Development of a General Aza-Cope Reaction Trigger Applied to Fluorescence Imaging of Formaldehyde in Living Cells

Article abstract of DOI:10.1021/jacs.6b12460

Formaldehyde (FA) is a reactive signaling molecule that is continuously produced through a number of central biological pathways spanning epigenetics to one-carbon metabolism. On the other hand, aberrant, elevated levels of FA are implicated in disease states ranging from asthma to neurodegenerative disorders. In this context, fluorescence-based probes for FA imaging are emerging as potentially powerful chemical tools to help disentangle the complexities of FA homeostasis and its physiological and pathological contributions. Currently available FA indicators require direct modification of the fluorophore backbone through complex synthetic considerations to enable FA detection, often limiting the generalization of designs to other fluorophore classes. To address this challenge, we now present the rational, iterative development of a general reaction-based trigger utilizing 2-aza-Cope reactivity for selective and sensitive detection of FA in living systems. Specifically, we developed a homoallylamine functionality that can undergo a subsequent self-immolative β-elimination, creating a FA-responsive trigger that is capable of masking a phenol on a fluorophore or any other potential chemical scaffold for related imaging and/or therapeutic applications. We demonstrate the utility of this trigger by creating a series of fluorescent probes for FA with excitation and emission wavelengths that span the UV to visible spectral regions through caging of a variety of dye units. In particular, Formaldehyde Probe 573 (FAP573), based on a resorufin scaffold, is the most red-shifted and FA sensitive in this series in terms of signal-to-noise responses and enables identification of alcohol dehydrogenase 5 (ADH5) as an enzyme that regulates FA metabolism in living cells. The results provide a starting point for the broader use of 2-aza-Cope reactivity for probing and manipulating FA biology.

Full text of DOI:10.1021/jacs.6b12460

Article
pubs.acs.org/JACS
Development of a General Aza-Cope Reaction Trigger Applied to
Fluorescence Imaging of Formaldehyde in Living Cells
Kevin J. Bruemmer,†,∇ Ryan R. Walvoord,†,∇,# Thomas F. Brewer,† Guillermo Burgos-Barragan,∥
Niek Wit,∥ Lucas B. Pontel,∥ Ketan J. Patel,∥,⊥ and Christopher J. Chang*,†,‡,§
‡
§
†Department of Chemistry, Department of Molecular and Cell Biology, and Howard Hughes Medical Institute, University of
California, Berkeley, Berkeley, California 94720, United States
∥MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom
⊥Department of Medicine, Addenbrooke’s Hospital, University of Cambridge, Cambridge CB2 2QQ, United Kingdom
S
* Supporting Information
ABSTRACT: Formaldehyde (FA) is a reactive signaling molecule that is continuously produced through a number of central
biological pathways spanning epigenetics to one-carbon metabolism. On the other hand, aberrant, elevated levels of FA are
implicated in disease states ranging from asthma to neurodegenerative disorders. In this context, fluorescence-based probes for
FA imaging are emerging as potentially powerful chemical tools to help disentangle the complexities of FA homeostasis and its
physiological and pathological contributions. Currently available FA indicators require direct modification of the fluorophore
backbone through complex synthetic considerations to enable FA detection, often limiting the generalization of designs to other
fluorophore classes. To address this challenge, we now present the rational, iterative development of a general reaction-based
trigger utilizing 2-aza-Cope reactivity for selective and sensitive detection of FA in living systems. Specifically, we developed a
homoallylamine functionality that can undergo a subsequent self-immolative β-elimination, creating a FA-responsive trigger that
is capable of masking a phenol on a fluorophore or any other potential chemical scaffold for related imaging and/or therapeutic
applications. We demonstrate the utility of this trigger by creating a series of fluorescent probes for FA with excitation and
emission wavelengths that span the UV to visible spectral regions through caging of a variety of dye units. In particular,
Formaldehyde Probe 573 (FAP573), based on a resorufin scaffold, is the most red-shifted and FA sensitive in this series in terms
of signal-to-noise responses and enables identification of alcohol dehydrogenase 5 (ADH5) as an enzyme that regulates FA
metabolism in living cells. The results provide a starting point for the broader use of 2-aza-Cope reactivity for probing and
manipulating FA biology.
metabolic enzyme systems, FA reaches a steady state level of
50−100 μM in the blood11 and 200−500 μM intracellularly.12
Even higher resting levels of FA have been found in a variety of
disease states, including neurodegenerative diseases,13 cancer,14
and asthma.15 To counteract the toxicity of FA, living
organisms have developed efficient metabolizing pathways for
FA. One predominant FA-metabolizing enzyme is cytosolic
alcohol dehydrogenase 5 (ADH5) (also known as FA
dehydrogenase and alcohol dehydrogenase 3), which oxidizes
FA to formate through a glutathione-dependent reaction.16
The dynamics of FA production and consumption in living
systems and its understudied consequences continues to
motivate the development of new methods for its detection
in biological specimens. Traditional detection methods for FA
rely upon mass spectrometry,17,18 high-performance liquid
INTRODUCTION
■
Formaldehyde (FA) is a reactive carbonyl species (RCS) that is
widely utilized in industrial applications1 as well as a protein
cross-linker for tissue fixation.2 Long classified as a toxin and
carcinogen,3 FA exposure can occur through a variety of natural
and anthropogenic sources including microbe emission, car
exhaust, and building materials.4 While traditionally thought of
as detrimental to living organisms, FA is an endogenously
produced biological metabolite that is continuously released
during essential biological pathways, including epigenetics and
one-carbon metabolism.5 For example, lysine and arginine
demethylase enzymes such as lysine specific demethylase 16 and
JmjC domain-containing proteins7 produce FA during
epigenetic regulation of histone tails.8,9 In addition, during
one-carbon metabolism, demethylation of choline metabolites
en route to production of glycine releases FA as a critical one-
carbon unit for the synthesis of important biological building
blocks.10 Governed by a complex homeostasis involving many
Received: December 2, 2016
© XXXX American Chemical Society
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chromotography,19,20 and preconcentration/chemical ioniza-
tion,21 which are highly sensitive but require harsh conditions
that are not suitable for live-specimen detection. In this context,
fluorescent probes offer a promising mode of FA detection as
they have been widely utilized to detect small-molecule
biological metabolites through recognition or reactivity-based
approaches.22−25 Indeed, reactivity-based methods have been
successfully used to visualize other carbonyl species such as
carbon monoxide26−30 and methylglyoxal,31 and our lab32,33
and others34−43 have developed new FA indicators suitable for
live-cell and live-animal imaging, based largely on 2-aza-Cope
or hydrazine condensation reactions.
Scheme 1. Self-Immolative Aza-Cope Strategy for FA-
Responsive Fluorescent Probes
These initial reports establish the promise of reactivity-based
fluorescent approaches for monitoring biological FA but leave
room for significant improvement. One key challenge to
address is that only a relatively small number of fluorescent
scaffolds have been reported for FA detection, because the vast
majority of fluorescent FA probes operate through direct
modification of the dye backbone to elicit a fluorescence
response. As such, efforts to improve FA reactivity and
selectivity tend to simultaneously perturb photophysical
properties of the dye. This synthetic limitation restricts the
ability to tune excitation/emission profiles, cellular localization,
and other properties independently of FA reactivity.
To address this outstanding issue, we now present the
development of a general 2-aza-Cope reaction trigger with a
self-immolative β-elimination linker that can be installed onto
any fluorophore containing a common phenol group, enabling
a wider range of fluorescent scaffolds to be functionalized for
FA detection. Iterative, rational design of triggers to optimize
structure−activity relationships for fast kinetics and fluorophore
release on a 4-OMe-Tokyo Green (TG) fluorescent scaffold44
can be expanded to deliver a variety of fluorescent FA
indicators with excitation and emission profiles spanning
wavelengths across the UV to visible spectrum. We further
establish the utility of these FA probes to detect changes in this
RCS in living cells. Moreover, the resorufin-based congener,
FAP573, gives the highest signal-to-noise response and the
most red-shifted excitation/emission profile, enabling visual-
ization of changes in the cellular metabolism of FA mediated by
ADH5 using genetic knockout models.
with FA through a 2-aza-Cope rearrangement to the rearranged
imine. Subsequent elimination of the two carbon linker via a β-
elimination would afford the uncaged phenolate an expected
increase in fluorescence in addition to the homoallylamine and
acrolein byproducts.45−48 Such a trigger could be applied to
phenolic fluorophores or related chemical probes, as well as to
amine-based dyes via carbamate linkers.
We initiated this study by preparing a general reagent for
caging nucleophilic fluorophores via a protected homoallyl-
amine with a β-leaving group. In a first working example,
aminoallylation of 4-hydroxybutanone, 1, afforded amino-
alcohol 2. Subsequent diazotransfer using Goddard-Borger’s
method49 and tosylation afforded the key caging group 3. We
explored the caging properties of 3 by appending it to a known
fluorescent scaffold, 2-methyl-4-methoxy-Tokyo Green (TG),
4. (Figure 1).44 This derivative has been utilized previously as a
platform for reactivity-based bioimaging purposes through
masking of its free phenolic group to limit conjugation through
the xanthenone portion of the dye.50−55 Accordingly, O-
alkylation of the fluorophore with Cs2CO3 and 3, followed by
azide reduction using a tin(II) chloride/thiophenol mixture,56
yielded weakly fluorescent, caged TG 6. Upon treatment with
FA in aqueous PBS buffer at physiological pH, the anion of 4 is
released, resulting in a ca. 10-fold fluorescence increase in
response to 100 μM FA within 2 h (Figure 1b). Moreover, the
probe also displayed selectivity for FA over other relevant RCS
and biological analytes (Figure 1c). Thus, this initial design of a
self-immolative 2-aza-Cope reactivity trigger shows a promising
approach to convert a phenol-containing fluorophore into a
selective FA-responsive fluorescent indicator.
RESULTS AND DISCUSSION
■
Design and Synthesis of a General 2-Aza-Cope
Reaction-Based Trigger. Previous work from our laboratory
reported FAP-1, a first-generation reaction-based fluorescent
probe for detecting FA through a turn-on response (Scheme
1).32 Specifically, we developed a reactivity-dependent FA
indicator through conversion of a homoallyl amine into an
aldehyde on a silicon rhodamine scaffold, where control of
spirocyclization gives rise to a turn-on fluorescence readout. In
attempts to generalize this reaction to develop fluorescent FA
probes with a broader range of excitation/emission colors and
maintain good insensitivity to pH, we were thwarted by the
need to directly functionalize the fluorophore backbone and
rely on often complex spirocyclization equilibria to achieve a
fluorescence response. We reasoned that separating the dye
scaffold and FA-dependent trigger unit into separate,
independent pieces might allow for a more general strategy
for developing a range of FA-responsive fluorophores.
