Palladium-Mediated Synthesis of a Near-Infrared Fluorescent K+ Sensor

Article abstract of DOI:10.1021/acs.joc.7b00845

Potassium (K⁺) exits electrically excitable cells during normal and pathophysiological activity. Currently, K⁺-sensitive electrodes and electrical measurements are the primary tools to detect K⁺ fluxes. Here, we describe the synthesis of a near-IR, oxazine fluorescent K⁺ sensor (K_NIR-1) with a dissociation constant suited for detecting changes in intracellular and extracellular K⁺ concentrations. K_NIR-1 treatment of cells expressing voltage-gated K⁺ channels enabled the visualization of intracellular K⁺ depletion upon channel opening and restoration of cytoplasmic K⁺ after channel closing.

Full text of DOI:10.1021/acs.joc.7b00845

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Palladium-mediated Synthesis of a Near-IR Fluorescent K Sensor
H. M. Dhammika Bandara, Zhengmao Hua, Mei Zhang, Steven M. Pauff,
Stephen C. Miller, Elizabeth A. Colby Davie, and William R Kobertz
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.7b00845 • Publication Date (Web): 30 Jun 2017
Downloaded from http://pubs.acs.org on July 1, 2017
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Palladium-mediated Synthesis of a Near-IR Fluorescent K+ Sensor
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H. M. Dhammika Bandara†, Zhengmao Hua†, Mei Zhang†, Steven M. Pauff†§, Stephen C. Milꢀ
ler†, Elizabeth A. Colby Davie‡, and William R. Kobertz*†
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†Department of Biochemistry and Molecular Pharmacology, Programs in Neuroscience and
Chemical Biology, University of Massachusetts Medical School, 364 Plantation Street, Worcesꢀ
ter, MA 01605, United States
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‡Department of Natural Sciences, Assumption College, 500 Salisbury Street, Worcester MA
01609, United States
ABSTRACT: Potassium (K+) exits electrically excitable cells during normal and pathophysioꢀ
logical activity. Currently, K+ꢀsensitive electrodes and electrical measurements are the primary
tools to detect K+ fluxes. Here we describe the synthesis of a nearꢀIR, oxazine fluorescent K+
sensor (KNIRꢀ1) with a dissociation constant suited for detecting changes in intracellular and exꢀ
tracellular K+ concentrations. KNIRꢀ1 treatment of cells expressing voltageꢀgated K+ channels
enabled the visualization of intracellular K+ depletion upon channel opening and restoration of
cytoplasmic K+ after channel closing.
Neuronal and muscle excitability, cardiac rhythmicity, vasodilation, insulin secretion, and
renal function all require the spatiotemporal release of potassium (K+) ions from cells and tisꢀ
sues1ꢀ6. During normal physical activity, the concentration of extracellular K+ surrounding human
muscle nearly triples, rising from 3.5 mM to 10 mM2,3. This substantial change in extracellular
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K+ is predicted to be even greater in the transverse tubules7ꢀ9, though current methods do not alꢀ
low for accurate measurements in these microscopic invaginations. Regulated K+ efflux is also
essential for the developing pancreas, kidney, and vestibular and cochlear endolymphs5,6,10. Acꢀ
cordingly, dysregulated exit of cellular K+ from tissues has been associated with a multitude disꢀ
ease states from epilepsy11, migraine auras12, hypokalemic periodic paralysis13, and cardiac arꢀ
rhythmias14 to neonatal diabetes15, hyperkalemia16 and congenital deafness17. Electrical measꢀ
urements and potassiumꢀsensitive electrodes provide pinpoint accuracy of K+ efflux, but both
approaches are invasive, technicallyꢀdemanding, and time consuming compared to the small
molecule18 and geneticallyꢀencodable fluorescentꢀbased sensors19,20 that have revolutionized the
intracellular signaling field. Although several small molecule fluorescent K+ sensors have been
synthesized21ꢀ25, the current cadre of water soluble, visibleꢀlight K+ sensors is not routinely used
to visualize K+ fluxes from cells, tissues or animal models. Part of the problem is that cells and
tissues absorb visible light26, resulting in cellular damage, loss of signal and unwanted autofluoꢀ
rescence. However, the major stumbling block has been the inefficient and limited routes to atꢀ
tach fluorophores to the K+ binding domain23ꢀ25, which has hampered the application of these
sensors for detecting K+ accumulation and depletion.
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Figure 1. NearꢀIR K+ sensor KNIRꢀ1 consists of a TAC K+ binding domain covalently linked to an oxazine
fluorophore. Binding of K+ to the TAC domain quenches photoinduced electron transfer (PET) from the oꢀ
alkoxyaniline moiety to the oxazine fluorophore, resulting in enhanced fluorescence emission.
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To overcome these barriers, we sought a synthesis of a nearꢀIR fluorescent K+ sensor
where fluorophore attachment to the K+ binding domain was facile. For the potassiumꢀselective
binding domain, we chose a triazacryptand (TAC) to detect physiological and pathophysiological
K+ concentrations for three reasons (Figure 1): (1) The TAC ion binding site is slightly larger
than the K+ ion diameter, yielding an apparent millimolar Kd for potassium23ꢀ25. (2) TAC does
not measurably bind to the smaller and physiologically abundant extracellular cations23ꢀ25: Na+,
Ca2+, and Mg2+. (3) The electronꢀrich aromatic groups of TAC enable K+ sensing via photoinꢀ
duced electron transfer (PET) with fluorophores. Given this flexibility, we opted to synthesize a
K+ sensor with an oxazine dye KNIR-1 (Figure 1) because of its compact structure,
photostability27, amenability to PET quenching28, and excitation and fluorescence emission
wavelengths in the nearꢀIR region (650 – 900 nm)29.
