c-di-GMP displays A monovalent metal ion-dependent polymorphism

Article abstract of DOI:10.1021/ja0449832

Cyclic diguanosine monophosphate (c-di-GMP) is known to be an important signaling molecule that regulates the formation of bacterial biofilms. We demonstrate here by UV, CD, and NMR that it displays a surprising polymorphism that varies with the monovalen

Full text of DOI:10.1021/ja0449832

Bioconjugate Chem. 2010, 21, 2147–2152
2147
Convenient Synthesis of a Propargylated Cyclic (3′-5′) Diguanylic Acid and
Its “Click” Conjugation to a Biotinylated Azide
Andrzej Grajkowski,*,‡ Jacek Cies´lak,‡ Alexei Gapeev,§ Christian Schindler,† and Serge L. Beaucage‡
Division of Therapeutic Proteins, Center for Drug Evaluation and Research, Food and Drug Administration,
8800 Rockville Pike, Bethesda, Maryland 20892, United States, Department of Microbiology & Immunology,
Columbia University, 701 West 168th Street, New York, New York 10032, United States, and Department of Chemistry and
Biochemistry, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250, United States.
Received August 26, 2010; Revised Manuscript Received September 16, 2010
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-cya-
noethyl)-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%.
these tools may also prove valuable in characterizing the re-
sponses associated with c-di-AMP (6).
INTRODUCTION
Cyclic (3′-5′)diguanylate (c-di-GMP) was first identified in
Acetobacter xylinum as a factor that allosterically regulates
membrane cellulose synthesis (1-3). More recently, enzymes
controlling the intracellular levels of c-di-GMP, particularly
those including the GGDEF (Gly-Gly-Asp-Glu-Phe) and EAL
(Glu-Ala-Leu) domains, which possess diguanylate cyclase and
phosphodiesterase activities, respectively, have been identified
in the majority of bacterial genomes (2). Consistent with these
findings, functional studies have determined that c-di-GMP
levels regulate important bacterial programs, such as those
associated with a switch from planktonic to biofilm growth and
those associated with virulence (2). Moreover, c-di-GMP has
been identified as a potent adjuvant, owing to its ability to induce
the expression of a number of inflammatory cytokines and
chemokines (e.g., TNF-R, IL-1, IFN-ꢀ, IP-10, and Rantes) (4, 5).
More recent studies have provided evidence for a cytosolic
sensor that detects c-di-GMP and initiates this inflammatory
response (5). However, the sensor responsible for eliciting this
inflammatory response, as well as many components in bacterial
c-di-GMP signaling cascades, remains unidentified. The devel-
opment of a biotinylated c-di-GMP conjugate provides an
important tool for the identification and characterization of the
proteins binding this ligand and their respective signaling
pathways, both in prokaryotes and in eukaryotes. Moreover,
Various synthetic strategies have been implemented for
the synthesis of c-di-GMP and its analogues. Indeed, in
additiontothehydroxybenzotriazolephosphotriestermethod(7,8),
combinations of phosphoramidite and phosphotriester (9-12),
phosphoramidite and H-phosphonate (13), or phosphotriester
and H-phosphonate (14, 15) methods have also been reported
for this purpose. Interestingly, an enzymatic approach and a
solid-phase approach to the synthesis of c-di-GMP have
recently been described in the literature (16, 17). We now
report a straightforward solution-phase synthesis of the
propargylated c-di-GMP 10 using exclusively, for the first
time, phosphoramidite chemistry. The synthetic strategy
consists of exploiting commercially available starting materi-
als (1 and 4) and clustering synthetic operations in order to
minimize the number of purification steps (Schemes 1-3).
As outlined in Scheme 4, synthesis of the biotinylated azide
13 from commercial aminoalkylated biotin amide 12 and
conversion of 13 to the biotinylated c-di-GMP 14 via a
copper(I)-catalyzed [3 + 2] azide-alkyne cycloaddition
reaction (CuAAC or click chemistry) (18, 19) are described
in detail in this report.
EXPERIMENTAL PROCEDURES
N2-Isobutyryl-3′-O-levulinyl-2′-O-propargyl Guanosine (3).