Specifically, we sought to cage phenolic fluorophores with a
homoallylamine aza-Cope trigger with a self-immolative linker,
envisioning that these O-alkylated fluorophores would react
Structure−Activity Studies to Optimize the FA
Responses of the Aza-Cope Reaction Trigger. The initial
probe 6 established the viability of the aza-Cope reactivity
trigger to selectively react with FA to induce a fluorescence
response, but the relatively slow conversion of 6 into the
uncaged fluorophore presaged potential limitations for its utility
in live-cell imaging studies of FA biology. As such, we next
explored several synthetic iterations of the trigger structure with
the aim of improving aza-Cope reactivity to increase
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Figure 1. (a) Reagents and conditions: (i) NH3, MeOH, 0 °C to rt, 30 min, then allyl pinacol boronate, rt, 24 h, 69%; (ii) diazo transfer reagent,
CuSO4·5H2O, K2CO3, MeOH, rt, 20 h; (iii) TsCl, Et3N, DMAP, CH2Cl2, rt, 16 h, 27% (2 steps); (iv) 3, Cs2CO3, DMF, 40 °C, 20 h, 62%; (v)
SnCl2, PhSH, Et3N, MeCN, rt, 12 h, 91%. (b) Fluorescence response and selectivity of 10 μM probe 6 to 100 μM FA. Data was acquired in 20 mM
PBS (pH 7.4) at 37 °C. Emission was collected between 500−600 nm (λex = 488 nm). Lines represent time points taken at 0 (black), 30 (light gray),
60 (gray), 90 (dark gray), and 120 min (red) after addition of 100 μM FA. (c) Fluorescence response of 10 μM probe 6 to RCS or relevant
biological analyte. Data was acquired in 20 mM PBS (pH 7.4) at 37 °C. Bars represent relative emission intensity responses to 100 μM analyte after
treatment for at 0 (black), 30 (light gray), 60 (gray), 90 (dark gray), and 120 min (red). Analytes were prepared as stated in the Selectivity Tests
section of the SI. Legend: (1) PBS, (2) FA, (3) acetaldehyde, (4) acrolein, (5) glucose (1 mM), (6) H2O2, (7) methylglyoxal, (8) dehydroascorbate
(9) pyruvate, (10) glucosone, (11) oxaloacetate, (12) 4-hydroxynonenal.
a
Scheme 2. Structural Variation of the Trigger at the 2 Position
a
Reagents and conditions: (i) TBSCl, imidazole, CH2Cl2, rt; (ii) 4-nitrotoluenesulfonyl chloride, Et3N, DMAP, CH2Cl2, 0 °C to rt, 73% (3 steps
from 1); (iii) MeI, K2CO3, DMF, rt; (iv) TBAF, THF, rt, 95% (2 steps); (v) TsCl, Et3N, DMAP, CH2Cl2, rt, 89%; (vi) 4, Cs2CO3, DMF, 50 °C,
30%; (vii) PhSH, K2CO3, MeCN, 50 °C, 42%; (viii) K2CO3, MeCN, DMF, rt, 20 h, 14%.
fluorophore signal-to-noise responses. In particular, we targeted
systematic modifications to the core trigger structure at the 2, 3,
and 4 positions and installed these respective triggers onto the
parent TG scaffold (Scheme 2).
Scheme 2 outlines the synthesis of probes 9 and 10, which
are modified at the 2 position. The direct monoalkylation of 6
proved surprisingly challenging. An alternative approach used
silyl protection and N-nosylation of 2 to yield sulfonamide 7,
which can be methylated readily. Conversion of the silyl ether
to the tosylate afforded key linker 8. It is noted that judicious
choice of protecting group strategies is critical, as attempts to
prepare analogues of 8 containing a secondary carbamate or
nosylate resulted in rapid intramolecular cyclizations. Con-
jugation of TG with 8 followed by nosyl group removal with
thiophenol provided N-methylated probe 9. An N-trifluoro-
ethylated analogue, 10, could be directly formed through
alkylation of 6 with the corresponding triflate.
Probe 14, which possesses a hydrogen at the 3-position in
place of the parent methyl group, was synthesized starting from
commercially available homoallylamino acid 11. Reduction of
the acid and Boc deprotection furnished aminoalcohol 12,
which was then converted to azido tosylate 13. Conjugation to
the TG fluorophore and azide reduction provided sterically
unencumbered variant 14. An alternate trigger modified at the
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a
Scheme 3. Structural Variation of the Trigger at the 3 Position
a
Reagents and conditions: (i) ethyl chloroformate, Et3N, THF, 0 °C, 30 min, then NaBH4, THF, H2O, rt, 2 h, 72%; (ii) HCl, Et2O, MeOH, 0 °C to
rt, 3 h; (iii) diazo transfer reagent, CuSO4·5H2O, K2CO3, MeOH, rt, 14 h; (iv) TsCl, Et3N, DMAP, CH2Cl2, rt, 24 h, 53% (3 steps); (v) 4, Cs2CO3,
DMF, 40 °C, 20 h, 58%; (vi) SnCl2, PhSH, Et3N, MeCN, rt, 2.5 h, 33%; (vii) NH3, MeOH, then allyl pinacol boronate; (viii) diazo transfer reagent,
CuSO4·5H2O, K2CO3, MeOH, rt, 78% (2 steps); (ix) TsCl, Et3N, DMAP, CH2Cl2, rt, quantitative; (x) 4, Cs2CO3, DMF, 45 °C, 33%; (xi) SnCl2,
PhSH, Et3N, MeCN, rt, 74%.
a
Scheme 4. Structural Variation of the Trigger at the 4 Position
a
Reagents and conditions: (i) H2PdCl4, tetrahydroxydiboron, DMSO, H2O, rt, 14 h; (ii) 1, NH3, MeOH 0 °C to rt, then 19, CHCl3, rt; (iii) diazo
transfer reagent, CuSO4·5H2O, K2CO3, MeOH, rt; (iv) TsCl, Et3N, DMAP, CH2Cl2, rt; (v) 4, Cs2CO3, DMF, 40 °C, 20 h; (vi) SnCl2, PhSH, Et3N,
MeCN, rt, 18 h, 11% (2 steps).
Figure 2. Relative fluorescence response rates of trigger structures appended to TG. Fluorescence response of 10 μM probe to 1 mM FA. Data was
acquired in 20 mM PBS (pH 7.4) at 37 °C. Emission was collected between 500−600 nm (λex = 488 nm). Relative fluorescence response rate
indicates the linear increase in fluorescence/min at λem (518 nm). Rate was determined by measuring a linear slope of the fluorescence intensity per
min before saturation kinetics were observed (Figure S1−S6).
3-position was constructed beginning with aminoalkylation of
ketone 15. The intermediate aminoalcohol was converted to
the azide through diazo transfer and then transformed via
tosylation to form 16. Fluorophore alkylation and azide
reduction then produced caged TG 17, which contains a
phenyl group at the 3-position (Scheme 3).
(FAP488, Scheme 4) corresponding to its excitation maximum
at 488 nm.
Effects of Trigger Modification on FA Probe
Sensitivity and Signal-to-Noise Responses. With this
family of TG derivatives caged with aza-Cope reactivity triggers
modified at the 2, 3, and 4 positions in hand, we compared their
relative responses to FA in vitro. Relative probe sensitivity was
determined based on the fluorescence response rate of the
probe relative to 6. Specifically, compounds 9, 10, 14, 17, and
FAP488 were treated with 1 mM FA in PBS buffer, and the
change in fluorescence was monitored over time (Figure 2). We
observed that modifications at the 2 position (9, 10) resulted in
a poorer fluorescence response relative to parent probe 6. We
speculate that this decrease in signal-to-noise sensitivity may
potentially result from increased steric encumbrance. Addition-
́
Recent work by Szabo on the addition of sterically hindered
allyl boronic acids to ketones57 provided a synthetic route for
the installation of alkyl groups at the 4-position of the aza-Cope
trigger. In particular, key aminoalcohol intermediate 20,
containing gem-dimethyl substitution at the 4-position, was
synthesized by treating the imine of 1 with prenyl boronic acid
19 formed in situ. The resulting aminoalcohol was similarly
converted to azido tosylate 21. Alkylation and azide reduction
yielded caged TG, herein named Formaldehyde Probe 488
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Scheme 5. Synthesis of a Series of FA-Responsive Fluorescent Probes
ally, the minimal response of 10 to FA may also be rationalized
through decreased nitrogen nucleophilicity due to the inductive
effects of the trifluoroethyl group, resulting in a slower initial
condensation reaction with FA. Probes modified at the 3
position produced similar (14) or reduced (17) fluorescent
responses relative to initial probe 6. In contrast, FAP488, which
possesses a gem-dimethyl group at the 4-position, displays
greatly enhanced sensitivity to FA, showing an order of
magnitude improvement in relative fluorescence response rate
compared to the parent trigger that lacks these methyl
substituents (Figure 2). We attribute this result to the gem-
disubstitution effect, thereby increasing the rate of the aza-Cope
reaction.58
In particular, substitution at the 4-position is anticipated to
favor the rearranged imine en route to fluorophore uncaging.
The ca. 10-fold fluorescence enhancement displayed by
FAP488 relative to parent probe 6 presages that probes
derived from aza-Cope trigger piece 21 would yield fluorescent
FA indicators with greater signal-to-noise responses and FA
sensitivity. As such, this improved aza-Cope trigger provides a
starting point for further fluorescent probe development.
Developing a Series of Fluorescent FA probes with
Varying Excitation/Emission Profiles with a General Aza-
Cope Trigger. With these results in hand, we then used the
gem-dimethyl aza-Cope trigger as a general caging group to
create a series of homologous FA probes with fluorescence
excitation and emission profiles that span the UV through
visible spectral regions (Scheme 5). The naming convention for
these FA indicators follows as Formaldehyde Probe (FAP) with
a number corresponding to its maximal excitation wavelength.
