Design and Synthesis of an Alkynyl Luciferin Analogue for Bioluminescence Imaging

Article abstract of DOI:10.1002/chem.201503944

Herein, the synthesis and characterization of an alkyne-modified luciferin is reported. This bioluminescent probe was accessed using C-H activation methodology and was found to be stable in solution and capable of light production with firefly luciferase. The luciferin analogue was also cell permeant and emitted more redshifted light than d-luciferin, the native luciferase substrate. Based on these features, the alkynyl luciferin will be useful for a variety of imaging applications.

Full text of DOI:10.1002/chem.201503944

DOI: 10.1002/chem.201503944
Communication
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Organic Chemistry |Hot Paper|
Design and Synthesis of an Alkynyl Luciferin Analogue for
Bioluminescence Imaging
Rachel C. Steinhardt,[a] Jessica M. O’Neill,[a] Colin M. Rathbun,[a] David C. McCutcheon,[a]
Miranda A. Paley,[a] and Jennifer A. Prescher *[a, b, c]
firefly luciferase (Fluc), catalyze the oxidation of d-luciferin (1)
Abstract: Herein, the synthesis and characterization of an
and release ꢀ500–600 nm light (Figure 1a).[2b,3] Wavelengths
alkyne-modified luciferin is reported. This bioluminescent
of this sort can penetrate the skin of small rodents and be de-
probe was accessed using CÀH activation methodology
tected by sensitive cameras, making insect luciferases attrac-
and was found to be stable in solution and capable of
tive for imaging in vivo.[6] Indeed, Fluc and related enzymes
light production with firefly luciferase. The luciferin ana-
have been expressed in a variety of tissue and cell types, and
logue was also cell permeant and emitted more redshifted
when exposed to d-luciferin, light is produced.[1] d-Luciferin is
light than d-luciferin, the native luciferase substrate. Based
on these features, the alkynyl luciferin will be useful for
extensively in preclinical models.[8]
a variety of imaging applications.
also sufficiently bioavailable in rodents[7] and has been used
Because of the sensitivity and user-friendly features of biolu-
minescence, there has been much interest in expanding the
Bioluminescence is a versatile imaging platform with
applications ranging from metabolite biosensing to
whole animal imaging.[1] At the heart of this technol-
ogy are enzymes (luciferases) that catalyze the oxida-
tion of small molecule substrates (luciferins).[2] During
each enzymatic transformation, an electronically ex-
cited oxyluciferin is generated that emits a photon of
light upon relaxation to the ground state.[3] Since
mammalian cells do not produce large numbers of
Figure 1. a) The luciferase-catalyzed oxidation of d-luciferin (1) produces visible light.
b) Retrosynthetic analysis of alkynyl luciferin (PG=protecting group).
photons in the absence of incident light, biolumines-
cence can provide an exquisitely sensitive readout on
biological processes in these environments.[4] Indeed,
luciferase–luciferin pairs have been widely used to report on
enzyme activities and gene expression patterns in live cells
and tissue lysates.[1] Additionally, since bioluminescence does
not require an excitation source, this technology is well suited
for noninvasive imaging in whole animals, where delivery of
excitation light is often inefficient or impractical.[1a,5]
scope of the technology.[5d,9] Several efforts have been directed
toward identifying other naturally occurring luciferase–luciferin
pairs for multicomponent imaging.[1a,10] The instability and
poor tissue penetrance of many luciferins have been prohibi-
tive in many cases. Other attempts have focused on generat-
ing luciferases that provide altered emission spectra. For exam-
ple, several insect luciferases have been engineered to emit
different colors of light (ranging from ꢀ500–650 nm) with d-
luciferin.[11] While these wavelengths can be adequately re-
solved in vitro, they cannot be easily discriminated in vivo,
where tissue absorption and scatter modulate the color of
light that ultimately reaches the detector.[6]
The most widely used luciferases for cell and animal imaging
originate from the insect family.[1b] These enzymes, including
[a] R. C. Steinhardt, J. M. O’Neill, C. M. Rathbun, Dr. D. C. McCutcheon,
Dr. M. A. Paley, Prof. Dr. J. A. Prescher
Department of Chemistry, University of California, Irvine, Irvine, CA (USA)
E-mail: jpresche@uci.edu
Compared to luciferase engineering efforts, there has been
less work invested in crafting new luciferins. Substrate engi-
neering is an obvious strategy to broaden the scope of biolu-
minescence technology, as the luciferin molecules can be
modified to emit different colors of light or be selectively uti-
lized by unique luciferases.[12,13] In some cases, the substrates
have proven remarkably cell and tissue permeant and, thus,
well suited for in vivo work.[14]
[b] Prof. Dr. J. A. Prescher
Department of Molecular Biology & Biochemistry
University of California, Irvine, Irvine, CA (USA)
[c] Prof. Dr. J. A. Prescher
Department of Pharmaceutical Sciences
University of California, Irvine, Irvine, CA (USA)
Supporting information and ORCID(s) from the author(s) for this article are
available on the WWW under http://dx.doi.org/10.1002/chem.201503944.
