Novel heterocyclic analogues of firefly luciferin

Article abstract of DOI:10.1021/bi301411d

Five novel firefly luciferin analogues in which the benzothiazole ring system of the natural substrate was replaced with benzimidazole, benzofuran, benzothiophene, benzoxazole, and indole were synthesized. The fluorescence, bioluminescence, and kinetic pr

Full text of DOI:10.1021/bi301411d

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Article
Novel Heterocyclic Analogs of Firefly Luciferin
Carolyn C Woodroofe, Poncho L Meisenheimer, Dieter H Klaubert, Yumi Kovic, Justin
Colin Rosenberg, Curran E. Behney, Tara L Southworth, and Bruce Robert Branchini
Biochemistry, Just Accepted Manuscript • DOI: 10.1021/bi301411d • Publication Date (Web): 19 Nov 2012
Downloaded from http://pubs.acs.org on November 24, 2012
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Biochemistry
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Novel Heterocyclic Analogs of Firefly Luciferin
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Byline
Carolyn C. Woodroofe†*, Poncho L. Meisenheimer†, Dieter H. Klaubert†, Yumi Kovic‡, Justin C.
Rosenberg‡, Curran E. Behney‡, Tara L. Southworth‡ and Bruce R. Branchini‡*
†Promega Biosciences, LLC, San Luis Obispo, California 93433
‡Department of Chemistry, Connecticut College, New London, CT 06320
Bruce R. Branchini‡*, Department of Chemistry, Connecticut College, New London, CT 06320.
Telephone: 860 439ꢀ2479. Fax: 860 439ꢀ2477. Eꢀmail: brbra@conncoll.edu
Carolyn C. Woodroofe†*, Promega Biosciences, LLC, San Luis Obispo, California 93433.
Telephone: 805ꢀ544ꢀ8524. Fax: 805ꢀ543ꢀ1531. Eꢀmail: carolyn.woodroofe@promega.com
Funding Source Statement
This work was supported by the National Science Foundation (MCB0842831), the Air Force
Office of Scientific Research (FA9550ꢀ10ꢀ1ꢀ0714), and the Hans & Ella McCollum ’21
Vahlteich Endowment.
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ABREVIATIONS
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BfLH2, Dꢀbenzofuranylluciferin; BiLH2, Dꢀbenzimidazolylluciferin; BoLH2, Dꢀ
benzoxazolylluciferin; BtLH2, Dꢀbenzothiophenylluciferin; InLH2, Dꢀindolylluciferin; LꢀAMP,
dehydroluciferyladenylate; LH2, Dꢀfirefly luciferin; Luc, Photinus pyralis luciferase (E.C.
1.13.12.7); PpyWT, recombinant Photinus pyralis luciferase containing the additional Nꢀ
terminal peptide GlyProLeuGlySer–.
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TEXTUAL FOOTNOTES
§During the preparation of this manuscript, the synthesis of benzimidazolylluciferin was
reported16.
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ABSTRACT
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Five novel firefly luciferin analogs were synthesized in which the benzothiazole ring
system of the natural substrate was replaced with: benzimidazole, benzofuran, benzothiophene,
benzoxazole and indole. The fluorescence, bioluminescence and kinetic properties of the
compounds were evaluated with recombinant Photinus pyralis wildꢀtype luciferase. With the
exception of indole, all of the substrates containing heterocycle substitutions produced readily
measurable flashes of light with luciferase. Compared to luciferin, the intensities ranged from
0.3% to 4.4% in reactions with varying pH optima and times to reach maximum intensity. The
heteroatom changes influenced both the fluorescence and bioluminescence emission spectra,
which displayed maxima of 479 nm to 528 nm and 518 nm to 574 nm, respectively. While there
were some interesting trends in the spectroscopic and bioluminescence properties of this group
of structurally similar substrate analogs, the most significant findings were associated with the
benzothiopheneꢀcontaining compound. This synthetic substrate produced slow decay glow
kinetics that increased the total light based specific activity of luciferase more than 4ꢀfold over
the luciferin value. Moreover, over the pH range 6.2 to 9.4 the emission maximum is 523 nm, an
unusual 37 nm blueꢀshift compared to the natural substrate. The extraordinary bioluminescence
properties of the benzothiophene luciferin should translate into greater sensitivity for analyte
detection in a wide variety of luciferase based applications.