To a solution of N2-isobutyryl-5′-O-(4,4′-dimethoxytrityl)-2′-
O-propargyl guanosine (1, 1.73 g, 2.50 mmol) in dry pyridine
(10 mL) was added levulinic anhydride (20, 21) (2.50 g, 10.0
mmol); the solution was stirred at ∼25 °C for 16 h and was
then evaporated to a gummy material under reduced pressure.
The residue was dissolved in CHCl3 (30 mL) and was vigorously
* To whom correspondence should be addressed. Tel: (301)-827-
1860. Fax: (301)-480-3256. E-mail: Andrzej.Grajkowski@fda.hhs.gov.
‡ Center for Drug Evaluation and Research.
§ University of Maryland.
† Columbia University.
10.1021/bc1003857  2010 American Chemical Society
Published on Web 10/13/2010

2148 Bioconjugate Chem., Vol. 21, No. 11, 2010
Grajkowski et al.
Scheme 1. 3′-O-Acylation of the Ribonucleoside 1 and
5′-O-Deprotection of 2a
mixture was concentrated under vacuum to an oil, which was
dissolved in CHCl3 (3 mL) and purified by chromatography on
silica gel (∼40 g) using a gradient of MeOH (0 f 3%) in
CHCl3. Fractions containing the ribonucleoside 3 were collected
and evaporated under reduced pressure to give a white solid
(1.12 mg, 2.28 mmol) in a yield of 91% based on the molar
amount of ribonucleoside 1 that was used as the starting material.
1H NMR (300 MHz, DMSO-d6): δ 12.1 (bs, 1H), 11.7 (bs, 1H),
8.29 (s, 1H), 5.90 (d, 1H, J ) 7.7 Hz), 5.38 (m, 2H), 4.83 (dd,
1H, J ) 7.7, 7.7 Hz), 4.14 (m, 3H), 3.63 (m, 2H), 3.41 (t, 1H,
J ) 2.4 Hz), 2.77 (m, 2H), 2.76 (sept, 1H, J ) 6.7 Hz), 2.56
(m, 2H), 2.13 (s, 3H), 1.12 (d, 6H, J ) 6.7 Hz). 13C NMR (75
MHz, DMSO-d6): δ 206.7, 180.1, 171.7, 154.6, 148.9, 148.4,
137.1, 119.8, 84.1, 83.9, 79.1, 78.7, 77.9, 71.4, 61.0, 57.6, 37.3,
34.7, 29.5, 27.5, 18.7. +ESI-HRMS: Calcd for C22H27N5O8
[M + H]+, 490.1932, found 490.1935.
a Keys: DMTr, 4,4′-dimethoxytrityl; Gua, guanin-9-yl; iBu, isobu-
tyryl; Lev2O, levulinic anhydride; Lev, levulinyl.
Propargylated Guanylyl(3′-5′)guanosine Phosphotriester 6.
To a stirred solution of pre-dried ribonucleoside 3 (970 mg,
2.00 mmol) and phosphoramidite 4 (1.94 g, 2.00 mmol) in
anhydrous MeCN (20 mL) was added solid 1H-tetrazole (210
mg, 3.00 mmol). 31P NMR analysis of the reaction mixture
showed, within 4 h at ∼25 °C, the appearance of two singlets
mixed with water (15 mL). The organic layer was collected
and evaporated under low pressure. The crude ribonucleoside
2 was dissolved in 80% AcOH (10 mL) in order to remove the
5′-protecting group; progress of the reaction was monitored by
TLC, which revealed complete cleavage of the 4,4′-dimethox-
ytrityl (DMTr) ether after a reaction time of 3 h. The reaction
Scheme 2. Preparation of the Diribonucleoside Phosphotriester 6a
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 11a
a Conditions: (i) (i-Pr2N)2POCH2CH2CN, 1H-tetrazole (1.0 equiv at the rate of 0.25 equiv/15 min), MeCN, 3 h; (ii) 1H-tetrazole (2 equiv),
MeCN, 16 h; (iii) conc aq NH3, 30 h, 25 °C; (iv) Et3N·3HF, 20 h, 25 °C. Keys: Gua, guanin-9-yl; iBu, isobutyryl; TBDMS, tert-butyldimethylsilyl.

Biotinylated c-di-GMP Conjugate
Bioconjugate Chem., Vol. 21, No. 11, 2010 2149
Scheme 4. Biotinylation of the Propargylated c-di-GMP 10a
a Keys: Gua, guanine-9-yl; TBTA, tris-(benzyltriazolylmethyl)amine.
