1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid

Catalog Number	ACM85233198-1
CAS	85233-19-8
Structure
Synonyms	BAPTA
IUPAC Name	2-[2-[2-[2-[Bis(carboxymethyl)amino]phenoxy]ethoxy]-N-(carboxymethyl)anilino]acetic acid
Molecular Weight	476.4
Molecular Formula	C22H24N2O10
Canonical SMILES	C1=CC=C(C(=C1)N(CC(=O)O)CC(=O)O)OCCOC2=CC=CC=C2N(CC(=O)O)CC(=O)O
InChI	InChI=1S/C22H24N2O10/c25-19(26)11-23(12-20(27)28)15-5-1-3-7-17(15)33-9-10-34-18-8-4-2-6-16(18)24(13-21(29)30)14-22(31)32/h1-8H,9-14H2,(H,25,26)(H,27,28)(H,29,30)(H,31,32)
InChI Key	FTEDXVNDVHYDQW-UHFFFAOYSA-N
Melting Point	177-179 °C
Purity	97%+
Complexity	613
Covalently-Bonded Unit Count	1
Defined Atom Stereocenter Count	0
Exact Mass	476.14309497
Heavy Atom Count	34
Hydrogen Bond Acceptor Count	12
Hydrogen Bond Donor Count	4
Monoisotopic Mass	476.14309497
Physical State	Powder
Rotatable Bond Count	15
Topological Polar Surface Area	174 Ų

Knowledge & Learning Case Study Q&A

Protective Effects of 1,2-Bis(2-aminophenoxy)Ethane-N,N,N',N'-Tetraacetic Acid Against Caffeine-Induced Apoptosis

Jang M-H, et al. Neuroscience Letters, 2004, 358(3),189-192.

This case study investigates the protective effects of 1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid (BAPTA-AM), an intracellular Ca2+ chelator, against caffeine-induced apoptosis in SK-N-MC human neuroblastoma cells. The study demonstrates that BAPTA-AM mitigates caffeine's cytotoxic effects by reducing apoptotic markers and caspase-3 activation.
Methods: SK-N-MC human neuroblastoma cells were treated with varying concentrations of caffeine (0.1, 0.5, 1, 5, 10 mM) and BAPTA-AM (1 μM, 10 μM). Cell viability was determined using the MTT assay. Apoptotic markers were analyzed using DAPI staining, TUNEL assay, flow cytometry, DNA fragmentation assay, and Western blot analysis. Caspase-3 activity was measured to assess the apoptotic pathway.
Results:
Cell Viability: Caffeine treatment significantly reduced cell viability in a dose-dependent manner, with the lowest viability at 10 mM caffeine. BAPTA-AM treatment improved cell viability in caffeine-treated cells.
Morphological Changes: Caffeine induced cell detachment, rounding, and cytoplasmic blebbing. These changes were less pronounced in cells treated with BAPTA-AM.
DAPI and TUNEL Assays: Caffeine caused nuclear condensation, DNA fragmentation, and formation of apoptotic bodies. BAPTA-AM reduced these apoptotic indicators.
Flow Cytometry: Caffeine increased the sub-G1 cell population, indicative of apoptosis, which was reduced by BAPTA-AM treatment.
DNA Fragmentation: Caffeine treatment led to a characteristic DNA ladder pattern, signifying apoptosis. This was attenuated by BAPTA-AM.
Protein Expression: Caffeine increased the expression of pro-apoptotic bax and decreased anti-apoptotic bcl-2 levels. BAPTA-AM reversed these changes, promoting cell survival.
Caspase-3 Activity: Caffeine significantly increased caspase-3 activity, which was reduced by BAPTA-AM treatment.
Conclusion: The findings indicate that caffeine induces apoptosis in neuroblastoma cells, characterized by increased caspase-3 activity and altered bax/bcl-2 expression. BAPTA-AM effectively mitigates these effects, demonstrating its protective role by reducing caspase-3 activation and maintaining the balance between pro- and anti-apoptotic proteins.
BAPTA-AM shows significant protective effects against caffeine-induced apoptosis in SK-N-MC human neuroblastoma cells. This suggests its potential as a therapeutic agent for preventing caffeine-induced cytotoxicity in neural cells.

Effects of 1,2-Bis(2-aminophenoxy)Ethane-N,N,N',N'-Tetraacetic Acid on Phospholipase C Activity

Hardie R.C. Cell Calcium, 2005, 38(6), 547-556.

