Exploiting the Inherent Photophysical Properties of the Major Tirapazamine Metabolite in the Development of Profluorescent Substrates for Enzymes That Catalyze the Bioreductive Activation of Hypoxia-Selective Anticancer Prodrugs

Article abstract of DOI:10.1021/acs.joc.7b03035

Hypoxia-selective cytotoxins (HSCs) seek to exploit the oxygen-poor nature of tumor tissue for therapeutic gain. Typically, HSCs require activation by one-electron bioreductive enzymes such as NADPH:cytochrome P450 reductase (CYPOR). Thus, successful clinical deployment of HSCs may be facilitated by the development and implementation of diagnostic probes that detect the presence of relevant bioreductive enzymes in tumor tissue. The work described here develops analogues of the well-studied HSC tirapazamine (3-amino-1,2,4-benzotriazine 1,4-di-N-oxide, TPZ) as profluorescent substrates of the one-electron reductases involved in bioactivation of HSCs. Hypoxic metabolism of TPZ or 7-fluoro-TPZ by one-electron reductases releases inherently fluorescent mono-N-oxide metabolites that may serve as indicators, probes, markers, or stains for the detection of the enzymes involved in the bioactivation of HSCs. In particular, profluorescent compounds of this type can provide a foundation for fluorescence-based bioassays that help identify tumors responsive to HSCs.

Full text of DOI:10.1021/acs.joc.7b03035

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Article
Exploiting the inherent photophysical properties of the major tirapazamine
metabolite in the development of profluorescent substrates for enzymes that
catalyze the bioreductive activation of hypoxia-selective anticancer prodrugs
Xiulong Shen, Charles H. Laber, Ujjal Sarkar, Fabio Gallazzi, Kevin M Johnson, Nathaniel Guy
Mahieu, Roman Hillebrand, Tarra Fuchs-Knotts, Charles L. Barnes, Gary A. Baker, and Kent S. Gates
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.7b03035 • Publication Date (Web): 20 Feb 2018
Downloaded from http://pubs.acs.org on February 24, 2018
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Exploiting the inherent photophysical properties of the
major tirapazamine metabolite in the development of
profluorescent substrates for enzymes that catalyze the
bioreductive activation of hypoxiaꢀselective anticancer
prodrugs
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‡
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Xiulong Shen , Charles H. Laber , Ujjal Sarkar , Fabio Galazzi , Kevin M. Johnson ,
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‡
‡
‡
Nathaniel G. Mahieu , Roman Hillebrand , Tarra FuchsꢀKnotts , Charles L. Barnes ,
‡
‡,§,
Gary A. Baker , and Kent S. Gates *
‡
University of Missouri
Department of Chemistry
125 Chemistry Building
Columbia, MO 65211
§
University of Missouri
Department of Biochemistry
125 Chemistry Building
Columbia, MO 65211
†
University of Missouri
Molecular Interaction Core
Columbia, MO 65211
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Address correspondence to: email: gatesk@missouri.edu; phone: (573) 882ꢀ6763
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Abstract: Hypoxiaꢀselective cytotoxins (HSCs) seek to exploit the oxygenꢀpoor nature
of tumor tissue for therapeutic gain. Typically, HSCs require activation by oneꢀelectron
bioreductive enzymes such as NADPH:cytochrome P450 reductase (CYPOR). Thus,
successful clinical deployment of HSCs may be facilitated by the development and
implementation of diagnostic probes that detect the presence of relevant bioreductive
enzymes in tumor tissue. The work described here develops analogs of the wellꢀstudied
HSC tirapazamine (3ꢀaminoꢀ1,2,4ꢀbenzotriazine 1,4ꢀdiꢀNꢀoxide, TPZ) as profluorescent
substrates of the oneꢀelectron reductases involved in bioactivation of HSCs. Hypoxic
metabolism of TPZ or 7ꢀfluoroꢀTPZ by oneꢀelectron reductases releases inherently
fluorescent monoꢀNꢀoxide metabolites that may serve as indicators, probes, markers, or
stains for the detection of the enzymes involved in the bioactivation of HSCs. In
particular, profluorescent compounds of this type can provide a foundation for
fluorescenceꢀbased bioassays that help identify tumors responsive to HSCs.