(δP ) 140.7 and 139.5 ppm) corresponding to the expected
diastereomeric diribonucleoside phosphite triesters 5. Addition
of 5.5 M tert-butyl hydroperoxide in decane (730 µL) to the
reaction mixture resulted in the conversion of the phosphite
triesters to their diastereomeric phosphate triester derivatives
within 30 min. The solution was evaporated under vacuum to
an oily residue, which was treated with a solution (40 mL) of
0.5 M hydrazine hydrate in pyridine/AcOH (3:2 v/v) for 15 min;
2,4-pentanedione (2 mL) was then added to the solution in order
to quench unreacted hydrazine hydrate. The volatiles were
removed under reduced pressure, and the reaction product was
partitioned between CHCl3 (40 mL) and H2O (20 mL). The
organic phase was collected and evaporated to dryness under
vacuum; the remaining material was dissolved in CHCl3 (5 mL)
and purified by chromatography on silica gel (∼40 g) using a
gradient of MeOH (0 f 5%) in CHCl3. Fractions containing
the 3′-deprotected dinucleoside phosphate triester were collected
and evaporated under low pressure to give a yellowish powder,
which was dissolved in of 80% AcOH (15 mL) and left stirring
for 3 h at ∼25 °C. Hexanes (25 mL) was added to the vigorously
stirred reaction mixture in order to extract the 4,4′-dimethox-
ytrityl alcohol side product. After phase separation, the lower
layer was collected and evaporated to dryness under vacuum.
The thoroughly dried material was dissolved in CH2Cl2 (5 mL),
and the solution was added dropwise to stirred hexanes (100
mL). The precipitate was filtered to give the diribonucleoside
phosphate triester 6 (2.25 g, 1.76 mmol) as a white solid in a
yield of 88% relative to the molar amount of ribonucleoside 3
that was used as starting material. 31P NMR (121 MHz, DMSO-
d6): δ -1.96, -2.38. +ESI-HRMS: Calcd for C40H56N11O14PSi
[M + H]+ 974.3588, found 974.3594.
was stirred for an additional 2 h at ∼25 °C, and 1H-tetrazole
(140 mg, 2.00 mmol) was then added to the reaction mixture,
which was left stirring for 16 h. Without any workup, tert-butyl
hydroperoxide (5.5 M in decane, 550 µL) was added to the
stirred solution. After 30 min, the reaction mixture was
concentrated under vacuum to an oil, which was dissolved in
CHCl3 (5 mL) and purified by chromatography on silica gel
(∼30 g). The product eluted from the silica gel column when
employing a gradient of MeOH (0 f 8%) in CHCl3. Fractions
containing the propargylated c-di-GMP triester 10 were checked
by TLC [Rf (CHCl3/MeOH 9:1 v/v) ) 0.33], collected, and
evaporated under reduced pressure to give a white solid (435
mg, 400 µmol) in a yield of 40%. The purified c-di-GMP triester
10 (109 mg, 100 µmol) was treated with concentrated aqueous
ammonia (3 mL) in a sealed glass vial for 30 h at ∼25 °C;
excess aqueous ammonia was then evaporated to dryness under
reduced pressure. The material left was dissolved in neat
triethylamine trihydrofluoride (4.00 mL, 24.5 mmol), and the
solution was allowed to stir for 20 h at ∼25 °C. The fully
deprotected product was purified by RP-HPLC 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% MeCN/min is pumped
at a flow rate of 3 mL/min for 40 min. Fractions containing the
product were collected and evaporated using a stream of air to
give the propargylated c-di-GMP 11 in a near-quantitative yield.