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INTRODUCTION
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Beetle bioluminescence is the result of two partial reactions in which the natural substrate
firefly (beetle) luciferin (LH2) is first converted into the corresponding luciferylꢀadenylate (LH2ꢀ
AMP) by firefly luciferase (Luc) as shown in Scheme 1. In the second halfꢀreaction, excited
state oxyluciferin is formed and subsequently emits a photon of light. A competing oxidative
dark reaction leads to the formation of a potent (Ki = 3.8 nM) luciferase inhibitor
dehydroluciferinꢀAMP (LꢀAMP), a likely cause of the rapid decay of light emission1. The
bioluminescence process catalyzed by Photinus pyralis luciferase is quite efficient having a
bioluminescence quantum yield (ΦBl) equal to 0.41 ± 0.0742. As advanced by White and
coworkers3 and shown in Equation 1, the ΦBl is dependent on the product of three yields: the
formation of oxyluciferin(ΦRx), the formation of oxyluciferin in the excited state (ΦES) and the
fluorescence quantum yield (ΦFl) of oxyluciferin.
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ΦBl = ΦRx
.
ΦFl
.
ΦES
(Equation 1)
While many luciferin analogs have been reported4ꢀ16 for a variety of applications, the
focus of this investigation is on substrates that react directly with luciferase and MgꢀATP to
produce light. The synthesis and evaluation of LH2 analogs began in the laboratory of E. H.
White4, 5 shortly after the structure proof and first synthesis of LH2 was accomplished17. The
first analogs contained variations in the position and number of hydroxyl groups on the luciferin
benzothiazole ring, and later analogs were made containing an additional methyl group8 and with
an amino functionality substituted for the 6´ꢀhydroxyl group6. Aminoluciferin proved to be an
interesting substrate analog mainly because it produced considerably bright redꢀshifted light
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emission (~605 nm) with Luc. Several recent reports10ꢀ14 have focused on introducing variations
to the aminoluciferin structure with quite interesting outcomes. This study is a continuation of a
previous one7, 9 that focused on LH2 analogs in which the benzothiazole moiety was replaced
with the naphthalene and quinoline aromatic systems, while maintaining the 2´ꢀ, 6´ꢀ substituent
pattern. Other than the substitution of the benzothiazole system with the coumarin moiety13, we
are unaware of any additional ring substitutions based on our original strategy§. Here we report
the preparation and evaluation with recombinant P. pyralis luciferase (PpyWT) of five novel LH2
analogs in which the benzothiazole ring system has been replaced with benzimidazole (BiLH2),
benzofuran (BfLH2), benzoxazole (BoLH2), benzothiophene (BtLH2) and indole (InLH2). The
properties of the new analogs whose structures are shown in Figure 1 were systematically
compared to those of the natural substrate LH2.
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One aim of these studies was to develop new reagents to increase the sensitivity and
diversity of enzyme assays based on luciferase bioluminescence measurements. While our
results with BtLH2 were quite promising, studies on the unusual properties of this substrate
analog have enabled us to gain a better understanding of the light emission kinetics of the
luciferaseꢀcatalyzed process.
MATERIALS AND METHODS
Materials. The following materials were obtained from the sources indicated: MgꢀATP
(bacterial source) from SigmaꢀAldrich (St. Louis, MO) and firefly luciferin (LH2) from Promega
(Madison, WI). Recombinant firefly luciferase (PpyWT) was prepared as described previously18,
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19. Synthetic procedures and characterization of novel compounds are provided in the supporting
information.
