Robust light emission from cyclic alkylaminoluciferin substrates for firefly luciferase

Article abstract of DOI:10.1021/ja104525m

Firefly luciferase utilizes the chemical energy of ATP and oxygen to convert its substrate, d-luciferin, into an excited-state oxyluciferin molecule. Relaxation of this molecule to the ground state is responsible for the yellow-green light emission. Synthetic cyclic alkylaminoluciferins that allow robust red-shifted light emission with the modified luciferase Ultra-Glo are described. Overall light emission is higher than that of acyclic alkylaminoluciferins, aminoluciferin, and the native substrate d-luciferin.

Full text of DOI:10.1021/ja104525m

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J Am Chem Soc. Author manuscript; available in PMC 2012 January 17.
Published in final edited form as:
J Am Chem Soc. 2010 October 6; 132(39): 13586–13587. doi:10.1021/ja104525m.
Robust light emission from cyclic alkylaminoluciferin substrates
for firefly luciferase
Gadarla Randheer Reddy, Walter C. Thompson, and Stephen C. Miller
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical
School, 364 Plantation St., Worcester, MA 01605
Stephen C. Miller: Stephen.miller@umassmed.edu
Abstract
Firefly luciferase utilizes the chemical energy of ATP and oxygen to convert its substrate, D-
luciferin, into an excited state oxyluciferin molecule. Relaxation of this molecule to the ground
state is responsible for the yellow-green light emission. Synthetic cyclic alkylaminoluciferins are
described that allow robust red-shifted light emission with the modified luciferase Ultra-Glo.
Overall light emission is higher than that of acyclic alkylaminoluciferins, aminoluciferin, and the
native substrate, D-luciferin.
Beetle luciferases chemically generate light by converting D-luciferin into an excited-state
1
,2
molecule of oxyluciferin (Figure 1). The energy difference between the excited state and
3
ground state determines the wavelength of the bioluminescence. Despite the fact that the
light emission properties of beetle luciferases are inherently limited by the luciferin
structure, there has been little work on the synthesis of modified luciferin substrates since
4
,5,6
the late sixties.
Here we describe conformationally-restricted cyclic alkylaminoluciferin
substrates that result in red-shifted and more intense light emission with Ultra-Glo
7
luciferase.
White and McElroy established that the 6’-hydroxyl of D-luciferin (LH ) can be replaced
2
8
with an amino group; 6’-aminoluciferin (6’-NH LH ) emits light at a longer wavelength
and has ~10-fold higher affinity for luciferase than LH . On the other hand, no light is
2
2
9
2
emitted when the 6’-hydroxyl is removed or replaced with 6’-methoxy, ethoxy, or N-
acetylamino groups. This is not due to a lack of binding – conversely, these and other bulky
1
0
modifications are well-tolerated. Rather, these modifications prevent light emission.
We hypothesized that replacement of the phenolic hydroxyl of luciferin with more strongly
electron-donating alkylamino groups would red-shift the spectral properties of the luciferin
chromophore while maintaining high affinity for the luciferase. Furthermore, we
hypothesized that restriction of the conformational flexibility of the alkylaminoluciferin by
Correspondence to: Stephen C. Miller, Stephen.miller@umassmed.edu.
SUPPORTING INFORMATION AVAILABLE: Experimental procedures and full spectroscopic data for all new compounds. This
material is available free of charge at http://pubs.acs.org

Reddy et al.
Page 2
cyclization of the aryl amine would maintain a high quantum yield of bioluminescence.
Conversely, free rotation around the aryl-nitrogen bond in acyclic alkylaminoluciferins was
predicted to result in a reduced quantum yield due to poor overlap of the nitrogen lone pair
with the conjugated system of the luciferin, particularly in the context of the luciferin
binding pocket.
We first synthesized acyclic alkylaminoluciferins from 1 (Scheme 1a). Reductive alkylation
of 3a followed by nucleophilic substitution with cyanide and condensation with D-cysteine
afforded 6’-MeNHLH (5a) and 6’-Me NLH (5b). However, attempts to construct cyclic
2
2
2
alkylamino modifications from 3a were stymied by the exclusive formation of the undesired
,7 isomer rather than the 5,6 ring fusion (Scheme 1b).
6
Thwarted by the positional reactivity preferences of 3a, we examined approaches to
construct the benzothiazole ring as a final step (Scheme 1c). Protection of 9 as a
trifluoroacetamide followed by reduction of the nitro group and oxidative ring formation
yielded the desired 2-aminobenzothiazole. Diazotization of the 2-amino group and
concomitant displacement by chlorine, followed by reductive removal of the
trifluoroacetamide protecting group afforded 14a. Introduction of the 2-cyano group and
subsequent treatment with D-cysteine afforded the respective luciferin analog 16a
(CycLuc1). The N-methylated derivative 16b (CycLuc2) was similarly constructed from
1
4b.
We next measured the emission wavelength of bioluminescence for our synthetic
aminoluciferin substrates. Relative to LH , 6’-NH LH exhibits red-shifted light emission
2
2
2
from wild-type firefly luciferase (593 nm vs. 557 nm). We found that 6’-MeNHLH2
extended this emission shift to 609 nm, while 6’-Me NLH was even more red-shifted to
2
2
6
23 nm. This emission wavelength is identical to the railroad worm beetle luciferase PxhRE,
1
1
the most red-shifted luciferase yet reported. Surprisingly, luciferase emission with the
cyclic aminoluciferins was less red-shifted than with their acyclic counterparts (599 nm for
CycLuc1 and 607 nm for CycLuc2; SI Table S1).
Measurement of the emission intensity as a function of wavelength revealed a significant
reduction in light output for all aminoluciferins relative to D-luciferin. The lower intensity
of light emission could arise from either kinetic factors (lower rate of enzymatic turnover or
product inhibition), or an inherently lower efficiency of light production (lower quantum
yield or failure to produce oxyluciferin), or both. We therefore performed a rapid injection
experiment in which the light output was measured during and immediately after mixing of
1
2
the luciferin and luciferase. All of the substrates gave a robust initial burst of light and are
thus capable of rapid conversion to a light-emitting oxyluciferin (Figure 2). However,
1
3
sustained light output is low after the initial burst, consistent with product inhibition.
1
Addition of coenzyme A, which has been shown to reduce product inhibition by L-AMP,
failed to prevent this effect (SI Figure S1). Encouragingly, the initial flash of light from
cyclic substrate CycLuc1 substantially exceeded that of all other aminoluciferins (Figure 2).
The burst kinetics of firefly luciferase, even with its native substrate D-luciferin, is well-
1
known (Figure 2). To provide a steady level of light emission, Promega has developed
Ultra-Glo, a highly mutated form of luciferase designed to be more stable and resistant to
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,14
inhibition during high-throughput screening assays.
When used with P450-Glo buffer,
this luciferase has also been shown to allow sustained light emission from substrates that led
5
,15
to product inhibition of wild-type firefly luciferase.
When tested with Ultra-Glo luciferase in P450-Glo buffer at a concentration of 1 μM,
CycLuc1 and CycLuc2 exhibited dramatically higher light output than any other substrate
(Figure 3). CycLuc1 emitted 5.7-fold more light than aminoluciferin, and 3.2-fold more light
J Am Chem Soc. Author manuscript; available in PMC 2012 January 17.