1H NMR (300 MHz, DMSO-d6): δ 10.78 (bs, 1H), 9.76 (bs,
1H), 8.00 (s, 1H), 7.95 (s, 1H), 6.59 (bs, 4H), 5.85 (d, 1H, J )
4.3 Hz), 5.73 (d, 1H, J ) 5.3 Hz), 4.88 (m, 1H), 4.73 (m 1H),
4.58 (t, 1H, J ) 4.0 Hz), 4.53 (t, 1H, J ) 5.2 Hz), 4.46 (dd,
1H, 2J ) 15.2 Hz, 4J ) 2.3 Hz), 4.38 (dd, 1H, 2J ) 15.2 Hz, 4J
) 2.3 Hz), 4.23-4.06 (m, 4H), 3.91 (m, 2H), 3.43 (t, 1H, 4J )
2.3 Hz). 13C NMR (75 MHz, DMSO-d6): δ 156.5, 153.9, 151.1,
150.7, 135.0, 134.7, 116.24, 116.16, 86.5, 85.1, 80.4 (d, JC-P
) 9.2 Hz), 80.2 (d, JC-P ) 5.7 Hz), 79.7, 78.4, 77.6, 72.6 (d,
JC-P ) 5.7 Hz), 72.0, 71.0, 62.8 (d, JC-P ) 5.7 Hz), 62.5 (d,
JC-P ) 5.7 Hz), 57.7, 45.4, 8.44. 31P NMR (121 MHz, DMSO-
Propargylated c-di-GMP 11. Vacuum-dried propargylated
diribonucleoside phosphotriester 6 (973 mg, 1.00 mmol) and
2-cyanoethyl tetraisopropylphosphordiamidite (301 mg, 1.00
mmol) were dissolved in dry MeCN (20 mL) to which was
added solid 1H-tetrazole (70 mg, 1.0 mmol) in four portions
over a period of 1 h under an inert gas atmosphere. The solution

2150 Bioconjugate Chem., Vol. 21, No. 11, 2010
Grajkowski et al.
d6): δ -0.29, -1.25. +ESI-HRMS: Calcd for C23H26N10O14P2
[M + H]+ 729.1178 found 729.1184.
Biotinylated Azide 13. To a solution of commercial N-D-
(+)-biotinyl-4,7,10,13,16-pentaoxa-1,19-diaminononadecane
(12, 50 mg, 94 µmol) in 1 mL of MeCN/C5H5N (9:1 v/v) was
added azidoacetic anhydride (35 mg, 0.19 mmol). The solution
was stirred at ∼25 °C for 1 h and was then concentrated to an
oil using a stream of argon. The crude product was purified by
chromatography on silica gel (2 g) using a gradient of MeOH
(0-10%) in CHCl3. Fractions containing the product were
collected and evaporated under vacuum to give 13 as a solid
(35 mg, 57 µmol, 61%). +ESI-HRMS: Calcd for C26H47N7O8S
[M + H]+ 618.3280, found 618.3283.
Biotinylated c-di-GMP 14. To the propargylated c-di-GMP
11 (150 OD260) was added a solution of 10 mM biotinylated
azide 13 (800 µL), 100 mM CuSO4 (120 µL), and 100 mM
tris-(benzyltriazolylmethyl)amine (TBTA, 240 µL), each in
DMSO/tert-BuOH (3:1 v/v). A solution of 400 mM sodium
ascorbate in H2O (175 µL) was then added to the reaction
mixture, which was left stirring at ∼25 °C for 16 h. The crude
reaction product was purified by RP-HPLC under conditions
identical to those described for the purification of the propar-
gylated c-di-GMP 11. Fractions containing the product were
collected and evaporated under a stream of air to give the
biotinylated c-di-GMP 14 (93 OD260) in an isolated yield of
62%. 31P NMR (121 MHz, D2O): δ -1.73, -2.06. +ESI-
HRMS: Calcd for C49H73N17O22P2S [M + H]+ 1346.4385, found
1346.4429; [M + 2H]2+ 673.7229, found 673.7229.
Figure 1. RP-HPLC analysis of the guanylyl(3′-5′)guanosine phos-
photriester 6. 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.
RESULTS AND DISCUSSION
Our solution-phase approach to the synthesis of the biotiny-
lated c-di-GMP conjugate 14 began with the preparation of the
ribonucleoside building block 3 (Scheme 1) from the com-
mercially available propargylated ribonucleoside 1, which was
3′-O-acylated by treatment with levulinic anhydride (20, 21) in
dry pyridine. After extractive workup, the fully protected
ribonucleoside 2 was treated with 80% AcOH in order to remove
the 5′-DMTr group. The 5′-deprotected ribonucleoside was
purified by chromatography on silica gel to give pure 3 in a
yield of 91%. The ribonucleoside was characterized by 1H and
13C NMR spectroscopies and by high-resolution mass spec-
trometry (HRMS).
