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Synthesis of Caged NAADP Antony Galione, Kai-Ting Chuang, Tim M. Funnell, Lianne C. Davis, Anthony J. Morgan, Margarida Ruas, John Parrington and Grant C. Churchill Cold Spring Harb Protoc; doi: 10.1101/pdb.prot076943 Email Alerting Service Subject Categories
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Protocol
Synthesis of Caged NAADP Antony Galione,1 Kai-Ting Chuang, Tim M. Funnell, Lianne C. Davis, Anthony J. Morgan, Margarida Ruas, John Parrington, and Grant C. Churchill Department of Pharmacology, University of Oxford, Oxford OX1 3QT, United Kingdom
Caged derivatives of Ca2+-mobilizing messengers, such as nicotinic acid adenine dinucleotide phosphate (NAADP), are particularly useful for establishing the effects of these messengers on Ca2+ signaling. Caged NAADP is no longer commercially available but can be synthesized in house, as described here. In brief, a stable precursor of the caging reagent is made and converted to an unstable reactive reagent immediately before addition to the compound to be caged.
MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol.
Reagents
Chloroform Diethylether Ethanol (95%) Glacial acetic acid Hydrazine monohydrate HCl (concentrated) MgSO4 (anhydrous) MnO2 NAADP (Sigma-Aldrich) 2-Nitroacetophenone Triethyl ammonium bicarbonate (TEAB) Equipment
MonoQ column Cotton wool Diatomaceous earth Erlenmeyer flask (250 mL) HPLC system pH paper or micro pH electrode 1
Correspondence: [email protected]
© 2014 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot076943
1105
Downloaded from http://cshprotocols.cshlp.org/ at COLUMBIA UNIVERSITY on October 1, 2014 - Published by Cold Spring Harbor Laboratory Press
A. Galione et al.
Pipette tip (5 mL) Reflux apparatus (a round-bottom flask and a condenser) Rotary evaporator Round bottom flask (250 mL) Separatory funnel (250 mL) Whatman No. 1 paper
METHOD
1. Synthesize 2-nitroacetophenone hydrazone. i. Place 100 mL of 95% ethanol, 10 g of 2-nitroacetophenone, 3.2 mL of glacial acetic acid, and 5.6 g of hydrazine monohydrate into a 250-mL round-bottom flask. ii. Heat the mixture under reflux for 3 h and then cool. Remove the ethanol by rotary evaporation. iii. Add 100 mL of double-distilled water and 100 mL of chloroform. Transfer solution to a separatory funnel (250 mL), shake, remove the upper (aqueous) layer and discard, and repeat twice more, each time add 100 mL of double-distilled water. iv. Place the chloroform phase into a 250-mL Erlenmeyer flask and dry by adding 3 g of anhydrous MgSO4. Swirl for a few minutes and then filter through Whatman No. 1 paper into a 250-mL round-bottom flask. v. Remove chloroform by rotary evaporation. Transfer product (a yellow oil) to a foil-wrapped container. Store at −20˚C until needed. We have successfully stored this product for 2 yr.