Previous syntheses of visibleꢀlight K+ sensors have directly coupled the fluorophore to
the TAC binding domain using aldehyde precursor 1a (TACꢀCHO, Scheme 1). Similar to the
reported low yields in the literature23ꢀ25, our attempts to form a nearꢀIR dye utilizing TACꢀCHO
were unsuccessful. Stymied by the inability to efficiently derivatize TACꢀCHO, we pursued a
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Scheme 1. Synthesis of near-IR fluorescent K+ sensor, KNIR-1
palladiumꢀmediated aryl coupling approach to append fluorophores to the TAC K+ binding doꢀ
main. Aryl halogenation of the TAC binding domain (Scheme 1) is ideal for selective modificaꢀ
tion of the oꢀalkoxyaniline PET donor because it is the only aromatic ring that is not blocked by
a methyl group in the para position. Treatment of 1b with bromine resulted in the selective and
near quantitative bromination of the aryl PET donor ring. Iodination of TAC also proceeds quanꢀ
titatively; however, the iodinated product is more challenging to purify and thus was not pursued
further.
With halogenated TAC 2 in hand, we initially envisioned attaching an intact oxazine
fluorophore via palladium–catalyzed crossꢀcoupling reactions (e.g. Sonogashira, Suzuki, Stille,
etc.). To synthesize the oxazine dye with a pendant propꢀ2ꢀynꢀ1ꢀyl group, aryl azobenzene 3 and
propꢀ2ꢀynꢀ1ꢀyl tetrahydroquinoline 4 were prepared separately, and then reacted together in acid
(Scheme 2). To prepare the aryl azobenzene 3, 7ꢀmethoxytetrahydroquinoline 5 was first Nꢀ
alkylated with iodoethane, and then subsequently reacted with 4ꢀnitrodiazobenzene. The tetrahyꢀ
droquinoline 4 was synthesized by Nꢀalkylation of 7ꢀhydroxytetrahydroquinoline 6 with 3ꢀ
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Scheme 2. Synthesis of oxazine dyes and precursors for palladium-based coupling reactions
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bromoꢀ1ꢀpropyne, followed by acetylation of the 7ꢀhydroxy group with acetic anhydride. Conꢀ
densation of aryl azobenzene 3 and propꢀ2ꢀynꢀ1ꢀyl tetrahydroquinoline 4 in acetic acid or 1.5%
hydrochloric acid yielded the desired oxazine dye 7. Synthesis of the borylated oxazine 8 was
accomplished by borylation of tetrahydroquinoline 4 with pinacol borane to generate boronate
ester 9, followed by condensation between 9 and arylazobenzene 3 in 1.5% hydrochloric acid.
Despite trying several different palladium ligands, coꢀcatalysts, counter anions, and solꢀ
vents, the Sonogashira reaction between 2 and propꢀ2ꢀynꢀ1ꢀyl oxazine dye 7 did not occur
(Scheme 1). Suzuki couplings between 2 and boronate ester oxazine dye 8 were also unsuccessꢀ
ful. To test whether the lack of reactivity was due to the seemingly cantankerous TAC ligand or
the oxazine dye, we repeated the palladiumꢀbased reactions with the oxazine dye precursors 4
and 9 shown in Scheme 2. Surprisingly, the Sonogashira reaction between 2 and 4 proceeded
smoothly, yielding TACꢀalkyne 10 (Scheme 1), lacking the acetate group, which falls off during
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the palladiumꢀmediated coupling. TACꢀBr 2 was also amenable to a Suzuki coupling with boroꢀ
nate ester 9 shown in Scheme 2 (data not shown), suggesting that the positiveꢀcharge and/or reꢀ
duction of the intact dye may impede palladiumꢀcatalyzed aryl coupling reactions. Palladium
impurities in the resultant alkyne were removed using a palladium scavenger (SiliaMetS® DMT)
and the purified alkyne was reduced by tosyl hydrazide hydrogenation. Failure to remove all of
the palladium impurities results in cleavage of the tetrahydroquinoline from the TAC K+ binding
domain during reduction. Oxazine dye formation between reduced 10 and aryl azobenzene 3
proceeded in acetic acid as previously reported30 and was purified by reverse phase HPLC. Inꢀ
terestingly, dye formation to afford TACꢀoxazine KNIRꢀ1 did not proceed in 1.5% hydrochloric,
trifluoroacetic, or formic acid, suggesting that protonation of the TAC K+ binding domain hinꢀ
ders oxazine dye formation.