Figure 2. RP-HPLC analysis of the conversion of the propargylated
c-di-GMP triester 10 to the propargylated c-di-GMP 11. (A) Chro-
matogram 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 normal-
ized to the highest peak, which was set to 1 arbitrary unit.
The commercial availability of the 2′-O-tert-butyldimethyl-
silyl (TBDMS) ribonucleoside phosphoramidite 4 (Scheme 2)
and the pervasive use of the click chemistry for labeling
nucleosides and oligonucleotides with fluorophores, carbohy-
drates, and other functional groups (22, 23) have been motiva-
tional in the development of our synthetic approach to the
synthesis of 14. Indeed, the reaction of 4 with the 5′-hydroxyl
function of ribonucleoside 3 in the presence of 1.5 molar equiv
of 1H-tetrazole in anhydrous MeCN produced the dinucleoside
phosphate triester 5 (Scheme 2). After P(III) oxidation by tert-
butyl hydroperoxide and upon concentration of the reaction
mixture to an oil, a solution of hydrazine hydrate in pyridine
and acetic acid was added in order to remove the levulinyl group
from the terminal 3′-hydroxyl of the diribonucleoside phosphate
triester. The hydrazinolysis reaction was stopped by the addition
of 2,4-pentanedione, and following an extractive workup, the
reaction product was purified by silica gel chromatography. The
5′-DMTr group of the purified material was then cleaved under
acidic conditions (80% AcOH) to give, after workup and precipita-
tion from hexanes, the propargylated diribonucleoside phosphate
triester 6 in a yield of 88% relative to the molar amount of
ribonucleoside building block 3 that was used as the starting
material. This yield is excellent given that out of four synthetic
operations only one purification step was performed. The dinucleo-
side phosphotriester 6 was characterized, as a mixture of two
P-diastereomers, by 31P NMR spectroscopy and HRMS (see
Experimental Procedures section). The purity of 6 was assessed
by RP-HPLC (Figure 1) and was determined to be 95%.
The preparation of the propargylated and fully protected c-di-
GMP derivative 10 (Scheme 3) was carried out in MeCN by
the phosphitylation of 6 with strictly 1 mol equiv of 2-cyanoethyl
tetraisopropylphosphordiamidite and solid 1H-tetrazole, which
was added at the rate of 0.25 mol equiv per 15 min under
rigorously anhydrous conditions. Such a stoichiometry presum-
ably favored the formation of the 5′-phosphoramidite 7 relative
to that of the 3′-phosphoramidite 8 and 3′,5′-bis-phosphoramidite
9, the formation of which cannot conclusively be ruled out. The
intramolecular cyclization of 7 and 8 was then performed by
adding two molar equivalents of solid 1H-tetrazole to the initial
phosphitylation reaction mixture. The concentration of the
reagents, with the exception of 1H-tetrazole, was sufficiently
low (∼50 mM) to favor intramolecular cyclization rather than
intermolecular polymerization. The intramolecular cyclization
reaction was allowed to proceed overnight (16 h) and was
followed by the addition of a solution of tert-butyl hydroper-
oxide in decane in order to convert the newly formed phosphite
triester function to its tetracoordinated phosphate triester state
and complete the synthesis of 10. The crude product was purified
by chromatography on silica gel to give an amorphous white

Biotinylated c-di-GMP Conjugate
Bioconjugate Chem., Vol. 21, No. 11, 2010 2151
solid in a yield of 40% based on the starting propargylated
dinucleoside phosphate triester 6.
The modest yield of intramolecular cyclization may be at-
tributed to a number of factors. The use of only 1 mol equiv of
2-cyanoethyl tetraisopropylphosphordiamidite is necessary to
minimize if not prevent the formation of 9, which otherwise
would result in a commensurate loss of cyclic product in addition
to residual unphosphitylated 6. Perhaps the most important factor
contributing to a lower yield of 10 may be related to the presence
of adventitious water in a relatively large volume of MeCN,
which would lead to hydrolysis of the 1H-tetrazole-activated
phosphoramidite functions of 7 and 8 and prevent intramolecular
cyclization. However, these limitations, which are inherent to
our approach to the synthesis of 10, are clearly outweighed by
the simplicity and convenience of the method.