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INTRODUCTION
Most tumors contain regions of poorly oxygenated (hypoxic) tissue.
selective cytotoxins (HSC) are a class of chemotherapeutic agents that seek to exploit
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,2
Hypoxiaꢀ
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tumor hypoxia for therapeutic gain.
Typically, these compounds are activated by oneꢀ
3ꢀ
electron enzymatic reductases to generate a critical oxygenꢀsensitive drug radical anion.
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Many different enzymes can be involved in these singleꢀelectron reduction
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processes.
In aerobic tissue, backꢀoxidation of the radical anion to the starting drug
is kinetically favored while, in an oxygenꢀpoor environment, the extended lifetime of the
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radical intermediate enables reactions that convert the parent drug to a cytotoxic agent.
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,7,11
There are at least two absolute requirements for efficacious use of HSCs: (i) the
tumor tissue must be hypoxic and (ii) the tumor tissue must express reductase enzyme(s)
required for bioactivation of the drug. Successful clinical deployment of HSCs may be
facilitated by the development and implementation of diagnostic probes that detect
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,12ꢀ23
hypoxia and the bioreductive enzymes required for drug activation in tumor tissue.
Here we describe efforts toward the development of profluorescent enzyme
substrates for the detection of the oneꢀelectron bioreductive enzymes involved in the
activation of HSCs. The substrates developed here are derivatives of the wellꢀstudied
24,25
HSC tirapazamine (TPZ, 1a, Scheme 1).
TPZ undergoes intracellular enzymatic oneꢀ
1
0
electron reduction to yield a oxygenꢀsensitive drug radical 2 that selectively
decomposes under hypoxic conditions to release a cytotoxic DNAꢀdamaging drug radical
26ꢀ31
or hydroxyl radical (HO•).
Many 1,2,4ꢀbenzotriazine 1,4ꢀdioxides with analogous
3
2ꢀ34
properties have been identified.
Importantly, hypoxic metabolism of 1,2,4ꢀ
benzotriazine 1,4ꢀdioxides typically generates the corresponding 1ꢀoxide (e.g. 4) as the
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26,27,35ꢀ37
major metabolite.
Our work exploits the fortuitous fluorescence properties of the
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1
ꢀoxide metabolite generated by TPZ.
We explore the use of TPZ and other 1,2,4ꢀ
benzotriazine 1,4ꢀdioxide analogs as profluorescent substrates for the selective detection
of oneꢀelectron reductases involved in bioreductive activation of HSCs.
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Scheme 1. Hypoxic metabolism of TPZ (1a) and analogs.
We synthesized and studied the florescence properties of twentyꢀsix 1,2,4ꢀ
benzotriazines and 1,2,4ꢀbenzotriazine 1ꢀoxides leading to the identification of two
analogs (7ꢀfluoro, 4b and 7ꢀOCH , 4c) with brightness superior to the TPZ metabolite 4a.
3
The photophysical properties including the fluorescence quantum yields,
absorption/emission spectra, and Stokes shifts of 4b and 4c were determined and
compared to 4a. LCꢀMS assays were used to characterize the enzymatic conversion of
the corresponding dioxides 1aꢀc to the 1ꢀoxide metabolites 4aꢀc under aerobic and
hypoxic conditions. Fluorescence assays were used to detect the enzymatic generation of
the fluorescent metabolites 4aꢀc. We found that hypoxic metabolism of TPZ (1a) or 7ꢀ
fluoroꢀTPZ (1b) by oneꢀelectron reductases releases easily detected fluorescent 1ꢀoxide
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metabolites that may serve as indicators, probes, markers, or stains for the detection of
enzymes involved in the bioactivation of HSCs.
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RESULTS AND DISCUSSION
Synthesis and Fluorescence Properties of 1,2,4-Benzotriazine 1-Oxides 4.
A
collection of twentyꢀsix 1,2,4ꢀbenzotriazine 1ꢀoxides was synthesized by published routes
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2,35,40ꢀ43
and minor modifications of published routes (Scheme 2).