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The fluorescence of TACꢀoxazine KNIRꢀ1 was initially tested in the presence of various
concentrations of physiological extracellular cations: Na+, Ca2+, Mg2+ and K+. Figure 2A shows
that KNIR-1 is essentially nonꢀresponsive to physiological concentrations of extracellular Na+
(150 mM), Ca2+ (1.5 mM), and Mg2+ (1 mM). In contrast, the fluorescence emission of KNIRꢀ1
was highly dependent on K+ concentration in the presence of these ions (Figure 2B) with an apꢀ
parent Kd of 12.1 ± 0.5 mM (Figure 2C). The dissociation constant of KNIRꢀ1 is ~ 5ꢀfold lower
than previous TACꢀbased K+ sensors where the fluorophore was directly conjugated to the K+
binding domain31. The peak emission wavelength of KNIRꢀ1 is 680 nm (Figure 2B) whereas the
peak absorbance was 670 nm with a molar extinction coefficient ɛ670 = 39,800 ± 100 L·molꢀ
1·cmꢀ1 (Figure 2D), enabling excitation with either visible or nearꢀIR light. K+ binding (200
mM) to KNIRꢀ1 had no discernable effect on absorbance (Figure 2D). Using oxazineꢀ170 as fluoꢀ
rescent reference standard32, the quantum yield for apo KNIRꢀ1: 0.0597 ± 0.0003 and K+ bound
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Figure 2. Fluorescence emission (Ex: 620 nm) and absorbance of KNIR-1 in 10 mM HEPES. (A) Fluoresꢀ
cence emission in the presence of Mg2+, Ca2+ and Na+. (B) Fluorescence emission at various K+ concentraꢀ
tions in the presence of Mg2+ (1 mM), Ca2+ (1.5 mM) and Na+ (150 mM). (C) Integrated fluorescence intenꢀ
sities at different K+ concentrations. F is the integrated emission intensity between 630 and 800 nm; F0 is the
integrated baseline fluorescence intensity (with 0 mM K+). (D) Absorbance spectra at 0 and 200 mM K+.
(E) Fluorescence intensity (F − F0)/F0 as determined in (C) in the presence of various cations [Na+ (150
mM), Ca2+ (1.5 mM), Mg2+ (1 mM), Zn2+ (0.3 mM), Fe3+ (50 ꢀM), K+ (5 mM), Rb+ (5 mM) and Cs+ (5
mM)] and at different pH values with 5 mM K+ present. Unless indicated, the pH was 7.4. (F) Stoppedꢀflow
experiments at various K+ concentrations; emission was collected at all wavelengths above 645 nm. Data
were averaged from three experiments; error bars are ± SEM. Data could not be fitted because the equilibriꢀ
um rate was faster than the mixing time (1.4 ms).
(200 mM): Φ = 0.289 ± 0.005 were determined from three experiments ± SEM (supporting inꢀ
formation). Much like K+ channels that bind to and permeate Rb+ and Cs+33, KNIRꢀ1 also reꢀ
sponds to these larger, but nonꢀphysiological cations (Figure 2E). As expected, smaller diameter
transition metals (Zn2+ and Fe3+) had no effect on KNIRꢀ1 fluorescence. The fluorescence emisꢀ
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sion of KNIRꢀ1 is stable at extracellular and intracellular physiological pH 6.8 – 7.8 (Figure 2E),
but begins to increase at pH 6.5 (Figure S1) as protonation starts to compete with K+ binding.
Thus, under suitably buffered conditions, the fluorescence of KNIRꢀ1 should faithfully report on
K+ accumulation and depletion during physiological and pathophysiological activity.
We next used stoppedꢀflow experiments to determine the K+ binding kinetics to TACꢀ
oxazine KNIRꢀ1. Figure 2F shows the fluorescence responses from rapid mixing (1.4 ms dead
time) of KNIRꢀ1 with different millimolar solutions of K+. Even with rapid mixing, the binding
between K+ and KNIRꢀ1 at all K+ concentrations reached equilibrium within the dead time of the
stoppedꢀflow instrument. The magnitude of the K+ꢀdependent fluorescent response confirmed
the Kd of KNIRꢀ1 (Figure 2C) and that the equilibrium between K+ and KNIRꢀ1 was reached withꢀ
in ~ 1 ms, which is faster than previously reported for TACꢀbased K+ sensors using fluorescence
correlation spectroscopy31. Thus, the subꢀmillisecond K+ association and disassociation kinetics
of KNIR-1 are sufficient to rapidly report on changes in K+ concentration due to ion channel acꢀ
tivity at the plasma membrane.
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Although the dissociation constant of KNIR-1 is somewhat better suited for extracellular
K+ detection, we determined whether the highly membrane permeant KNIR-1 could detect intraꢀ
cellular K+ depletion upon K+ channel opening (Figure 3). Chinese hamster ovary (CHO) cells
transiently transfected with empty vector (pCDNA3.1) or a voltageꢀgated K+ channel (Shakerꢀ
IR) were treated with 50 ꢀM KNIR-1 for 3 min and the excess reagent removed before forming a
gigaseal with a single cell (Figure 3A, dotted circles). Whole cell patch clamp fluorometry
(Figure 3B) with untransfected cells (−) showed no voltageꢀdependent currents (I) or change in
fluorescence (ꢁF/F0). In contrast, ShakerꢀIR expressing cells showed robust outward currents
with depolarizing voltages ≥ 0 mV (Figure 3C). Simultaneous fluorescent imaging (635 nm exꢀ
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Figure 3. Fluorescent detection of K+ depletion in cells. (A) Wide field images of CHO cells transiently
transfected with empty pCDNA3.1 plasmid (−) or ShakerꢀIR DNA and labeled with 50 ꢀM KNIR-1. Yellow
dotted circles indicate the patch clamped cell in the field of view; fluorescent image scale bars represents 10
ꢀm. Voltageꢀclamp fluorometry traces for (B) pCDNA3.1 (−) and (C) ShakerꢀIR: cells were held at − 80 mV
and currents (I) were elicited from 2ꢀs command voltages from 0 to 100 mV in 20ꢀmV increments. Total celꢀ
lular fluorescence was simultaneously collected at 10 Hz by 10ꢀms excitation at 635 nm; F0 is the average
fluorescence intensity of the first ten data points before the test depolarization. Voltage clamp fluorometry
scale bars are for both (B) and (C); dotted line indicates zero current or change in fluorescence.