This case study explores the role of 1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid (BAPTA) in modulating phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis by phospholipase C (PLC) in Drosophila photoreceptors. The investigation utilizes electrophysiological biosensors to assess both light-induced and basal PLC activity, highlighting BAPTA's dual function as a Ca²⁺ chelator and an inhibitor of PLC.
Methods:
Electrophysiological Monitoring: Light-induced and basal hydrolysis of PIP2 by PLC was monitored in Drosophila photoreceptors.
Ca²⁺ Buffering: Cells were loaded with different concentrations of Ca²⁺ buffered by 4 mM free BAPTA.
BAPTA Concentration Variation: The total BAPTA concentration was varied while maintaining constant free [Ca²⁺].
Inhibition Analysis: The inhibitory effects of BAPTA and its analog, di-bromo BAPTA (DBB), on PLC activity were assessed. EGTA was used as a comparative Ca²⁺ chelator.
Results:
Ca²⁺ Dependence: Light-induced PLC activity displayed a bell-shaped dependence on free Ca²⁺, peaking at around 100 nM. At <10 nM or ~1 μM Ca²⁺, PLC activity was inhibited approximately tenfold.
BAPTA Inhibition: Varying BAPTA concentrations while keeping free [Ca²⁺] constant showed that PLC inhibition at higher Ca²⁺ levels (>100 nM) was due to BAPTA itself, with an IC50 of approximately 8 mM.
DBB Potency: DBB was a more potent inhibitor of PLC activity, with an IC50 of around 1 mM.
Basal PLC Activity: Both BAPTA and DBB modestly inhibited basal PLC activity.
EGTA Comparison: EGTA did not inhibit PLC activity when pre-loaded with Ca²⁺, but like BAPTA, inhibited both basal and light-induced PLC activity when introduced without Ca²⁺.
Conclusion:
The study reveals that BAPTA and DBB inhibit PLC activity independently of their Ca²⁺ chelation properties. This inhibition is evident at non-physiologically low Ca²⁺ levels (<100 nM), which suppress both basal and light-induced PLC activity. These findings indicate that BAPTA and DBB can directly modulate PLC activity, apart from their primary role in buffering intracellular Ca²⁺. BAPTA significantly impacts PLC activity in Drosophila photoreceptors, acting not only as a Ca²⁺ chelator but also as a direct inhibitor of PLC.

Evaluation of 1,2-Bis(2-aminophenoxy)Ethane-N,N,N',N'-Tetraacetic Acid as A Calcium Chelator

Britigan BE, et al. Biochemical Pharmacology, 1998, 55(3), 287-295.

This case study explores the involvement of Ca²⁺ in this process by assessing the effects of two Ca²⁺ chelating agents: Fura-2 and 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA).
Intracellular calcium (Ca²⁺) is implicated in the pathophysiology of various oxidant-mediated cell injuries. Recent studies have highlighted the role of iron bound to the Pseudomonas aeruginosa siderophore, pyochelin, in exacerbating oxidant-mediated endothelial cell injury by catalyzing the formation of hydroxyl radicals (HO·).
In the experiments, BAPTA, but not Fura-2, demonstrated a protective effect against H₂O₂/ferripyochelin-mediated injury. The protective mechanism of BAPTA appears to be linked to its iron-chelating properties rather than its ability to chelate Ca²⁺. Spectrophotometric analysis showed that BAPTA forms complexes with both ferrous (Fe²⁺) and ferric (Fe³⁺) iron, with an affinity hierarchy of Fe³⁺ > Ca²⁺ > Fe²⁺. This iron-binding capacity of BAPTA significantly reduced the iron-catalyzed production of HO· and ferryl species, as evidenced by spin trapping analysis.
Although our findings do not definitively confirm that BAPTA protects endothelial cells from ferripyochelin-associated damage through iron chelation, they suggest that the observed protection is likely due to this mechanism. Consequently, our data highlight the need for caution when interpreting the protective effects of intracellular Ca²⁺ chelating agents. Specifically, the use of these agents as evidence for the involvement of Ca²⁺ in cell injury must account for potential concurrent iron-mediated oxidant production.
In conclusion, while BAPTA's protective effect against H₂O₂/ferripyochelin-mediated endothelial cell injury underscores its iron-chelating capabilities, further studies are needed to fully elucidate its mechanism of action. This case study emphasizes the importance of considering alternative explanations when assessing the role of Ca²⁺ in cellular injury, particularly in scenarios where iron-mediated oxidative stress is a factor.

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