Chemistry Reference
In-Depth Information
COO
COO
X
1
X
2
Y
n
S
S
S
S
aTAM
1
H
H
H
3
S
S
S
S
aTAM
2
H
H
3-n
2
tBu
3-n
C
C
aTAM
3
H
n
H
n
1
tBu
S
S
S
S
aTAM
4
Et
H
1
Et
S
S
aTAM
5
Et
Et
D
S
S
1
CY
2
N
H
cTAM
1
(n
=
1)
X
1
cTAM
2
(n
=
2)
X
2
Scheme 16.7
Chemical structures of TAM derivatives containing carboxyl groups, cTAM
1
and cTAM
2
,amino
groups, aTAM
1
, and both carboxyl and amino groups, aTAM
2
-aTAM
5
pH range from 2 to 4, which still could be useful, for example, for the studies of the stomach acidity.
18
However, another limitation of this particular pH probe is its frequency-dependent pH shift, which becomes
impractically small at low field (about 30 mG at L-band). Note that pH-sensitive NRs keep their sensitivity
at low field EPR due to frequency-independent pH effect on the hyperfine splitting (a
N
). A similar effect
was observed for carboxyl group containing TAM derivatives (Scheme 16.7) with one (cTAM
1
)ortwo
(cTAM
2
) protons attached to the aryl groups. cTAM
1
and cTAM
2
demonstrate doublet and triplet EPR
spectral patterns, respectively, with the hydrogen hyperfine splitting (a
H
) reversibly changed in acidic pH
range owing to reversible deprotonation of carboxyl groups.
46
The observed a
H
changes were comparatively
small (about 10 - 20 mG) in agreement with the long distance between COOH group and hydrogen atom,
being located at different phenyl rings. Nevertheless, the data supported the principal of using hyperfine
splitting parameter of TAM derivatives as a pH marker.
Recently synthesized TAMs containing amino groups
47
represent the first pH-sensitive trityl probes with
reasonably valuable spectral properties for application in physiological range of pH from 6.8 to 9.0. The
presence of nitrogen and hydrogen atoms in direct proximity to protonatable amino groups resulted in strong
pH-induced changes of the corresponding hyperfine splittings,
300 - 1000 mG (Figure 16.13).
The superposition of two forms of the aTAM
4
radical at pH around
pK
a
is particularly obvious for
the outermost spectral components, as shown in Figure 16.14a. The high field fractions of the integrated
EPR spectra of aTAM
4
shown in Figure 16.14b demonstrate isosbestic point characteristics for a chemical
equilibrium between two forms of the radical. The pH dependence of the fraction of the aTAM
4
protonated
form shown in Figure 16.14c represents a typical titration curve with
pK
a
=
hf s
≈
1at37
◦
C. The ratio of
the spectral amplitudes of the high field components shown in Figure 16.14a is a convenient experimental
parameter for aqueous acidity measurements using aTAM
4
pH probe allowing for pH measurement in
physiologically relevant pH range of 6.8 to 9.0.
47
Note that changes in oxygen concentration do not
influence the [RH
+
]/[R] ratio of aTAM
4
but result in line broadening in a linearly dependent manner on
oxygen concentration (6 mG/per % [O
2
]).
47
The independent character of pH and [O
2
] effects on the EPR
spectra of aTAM
4
provides dual functionality to this probe, allowing an extraction of both parameters from
asinglespectrum.
TAM radicals, such as Oxo63, were found to be useful probes for EPR oximetry due to both their
high sensitivity to oxygen-induced line broadening (
8
.
0
±
0
.
3mG per % [O
2
]
21
) and the presence of a single
spectral line. The appearance of complex spectral patterns for dual function probes, for example, for aTAM
4
,
decreases their value for oxygen measurements, particularly for oxygen mapping. Further simplification
≈
5
.
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