For example, we utilized a trifluoromethyl-substituted coumarin
with an excitation wavelength of 385 nm to produce FAP385 as
a blue-emitting probe. FAP498 is derived from a green-emitting
trifluoroethyl rhodol scaffold that exhibits an excitation
wavelength of 498 nm. A carbofluorescein scaffold with the
carboxylic acid substituted for a trifluoromethyl group was
prepared for an orange-emitting FAP555 with an excitation
wavelength of 555 nm.59 Finally, the red-emitting FAP573 is
based on a resorufin scaffold with a corresponding excitation
wavelength of 573 nm.
the azide through a tin(II) chloride/thiophenol mixture gives
the homoallylamine-trigger conjugated to the fluorophore.
Spectroscopic Responses to FA and FA Selectivity. We
next evaluated the fluorescence responses of the FAP series to
FA in aqueous solution buffered to physiological pH (Figure 3).
FAP385 is initially weakly fluorescent (ϕfl = 0.11, ε385 = 1750
M−1 cm−1) and exhibits a ca. 4.5-fold fluorescence turn-on
response to 100 μM FA after 2 h. FAP498 (ϕfl = 0.23, ε480
=
1818 M−1 cm−1) shows only a ca. 2.2-fold response to 100 μM
FA after 2 h due to the higher initial fluorescent background
signal, which is likely a result of the more favorable equilibrium
of the open-form alcohol over the closed-form lactone in
aqueous solution at near neutral pH. FAP555 (ϕfl = 0.50, ε573
=
12 175 M−1 cm−1) displays a large bathochromic shift upon
elimination of the trigger, leading to a ca. 10-fold increase in
fluorescence response to FA after 2 h. Similar to FAP385,
FAP573 displays weak initial fluorescence (ϕfl = 0.18, ε573
=
1369 M−1 cm−1) and gives a ca. 4-fold increase in fluorescence
response to FA after 2 h. At 10 μM probe after 2 h incubation,
all probes display a 10 μM limit of detection for FA (Figure
S7). The pseudo first order kinetic plots of FAP498, FAP555,
and FAP573 all display similar rate constants of k = 7 × 10−4
s−1, which can be converted into bimolecular rate constants of k
= 0.14(1) M−1 s−1 by taking into account the concentration of
FA (5 mM) used in high excess for these experiments (Figure
S8).
The data show that this gem-dimethyl aza-Cope trigger
allows for the rational and general functionalization of a wide
variety of fluorophores for FA detection by separating the dye
scaffold from the FA-reactive cage. As expected from the
demonstrated FA specificity of the aza-Cope reaction trigger for
FA, FAP385, FAP498, FAP555, and FAP573 all exhibit high
selectivity for FA over a panel of biologically relevant RCS such
as methylglyoxal, 4-hydroxynonenal, and acetaldehyde. More-
over, these aldehydes do not interfere with the turn-on
response of these reagents to FA (Figure S9). The probes
also do not respond to addition of reagents that induce higher
levels of oxidizing (H2O2) and reducing (glutathione)
equivalents in the cell.60 Taken together, the data establish
that this 2-aza-Cope reactivity trigger can be generally applied
to develop probes with a selective response to FA with varying
excitation and emission colors.
Notably, all four of these probes, along with FAP488, feature
distinctly different fluorescent scaffolds with varying excitation/
emission profiles but can all be synthesized using the same
trigger conjugation strategy. Briefly, treatment of the
fluorophore with Cs2CO3 and tosylate 21 allows for the
conjugation of the azide-trigger to the parent dye. Reduction of
Application of FAP Reagents to Turn-on Fluorescence
Detection of Changes in FA Levels in Living Cells. After
confirming all four FAP probes are responsive and selective for
FA in aqueous buffer, we next assessed their capability for
detecting changes in FA levels in living cells. Incubation of
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Figure 3. Fluorescence responses and selectivities of FA probes. (a−c) Fluorescence responses of 10 μM (a) FAP385, (b) FAP498, (c) FAP555, or
(d) FAP573 to 100 μM FA. Data were acquired in 20 mM PBS (pH 7.4) at 37 °C. Emission was collected between (a) 450−535 nm (λex = 385
nm), (b) 505−645 nm (λex = 498 nm), (c) 560−625 nm (λex = 555 nm) or (d) 578−650 nm (λex = 573 nm). Lines represent time points taken at 0
(lightest gray), 30 (light gray), 60 (gray), 90 (dark gray), and 120 min (colored) after addition of 100 μM FA. (e−h) Fluorescence responses of 10
μM probe to RCS or relevant biological analyte. Bars represent emission intensity responses to 100 μM analyte unless otherwise stated for 0 (lightest
gray), 30 (light gray), 60 (gray), 90 (dark gray), and 120 (darkest gray) min, except FA, which is shown in colored bars. Analytes were prepared as
stated in the Selectivity Tests section of the SI. Legend: (1) PBS, (2) FA, (3) acetaldehyde, (4) glucose (1 mM), (5) 4-hydroxynonenal, (6)
dehydroascorbate, (7) glucosone, (8) pyruvate, (9) oxaloacetate, (10) acrolein, (11) methylglyoxal, (12) H2O2, (13) glutathione (5 mM).
HEK293T cells with 10 μM FAP385 (Figure S10), 5 μM
FAP498 (Figure 4), 10 μM FAP555 (Figure 5), or 10 μM
FAP573 (Figure 6) for 30 min at 37 °C in BSS buffer, followed
by a wash into fresh buffer to remove excess probe, allowed for
initial probe staining of cells with low background. Exogenous
additions of 200−1000 μM FA for 30 and 60 min resulted in a
linear increase for green, orange, and red intracellular
fluorescence for FAP498, FAP555, and FAP573, respectively,
establishing the utility of these three indicators for live-cell FA
imaging However, cells incubated with FAP385 display a
background increase in the surrounding media over the course
of the FA treatment, suggesting that the lower fluorescence
response for live-cell imaging may be due to poor cellular
retention of the probe or its fluorescent product (Figure S10).
Flow cytometry experiments were performed to verify probe
turn-on in cells (Figure S11−S13). In all cases, nuclear staining
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Figure 4. Confocal microscopy images of FAP498 in response to
exogenous FA addition in HEK293T cells. Cells were treated with 5
μM FAP498 in BSS buffer for 30 min, exchanged into fresh buffer, and
then treated with (a) vehicle, (b) 200, (c) 500, or (d) 1000 μM FA.
Images were taken after 60 min. (e) Bright field image of cells in (d)
overlaid with 1 μM Hoechst 33342. (f) Mean fluorescent intensities of
cells in (a−d) 60 min after addition of FA relative to mean
fluorescence intensity before addition of vehicle or FA; error bars
denote SEM (n = 3). *P < 5 × 10−3, ***P < 5 × 10−5. Scale bar
represents 50 μm in all images.
Figure 6. Confocal microscopy images of FAP573 in response to
exogenous FA addition in HEK293T cells. Cells were treated with 10
μM FAP573 in BSS buffer for 30 min, exchanged into fresh buffer, and
then treated with (a) vehicle, (b) 200, (c) 500, or (d) 1000 μM FA.
Images were taken after 60 min. (e) Bright field image of cells in (d)
overlaid with 1 μM Hoechst 33342. (f) Mean fluorescent intensities of
cells in (a−d) 60 min after addition of FA relative to mean
fluorescence intensity before addition of vehicle or FA; error bars
denote SEM (n = 3). ***P < 5 × 10−5. Scale bar represents 50 μm in
all images.
FAP573 Enables Detection of FA Fluxes via ADH5
Metabolism. We next sought to establish the potential value
of FAP573 for monitoring intracellular metabolism of FA to
identify sources and targets of this RCS. In this context, alcohol
dehydrogenase 5 (ADH5) is a major enzyme responsible for
cellular FA metabolism, and the recent development of two
ADH5 knockout (KO) cell lines, mouse endothelial
fibroblasts61 (MEF) and near-haploid cells (HAP1) (Figure
S15), offers the possibility to directly evaluate whether FAP
reagents can monitor potential changes in FA levels regulated
by this enzyme in living cells. Along these lines, we reasoned
that ADH5 KO cells would exhibit impaired FA clearance
compared to wild-type (WT) counterparts. To test this
hypothesis, WT and KO MEF (Figure S16) or KO HAP1
(Figure 7) cells were loaded with 10 μM FAP573 in BSS buffer
Figure 5. Confocal microscopy images of FAP555 in response to
exogenous FA addition in HEK293T cells. Cells were treated with 10
μM FAP555 in BSS buffer for 30 min, exchanged into fresh buffer, and
then treated with (a) vehicle, (b) 200, (c) 500, or (d) 1000 μM FA.
Images were taken after 60 min. (e) Bright field image of cells in (d).
(f) Mean fluorescent intensities of cells in (a−d) 60 min after addition
of FA relative to mean fluorescence intensity before addition of vehicle
or FA; error bars denote SEM (n = 3). *P < 5 × 10−3, ***P < 5 ×
10−5. Scale bar represents 50 μm in all images.
and/or flow cytometry experiments confirmed that the cells
remain viable throughout the course of the experiment (Figure
S14). For the case of FAP555, nuclear staining was not used
due to the broad excitation of both the caged (λex = 420 nm)
and uncaged probe overlapping with commonly available
nuclear dyes.
While three probes are capable of detecting changes in
intracellular FA levels with exogenously added FA, we observed
that FAP498 and FAP555 exhibit a lower fluorescence turn-on
response to FA compared to FAP573, which displays the best
response to FA in this series owing to its relatively
homogeneous staining and good cellular retention throughout
the imaging experiments. These properties led us to utilize
FAP573 in further experiments to study endogenous FA
metabolism in living cells.
Figure 7. Confocal microscopy images of FAP573 in response to FA
metabolism in ADH5 KO (a,b) or WT (c,d) HAP1 cells. Cells were
treated with 10 μM FAP573 for 30 min, exchanged into fresh buffer,
and then treated with vehicle (b,d) or 100 μM FA (a,c). Images were
taken after 60 min. (e) Bright field image of (a) overlaid with 1 μM
Hoechst 33342. (f) Mean fluorescent intensities of cells in (a−d) 60
min after addition of FA relative to mean fluorescence intensity before
addition of vehicle or FA; error bars denote SEM (n = 3). *P < 5 ×
10−3. Scale bar represents 50 μm in all images.
