Convenient synthesis of a propargylated cyclic (3'-5') diguanylic acid and its "click" conjugation to a biotinylated azide

Abstract

The ribonucleoside building block, N²-isobutyryl-2'-O-propargyl-3'-O-levulinyl guanosine, was prepared from commercial N²-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-O-propargyl guanosine in a yield of 91%. The propargylated guanylyl(3'-5')guanosine phosphotriester was synthesized from the reaction of N²-isobutyryl-2'-O-propargyl-3'-O-levulinyl guanosine with N²-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-O-tert-butyldimethylsilyl-3'-O-[(2-cyanoethyl)-N,N-diisopropylaminophosphinyl] guanosine and isolated in a yield of 88% after P(III) oxidation, 3'-/5'-deprotection, and purification. The propargylated guanylyl(3'-5')guanosine phosphotriester was phosphitylated using 2-cyanoethyl tetraisopropylphosphordiamidite and 1H-tetrazole and was followed by an in situ intramolecular cyclization to give a propargylated c-di-GMP triester, which was isolated in a yield of 40% after P(III) oxidation and purification. Complete N-deacylation of the guanine bases and removal of the 2-cyanoethyl phosphate protecting groups from the propargylated c-di-GMP triester were performed by treatment with aqueous ammonia at ambient temperature. The final 2'-desilylation reaction was effected by exposure to triethylammonium trihydrofluoride affording the desired propargylated c-di-GMP diester, the purity of which exceeded 95%. Biotinylation of the propargylated c-di-GMP diester was easily accomplished through its cycloaddition reaction with a biotinylated azide derivative under click conditions to produce the biotinylated c-di-GMP conjugate of interest in an isolated yield of 62%.

Figure 1

RP-HPLC analysis of the guanylyl(3′-5′)guanosine phosphotriester 6. ^{a a}Analytical chromatographic conditions are described in the Supporting Information under Materials and Methods. Peak heights were normalized to the highest peak, which was set to 1 arbitrary unit.

Figure 2

RP-HPLC analysis of the conversion of the propargylated c-di-GMP triester 10 to the propargylated c-di-GMP 11. A: Chromatogram of the silica gel-purified propargylated c-di-GMP triester 10. B: Chromatogram of the propargylated c-di-GMP 11 that was obtained from silica gel-purified 10 after complete deprotection, which was effected by treatment with: (i) concentrated aqueous ammonia for 30 h at 25 °C; and (ii) triethylamine trihydrofluoride for 20 h at 25 °C. Analytical chromatographic conditions are described in the Supporting Information under Materials and Methods. Peak heights were normalized to the highest peak, which was set to 1 arbitrary unit.

Figure 3

RP-HPLC analysis of the “click” conjugation of the propargylated c-di-GMP 11 with the biotinylated azide 13. A: Chromatogram of the propargylated c-di-GMP 11. B: Chromatogram of the purified biotinylated c-di-GMP 14 obtained from “click” conjugation of 11 with 13 under the conditions described in the Experimental section. Analytical chromatographic conditions are described in the Supporting Information under Materials and Methods.

Scheme 1

3′-O-Acylation of the ribonucleoside 1 and 5′-O-deprotection of 2.^{a a} Keys: DMTr, 4,4′-dimethoxytrityl; Gua, guanin-9-yl; iBu, isobutyryl; Lev₂O, levulinic anhydride; Lev, levulinyl.

Scheme 2

Preparation of the diribonucleoside phosphotriester 6.^{a a} Conditions: (i) 3, 1H-tetrazole, MeCN, 2 h, 25°C; (ii) tert-BuOOH/decane, 30 min; (iii) hydrazine hydrate/AcOH/C₅H₅N, 15 min, then 2,4-pentanedione, 5 min; (iv) chromatography on silica gel; (v) 80% AcOH, 3 h, 25°C. Keys: DMTr, 4,4′-dimethoxytrityl; Gua, guanin-9-yl; iBu, isobutyryl; TBDMS, tert-butyldimethylsilyl; Lev, levulinyl.

Scheme 3

Conversion of the diribonucleoside phosphotriester 6 to the propargylated c-di-GMP 11.^{a a} Conditions: (i) (i-Pr₂N)₂POCH₂CH₂CN, 1H-tetrazole (1.0 equiv at the rate of 0.25 equiv/15 min), MeCN, 1 h; (ii) 1H-tetrazole (2 equiv), MeCN, 16 h; (iii) concd aq NH₃, 30 h, 25°C; (iv) Et₃N•3HF, 20 h, 25°C. Keys: Gua, guanin-9-yl; iBu, isobutyryl; TBDMS, tert-butyldimethylsilyl.

Scheme 4

Biotinylation of the propargylated c-di-GMP 10.^{a a} Keys: Gua, guanine-9-yl; TBTA, tris-(benzyltriazolylmethyl)amine.