An improved strategy for the chemical synthesis of 3’, 5’-cyclic diguanylic acid

Abstract

The physiological functions of c-di-GMP and its involvement in many key processes led to its recognition as a major and ubiquitous bacterial second messenger. Aside from being a bacterial signaling molecule, c-di-GMP is also an immunostimulatory molecule capable of inducing innate and adaptive immune responses through maturation of immune mammalian cells. Given the broad biological functions of c-di-GMP and its potential applications as a nucleic-acid-based drug, the chemical synthesis of c-di-GMP has drawn considerable interest. An improved phosphoramidite approach to the synthesis of c-di-GMP is reported herein. The synthetic approach is based on the use of a 5’-O-formyl protecting group, which can be timely and chemoselectively cleaved from a key dinucleotide phosphoramidite intermediate to enable a cyclocondensation reaction leading to a fully protected c-di-GMP product in a yield of near 80%. The native c-di-GMP is isolated, after complete deprotection, in an overall yield of 36% based on the commercial ribonucleoside used as starting material.

INTRODUCTION

The multiple functions of c-di-GMP involve regulating highly complex signaling processes; high levels of c-di-GMP are known to promote biofilm formation, whereas low levels promote motility, virulence and persistence in bacterial cells (Chen & Schaap, 2016; Caly, Bellini, Walsh, Dow & Ryan, 2015). Biofilm formation occurs through a multifaceted cascade of events including activation of cellulose biosynthesis, secretion of extracellular polysaccharides, inhibition of flagella- and pili-based motility, biosynthesis of adhesins, and regulated proteolysis; many pathogen-produced biofilms are responsible for human diseases (Clivio, Coantic-Castex & Guillaume, 2013).

Innate and adaptive immune responses in mammalian cells are induced by c-di-GMP (Karaolis et al., 2007) through activation of type I interferon (IFN) production and binding to the transmembrane protein STING (Burdette et al., 2011). STING agonists are not only able to elicit IFN production and IFN-stimulated gene expression in T-cells, they are also competent at activating cell death pathways (Larkin et al., 2017), which can be utilized for cancer immunotherapeutic indications (Tang et al., 2016). The wide-ranging biological functions of c-di-GMP and its potential utility, as a nucleic-acid-based drug, led to the development of innovative approaches to the chemical synthesis of c-di-GMP and its analogues. A number of those synthetic methods have been reported and comprehensively reviewed in the literature (Clivio, Coantic-Castex & Guillaume, 2013). The major challenge in the chemical synthesis of c-di-GMP remains to develop strategies for adequate protection of ribonucleosidic hydroxyl functions; one hydroxyl protecting group must be chemoselectively removed prior to performing cyclocondensation of the c-di-GMP linear precursor. Although the preparation of a 2′-O-propargylated c-di-GMP analog has been reported earlier (Grajkowski, Cieślak, Gapeev, Schindler & Beaucage, 2010; and Grajkowski, Cieślak, Abdul-Sater, Schindler & Beaucage, 2013) from the cyclocondensation of a mixture of 5’-O- or 3’-O-mono-phosphoramidite intermediates, the presence of bis-(3’,5’)-phosphoramidite contaminants might have contributed, during the course of the cyclocondensation reaction, to the relatively low isolated yield (40%) of 2′-O-propargylated c-di-GMP. This limitation is the outcome of a lack of compatible protecting groups for hydroxyl, exocyclic amino and phosphate functions to enable an efficient phosphoramidite-driven cyclization reaction. An improved chemical synthesis of c-di-GMP from phosphoramidite intermediates is delineated here. Specifically, Basic Protocol 1 outlines the 5’-O-formylation of a commercial, suitably protected, guanosine ribonucleoside to provide a 5’-O-formylated ribonucleoside intermediate, which upon its exposure to acidic conditions results in the cleavage of its 3’-hydroxyl protecting group. Basic Protocol 2 describes the 5’-O-phosphitylation of the commercial guanosine ribonucleoside to provide a fully protected dinucleoside phosphate triester, as detailed in Basic Protocol 3. The fully protected dinucleoside phosphate triester is 3’-O-deprotected and phosphitylated to give a 3’-O-phosphoramidite derivative of the 5’-O-formyl dinucleoside phosphate triester, as presented in Basic Protocol 4. The chemoselective 5’-O-deprotection of the formyl ester from the dinucleoside phosphate triester 3’-O-phosphoramidite derivative and its cyclocondensation reaction to produce the fully protected c-di-GMP intermediate is delineated in Basic Protocol 5 along with the complete removal of all functional groups protecting the nucleobase, 2’-hydroxyls and phosphates. With the intent of providing the novice experimenters with comprehensive information on the work recently published in the literature (Grajkowski, Takahashi, Kaczyński, Srivastava & Beaucage, 2019), figures and textual materials have been taken from this article and adapted into step-by-step procedures to facilitate reproduction of the original work; this resulted in unavoidable but necessary circumstantial similarities.

BASIC PROTOCOL 1

SYNTHESIS OF THE RIBONUCLEOSIDE 3

The preparation of ribonucleoside 3 is achieved through the 5’-O-formylation of commercial N²-isobutyryl-3’-O-(4,4’-dimethoxytrityl)-2’-O-(tert-butyldimethylsilyl)guanosine (1) upon reaction with cyanomethyl formate in the presence of imidazole (Deutsch, Duczek, & Niclas, 1996) to provide the 5’-formyl ester intermediate 2. Exposure of 2 to acidic conditions produces the 3’-hydroxylated ribonucleoside 3 (Fig. 1)

Figure 1.

Preparation of N²-isobutyryl-5’-O-formyl-2’-O-(tert-butyldimethylsilyl)guanosine (3). DMTr, 4,4’dimethoxytrityl; TBDMS, tert-butyldimethylsilyl; Gua^ibu, N²-isobutyrylguanine; TFA, trifluoroacetic acid.

Materials

N²-Isobutyryl-3’-O-(4,4’-dimethoxytrityl)-2’-O-(tert-butyldimethylsilyl)guanosine (1, ChemGenes Corporation)

Imidazole (MilliporeSigma)

(Formyloxy)acetonitrile (cyanomethyl formate, MilliporeSigma)

Calcium sulfate with indicator, 8 mesh (Drierite, MilliporeSigma)

Chloroform (CHCl₃, Fisher Scientific)

Dichloromethane (CH₂Cl₂, Fisher Scientific)

Trifluoroacetic acid (TFA, MilliporeSigma)

Anhydrous acetonitrile (CH₃CN, Acros)

Acetonitrile (Fisher Scientific)

Silica gel (60-Å, 230 to 400 mesh, MilliporeSigma)

Water bath (Fisher Scientific)

Magnetic stirrer hot plate and stir bars (Thermo Fisher Scientific)

Rubber septa for 14/20 glass joints (MilliporeSigma)

Liebig glass condenser (Kimble)

Drying tube (Thomas Scientific) 10-mL glass pipette (Kimble) & Pipettor (Drummond)

Pipettor (Corning) & disposable plastic 1000-µL pipette tips (Fisher Scientific)

Pasteur pipettes & 2-mL Pasteur pipette bulbs (Fisher Scientific)

100- and 250-mL round-bottom flasks (Kontes) 50-mL graduated glass cylinder (Kimble)

2.5 × 20-cm disposable Flex chromatography columns (Kontes)

13 × 100 mm borosilicate glass tubes (Fisher Scientific)

2.5 × 7.5-cm EMD TLC plates pre-coated with a 250-μm layer of silica gel 60 F₂₅₄ (MilliporeSigma)

NMR spectrometer (Bruker) & 5-mm NMR tubes with caps (Wilmad)

Fraction collector (Gilson)

Rotary evaporator equipped with a dry ice condenser and connected to a diaphragm vacuum pump (~20 mmHg, Büchi) or a high-vacuum oil pump (~0.01 mmHg, Edwards)

Hand-held UV₂₅₄ lamp (UVP)

Additional reagents and equipment for column chromatography (Meyers, 2001, APPENDIX 3E) and TLC (Meyers and Meyers, 2008, APPENDIX 3D)

Prepare the ribonucleoside 3

• 1Place N²-isobutyryl-3’-O-(4,4’-dimethoxytrityl)-2’-O-(tert-butyldimethylsilyl)guanosine (1, 2.31 g, 3.00 mmol), imidazole (0.27 g, 4.0 mmol), cyanomethyl formate (0.34 g, 4.0 mmol) and a magnetic stir bar in a flame-dried 100-mL round-bottom flask. Seal the flask with a Liebig glass condenser and a drying tube filled with 8-mesh Drierite.
• 2Remove the drying tube. Add anhydrous CH₃CN (10 mL) through the condenser using a 10-mL glass pipette and a pipettor. Cap the glass condenser with the drying tube. Stir with a magnetic stirrer hot plate until a solution is obtained.
• 3Place the flask in a water bath pre-heated to ~55 °C on a magnetic stirrer hot plate. Stir the solution over a period of 20 hr. Remove the drying tube and Liebig condenser. Rotoevaporate the solution to a syrup under reduced pressure (~20 mmHg).
• 4
  Add a solution of 3% TFA in CH₂Cl₂ (30 mL) using a 50-mL graduated glass cylinder to cleave the 3′-O-DMTr protecting group.