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General Methods. Protein concentrations were determined with the BioꢀRad Protein
Assay system using bovine serum albumin as the standard. Ultraviolet (UV)ꢀvisible spectra
were recorded in 25 mM glycylglycine buffer, pH 7.8, with a Perkin Elmer Lambda 25
spectrometer. Enzyme activity assays were performed with PpyWT, and all data were replicated
in triplicate and are reported as the mean ± standard deviation.
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Luciferase Activity Assays. Bioluminescence activity assays were performed with a
customꢀbuilt luminometer assembly containing a Hamamatsu R928 PMT and a C6271 HV
power supply socket assembly that was described in detail previously20. Reactions (0.525 mL
final volume) were initiated by the injection of 0.120 mL of 9.0 mM ATP into 8 x 50ꢀmm
polypropylene tubes containing 0.4 mL of buffer with luciferin or substrate analogs and 5 ꢀL
(0.4 – 4 ꢁg) of enzyme in 20 mM TrisꢀHCl (pH 7.4 at 4 °C) containing 150 mM NaCl, 1 mM
EDTA, 1 mM DTT, 0.8 M ammonium sulfate and 2% glycerol. Assay buffers were: 25 mM
glycylglycine, pH 8.2; and 50 mM 2ꢀaminoꢀ2ꢀmethylꢀ1,3ꢀpropanediol (AMPD), pH 8.5 or pH
9.1 for BtLH2. The final LH2 and analog concentrations were ~6 times their Km values. Peak
height and integrated intensity values were recorded and corrected for the spectral response of
the detector.
Bioluminescence Emission Spectra. Emission spectra were obtained using a Horiba
JobinꢀYvon iHR imaging spectrometer equipped with a liquid N2 cooled CCD detector and the
excitation source turned off. Data were collected at 25 °C in a 0.8 ml quartz cuvette over the
wavelength range 400–935 nm with the emission slit width set to 10 nm. Reactions (0.525 mL
final volume) were initiated by addition of 5 ꢀL of PpyWT stock solution (15 ꢀ 150 nM final
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concentration) to cuvettes containing substrate (150 ꢀM) and MgꢀATP (2 mM) in 25 mM 2[Nꢀ
morpholino]ethanesulfonic acid (MES), pH 6.2; 25 mM 4ꢀ(2ꢀhydroxyethyl)ꢀ1ꢀpiperazineethaneꢀ
sulfonic acid (HEPES), pH 7.0; 25 mM glycylglycine buffer, pH 7.8 or 8.2; 50 mM AMPD, pH
8.5, 8.6, 9.1 or 9.4. The pH values were confirmed before and after spectra were obtained. All
spectra were corrected for the spectral response of the CCD using a correction curve provided by
the manufacturer.
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Relative Bioluminescence Quantum Yields. Bioluminescent quantum yields for LH2
and the substrate analogs were determined from bioluminescence activity assays in which a
limiting amount of compound was reacted with an excess of luciferase under single turnover
conditions. Into 0.4 mL of buffer at the optimal pH for each compound (see above) containing
65 nM substrate and 3.25 ꢀM PpyWT, 0.12 mL of 9 mM MgꢀATP in the same buffer was
injected. The light output was monitored until the initial signal intensity decreased by 99% at a
sampling rate of 100 Hz. An additional aliquot of enzyme was added to the spent mixtures and
emission intensity was monitored to ensure that the reactions were completed. The total
integrated light intensities were reported relative to the value obtained with LH2 at pH 8.2.
Steady-State Kinetic Constants. Values of Km and Vmax for the substrate analogs were
determined as previously described21 from bioluminescence activity assays in which
measurements of maximal light intensities were taken as estimates of initial velocities. Activity
assays were performed at the pH optimum for each compound (Table 1). Briefly, the
measurements were made with MgꢀATP at a saturating level (2 mM) and varying concentrations
of substrate: LH2, 2 – 500 ꢁM; BtLH2, 3 ꢀ750 ꢁM; BoLH2, 0.6 – 1.0 mM; BiLH2, 5 – 200 ꢁM
and BfLH2, 5 – 600 ꢁM. Data were collected and analyzed using Enzyme Kinetics Pro software
(Syntex, Palo Alto, CA).