The compounds in the
collection contained various electronꢀdonating and electronꢀwithdrawing substituents.
We performed preliminary measurements to determine whether any of the compounds
displayed desirable photophysical properties such as high fluorescence quantum yields
(Φ), large extinction coefficients (ε), and large brightness values (Φ•ε) relative to the
TPZ metabolite 4a. This survey revealed that most analogs had brightness values lower
than that of 4a (Figure S1) We did, however, identify a small number of analogs with
fluorescence brightness comparable to 4a (Figure S1). Of these, 4b and 4c were selected
for further study based on their brightness, the anticipated synthetic accessibility of the
requisite 1,4ꢀdioxide analogs, the retention of fluorescence properties in water, and due to
the fact that previous results indicated that these analogs displayed hypoxiaꢀselective
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2
cytotoxicity (and presumably cell permeability) against cancer cells in culture.
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Scheme 2. Synthesis of TPZ and analogs.
Fluorescence Properties of 1,2,4-Benzotriazine Oxides 1a-c and 4a-c. We next
undertook more complete photophysical characterization of the dioxides 1b and 1c and
their putative monoꢀNꢀoxide metabolites 4b and 4c, in comparison to TPZ (1a) and its
metabolite 4a (Table 1). The dioxides 1aꢀc were synthesized as shown in Scheme 2 via
oxidation of the monoꢀNꢀoxide derivatives with a mixture of trifluoroacetic anhydride
32,35,40
and H O .
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a
b
c
Compound λabs/nm log10
ε
λ /nm
Φ /%
fl
em
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3.765
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16.86±1.40
29.94±2.09
47.22±5.07
3.67±0.21
2.44±0.21
8.68±1.00
0.60±0.01
0.77±0.02
2.27±0.35
0.33±0.01
0.30±0.01
1.24±0.16
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542
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c
ε in units of L—mol —cm . For excitation at 421 nm. Determined using coumarin
53 (C153) in ethanol (Φre ef = 38%) as the fluorescence quantum yield standard.
Measured in acetonitrile. Measured in water.
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Table 1. Static photophysical properties of 1,2,4ꢀbenzotriazine oxides.
The extinction coefficients for all compounds 1aꢀc and 4aꢀc varied by only about
0% (Table 1). The dioxides 1aꢀc displayed weak fluorescence in water and various nonꢀ
3
aqueous solvents, with quantum yields in the range of 0.6–2.2% in acetonitrile and 0.3–
.2% in water (Table 1). The monoꢀNꢀoxides displayed substantially stronger
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fluorescence quantum yields in the range of 16–47% in acetonitrile and 2–8% in water
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(
Table 1). The monoꢀNꢀoxides 4aꢀc displayed large Stokes shifts, with separations
between the absorption and emission maxima of 71, 76, and 79 nm in acetonitrile and
104, 106, and 106 nm in water, for 4aꢀc, respectively (Table 1, Figures S2ꢀ4). The
absorption/emission maxima in water for the 7ꢀfluoro analog 4b (422/526 nm) and the 7ꢀ
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OCH derivative 4c (441/547 nm) were located at longer wavelengths than those for the
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TPZ metabolite 4a (415/521 nm). Longer emission wavelengths are preferred for
biological and biochemical applications to avoid interference arising from
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autofluorescence of biological materials such as flavins and NADPH. The fluorescence
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brightness (Φ•ε) of the 7ꢀfluoro analog 4b (1917 M cm ) is 2.0ꢀfold larger, and the 7ꢀ
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–
OCH derivative 4c (2089 M cm ) 2.2ꢀfold larger, than the TPZ metabolite 4a (944 M
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–1
cm ) in acetonitrile.
Figure 1. Fluorescence quantum yields and emission maxima measured for 4b and 4c in
water in the presence of increasing levels of bovine serum albumin (BSA). Error bars
depict standard deviation in the measurements.