  Progress of the cleavage reaction is monitored by TLC (Meyers and Meyers, 2008, APPENDIX 3D) on a 2.5 × 7.5-cm EMD silica gel 60 F₂₅₄ TLC plate using 1:1 (v/v) CHCl₃/CH₃CN as the eluent. Cleavage of the 4,4′-dimethoxytrityl ether is complete after a reaction time of 1 hr at ∼25 °C, as visualized on the TLC plate placed under a hand-held UV₂₅₄ lamp.

Purify and isolate the ribonucleoside 3

• 5Rotoevaporate the orange solution to dryness under reduced pressure (~20 mmHg). Dissolve the crude material by adding 3 mL of 4:1 (v/v) CHCl₃/CH₃CN using a pipettor and a 1000-µL plastic tip. Use a Pasteur pipette to evenly spread the solution on the top of a 2.5 × 20 cm chromatography column (Meyers, 2001, APPENDIX 3E) packed with silica gel (~40 g) pre-equilibrated in 4:1 (v/v) CHCl₃/CH₃CN.
• 6Elute the product off the column using a gradient of 4:1 (v/v) CHCl₃/CH₃CN → 3:1 (v/v) CHCl₃/CH₃CN. Collect 7 mL fractions in 13 × 100 mm borosilicate glass tubes using a fraction collector.
• 7
  Analyze the fractions by TLC (Meyers and Meyers, 2008, APPENDIX 3D) on a 2.5 × 7.5-cm EMD silica gel 60 F₂₅₄ TLC plate using 1:1 (v/v) CHCl₃/CH₃CN as the eluent. Fractions containing the pure ribonucleoside 3 are pooled together in a 250-mL round bottom flask and rotoevaporated under reduced pressure (~20 mm Hg) to give a white solid (1.18 g, 2.38 mmol) in a yield of 78% relative to the molar amount of ribonucleoside 1 used as the starting material.

  TLC spots are visualized using a hand-held UV₂₅₄ lamp.

  N²-Isobutyryl-5’-O-formyl-2’-O-(tert-butyldimethylsilyl)guanosine (3): R_f [1:1 (v/v) CHCl₃/CH₃CN] = 0.30. ¹H NMR (300 MHz, DMSO-d₆): δ 12.1 (br s, 1H), 11.6 (br s, 1H), 8.30 (s, 1H), 8.25 (s, 1H), 5.88 (d, J = 6.3 Hz, 1H), 5.30 (d, J = 4.8 Hz, 1H), 4.66 (dd, J = 4.7, 6.0 Hz, 1H), 4.43−4.32 (m, 2H), 4.16−4.15 (m, 2H), 2.82−2.73 (m, 1H), 1.14 (s, 3H), 1.12 (s, 3H), 0.75 (s, 9H), 0.00 (s, 3H), −0.13 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆) δ 180.5, 162.4, 155.2, 149.4, 148.6, 138.1, 120.5, 80.8, 82.7, 75.5, 70.8, 63.9, 35.2, 25.9 (3 CH₃), 19.3, 19.2, 18.2, −4.42, −4.91. +ESI-HRMS: Calcd for C₂₁H₃₄N₅O₇Si (M+H)⁺ 496.2228, Found 496.2235.

BASIC PROTOCOL 2

SYNTHESIS OF the RIBONUCLEOSIDE PHOSPHORAMIDITE 4

This protocol provides a procedure for the preparation of ribonucleoside phosphoramidite 4 from commercial N²-isobutyryl-2’-O-(tert-butyldimethylsilyl)-3’-O-(4,4’-dimethoxytrityl)guanosine (1), N,N,N’,N’-tetraisopropyl-O-[2-(N-formyl-N-methyl)aminoethyl]phosphordiamidite (Grajkowski, Wilk, Chmielewski, Phillips & Beaucage, 2001) and 1H-tetrazole (Fig. 2).

Figure 2.

Preparation of the ribonucleoside phosphoramidite 4. DMTr, 4,4’-dimethoxytrityl; TBDMS, tert-butyldimethylsilyl; Gua^ibu, N²-isobutyrylguanine.

Materials

N²-Isobutyryl-3’-O-(4,4’-dimethoxytrityl)-2’-O-(tert-butyldimethylsilyl)guanosine (1, ChemGenes Corporation)

N,N,N’,N’-tetraisopropyl-O-[2-(N-formyl-N-methyl)aminoethyl]phosphordiamidite (see Grajkowski, Wilk, Chmielewski, Phillips & Beaucage, 2001)

0.45 M 1H-tetrazole in CH₃CN (Glen Research)

Anhydrous acetonitrile (CH₃CN, Glen Research)

Anhydrous benzene (MilliporeSigma)

Dichloromethane (CH₂Cl₂, Fisher Scientific)

Phosphorus pentoxide (P₂O₅, MilliporeSigma)

Triethylamine (Et₃N, MilliporeSigma)

Trifluoroacetic acid (TFA, MilliporeSigma)

Silica gel (60-Å, 230 to 400 mesh; MilliporeSigma)

Argon or nitrogen source 10-mL glass pipettes (Kimble) & Pipettor (Drummond)

Pipettor (Corning) & disposable plastic 1000-µL pipette tips (Fisher Scientific)

2.5- and 10-mL glass luer-tipped syringes (Hamilton)

50-,100- and 250-mL round-bottom flasks (Kontes)

13 × 100 mm borosilicate glass tubes (Fisher Scientific)

Polycarbonate dessicator (Thermo Fisher Scientific)

Oil bubbler apparatus (Ace Glass)

Pasteur pipettes & 2-mL Pasteur pipette bulbs (Fisher Scientific)

16-G- and 21-G stainless steel syringe needles (Thomas Scientific)

Dry ice/acetone bath Magnetic stirrer and stir bars (VWR)

Rubber septa for 14/20-glass joints (MilliporeSigma)

Tygon flexible tubing (Fisher Scientific)

Spatula (Fisher Scientific)

2.5 × 20-cm disposable Flex chromatography columns (Kontes)

2.5 × 7.5-cm EMD TLC plates pre-coated with a 250-μm layer of silica gel 60 F₂₅₄ (MilliporeSigma)

Fraction collector (Gilson)

NMR spectrometer (Bruker) & 5-mm NMR tubes with caps (Wilmad)

Rotary evaporator equipped with dry ice condenser and connected to a diaphragm vacuum pump (~20 mmHg, Büchi,) or a high-vacuum oil pump (~0.01 mmHg, Edwards,)

Hand-held UV₂₅₄ lamp (UVP)

Lyophilizer (Cole-Parmer)

Additional reagents and equipment for column chromatography (Meyers, 2001, APPENDIX 3E) and TLC (Meyers and Meyers, 2008, APPENDIX 3D)

Prepare the ribonucleoside phosphoramidite 4

• 1
  Place N²-isobutyryl-3’-O-(4,4’-dimethoxytrityl)-2’-O-(tert-butyldimethylsilyl)guanosine (1, 2.50 g, 3.25 mmol) and a magnetic stir bar in a flame-dried 50-mL round-bottom flask. Seal the flask with a rubber septum.

