Supplementary Material

Quantification of cAMP and cGMP analogs in intact cells:

Pitfalls in enzyme immunoassays for cyclic nucleotides

Naunyn- Schmiedeberg`s Archives of Pharmacology

Katharina Werner, Frank Schwede, Hans-Gottfried Genieser, Jörg Geiger, and Elke Butt

Corresponding author

Prof. Dr. Elke Butt

University of Wuerzburg

Institute of Clinical Biochemistry

Grombuehlstr. 12

D-97080 Wuerzburg

Tel.: 0049-931-888-83174

FAX: 0049-931-3293630

Supplementary Fig.S1:

cAMP-analogs

Adenosine-3`,5`- cyclic monophosphate / cAMP
1 / N6-Monobutyryladenosine-3`,5`-cyclic monophosphate / 6-MB-cAMP
2 / N6-Benzoyladenosine-3`,5`-cyclic monophosphate / 6-Bnz-cAMP
3 / 7-Deazaadenosine-3`,5`-cyclic monophosphate / 7-CH-cAMP
4 / 8-Bromoadenosine-3`,5`-cyclic monophosphate / 8-Br-cAMP
5 / 8-(4- Chlorophenylthio)adenosine-3`,5`-
cyclic monophosphate / 8-pCPT-cAMP
6 / 2`-Deoxyadenosine- 3`,5`- cyclic monophosphate / 2`-dcAMP
7 / Adenosine- 3`,5`- cyclic monophosphorothioate, Rp-isomer / Rp-cAMPS
8 / Adenosine- 3`,5`- cyclic monophosphorothioate, Sp-isomer / Sp-cAMPS
9 / Adenosine-1-N- oxide- 3`,5`- cyclic monophosphate / 1-NO-cAMP
10 / 8-(4- Chlorophenylthio)-2`-O- methyladenosine- 3`,5`-
cyclic monophosphate / 8-pCPT-2`-O-Me-cAMP
11 / 5,6- Dichloro-1- β- D- ribofuranosylbenzimidazole- 3`,5`- cyclic monophosphorothioate, Rp-isomer / Sp-5,6-DCl-cBIMPS
12 / 8-Bromoadenosine- 3`,5`- cyclic monophosphorothioate, Rp-isomer / Rp-8-Br-cAMPS

Supplementary Fig.S2:

cGMP analogs

Guanosine- 3`,5`- cyclic monophosphate / cGMP
13 / 6-Thioguanosine- 3`,5`- cyclic monophosphate / 6-SH-cGMP
14 / 7-Deazaguanosine- 3`,5`- cyclic monophosphate / 7-CH-cGMP
15 / 8-Bromoguanosine- 3`,5`- cyclic monophosphate / 8-Br-cGMP
16 / 8-(4- Chlorophenylthio)guanosine- 3`,5`-
cyclic monophosphate / 8-pCPT-cGMP
17 / 2`-Deoxyguanosine- 3`,5`- cyclic monophosphate / 2`-dcGMP
18 / Guanosine- 3`,5`- cyclic monophosphorothioate, Rp-isomer / Rp-cGMPS
19 / Guanosine- 3`,5`- cyclic monophosphorothioate, Sp-isomer / Sp-cGMPS
20 / Purin-1- β-D- ribofuranoside- 3`,5`- cyclic monophosphate / cPuMP
21 / 2- Aminopurinriboside- 3`,5`- cyclic monophosphat / 2-NH2-cPuMP
22 / β- Phenyl-1,N2- etheno-8- Bromoguanosine- 3`,5`- cyclic monophosphate / 8-Br-PET-cGMP
23 / β- Phenyl-1,N2- etheno-8- Bromoguanosine- 3`,5`- cyclic monophosphorothioate, Rp-isomer / Rp-8-Br-PET-cGMPS

Supplementary Fig.S3:

Phosphodiesterase inhibitors

24 / 3- Isobutyl-1- methylxanthine / IBMX
25 / 1,3- dimethyl- 7H- purine- 2,6- dione / Theophylline
26 / 3-(6-aminopurin-9-yl)nonan-2-ol hydrochloride / EHNA

Supplementary Fig.S4:

Binding curves of selected cyclic nucleotide analogs with thecAMP and cGMP-EIA.

The data represent means (SD) of three experiments in duplicate.

Supplementary Table 1

Binding specificities of cyclic nucleotide analogs measured with the cAMP-EIA.

Analog / Absolute binding
EC50 [M] / Relative binding
EC50cAMP/EC50Analog
cAMP / 7.62 ∙10-10 / 1.0000
cPuMP (20) / 9.52 ∙10-10 / 0.8000
Rp-cAMPS (7) / 1.11 ∙10-09 / 0.6865
2`-dcAMP (6) / 2.23 ∙10-08 / 0.0342
6-Bnz-cAMP (2) / 1.27 ∙10-07 / 0.0060
1-NO-cAMP (9) / 1.38 ∙10-07 / 0.0055
8-Br-cAMP (4) / 1.78 ∙10-07 / 0.0043
6-MB-cAMP (1) / 1.76 ∙10-07 / 0.0043
Rp-8-Br-cAMPS (12) / 2.78 ∙10-07 / 0.0027
Sp-cAMPS (8) / 3.87 ∙10-07 / 0.0020
8-pCPT-cAMP (5) / 1.64 ∙10-06 / 0.0005
8-pCPT-2`-O-Me-cAMP(10) / 2.50 ∙10-06 / 0.0003
Sp-5,6-DCl-cBIMPS (11) / >10-05 / <10-05
7-CH-cAMP (3) / >10-05 / <10-05
8-Br-cGMP(15) / >10-05 / <10-05
IBMX (24) / >10-03 / <10-06
Theophylline (25) / >10-03 / <10-06
EHNA (26) / >10-03 / <10-06

The EC50 values represent means (SEM) of three independent experiments in duplicate

Supplementary Table 2

Binding specificities of cyclic nucleotide analogs measured with the cGMP-EIA.

Analog / Absolute binding
EC50 [M] / Relative binding
EC50cGMP/EC50Analog
cGMP / 2.27 ∙10-10 / 1.0000
8-Br-cGMP (15) / 4.56 ∙10-11 / 4.9781
8-pCPT-cGMP (16) / 9.40 ∙10-10 / 2.4149
7-CH-cGMP (14) / 2.25 ∙10-09 / 0.1009
2`-dcGMP (17) / 4.36 ∙10-09 / 0.0521
6-SH-cGMP (13) / 5.25 ∙10-09 / 0.0432
8-Br-PET-cGMP (22) / 2.25 ∙10-09 / 0.1008
2-NH2-cPuMP (21) / 2.98 ∙10-08 / 0.0076
Rp-cGMPS (18) / 8.28 ∙10-08 / 0.0027
8-Br-cAMP(4) / 9.12 ∙10-08 / 0.0024
Rp-8-Br-PET-cGMPS (23) / 1.23 ∙10-07 / 0.0018
6-Bnz-cAMP (2) / 3.34 ∙10-07 / 0.0007
8-pCPT-2`-O-Me-cAMP (10) / 1.04 ∙10-06 / 0.0002
cPuMP (20) / 1.47 ∙10-06 / 0.0002
6-MB-cAMP (1) / 1.88 ∙10-06 / 0.0001
Sp-cGMPS (8) / 8.34 ∙10-05 / 0.00002
IBMX (24) / >10-03 / <10-06
Theophylline (25) / >10-03 / <10-06
EHNA (26) / >10-03 / <10-06

The EC50 values represent means (SEM) of three experiments in duplicate.