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citation) of the whole cell at 10 Hz revealed a concomitant loss in fluorescence that was comꢀ
mensurate with the outward K+ currents during the 2ꢀs depolarization. The kinetics of the fluoꢀ
rescence signals were substantially slower than the currents, which was consistent with cytoꢀ
plasmic K+ depletion at the ShakerꢀIR intracellular vestibule. Upon channel closing by repolariꢀ
zation to – 80 mV, the current rapidly returned to zero (< 100 ms) whereas it required seconds for
the fluorescent signal to return to the preꢀdepolarization values. Based on the subꢀmillisecond
association and disassociation kinetics between K+ and KNIR-1 in stoppedꢀflow experiments
(Figure 2F), the ꢁF/F0 kinetics in Figure 3C are consistent with intracellular K+ depletion during
channel opening followed by reestablishment of the cytoplasmic K+ concentration after channel
closing. The extremely long time to restore cellular K+ to steady state concentrations highlights
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the importance of the Na+/K+ꢀATPase in maintaining the cellular K+ and Na+ gradients in electriꢀ
cally excitable cells.
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Our synthesis of TACꢀoxazine KNIRꢀ1 has several advantages over previous syntheses of
visibleꢀlight K+ sensors. First, selective halogenation of the PET donor greatly expands the repꢀ
ertoire of chemistries to attach a fluorophore to the TAC ion binding domain. For example, the
Sonogashira coupling described herein should enable the synthesis of a wide palette of fluoresꢀ
cent K+ sensors using alkynylꢀderivatized fluorophores that have become commerciallyꢀavailable
due to the emergence of bioorthogonal Huisgen 1,3ꢀdipolar cycloadditions (click chemistry).
Second, the absorption and emission spectra of TACꢀoxazine KNIRꢀ1 enable K+ detection using
nearꢀIR light, which has better tissue penetration and reduced autofluorescence compared to visꢀ
ible light26. In addition, TACꢀoxazine KNIRꢀ1 can be utilized with the standard repertoire of visiꢀ
bleꢀlight calcium sensors18ꢀ20 and channelrhodopsins34,35 – negating the need for electrodes and
enabling the visualization of multiple ion activities over the landscape of a cell, neuronal circuit,
or brain slice. Lastly, the modular synthesis of the oxazine dye will enable the synthesis of
chemicallyꢀreactive derivatives of KNIRꢀ1 that can be conjugated to membrane proteins36 and/or
the cell surface37 to detect extracellular K+ accumulation and depletion.
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EXPERIMENTAL SECTION
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General experimental procedures. Materials were obtained from commercial suppliers and
were used without further purification. The final oxazine compound was purified by RP HPLC.
NMR spectra were recorded in CDCl3, CD3OD or DMSOꢀd6 on a 400 MHz spectrometer.
HRMS were obtained on a QꢀTOF mass spectrometer equipped with an ESI source.
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UV-visible and fluorescence experiments. Unless otherwise stated, all UVꢀvisible and fluoresꢀ
cence experiments were performed at 14 ꢁM KNIR-1 in 10 mM HEPES solutions (pH 7.4) using
acetonitrile as a vehicle (0.5% final concentration) in 3 mL quartz cuvettes (1 cm optical path
length). Excitation wavelength (λex) was 620 nm and slit width was 3 nm. All measurements
were corrected for dilution when aliquots of the different test cation solutions significantly
changed the total volume. Stock solutions of MgCl2 (0.30 M), CaCl2 (0.30 M), NaCl (5.4 M),
KCl (4.0 M) ZnCl2 (30 mM), FeCl3 (0.5 mM), RbCl (0.5 M), and CsCl (0.5 M) were used.
Stopped-flow fluorescence measurements. A HiꢀTech Scientific SFꢀ61DX2 stoppedꢀflow sysꢀ
tem was used to estimate the time course of K+ binding to KNIR-1. KNIR-1 (150 ꢀL of a 14 ꢁM
solution in 10 mM HEPES pH 7.4) was mixed with various concentrations (6 – 200 mM) of K+
in 10 mM HEPES pH 7.4 and injected into the light path of the flow cell within 1.4 ms using a
pneumatic drive system. Excitation was at 620 nm (5nm slit width); emission of all wavelengths
above 645 nm was collected with a photomultiplier. The flow cell and solution reservoirs were
at room temperature. Only steady state fluorescence was observed, indicating the equilibrium
binding between KNIR-1 and K+ occurs within 1ms.