  The commercial ribonucleoside 1 is meticulously dried overnight, over phosphorus pentoxide in a dessicator connected to a high-vacuum oil pump, prior to use.

• 2
  Add, under a positive pressure of argon, anhydrous CH₃CN (20 mL) through the rubber septum using a 10-mL glass luer-tipped syringe connected to a 21-G stainless steel needle. Add N,N,N’,N’-tetraisopropyl-O-[2-(N-formyl-N-methyl)aminoethyl]phosphordiamidite (1.16 g, 3.50 mmol) using a 2.5-mL glass luer-tipped syringe connected to a 16-G stainless steel needle. Rinse the syringe twice by syringing in and out ~2 mL of solution.

  A positive pressure of argon is created by piercing the rubber septum of the 50-mL round-bottom flask with a 16-G needle tied to one end of a Tygon flexible tube while the other end is connected to an argon source. The system is vented through the rubber septum using a 16-G needle tied to one end of a Tygon flexible tube while the other end is coupled to an oil bubbler apparatus.

  N,N,N’,N’-tetraisopropyl-O-[2-(N-formyl-N-methyl)aminoethyl]-phosphordiamidite is prepared as reported by Grajkowski et al., 2001 and was dried over phosphorus pentoxide in a dessicator connected to a high-vacuum oil pump over a period of 2 hr at ~ 25 °C prior to use.

• 3
  Remove the rubber septum. Rapidly add solid 1H-tetrazole (231 mg, 3.28 mmol) using a spatula at the rate of 77 mg per 20 min while removing and capping back the flask with the rubber septum after each addition. Allow the solution to stir under an argon atmosphere for a total time of 3 hr at ∼25 °C.

  Solid 1H-tetrazole is obtained by rotary evaporation of its commercial solution in CH₃CN under reduced pressure and is further dried over phosphorus pentoxide in a dessicator connected to a high-vacuum oil pump for 2 hr prior to use.

  Caution: Can explode if exposed to shock or heat from friction or fire. Wear appropriate personal protective equipment when handling solid 1H-tetrazole.

• 4Remove the rubber septum. Add triethylamine (1 mL) using a pipettor and a 1000-µL plastic tip; immediately concentrate the reaction mixture to an oil using a rotary evaporator connected to a vacuum pump (~20 mm Hg).

Purify and isolate the ribonucleoside phosphoramidite 4

• 5Dissolve the oily material by pipetting 3 mL of 9:1 (v/v) benzene/Et₃N using a pipettor and a 1000-uL pipette tip. Use a pasteur pipette to evenly spread the solution on the top of a 2.5 × 20 cm chromatography column (Meyers, 2001, APPENDIX 3E) packed with silica gel (~35 g) pre-equilibrated in 9:1 (v/v) benzene/Et₃N.
• 6Elute the product off the column using a gradient of 9:1 (v/v) benzene/Et₃N → 5:4:1 (v/v/v) benzene/CH₂Cl₂/Et₃N. Collect 7 mL fractions in 13 ×100 mm borosilicate glass tubes using a fraction collector.
• 7
  Analyze the fractions by TLC (Meyers and Meyers, 2008, APPENDIX 3D) on a 2.5 × 7.5-cm EMD silica gel 60 F₂₅₄ TLC plate using 9:1 (v/v) dichloromethane/triethylamine as the eluent. Pool the fractions containing the product in a 250-mL round-bottom flask and remove the volatiles by rotary evaporation under reduced pressure (~20 mmHg).

  TLC spots are visualized using a hand-held UV₂₅₄ lamp

• 8
  Dissolve the oily material with 10 mL dry benzene using a 10-mL pipette and a pipettor. Pipet the solution into a 50-mL round-bottom flask. Swirl the flask in a dry-ice-acetone bath (−78 °C) until the solution is frozen. Lyophilize the frozen solution overnight using a commercial lyophilizer. The ribonucleoside phosphoramidite 4 (2.70 g, 2.69 mmol) is isolated as a triethylamine-free white powder in 83% yield.

  5’-O-(N,N-diisopropylamino-O-[2-(N-formyl-N-methyl)aminoethyl]) phosphoramidite derivative of N²-isobutyryl-3’-O-(4,4’-dimethoxytrityl)-2’-O-(tert-butyldimethylsilyl)guanosine 4: R_f [9:1 (v/v) dichloromethane/triethylamine] = 0.32,0.44 [P(III)diastereomers]. ³¹P NMR (121 MHz, CH₃CN): δ 149.5, 148.9, 148.8, 148.3. +ESI-HRMS: Calcd for C₅₁H₇₃N₇O₁₀PSi (M+H)⁺ 1002.4920, Found 1002.4926. The four ³¹P-NMR signals correspond to the P(III) diastereomers, R_p and S_p and the two cis-trans N-methylformamidic rotamers of each diastereomer.

BASIC PROTOCOL 3

SYNTHESIS OF the diribonucleoside phosphate triesters 5 and 6

Activation of the ribonucleoside phosphoramidite 4 with 1H-tetrazole in the presence of ribonucleoside 3 affords, upon treatment with tert-butyl hydroperoxide, the fully protected diribonucleoside phosphate triester 5. Exposure of 5 to acidic conditions yields the diribonucleoside phosphotriester 6 (Fig. 3).

Figure 3.

Preparation of the fully protected guanylyl-(3′,5′)-guanosine phosphate triester 5 and its 3’-hydroxylated derivative 6. Gua^ibu, N²-isobutyrylguanine DMTr, 4,4’dimethoxytrityl; TBDMS, tert-butyldimethylsilyl; t-BuOOH, tert-butyl hydroperoxide.

Materials

N²-Isobutyryl-5’-O-formyl-2’-O-(tert-butyldimethylsilyl)guanosine (3, Basic Protocol 1)

5’-O-(N,N-diisopropylamino-O-[2-(N-formyl-N-methyl)aminoethyl]) phosphoramidite derivative of N²-isobutyryl-3’-O-(4,4’-dimethoxytrityl)-2’-O-(tert-butyldimethylsilyl)guanosine (4, Basic Protocol 2)

0.45 M 1H-tetrazole in CH₃CN (Glen Research)

Acetone (Fisher Scientific)

Anhydrous acetonitrile (CH₃CN, Glen Research)

Acetonitrile (Fisher Scientific)

5.5 M tert-butyl hydroperoxide in decane (MilliporeSigma)

Dichloromethane (CH₂Cl₂, Fisher Scientific)

Phosphorus pentoxide (P₂O₅, MilliporeSigma)

Trifluoroacetic acid (TFA, MilliporeSigma)

Silica gel (60-Å, 230 to 400 mesh; MilliporeSigma)

Argon gas source

Magnetic stirrer and stir bars (VWR)

Rubber septa for 14/20-glass joints (MilliporeSigma)

5- and 10-mL glass pipettes (Kimble) & a pipettor (Drummond)

1- and 10-mL glass luer-tipped syringes (Hamilton)

Spatula (Fisher Scientific)

Polycarbonate dessicator (Thermo Fisher Scientific)

Pasteur pipettes & 2-mL Pasteur pipette bulbs (Fisher Scientific)

100- and 250-mL round-bottom flasks (Kontes)

50-mL graduated glass cylinder (Kimble)

21-G stainless steel syringe needles (Thomas Scientific)

2.5 × 20-cm disposable Flex chromatography columns (Kontes)

13 × 100 mm borosilicate glass tubes (Fisher Scientific)

2.5 × 7.5-cm EMD TLC plates pre-coated with a 250-μm layer of silica gel 60 F₂₅₄ (MilliporeSigma)

NMR spectrometer (Bruker) & 5-mm NMR tubes with caps (Wilmad)

Fraction collector (Gilson)

Rotary evaporator equipped with dry ice condenser and connected to a diaphragm vacuum pump (~20 mmHg, Büchi) or a high-vacuum oil pump (0.01 mmHg, Edwards)

Hand-held UV₂₅₄ lamp (UVP)

Additional reagents and equipment for column chromatography (Meyers, 2001, APPENDIX 3E) and TLC (Meyers and Meyers, 2008, APPENDIX 3D)

Prepare the fully protected guanylyl-(3′,5′)-guanosine phosphate triester 5 and its 3’-hydroxylated derivative 6

• 1
  Place in a flame-dried 100-mL round bottom flask, under a positive pressure of argon, the dry ribonucleoside phosphoramidite 4 (2.51 g, 2.50 mmol), ribonucleoside 3 (1.01 g, 2.02 mmol) and a stir bar. Seal the flask with a rubber septum.