Given the intended use of these compounds in biochemical systems, we examined
the effects of added protein on the photophysical properties of 4b and 4c. We found that
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the fluorescence emission maxima and quantum yields for these two compounds are not
markedly influenced by the presence of the protein bovine serum albumin (BSA), a
plasma protein which displays ubiquitous nonꢀspecific binding with small molecules
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(Figure 1).
LC-MS Analysis of In Vitro Hypoxic Metabolic Conversion 1a-c To The
Corresponding Mono-N-oxides 4a-c. Having determined that 4b and 4c possess
promising photophysical properties, it was important to examine whether the
corresponding diꢀNꢀoxides 1b and 1c undergo the expected hypoxiaꢀselective, enzymatic
conversion to the fluorescent monoxide metabolites 4b and 4c. We expected clean
conversion to the monoꢀNꢀoxides, but recognized that this expectation was based on
results from only a handful of 1,2,4ꢀbenzotriazine 1,4ꢀdioxide analogs for which
2
6,27,35ꢀ37,46
metabolism had been studied.
For these assays, we used NADPH:cytochrome
P450 oxidoreductase (CYPOR) and xanthine oxidase (XO) because these enzymes are
commonly involved in oneꢀelectron bioreductive activation of HSCs both in vivo and in
1
0,13,26,28,31,47ꢀ50
vitro.
We used HPLC and LCꢀMS analysis to characterize the in vitro
metabolism of 1b and 1c, under aerobic and hypoxic conditions, in comparison with the
parent compound 1a (Figures 2, 3, S5, and S6).
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Figure 2. Yields of monoꢀNꢀoxides (4a-c) generated by in vitro metabolism of 1a, 1b,
and 1c under various conditions. The standard deviation was calculated from two
replicates.
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Figure 3. LCꢀMS analysis of the in vitro hypoxic metabolism of 1b catalyzed by
NADPH/CYPOR. (A) Total ion chromatogram of the aerobic assay; (B) Total ion
chromatogram of the hypoxic assay; (C) LCꢀMS data for 4b; (D) LCꢀMS data for 1b.
Hypoxic assays were carried out in an inert atmosphere glove bag using degassed
solutions. We found that incubation of 1aꢀc (500 ꢀM) with CYPOR (0.13 units/mL) and
NADPH (2 mM, 4 equiv) in sodium phosphate buffer (50 mM, pH 7) under hypoxic
conditions for 4 h at 24 °C generated substantial yields of the monoxide metabolites 4aꢀc
(Figure 2). Representative LCꢀMS data for the in vitro metabolism of 1b are shown in
Figure 3. The retention times and mass spectra of the metabolites 4 matched those of
authentic monoxides prepared by chemical synthesis. In an identical assay, except under
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aerobic conditions, in vitro metabolism of 1a produced an 18ꢀfold lower yield of the
monoꢀNꢀoxide 4a compared to the hypoxic assay (Figure 2). Metabolism of 1b was
similarly inhibited under aerobic conditions, giving an 8ꢀfold lower yield of the monoꢀNꢀ
oxide 4b. In contrast, the metabolism of 1c proved to be poorly selective for hypoxia.
Indeed, 1c underwent large amounts of aerobic metabolism, relative to 1a and b. Similar
results were observed for the enzyme XO (Figure 2). Possible reasons for the aerobic
metabolism of 1c are considered in the Conclusions.
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We also examined the in vitro metabolism of 1aꢀc by the obligate twoꢀelectron
5
1
reductase NADPH:quinone oxidoreductase (DTꢀdiaphorase) and by NADPH alone.
We found that 1a and 1b were not metabolized to a significant extent under these
conditions. On the other hand, NADPH alone and in concert with DTꢀdiaphorase
mediated significant conversion of 1c to 4c (Figure 2). Control experiments showed that
the DTꢀdiaphorase system was effective in reducing the archetypal substrates 1,4ꢀ
naphthoquinone and 2,3ꢀdimethoxyꢀ1,4ꢀnaphthoquinone.
Fluorimetric Analysis of the In Vitro Metabolic Conversion of 1a-c to 4b-c.