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at 37 °C for 30 min. The cells were washed with fresh buffer to
remove excess probe, and then incubated with 0 or 100 μM FA
for 60 min. Nuclear staining and flow cytometry experiments
confirm cell viability during the experiment (Figure S14).
Interestingly, FAP573 showed patently higher fluorescence
signals in both MEF KO and HAP1 KO cells compared to their
WT congeners upon FA incubation by both confocal
microscopy and flow cytometry (Figure S17), establishing
that FA metabolism is impeded in the KO cells. Moreover,
addition of a FA scavenger, NaHSO3,35 showed a lower level of
fluorescence under the same conditions, confirming the
fluorescence increase due to lower FA metabolism (Figure
S18). Furthermore, FAP573 was able to show a higher level of
fluorescence to basal levels of FA in KO cells compared to WT
and KO cells with NaHSO3 by confocal microscopy and flow
cytometry (Figure 8). Elimination of ADH5 results in a loss of
separating the FA-reactive cage from the fluorophore backbone,
FA responses can be tuned in a dye-independent manner to
create a broad range of FA probes in a convergent fashion. We
have applied this trigger to produce a set of fluorescent probes
for FA detection with high and predictable selectivity and
sensitivity for monitoring FA in solution and in cells with
excitation and emission colors that span from blue to green to
orange to red, either by flow cytometry and/or live-cell
imaging. Resorufin-based FAP573 exhibits the best signal-to-
noise response and most red-shifted excitation and emission
profile enables identification of ADH5 as a key enzyme for
endogenous metabolism of FA in living cells using ADH5 KO
fibroblast and haploid cell models. Current efforts are underway
to further expand the scope of this aza-Cope reactivity trigger
for developing FA probes with expanded color palettes, cellular
and subcellular targeting capabilities, and application to
different chemical strategies for FA imaging and therapeutic
applications. Because this trigger is potentially applicable to any
alcohol or amine scaffold by a direct connection or through an
additional self-immolative linkage, we envision the broader
utility of this chemistry in elucidating new sources and targets
of FA metabolism in biological pathways related to cell viability
and disease state progression, akin to the boronate trigger that
has proven useful for studying the chemical biology of
hydrogen peroxide.62,63 Of particular interest is identifying
and elucidating the contributions of FA in biological stress
mechanisms during carcinogenesis, asthma progression, and
neurodegenerative disorders.
EXPERIMENTAL DETAILS
■
Figure 8. Confocal microscopy images (a−c) and flow cytometry
histograms (e) of FAP573 in response to basal FA levels in (a) WT,
(b) ADH5 KO, and (c) ADH5 KO HAP1 cells with 200 μM NaHSO3.
(a,b) Cells were treated with 1 μM FAP573 for 30 min, then
exchanged into fresh buffer. (c) Cells were treated with 200 μM
NaHSO3 for 30 min, and then as in (a,b) keeping 200 μM NaHSO3
present in all buffers. Images were taken after 60 min. (d) Mean
fluorescent intensities of cells in (a−c) 60 min after addition of
FAP573 to mean fluorescence intensity of cells in (a). (e)
Representative flow cytometry experiment of 1 μM FAP573 incubated
in WT (blue), ADH5 KO (red) or KO cells with NaHSO3 (orange)
for 1 h. (f) Mean fluorescence intensity of median PE-A fluorescence
relative to fluorescence intensity of median PE-A in the WT cells; error
bars denote SEM (n = 3). *P < 5 × 10−3, **P < 5 × 10−4. ***P < 5 ×
10−5. Scale bar represents 50 μm in all images.
Synthetic Materials and Methods. Reactions utilizing air- or
moisture-sensitive reagents were performed in oven- or flame-dried
glassware under an atmosphere of dry N2. Reagents from commercial
sources were used without further purification. 4-Hydroxynonenal
solution was purchased from Cayman Chemical (Ann Arbor, MI);
2,2,2-trifluoroethyl trifluoromethanesulfonate was purchased from AK
Scientific (Union City, CA); glucosone was purchased from Santa
Cruz Biotech (Dallas, TX); thiophenol was purchased from Oakwood
Chemical (Estill, SC); 3-((tert-butoxycarbonyl)amino)hex-5-enoic acid
was purchased from ChemPep (Wellington, Fl); and all other reagents
were purchased from Sigma-Aldrich (St. Louis, MO). 1-(Azidosulfon-
yl)-1H-imidazol-3-ium chloride (Goddard’s diazo transfer reagent) was
prepared according to published procedures.49 Prenylboronic acid
solution was prepared through a slightly modified literature
procedure.64 3,6-Bis((tert-butyldimethylsilyl)oxy)-10,10-dimethylan-
thracen-9(10H)-one was prepared according to published proce-
dures.59 (Z)-9-(2-(Hydroxymethyl)phenyl)-3-((2,2,2-trifluoroethyl)-
imino)-3H-xanthen-6-ol was prepared according to published
procedures.65 Silica gel P60 (SiliCycle) was used for all column
chromatography purifications and SiliCycle 60 F254 silica gel
(precoated sheets, 0.25 mm thick) was used for thin layer
a key FA metabolism pathway and excess buildup of this RCS,
which has been shown to be a metabolic carcinogen and a
hemoatopoietic stem cell genotoxin related to leukemia models.
These data provide a unique example of a chemical tool that
enables direct visualization of endogenous FA in living systems
through fluorescence imaging with identification of a specific
molecular source for its metabolism.
1
chromatography. H NMR and 13C NMR spectra were collected in
CDCl3 or CD3OD (Cambridge Isotope Laboratories, Cambridge,
MA) at 25 °C on Bruker AV-500 and AV-300 (used for H NMR
1
only), AVB-400, and AVQ-400 with 13C operating frequencies of 101
MHz at the College of Chemistry NMR Facility at the University of
California, Berkeley. Chemical shifts are reported in the standard δ
notation of parts per million relative to residual solvent peak at 7.26
(CDCl3) or 3.31 (CD3OD) for 1H and 77.16 (CDCl3) or 49.00
(CD3OD) for 13C as an internal reference. Splitting patterns: br,
broad; s, singlet; d, doublet; t, triplet; m, multiplet; dd, doublet of
doublets; dt, doublet of triplets; qt, quartet of triplets; ddt, doublet of
doublet of triplets. Low-resolution electrospray mass spectral analyses
were performed using a LC−MS (Advion Expression-L Compact MS,
ESI source). High resolution mass spectral analyses (ESI-MS) were
CONCLUDING REMARKS
■
We have presented the development of a general 2-aza-Cope
reactivity trigger and its application to produce fluorescent
probes for imaging FA in living cells. Rational, iterative
optimization on a Tokyo Green platform affords a gem-
dimethyl derivative bearing a self-immolative β-elimination
linker that can be installed onto any fluorescent dye scaffold
that bears a phenol functionality. Notably, this relatively modest
chemical change enables a ca. 10-fold increase in fluorescence
turn-on response over the parent homoallylamine trigger. By
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performed at the College of Chemistry Mass Spectrometry Facility at
the University of California, Berkeley.
5.12 (m, 2H), 4.21 (t, J = 6.6 Hz, 2H), 3.89 (s, 3H), 2.38 (d, J = 7.2
Hz, 2H), 2.04 (s, 5H), 1.37 (s, 3H). LRMS calcd. for C28H27N3O4 (M
+ H) 470.20, found 470.5.
General Synthesis I for Conjugation of FA Trigger to
Fluorophores. To an oven-dried 2-neck round-bottomed flask was
added phenolic fluorophore (1.2 equiv) and Cs2CO3 (1.2 equiv)
dissolved in anyhydrous DMF. The FA trigger (1 equiv) was added
dropwise, and the solution was stirred under N2 at 40 °C for 12 h. The
DMF was removed under high vacuum, and purification by silica
chromatography (EtOAc/hexanes) afforded the fluorophore con-
jugated to the azido-trigger.
General Synthesis II for Azide Reduction. To an oven-dried 2-
neck round-bottomed flask was added SnCl2 (1.5 equiv) and a solution
of PhSH (4.5 equiv) and Et3N (4.5 equiv) in MeCN. The reaction
mixture was stirred for 15 min at room temperature, then azide-
conjugated fluorophore (1 equiv) was added as a solution in MeCN
and stirred at room temperature for an additional 12 h. The reaction
was concentrated under reduced pressure, diluted in CH2Cl2, and
poured into 2 M NaOH. The organic layer was extracted with CH2Cl2,
dried over Na2SO4, filtered, and concentrated under reduced pressure.
Purification by silica chromatography (EtOAc/hexanes) afforded the
formaldehyde probe.
6-((3-Amino-3-methylhex-5-en-1-yl)oxy)-9-(4-methoxy-2-
methylphenyl)-3H-xanthen-3-one (6). Synthesized via general
1
synthesis II described above (6.3 mg, 91%). H NMR (400 MHz,
MeOD) δ 7.29 (d, J = 2.3 Hz, 1H), 7.27−7.12 (m, 3H), 7.11−6.96 (m,
3H), 6.63 (dd, J = 9.6, 1.9 Hz, 1H), 6.48 (d, J = 1.9 Hz, 1H), 6.01−
5.86 (m, 1H), 5.40−5.26 (m, 2H), 4.39 (t, J = 5.8 Hz, 2H), 3.90 (s,
3H), 2.52 (d, J = 7.4 Hz, 2H), 2.23 (t, J = 5.7 Hz, 2H), 2.03 (s, 3H),
1.45 (s, 3H). LRMS calcd. for C28H29NO4 (M + H) 444.21, found
444.2.
N-(1-((tert-Butyldimethylsilyl)oxy)-3-methylhex-5-en-3-yl)-4-
nitrobenzenesulfonamide (7). To a solution of 2 in 13 mL CH2Cl2
in a 50 mL round-bottomed flask was added pyridine (0.22 mL, 2.7
mmol) followed by 4-Nitrobenzenesulfonyl chloride (0.60 g, 2.7
mmol) to form a deep orange solution. After stirring at room
temperature for 24 h, the reaction was poured into sat. NH4Cl (50
mL), extracted with CH2Cl2 (3×), dried over Na2SO4, filtered, and
concentrated under reduced pressure. Purification via column
chromatography (0 → 30% EtOAc/hexanes gradient) afforded 7 as
a dark yellow oil (65 mg, 6% from 2). 1H NMR (300 MHz, CDCl3) δ
8.37−8.24 (m, 2H), 8.09−7.98 (m, 2H), 6.78 (s, 1H), 5.65 (ddt, J =
16.4, 10.6, 7.3 Hz, 1H), 5.11−4.97 (m, 2H), 3.82 (pt, J = 6.7, 3.4 Hz,
2H), 2.60−2.39 (m, 2H), 1.78−1.49 (m, 3H), 1.22 (s, 4H), 0.10 (d, J
= 10.9 Hz, 8H). LRMS calcd. for C19H32N2O5SSi (M−H) 427.18,
found 427.1.