Part of a Special Issue “Women in Chemistry” to celebrate International
Women’s Day 2016. To view the complete issue, visit: http://dx.doi.org/
chem.v22.11.
Continued efforts to develop unique bioluminescent tools
would benefit from rapid access to diverse collections of light-
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emitting luciferins. These scaffolds have been notoriously diffi-
cult to synthesize owing to their electron-rich and highly sub-
stituted cores. Late-stage modifications to luciferin molecules
are also complicated. For example, most attempts to derivatize
d-luciferin (1) have focused on altering the 6’-position by alkyl-
ation or acylation chemistries.[7a,15] While facile, these strategies
have produced scaffolds that are somewhat limited in scope.
Electron donation is required for robust emission and, thus,
the 6’-position is particularly sensitive to modification.
We aimed to develop a bioluminescent probe modified at
an alternative ring position. We were initially drawn to the 5’-
alkyne derivative (2) shown in Figure 1b. Previous work estab-
lished that 5’-fluoro and other small substituents were well tol-
erated by Fluc and minimally perturbing to the bioluminescent
reaction.[12b] Modeling analyses suggested that the alkyne
would be similarly accommodated in the luciferase active site
(Figure 2a). Furthermore, computational data[16] indicated that
2 would be a viable light emitter (Figure 2b).
We were further attracted to alkyne 2 as its benzothiazole
core could be accessed using CÀH activation chemistry previ-
ously reported by our group.[17] However, the functionalized lu-
ciferin still presented some synthetic challenges. Electron-rich
heterocycles like 2 are susceptible to nonspecific oxidation and
are thus difficult to handle and prepare on scale. Methods to
produce highly substituted benzothiazoles are also rare.
Figure 2. In silico analyses of d-luciferin. a) Overlay of 2 with firefly luciferase
(PDB ID: 4G36) suggests that the alkyne motif will be tolerated. b) B3LYP/6-
311** MO predictions[16] of the HOMO (middle) and LUMO (bottom) of the
oxidzed product (top).
To access the desired heterocycle, we began with trisubsti-
tuted phenol 3. The hydroxy substituent was first protected
with a mesyl group (Scheme 1).[18] Other classic phenol protect-
ing groups (e.g., silyl and methyl) were explored, but most
proved either incompatible with subsequent transformations
(in the case of bulky silyl groups) or difficult to remove later
on in the synthesis (in the case of methyl groups). Mesylate 4
was ultimately subjected to Sonogashira conditions for alkyne
installation. Notably, this reaction was readily scalable and pro-
vided decagram quantities of 5 (Scheme S1, Supporting Infor-
mation). The nitro group of 5 was reduced using iron filings
and glacial acetic acid[19] to reveal aniline 6 in good yield and
purity. Compound 6 was then treated with Appel’s salt 7, and
the resulting adduct was fragmented with resin-linked PPh3 to
yield thioamide 9 (Scheme 2).[20] It should be noted that while
other bulky nucleophiles (e.g., DBU and DBA)[21] can be used
for such fragmentations, they resulted in premature deprotec-
tion of the mesyl group and reduced overall yields in this case.
