.
Angewandte
Communications
DOI: 10.1002/anie.201105653
Bioluminescence
A Selenium Analogue of Firefly d-Luciferin with Red-Shifted
Bioluminescence Emission**
Nicholas R. Conley, Anca Dragulescu-Andrasi, Jianghong Rao, and W. E. Moerner*
Bioluminescence imaging (BLI) makes use of light-generat-
ing luciferase enzymes, most commonly firefly luciferase
(FLuc), Renilla reniformis luciferase, or Gaussia luciferase in
combination with their appropriate substrates.[1] Noninvasive
BLI of living subjects has become a routine technique in
cancer biology research because it enables the monitoring of
gene expression, gene delivery, tumor growth, enzyme
activity, response to experimental drug therapies, and pro-
tein–protein interactions.[2]
the emission maximum because of the polar effect of the
selenium atom, which has been previously reported in
fluorophores.[7] Furthermore, it is known that several enzymes
recognize selenium analogues equally as well as the natural
sulfur-containing substrates,[8] which makes it likely that the
derivative would be an efficient luciferase substrate. Finally,
on account of the red shift induced by the 6’-amino
substituent in 3b, and the usefulness of the amino group in
the preparation of bioluminogenic substrates,[2d,9] we chose to
preserve this functionality in our design.
The major limitation in in vivo BLI experiments is
absorption and scattering of light by tissue, which results in
strong attenuation of BL signals that are emitted below
600 nm.[3] The yellow-green BL emission from the native
FLuc (d-luciferin substrate, lem,max = 553–559 nm),[4] which is
the most red-shifted native bioluminescent system, decreases
substantially with tissue depth. Consequently, the applications
of this system are restricted mainly to small animals at
superficial depths. To overcome this limitation, red-shifted
variants of native FLuc have been selected by using random
mutagenesis.[5] Considerable efforts have also been directed
toward the development of analogues of d-luciferin[6] that
produce a longer wavelength of light, which is an orthogonal
approach to enzyme engineering. A recent report described
the development of a d-luciferin analogue that emits in the
near-infrared region.[6e] This analogue is an aminoluciferin-
Cy5 conjugate and is based on bioluminescence resonance
energy transfer (BRET). This type of modification, however,
alters the cellular uptake properties of the substrate and likely
changes its biodistribution in vivo. Herein, we describe
a simple modification to d-luciferin, the production of
a selenium analogue, which exhibits red-shifted BL emission,
and we demonstrate its use for in vivo BLI.
The synthesis of native d-luciferin (3a)[10b–d] and its
analogue 6’-amino-d-luciferin (3b)[6b] is straightforward and
involves a condensation reaction[10] between 2-cyanobenzo-
thiazole derivative 1a,b and cysteine (2a) (Scheme 1). Com-
pound 3b is a competent substrate for FLuc and exhibits red-
shifted BL emission with lem,max = 578 nm.[11] The only known
d-luciferin analogues that are tolerated by native FLuc
contain 4’- or 6’-substitutions on the benzothiazole ring[6a,b]
or naphthalene or quinoline in place of the benzothiazole,[12]
although aliphatic[6c] and cyclic alkylaminoluciferin[6f] sub-
strates can generate light with mutated variants of FLuc. We
coupled seleno-d-cysteine (2b) with 2-cyano-6-aminobenzo-
thiazole (1b) at room temperature in 0.5m Tris-HCl buffer
(pH 7.5) that contained 13% v/v DMSO to afford amino-
seleno-d-luciferin (3c). The product was isolated by HPLC in
78% yield. Because the l enantiomer of luciferin generates
no BL signal but inhibits FLuc,[13] it is desirable to use
enantiomerically pure 2b in the synthesis, which can be
prepared from elemental selenium.[14] In this manner, [75Se]3c
and [77Se]3c for dual modality BLI/PET and BLI/MRI,
respectively, are readily accessible. Finally, we note that
other chalcogen-substituted cysteine derivatives, such as
tellurocysteine (2c), which is known to react analogously to
2a, might also be successfully incorporated in the reaction
shown in Scheme 1.
We designed a d-luciferin analogue that contains a sele-
nium atom in place of the native sulfur atom at position 1
(3c). We hypothesized that this replacement would red-shift
The absorbance spectra of 3b and its selenium analogue,
3c, are nearly identical; both contain a local maximum at
350 nm in 50 mm Tris-HCl buffer (pH 7.5), and the molar
absorptivities are 15100mÀ1 cmÀ1 and 15500mÀ1 cmÀ1, respec-
tively. The value for 3b is in good agreement with a previous
measurement in 95% ethanol (15500mÀ1 cmÀ1).[6a]
[*] Dr. N. R. Conley,[+] Prof. W. E. Moerner
Department of Chemistry, Stanford University
Stanford, CA 94305-5080 (USA)
E-mail: wmoerner@stanford.edu
Dr. A. Dragulescu-Andrasi,[+] Prof. J. Rao
Department of Radiology, Molecular Imaging Program at Stanford
Stanford University School of Medicine (USA)
[+] These authors contributed equally to this work.
[**] This work was supported by grants 1R01GM086196-01, 1P20-
HG003638, R01CA135294, and ICMIC P50CA114747 from the NIH.
We thank Gaolin Liang and Winston Wei for their assistance with
mice imaging and Eric Johnson at Bruker BioSpin for his assistance
with NMR experiments.
Supporting information for this article is available on the WWW
Scheme 1. Structure and synthesis of d-luciferin (3a), amino-d-luciferin
(3b), and designed aminoseleno-d-luciferin (3c).
3350
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2012, 51, 3350 –3353