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RESULTS AND DISCUSSION
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Luciferin analog synthesis and characterization. The novel luciferin analogs were
synthesized as shown in Scheme 2. All were accessed via reaction of Dꢀcysteine with the
corresponding nitrile precursor under mild aqueous conditions. While the appropriate precursor
for benzoxazole luciferin BoLH2 was prepared directly by treating aminoresorcinol with Appel’s
salt22, the other analogs made use of a phenolic methyl ether protecting group which was
removed by brief treatment with molten pyridinium hydrochloride. Synthesis of the indole core
has been previously reported and was achieved via a similar route23. The nitrile intermediates for
the benzofuran and benzothiophene luciferins were approached from a common 4ꢀ
methoxysalicylaldehyde precursor. In the case of the benzofuran luciferin, direct reaction with a
haloacetonitrile and subsequent condensation of the activated methylene with the proximal
aldehyde afforded the methoxy benzofuranonitrile. The latter transformation was somewhat
sensitive to reaction conditions, as the nitrile was prone to hydration to form the amide; however
potassium carbonate in anhydrous DMF at high temperature proved quite effective24. The
benzothiophene core was accessed by thiocarbamoylation of the same salicylaldehyde, followed
by a heatꢀinduced rearrangement25 and subsequent hydrolysis to furnish the thiophenol. The
conditions used to alkylate the thiophenol intermediate resulted in spontaneous condensation of
the aldehyde with the activated methylene to yield the desired benzothiophene nitrile. Finally,
the benzimidazole derivative BiLH2 was synthesized from the commercially available 2ꢀ
thiobenzimidazole via methylation, oxidation to the sulfone, and subsequent displacement with
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potassium cyanide. The methoxybenzimidazoleꢀ2ꢀcarbonitrile was elaborated to BiLH2 by
standard deprotection and cyclization with Dꢀcysteine. Additionally, chiral HPLC analysis
suggested that the substrates were obtained in > 99% enantiomeric excess (ee). Racemization
occurred very slowly in solid form, such that, for example, a sample of BoLH2 still retained an ee
of > 95% after more than 5 years of storage at ꢀ80 °C.
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As expected16, the H1ꢀ and C13ꢀNMR spectra of the BiLH2 analog displayed peak
broadening of the resonances due to rapid tautomerization of the benzimidazole ring in solution.
It was not possible to determine to what extent BiLH2 exists as the 5´ or 6´ꢀ hydroxy substituted
compound at the luciferase active site. While this ambiguity is problematic, especially because
the 5´ꢀ substituted regioisomer is expected to be inactive in light production, the results and
interpretations that follow were made using the assumption that BiLH2 exists solely as the 6´ꢀ
hydroxy substituted analog.
Kinetic Properties with PpyWT. Prior to determining the performance of the substrate
analogs with PpyWT, we first determined the optimum assay pH (Table 1) based on the intensity
of the flash height produced with MgꢀATP. We then compared the properties of the compounds
at their respective pH optima, which ranged from 8.2 to 9.1, throughout the rest of the study.
Little variation in the Km values was observed with the exception that the value for BoLH2 was
unexpectedly 6ꢀfold higher than that of LH2. The parameters Km, kcat and kcat/Km were
determined from assays performed at the respective pH optima (Table 1) in which the peak
intensities were taken as measures of initial velocity. For PpyWT the highest kcat and kcat/Km
values, measures of the catalytic efficiency of PpyWT with the analogs, were obtained with
BtLH2 (Table 2). Additionally, the emission kinetics (rise and decay times) varied significantly
(Table 2). While LH2, BoLH2 and BiLH2 with Y = N (Figure 1), reached maximum intensity
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from 0.35 to 0.6 s, BtLH2 and BfLH2 with Y = CH had rise times of 6 s and 4 s, respectively.
BtLH2 had the most sustained emission as indicated by a decay time of 95 min greatly exceeding
the 23 s value of LH2. This “glow” type kinetic profile is unusual for PpyWT, which typically
displays strong product inhibition and flash kinetics, and contributed to the high relative
bioluminescence quantum yield of BtLH2 as discussed below.
