Imidazol-1-ylalkanoic acids as extrinsic 1H NMR probes for the determination of intracellular pH, extracellular pH and cell volume.
The synthesis and biological evaluation of a novel series of extrinsic probes for intracellular pH (pHi), extracellular pH (pHo) and cell volume determination by 1H NMR is described. Imidazol-1-ylacetate, malonate, 3-glutarate and 2-succinate esters were synthesized by reaction of imidazole with alpha-bromo esters or with alpha, beta-unsaturated esters. The corresponding acids were prepared by hydrolysis. Rat erythrocytes were readily permeable to methyl imidazol-1-ylacetate, moderately permeable to diethyl 2-imidazol-1-ylsuccinate and impermeable to diethyl 3-imidazol-1-yl-glutarate esters. Imidazol-1-ylacetic acid was the only acid derivative which penetrated the erythrocyte interior when added directly to the incubation medium. Transport of the permeable compounds to the erythrocyte interior was non-saturable up to 200 mM added compound. Addition of methyl imidazol-1-ylacetate or diethyl 2-imidazol-1-ylsuccinate esters to erythrocyte suspensions, resulted in hydrolysis to imidazol-1-ylacetic acid and 2-imidazol-1-ylsuccinic acid mono-ethyl ester in the intracellular and extracellular spaces, respectively. pHi and pHo were determined from the different chemical shifts of the H-2 proton of the acid derivatives in the intracellular (H-2i) and extracellular (H-2o) compartments. In addition, the relative intracellular and extracellular volumes could be calculated from the areas of the intracellular and extracellular H-2 resonances.
Gil
S
S
Departamento de Química Orgánica y Biología, Facultad de Ciencias, UNED, Madrid, Spain.
Zaderenzo
P
P
Cruz
F
F
Cerdán
S
S
Ballesteros
P
P
D000818:Animals; D048429:Cell Size; D004912:Erythrocytes / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism*; D006859:Hydrogen / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000138:chemical synthesis* / Q000737:chemistry; D066298:In Vitro Techniques; D007424:Intracellular Fluid / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D015335:Molecular Probes; D051381:Rats; D017207:Rats, Sprague-Dawley
D007093:Imidazoles; D015335:Molecular Probes; D006859:Hydrogen