Whole cell patch clamp fluorometry. CHO cell maintenance and transient transfection protoꢀ
col are listed in the supporting information. Transfected CHO cells were trypsinized and seeded
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on a glass bottom culture dish for 2 h and then labeled with KNIR-1 (50 ꢀM) in PBS (0.5% final
acetonitrile concentration) at R.T. for 3min; transfected cells were identified using an inverted
light microscope (Axiovert 40 CFL; Carl Zeiss, Inc.) and the currents were recorded in the whole
cell patch configuration at room temperature (24 ± 2°C) using glass pipettes (electrode reꢀ
sistance: 2.0 – 3.0 Mꢂ) filled with internal solution (in mM): 126 KCl, 2 MgSO4, 0.5 CaCl2, 5
EGTA, 4 K2ꢀATP, 0.4 GTP and 25 HEPES at pH = 7.2 adjusted with KOH. Bath solution conꢀ
tained (in mM): 145 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES at pH=7.4 adꢀ
justed with NaOH. Cells were imaged at 10 Hz using a CoolLED pEꢀ4000 light source (expoꢀ
sure time = 10 ms), 63 x 1.4 N.A. oil immersion objective, a Chroma Quad filter set (89402:
391ꢀ32/479ꢀ33/554ꢀ24/638ꢀ31) and a Zyla sCMOS camera (ANDOR). GFP and KNIR were exꢀ
cited (10ꢀms) at 460 nm channel (light power = 30%) and 635 nm channel (light power = 10%),
respectively. The patch clamp (Axopatch 200B), light source, and camera were controlled with
Clampex 10.5 (Molecular Devices); fluorescent images were collected (10 Hz, 4 x 4 binning)
and processed using open source software (microꢀmanager and ImageJ). F0 is the average fluoꢀ
rescence intensity of the first ten data points before the test depolarization.
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Synthetic methods and compound characterization:
TACꢀBr (2). Bromination of triazacryptand 1 was carried out according to a previously reported
aryl bromination procedure38. Triazacryptand 123 (220 mg, 0.32 mmol, 1.0 eq) was dissolved in
CH2Cl2 (5 mL) and cooled to 0 °C in an ice bath. A 0.55 M stock solution of bromine in dry
CH2Cl2 was added dropwise by syringe (0.63 mL, 0.35 mmol, 1.1 eq) to above solution over a 5
min period. After 10 minutes of stirring, the ice bath was removed and the mixture was stirred at
room temperature for an additional 1.25 h. The reaction was cooled again to 0 °C in an ice bath
and another aliquot of the 0.55 M stock solution of bromine in dry CH2Cl2 was added via syringe
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(0.63 mL, 0.35 mmol, 1.1 eq). After 10 minutes of stirring, the ice bath was removed and the
mixture was stirred at ambient temperature for an additional 1.25 h. The reaction was quenched
by adding 10% aqueous NaOH (10 mL) and saturated aqueous Na2S2O3 solution (10 mL). The
organic layer was washed with water (10 mL), dried with Na2SO4, filtered, and concentrated unꢀ
der reduced pressure to give 2 as a brown solid (237 mg, 95%) which was used in the next step
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without further purification. H NMR (CDCl3, 400 MHz) δ: 6.96ꢀ7.01 (m, 3H), 6.87 (d, J = 7.8,
2H), 6.65 (d, J = 7.8, 2H), 6.56 (s, 2H) 4.12ꢀ4.18 (m, 2H), 3.98ꢀ4.01 (m, 4H), 3.89ꢀ3.92 (m, 4H),
3.78ꢀ3.80 (m, 2H) 3.59ꢀ3.73 (m, 16H), 3.46 (s, 3H), 3.40ꢀ3.45 (m, 4H), 3.26ꢀ3.33 (m, 4H), 2.24
13
(s, 6H). C NMR (CDCl3, 100 MHz): δ 153.3, 153.1, 138.3, 137.7, 132.8, 124.6, 123.0, 121.6,
121.2, 117.4, 114.5, 114.1, 71.4, 71.2, 70.2, 68.3, 67.2, 59.3, 54.1, 53.2, 21.3. HRMS (ESI): m/z
calculated for C39H55BrN3O8 ([M+H]+): 772.3173; found: 772.3169.
1ꢀEthylꢀ7ꢀmethoxyꢀ1,2,3,4ꢀtetrahydroquinoline (11). Potassium carbonate (1.2 g, 8.4 mmol, 1.5
eq) was added to a solution of 7ꢀmethoxyꢀ1,2,3,4ꢀtetrahydroxyquinoline (5)30 (0.91 g, 5.6 mmol,
1.0 eq) in N,Nꢀdimethylformamide (DMF) (5 mL). Ethyl iodide (0.54 mL, 6.8 mmol, 1.2 eq) was
added and the reaction was stirred at 65 °C for 18 h. The mixture was allowed to cool to room
temperature, diluted with water (80 mL), and acidified to pH 3 with 1 M HCl. The aqueous phase
was extracted with ethyl acetate (1 × 80 mL, 5 × 50 mL) and the combined organic phases were
washed with water (3 × 60 mL) and brine (80 mL) and dried over Na2SO4. The solvent was
evaporated under vacuum and the crude product was purified by flash column chromatography
on silica (100% hexanes to acetone/hexanes 5:95) to yield 11 as a yellow oil (0.91 g, 85%). Rf =
1
0.58 (silica/ethyl acetate/hexanes 1:19). HꢀNMR (CDCl3): δ 6.84 (d, 1H, J = 8.1 Hz), 6.18 (d,
1H, J = 2.1 Hz), 6.13 (dd, 1H, J = 2.4 Hz, 8.1 Hz), 3.78 (s, 3H), 3.32 (q, 2H, J = 7.1 Hz), 3.24 (t,
13
2H, J = 5.6 Hz), 2.68 (t, 2H, J = 6.4 Hz), 1.96ꢀ1.9 (m, 2H), 1.14 (t, 3H, J = 7.1Hz). CꢀNMR
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(CDCl3): δ 159.6, 146.1, 129.7, 115.6, 99.8, 97.6, 55.4, 48.6, 45.7, 27.8, 22.8, 11.1. HRMS (ESI)
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m/z calculated for C12H18NO2: 192.1388 ([M+H]+); found: 192.1379.
