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OH
O
2,3
end
C
6
Inside H
3
Outside H
2,4
O
C
4
C
2
C
5
O
C
1
HO
C
3
Inside H
5
Outside H
1
OH
O
O
6
end
SCHEME 9.6
A macrocyclic oligosaccharide cyclodextrin forming a torus-shaped structure of cyclodex-
trin with rigid lipophilic cavities. (Adapted from Polyakov, N.E.,
Free Rad. Biol. Med
., 36, 872, 2004. With
permission.)
9.8 CAROTENOID INTERACTION WITH SURROUNDINGS: ESEEM METHOD
Advanced EPR techniques such as CW and pulsed ENDOR, electron spin-echo envelope modula-
tion (ESEEM), and two-dimensional (2D)-hyperi ne sublevel correlation spectroscopy (HYSCORE)
have been successfully used to examine complexation and electron transfer between carotenoids
and the surrounding media in which the carotenoid is located.
Resolvable modulation is detected on a three-pulse echo decay spectrum of predeuterated
β
-carotene radical (Gao et al. 2005) as a function of delay time,
T
. The resulting modulation is known
as ESEEM. Resolvable modulation will not be detected for nondeuterated
-carotene radical since
the proton frequency is six times larger. The modulation signal intensity is proportional to the square
root of phase sensitive detection and interfering two-pulse echoes and suppressed by phase-cycling
technique (Gao et al. 2005). Analysis of the ESEEM spectrum yields the distance from the radical to
the D nucleus,
a
the deuterium coupling constant, and the number of equivalent interacting nuclei (D).
The details related to the analysis of the ESEEM spectrum are presented in Gao et al. 2005.
Pulsed ENDOR spectra can be recorded with a Bruker E-560 pulsed ENDOR acces-
sory and an A-500 RF power amplii er using the Davies (
β
π−
T
−π
/2
−τ−π−τ−
echo) and Mims
(
π
/2
−τ−π
/2
−
T
−π
/2
−τ−
echo) sequences where the additional RF
π
-pulse was applied during separa-
tion time,
T
. For the initial
664 ns for the Davies sequence of pulses, the proton couplings less
than 1 MHz are not resolved, and the Davies ENDOR spectrum lacks spectral lines ±0.5 MHz
around the free proton frequency. On the other hand, the Mims ENDOR will miss spectral lines
due to couplings of 5, 10, and 15 MHz due to
τ =
200 ns. The Mims pulsed ENDOR sequence aids in
detecting powder ENDOR spectra containing couplings less than 10 MHz where powder averaging
may broaden and make the large couplings difi cult to detect. Pulsed ENDOR hyperi ne coupling
simulations were carried out using the SimBud/SpecLab spectral simulation programs.
HYSCORE, is a 2D four-pulse ESEEM technique which provides correlation between nuclear
frequencies originating from different electron manifolds. The sequence of four microwave pulses
is
τ =
π
/2
−τ−π
/2
−
t
1
−π−
t
2
−π
/2
−τ−
echo where the echo amplitude is measured as a function of
t
1
and
t
2
at i xed
-proton anisotropic couplings can be detected by this technique (Konovalova et al.
2001a, Focsan et al. 2008).
τ
. The
α
9.8.1 B
ONDING
OF
b
-C
AROTENE
TO
C
U
2
+
IN
C
U
-MCM-41
ESEEM measurements of perdeuterated all-
trans
-
-carotene imbedded in activated Cu-substituted
MCM-41 molecular sieve revealed (Gao et al. 2005) that two deuterons of the carotenoid interact
with the Cu
2+
at a distance of 3.3 Å. Possible double bonds of
β
β
-carotene with one deuterium at each
carbon that could interact with Cu are C7
=
C8, CH-C12, C15
=
C15, C12
′
- C11
′
, and C8
′ =
C7.
The narrow, resolved lines observed in the
1
H-ENDOR spectrum of
β
-carotene indicated that
the methyl protons at the 5 or 5
′
and the 9 or 9
′
carbon positions were undergoing rapid rotation.
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