  The ribonucleoside 3 and the ribonucleoside phosphoramidite 4 are thoroughly dried over phosphorus pentoxide overnight in a dessicator connected to a high-vacuum oil pump prior to use.

  The positive pressure of Argon is created as described in step 2 of Basic Protocol 2.

• 2Add 20 mL anhydrous acetonitrile under argon through the rubber septum using a 10-mL glass luer-tipped syringe connected to a 21-G stainless steel needle. Stir to a solution using a magnetic stirrer.
• 3
  Remove the rubber septum and rapidly add solid 1H-tetrazole (700 mg, 10.0 mmol) using a spatula. Seal the flask with the rubber septum. Allow the solution to stir under a positive pressure of argon for 5 hr at ∼25 °C.

  See the annotation to step 3 of Basic Protocol 2 when handling solid 1H-tetrazole.

• 4Add through the rubber septum 750 μL of 5.5 M tert-butyl hydroperoxide in decane using a 1-mL glass luer-tipped syringe equipped with a 21-G stainless steel needle. Allow the reaction mixture to stir for 30 min at ∼25°C. Remove the rubber septum and rotoevaporate the volatiles under reduced pressure (~20 mm Hg) to obtain the gummy reaction product 5.
• 5Dissolve the crude product 5 by adding 30 mL 3% TFA in CH₂Cl₂ using a 50-mL glass cylinder. Cap the flask with a rubber septum. Stir the orange solution at ∼25 °C for 30 min. Remove the rubber septum and rotoevaporate the solution to dryness under reduced pressure (~20 mm Hg).

Purify and isolate the 3’-hydroxylated guanylyl-(3′,5′)-guanosine phosphate triester 6

• 6Dissolve the crude product 6 by adding 5 mL CH₃CN using a 5-mL glass pipette and a pipettor. Use a Pasteur pipette to evenly spread the solution on the top of a 2.5 × 20 cm chromatography column (Meyers, 2001, APPENDIX 3E) packed with silica gel (~40 g) pre-equilibrated in CH₃CN.
• 7Elute the product off the column using a gradient of acetone (0→33%) in CH₃CN. Collect 7 mL fractions in 13 × 100 mm borosilicate glass tubes using a fraction collector.
• 8
  Analyze the fractions by TLC (Meyers and Meyers, 2008, APPENDIX 3D) on a 2.5 × 7.5-cm EMD silica gel 60 F₂₅₄ TLC plate using 2:1 (v/v) acetonitrile/acetone as the eluent. Pool the fractions containing the product in a 250-mL round-bottom flask and remove the volatiles by rotary evaporation under low pressure (~20 mm Hg) to provide the 3’-hydroxylated diribonucleoside phosphate triester 6 (1.74 g, 1.57 mmol) as a white solid in a yield of 78% relative to the molar amount of ribonucleoside phosphoramidite 4 used as the starting material.

  TLC spots are visualized using a hand-held UV₂₅₄ lamp. The 3’-hydroxylated guanylyl-(3′,5′)-guanosine phosphate triester 6: R_f [2:1 (v/v) acetonitrile/acetone] = 0.25. Proton-decoupled ³¹P NMR (121 MHz, CH₃CN): δ −1.83, −1.90, −1.94, −1.99. +ESI-HRMS: Calcd for C₄₅H₇₃N₁₁O₁₂PSi₂ (M+H)⁺ 1110.4507, Found 1110.4518.

  The four ³¹P-NMR signals correspond to the P(V) R_p and S_p diastereomers and the two cis-trans N-methylformamidic rotamers of each diastereomer.

BASIC PROTOCOL 4

SYNTHESIS OF the pre-cyclocondensation phosphoramidite 8

The 3’-hydroxylated guanylyl-(3′,5′)-guanosine phosphate triester 6 is reacted with N,N,N’,N’-tetraisopropyl-O-[2-(N-formyl-N-methyl)aminoethyl]phosphordiamidite in the presence of 1H-tetrazole to provide the fully protected phosphoramidite derivative 7, which after chemoselective cleavage of the 5’-O-formyl ester gives the pre-cyclocondensation phosphoramidite 8 (Fig. 4).

Figure 4.

Preparation of the 3’-O-phosphoramidite derivative of fully protected guanylyl-(3′,5′)-guanosine phosphate triester 7 and of its 5’-hydroxylated derivative 8. Gua^ibu, N²-isobutyrylguanine; TBDMS, tert-butyldimethylsilyl; DBU, 1,8-Diazabicyclo[5.4.0]undec-7-ene.

Materials

The 3’-hydroxylated guanylyl-(3′,5′)-guanosine phosphate triester 6 (see Basic protocol 3)

N,N,N’,N’-tetraisopropyl-O-[2-(N-formyl-N-methyl)aminoethyl]phosphordiamidite (see Grajkowski, Wilk, Chmielewski, Phillips & Beaucage, 2001)

0.45 M 1H-tetrazole in CH₃CN (Glen Research)

Acetone (Fisher Scientific)

Anhydrous acetonitrile (CH₃CN, Glen Research)

1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU, MilliporeSigma)

Dichloromethane (CH₂Cl₂, Fisher Scientific)

Phosphorus pentoxide (P₂O₅, MilliporeSigma)

Trifluoroacetic acid (TFA, MilliporeSigma)

Triethylamine (Et₃N, MilliporeSigma)

Silica gel (60-Å, 230 to 400 mesh; MilliporeSigma)

Magnetic stirrer and stir bars (VWR)

Spatula (Fisher Scientific)

Rubber septa for 14/20-glass joints (MilliporeSigma)

5-mL glass pipettes (Kimble) & a pipettor (Drummond)

1-and 10-mL glass luer-tipped syringes (Hamilton)

Pasteur pipettes & 2-mL Pasteur pipette bulbs (Fisher Scientific)

50- and 250-mL round-bottom flasks (Kontes)

16- and 21-G stainless steel needles (Thomas Scientific)

2.5 × 20-cm disposable Flex chromatography columns (Kontes)

13 × 100 mm borosilicate glass tubes (Fisher Scientific)

2.5 × 7.5-cm EMD TLC plates pre-coated with a 250-μm layer of silica gel 60 F₂₅₄ (MilliporeSigma)

Argon gas source Polycarbonate dessicator (Fisher Scientific)

NMR spectrometer (Bruker) & 5-mm NMR tubes with caps (Wilmad)

Fraction collector (Gilson)

Rotary evaporator equipped with dry ice condenser and connected to a diaphragm vacuum pump (~20 mmHg, Büchi) or a high-vacuum oil pump (~0.01 mmHg, Edwards)

Hand-held UV₂₅₄ lamp (UVP)

Additional reagents and equipment for column chromatography (Meyers, 2001, APPENDIX 3E) and TLC (Meyers and Meyers, 2008, APPENDIX 3D)

Prepare the 3’-O-phosphoramidite derivative of the 3’-hydroxylated guanylyl-(3′,5′)-guanosine phosphate triester 6 (7)

• 1
  Place under a positive pressure of argon, dry N,N,N’,N’-tetraisopropyl-O-[2-(N-formyl-N-methyl)aminoethyl]phosphordiamidite (503 mg, 1.50 mmol) using a 1-mL glass luer-tipped syringe connected to a 16-G stainless steel needle, dry 3’-hydroxylated guanylyl-(3′,5′)-guanosine phosphate triester 6 (1.54 g, 1.35 mmol) and a stir bar in a flame-dried 50-mL round-bottom flask. Seal the flask with a rubber septum.

  N,N,N’,N’-tetraisopropyl-O-[2-(N-formyl-N-methyl)aminoethyl]phosphordiamidite and the diribonucleoside phosphate triester 6 have been separately dried overnight under phosphorus pentoxide in a dessicator connected to a high-vacuum oil pump.

  The positive pressure of Argon is created as described in step 2 of Basic Protocol 2.