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(1ꢀEthylꢀ7ꢀmethoxyꢀ1,2,3,4ꢀtetrahydroquinolinꢀ6ꢀyl)ꢀ(4ꢀnitrophenyl)ꢀdiazene
(3).
4ꢀ
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Nitrobenzenediazonium tetrafluoroborate (0.57 g, 2.4 mmol, 1.0 eq) was suspended in 10%
aqueous H2SO4 (1.6 mL) and added dropwise to a solution of 11 (0.46 g, 2.4 mmol, 1.0 eq) in
methanol (2 mL). The resulting dark purple solution was stirred at room temperature for 1 h and
cooled in an ice bath. The reaction was neutralized with aqueous NH4OH (28ꢀ30%) and the reꢀ
sulting precipitate was isolated by filtration and washed with water (300 mL) to give a dark red
solid. The crude product was recrystallized from 1ꢀbutanol/water to yield 3 as dark purple crysꢀ
1
talline solid (0.52 g, 64%). Rf = 0.14 (silica/ethyl acetate/hexanes 1:19). HꢀNMR (CDCl3): δ
8.26 (d, 2H, J = 8.9 Hz), 7.84 (d, 2H, J = 8.9 Hz), 7.58 (s, 1H), 6.11 (s, 1H), 4.00 (s, 3H), 3.46
(q, 2H, J = 7.1 Hz), 3.38 (t, 2H, J = 5.6 Hz), 2.71 (t, 2H, J = 6.0 Hz), 1.95(p, 2H, J = 5.6 Hz, 6.1
Hz), 1.26 (t, 3H, J = 7.1Hz). 13CꢀNMR (CDCl3): δ 160.3, 158, 151.4, 146.7, 133.6, 124.9, 122.5,
117.7, 116.5, 92.8, 56.5, 49.2, 46.3, 27.5, 22.2, 11.5. HRMS (ESI) m/z calculated for
C18H21N4O3: 341.1614 ([M+H]+); found: 341.1625.
1ꢀ(propꢀ2ꢀynꢀ1ꢀyl)ꢀ1,2,3,4ꢀtetrahydroquinolinꢀ7ꢀol (12). 7ꢀHydroxyꢀ1,2,3,4ꢀtetrahydroquinoline
(6)30 (0.60 g, 4.0 mmol, 1.0 eq) and K2CO3 (0.66 g, 4.8 mmol, 1.2 eq) were combined in DMF (5
mL). 3ꢀbromoꢀ1ꢀpropyne (0.9 mL of an 80 wt% solution in toluene, 6.0 mmol, 1.5 eq) was addꢀ
ed, and the reaction was stirred at 60 °C for 1.5 h. The reaction was cooled, and partitioned beꢀ
tween ethyl acetate (100 mL) and water (20 mL). The aqueous layer was washed with ethyl aceꢀ
tate (3 × 20 mL), organic layers were combined, dried over Na2SO4, filtered, and concentrated
under vacuum. The crude product was purified by flash chromatography on silica (ethyl aceꢀ
tate/hexanes 1:4) to yield 12 as a brown oil which acquired a light pink color upon standing (0.52
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g, 69%). Rf = 0.35 (silica/ethyl acetate/hexanes 1:4). 1H NMR (CDCl3, 400 MHz) δ: 6.82 (d, J =
8.0 Hz, 1H), 6.24 (d, J = 3.6 Hz), 6.16 (dd, J = 8.0 Hz, J = 3.6 Hz, 1H), 4.68 (s, 1H), 3.97 (d, J =
2.4 Hz, 2H), 3.27 (t, J = 5.6 Hz, 2 H), 2.69 (t, J = 6.4 Hz, 2H), 2.17 (t, J = 2.4 Hz, 1H), 2.01 –
1.94 (m, 2H). 13C NMR (CDCl3, 100 MHz): δ 154.7, 145.5, 129.8, 116.4, 104.2, 99.3, 79.5, 71.8,
49.1, 40.8, 26.9, 22.5. HRMS (ESI): m/z calculated for C12H14NO ([M+H]+): 188.1070; found
188.1065.