Compounds 1aꢀc were incubated with CYPOR, XO, or DTꢀdiaphorase enzyme systems
under aerobic or hypoxic conditions, the reaction mixtures extracted with ethyl acetate,
and the fluorescence of the extracts determined at the excitation/emission maxima of the
corresponding monoꢀNꢀoxide metabolite (4aꢀc) anticipated for each reaction.
Compounds 1a and 1b gave easily detected fluorescence signals in the presence of the
oneꢀelectron reducing enzymes CYPOR or XO under hypoxic conditions. Relatively
weak fluorescence output was produced by incubation of 1a and 1b with CYPOR and
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XO under aerobic conditions or when DTꢀdiaphorase or NADPH alone was used. The
fluorescence signals produced by the 7ꢀfluoro analog 1b were 16ꢀfold and 24ꢀfold above
background in the presence of XO and CYPOR enzyme systems, respectively (Figure 4).
Qualitatively similar results were obtained by direct fluorimetric analysis in aqueous
buffer (Figure S7). In addition, the dioxide 1b can be metabolized in a hypoxic cell
extract and fluorescence from the resulting monoꢀNꢀoxide can be detected against
background autofluorescence (Figure S8).
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Figure 4. Fluorimetric analysis of metabolites generated in the aerobic and hypoxic metabolism of 1a, 1b,
and 1c by CYPOR, XO, and DTꢀdiaphorase. The results of aerobic assays are shown with blue bars; the
results of anaerobic assay are shown with red bars. (A) Results for 1a; (B) Results for 1b; (C) Results for
1
c. Assays were extracted with ethyl acetate and fluorescence of the organic extract measured. Reaction
conditions: X/XO assays: diꢀNꢀoxide (500 ꢁM), xanthine (2 mM), xanthine oxidase (0.04 U/mL), sodium
phosphate buffer (50 mM, pH 7.0); CYPOR assays: diꢀNꢀoxide (500 ꢁM), NADPH (2 mM), cytochrome
P450 reductase (0.13 U/mL), sodium phosphate buffer (50 mM, pH 7.0); noꢀenzyme control assays: diꢀNꢀ
oxides (500 ꢁM), NADPH (2 mM), sodium phosphate buffer (50 mM, pH 7.0); DTꢀdiaphorase assays: diꢀ
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Nꢀoxides (500 ꢁM), NADPH (2 mM), DTꢀdiaphorase (0.01 U/mL), sodium phosphate buffer (50 mM, pH
7
.0). Error bars represent the standard deviation.
In vitro metabolism of the 7ꢀOCH derivative 1c by CYPOR and XO produced
3
strong fluorescence signals under hypoxic conditions, but also generated rather high
fluorescence responses under aerobic conditions. More significantly, in vitro metabolism
of 1c by NADPH alone and in the presence of DTꢀdiaphorase produced substantial
fluorescence under both hypoxic and aerobic conditions. The fluorescence results are
consistent with the HPLC and LCꢀMS analyses described above.
0
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CONCLUSIONS
Profluorescent enzyme substrates are important in chemical biology and clinical
5
2,53
diagnostics.
Our work exploits the fortuitous fluorescence properties of the 1ꢀoxide
38,39
metabolite generated by hypoxic metabolism of the HSC agent TPZ.
Indeed, we
found that hypoxic metabolism of TPZ (1a) and its fluoroꢀanalog 1b generates easily
detectable fluorescent metabolites 4a and 4b. The fluoroꢀTPZ analog 1b developed here
generates substantially brighter fluorescence output than the parent compound TPZ (1a).
In addition to its superior brightness, the metabolite 4b emits fluorescence at longer
(green) wavelengths (491 nm in CH CN and 529 nm in H O), a feature which may be
3
2
44
advantageous in biochemical and biological applications. Importantly, 1a and 1b are
not substrates for the twoꢀelectron reductase DTꢀdiaphorase. This observation is
1
3
consistent with inferences drawn previously from studies of TPZ in cell culture. It is
critical that probes of this sort respond selectively to the oneꢀelectron reductases involved
in the oxygenꢀsensitive bioactivation of HSCs.