Subsequent cyclization of thioamide 9 by palladium- and
copper-catalyzed CÀH activation[22] provided 10 in 61% yield.
Attempts to isolate 10 directly from 8 by thermal cyclization
resulted in product decomposition and were not further pur-
sued. The desired alkyne luciferin 2 was ultimately isolated fol-
lowing mesyl-group removal[23] and cysteine condensation. Im-
portantly, luciferin 2 was stable for weeks as a solid material
and in aqueous solution.
Scheme 1. Installation of the alkyne substituent. TEA=triethylamine.
Scheme 2. Synthesis of alkyne luciferin 2 by using CÀH activation chemistry. LiHMDS=lithium hexamethyl disilazide; TBAB=tetrabutylammonium bromide.
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Figure 4. Normalized bioluminescence emission spectra for alkynyl luciferin
2 (lmax 610 nm) and d-luciferin 1 (lmax 565 nm). Samples (100 mm) were com-
bined with Fluc (10 mg) and monitored at 258C.
Figure 3. a) Alkynyl luciferin 2 produces light upon incubation with Fluc. Sol-
utions of 2 (0.5–100 mm) were mixed with Fluc, ATP, and CoA in pH 8 buffer
in 96-well plates. Light emission was measured using a cooled CCD camera.
Sample images are shown in the inset. b) Analogue 2 exhibits sustained
light emission. Compound 2 (100 mm) was incubated with Fluc, ATP, and
CoA. Light emission was measured over time, and sample images are
shown. For a–b, error bars represent the standard deviation of the mean for
three replicate experiments.
Luciferin 2 was also found to be a viable substrate for firefly
luciferase (Fluc). When 2 was incubated with Fluc in the pres-
ence of ATP, bioluminescent light was observed. As shown in
Figure 3, light emission was both concentration-dependent
and sustained. The overall photon output from 2 is weaker
than that observed with d-luciferin (the native substrate), but
on par with other luciferin analogues used in biological assays
(Figure S1, Supporting Information).[12b] The measured Km value
was 8.5Æ1 mm, and the apparent Vmax was 130Æ5”
106 photonssÀ1 (Figure S5, Supporting Information). Interest-
ingly, the bioluminescence emission spectrum of 2 was sub-
stantially redshifted compared to d-luciferin (lmax =610 nm at
258C, Figure 4). In fact, the alkynyl luciferin spectrum is similar
to those of aminoluciferins used in bioluminescence imag-
ing.[14,15b,12e]
Figure 5. a) Alkynyl luciferin 2 produces light when incubated with HEK293
cells. Analogue 2 (25–250 mm in PBS) was added to cells (100,000 cells per
well). Sample images are shown (inset). b) Analogue 2 exhibits sustained
light emission with HEK293 cells. Analogue 2 (250 mm) was incubated with
HEK293 cells (100,00 cells per well) and photon production was monitored
over time. For a, error bars represent the standard deviation of the mean for
6 replicate experiments. For b, error bars represent the standard deviation
of the mean for 3 replicate experiments.
We further evaluated the luciferin analogue in live cells.
Fluc-expressing HEK293 cells were incubated with 2, and biolu-
minescent images were acquired. As shown in Figure 5a, dose-
dependent light emission was observed, indicating that the al-
kynyl luciferin is cell permeable. The photon outputs from cul-
tures treated with 2 were weaker than cultures treated with d-
luciferin (Figure S7, Supporting Information). However, the in-
tensities observed are similar to other luciferin analogues and
sufficient for some cellular imaging applications.[12b–d] Impor-
tantly, the light emission from cells treated with 2 was also sus-
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[1] a) M. A. Paley, J. A. Prescher, MedChemComm 2014, 5, 255–267; b) J. A.
Prescher, C. H. Contag, Curr. Opin. Chem. Biol. 2014, 18, 80–89.