• 2
  Add anhydrous acetonitrile (12 mL), under argon, through the rubber septum using a 10-mL glass luer-tipped syringe equipped with a 21-G stainless steel needle. Remove the septum and add solid 1H-tetrazole (105 mg 1.50 mmol) using a spatula. Rapidly seal the flask with the rubber septum. Magnetically stir the solution under argon for 12 hr at ~ 25 °C to provide the fully protected diribonucleoside phosphoramidite 7.

  Solid 1H-tetrazole is added in three portions at the rate of 0.5 mmol (35 mg)/20 min. The rubber septum is quickly removed and replaced before and after each addition. The fully protected diribonucleoside phosphoramidite 7 is used without isolation in the next step.

  See annotation to step 3 of Basic Protocol 2, when handling solid 1H-tetrazole

Prepare the 5’-hydroxylated derivative of the fully protected diribonucleoside phosphoramidite 7 (8)

• 3
  Add DBU (930 µL) to the reaction mixture of step 2, through the rubber septum, using a 1-mL glass luer-tipped syringe equipped with a 21-G stainless steel needle. Stir the reaction mixture under argon for 1 hr at ~25 °C. Remove the rubber septum and rotoevaporate the reaction mixture to a syrup under reduced pressure (~20 mm Hg).

  The addition of DBU leads to the chemoselective cleavage of the 5’-O-formyl ester from the fully protected diribonucleoside phosphoramidite 7 to give the 5’-hydroxylated diribonucleoside phosphoramidite 8.

Purify and isolate the 5’-hydroxylated diribonucloside phosphoramidite 8

• 4Dissolve the material of step 3 with 5 mL of 9:1 (v/v) CH₂Cl₂/Et₃N using a 5-mL glass pipette and a pipettor. Use a Pasteur pipette to evenly spread the solution on the top of a 2.5 × 20 cm chromatography column (Meyers, 2001, APPENDIX 3E) packed with silica gel (~30 g) pre-equilibrated in 9:1 (v/v) CH₂Cl₂/Et₃N.
• 5Elute the product off the column using 4:5:1, (v/v/v) CH₂Cl₂:acetone:Et₃N as the eluent. Collect 7 mL fractions in 13 × 100 mm borosilicate glass tubes using a fraction collector.
• 6
  Analyze the fractions by TLC (Meyers and Meyers, 2008, APPENDIX 3D) on a 2.5 × 7.5-cm EMD silica gel 60 F₂₅₄ TLC plate using 4:5:1, (v/v/v) CH₂Cl₂/acetone/Et₃N as the eluent. Pool the fractions containing the product in a 250-mL round-bottom flask and remove the volatiles by rotary evaporation under low pressure (~20 mm Hg) to afford the 5’-hydroxylated diribonucleoside phosphoramidite 8 (1.25g, 0.950 mmol) as a white amorphous powder in a yield of 70%, based on the amount of 6 used as the starting material.

  The 5’-hydroxylated diribonucleoside phosphoramidite 8: R_f [4:5:1, (v/v/v) CH₂Cl₂:acetone:Et₃N] = 0.31, 0.39 (diastereomeric mixture). ³¹P NMR (121 MHz, CH₃CN): δ 150.8−147.7 ppm corresponding to the P(III) portion of 8 and −1.31 to −2.27 corresponding to the P(V) portion of 8. +ESI-HRMS: Calcd for C₅₄H₉₄N₁₃O₁₇P₂Si₂ (M+H)⁺ 1314.5899, Found 1314.5913.

  The expected number of ³¹P NMR signals for 8 is based on the following pairs of diastereomers: R_p(V)-R_p(III); R_p(V)-S_p(III); S_p(V)-R_p(III); S_p(V)-S_p(III).There are two cis-trans N-methylformamidic rotamers for each diastereomer accounting for a theoretical number of 16 ³¹P NMR signals.

BASIC PROTOCOL 5

SYNTHESIS OF the native 3’,5’-cyclic diguanylic acid 10

Activation of the 5’-hydroxylated diribonucleoside phosphoramidite 8 by 1H-tetrazole has efficiently led, after P(III) oxidation, to the formation of a fully protected 3’,5’-diguanylic acid phosphate triester intermediate (9, Fig. 5). The deprotection of 9 involves a treatment with an acetonitrile solution of ethylenediamine at ∼25 °C to remove the guanine protecting groups; this is followed by exposure of the partially deprotected product to a solution of 40% acetonitrile in 0.1 M triethylammonium acetate buffer (pH 7.0) to cleanly cleave the thermolytic phosphate protecting groups at elevated temperature. The final deprotection step consists of a treatment with neat triethylamine trihydrofluoride to remove the 2’-O-(tert-butyldimethylsilyl) protecting groups leading to the production of the native 3’,5’-cyclic diguanylic acid (10, Fig 5).

Figure 5.

Preparation of the (3,5’)-cyclic diguanylic acid 10 from the linear 5’-hydroxylated diribonucleoside phosphoramidite 8 and its precursor 9. t-BuOOH, tert-butyl hydroperoxide; EDA, ethylenediamine; TEAA, triethylammonium acetate (pH 7.0); TEA.3HF, triethylamine trihydrofluoride; TBDMS, tert-butyldimethylsilyl; Gua^ibu, N²-isobutyryl guanine; Gua, guanin-9-yl.

Materials

5’-Hydroxylated diribonucleoside phosphoramidite 8 (see Basic Protocol 4)

0.45 M 1H-tetrazole in CH₃CN (Glen Research)

Anhydrous acetonitrile (CH₃CN, Glen Research)

Phosphorus pentoxide (P₂O₅, MilliporeSigma)

Ethylenediamine (MilliporeSigma)

2.0 M Triethylamine Acetate solution (Thermo Fisher Scientific)

5.5 M tert-butyl hydroperoxide (MilliporeSigma)

Magnetic stirrer and stir bars (VWR)

Spatula (Fisher Scientific)

Rubber septa for 14/20-glass joints (MilliporeSigma)

1- and 5-mL glass luer-tipped syringes (Hamilton)

13 × 100 mm borosilicate glass tubes (Fisher Sientific)

Gas-tight 100 µL-glass luer-tipped syringe (Hamilton)

Pasteur pipettes & 2-mL Pasteur pipette bulbs (Fisher Scientific)

Pipettor (Corning) & disposable plastic 1000-µL pipette tips (Fisher Scientific)

15-mL polypropylene centrifuge tubes (Corning)

25-mL round-bottom flasks (Kontes)

21-G stainless steel needles (Thomas Scientific)

4-mL screw-capped glass vials with caps (Fisher Scientific)

Polycarbonate dessicator (Fisher Scientific)

Heat block (VWR)

NMR spectrometer (Bruker) & 5-mm NMR tubes with caps (Wilmad)

SpeedVac concentrator (Thermo Fisher Scientific)

Rotary evaporator equipped with dry ice condenser and connected to a diaphragm vacuum pump (~20 mmHg, Büchi) or a high-vacuum oil pump (~0.01 mmHg, Edwards)

HPLC system equipped with a diode array Vis/UV detector (Agilent technologies)

25 cm × 10 mm 5 μm Supelcosil LC-18S RP-HPLC column (Supelco)

Prepare the fully protected c-di-GMP 9

• 1
  Place under a positive pressure of argon dry 5’-hydroxylated diribonucleoside phosphoramidite 8 (128 mg, 97.3 µmol) and a stir bar in a flame-dried 25-mLround bottom flask. Seal the flask with a rubber septum.

  The solid phosphoramidite 8 has been dried overnight over phosphorus pentoxide in a dessicator connected to a high-vacuum oil pump.

  The positive pressure of Argon is created as described in step 2 of Basic Protocol 2.

• 2
  Add, under argon, anhydrous acetonitrile (2 mL) through the rubber septum using a 5-mL glass luer-tipped syringe connected to a 21-G stainless steel needle. Magnetically stir to a solution. Remove the rubber septum and quickly add solid 1H-tetrazole (35 mg, 0.50 mmol) in one portion using a spatula. Seal the flask with the rubber septum and allow the solution to stir under argon for 2 hr at ~25 °C.

