in reaction buffer (40 mm Tris-HCl, ph 8.0, 100 mm NaCl and 10 mm MgCl 2 ). After

Transcription

1 Supplementary Notes Enzymatic Assays a. Synthesis of 32 P-c-di-AMP 32 P-c-di-AMP synthesis: 333 nm 32 P-ATP and 5 μm DisA (Bacillus subtilis) were mixed in reaction buffer (40 mm Tris-HCl, ph 8.0, 100 mm NaCl and 10 mm MgCl 2 ). After overnight incubation, reaction was stopped by heating up to 95 C for 5 min. b. CdnP cleavage of c-di-amp 24 nm 32 P-c-di-AMP, 2 μm CdnP and various concentrations of c-di-amp were mixed in reaction buffer (50 mm Tris-HCl, ph 9.0 and 1 mm MgCl 2 ). At different time points, reaction mixture was spotted on a TLC plate (EMD Millipore TLC Cellulose). The mobile phase used for the TLC was saturated (NH 4 ) 2 SO 4 : 1.5 M KH 2 PO 4 = 1:1.5. c. CdnP cleavage of c-di-amp at different phs 24 nm 32 P-c-di-AMP and 2 μm CdnP were used in 50 mm Tris-HCl buffers with different phs (ph 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 and 9.0) containing 1 mm MgCl 2. At different time points, reaction mixture was spotted on a TLC plate (EMD Millipore TLC Cellulose). The mobile phase used for the TLC was saturated (NH 4 ) 2 SO 4 : 1.5 M KH 2 PO 4 = 1:1.5. d. CdnP cleavage of c-di-amp in the presence of different cations 24 nm 32 P-c-di-AMP and 2 μm CdnP were used in 50 mm Tris-HCl, ph 9.0. Different cations (Li +, Mg 2+, Mn 2+ and Ca 2+ ) were added at a concentration of 5 mm to the reaction mixture to test the effect of these cations on enzymatic activity. 1

2 e. Cleavage of c-di-amp by ENPP1 24 nm 32 P-c-di-AMP and 2.5 μm cold c-di-amp was cleaved by 2 μm ENPP1. Aliquots of the reactions were taken at various time points and analyzed by TLC. ENPP1 reaction was carried out in a buffer containing 20 mm Tris-HCl, ph 9.0, 2 mm CaCl 2, 0.2 mm ZnCl 2 and 0.01% Triton X-100. f. Kinetics of c-di-amp Cleavage by CdnP with or without Inhibitor(s) 40 nm Rv2837c was incubated with 24 nm 32 P-c-di-AMP plus different concentrations of cold c-di-amp. For the inhibition studies, 50 μm Ap(S)A was added. The reaction progress was analyzed by TLC. Initial reactions velocities were calculated and fit with Michaelis Menten kinetics equation. g. Cleavage of c-di-amp with inhibitors 32 P-c-di-AMP was generated from 32 P-ATP by DisA. 24 nm 32 P-c-di-AMP, 2.5 μm c-di- AMP, 2 μm CdnP and 250 μm inhibitors were mixed and incubated at 37 C. The reaction progress was analyzed using polyethyleneimine cellulose TLC plate (EMD). Mobile phase used for the TLC was a mixture of 4mL saturated (NH 4 ) 2 SO 4 and 6mL 1.5 M KH 2 PO 4. The following equations were used to calculate percent of c-di-amp and percent inhibition: Where I = intensity of radioactive spot on the TLC plate. 2

3 HPLC analysis for identification and verification of reaction products a. CdnP cleavage of c-di-amp, c-di-gmp, 2 3 -cgamp and ApA analogs 2 μm CdnP and 100 μm c-di-amp or c-di-gmp or 2 3 -cgamp or ApA analogs were incubated overnight in 50 mm Tris-HCl, ph 7.5 and 1 mm MnCl 2. Separation was performed on Varian ProStar HPLC using Purospher STAR RP-18 (5 µm) LiChroCART column. The mobile phases are buffer A: 0.1 M triethylammonium acetate (TEAA) in water and buffer B: acetonitrile. Samples were eluted with 95% 80 % A at 0-16 min and 80% 10 % A at min and kept 10% A at min. Signals were detected at room temperature with a 254 nm UV detector. b. Cleavage of 2 3 -cgamp by different PDEs 10 μm CdnP or 10 μm RocR (Pseudomonas aeruginosa) or 10 μm YybT (Bacillus subtilis) or 10 μm GdpP (Staphylococcus aureus) or 1.7 μm hpde12 (Origene) or 2.8 μm hrexo2 (Origene) were incubated over-night with 100 μm 2 3 -cgamp (Invivogen) in reaction buffers. RocR reaction buffer is 10 mm Tris-HCl, ph 8.0, 100 mm NaCl and 5 mm MgCl 2. YybT buffer is 100 mm Tris-HCl, ph 8.3, 10 mm KCl and 500 μm MnCl 2. GdpP buffer is 50 mm Tris-HCl ph 8.5, 10 mm KCl and 100 μm MnCl 2. hpde12 buffer is 100 mm Tris-HCl, ph7.5 and 10 mm MgCl 2. hrexo2 buffer is 50 mm HEPES-KOH, ph 7.4, 50 mm KCl, 2% glycerol, 0.01% Triton X-100 and 10 mm MnCl 2. Separation was performed on Varian ProStar HPLC using Cosmosil C18-MS-II column. The mobile phases are buffer A: 0.1 M TEAA in water and buffer B: acetonitrile. Samples were eluted with 99% 87 % A at 0-16 min and 87% 10 % A at min, kept 10% A at 3