[2] a) V. R. Viviani, Cell. Mol. Life Sci. 2002, 59, 1833–1850; b) J. W. Hastings,
Gene 1996, 173, 5–11.
[3] E. H. White, J. D. Miano, M. Umbreit, J. Am. Chem. Soc. 1975, 97, 198–
200.
[4] B. W. Rice, M. D. Cable, M. B. Nelson, J. Biomed. Opt. 2001, 6, 432–440.
[5] a) K. L. Tinkum, L. S. White, L. Marpegan, E. Herzog, D. Piwnica-Worms,
H. Piwnica-Worms, J. Biol. Chem. 2013, 288, 27999–28008; b) H. Taka-
kura, M. Hattori, M. Takeuchi, T. Ozawa, ACS Chem. Biol. 2012, 7, 901–
910; c) M. Hattori, S. Haga, H. Takakura, M. Ozaki, T. Ozawa, Proc. Natl.
Acad. Sci. USA 2013, 110, 9332–9337; d) R. S. Dothager, K. Flentie, B.
Moss, M. H. Pan, A. Kesarwala, D. Piwnica-Worms, Curr. Opin. Biotechnol.
2009, 20, 45–53.
[6] a) B. W. Rice, C. H. Contag, Nat. Biotechnol. 2009, 27, 624–625; b) H.
Zhao, T. C. Doyle, O. Coquoz, F. Kalish, B. W. Rice, C. H. Contag, J.
Biomed. Opt. 2005, 10, 041210.
[7] a) R. Shinde, J. Perkins, C. H. Contag, Biochemistry 2006, 45, 11103–
11112; b) F. Berger, R. Paulmurugan, S. Bhaumik, S. S. Gambhir, Eur. J.
Nucl. Med. Mol. Imaging 2008, 35, 2275–2285.
[8] a) C. H. Contag, M. H. Bachmann, Annu. Rev. Biomed. Eng. 2002, 4, 235–
260; b) A. Sacco, R. Doyonnas, P. Kraft, S. Vitorovic, H. M. Blau, Nature
2008, 456, 502–506; c) J. A. Olson, R. Zeiser, A. Beilhack, J. J. Goldman,
R. S. Negrin, J. Immunol. 2009, 183, 3219–3228; d) H. Liu, M. R. Patel,
J. A. Prescher, A. Patsialou, D. Qian, J. Lin, S. Wen, Y. F. Chang, M. H.
Bachmann, Y. Shimono, P. Dalerba, M. Adorno, N. Lobo, J. Bueno, F. M.
Dirbas, S. Goswami, G. Somlo, J. Condeelis, C. H. Contag, S. S. Gambhir,
M. F. Clarke, Proc. Natl. Acad. Sci. USA 2010, 107, 18115–18120.
[9] a) V. Villalobos, S. Naik, D. Piwnica-Worms, Annu. Rev. Biomed. Eng. 2007,
9, 321–349; b) Y. Q. Sun, J. Liu, P. Wang, J. Zhang, W. Guo, Angew.
Chem. Int. Ed. 2012, 51, 8428–8430; Angew. Chem. 2012, 124, 8554–
8556.
tained (Figure 5b). Prolonged emission is desirable for routine
imaging experiments.
We also recognized that 2 could be further “clicked”[24] with
azido appendages by copper-catalyzed azide–alkyne cycloaddi-
tion (CuAAC). This transformation could potentially expedite
the production of new luciferin analogues by using 2 as a plat-
form for late-stage modification. Model reactions with 2 and
various azido compounds suggested that the CuAAC diversifi-
cation strategy is feasible (Figures S2–S3 and S5, Supporting In-
formation). Notably, the cycloaddition can proceed in aqueous
solvents and in the absence of copper chelators (Figure S6,
Supporting Information). We envision using CuAAC to produce
different classes of luciferins that can be screened for selective
processing by mutant luciferases. Recent crystallographic anal-
yses have revealed Fluc amino acids in close proximity to the
5’ carbon of a bound luciferin intermediate.[11h,25] These amino
acids could potentially be mutated to complement more
bulky, steric appendages on the luciferin ring, thereby facilitat-
ing the development of substrate-specific (i.e., orthogonal) bio-
luminescent tools.