  See the annotation to step 3 of Basic Protocol 2 when handling solid 1H-tetrazole

• 3Add through the rubber septum 55-µL 5.5 M tert-butyl hydroperoxide in decane using a gas-tight 100 µL-glass luer-tipped syringe coupled to a 21-G stainless steel needle. Stir the solution at ~25 °C for 30 min. Rotoevaporate to dryness under reduced pressure (~20 mm Hg) to produce the fully protected c-di-GMP phosphate triester 9.

Deprotect the c-di-GMP phosphotriester 9

• 4Add 0.5 mL of 1:4 (v/v) ethylenediamine/acetonitrile to the material of step 3 using a pipettor and a 1000-µL pipette tip. Transfer the solution to a 4-mL screw-capped glass vial using the pipettor and a 1000-µL pipette tip. Cap the glass vial and allow the solution to stand for 20 hr at ~25 °C. Uncap the vial and evaporate the solution using a SpeedVac concentrator.
• 5Dissolve the material in a 0.5 mL 40% CH₃CN in 0.1 M triethylammonium acetate buffer (pH 7.0) using a pipettor and a 1000-µL pipette tip. Cap the glass vial and place it in a heat block pre-heated at 90 °C. Allow the glass vial to stand at 90 °C for 3 hr. Allow the vial to cool to ~25 °C; remove the cap and evaporate the solution using a SpeedVac concentrator.
• 6Add 0.5mL neat triethylamine trihydrofluoride using a pipettor and a 1000-µL pipette tip to the glass vial and a stir bar. Cap the vial and allow the solution to magnetically stir for 20 h at ∼25 °C.

Purify and isolate the native c-di-GMP 10.

• 7Purify the fully deprotected product by RP-HPLC using a semi-preparative 5-μm Supelcosil LC-18S column (25 cm × 10 mm). Inject the solution using the instrument’s auto-injector. Start pumping 0.1 M triethylammonium acetate (pH 7.0) and a linear gradient of 2.5% CH₃CN/min at a flow rate of 3 mL/min for 40 min. Collect the fractions eluting at a retention time of 9.5 min in 15-mL polypropylene centrifuge tubes.

1. Evaporate the fractions using a SpeedVac concentrator to yield the 3’,5’-cyclic diguanylic acid 10 (52.4 mg, 75.9 µmol) in a yield of 78% based on the molar amount of 5’-hydroxylated diribonucleoside phosphoramidite 8 used as the starting material.

3’,5’-cyclic diguanylic acid 10: ¹H NMR (300 MHz, DMSO-d₆): δ 10.6 (s, 2H), 8.01 (br s, 2H), 7.95 (s, 2H), 6.57 (br s, 4H), 5.76 (d, J = 8.1 Hz, 2H), 4.65−4.57 (m, 4H), 4.18−4.13 (dd, J = 4.6, 10.7 Hz, 2H), 4.03−3.95 (m, 2H), 3.85−3.71 (m, 2H). ³¹P NMR (121 MHz, DMSO-d₆): δ 0.25. -ESI-HRMS: Calcd for C₂₀H₂₂N₁₀O₁₅P₂ (M-H)^-, 689.0865, Found 689.0888.

RP-HPLC analysis of 10 has been performed using a 5 µm Supelcosil LC-18S column (25 cm × 10 mm) according to the following conditions: starting from 0.1 M triethylammonium acetate pH 7.0, a linear gradient of 2.5% CH₃CN/min is pumped at a flow rate of 3 mL/min for 40 min. Under these chromatographic conditions the retention time (t_R) of 10 is 9.5 min.

REAGENTS AND SOLUTIONS

Use deionized distilled water for all recipes.

0.1 M Triethylammonium acetate buffer, pH 7.0

In a 1-L volumetric flask (Chemglass) add 50-mL 2.0 M Triethylamine Acetate solution (pH 7.0, Thermo Fisher Scientific) followed by deionized distilled water up to the calibration mark of the flask. Filter the solution through a 0.45 micron nylon filter membrane (MilliporeSigma) using a vacuum filtration assembly (MilliporeSigma) prior to use for HPLC purification. Prepare weekly or as needed.

40% CH₃CN in 0.1 M triethylammonium acetate buffer (pH 7.0)

In a 2-L Erlenmeyer, add 600 mL 0.1 M triethylammonium acetate buffer (pH 7.0) and 400 mL acetonitrile. Manually stir the solution and filter it through a 0.45 micron nylon filter membrane using a vacuum filtration assembly (MilliporeSigma) prior to use for HPLC purification. Prepare weekly or as needed.

COMMENTARY

Background Information

The major hurdle to overcome prior to initiating the chemical synthesis of c-di-GMP is to define a strategy whereby a suitable ribonucleoside hydroxyl protecting groups can be chemoselectively cleaved prior to performing the final cyclization of the linear c-di-GMP precursor product.

Chemical syntheses of c-di-GMP have been reported, employing phosphotriester-based cyclocondensation reactions (Ross et al., 1987; Ross et al., 1990). However, the efficiency of those syntheses, in terms of the yields of native c-di-GMP being produced, remains unknown. More than 10 years later, the synthesis of c-di-GMP using a combination of phosphoramidite/phosphotriester intermediates and a phosphotriester-mediated cyclization reaction had been reported to provide a fully protected c-di-GMP product in an isolated yield of 75% (Hayakawa, Nagata, Hirata, Hyodo & Kawai 2003); complete deprotection the cyclic dinucleotide phosphotriester afforded the native c-di-GMP product in a yield of 77% or 58% based on its immediate cyclic precursor. Ching, Tan, Chua & Lam (2010) reported a similar method for the preparation of a fully protected c-di-GMP via a phosphotriester-driven cyclization reaction; the fully protected c-di-GMP product was isolated in a yield of 40%. However, the isolated yield of the native c-di-GMP was not better than 84% or 35% based on the fully protected c di-GMP precursor.

Multiple syntheses of native c-di-GMP and c-di-GMP analogues have been described by Zheng & Jones (1996); Gaffney, Veliath, Zhao & Jones (2010) and Gaffney & Jone (2012); a combination of phosphoramidite/H-phosphonate intermediates and H-phosphonate-assisted cyclization reactions have been employed for this purpose. Along similar lines, a H-phosphonate-mediated cyclocondensation of H-phosphonate intermediates has been carried out by Yan & Aguilar (2007) and Yan et al., (2009) for the preparation of native c-di-GMP with the purpose of disclosing its adjuvant properties. The synthesis of a 2’-O-propargylated c-di-GMP analogue has been performed using a mixture of 5’- or 3’-diribonucleoside phosphoramidites (Grajkowski, Cieślak, Gapeev, Schindler & Beaucage, 2010 and Grajkowski, Cieślak, Abdul-Sater, Schindler & Beaucage, 2013). The 2’-O-propargylated c-di-GMP analogue has been isolated in a relatively low yield of 40%; this was largely caused by the lack of orthogonal hydroxyl protecting groups that can be removed, when needed, without affecting the stability of other hydroxyl, amine and phosphate protecting groups in a phosphoramidite-mediated cyclization reaction. Such a limitation has prompted the development of an improved chemical synthesis of c-di-GMP from phosphoramidite intermediates (Grajkowski, Takahashi, Kaczyński, Srivastava & Beaucage, 2019). This improved synthesis is based on the unprecedented 5’-O-formylation of a commercial guanosine ribonucleoside (Srivastava, Pandey, Srivastava & Bajpai, 2008; Srivastava, Pandey, Srivastava & Bajpai, 2011), which had its 2’- and 3’-hydroxyl functions protected with a tert-butyldimethylsilyl and a 4,4’-dimethoxytrityl group, respectively; the exocyclic amine of the guanine moiety had been protected with a traditional isobutyryl group. The 5’-O-formylation of ribonucleoside 1 has been performed, as reported, by adding cyanomethyl formate to a solution of the ribonucleoside and imidazole at elevated temperature (Deutsch, Duczek & Niclas,1996), to afford the fully protected ribonucleoside intermediate 2 (Fig. 1). Exposure of this intermediate to an acidic solution led to complete cleavage of the 3’-O-(4,4’-dimethoxytrityl) ether function to provide, after purification, the 5’-O-formyl ribonucleoside 3 in a yield of 78% based on the molar amount of 1 used as the starting material. The characterization of 3 has been made based on ¹H- and ¹³C-NMR data and accurate mass determination by high-resolution mass spectrometry. Given that the synthesis of the fully protected guanosine 5’-O-[2-(N-formyl-N-methyl)]-N,N-diisopropylphosphoramidite 4 is required for the assembly of a key intermediate in the preparation of c-di-GMP, the monomeric phosphoramidite has been prepared by phosphitylation of the commercial guanosine ribonucleoside 1 using N,N,N’,N’-tetraisopropyl-O-[2-(N-formyl-N-methyl)aminoethyl]phosphordiamidite in the presence of 1H-tetrazole (Basic Protocol 2, Fig.2). The ribonucleoside phosphoramidite 4 has been isolated as a solid in a yield of 83%, after silica gel purification and lyophilization. Characterization of the product by ¹H-NMR spectroscopy has been challenging given its P(III) chirality and associated R_p and S_p diastereomers. Moreover, each P(III) diastereomer exists as two cis-trans amide bond rotamers (Laursen, Engel-Andreasen, Fristrup, Harris & Olsen, 2013) leading to a complex ¹H-NMR spectrum showing essentially overlapping multiplets, which are of limited usefulness. With the intent of mitigating this quandary, images of the NMR spectra are provided below (Figs. 6 and 7) to better support reproducibility of the work by visual comparison of the NMR signals.