Reddy et al.
Page 3
than D-luciferin. Similarly, CycLuc2 emitted 4.7-fold more light than aminoluciferin, and
.6-fold more light than D-luciferin. The superior light output of CycLuc1 held true across a
2
wide concentration range (1 – 100 μM; SI Figure S2), indicating that this behavior is not
simply due to higher affinity for the luciferase. Even when tested at exorbitantly high
substrate concentrations (750 μM), the light emission from CycLuc1 greatly exceeds all
other aminoluciferins and is equivalent to that of D-luciferin (SI Figure S2). In contrast, the
acyclic substrate 6’-MeNHLH emitted ~50% more light than aminoluciferin at 1 μM but
2
was no better at concentrations over 100 μM, and 6’-Me NLH had uniformly lower light
2
2
emission at all tested concentrations (SI Figure S2).
The superior light output of CycLuc1 and CycLuc2 demonstrates that they are more efficient
light emitters than their respective acyclic counterparts and that both monoalkyl and
dialkylaminoluciferins can be efficient substrates (Figure 3). We hypothesize that this is due
to the increased rigidity and restricted bond rotation of the cyclic alkylaminoluciferins,
1
6
leading to an increase in the relative quantum yield. In the context of wild-type firefly
luciferase, product inhibition overshadowed this effect and limited light output (Figure 2).
The use of cyclic aminoluciferin substrates or pro-substrates for in vitro assays with Ultra-
1
7
Glo is expected to improve the sensitivity of detection. However, despite its utility for
high-throughput screening applications, Ultra-Glo is not available as a genetic construct that
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,18
can be introduced into living organisms for bioluminescence imaging.
Future work will
be directed toward mutation of luciferase in order to relieve product inhibition and allow
sustained light emission from cyclic aminoluciferins under physiological conditions, using a
luciferase that can be readily expressed within cells and whole organisms.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
This work was supported by a grant from the NCI (NIH R21CA127196). We thank Bill Kobertz for critical reading
of the manuscript.
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3. While it was qualitatively apparent that these substrates had higher affinity for luciferase than LH
3
1
1
1
1
2
or 6’-NH LH , their kinetic behavior prevented accurate determination of their true K values.
2
2
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Figure 1.
Beetle luciferases catalyze the conversion of D-luciferin (R = OH) or 6’-aminoluciferin (R =
NH ) into the corresponding oxyluciferin. Light emission occurs from the oxyluciferin
2
excited state.
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Reddy et al.
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Figure 2.
Light emission from 10 μM luciferin substrate after rapid injection of wild-type firefly
luciferase at the 30 second time point.
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Reddy et al.
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Figure 3.
Ultra-Glo light output with 1 μM luciferin substrate.
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Reddy et al.
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Scheme 1.
Synthesis of alkylaminoluciferins. a) Reductive alkylation of intermediate 3a afforded
acyclic alkylaminoluciferins; b) Attempts to form cyclic alkylaminoluciferins from 3a gave
exclusive formation of the undesired 6’,7’-fused isomer (e.g., 8); c) Synthesis of 5’,6’-fused
cyclic alkylaminoluciferins 16 required reversal of the order of ring formation. i) HNO3,
H SO ; ii) SnCl -2H O, EtOH, reflux; iii) NaBH(OAc) , 37% HCHO, DCE, HOAc; iv)
2
4
2
2
3
KCN, DMSO, 130°C; v) D-Cys, aq. MeOH, pH 8; vi) 5 mol% In(OTf) , acetone, rt; vii)
3
TFAA, TEA, rt; viii) KSCN, Br , AcOH, rt; ix) t-BuONO , CuCl, CH CN; x) NaBH ,
2
2
3
4
EtOH.
J Am Chem Soc. Author manuscript; available in PMC 2012 January 17.