Conclusion
In conclusion, we identified an alkyne-modified luciferin 2 for
use in bioluminescence assays. This scaffold is isolable in rea-
sonable quantities and is a functional light emitter with lucifer-
ase. The alkynyl probe can also be selectively modified with
azido appendages by CuAAC. Such designer luciferins are ap-
plicable to multicomponent imaging or biosensing in cells and
live organisms.[26] Based on the accessibility and unique fea-
tures of 2, we anticipate that the alkynyl probe will find use in
various imaging assays and further expand the scope of biolu-
minescence technology.
[10] a) B. A. Tannous, D. E. Kim, J. L. Fernandez, R. Weissleder, X. O. Breake-
field, Mol. Ther. 2005, 11, 435–443; b) S. Bhaumik, S. S. Gambhir, Proc.
Natl. Acad. Sci. USA 2002, 99, 377–382.
[11] a) B. R. Branchini, D. M. Ablamsky, A. L. Davis, T. L. Southworth, B. Butler,
F. Fan, A. P. Jathoul, M. A. Pule, Anal. Biochem. 2010, 396, 290–297;
b) B. R. Branchini, D. M. Ablamsky, M. H. Murtiashaw, L. Uzasci, H. Fraga,
T. L. Southworth, Anal. Biochem. 2007, 361, 253–262; c) B. R. Branchini,
T. L. Southworth, N. F. Khattak, E. Michelini, A. Roda, Anal. Biochem.
2005, 345, 140–148; d) S. T. Gammon, W. M. Leevy, S. Gross, G. W.
Gokel, D. Piwnica-Worms, Anal. Chem. 2006, 78, 1520–1527; e) B. A. Ra-
binovich, Y. Ye, T. Etto, J. Q. Chen, H. I. Levitsky, W. W. Overwijk, L. J.
Cooper, J. Gelovani, P. Hwu, Proc. Natl. Acad. Sci. USA 2008, 105, 14342–
14346; f) J.-B. Kim, K. Urban, E. Cochran, S. Lee, A. Ang, B. Rice, A. Bata,
K. Campbell, R. Coffee, A. Gorodinsky, Z. Lu, H. Zhou, T. K. Kishimoto, P.
Lassota, PLoS ONE 2010, 5, e9364; g) L. Mezzanotte, I. Que, E. Kaijzel, B.
Branchini, A. Roda, C. Lowik, PLoS ONE 2011, 6, e19277; h) T. Nakatsu, S.
Ichiyama, J. Hiratake, A. Saldanha, N. Kobashi, K. Sakata, H. Kato, Nature
2006, 440, 372–376.
[12] a) B. R. Branching, M. M. Hayward, S. Bamford, P. M. Brennan, E. J. Laji-
ness, Photochem. Photobiol. 1989, 49, 689–695; b) H. Takakura, R.
Kojima, T. Ozawa, T. Nagano, Y. Urano, ChemBioChem 2012, 13, 1424–
1427; c) A. P. Jathoul, H. Grounds, J. C. Anderson, M. A. Pule, Angew.
Chem. Int. Ed. 2014, 53, 13059–13063; Angew. Chem. 2014, 126, 13275–
13279; d) R. Kojima, H. Takakura, T. Ozawa, Y. Tada, T. Nagano, Y. Urano,
Angew. Chem. Int. Ed. 2013, 52, 1175–1179; Angew. Chem. 2013, 125,
1213–1217; e) D. M. Mofford, G. R. Reddy, S. C. Miller, J. Am. Chem. Soc.
2014, 136, 13277–13282.
Experimental Section
Experimental details are available in the Supplementary Informa-
tion.
