Chemistry Reference
In-Depth Information
Table 1 Relative
photostabilities (
F
/
F
0
) and
quantum yields (
Dye
F
/
F
0
F
F
F
/
F
0
F
F
Cy5
1.0
0.20
0.20
F
F
) in water
3a-dextran
0.95
0.14
0.13
[
2
]
3a
0.53
0.12
0.06
3b
0.41
0.11
0.05
3c
0.13
0.07
0.01
3d
0.53
0.13
0.06
4a
9.1
0.06
0.55
4b
1.6
0.10
0.16
4c
0.44
0.14
0.06
4d
20
0.07
1.40
In general, the aggregates of cyanine dyes photobleach faster than the mono-
meric (nonaggregated) dyes and they also display lower fluorescence quantum
yields (
F
F
)[
2
]. In the series 3a-3d shown in Table
1
, the dye hydrophobicity
increases as the side chain is lengthened causing increased dye aggregation in
aqueous solution. Importantly, the photostability and quantum yields decrease in
the same order: 3a
3c. The water soluble 3a-dextran (not CD)
conjugate exhibits a similar photostability as Cy5 (GE Healthcare) suggesting
that the electronic differences between the sulfonated (Cy5) and nonsulfonated
(3a) chromophores do not have a significant effect on their relative rates of
photobleaching [
2
]. Coupling of dyes 3a-d with 6-amino-6-deoxy-
b
-CD yields
the water-soluble conjugates 4a-d which are more photostable than their parent
dyes (Table
1
). The increase in photostability can be attributed to the reduced
tendency to form aggregates and the protective effect of the CD. Conjugates 4a and
4d are able to emit approximately 2.7- and 7-fold, respectively, more photons than
Cy5, before they are decomposed. When injected into live
Swiss 3T3
cells, con-
jugates 4a and 4d were found to be about 7- and 8-times more photostable than
3a-dextran, which exhibited a similar photostability to Cy5 conjugated to a non-
specific antibody. Cyanine-
3d
>
3b
>
-CD conjugates therefore can be considered a viable
approach to the preparation of fluorescent markers having improved photostability.
The complexation with CDs also results in spectral shifts of the absorption and
emission maxima of cyanine dyes. The complexation of cyanine dyes 1 (X
b
¼
S,
R
¼
Et,
n
¼
1-3) with
b
-CD red-shifts the emission bands [
25
].
-CD, does not form
complexes (pseudorotaxanes) with indocyanines. However, a heptamethine indo-
cyanine dye rotaxane 5
Importantly,
a
-CD
, which has a smaller cave compared to
b
a
-CD
PF
6
, consisting of the symmetrical cyanine 5,
threaded through an
-CD can easily be synthesized on a gram scale in one step
from readily available starting materials - reacting glutaconaldehyde acetate with a
tetramethyl indolium salt in the presence of a
a
-CD in aqueous potassium acetate
(Fig.
4
)[
26
]. The indolenine terminal groups of the cyanine dye were found to be
sufficiently bulky to lock the
a
a
-CD permanently over the center of the chromo-
phore, without the need for any additional bulky stopper groups.
Compared to the absorption and emission spectra of the free dye, the spectra of
the rotaxane 5
a
-CD
PF
6
are sharper and red-shifted. The absorption maximum
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