New Calcium Indicators and Buffers with High Selectivity against Magnesium and Protons : Design , Synthesis , and Properties of Prototype Structures ?

A new family of high-affinity buffers and optical indicators for Ca2+ is rationally designed and synthesized. The parent compound is 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), a relative of the well-known chelator EGTA [ethylene glycol bis(/3-aminoethyl ether)N,N,N',N'-tetraacetic acid] in which methylene links between oxygen and nitrogen are replaced by benzene rings. BAPTA and its derivatives share the high (>lo5) selectivity for Ca2+ over Mg2+ of EGTA but are very much less affected by pH changes and are faster at taking up and releasing Ca2+. The affinity of the parent compound for Ca2+ (dissociation constant 1.1 X lo-' M in 0.1 M KC1) may be strengthened or weakened by electron-releasing or -withdrawing substituents on the I t is scarcely necessary any longer to stress the importance of intracellular free Ca2+ as a second messenger for external stimuli and as a regulatory ion. There is vast literature on this topic, and several recent symposia have been devoted to the cellular functions of Ca2+ (Duncan, 1976; Scarpa & Carafoli, 1978). A major technical challenge has been to devise satisfactory means for nondestructively measuring intracellular free Ca2+ with good time resolution [for reviews see Kretsinger & Nelson (1976) and Ashley & Campbell (1979)l. The most popular technique has been to use dyes or proteins which change their absorption or luminescence upon binding Ca2+ ions. In the past, these indicators have suffered from several problems (Brown et al., 1977; Thomas, 1979; Moisescu et al., 1975; Blinks et al., 1976, 1978): ( I ) insufficient selectivity against competing cations, particularly H+ and Mg2+; (2) complex stoichiometries of interaction with Ca2+, for example, 1 Ca2+/2 dyes or several Ca2+/1 protein; (3) inflexibility of From the Physiological Laboratory, University of Cambridge, Cambridge CB2 3EG, England. Receiced October 26, 1979. This work was supported by grants from the Science Research Council. 0006-2960/80 /0419-2396$01 . OO/O aromatic rings. The Ca2+ and Mg2+ affinities may further be altered by replacing the ether oxygens by heterocyclic nitrogen atoms. The compounds described are fluorescent Ca2+ indicators absorbing in the ultraviolet region; the very large spectral shifts observed on binding Ca2+ fit the prediction that complexation should hinder the conjugation of the nitrogen lone-pair electrons with the aromatic rings. Derivatives with quinoline nuclei are notable for their high sensitivity of fluorescent quantum yield to the binding of ea2+ but not of Mg2+. Preliminary biological tests have so far revealed little or no binding to membranes or toxic effects following intracellular microinjection. molecular design, or the difficulty of rationally adjusting inicator properties by tinkering with the molecular structure. This paper demonstrates that these problems can be largely overcome by new rational designs and chemical syntheses. The resulting simple compounds are both fluorescent indicators, absorbing in the ultraviolet region, and buffers with distinct advantages over previous buffers. Later papers will describe substantial progress toward the goal of superior visible Ca2+ indicators which can give fast quantitative readout of intracellular free Ca2+ concentrations in a wide variety of interesting physiological systems. Consider EGTAI (ethylene glycol bis(0-aminoethyl ether)-N,N,N',N'-tetraacetic acid) (1). This compound is the only buffer with high selectivity for Ca2+ over Mg2+ to have found significant use in biological research. The Ca2+/Mg2+ I Abbreviations used: EGTA, ethylene glycol bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid; BAPTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; Mops, 3-(N-morpholino)propanesulfonic acid; Hepes. N-2-hydroxyethylpiperazine-N'2-ethanesulfonic acid. D 1980 American Chemical Society N E W I N D I C A T O R S A N D B U F F E R S F O R C A L C I U M