3-Methyl-3-((N-methyl-4-nitrophenyl)sulfonamido)hex-5-
en-1-yl 4-methylbenzenesulfonate (8). A solution of 7 (0.122 g,
0.274 mmol) in 1 mL DMF was added to a 10 mL round-bottomed
flask. Then, K2CO3 (0.075 g, 0.55 mmol) was added followed by
dropwise addition of methyl iodide (0.025 mL, 0.38 mmol). The
reaction was stirred overnight at room temperature. The reaction
mixture was partitioned between EtOAc and H2O, washed with H2O
(3×), dried over Na2SO4, filtered, and concentrated under reduced
pressure.
1-((tert-Butyldimethylsilyl)oxy)-3-methylhex-5-en-3-amine
(2). A 2-neck flask was charged with 1 (213 μL, 2.47 mmol), cooled to
0 °C and ammonia was added (5.0 mL, 7.0 M in MeOH). After
stirring for 30 min, allyl pinacol boronate (550 μL, 2.96 mmol) was
added dropwise, and the resulting solution stirred at room temperature
for 24 h. The volatiles were then removed under reduced pressure, and
the residue was taken up in 1 M HCl and washed with Et2O (3×). The
aqueous layer was then basified with KOH and extracted with CH2Cl2
(3×). The combined organic layer was dried over Na2SO4, filtered and
concentrated under reduced pressure to provide 2 as a colorless oil
1
(221 mg, 69%). H NMR (400 MHz, CDCl3) δ 5.83 (ddt, J = 17.6,
10.3, 7.5 Hz, 1H), 5.15−5.01 (m, 2H), 3.77 (t, J = 6.7 Hz, 2H), 2.13
(d, J = 7.5 Hz, 2H), 1.96 (s, 2H), 1.61 (t, J = 6.7 Hz, 2H), 1.23 (s,
2H), 1.08 (s, 3H), 0.06 (d, J = 10.8 Hz, 9H). LRMS calcd. for
C13H29NOSi (M + H) 244.20, found 244.2.
3-Azido-3-methylhex-5-en-1-ol (2-ii). To a solution of CuSO4·
5H2O (2.0 mg, 7.7 μmol), K2CO3 (53.0 mg, 0.39 mmol), MeOH (4.0
mL) and 2 (100 mg, 0.774 mmol) at room temperature was added
Goddard’s diazo transfer reagent (161 mg, 0.929 mmol), resulting in a
change in color from blue to teal. The resulting slurry stirred at room
temperature for 20 h, after which the volatiles were removed under
reduced pressure, and the residue was taken up in 1 M HCl and
extracted with EtOAc (3×). The combined organic layer was dried
over Na2SO4, filtered, and concentrated to afford crude 2-ii as a
The concentrated material was resuspended in THF, placed in a 10
mL round-bottomed flask, and cooled to 0 °C. Then, tetra-n-
butylammonium fluoride (1.0 M in THF, 0.27 mL, 0.28 mmol) was
added dropwise to form a yellow solution that was warmed to room-
temperature and stirred for 16 h. The reaction was poured into sat.
NH4Cl (20 mL), extracted with CH2Cl2 (3×), dried over Na2SO4,
filtered, and concentrated under reduced pressure. Purification via
column chromatography (45% EtOAc/hexanes) afforded a yellow oil.
The oil was dissolved CH2Cl2 (1.5 mL) and added to a 10 mL
round-bottomed flask. Then, TsCl (43 mg, 0.23 mmol), DMAP (2.5
mg, 0.021 mmol), and Et3N (30 μL, 0.23 mmol). After stirring for 16 h
at room temperature, the solution was poured into sat. aqueous
NH4Cl and extracted with CH2Cl2 (3×). The combined organic layer
was dried over Na2SO4, filtered, and concentrated under reduced
pressure. Purification via column chromatography (0 → 35% EtOAc/
hexanes gradient) afforded 8 as a colorless oil (91 mg, 9.4% from 2).
1H NMR (300 MHz, CDCl3) δ 8.41−8.21 (m, 2H), 8.00−7.89 (m,
2H), 7.82−7.69 (m, 2H), 7.35 (d, J = 8.0 Hz, 2H), 5.49 (ddt, J = 17.2,
10.2, 7.2 Hz, 1H), 5.13−4.94 (m, 2H), 4.21−3.97 (m, 3H), 2.94 (s,
3H), 2.56 (dd, J = 14.2, 6.8 Hz, 1H), 2.45 (s, 3H), 2.32 (ddt, J = 21.3,
14.1, 6.9 Hz, 3H), 2.04 (s, 1H), 1.96 (dd, J = 14.7, 7.3 Hz, 1H), 1.33
(s, 3H), 1.25 (t, J = 7.1 Hz, 1H).
N-(1-((9-(4-Methoxy-2-methylphenyl)-3-oxo-3H-xanthen-6-
yl)oxy)-3-methylhex-5-en-3-yl)-N-methyl-4-nitrobenzenesulfo-
namide (8-vi). Synthesized via general synthesis I described above.
1H NMR (300 MHz, MeOD) δ 8.16 (dd, J = 8.9, 2.5 Hz, 2H), 8.04 (d,
1
colorless oil (crude, 116 mg). H NMR (300 MHz, CDCl3) δ 5.81
(ddt, J = 15.3, 11.0, 7.3 Hz, 1H), 5.23−5.10 (m, 1H), 5.01 (s, 1H),
3.92 (s, 1H), 3.79 (t, J = 6.4 Hz, 1H), 3.06 (s, 1H), 1.89−1.65 (m,
1H), 1.29 (d, J = 25.1 Hz, 3H). LRMS calcd. for C7H13N3O (M + H)
155.10, found 155.2.
3-Azido-3-methylhex-5-en-1-yl 4-methylbenzenesulfonate
(3). To a solution of crude 2-ii (116 mg) in CH2Cl2 (3.75 mL) was
added TsCl (172 mg, 0.900 mmol), DMAP (9.0 mg, 0.075 mmol),
and Et3N (125 μL, 0.900 mmol). After stirring for 16 h at room
temperature, the solution was poured into sat. aqueous NH4Cl and
extracted with CH2Cl2 (3×). The combined organic layer was dried
over Na2SO4, filtered, and concentrated under reduced pressure.
Purification via column chromatography (0 → 10% EtOAc/hexanes
gradient) afforded 4 as a colorless oil (64 mg, 27% from 2). 1H NMR
(300 MHz, CDCl3) δ 7.79 (d, J = 8.1 Hz, 2H), 7.36 (d, J = 8.1 Hz,
2H), 5.85−5.64 (m, 1H), 5.21−5.04 (m, 2H), 4.13 (t, J = 6.9 Hz, 2H),
2.45 (s, 4H), 2.25 (d, J = 7.3 Hz, 2H), 1.83 (t, J = 8.1 Hz, 2H), 1.25 (s,
3H). 13C NMR (75 MHz, CDCl3) δ 162.44, 145.09, 132.91, 132.05,
130.05, 128.04, 119.97, 66.54, 44.45, 37.84, 23.47, 21.81. LRMS calcd.
for C14H19N3O3S (M + H) 310.11, found 310.4.
J = 8.8 Hz, 2H), 7.25−6.98 (m, 5H), 6.90 (t, J = 2.3 Hz, 1H), 6.78
(ddd, J = 8.9, 5.2, 2.4 Hz, 1H), 6.63 (dd, J = 9.6, 2.1 Hz, 1H), 6.49 (t, J
= 1.8 Hz, 1H), 5.80 (dtd, J = 15.7, 8.0, 6.1 Hz, 1H), 5.25−5.05 (m,
2H), 3.90 (s, 5H), 3.13 (d, J = 1.2 Hz, 3H), 2.76 (dt, J = 13.8, 6.8 Hz,
1H), 2.59−2.43 (m, 1H), 2.34 (dd, J = 14.2, 7.5 Hz, 1H), 2.09−1.97
(m, 4H), 1.53 (s, 3H), 1.28 (s, 1H), 0.98−0.82 (m, 2H). LRMS calcd.
for C35H34N2O8S (M + H) 643.20, found 643.3.
6-((3-Azido-3-methylhex-5-en-1-yl)oxy)-9-(4-methoxy-2-
methylphenyl)-3H-xanthen-3-one (5). Synthesized via general
1
synthesis I described above (46.8 mg, 56%). H NMR (400 MHz,
CDCl3) δ 7.01 (ddd, J = 38.6, 20.6, 11.2 Hz, 6H), 6.76 (d, J = 6.6 Hz,
1H), 6.58 (d, J = 9.7 Hz, 1H), 6.45 (s, 1H), 5.96−5.72 (m, 1H), 5.29−
I
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9-(4-Methoxy-2-methylphenyl)-6-((3-methyl-3-
(methylamino)hex-5-en-1-yl)oxy)-3H-xanthen-3-one (9). To a
solution of 8-vi (6.0 mg, 9.0 μmol) in MeCN, was added K2CO3 (5.5
mg, 14 μmol) and thiophenol (1.4 μL, 14 μmol). The reaction mixture
was heated at 50 °C for 16 h. The reaction was then cooled, and
directly purified by column chromatography (0 → 10% MeOH/
1.88 (dddd, J = 14.7, 8.3, 6.3, 3.7 Hz, 1H), 1.62 (ddt, J = 14.6, 9.6, 4.6
Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 145.13, 132.90, 132.75,
130.02, 127.97, 119.10, 67.08, 58.03, 38.82, 33.23, 21.72. LRMS calcd.
for C13H17N3O3S (M + H) 296.09, found 296.4.