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” con-
jugation of 11 with 13 under the conditions described in the Experi-
mental Procedures section. 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.
The copper(I)-catalyzed [3 + 2] cycloaddition of 13 with the
propargylated c-di-GMP 11 was performed essentially as
reported by Berndl et al. (25). The biotinylated c-di-GMP 14
was purified by RP-HPLC and was isolated in a yield of 62%,
which is based on the molar amount of 11 used as the starting
material. Figure 3 illustrates the “click” conjugation of the
biotinylated azide 13 with the propargylated c-di-GMP 11.
RP-HPLC analysis of 10 revealed the expected presence of
four diastereomers, two for each chiral phosphate function
(Figure 2A). The asymmetry of 10 in addition to the chirality
of its two tetracoordinated phosphate esters contributed to the
CONCLUSION
The synthetic strategy for the synthesis of the propargylated
c-di-GMP 10 reported herein is based on the use of commercial
starting materials including the propargylated ribonucleoside 1
and the 2′-O-TBDMS ribonucleoside phosphoramidite 4. The
absence of a vicinal hydroxyl function in 4 precluded the
notorious migration of the TBDMS group (26) and formation
of any isomeric side product when using this reagent. Phos-
phitylation of the propargylated diribonucleoside phosphate
triester 6 when employing only 1 mol equiv of commercial
2-cyanoethyl tetraisopropylphosphordiamidite led to the forma-
tion of 10, for the first time through exclusively phosphoramidite
chemistry. The yield of 10 is perhaps lower than those reported
using other cyclization procedures, but the convenience and
simplicity of our synthetic strategy prevail over other preparative
synthetic approaches in terms of the availability of starting
materials, minimization of potential side reactions, and number
of necessary purification steps. As mentioned above, the bio-
logical implications of the biotinylated c-di-GMP 14, in regard
to identification and characterization of the proteins recognizing
and binding to c-di-GMP, are currently being investigated and
may provide valuable insight into their signaling pathways and
bioactivity in prokaryotes and eukaryotes. The results of these
studies are beyond the scope of this report and will be
communicated elsewhere. In this context, our straightforward
approach to the synthesis of the propargylated c-di-GMP 11 is
being exploited in the preparation of structural analogues of 11
having either one or two (2′-5′)phosphodiester linkages instead
of the native (3′-5′)phosphodiester functions in order to assess
whether their biotinylated conjugates may permit the identifica-
tion of proteins that may or may not be similar to those
associated with the recognition and binding of 11. This work is
again based on the commercial availability of both the 3′-O-
propargylated and 3′-O-TBDMS isomers of ribonucleoside 1
1
complexity of its H, 13C, and 31P NMR spectra. Indeed, the
proton-decoupled 31P NMR analysis of 10, which revealed six
out of theoretically eight signals, underscores the challenging
characterization of 10 by spectroscopic techniques. In order to
mitigate this difficulty, the strategy of converting 10 to the c-di-
GMP 11 was adopted. Complete deprotection of 10 consisted
of removing the N2-isobutyryl and 2-cyanoethyl phosphate
protecting groups first by treatment with concentrated aqueous
ammonia in a sealed glass vial over a period of 30 h at ambient
temperature. Lastly, the cleavage of the 2′-O-TBDMS group
was effected by reaction with triethylamine trihydrofluoride to
give, nearly quantitatively, the propargylated c-di-GMP 11, as
judged by RP-HPLC analysis of the deprotection reaction
(Figure 2B). This analysis also revealed the purity of 11, which
was determined to be 95%. The propargylated c-di-GMP 11
1
was adequately characterized by H, 13C, and 31P NMR spec-
troscopies and HRMS (see Experimental Procedures section).
The preparation of the biotinylated azide 13 was required
for the biotinylation of 11 (Scheme 4). Acylation of the
commercial aminoalkylated biotin derivative 12 with azidoacetic
anhydride in a solution of 10% pyridine in MeCN over a period
of 1 h at ambient temperature afforded 13 in moderate yield
after purification. Azidoacetic anhydride was generated in situ
from azidoacetic acid and N,N′-dicyclohexylcarbodiimide in
THF under conditions similar to those employed for the
preparation of levulinic anhydride (20, 21). Azidoacetic acid
was prepared from the reaction of bromoacetic acid and sodium
azide, as described in the literature (24). Given the inherent
complexity of the 1H and 13C NMR spectra of biotinylated azide
13, its characterization by HRMS was found to be the most
informative.

