Sd. Clarke et al., DESIGN AND PROPERTIES OF NEW F-19 NMR CA2- MODULATION OF THE AFFINITIES OF BAPTA DERIVATIVES VIA ALKYLATION( INDICATORS ), Perkin transactions. 2, (6), 1993, pp. 1187-1194
The effects of alkyl substitution in the aminodiacetic groups of ,2-bi
s(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) have been e
xamined systematically with the aim of increasing the affinity for Ca2
+ of novel F-19 NM R and fluorescent Ca2+ indicators based on the BAPT
A structure into the range required to measure the cytosolic free Ca2 concentration ([Ca2+]i). Single methylation on the beta-carbon of eac
h aminodiacetic acid group of BAPTA derivatives gave increases in the
association constant for Ca2+ [K(Ca2+)] of up to 22-fold, depending on
the structure of the parent BAPTA derivative. This was shown to be th
e most effective alkylation for BAPTA derivatives to raise K(Ca2+) wit
h a minimal increase in pK(a), and no loss of selectivity for Ca2+, so
that the insensitivity of Ca2+ binding to pH and [Mg2+] over the phys
iological ranges was maintained. (1) Single methylation on the beta-ca
rbon of each aminodiacetic acid group of BAPTA derivatives symmetrical
ly substituted with single fluorines at the 4-position (4FBAPTA) or at
the 5-position (5FBAPTA) raised log K(Ca2+) from 5.61 to 6.81 and 6.1
2 to 7.47, respectively. The corresponding pK(av) values (average of t
he two highest pK(a) values in the molecule) were increased from 4.50
to 6.02 and 5.85 to 6.59. The F-19 NMR spectroscopic properties of the
methylated derivatives were unchanged compared with the parent compou
nds except for slower exchange rates. These new methylated derivatives
of 4FBAPTA and 5FBAPTA therefore extend the range of K(Ca2+) availabl
e for F-19 NMR indicators of [Ca2+]i. (2) Single methylation on the be
ta-carbon of each aminodiacetic acid group of BAPTA derivatives symmet
rically substituted at the 5-position on the aromatic rings with trifl
uoromethyl groups increased log K(Ca2+) from 4.83 to 6.14 and the pK(a
v) from 5.39 to 5.90, respectively. There is a Ca2+-induced chemical s
hift of the F-19 NMR resonance of 0.81 ppm upfield with slow exchange
at 376 MHz and 30-degrees-C. This compound was selected as a prototype
for F-19 NMR indicators with enhanced sensitivity through the incorpo
ration of two trifluoromethyl groups to replace the two single fluorin
e substituents in the current F-19 NMR indicators.