INQUIMAE   12526
INSTITUTO DE QUIMICA, FISICA DE LOS MATERIALES, MEDIOAMBIENTE Y ENERGIA
Unidad Ejecutora - UE
artículos
Título:
Masked Rhodamine Dyes of Five Principal Colors Revealed by Photolysis of a 2-Diazo-1-Indanone Caging Group: Synthesis, Photophysics, and Light Microscopy Applications
Autor/es:
BELOV, VLADIMIR N.; MITRONOVA, GYUZEL YU. ; BOSSI, MARIANO LUIS; BOYARSKIY, VADIM P. ; HEBISCH, ELKE; GEISLER, CLAUDIA; KOLMAKOV, KIRILL; WURM, CHRISTIAN A ; WILLIG, KATRIN I.; HELL, STEFAN W.
Revista:
CHEMISTRY-A EUROPEAN JOURNAL
Editorial:
WILEY-V C H VERLAG GMBH
Referencias:
Lugar: Weinheim; Año: 2014 vol. 20 p. 13162 - 13173
ISSN:
0947-6539
Resumen:
Caged rhodamine dyes (Rhodamines NN) of five basic colors were
synthesized and used as ?hidden? markers in subdiffractional and
conventional light microscopy. These masked fluorophores with a
2-diazo-1-indanone group can be irreversibly photoactivated, either by
irradiation with UV- or violet light (one-photon process), or by
exposure to intense red light (λ∼750 nm; two-photon mode). All
dyes possess a very small 2-diazoketone caging group incorporated into
the 2-diazo-1-indanone residue with a quaternary carbon atom (C-3) and a
spiro-9H-xanthene fragment. Initially they are non-colored (pale yellow), non-fluorescent, and absorb at λ=330?350 nm (molar extinction coefficient (ε)≈104 M−1 cm−1) with a band edge that extends to about λ=440 nm. The absorption and emission bands of the uncaged derivatives are tunable over a wide range (λ=511?633 and 525?653 nm, respectively). The unmasked dyes are highly colored and fluorescent (ε= 3?8×104 M−1 cm−1 and fluorescence quantum yields (ϕ)=40?85
% in the unbound state and in methanol). By stepwise and orthogonal
protection of carboxylic and sulfonic acid groups a highly water-soluble
caged red-emitting dye with two sulfonic acid residues was prepared.
Rhodamines NN were decorated with amino-reactive N-hydroxysuccinimidyl
ester groups, applied in aqueous buffers, easily conjugated with
proteins, and readily photoactivated (uncaged) with λ=375?420 nm light or intense red light (λ=775 nm). Protein conjugates with optimal degrees of labeling (3?6) were prepared and uncaged with λ=405 nm light in aqueous buffer solutions (ϕ=20?38
%). The photochemical cleavage of the masking group generates only
molecular nitrogen. Some 10?40 % of the non-fluorescent (dark)
byproducts are also formed. However, they have low absorbance and do not
quench the fluorescence of the uncaged dyes. Photoactivation of the
individual molecules of Rhodamines NN (e.g., due to reversible or
irreversible transition to a ?dark? non-emitting state or
photobleaching) provides multicolor images with subdiffractional optical
resolution. The applicability of these novel caged fluorophores in
super-resolution optical microscopy is exemplified.