2. In preparation for the actual caging reaction, dissolve 10 mg of NAADP in 1 mL of doubledistilled water. Adjust pH to 1.5 using concentrated HCl (if a salt form of NAADP is used, 7 µL is required) and either pH paper or a micro pH electrode. Bring volume of solution up to 2 mL with double-distilled water. 3. Synthesize 1(2-nitrophenyl)diazoethane: Into a 5-mL round-bottom flask, place (in this order) a magnetic stir bar, 20 mL of 2-nitroacetophenone hydrazone, 2 mL of diethylether, and 60 mg of MnO2 while stirring vigorously. Stir for 20 min. Perform this reaction immediately before the caging reaction in a fume hood. Note that the product of this reaction, like all diazoethane derivatives, is thermally unstable and light sensitive, and can explode. This danger is minimal if it is used in low amounts (
Synthesis of Caged NAADP Antony Galione, Kai-Ting Chuang, Tim M. Funnell, Lianne C. Davis, Anthony J. Morgan, Margarida Ruas, John Parrington and Grant C. Churchill Cold Spring Harb Protoc; doi: 10.1101/pdb.prot076943 Email Alerting Service Subject Categories
Receive free email alerts when new articles cite this article - click here. Browse articles on similar topics from Cold Spring Harbor Protocols. Cell Biology, general (1064 articles) Signal Transduction (44 articles)
To subscribe to Cold Spring Harbor Protocols go to:
http://cshprotocols.cshlp.org/subscriptions
© 2014 Cold Spring Harbor Laboratory Press
Downloaded from http://cshprotocols.cshlp.org/ at COLUMBIA UNIVERSITY on October 1, 2014 - Published by Cold Spring Harbor Laboratory Press
Protocol
Synthesis of Caged NAADP Antony Galione,1 Kai-Ting Chuang, Tim M. Funnell, Lianne C. Davis, Anthony J. Morgan, Margarida Ruas, John Parrington, and Grant C. Churchill Department of Pharmacology, University of Oxford, Oxford OX1 3QT, United Kingdom
Caged derivatives of Ca2+-mobilizing messengers, such as nicotinic acid adenine dinucleotide phosphate (NAADP), are particularly useful for establishing the effects of these messengers on Ca2+ signaling. Caged NAADP is no longer commercially available but can be synthesized in house, as described here. In brief, a stable precursor of the caging reagent is made and converted to an unstable reactive reagent immediately before addition to the compound to be caged.
MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol.
Reagents
Chloroform Diethylether Ethanol (95%) Glacial acetic acid Hydrazine monohydrate HCl (concentrated) MgSO4 (anhydrous) MnO2 NAADP (Sigma-Aldrich) 2-Nitroacetophenone Triethyl ammonium bicarbonate (TEAB) Equipment
MonoQ column Cotton wool Diatomaceous earth Erlenmeyer flask (250 mL) HPLC system pH paper or micro pH electrode 1
Correspondence: [email protected]
© 2014 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot076943
1105
Downloaded from http://cshprotocols.cshlp.org/ at COLUMBIA UNIVERSITY on October 1, 2014 - Published by Cold Spring Harbor Laboratory Press
A. Galione et al.
Pipette tip (5 mL) Reflux apparatus (a round-bottom flask and a condenser) Rotary evaporator Round bottom flask (250 mL) Separatory funnel (250 mL) Whatman No. 1 paper
METHOD
1. Synthesize 2-nitroacetophenone hydrazone. i. Place 100 mL of 95% ethanol, 10 g of 2-nitroacetophenone, 3.2 mL of glacial acetic acid, and 5.6 g of hydrazine monohydrate into a 250-mL round-bottom flask. ii. Heat the mixture under reflux for 3 h and then cool. Remove the ethanol by rotary evaporation. iii. Add 100 mL of double-distilled water and 100 mL of chloroform. Transfer solution to a separatory funnel (250 mL), shake, remove the upper (aqueous) layer and discard, and repeat twice more, each time add 100 mL of double-distilled water. iv. Place the chloroform phase into a 250-mL Erlenmeyer flask and dry by adding 3 g of anhydrous MgSO4. Swirl for a few minutes and then filter through Whatman No. 1 paper into a 250-mL round-bottom flask. v. Remove chloroform by rotary evaporation. Transfer product (a yellow oil) to a foil-wrapped container. Store at −20˚C until needed. We have successfully stored this product for 2 yr.
2. In preparation for the actual caging reaction, dissolve 10 mg of NAADP in 1 mL of doubledistilled water. Adjust pH to 1.5 using concentrated HCl (if a salt form of NAADP is used, 7 µL is required) and either pH paper or a micro pH electrode. Bring volume of solution up to 2 mL with double-distilled water. 3. Synthesize 1(2-nitrophenyl)diazoethane: Into a 5-mL round-bottom flask, place (in this order) a magnetic stir bar, 20 mL of 2-nitroacetophenone hydrazone, 2 mL of diethylether, and 60 mg of MnO2 while stirring vigorously. Stir for 20 min. Perform this reaction immediately before the caging reaction in a fume hood. Note that the product of this reaction, like all diazoethane derivatives, is thermally unstable and light sensitive, and can explode. This danger is minimal if it is used in low amounts (