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Spectral Emission Properties. The fluorescence emission spectra (Figure 2) of the
substrate analogs were recorded using a common excitation wavelength of 330 nm; the
fluorescence emission maxima and quantum yields (ΦFl) are summarized in Table 1. The
emission maxima varied from 429 nm (InLH2) to 537 nm (LH2) and the quantum yields
(measured at pH 11.0) from 0.01 (InLH2) to 0.83 (LH2). Additionally, the fluorescence emission
maxima of the analogs varied only slightly (≤ 10 nm) over the pH range 7.8 – 11.0. With respect
to emission maxima at optimum pH, the substrates fell into two groups: (1) LH2, BoLH2, and
BiLH2 that have λmax = ~530 nm (range 528 nmꢀ537 nm) and (2) InLH2, BfLH2 and BtLH2 that
have λmax values in the range 429 nm to 485 nm. The clear distinguishing factor is the absence or
presence of a nitrogen atom (Y = N in Figure 1) that is associated with longer wavelength
emission.
The range of bioluminescence emission maxima (Table 3 and Figure 3) at the respective
pH optima for the substrateꢀluciferase reactions varied 56 nm from 518 nm (BfLH2) to 574 nm
(BiLH2). When Y = CH, green emission (518 nm – 523 nm) is observed for BtLH2, and BfLH2.
Yellowꢀgreen light (556 nm – 574 nm) was observed with LH2, BiLH2, and BoLH2 that have Y =
N. Data could not be obtained for InLH2 because of extremely low bioluminescent activity, and
this analog was not further evaluated.
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The spectral shift of approximately 50 nm over the pH range 6.2 to 9.4 with LH2 is an
example of the pH sensitivity of the true firefly luciferases (Table 3). Only BoLH2 and BiLH2
show very similar pH behavior to LH2, while BtLH2 and BfLH2 are almost insensitive to pH
change. Apparently, fluorescence and bioluminescence emission as well as pH sensitivity and
reaction rise times are related to whether Y = CH or Y = N. In the crystal structures of L.
cruciata and P. pyralis luciferases26, 27 in complex with an inhibitor structurally similar to Lꢀ
AMP, the N atom at the Y position of the inhibitor is hydrogen bonded to S347 through an
intervening water molecule at the active site. A disruption of this interaction may be responsible
for the resistance to redꢀshifting emission at low pH, the long wavelength bioluminescence and
slow rise times.
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Relative Bioluminescence Quantum Yields (ΦBl). The ΦBl values (relative to the value
obtained with LH2) were measured with excess enzyme under single turnover conditions (Table
1). The results also fall into two groups: (1) those with high (≥70%) values (LH2, BtLH2) and (2)
those with very low (≤14%) values (BiLH2, BfLH2, BoLH2, and InLH2). Here there is a
correlation with the electronegativity of the X position atom (Figure 1). Higher values are
associated with the low electronegativity of S, while O and N produce lower ΦBl values. This
effect is especially powerful as one might expect that the ΦBl values would follow the
fluorescence quantum yield (ΦFl) trend (Table 1) suggesting, for example, that BoLH2 (ΦFl =
0.71) should have the highest ΦBl value among the analogs. Instead, BoLH2 produced little light
(~1% compared to LH2). Evidently, the high electronegativity of the oxygen atom interferes
with the efficient formation of the excited state analog oxyluciferins (ΦES) and/or causes the
corresponding adenylates to form dehydroluciferinꢀlike product in great excess over oxyluciferin
(ΦRx). Possibly too, adenylate formation is severely reduced (Scheme 1). In marked contrast,
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BtLH2 efficiently produces light with a relative ΦBl equal to 70% that of LH2 despite having a ΦFl
= 0.33, only 40% of the LH2 value. This analog may be capable of producing a greater
percentage of analog oxyluciferin in the excited state (ΦES) and/or with a higher analog
oxyluciferin/LꢀAMP ratio (ΦRx). The latter is likely occurring as evidenced by the extended
decay when substrate is in excess. The slow decay may be due to slower PpyWT inhibition
because less analog LꢀAMP is formed and/or because it has a higher Ki value than LꢀAMP.