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The behavior of the 7ꢀOCH analog 1c was surprising. This compound was
3
previously reported to display hypoxiaꢀselective cytotoxicity similar to the 7ꢀfluoro
3
2
analog 1b. Thus, we expected that both 1b and 1c would undergo hypoxiaꢀselective
metabolism by oneꢀelectron reductases and would be refractory to processing by a twoꢀ
electron reductase like DTꢀdiaphorase. As described above, the 7ꢀfluoro derivative 1b
1
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met these expectations. In stark contrast, the 7ꢀOCH analog 1c underwent significant
3
amounts of aerobic metabolism by the oneꢀelectron reductases CYPOR and XO and also
was a substrate for the twoꢀelectron reductase DTꢀdiaphorase. As a result, compound 1c
will likely not be a particularly useful probe for the selective detection of oneꢀelectron
reductases. We posit that the electronꢀdonating OCH substitutent may favor protonation
3
of the activated intermediate 2/3 and/or increase the fragmentation rate constant (kf,
Scheme 1), leading to diminished oxygenꢀsensitivity in the production of the 1ꢀoxide
1
1,30,31
metabolite 1c.
Reasons for the different hypoxia selectivities observed in our
3
2
assays compared to the cell culture assays reported previously deserve further study.
Overall, we found that hypoxic metabolism of the substrates 1a and 1b generated
easily detected fluorescent metabolites that have the potential to serve as markers for the
oneꢀelectron reductases required for the hypoxiaꢀselective bioactivation of HSCs. Probes
of this type enable simultaneous functional determination of the total oneꢀelectron
1
8
enzymatic reducing capacity in a biological sample. This may provide a more direct
and perhaps simpler approach to the assessment of oneꢀelectron reductase activity than
transcriptomic measurements of gene expression or proteomic measurements protein
expression. The benzotriazineꢀbased probes clearly are well positioned to detect the
enzymes relevant to the oneꢀelectron bioactivation of 1,2,4ꢀbenzotriazine 1,4ꢀdioxide
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9,13
HSCs.
Importantly, compounds such as 1b could be generally applicable because the
enzymes involved in bioactivation of 1,2,4ꢀbenzotriazine 1,4ꢀdioxides are likely to be
8
,9,13
major players in the bioactivation of many HSCs.
Thus, profluorescent compounds
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such as 1b may provide a foundation for fluorescenceꢀbased bioassays that aid in the
18
identification of tumors responsive to HSCs.
EXPERIMENTAL METHODS
Materials. Materials were of the highest purity available and were obtained from the
following sources: NADPH:cytochrome P450 reductase, DTꢀdiaphorase, NADPH,
xanthine, 1,4ꢀnaphthoquinone, 2,3ꢀdimethoxyꢀ1,4ꢀnaphthoquinone, sodium phosphate,
DMSO, silica gel (0.04–0.063 mm pore size), and silica gel plates for thin layer
chromatography from Sigma Chemical Co. (St. Louis, MO); xanthine oxidase from
Roche Applied Science (Indianapolis, IN); HPLC grade solvents (acetonitrile, methanol,
ethyl acetate, hexane, trifluoroacetic acid and acetic acid) from Fischer (Pittsburgh, PA);
deuterated NMR solvents were from Cambridge Isotope Laboratories (Andover, MA).
General procedure for in vitro metabolism reactions. In a typical hypoxic assay, the
1
,2,4ꢀbenzotriazine 1,4ꢀdioxide (500 ꢁM) was incubated with NADPH (2 mM) or
xanthine (2 mM) and cytochrome P450 reductase (0.13 U/mL) or xanthine oxidase (0.04
U/mL, or DTꢀdiaphorase (0.005 U/mL), or no enzyme in sodium phosphate buffer (50
mM, pH 7.0) containing DMSO (0.5% v/v) at 24 °C for 4 h. All components of the
reactions except enzymes and NADPH were degassed by three freeze−pump−thaw
cycles. Enzymes and NADPH were diluted with degassed water in an argonꢀfilled
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glovebag to prepare stock solutions. Reactions were initiated by the addition of enzymes,
wrapped in aluminum foil to prevent exposure to light, and incubated in an argonꢀfilled
glovebag. The aerobic controls were performed in an identical manner except with
aerobic stock solutions and solvents. Following incubation, the reactions were opened to
air and diluted to 1 mL by the addition of 800 ꢀL of aerobic sodium phosphate buffer (50
mM, pH 7.0) and extracted with ethyl acetate (1 mL). The materials in the organic layer
were analyzed using fluorescence spectroscopy or HPLC analysis. The brightness (Φ•ε)
1
1
1
1
1
1
1
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1
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0
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–
1
–1
–1
–1
of 4b and 4c in ethyl acetate is 1475 M cm and 1742 M cm , respectively. These
–1
values are comparable to the brightness of these compounds in acetonitrile (4b, 1917 M
–1
–1
–1
cm and 4c, 2089 M cm ).