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1ꢀ(propꢀ2ꢀynꢀ1ꢀyl)ꢀ1,2,3,4ꢀtetrahydroquinolinꢀ7ꢀyl acetate (4). The product 12 (200 mg, 1.1
mmol, 1.0 eq), acetic anhydride (402 µL, 4.4 mmol, 4.0 eq) and triethylamine (306 µL, 2.2
mmol, 2.0 eq) were dissolved in CH2Cl2 (5 mL), and the reaction was stirred at room temperaꢀ
ture for 18 hrs. The reaction was washed with saturated aqueous Na2CO3 (20 mL) and 0.5 M HCl
(20 mL), dried over MgSO4, filtered and solvent was removed. Purification by flash chromatogꢀ
raphy on silica (ethyl acetate/hexanes 5:95) yielded 4 as a yellow oil which became a white solid
1
upon standing (192 mg, 71%). Rf = 0.33 (silica/ethyl acetate/hexanes 1:19). H NMR (CDCl3,
400 MHz) δ: 6.97 (d, J = 8.0 Hz, 1H), 6.42 (d, J = 2.0 Hz, 1H), 6.40 (dd, J = 8.0 Hz, J = 2.0 Hz),
3.97 (d, J = 2.4 Hz, 2H), 3.29 (t, J = 5.6 Hz, 2 H), 2.74 (t, J = 6.4 Hz, 2H), 2.29 (s, 3H), 2.20 (t, J
13
= 2.4 Hz, 1H), 2.02 – 1.96 (m, 2H). C NMR (CDCl3, 100 MHz): δ 169.9, 149.9, 145.4, 129.5,
121.4, 110.0, 105.1, 79.3, 72.0, 48.9, 40.7, 27.2, 22.2, 21.2. HRMS (ESI): m/z calculated for
C14H16NO2 ([M+H]+): 230.1181; found: 230.1171.
11ꢀethylꢀ1ꢀ(propꢀ2ꢀynꢀ1ꢀyl)ꢀ2,3,4,8,9,10ꢀhexahydroꢀ1Hꢀdipyrido[3,2ꢀb:2',3'ꢀi]phenoxazinꢀ11ꢀ
ium chloride (7). The product 3 (20 mg, 0.59 mmol, 1.0 eq) and the product 4 (13 mg, 0.59
mmol, 1.0 eq) were combined in 0.5 mL of 1.5% HCl (37% HCl/ethanol/water 0.5 mL:10 mL:1
mL) and stirred at 80 °C for 4 h. The resulting dark green reaction was concentrated under vacuꢀ
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um. Purification by column chromatography on silica (methanol/CH2Cl2 1:10) yielded the chloꢀ
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ride salt of 7 as a dark blue solid (13 mg, 59%). Rf = 0.59 (silica/MeOH/CH2Cl2 1:9). H NMR
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(CDCl3, 400 MHz) δ: 7.47 (s, 1H), 7.36 (s, 1H), 7.29 (s, 1H), 6.51 (s, 1H), 3.72 – 3.68 (m, 2H),
3.55 (t, J = 6.8 Hz, 4H), 3.49 (s, 2H), 2.87 (t, J = 6.0 Hz, 4H), 2.17 (s, 1H), 2.08 – 1.97 (m, 4H),
1.34 (t, J = 7.2 Hz, 3H). 13C NMR (CDCl3, 100 MHz): δ 155.1, 153.4, 146.3, 132.8, 130.7,
130.4, 130.3, 130.2, 128.5, 95.6, 94.9, 76.5, 73.7, 60.9, 57.8, 50.1, 26.9, 26.8, 20.7, 20.4, 10.4.
HRMS (ESI): m/z calculated for C23H24N3O (M+): 358.1914; found: 358.1917.
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(E)ꢀ1ꢀ(3ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)allyl)ꢀ1,2,3,4ꢀtetrahydroquinolinꢀ7ꢀyl aceꢀ
tate (9). The product 4 (220 mg, 0.74 mmol, 1.0 eq), pinacolborane (110 mg, 0.89 mmol, 1.2 eq)
and triethylamine (8.6 µL, 0.060 mmol, 0.086 eq) were combined in a sealable reaction vessel
and the vessel was purged with Ar for 10 min. ZrCp2HCl (8.1 mg, 0.031 mmol, 0.042 eq) was
quickly added, vessel was sealed and the reaction was stirred at 60 °C for 15 h under Ar. Reacꢀ
tion contents were dissolved in ethyl acetate/hexanes (1:9) and the product was purified by flash
chromatography on silica (ethyl acetate/hexanes 1:99 to 3:97) to yield 9 as a yellow oil (127 mg,
48%). Rf = 0.15 (silica/ethyl acetate/hexanes 1:19). 1H NMR (CD3OD, 400 MHz) δ: 6.79 (dd, J =
12 Hz, J = 8 Hz, 1H), 6.09 – 6.01 (m, 2H), 5.62 (d, J = 18 Hz, 1H), 4.86 (m, 2H), 3.20 – 3.18 (m,
2H), 2.64 – 2.61 (m, 2H), 2.06 (s, 3H), 1.94 – 1.86 (m, 2H), 1.24 (d, 12H).13C NMR (CD3OD,
100 MHz): δ 167.9, 155, 145.7, 136.2, 129.5, 124.7, 113.1, 104.3, 100.9, 82.4, 74.5, 71.8, 50.9,
41.4, 23.4, 23.5, 22.3, 17.0. HRMS (ESI): m/z calculated for C20H29BNO4 ([M+H]+): 358.2190;
found: 358.2186.