2152 Bioconjugate Chem., Vol. 21, No. 11, 2010
Grajkowski et al.
(11) Hyodo, M., and Hayakawa, Y. (2004) An improved method
for synthesizing cyclic bis(3′-5′)diguanylic acid (c-di-GMP). Bull.
Chem. Soc. Jpn. 77, 2089–2093.
and ribonucleoside phosphoramidite 4, respectively. The results
of these studies will be reported in due course.
(12) Hyodo, M., Sato, Y., and Hayakawa, Y. (2006) Synthesis of
cyclic bis(3′-5′)diguanylic acid (c-di-GMP) analogs. Tetrahedron
62, 3089–3094.
(13) Zhang, Z., Gaffney, B. L., and Jones, R. A. (2004) c-di-GMP
displays a monovalent metal ion-dependent polymorphism. J. Am.
Chem. Soc. 126, 16700–16701.
(14) Humes, E., and Yan, H. (2006) Convenient synthesis of 3′,5′-
cyclic diguanylic acid (cdiGMP). Nucleic Acids Symp. Ser. 50,
5–6.
(15) Yan, H., and Lo´pez Aguilar, A. (2007) Synthesis of 3′,5′-
cyclic diguanylic acid (cdiGMP) using 1-(4-chlorophenyl)-4-
ethoxypiperidin-4-yl as a protecting group for 2′-hydroxy
functions of ribonucleosides. Nucleosides, Nucleotides Nucleic
Acids 26, 189–204.
ACKNOWLEDGMENT
This work was supported in part by NIH grants R21
ES016835 and RO1 AI058211 (C.S.).
Supporting Information Available: Materials and methods;
1H and 13C NMR spectra of the propargylated ribonucleoside 3
and c-di-GMP 11; 31P NMR spectra of the propargylated
guanylyl(3′-5′)guanosine phosphotriester 6, fully protected c-di-
GMP 10, c-di-GMP 11 and biotinylated c-di-GMP 14; mass
spectra of 3, 6, 11, 13, and 14; TLC analysis of 10 and detailed
preparations of levulinic anhydride and azidoacetic anhydride.
This material is available free of charge via the Internet at http://
pubs.acs.org.
(16) Rao, F., Pasunooti, S., Ng, Y., Zhuo, W., Lim, L., Liu, A. W.,
and Liang, Z.-X. (2009) Enzymatic synthesis of c-di-GMP using
a thermophilic diguanylate cyclase. Anal. Biochem. 389, 138–
142.
LITERATURE CITED
(17) Kiburu, I., Shurer, A., Yan, L., and Sintim, H. O. (2008) A
simple solid-phase synthesis of the ubiquitous bacterial signaling
molecule, c-di-GMP and analogues. Mol. BioSyst. 4, 518–520.
(18) Rostovtsev, V. V., Green, L. G., Fokin, V. V., and Sharpless,
K. B. (2002) A stepwise Huisgen cycloaddition process: Cop-
per(I)-catalyzed regioselective “ligation” of azides and terminal
alkynes. Angew. Chem., Int. Ed. 41, 2596–2599.
(19) Tornøe, C. W., Christensen, C., and Meldal, M. (2002)
Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific
copper(I)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes
to azides. J. Org. Chem. 67, 3057–3064.
(1) Ross, P., Weinhouse, H., Aloni, Y., Michaeli, D., Weinberger-
Ohana, P., Mayer, R., Braun, S., de Vroom, E., van der Marel,
G. A., van Boom, J. H., and Benziman, M. (1987) Regulation
of cellulose synthesis in Acetobacter xylinum by cyclic diguanylic
acid. Nature 325, 279–281.
(2) Hengge, R. (2009) Principles of c-di-GMP signalling in bacteria.
Nat. ReV. Microbiol. 7, 263–273.
(3) Yan, H., and Chen, W. (2010) 3′,5′-Cyclic diguanylic acid: a
small nucleotide that makes big impacts. Chem. Soc. ReV. 39,
2914–2924.