6-((3-Azidohex-5-en-1-yl)oxy)-9-(4-methoxy-2-methylphen-
yl)-3H-xanthen-3-one (13-v). Synthesized via general synthesis I
1
1
CH2Cl2) to give a yellow oil (1.75 mg, 42%). H NMR (400 MHz,
described above (46.3 mg, 58%). H NMR (300 MHz, CDCl3) δ
CDCl3) δ 7.11−6.96 (m, 3H), 6.91 (d, J = 10.7 Hz, 2H), 6.78 (d, J =
9.0 Hz, 1H), 6.60 (d, J = 9.7 Hz, 1H), 6.48 (s, 1H), 5.84 (dd, J = 16.8,
8.9 Hz, 1H), 5.28−5.09 (m, 2H), 4.26 (t, J = 6.8 Hz, 2H), 3.90 (s,
2H), 2.45 (s, 2H), 2.36 (d, J = 7.4 Hz, 1H), 2.04 (s, 4H), 1.26 (s, 7H).
LRMS calcd. for C29H31NO4 (M + H) 458.22, found 458.3.
7.15−6.83 (m, 6H), 6.77 (dd, J = 8.9, 2.5 Hz, 1H), 6.57 (dd, J = 9.7,
1.9 Hz, 1H), 6.44 (d, J = 2.0 Hz, 1H), 5.85 (ddt, J = 17.1, 10.2, 7.0 Hz,
1H), 5.29−5.07 (m, 2H), 4.32−4.11 (m, 2H), 3.89 (s, 3H), 3.79−3.55
(m, 1H), 2.53−2.31 (m, 2H), 2.04 (s, 3H), 1.89 (ddt, J = 14.5, 9.6, 4.9
Hz, 1H). LRMS calcd. for C27H25N3O4 (M + H) 456.18, found 456.2.
6-((3-Aminohex-5-en-1-yl)oxy)-9-(4-methoxy-2-methyl-
phenyl)-3H-xanthen-3-one (14). Synthesized via general synthesis
9-(4-Methoxy-2-methylphenyl)-6-((3-methyl-3-((2,2,2-
trifluoroethyl)amino)hex-5-en-1-yl)oxy)-3H-xanthen-3-one
(10). A solution of 6 in 0.4 mL DMF was added to a dry 5 mL round-
bottomed flask. Then, K2CO3 (14.8 mg, 0.107 mmol) was added,
followed by 2,2,2-trifluoroethyl trifluoromethanesulfonate (74 μL of a
7 M solution in MeCN, 0.052 mmol). The reaction was stirred for 5 h
at room temperature. Additional 2,2,2-trifluoroethyl trifluoromethane-
sulfonate (74 μL of a 7 M solution in MeCN, 0.052 mmol) was added,
and the reaction was stirred for 24 h. The solvent was removed under
reduced pressure, and purification via column chromatography (0 →
15% MeOH/CHCl3 gradient) afforded 10 as a yellow solid (5.1 mg,
23%) 1H NMR (400 MHz, CDCl3) δ 7.18−6.86 (m, 5H), 6.78 (dd, J
= 8.9, 2.4 Hz, 1H), 6.61 (dd, J = 9.7, 2.0 Hz, 1H), 6.50 (d, J = 1.9 Hz,
1H), 5.80 (ddt, J = 17.5, 10.5, 7.1 Hz, 1H), 5.24−5.11 (m, 2H), 4.37−
4.11 (m, 2H), 3.90 (s, 3H), 3.28 (dq, J = 14.4, 9.3 Hz, 1H), 3.12 (dq, J
= 14.5, 9.3 Hz, 1H), 2.97 (p, J = 5.8 Hz, 1H), 2.28 (h, J = 7.1, 6.5 Hz,
2H), 2.05 (s, 4H), 1.88−1.68 (m, 5H), 1.26 (s, 2H). LRMS calcd. for
C29H28F3NO4 (M + H) 526.21, found 526.0.
1-Hydroxyhex-5-en-3-aminium chloride (12). A solution of 11
((S)-3-Boc-amino-5-hexenoic acid) (1.40 g, 6.11 mmol) was dissolved
in dry THF (21 mL) and cooled to 0 °C. To the resulting solution was
added Et3N (1.70 mL, 12.2 mmol) and ethyl chloroformate (0.70 mL,
7.3 mmol), causing a white precipitate to form, and the reaction was
filtered after stirring for 30 min at 0 °C. After cooling the filtrate to 0
°C, NaBH4 (462 mg, 12.2 mmol) and H2O (2 mL) were added, and
the resulting mixture stirred for 2 h at room temperature. The reaction
was quenched with sat aq NH4Cl, the THF was removed under
reduced pressure, and the resulting solution was extracted with EtOAc
(3×). The combined organic layer was washed with H2O, 1 M aq
NaOH, and brine, then dried over Na2SO4, filtered, and concentrated
under reduced pressure. Purification via column chromatography (5 →
20% EtOAc/hexanes) provided 12 as a colorless oil (0.944 g, 72%).
1H NMR (400 MHz, MeOD) δ 5.93−5.72 (m, 1H), 5.33−5.18 (m,
1
II described above (7.2 mg, 33%). H NMR (400 MHz, MeOD) δ
7.15−6.94 (m, 5H), 6.85−6.70 (m, 2H), 6.60 (dd, J = 9.3, 2.2 Hz,
1H), 6.54 (d, J = 2.2 Hz, 1H), 5.87 (ddt, J = 17.2, 10.0, 7.0 Hz, 1H),
5.21−5.01 (m, 2H), 3.95 (s, 1H), 3.89 (s, 3H), 3.73−3.55 (m, 2H),
2.55−2.28 (m, 2H), 2.02 (d, J = 1.3 Hz, 3H), 1.90 (dddd, J = 14.1, 8.0,
6.1, 4.7 Hz, 1H), 1.76 (ddt, J = 14.0, 8.4, 5.4 Hz, 1H). LRMS calcd. for
C27H27NO4 (M + H) 430.19, found 430.2.
3-Azido-3-phenylhex-5-en-1-ol (15-viii). To an oven-dried 25
mL 2-neck round-bottomed flask was added 4 mL of a 7 M NH3
solution in MeOH. The solution was cooled to 0 °C and 15 (0.53 mg,
2.77 mmol) was added. The reaction was warmed to room
temperature and stirred for 30 min. The reaction was again cooled
to 0 °C, and then allyl pinacol boronate was added as a solution in
CHCl3. The reaction mixture was stirred at room temperature for 12
h. The reaction was concentrated under reduced pressure, then diluted
in 0.1 M NaOH, and extracted with Et2O (3×). The combined organic
layers were washed with brine, dried over Na2SO4, filtered, and
concentrated under reduced pressure to afford a light yellow oil.
The oil was dissolved in 12 mL MeOH and added to a 100 mL
round-bottomed flask. Then, CuSO4·5H2O (7 mg, 0.03 mmol) and
K2CO3 (0.651 g, 4.71 mmol), followed by Goddard’s diazo transfer
reagent (0.7 g, 3.32 mmol) was added. After stirring for 12 h, the
reaction was concentrated under reduced pressure, partitioned
between EtOAc and 1 M HCl, and extracted with EtOAc (3×). The
combined organic layers were washed with 1 M HCl, brine, dried over
Na2SO4, and concentrated under reduced pressure. Purification by
silica gel chromatography (25% EtOAc/hexanes) afforded a clear oil
1
(0.47 g, 78%). H NMR (400 MHz, CDCl3) δ 7.41 (d, J = 4.3 Hz,
4H), 7.32 (h, J = 4.2 Hz, 1H), 5.66 (ddt, J = 17.2, 10.2, 7.1 Hz, 1H),
5.26−5.06 (m, 2H), 3.61 (ddd, J = 11.0, 8.0, 6.2 Hz, 1H), 3.46 (ddd, J
= 10.9, 8.0, 5.8 Hz, 1H), 2.81 (t, J = 7.3 Hz, 3H), 2.24 (qdd, J = 14.1,
8.0, 6.0 Hz, 2H).
2H), 5.00−4.86 (m, 3H), 3.89−3.66 (m, 2H), 3.42 (t, J = 6.8 Hz, 1H),
3.33−3.27 (m, 1H), 2.55−2.37 (m, 2H), 1.95−1.71 (m, 2H). LRMS
calcd. for C6H14ClNO (M + H) 152.07, found 152.1.
3-Azido-3-phenylhex-5-en-1-yl 4-methylbenzenesulfonate
(16). To an oven-dried 25 mL 2-neck round-bottomed flask was
added 3-azido-3-phenylhex-5-en-1-ol (0.240 g, 1.1 mmol) as a solution
in 6 mL CH2Cl2, p-Toluenesulfonyl chloride (0.316 g, 1.6 mmol),
DMAP (0.068 g, 0.55 mmol), and NEt3 (0.23 mL, 1.6 mmol). The
reaction mixture was stirred for 16 h at room temperature. The
reaction mixture was concentrated under reduced pressure, and
purification by silica column chromatography (5% EtOAc/hexanes)
3-Azidohex-5-en-1-yl 4-methylbenzenesulfonate (13). To a
solution of CuSO4·5H2O (7.0 mg, 0.03 mmol), K2CO3 (850 mg, 6.16
mmol), MeOH (12 mL) and 12 at room temperature was added
Goddard’s diazo transfer reagent (573 mg, 2.74 mmol), resulting in a
change in color from blue to teal. The resulting slurry stirred at room
temperature for 14 h, after which the volatiles were removed under
reduced pressure, and the residue was taken up in 1 M HCl and
extracted with EtOAc (3×). The combined organic layer was dried
over Na2SO4, filtered, and concentrated under reduced pressure to
afford crude 12-iii as a colorless oil that was used directly in the next
step.
1
afforded 16 (0.41 g, quantitative). H NMR (400 MHz, CDCl3) δ
7.77−7.70 (m, 2H), 7.47−7.25 (m, 7H), 5.58 (ddt, J = 17.2, 10.2, 7.0
Hz, 1H), 5.20−5.07 (m, 2H), 4.10−3.99 (m, 1H), 3.91−3.80 (m, 1H),
2.74 (d, J = 7.1 Hz, 2H), 2.46 (s, 3H), 2.34 (ddd, J = 8.1, 6.5, 1.6 Hz,
2H). 13C NMR (101 MHz, CDCl3) δ 144.85, 139.84, 132.64, 131.39,
129.82, 128.68, 127.80, 127.68, 125.66, 125.60, 119.74, 67.22, 66.26,
44.46, 44.36, 38.08, 21.57. LRMS calcd. for C19H21N3O3S (M + H)
372.13, found 372.2.
