Biology Reference
In-Depth Information
Administration for clinical use and is considered as a “gold standard” for
fluorophores for in vivo applications.
Cyanines are formally compounds with two nitrogen atoms linked by an
odd number of methene units. 26-28 The nitrogen atoms are parts of the
heterocyclic units (such as indole, benzoxazol, or benzothiazol). The
structures and optical properties of representative cyanine dyes used for
in vivo imaging are presented in Chart 3.1 . Cyanines are characterized by
long wavelength, tunable absorption and emission, very high extinction
coefficient (up to 300,000 M -1 cm -1 ), good water solubility, and relatively
straightforward synthesis. The wavelengths of absorption and emission in
cyanines can be tuned and shifted toward longer wavelengths either by
changing the number of carbon atoms in the polymethine chain or by
expanding the aromatic part of the terminal heterocyclic units. The
increase of polymethine chain by two carbon atoms shifts bathochromically
the absorption band by
100 nm, whereas fusing the benzo ring at the
terminal indole moiety shifts the absorption band by about 30 nm (see data
in Chart 3.1 , as well as Refs. 29 and 30 ). The long-wavelength absorbing
and emitting cyanine fluorophores suitable for in vivo applications are (a)
pentamethine cyanines with an additional benzene ring fused to the
terminal indole moieties (e.g., Cy5.5, Chart 3.1 ), with absorption and
emission at
675/695 nm 29 ; (b) heptamethine cyanines, with an indole
terminal moiety, absorbing/emitting at 750-790 and 780-820 nm,
respectively (e.g., Cy7, 30 cybate, 34,35 and NIR-820 31 ); and (c)
heptamethine cyanines with benzoindole as terminal moieties absorbing/
emitting around 780-822 and 810-847 nm (e.g., ICG, 32,33 cypate 34,35 ,and
CyTE-822 36 ). The main design and synthetic efforts have been recently
dedicated to preparing derivatives suitable for further modifications (e.g.,
mono- and polyvalent bioconjugatable cyanines) and methods to improve
water solubility and chemical and photochemical stability of cyanines.
2.2. Bioconjugation
In vivo application of any fluorophores often requires attaching a targeting
group, or conjugation to biomolecules (such as antibodies), which ensures
selective localization of fluorophores in the target cells, tissues, or organs. 14
Therefore, availability of fluorophore derivatives with reactive functional
groups that are suitable for bioconjugation is an important issue in designing
new fluorophores. Bioconjugation of any molecule is usually achieved by
attaching an amino-reactive N -hydroxysuccinimide ester
(formed by
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