(4) Karaolis, D. K. R., Means, T. K., Yang, D., Takahashi, M.,
Yoshimura, T., Muraille, E., Philpott, D., Schroeder, J. T., Hyodo,
M., Hayakawa, Y., Talbot, B. G., Brouillette, E., and Malouin,
F. (2007) Bacterial c-di-GMP is an immunostimulatory molecule.
J. Immunol. 178, 2171–2181.
(5) McWhirter, S. M., Barbalat, R., Monroe, K. M., Fontana, M. F.,
Hyodo, M., Joncker, N. T., Ishii, K. J., Akira, S., Colonna, M.,
Chen, Z. J., Fitzgerald, K. A., Hayakawa, Y., and Vance, R. E.
(2009) A host type I interferon response is induced by cytosolic
sensing of the bacterial second messenger cyclic-di-GMP. J. Exp.
Med. 206, 1899–1911.
(6) Woodward, J. J., Iavarone, A. T., and Portnoy, D. A. (2010)
c-di-AMP secreted by intracellular Listeria monocytogenes
activates a host type I interferon response. Science 328, 1703–
1705.
(7) Ross, P., Mayer, R., Weinhouse, H., Amikam, D., Huggirat,
Y., Benziman, M., de Vroom, E., Fidder, A., de Paus, P.,
Sliedregt, L. A. J. M., van der Marel, G. A., and van Boom,
J. H. (1990) The cyclic diguanylic acid regulatory system of
cellulose synthesis in Acetobacter xylinum. J. Biol. Chem. 265,
18933–18943.
(8) Amiot, N., Heintz, K., and Giese, B. (2006) New approach for
the synthesis of c-di-GMP and its analogues. Synthesis 4230–
4236.
(9) Hayakawa, Y., Nagata, R., Hirata, A., Hyodo, M., and Kawai,
R. (2003) A facile synthesis of cyclic bis(3′f5′)diguanylic acid.
Tetrahedron 59, 6465–6471.
(10) Kawai, R., Nagata, R., Hirata, A., and Hayakawa, Y. (2003)
A new synthetic approach to cyclic bis(3′f5′)diguanilic acid.
Nucleic Acids Res. Suppl. 3, 103–104.
(20) Hassner, A., Strand, G., Rubinstein, M., and Patchornik, A.
(1975) Levulinic esters. Alcohol protecting group applicable to
some nucleosides. J. Am. Chem. Soc. 97, 1614–1615.
(21) Guo, Z., and Xue, J. (2009) LeVulinic anhydride. Encyclopedia
of Reagents for Organic Synthesis, 2nd ed. (Paquette, L. A.,
Crich, D., Fuchs, P. L., and Molander, G., Eds.) pp 5961-5963,
John Wiley & Sons, Hoboken, NJ.
(22) El-Sagheer, A. H., and Brown, T. (2010) Click chemistry with
DNA. Chem. Soc. ReV. 39, 1388–1405, and references therein.
(23) Gramlich, P. M. E., Wirges, C. T., Manetto, A., and Carell,
T. (2008) Postsynthetic DNA modification through the copper-
catalyzed azide-alkyne cycloaddition reaction. Angew. Chem.,
Int. Ed. 47, 8350–8358, and references therein.
(24) Dyke, J. M., Groves, A. P., Morris, A., Ogden, J. S., Dias,
A. A., Oliveira, A. M. S., Costa, M. L., Barros, M. T., Cabral,
M. H., and Moutinho, A. M. C. (1997) Study of the thermal
decomposition of 2-azidoacetic acid by photoelectron and matrix
isolation infrared spectroscopy. J. Am. Chem. Soc. 119, 6883–
6887.
(25) Berndl, S., Herzig, N., Kele, P., Lachmann, D., Li, X.,
Wolfbeis, O. S., and Wagenknecht, H.-A. (2009) Comparison
of a nucleosidic vs non-nucleosidic postsynthetic “Click”modi-
fication of DNA with base-labile fluorescent probes. Bioconjugate
Chem. 20, 558–564.
(26) Beaucage, S. L., and Reese, C. B. (2009) Recent AdVances in
the Chemical Synthesis of RNA. Current Protocols in Nucleic
Acid Chemistry. (Beaucage, S. L., Bergstrom, D. E., Herdewijn,
P., Matsuda, A., Eds.) pp 2.16.1-2.16.31, Chapter 2, Vol. 1,John
Wiley & Sons, Hoboken, NJ.
BC1003857