To a solution of crude 12-iii in CH2Cl2 (10 mL) was added TsCl
(625 mg, 3.28 mmol), DMAP (107 mg, 0.876 mmol), and Et3N (457
μL, 3.28 mmol). After stirring for 24 h at room temperature, the
solution was poured into sat. aqueous NH4Cl and extracted with
CH2Cl2 (3×). The combined organic layer was dried over Na2SO4,
filtered, and concentrated under reduced presuure. Purification via
column chromatography (0 → 20% EtOAc/hexanes gradient) afforded
6-((3-Amino-3-phenylhex-5-en-1-yl)oxy)-9-(4-methoxy-2-
methylphenyl)-3H-xanthen-3-one (17). Synthesized via general
1
synthesis I (48 mg, 33%) and II (16.8 mg, 74%) described above. H
NMR (400 MHz, CDCl3) δ 7.79−7.70 (m, 2H), 7.43−7.23 (m, 7H),
5.58 (ddt, J = 17.2, 10.2, 7.0 Hz, 1H), 5.26−5.04 (m, 2H), 4.16−3.96
(m, 1H), 3.93−3.79 (m, 1H), 2.74 (d, J = 7.1 Hz, 2H), 2.46 (s, 3H),
2.34 (ddd, J = 8.1, 6.5, 1.6 Hz, 2H). LRMS calcd. for C33H31NO4 (M +
H) 506.23, found 506.2.
1
13 as a colorless oil (353 mg, 53% from 11-i). H NMR (400 MHz,
CDCl3) δ 7.79 (d, J = 8.2 Hz, 2H), 7.35 (d, J = 8.1 Hz, 2H), 5.74 (ddt,
J = 16.4, 10.6, 7.0 Hz, 1H), 5.21−5.07 (m, 2H), 4.17−4.03 (m, 2H),
3.50 (dtd, J = 10.1, 6.4, 3.7 Hz, 1H), 2.44 (s, 3H), 2.36−2.22 (m, 2H),
J
DOI: 10.1021/jacs.6b12460
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Journal of the American Chemical Society
Article
3-Amino-3,4,4-trimethylhex-5-en-1-ol (20). To an oven-dried
250 mL 2-neck round-bottomed flask equipped with a 100 mL
addition funnel was cannulated 60 mL of a 7 M NH3 solution in
MeOH. The solution was cooled to 0 °C and the ketone (1.7 mL, 20
mmol) was added. The reaction was warmed to room temperature and
stirred for 1 h. The reaction was again cooled to 0 °C, and the
previously prepared (3-methylbut-2-en-1-yl)boronic acid was added as
a solution in CHCl3. The reaction mixture was stirred at room
temperature for 24 h. The reaction was diluted in 1 M HCl (100 mL),
washed with EtOAc (3×), basified with NaOH, and extracted with
DCM (3×). The combined organic layers were dried over Na2SO4,
filtered, and concentrated under reduced pressure to afford a clear oil
(2.31 g, 74%). 1H NMR (400 MHz, CDCl3) δ 5.71 (dd, J = 17.5, 10.9
Hz, 1H), 4.91−4.69 (m, 2H), 3.68 (t, J = 12.5 Hz, 1H), 3.45 (d, J = 7.8
Hz, 1H), 2.74 (s, 2H), 1.50 (t, J = 15.0 Hz, 1H), 1.23 (d, J = 17.1 Hz,
1H), 0.89 (s, 3H), 0.77 (s, 6H). 13C NMR (101 MHz, CDCl3) δ
144.29, 113.24, 59.62, 43.79, 40.51, 35.12, 21.64, 20.86, 19.60. LRMS
calcd. for C9H19NO (M + H) 158.15, found 158.3.
1.10 (s, 3H), 1.06 (s, 6H). HRMS calcd. for C19H22F3NO3 (M + H)
370.1552, found 370.1621.
FAP498 Azide. Synthesized via general synthesis I described above
1
(50.1 mg, 99%). H NMR (400 MHz, CDCl3) δ 7.37 (d, J = 5.5 Hz,
1H), 6.95−6.75 (m, 1H), 6.71 (d, J = 2.5 Hz, 0H), 6.59 (dd, J = 8.9,
2.5 Hz, 0H), 6.52−6.46 (m, 0H), 6.38 (d, J = 8.6 Hz, 0H), 5.85 (ddt, J
= 16.1, 10.9, 7.3 Hz, 0H), 5.27 (s, 1H), 5.23−5.13 (m, 1H), 4.11 (t, J =
6.6 Hz, 1H), 3.85−3.71 (m, 1H), 3.54−3.44 (m, 3H), 2.37 (d, J = 7.3
Hz, 1H), 2.09−1.93 (m, 1H), 1.36 (s, 1H), 1.22 (t, J = 7.0 Hz, 4H).
FAP498. Synthesized via general synthesis II described above (20.6
mg, 46%).1H NMR (400 MHz, CDCl3) δ 7.52 (d, J = 13.4 Hz, 2H),
7.40 (d, J = 7.2 Hz, 1H), 7.03 (d, J = 7.3 Hz, 1H), 6.93 (s, 1H), 6.82
(s, 1H), 5.91 (dd, J = 17.3, 10.8 Hz, 1H), 5.35−5.14 (m, 2H), 4.54 (s,
1H), 4.35 (d, J = 20.0 Hz, 2H), 3.96 (s, 2H), 3.62 (s, 1H), 3.44 (s,
7H), 2.07 (dt, J = 14.8, 5.6 Hz, 1H), 1.36 (s, 3H), 1.15 (s, 6H). HRMS
calcd. for C31H33F3N2O3 (M + H) 539.2443, found 539.2512.
7-Hydroxy-9,9-dimethyl-10-(2-(trifluoromethyl)phenyl)-
anthracen-2(9H)-one. To
a solution of 1-bromo-2-
(trifluoromethyl)benzene in 1 mL anhydrous THF was added tBuLi
(0.24 mL, 1.7 M) dropwise at −78 °C and stirred for 1 h at that
temperature. 3,6-bis((tert-butyldimethylsilyl)oxy)-10,10-dimethylan-
thracen-9(10H)-one (50 mg, 0.104 mmol) as a solution in 4 mL
THF was added dropwise at −78 °C and the reaction was warmed to
room temperature and stirred for 3 h. The reaction was quenched with
10 mL 1 M HCl, stirred for 20 more minutes, and then 25 mL hexanes
was added to form dark red crystals (76 mg, 52%). LRMS calcd. for
C23H17F3O2 (M + H) 383.38, found 383.4.
FAP555 Azide. Synthesized via general synthesis I described above
(10 mg, 18%). 1H NMR (500 MHz, Chloroform-d) δ 7.88 (q, J = 7.3
Hz, 1H), 7.69 (dt, J = 24.5, 7.8 Hz, 2H), 7.28 (s, 3H), 7.19 (dd, J =
6.6, 4.2 Hz, 1H), 6.86−6.59 (m, 4H), 6.31 (d, J = 9.7 Hz, 1H), 5.99
(dd, J = 17.4, 11.0 Hz, 1H), 5.12 (dt, J = 28.4, 11.9 Hz, 2H), 4.37−
4.15 (m, 2H), 1.74 (d, J = 13.1 Hz, 3H), 1.58 (s, 3H), 1.27 (s, 3H),
1.15−1.06 (m, 6H).
FAP555. Synthesized via general synthesis II described above (4.4
mg, 46%). 1H NMR (500 MHz, Chloroform-d) δ 7.88 (q, J = 7.3 Hz,
1H), 7.69 (dt, J = 24.5, 7.8 Hz, 2H), 7.28 (s, 3H), 7.19 (dd, J = 6.6, 4.2
Hz, 1H), 6.86−6.59 (m, 4H), 6.31 (d, J = 9.7 Hz, 1H), 5.99 (dd, J =
17.4, 11.0 Hz, 1H), 5.12 (dt, J = 28.4, 11.9 Hz, 2H), 4.37−4.15 (m,
2H), 2.12−1.89 (m, 2H), 1.74 (d, J = 13.1 Hz, 3H), 1.58 (s, 3H), 1.27
(s, 3H), 1.15−1.06 (m, 6H). HRMS calcd. for C32H34F3NO2 (M + H)
522.2541, found 522.2618.
FAP573 Azide. Synthesized via general synthesis I described above
(7.0 mg, 20%). 1H NMR (400 MHz, CDCl3) δ 7.72 (d, J = 8.9 Hz,
1H), 7.44 (d, J = 9.8 Hz, 1H), 6.95 (d, J = 8.9 Hz, 1H), 6.84 (d, J = 1.6
Hz, 2H), 6.36 (s, 1H), 5.99 (dd, J = 17.5, 10.9 Hz, 1H), 5.21−5.03 (m,
2H), 4.21 (t, J = 6.8 Hz, 2H), 2.22−1.93 (m, 3H), 1.41 (s, 3H), 1.25
(s, 4H), 1.12 (s, 5H).
3-Azido-3,4,4-trimethylhex-5-en-1-ol (20-iii). To an oven-dried
250 mL 2-neck round-bottomed flask was added CuSO4·5H2O (33.9
mg, 0.136 mmol) and K2CO3 (2.82 g, 20.4 mmol) dissolved in MeOH
(130 mL). Then, 20 was added as a solution in 10 mL MeOH,
followed by Goddard’s diazo transfer reagent (3.41 g, 16.3 mmol). The
resulting teal slurry was stirred at room temperature for 18 h. The
solvent was removed under reduced pressure. The residue was
partitioned between EtOAc (75 mL) and 1 M HCl (75 mL) and
extracted with EtOAc (2 × 100 mL). The combined organic layers
were dried over Na2SO4, filtered, and concentrated under reduced
pressure. Purification by silica column chromatography (20% EtOAc/
1
hexanes) afforded a colorless liquid (720 mg, 29%). H NMR (400
MHz, CDCl3) δ 5.92 (dd, J = 17.4, 10.9 Hz, 1H), 5.12−4.94 (m, 2H),
3.73 (qt, J = 10.7, 6.9 Hz, 2H), 2.69 (s, 1H), 1.85 (dt, J = 14.1, 7.0 Hz,
1H), 1.73−1.55 (dt, 1H), 1.29 (s, 3H), 1.04 (s, 6H). 13C NMR (101
MHz, CDCl3) δ 143.81, 113.80, 67.71, 59.26, 45.53, 37.94, 22.37,
22.22, 17.80. LRMS calcd. for C9H17N3O (M + H) 184.14, found
184.3.