Lower product inhibition by the oxyluciferin analog may also contribute. These arguments, like
those above, assume that the heterocyclic analogs bind in the same relative orientation as LH2
with respect to rotation about the C2ꢀC2´ bond. Otherwise, the relative position of the 6´ꢀ
hydroxyl at the active site will be altered and would likely diminish light emission.
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BtLH2 is a promising Luc substrate. Firefly luciferase assays based on ATP detection,
reporter gene detection and in vivo bioluminescence imaging can be performed more
conveniently and with greater sensitivity when the signal decays slowly displaying “glow” rather
than “flash” kinetics. Interestingly, although the initial intensity of BtLH2 is only ~4% of that
produced with LH2, this intensity decays so slowly (Figure 4) that over 1.5 hours, ~4ꢀfold greater
total light is emitted than with the natural substrate (Table 2). To demonstrate the importance of
the sustained light emission of BtLH2, we recorded images of reactions with identical amounts of
PpyWT and MgꢀATP and saturating concentrations of substrates under optimal conditions, pH
8.2 for LH2 and pH 9.1 for the analog. Initially the LH2ꢀcontaining reaction is brighter, but after
5 min the reactions are of approximately equal intensity (Figure 4 inset). However, the bright
emission with BtLH2, but not LH2, persisted even after 30 min (Figure 4 inset). Also, the blue
shifted emission of PpyWT with BtLH2 will provide better signal separation with red
bioluminescence signals than is possible with LH2. Additionally, we evaluated the potential of
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BtLH2 for imaging studies by using it to visualize single E. coli colonies expressing PpyWT
(Figure 5). Surprisingly, despite the higher pH optimum of bioluminescence with BtLH2, the
signal intensities produced in bacteria with the analog and LH2 are very similar.
We have developed synthetic methods for the preparation of five novel LH2 analogs.
Among them, BtLH2 displays the most promise as an alternative substrate for Luc in application
where it is advantageous to have blueꢀshifted and longer lived emission than is typically
observed with the natural substrate. These attributes have been achieved without the necessity of
chemical additives. Studies are in progress to develop firefly luciferases that selectively enhance
the properties of BtLH2 compared to the natural substrate.
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SUPPORTING INFORMATION
Synthetic procedures; procedures for determining substrate pKa values, bioluminescence
reaction pH optima, and fluorescence spectra; and figure of dependence of bioluminescence
activity on pH. This material is available free of charge via the Internet at http://pubs.acs.org.
ACKNOWLEDGEMENT
We thank Danielle M. Fontaine and Kelsey S. Row for technical assistance and Jennifer Prescher
for the generous gift of BiLH2. Compound characterization and preliminary bioluminescence
studies were performed by the Promega Biosciences Analytical Services Laboratory.
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Table 1. Fluorescence and bioluminescence properties of LH2 and analogs
d
fluorescence ΦFl
relative
bioluminescence
ΦBl
pH
optimab
8.2
9.1
8.5
fluorescence
pH
a
c
substrate
LH2
BtLH2
BoLH2
BiLH2
BfLH2
InLH2
pKa
λmax
pH 11.0
0.83
optima
0.71
0.28
0.57
0.02
0.07
0.01
8.6
9.1
8.8
9.6
9.2
537
485
528
533
479
429
100 ± 1.5
70 ± 2.5
0.9 ± 0.1
14 ± 1.0
1.9 ± 0.1
< 0.1%
0.33
0.71
0.03
0.08
8.5
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8.5
10.0
NDe
0.02
apKa values are within error of ± 0.1. bpH optima of reactions with PpyWT values are
within error of ± 0.1. cFluorescence λmax values are within error of ± 1. dFluorescence
quantum yields were measured at pH 11.0 and reaction pH optima with 370 nm
excitation and values are within error of ± 5%. Data could not be obtained for InLH2
because of low bioluminescent activity.