Figure 6.

300 MHz ¹H NMR spectrum of the fully protected guanosine 5’-O-[2-(N-formyl-N-methyl)]-N,N-diisopropylphosphoramidite 4 in DMSO-d₆. DMTr, 4,4’-dimethoxytrityl; TBDMS, tert-butyldimethylsilyl; Gua^ibu, N²-isobutyrylguanine.

Figure 7.

Limited expansion of the 300 MHz ¹H NMR spectrum of 4. DMTr, 4,4’-dimethoxytrityl; TBDMS, tert-butyldimethylsilyl; Gua^ibu, N²-isobutyrylguanine.

The ribonucleoside phosphoramidite 4 has also been characterized by proton-decoupled ³¹P-NMR spectroscopy and accurate mass determination; the characterization data are annotated in step 8 of Basic Protocol 2. The 1H-tetrazole-mediated coupling of the ribonucleoside phosphoramidite 4 with ribonucleoside 3 led to the fully protected diribonucleoside phosphate triester 5 upon P(III) oxidation by tert-butyl hydroperoxide. The fully protected diribonucleoside phosphate triester 5 had then been exposed to acidic conditions to cleave the 3’-hydroxyl protecting group affording the 3’-hydroxylated guanylyl-(3′,5′)-guanosine phosphate triester 6 (Basic Protocol 3, Fig. 3) in a post-purification yield of 80% based on the molar amount of 4 used as the starting material. The product has been characterized by ¹H NMR spectroscopy. Images of the spectra (Figs 8 and 9) are shown below to support reproducibility of the work through visual comparability of the NMR signals, the complexity of which arose from the P(V) diastereomers and their cis-trans N-methylformamidic rotamers.

Figure 8.

300 MHz ¹H NMR spectrum of the diribonucleoside phosphotriester 6 in DMSO-d₆. TBDMS, tert-butyldimethylsilyl; Gua^ibu, N²-isobutyrylguanine.

Figure 9.

Limited expansion of the 300 MHz ¹H NMR spectrum of 6.

Proton-decoupled ³¹P-NMR spectroscopy and accurate mass determination characterization data for the product 6 are annotated in step 8 of Basic Protocol 3. The 3’-hydroxylated guanylyl-(3′,5′)-guanosine phosphate triester 6 has then been phosphitylated, under conditions like those employed for the phosphitylation of commercial guanosine ribonucleoside 1, to produce the fully protected phosphoramidite derivative 7. Chemoselective DBU-mediated cleavage of the 5’-O-formate ester from 7 gave the pre-cyclocondensation diribonucleoside phosphoramidite 8 in a post-purification yield of 70%, based on the molar amount of starting material 6 (Basic Protocol 4, Fig. 4). The product 8 has been characterized by both ¹H- and proton-decoupled ³¹P-NMR spectroscopies and by accurate mass spectrometry. Images of the ¹H- and ³¹P-NMR spectra are provided below (Figs. 10 through 14) to serve as references for visual comparability of the NMR signals, should reproducibility of the work be evaluated.

Figure 10.

300 MHz ¹H NMR spectrum of the pre-cyclocondensation diribonucleoside phosphoramidite 8 in DMSO-d₆. TBDMS, tert-butyldimethylsilyl; Gua^ibu, N²-isobutyrylguanine.

The proton-decoupled ³¹P-NMR signals and accurate mass results are annotated in step 6 of Basic Protocol 4. Activation of the 5’-hydroxylated diribonucleoside phosphoramidite 8 by1H-tetrazole resulted in its intramolecular cyclization; oxidation of the phosphite triester intermediate produced the fully protected c-di-GMP product 9 (Basic Protocol 5, Fig. 5), which has been fully deprotected upon treatment with ethylenediamine to remove the isobutyryl groups protecting the exocyclic amines of both guanines. The phosphate protecting groups of the partially deprotected material have then been subjected to thermolytic cleavage upon heating at 90 °C in an aqueous acetonitrile-containing buffer at pH 7.0. Exposure of the nucleobase- and phosphate-deprotected 9 to neat triethylamine trihydrofluoride at ~25 °C, to remove the 2’-O-(tert-butyldimethylsilyl) protecting groups, afforded the c-di-GMP 10 in a yield of 78% relative to the molar amount of phosphoramidite 8 used for the intramolecular cyclization reaction. The native c-di-GMP 10 has been characterized by both ¹H-NMR and proton-decoupled ³¹P-NMR spectroscopies and by accurate mass measurement. The characterization data are provided in an annotation to step 8 of Basic Protocol 5.

The all phosphoramidite approach to the synthesis of c-di-GMP has indeed been significantly improved over our previous phosphoramidite-driven synthesis of a biotinylated c-di-GMP conjugate (Grajkowski, Cieślak, Gapeev, Schindler & Beaucage, 2010 and Grajkowski, Cieślak, Abdul-Sater, Schindler & Beaucage, 2013); the intramolecular cyclization coupling yield of the c-di-GMP linear precursor did not exceed 40%. As reported herein, cyclization of the c-di-GMP linear precursor has now been achieved in an unprecedented yield of ~80%. Such an improvement arose from a number of modifications to the method including implementation of a formic acid ester as a 5’-hydroxyl protecting group for the commercial ribonucleoside 1 (Basic Protocol 1, Fig. 1), the use of DBU for chemoselective removal of the 5’-O-formate ester from the diribonucleoside phosphoramidite 7 (Basic Protocol 4, Fig. 4) and implementation of the 2-(N-formyl-N-methylaminoethyl) group as a thermosensitive P(V) protecting group. This thermolytic group largely contributed to the quantitative deprotection of the c-di-GMP cyclic precursor to provide the highest overall yield of native c-di-GMP.

The conversion of a phosphoramidite function to a H-phosphonate or phosphodiester function, as reported by others (Zheng & Jones, 1996; Gaffney, Veliath, Zhao & Jones, 2010; Gaffney & Jone, 2012), is not needed for the method described in this report; the native c-di-GMP is produced in an overall yield of 36%, when calculated from the molar amount of ribonucleoside 1 used as the starting material. The overall yields of native c-di-GMP produced by either phosphotriester or H-phosphonate-driven cyclocondensation reactions are generally lower. Indeed, a similar c-di-GMP, prepared from a combination of phosphoramidite/H-phosphonate intermediates and a H-phosphonate-mediated cyclocondensation reaction, was isolated in overall yields not exceeding 30% (Gaffney, Veliath, Zhao & Jones, 2010; Gaffney & Jone, 2012). Given that an intramolecular cyclization reaction is generally required for the production of cyclic dinucleotide analogues, one can realistically claim the suitability of the improved phosphoramidite method, outlined here, for the synthesis of numerous c-di-GMP analogues.

Critical Parameters and Troubleshooting

Although the protocols of this article are quite straightforward, one must nonetheless be aware of not using any primary, secondary or tertiary alcohols in aprotic solvent, as an eluent when performing the purification of ribonucleoside 3 (see Basic Protocol 1) and diribonucleoside phosphate triester 6 (see Basic Protocol 3) on silica gel. This action is necessary to prevent the 2’↔3’-isomerization of the tert-butyldimethylsilyl group (Ogilvie & Entwistle, 1981), which can be problematic when it happens.