3-Azido-3,4,4-trimethylhex-5-en-1-yl 4-methylbenzenesul-
fonate (21). To an oven-dried 100 mL 2-neck round-bottomed
flask was added 3-Azido-3,4,4-trimethylhex-5-en-1-ol (700 mg, 3.81
mmol) as a solution in 50 mL CH2Cl2, p-Toluenesulfonyl chloride
(1.09 g, 5.72 mmol), DMAP (186 mg, 1.52 mmol), and Et3N (0.79
mL, 5.7 mmol). The reaction mixture was stirred for 16 h at room
temperature. The mixture was poured into saturated NH4Cl (60 mL)
and extracted with CH2Cl2 (3×). The combined organic layers were
dried over Na2SO4, filtered, and concentrated under reduced pressure.
Purification by silica column chromatography (5% EtOAc/hexanes)
afforded a colorless oil (187 mg, 15%). 1H NMR (400 MHz, CDCl3) δ
7.77 (d, J = 8.1 Hz, 2H), 7.33 (d, J = 8.0 Hz, 2H), 5.84 (dd, J = 17.4,
10.9 Hz, 1H), 5.09−4.90 (m, 2H), 4.20−4.04 (m, 2H), 2.42 (s, 3H),
1.81 (ddt, J = 42.9, 14.5, 6.9 Hz, 2H), 1.23 (s, 3H), 0.99 (s, 6H). 13C
NMR (101 MHz, CDCl3) δ 144.95, 143.26, 129.91, 127.87, 114.14,
67.42, 66.78, 45.52, 34.63, 22.23, 22.03, 21.60, 17.72. LRMS calcd. for
C16H23N3O3S (M + H) 338.15, found 338.2.
FAP488. Synthesized via general synthesis I (11.8 mg, 24%) and II
(6.8 mg, 33%) described above. 1H NMR (400 MHz, MeOD) δ 7.65−
7.51 (m, 3H), 7.32 (dd, J = 9.3, 2.3 Hz, 1H), 7.24 (dd, J = 8.4, 1.0 Hz,
1H), 7.21−7.11 (m, 3H), 7.07 (dd, J = 8.5, 2.5 Hz, 1H), 6.07 (dd, J =
17.3, 10.8 Hz, 1H), 5.40−5.25 (m, 2H), 4.56 (t, J = 6.2 Hz, 2H), 3.93
(s, 3H), 2.46−2.26 (m, 2H), 2.04 (s, 3H), 1.47 (s, 3H), 1.23 (s, 6H).
LRMS calcd. for C30H33NO4 (M + H) 472.24, found 472.2.
FAP385 Azide. Synthesized via general synthesis I described above
(21.3 mg, 61%). 1H NMR (400 MHz, CDCl3) δ 7.62 (d, J = 10.7 Hz,
1H), 6.99−6.85 (m, 2H), 6.62 (s, 1H), 5.98 (dd, J = 17.4, 10.9 Hz,
1H), 5.19−5.02 (m, 2H), 4.18 (t, J = 6.8 Hz, 2H), 2.23−1.84 (m, 2H),
1.40 (s, 3H), 1.11 (s, 6H).
FAP573. Synthesized via general synthesis II described above (1.3
1
mg, 11%). H NMR (400 MHz, CDCl3) δ 7.69 (d, J = 8.4 Hz, 1H),
7.41 (d, J = 9.8 Hz, 1H), 6.98 (d, J = 10.2 Hz, 1H), 6.88 (s, 1H), 6.82
(d, J = 11.3 Hz, 1H), 6.31 (s, 1H), 5.92 (dd, J = 17.3, 10.8 Hz, 1H),
5.34−5.18 (m, 2H), 4.29 (s, 2H), 3.36 (s, 1H), 2.34−2.24 (m, 1H),
2.10 (d, J = 8.6 Hz, 1H), 1.38 (s, 3H), 1.17 (s, 5H). HRMS calcd. for
C21H24N2O3 (M + H) 353.1787, found 353.1858.
Spectroscopic Materials and Methods. All spectroscopic
measurements were performed in 20 mM PBS, pH 7.4. Fluorescence
spectra were recorded using a Photon Technology International
Quanta Master 4 L-format scan spectrofluorometer equipped with an
LPS- 220B 75-W xenon lamp and power supply, A-1010B lamp
housing with integrated igniter, switchable 814 photocounting/analog
photomultiplier detection unit, and MD5020 motor driver. Samples
were contained in 1 cm × 1 cm quartz cuvettes during measurement
(1.4 mL volume, Starna).
Quantum Yield Determination. All absorbance spectra were
measured with an absorbance below 0.1. Quantum yield for each
fluorophore was determined using the equation (ϕ = quantum yield, y
= emission intensity versus absorbance, and η = refractive index):
FAP385. Synthesized via general synthesis II described above (20.2
mg, 99%). 1H NMR (400 MHz, CDCl3) δ 7.66−7.56 (m, 1H), 6.97−
6.86 (m, 2H), 6.60 (s, 1H), 5.99 (dd, J = 17.5, 10.9 Hz, 1H), 5.19−
4.98 (m, 2H), 4.37−4.15 (m, 2H), 2.08−1.82 (m, 2H), 1.42 (s, 2H),
2
ϕ
= ϕstandard(ysample/y
)(ηsample/η
)
sample
standard
standard
K
DOI: 10.1021/jacs.6b12460
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
FAP385 quantum yield was determined using harmaline in 0.005 M
H2SO4 (ϕ = 0.32) as a reference according to published procedures.66
FAP498 quantum yield was determined using fluorescein in 0.1 M
NaOH (ϕ = 0.91) as a reference according to published procedures.67
FAP555 and FAP573 quantum yield was determined using rhodamine
6G in ethanol (ϕ = 0.94) as a reference according to published
procedures.68
Cell Culture Procedures. HEK293T cells were maintained in
exponential growth as a monolayer in Dulbecco’s Modified Eagle
Medium, high glucose, (DMEM, Invitrogen) supplemented with
glutamax (Gibco), 10% fetal bovine serum (FBS, Hyclone) and 1%
nonessential amino acids (NEAA, Gibco), and incubated at 37 °C in
5% CO2. One day before imaging, the cells were passaged and plated
in DMEM with glutamax (phenol red-free) supplemented with 10%
FBS on poly D-lysine-coated 4-well Lab Tek borosilicate chambered
coverglass slides (Nunc) at 1.8 × 105 per well.
cytometry conditions and experiments, in vitro fluo-
rescence response data of 6, 9, 10, 14, 17, and FAP488,
limit of detection, kinetics, cell viability, and NMR
spectra (PDF)
AUTHOR INFORMATION
Corresponding Author
*chrischang@berkeley.edu
ORCID
■
Christopher J. Chang: 0000-0001-5732-9497
Present Address
#Department of Chemistry, Ursinus College, 601 E. Main
Street, Collegeville, Pennsylvania 19426, United States.
ADH5 knockout HAP1 and genetically matched WT HAP1 cells
were maintained in exponential growth as a monolayer in Iscove’s
Modified Dulbecco’s Medium, high glucose, (IMDM, Invitrogen)
supplemented with 10% FBS and incubated at 37 °C in 5% CO2. One
day before imaging, the cells were passaged and plated in DMEM with
glutamax (phenol red-free) supplemented with 10% FBS on poly D-
lysine-coated 4-well Lab Tek borosilicate chambered coverglass slides
at 75% confluence.
ADH5 knockout MEF and genetically matched WT MEF cells were
maintained in exponential growth as a monolayer in DMEM
supplemented with 10% FBS and incubated at 37 °C in 5% CO2.
One day before imaging, the cells were passaged and plated in DMEM
with glutamax (phenol red-free) supplemented with 10% FBS on poly
D-lysine-coated 4-well Lab Tek borosilicate chambered coverglass
slides at 75% confluence.
Author Contributions
∇K.J.B. and R.R.W. contributed equally to this work and are
listed in alphabetical order.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
K.J.B. was partially supported by an NSF graduate fellowship.
T.F.B. was partially supported by a Chemical Biology Training
Grant from the NIH (T32 GM066698). C.J.C. is an
Investigator of the Howard Hughes Medical Institute. We
thank Alison Killilea and Carissa Tasto (UC Berkeley Tissue
Culture Facility) for expert technical assistance.
Confocal Fluorescence Imaging Experiments. Confocal
fluorescence imaging studies were performed with a Zeiss laser
scanning microscope 710 with a 20× objective lens using Zen 2009
software (Carl Zeiss). FAP573 was excited using a 561 diode (for
experiments in Figure 8) or 594 nm HeNe laser (for all other
experiments with FAP573), and emission was collected using a META
detector between 573 to 682 nm. The 561 nm laser was utilized on a
Zeiss laser scanning microscope 710 located in the Molecular Imaging
Center at UC Berkeley. FAP555 was excited using a 543 nm HeNe
laser, and emission was collected using a META detector between 560
to 669 nm. FAP498 was excited using a 488 nm HeNe laser, and
emission was collected using a META detector between 493 to 630
nm. FAP385 was excited with a 405 nm diode laser, and emission was
collected using a META detector between 450 and 540 nm. Hoechst
33342 was excited with a 405 nm diode laser, and emission was
collected using a META detector between 400 and 450 nm. BSS
(136.9 mM NaCl, 5.37 mM KCl, 1.26 mM CaCl2, 0.81 mM MgSO4,
0.44 mM KH2PO4, 0.335 mM Na2HPO4, 10 mM PIPES; pH to 7.2
with NaOH) was used as the imaging buffer for all confocal
experiments. The cells were imaged at 37 °C throughout the course
of the experiment. Image analysis and quantification was performed
using ImageJ (National Institutes of Health). Quantification of
fluorescence intensity was performed using three fields of cells in
the same well by generating a region of interest (ROI) around each
image. The mean fluorescence intensity of each cell was measured
(using “Measure” function) and averaged across the three ROIs. For
each condition, multiple wells (reported as n) were analyzed using this
process, and the values were averaged across independent experiments
for statistical analysis. Statistical analyses for multiple comparisons
were performed using one-way ANOVA with the Bonferroni
correction in the statistical analysis software, R.
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.6b12460.
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