LC-MS analysis of the in vitro enzymatic metabolism of 3-amino-1,2,4-benzotriazine
,4-dioxides. Following incubation, the samples were dried in a speedꢀvac concentrator
1
under vacuum at 24 °C, dissolved in DMSO (1 mL), and analyzed by LCꢀMS employing
a C18 reverse phase Betabasic column (5 ꢁm particle size, 150 Å pore size, 25 cm length,
and 4.6 mm i.d.) eluted with a gradient starting from 5% B (0.1% trifluoacetic acid in
acetonitrile) and 95% A (0.1% trifluoacetic acid in water) for 5 min, followed by linear
increase to 35% B over 60 min, a linear increase to 80% of B over 5 min, and finally a
wash with 80% of B for 10 min. A flow rate of 1.0 mL/min was used, and the products
were monitored by their UVꢀabsorbance at 254 nm. LC/ESIꢀMS experiments were
carried out using an ion trap mass analyzer, on a LCQ FLEET instrument (Thermo Fisher
Scientific). Positive ion electrospray was used as the means of ionization. The heated
inlet capillary temperature was 375 °C, and electrospray needle voltage was 5 kV.
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Nitrogen sheath gas was supplied at 45 psi, and the LC/ESIꢀMS analysis was performed
in the positive ion mode.
0
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Quantum yield determination. Fluorescence quantum yields (Φ ) were measured and
u
5
4
calculated by the ParkerꢀRees method employing the relationship:
2
2
r
Φ = Φ (A /L )(L /A )(η /η )
u
ref
r
r
u
u
u
In the expression above, Φref denotes the quantum yield of a wellꢀknown fluorophore;
which in this case was coumarin 153 (Φref = 38% in ethanol). A represents the
u
absorbance of the unknown sample at the desired excitation wavelength, A is the
r
reference fluorophore’s absorbance at the same excitation wavelength, L designates the
u
total integrated luminescence intensity of the unknown sample when excited at the same
excitation wavelength and L is the total integrated luminescence intensity of the
r
reference fluorophore when excited at the same excitation wavelength. The refractive
indices of the solvents in which the unknown and reference emissions are measured in are
5
5
denoted as η and η , respectively.
u
r
HPLC calibration curves. Standard solutions of each analyte (10, 20, 30, 50, 100 ꢁM in
DMSO) were prepared using serial dilution from higher concentration stock solutions and
analyzed using a C18 reverseꢀphase column (particle size 5 ꢀm, 150 Å pore size, 25 cm
length, and 4.6 mm i.d.) eluted with a mixture of water:methanol:acetic acid (74:25:1) for
4
5 min at a flow rate of 0.9 mL/min, while products were detected by their UVꢀ
absorbance at 254 nm. Quinone compounds were analyzed using the same system eluted
with an isocratic solvent composed of water:methanol:acetic acid (59:40:1) for 45 min at
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a flow rate of 0.9 mL/min, while the compounds were detected by their absorbance at 260
nm.
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Supporting Information
Brightness of twentyꢀsix different benzotriazine and benzotriazine 1ꢀoxides,
photophysical properties of 4aꢀc, LCꢀMS analysis of hypoxic metabolism, fluorimetric
analysis of hypoxic metabolism, and HPLC analysis of 4aꢀc. This material is available
free of charge via the Internet at http://pubs.acs.org.
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