As previously discussed, precautions must be taken when preparing and working with phosphordiamidites and phosphoramidites. Indeed, the reaction of ribonucleoside 1 or diribonucleoside phosphotriester 6 with N,N,N’,N’-tetraisopropyl-O-[2-(N-formyl-N-methyl)aminoethyl]phosphordiamidite to provide the ribonucleoside phosphoramidite 4 (see Basic Protocol 2) and the diribonucleoside phosphoramidite 8 (see Basic protocol 4) is to be performed under strictly anhydrous conditions. All glassware should be flame-dried and allowed to cool in a desiccator over P₂O₅ prior to use. Each reactant (1, 4, 6 and 8) including solid 1H-tetrazole should be dried under high vacuum overnight at ambient temperature prior to being dissolved in commercial anhydrous MeCN that is stored in an amber glass bottle over activated 4Å (8 to 12 mesh) molecular sieves. Readers are also referred to the Critical Parameters and Troubleshooting sections of UNITS 3.17 & 2.7 [*Copy editor: Please reference the units as journal articles using the Rosetta Stone document.], which address the critical issues associated with the preparation and use of nucleoside phosphoramidites.

Understanding Results

The reaction of commercial N²-isobutyryl-2′-O-tert-butyldimethylsilyl-3′-O-(4,4′-dimethoxytrityl) guanosine (1) with cyanomethyl formate in the presence of imidazole in dry CH₃CN provides the ribonucleoside 2, which has not been isolated. Exposure of 2 to an acidic solution (3% TFA in CH₂Cl₂) gives the ribonucleoside 3 in a post-purification yield of 78%. It is important to mention that the presence of imidazole is absolutely required for this reaction to proceed. As mentioned in the previous section, purification of the 3’-hydroxylated ribonucleoside 3 on silica gel should be performed using an eluent that is free of protic solvents (i.e., any alcohols) to prevent formation of the unwanted 3’-O-TBDMS ribonucleoside side-product. It should also be noted that the formation of intermediate ribonucleoside 2 has not been optimized; better yields of 2 would lead to higher yields of 3.

The phosphitylation of the commercial ribonucleoside 1 with N,N,N’,N’-tetraisopropyl-O-[2-(N-formyl-N-methyl)aminoethyl]phosphordiamidite is expected to proceed smoothly as long as the glassware and reagents are dry and the reaction is performed under an inert atmosphere. The ribonucleoside phosphoramidite 4 is isolated as a triethylamine-free solid in a yield of 83%. It is critically important to have the ribonucleoside phosphoramidite 4 free from residual moisture and triethylamine; these contaminants can and will affect the coupling efficiency of 4. The presence of moisture and triethylamine can be detected by ¹H NMR spectroscopy. Should the level of triethylamine be questionable, it is often preferable to dissolve 4 in a minimal amount of CH₂Cl₂ and adding the solution to cold hexanes (−78°C). Lyophilization of the precipitate from dry benzene ensures complete removal of residual moisture and triethylamine from the ribonucleoside phosphoramidite 4. The 1H-tetrazole activation of 4 in the presence of ribonucleoside 3 affords, after oxidation of the phosphite triester with tert-butyl hydroperoxide, the diribonucleoside phosphate triester 5. Treatment of the oxidized product with 3% TFA in CH₂Cl₂ gives after purification on silica gel the 3’-hydroxylated diribonucloside phosphate triester 6 in a yield of 78% relative to the molar amount of 3 used as starting material. As discussed above, it is important to use an eluent that is free of protic solvents (i.e., any alcohols) when purifying 6 by silica gel chromatography to prevent the formation of unwanted 3’-O-TBDMS diribonucleoside phosphate triester side-product.

Phosphitylation of the diribonucleoside phosphate triester 6 with N,N,N’,N’-tetraisopropyl-O-[2-(N-formyl-N-methyl)aminoethyl]phosphordiamidite is expected to proceed as reported above for the preparation of the ribonucleoside phosphoramidite 4, when applying the same criteria to ensure rigorous exclusion of moisture. The fully protected diribonucleoside phosphoramidite 7 is then treated with DBU to induce the cleavage of the 5’-formyl ester leading to the production of the 5’-hydroxylated diribonucleoside phosphoramidite 8 in a post-purification yield of 70% based on the molar amount of 6 used as the staring material. It is critically important to make sure that the phosphoramidite 8 is free of residual moisture and DBU. Should this be necessary, the process used for eliminating moisture and triethylamine from the phosphoramidite 4 can also be used for the phosphoramidite 8, as long as the lyophilization step is performed with a frozen solution of 8 in dry 1,4-dioxane. The 1H-terazole-mediated cyclocondensation of phosphoramidite 8 with its 5’-hydroxyl has led to the formation of the fully protected cyclic di-GMP product 9 after tert-butyl hydroperoxide oxidation of its phosphite triester function. Without isolation, the product 9 has been subjected to treatment with ethylenediamine to cleave the isobutyryl groups protecting the amine function of both guanines. It is important to perform this deprotection step at ~25°C to preserve the integrity of the phosphate and 2’-hydroxyl protecting groups. The partially deprotected c-di-GMP product is then heated at 90 °C in an aqueous buffer (40% CH₃CN in 0.1 M triethylammonium acetate buffer, pH 7.0) to thermolytically cleave the phosphate protecting groups. Complete deprotection of the cyclic-di-GMP product is achieved last, by treatment with neat triethylamine trihydrofluoride to remove the tert-butyldimethylsilyl groups protecting the 2’-hydroxyls of cyclic-di-GMP. Cleavage of the TBDMS protecting group must be performed last, to prevent unwanted intramolecular linearization of c-di-GMP. The fully deprotected product is purified by RP-HPLC to yield the native c-di-GMP in 78% based on the molar amount of 5’-hydroxylated diribonucleoside phosphoramidite 8 used as the starting material.

Time Considerations

Preparation of the ribonucleoside 3 including 3’-O-deprotection of intermediate 2, work up, purification and rotoevaporation of volatiles takes 30 hr. Preparation and purification of the ribonucleoside phosphoramidite 4 from the commercial ribonucleoside 1, including drying and lyophilization time can be achieved within 32 hr. Preparation and purification of the 3’-hydroxylated guanylyl-(3′,5′)-guanosine phosphate triester 6 from the ribonucleoside 3, ribonucleoside 5’-phosphoramidite 4 and 3’-O-deprotection of the diribonucleoside phosphate triester 5, take 16 hr. Preparation and purification of the 5’-hydroxylated diribonucleoside phosphoramidite 8 from the 3’-hydroxylated diribonucloside phosphate triester 6 and the 5’-O-deprotection of the diribonucleoside phosphate triester 7 is completed within 26 hr. The cyclization of the 5’-hydroxylated diribonucleoside phosphoramidite 8 to provide the fully protected cyclic-di-GMP 9 followed by its complete deprotection to the unpurified native cyclic di-GMP 10 can be done within 50 hr. Purification of the crude c-di-GMP 10 by RP-HPLC, followed by removal of the elution buffer requires 16 hr to complete.

Figure 11.

Limited expansion of the 300 MHz ¹H NMR spectrum of the pre-cyclocondensation diribonucleoside phosphoramidite 8.

Figure 12.

121 MHz ³¹P NMR spectrum of the pre-cyclocondensation diribonucleoside phosphoramidite 8 in CH₃CN.

Figure 13.

Expansion of the ³¹P(III) NMR signals recorded for the pre-cyclocondensation diribonucleoside phosphoramidite 8.

Figure 14.

Expansion of the ³¹P(V) NMR signals recorded for the pre-cyclocondensation diribonucleoside phosphoramidite 8.

FIGURE LEGENDS

The figures of this article are: “Reprinted from Tetrahedron Letters, Vol. 60, A. Grajkowski, M. Takahashi, T. Kaczyński, S. C. Srivastava, S. L. Beaucage, An improved phosphoramidite approach for the chemical synthesis of 3’, 5’-cyclic diguanylic acid, pp. 452–455., Copyright (2019), with permission from Elsevier”