Published on Web 03/06/2008
Syntheses of Highly Fluorescent GFP-Chromophore
Analogues
Liangxing Wu and Kevin Burgess*
Department of Chemistry, Texas A & M UniVersity, Box 30012, College Station, Texas 77841
Received December 4, 2007; E-mail: burgess@tamu.edu
Abstract: Eight B-containing compounds, i.e., 1a-h, were prepared as mimics of the green fluorescent
protein (GFP) fluorophore. The underlying concept was that synthetic GFP chromophore analogues are
not fluorescent primarily because of free rotation about an aryl-alkene bond (Figure 1b). This rotation is
not possible in the â-barrel of GFP; hence, the molecule is strongly fluorescent. In compounds 1a-h,
radiationless decay via this mechanism is prevented by complexation of the BF2 entity. The target materials
were prepared via two methods; most were obtained according to the novel route shown in Scheme 1b,
but compound 1f was made via the procedure described in Scheme 2. Both syntheses involved formation
of undesired compounds E-4a-h that formed simultaneously with the desired isomeric intermediates Z-4a-
h. Both compounds form BF2 adducts, i.e., 1a-h and 5a-h, respectively. Methods used for spectroscopic
characterization and differentiation of compounds in the series 1 and 5 are discussed, and these are
supported by single-crystal X-ray diffraction analyses for compounds 1c, 5c, 1f, and 5f. Electronic spectra
of compounds 1a-h and 5a-h were studied in detail. Those in the 5 series were shown to be only weakly
fluorescent, but the 1 series were strongly fluorescent compounds (comparable to the boraindacene,
BODIPY, dyes). Compounds 1g and 1h are water soluble, and 1h has particularly significant potential as
a probe, since it also has a carboxylic acid group for attachment to biomolecules.
solution.11,12 They fluoresce at somewhat shorter wavelengths
Introduction
than their parent proteins and with extremely low quantum yields.
Expression of proteins attached to green fluorescent protein
(GFP; Figure 1a) is one of the most widely used strategies for
labeling inside live cells.1-5 Most of the GFP molecule is not
directly useful for the fluoresence of this material; in fact, the
chromophore is relatively small (Figure 1b).6,7 The GFP
chromophore is formed via autocatalytic dehydration of a Ser-
Tyr-Gly tripeptide motif to give an imidazolinone that is then
air-oxidized (Figure 1c).8 Chromophores in GFP analogues are
formed via similar condensation/oxidation routes from other
tripeptides (e.g., blue fluorescent protein, BFP, from Ser-His-
Gly; cyan fluorescent protein, CFP, from Ser-Trp-Gly).1,8
Molecules representing the chromophores of GFP (A9 and
B6) and point mutants (C for Y66F mutant of GFP; D for cyan
fluorescent protein CFP representing the Y66W mutant of GFP;
E for blue fluorescent protein BFP representing the Y66H
mutant of GFP)9 or naturally occurring analogues (F for red
fluorescent protein RFP)10 have been prepared and studied in
This striking difference in quantum yields has been much
discussed in the literature, but the consensus opinion is relatively
simple.13 Loss of fluorescence energy from the synthetic
chromophores in solution is mainly the result of radiationless
transfer arising from free rotation about the aryl-alkene bond
(blue arrows, Figure 2). Isomerization of the alkene (red arrows)
and the polar nature of aqueous media relative to the apolar
environment within the â-barrel protein structures may also be
contributing factors, but the main one is that free rotation
parameter. Steric and electronic factors prevent free rotation of
the aryl substituents when the chromophores are encapsulated
in the proteins. The environment also disfavors E/Z-isomeriza-
tion and provides an apolar medium that is often conducive to
fluorescence.
The research discussed here explores a hypothesis that highly
fluorescent analogues of the GFP chromophore could be made
by including boron. The conceptual genesis of these analogues
1 and the particular compounds that in fact have now been made
are represented in Figure 3. It was supposed that inclusion of
the boron atom would preclude free rotation of the aryl-alkene
bond, so these chromophore analogues would be highly
fluorescent in solution. Further, the structures of the target
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10.1021/ja710388h CCC: $40.75 © 2008 American Chemical Society
J. AM. CHEM. SOC. 2008, 130, 4089-4096
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