4 min and washed back to 1% A at min. Signals were detected by a 254 nm UV detector. c. Cleavage of pgp(2-5 )A 1.7 μm PDE12 was incubated overnight with 100 μm pgp(2-5 )A in 100 mm Tris-HCl, ph7.5 and 10 mm MgCl 2. Separation was performed on Varian ProStar HPLC using Cosmosil C18-MS-II column. The mobile phases are buffer A: 0.1 M TEAA in water and buffer B: acetonitrile. Samples were eluted with 99% 87% A at 0-16 min and 87% 10 % A at min, kept 10% A at min and washed back to 1% A at min. Signals were detected by a 254 nm UV detector. d. Kinetics of 2 3 -cgamp cleavage by CdnP 10 μm Rv2837c was incubated with different concentrations of 2 3 -cgamp. At various time points, aliquots of the reactions were stopped by heating up to 95 C for 5 min and enzymes were removed by filteration. After HPLC analysis, initial reactions velocities were calculated and fit with Michaelis Menten kinetics equation. e. Cleavage of 2 3 -cgamp by ENPP1 2 μm ENPP1 was incubated with 100 μm 2 3 -cgamp for 2 hrs in 20 mm Tris-HCl, ph 9.0, 2 mm CaCl 2, 0.2 mm ZnCl 2 and 0.01% Triton X-100. Reaction product was analyzed using Cosmosil C18-MS-II column. Signals were detected by a 254 nm UV detector. f. Inhibition of ENPP1 activity by Ap(S)A and Sildenafil 4

5 100 nm ENPP1 was incubated with 50 μm 2 3 -cgamp in 20 mm Tris-HCl, ph 9.0, 2 mm CaCl 2, 0.2 mm ZnCl 2 and 0.01% Triton X-100. For the inhibition studies, 100 μm Ap(S)A or Sildenafil was added. Reaction product was analyzed by HPLC and signals were detected by a 254 nm UV detector. 5

6 Synthesis of nucleotide analogs Possible products from the cleavage of 2 3 -cgamp Structures of ApA and analogs used in this Study 6

7 Solid-phase synthesis of dinucleotides All synthesis were done by Programmed automated synthesis using Applied Biosystem 392 DNA/RNA Synthesizer. PG = protecting group, in details, benzoyl (Bz) protection for amine at 6 th position in adenine base, isobutyryl (ibu) protection for amine at 2 nd position in guanine base. a) Synthesis of Gp(2'-5 )Ap 7

8 b) Synthesis of pgp(2-5 )A 8

9 c) Synthesis of ApGp(2') 9

10 d) Synthesis of papg 10

11 e) Synthesis of ApA and Ap(S)A 11

12 f) Synthesis of dap(carboxylate)da and dap(carboxylate)a Synthesis of 3 -phosphate analogs (synthesis scheme a-d) On Applied Biosystem 392 DNA/RNA synthesizer, the first phosphoramidite was coupled to sulfonylethyl controlled pore glass (CPG), followed by the second phosphoramidite (Note- the PG on the nucleobase = protecting group. Benzoyl (Bz) for A and isobutyl (ibu) for G). The dinucleotide was then cleaved from the solid support by 30% NH 4 OH at room temperature for 12 hrs. The TBS group was then deprotected 12

13 using Et 3. 3HF at 55 o C for 1 hr. The reaction mixture was washed with acetone (50 ml X3) and subjected to HPLC purification. Synthesis of 3 -OH Analogs (synthesis scheme e-f) On Applied Biosystem 392 DNA/RNA synthesizer, the second nucleobase was coupled to a first nucleobase, which is on a CPG (Note- the PG on the nucleobase = protecting group. Bz for A and ibu for G). For phosphorothioate, Beaucage reagent was used for the oxidation step. The dinucleotide was then cleaved from the solid support with 30% NH 4 OH at room temperature for 12 hrs. For the carboxylate analog, deprotection was achieved with 15% DBU in dry MeCN (ACN) for 2 hrs followed by 40% methylamine in water. The TBS group was then deprotected using Et. 3 3HF at 55 o C for 1 hr. The reaction mixture was washed with acetone (50 ml X 3) and subjected to HPLC purification High resolution mass spectrometry (HRMS) of the synthesized dinucleotides Gp(2-5 )Ap (1): ESI - /MS for [C 20 H 25 N 10 O 14 P 2 ] - : calculated , found pgp(2-5 )A (2): ESI - /MS for [C 20 H 25 N 10 O 14 P 2 ]- : calculated , found ApGp(2 ) (3): ESI - /MS for [C 20 H 25 N 10 O 14 P 2 ] - : calculated , found papg (4): ESI - /MS for [C 20 H 25 N 10 O 14 P 2 ] - : calculated , found ApA (5), ESI - /MS for [C 20 H 24 N 10 O 10 P] - : calculated , found Ap(S)A (6), ESI - /MS for [C 20 H 24 N 10 O 0 PS] - : calculated , found dap(carboxylate)da (7), ESI - /MS for [C 20 H 26 N 10 O 9 P] - : calculated , found 13

14 dap(carboxylate)a (8), ESI - /MS for [C 20 H 26 N 10 O 10 P] - : calculated , found The p(carboxylate) center is chiral and because the oxidation step is not diastereoselective, two compounds were generated denoted as (faster eluting) and (slower eluting). Since, the absolute stereocenters were not determined, and nomenclature were used to reflect the elution profile on reverse-phase C18 column. Thus α-dap(carboxylate)a and β-dap(carboxylate)a are diastereomers having same mass. α-dap(carboxylate)da and β-dap(carboxylate)da are also diastereomers having same mass. 14