Acknowledgements
This work was supported by the National Institutes of Health
(NIH, R01M107630 to J.A.P.). Some experiments were per-
formed in the Laboratory for Fluorescence Dynamics (LFD) at
UC Irvine. The LFD is supported jointly by the National Institute
of General Medical Sciences of the NIH (8P41M103540) and UC
Irvine. We also thank members of the Jarvo, Chamberlin, and
Overman laboratories for providing reagents and experimental
advice. We thank Krysten Jones for kindly preparing samples
for cellular assays. Finally, we thank members of the Prescher
laboratory for assistance with manuscript preparations.
[13] K. R. Harwood, D. M. Mofford, G. R. Reddy, S. C. Miller, Chem. Biol. 2011,
18, 1649–1657.
[14] M. S. Evans, J. P. Chaurette, S. T. Adams Jr, G. R. Reddy, M. A. Paley, N.
Aronin, J. A. Prescher, S. C. Miller, Nat. Methods 2014, 11, 393–395.
[15] a) C. C. Woodroofe, J. W. Shultz, M. G. Wood, J. Osterman, J. J. Cali, W. J.
Daily, P. L. Meisenheimer, D. H. Klaubert, Biochemistry 2008, 47, 10383–
10393; b) G. R. Reddy, W. C. Thompson, S. C. Miller, J. Am. Chem. Soc.
2010, 132, 13586–13587.
[16] SPARTAN Student v. 5.0.2 Wavefunction, Inc.
Keywords: bioluminescence imaging
luciferase · luciferin · Sonogashira coupling
·
CÀH activation
·
[17] a) R. Appel, H. Janssen, M. Siray, F. Knoch, Chem. Ber. 1985, 118, 1632–
1643; b) D. C. McCutcheon, M. A. Paley, R. C. Steinhardt, J. A. Prescher, J.
Am. Chem. Soc. 2014, 136, 7604–7607; c) T. Besson, J. Guillard, C. W.
Chem. Eur. J. 2016, 22, 3671 – 3675
www.chemeurj.org
3674
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Communication
Rees, J. Chem. Soc. Perkin Trans. 1 2000, 563–566; d) P. A. Koutentis, M.
Koyioni, S. S. Michaelidou, Org. Biomol. Chem. 2013, 11, 621–629; e) H.
Akhavan-Tafti, R. De Silva, G. Wang, R. A. Eickholt, R. Gupta, L. S. Kaanu-
malle, 2011/0014599 A1 US.
Prescher, S. T. Laughlin, N. J. Agard, P. V. Chang, I. A. Miller, A. Lo, J. A.
Codelli, C. R. Bertozzi, Proc. Natl. Acad. Sci. USA 2007, 104, 16793–
16797; c) D. M. Patterson, L. A. Nazarova, J. A. Prescher, ACS Chem. Biol.
2014, 9, 592–605.
[18] J. H. Looker, D. N. Thatcher, J. Org. Chem. 1954, 19, 784–788.
[19] M. Shen, T. G. Driver, Org. Lett. 2008, 10, 3367–3370.
[20] T. Besson, K. Emayan, C. W. Rees, J. Chem. Soc. Chem. Commun. 1995,
1419–1420.
[21] S. S. Michaelidou, P. A. Koutentis, Synthesis 2009, 4167–4174.
[22] K. Inamoto, C. Hasegawa, K. Hiroya, T. Doi, Org. Lett. 2008, 10, 5147–
5150.
[25] J. A. Sundlov, D. M. Fontaine, T. L. Southworth, B. R. Branchini, A. M.
Gulick, Biochemistry 2012, 51, 6493–6495.
[26] a) J. Li, L. Chen, L. Du, M. Li, Chem. Soc. Rev. 2013, 42, 662–676; b) H.
Yao, M. K. So, J. Rao, Angew. Chem. Int. Ed. 2007, 46, 7031–7034;
Angew. Chem. 2007, 119, 7161–7164.
[23] T. Ritter, K. Stanek, I. Larrosa, E. M. Carreira, Org. Lett. 2004, 6, 1513–
1514.
Received: October 1, 2015
[24] a) H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew. Chem. Int. Ed. 2001, 40,
2004–2021; Angew. Chem. 2001, 113, 2056–2075; b) J. M. Baskin, J. A.
Published online on January 19, 2016
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