Copyright 2010 American Chemical Society. View Image -
Figure 14.9.4 Synthesis of the propargylated dinucleoside phosphate triester 17 . Abbreviations: DMTr, 4,4′‐dimethoxytrityl; Lev, levulinyl; TPS, 2,4,6‐triisopropylbenzenesulfonyl; TBDMS, tert ‐butyldimethylsilyl; DMAP, 4‐(dimethylamino)pyridine; iBu, isobutyryl; GuaiBu , 2‐( N ‐isobutyryl)guanin‐9‐yl; NMP, 1‐methylpyrrolidine. View Image -
Figure 14.9.5 Synthesis of the propargylated c‐di‐GMP 21 from the propargylated dinucleoside phosphate triester 17 . Abbreviations: GuaiBu , 2‐( N ‐isobutyryl)guanin‐9‐yl; iBu, isobutyryl; TBDMS, tert ‐butyldimethylsilyl; Gua, guanin‐9‐yl. View Image -
Figure 14.9.6 Click synthesis of the biotinylated c‐di‐GMP conjugate 22 from the biotinylated azide 11 and mono‐6‐thioated analog of 2′‐ O ‐propargylated cyclic‐di‐GMP (21 ). Abbreviation: Gua, guanin‐9‐yl. View Image -
Figure 14.9.7 Chemical structure of a 3′,5′‐bis‐phosphoramidite, as a potential side‐product of the phosphitylation of 5 . Abbreviations: GuaiBu , 2‐( N ‐isobutyryl)guanin‐9‐yl; TBDMS, tert ‐butyldimethylsilyl. Adapted with permission from Grajkowski et al. (). Copyright 2010 American Chemical Society. View Image -
Figure 14.9.8 RP‐HPLC analysis of the conversion of the propargylated c‐di‐GMP triester 8 to the propargylated c‐di‐GMP 9 . (A ) Chromatogram of the silica gel‐purified propargylated c‐di‐GMP triester 8 . (B ) Chromatogram of the propargylated c‐di‐GMP 9 , which is obtained from silica gel‐purified 8 after treatment with: (1) concentrated aqueous ammonia for 30 hr at 25°C; and (2) triethylamine trihydrofluoride for 20 hr at 25°C. Analytical RP‐HPLC analyses are performed using a 5‐µm Supelcosil LC‐18S column (25 cm × 4.6 mm) according to the following conditions: starting from 0.1 M triethylammonium acetate, pH 7.0, a linear gradient of 2.5% MeCN/min is pumped at a flow rate of 1 mL/min for 40 min. Peak heights are normalized to the highest peak, which is set to 1 arbitrary unit. Reprinted with permission from Grajkowski et al. (). Copyright 2010 American Chemical Society View Image
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