e
Table 2. Kinetic properties of LH2 and analogsa
relative specific activityd
flash
height
kcat/Km
rise timec
decay time
to 10%c (s)
23
substrate
LH2
Km (ꢀM)
28 ± 2
kcatb (s-1)
1.9E-01
6.7E-03
1.8E-03
5.1E-04
6.3E-04
(mM-1 s-1)
(s)
0.36
6
0.35
0.6
4
integration
100
6.78 ± 0.14
0.11 ± 0.002
0.01 ± 0.001
0.03 ± 0.001
0.01 ± 0.001
100
4.4
1.0
0.3
0.6
BtLH2
BoLH2
BiLH2
BfLH2
61 ± 2
176 ± 26
20 ± 5
5700
500
400
150
421
13
0.4
1.7
70 ± 4
b
aData could not be obtained for InLH2 because of low bioluminescent activity. kcat values are within
error of ± 5%. cRise (time to reach maximum intensity) and decay times are within error of ± 10%.
dRelative specific activity values are within error of ± 10%.
Table 3. Bioluminescence emission spectraa
bioluminescence λmax (nm)
substrate
LH2
BtLH2
BoLH2
BiLH2
BfLH2
pH 6.2
613 (65)
525 (61)
604 (63)
614 (96)
NDb
pH 7.0
562 (89)
522 (56)
580 (94)
600(103)
527 (47)
pH 7.8
pH 8.6
pH 9.4
pH optima
560 (71)
523 (57)
557 (77)
574 (80)
518 (57)
561 (73)
523 (57)
561 (86)
577 (96)
519 (57)
560 (68)
523 (57)
557 (75)
574 (80)
518 (58)
559 (69)
523 (57)
557 (71)
570 (78)
516 (62)
aBioluminescence λmax values are within error of ± 1. Values in parentheses are
bandwidths (nm) measured at half-maximum intensity. bData could not be
obtained because of low bioluminescent activity.
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Scheme 1. The reactions of firefly bioluminescence
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Scheme 2. Synthetic Pathways to Luciferin Analogs
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FIGURE LEGENDS
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Figure 1. Chemical structures of firefly luciferin (LH2) and substrate analogs.
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Figure 2. Normalized fluorescence emission spectra of LH2 (black) and analogs (InLH2,
cyan; BfLH2, green; BtLH2, blue; BoLH2, orange; BiLH2, magenta) recorded at the pH optima
for bioluminescence (Table 1). Spectra were acquired with 330 nm excitation as described
in detail in the Supporting Information.
Figure 3. Normalized bioluminescence emission spectra of LH2 (black) and the analogs
(BfLH2, green; BtLH2, blue; BoLH2, orange; BiLH2, magenta) recorded at the pH optima for
bioluminescence (Table 1). Additional experimental details are described in the Materials
and Methods.
Figure 4. Light emission time courses for PpyWT catalyzed bioluminescence reactions of
LH2 at pH 8.2 and BtLH2 at pH 9.1 recorded as described in Materials and Methods. Top
right inset: Bioluminescence emission images of in vitro reactions of LH2 (top) and BtLH2
(bottom) taken (A) 5 s, (B) 5 min, and (C) 30 min after initiation of light reactions. Assays
(0.130 mL volume) in 25 mM glycylglycine buffer, pH 8.2 for LH2, or 50 mM AMPD, pH 9.1
for BtLH2, contained 2.0 mM Mg-ATP, 1 μg of enzyme, and substrate concentrations ~6
times their Km values. All images were obtained with a ChromaScan Lite Imaging System.
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Figure 5. Bioluminescence imaging of E. coli colonies on nitrocellulose filters expressing
PpyWT. Light reactions were initiated by soaking nitrocellulose filters with 0.6 mL of 1 mM
solutions of LH2 (left) and BtLH2 (right) in 0.1 M sodium citrate buffer, pH 5.5. After ~3
min, images were obtained with a ChromaScan Lite Imaging System.
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