(E)ꢀ11ꢀethylꢀ1ꢀ(3ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl)allyl)ꢀ2,3,4,8,9,10ꢀhexahydroꢀ
1Hꢀdipyrido[3,2ꢀb:2',3'ꢀi]phenoxazinꢀ11ꢀium chloride salt (8). The product 3 (28 mg, 0.095
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mmol, 1.0 eq) and the borane ester 9 (40 mg, 0.095 mmol, 1.0 eq) were combined in 0.5 mL of
1.5% HCl (37% HCl/ethanol/water 0.5 mL:10 mL:1 mL) and stirred at 80 °C for 4 h. The resultꢀ
ing dark green reaction was concentrated under vacuum. Purification by column chromatography
on silica (methanol/CH2Cl2 1:10) yielded 8 as a dark blue solid (14 mg, 29%). Rf = 0.57 (siliꢀ
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ca/MeOH/CH2Cl2 3:17). H NMR (CDCl3, 400 MHz) δ: 6.96 (s, 1H), 6.94 (s, 1H), 6.41 – 6.37
(m, 4H), 3.97 (d, J = 2.7 Hz, 2H), 3.29 (m, 4H), 2.72 (t, J = 6.8 Hz, 4H), 2.28 (s, 12H). 2.17 (m,
3H), 2.00 – 1.97 (m, 4H). 13C NMR (CDCl3, 100 MHz): δ 155.2, 153.5, 148.0, 130.8, 130.3,
128.7, 121.3, 111.1, 74.8, 73.8, 61.1, 57.9, 50.2, 27.1, 26.9, 20.8, 20.6, 10.5. HRMS (ESI): m/z
calculated for C29H37BN3O3 (M+): 484.9222; found: 484.9227.
TACꢀNꢀpropargylꢀ1,2,3,4ꢀtetrahydroquinolinꢀ7ꢀol (10). A reaction vessel containing TACꢀBr 2
(271 mg, 0.35 mmol, 1.0 eq) and product 4 (250 mg, 1.1 mmol, 3.1 eq) was purged with Ar for
10 min and the Ar was removed by applying high vacuum. The above procedure was repeated
two more times. DMF (1.0 mL) and pyrrolidine (1.0 mL) were injected into the reaction vessel
and Ar was bubbled through the resulting solution for 10 min. Pd(PPh3)4 (66 mg, 0.058 mmol,
0.17 eq) and CuI (9.5 mg, 0.050 mmol, 0.014 eq) were quickly added, reaction was purged with
Ar for 5 min and stirred at 80 ºC for 18 hrs. Solvents were removed under high vacuum and the
resulting brown residue was purified by flash chromatography on neutral alumina (CH2Cl2 to
methanol/CH2Cl2 1:9). The purified product was dissolved in 1:2 methanol/CH2Cl2 (6 mL), Siliꢀ
aMet® DMT (450 mg of silica with 0.60 mmol/g scavenger loading, 5 eq) was added and the reꢀ
action was refluxed at 80 ºC for 4 h. The silica was filtered off and the solvent removed to yield
10 as a brown oil (108 mg, 35%). A 30 mg aliquot of 10 was purified by preparative HPLC acꢀ
cording to the conditions below to obtain a sample with sufficient purity for spectroscopic charꢀ
acterization. Method started at 30% solvent B (0.1% trifluoroacetic acid in acetonitrile) and 70%
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solvent A (0.1% trifluoroacetic acid in water), then 30ꢀ60% solvent B over 15 min, then 60ꢀ
100% solvent B over 1 min, then 100% solvent B for 5 min. HPLC absorbance detector was set
at 254 nm. The product eluted at 15.0 min. HPLC fractions were combined and lyophilized to
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yield 10 as a brown oil. Rf = 0.30 (neutral alumina/methanol/CH2Cl2 1:49). H NMR (CDCl3,
400 MHz) δ: 7.08 (d, J = 8.0 Hz, 1H), 6.96 – 6.82 (m, 6H), 6.75 (m, 2H), 6.20 (dd, J = 6.0 Hz, J
= 2.4 Hz, 1H), 6.03 (d, J = 2.4 Hz, 1H), 4.21 (t, J = 4.2 Hz, 4H), 4.10 (t, J = 4.4 Hz, 2H), 3.79 –
3.61 (m, 27H), 3.40 (t, J = 4.8 Hz, 1H), 3.35 (s, 3H), 3.29 – 3.26 (m, 8H), 2.69 (t, J = 6.4 Hz,
4H), 2.36 (s, 6H), 1.94 – 1.88 (m, 2H). 13C NMR (CDCl3, 100 MHz): δ 153.3, 150.4, 148.8,
131.8, 129.5, 127.4, 121.6, 118.6, 112.0, 109.5, 102.3, 98.5, 81.9, 68.2, 68.1, 67.6, 65.3, 64.2,
51.0, 50.5, 49.9, 39.2, 28.5, 23.7, 19.9, 18.5, 18.2. HRMS (ESI): m/z calculated for
C51H66N4O9Na ([M+Na]+): 901.4727; found: 901.4726.
TACꢀNꢀpropanꢀ1,2,3,4ꢀtetrahydroquinolinꢀ7ꢀol (13). The TACꢀalkyne 10 (104 mg, 0.12 mmol,
1.0 eq) and tosyl hydrazide (440 mg, 2.4 mmol, 20 eq) were dissolved in 1,2ꢀdimethoxyethane
(1.0 mL) and stirred at 80 ºC. A solution of sodium acetate (190 mg, 2.4 mmol, 20 eq) in water
(1.0 mL) was added dropwise to the above solution over a period of 6 h. The resulting mixture
was stirred at 80 ºC for 18 h and then stirred at room temperature for another 4 h. The reaction
was partitioned between ethyl acetate (10 mL) and water (5 mL), and the aqueous phase was exꢀ
tracted with ethyl acetate (3 × 10 mL). The organic layers were combined, dried over MgSO4,
filtered and solvent was removed. Purification by flash chromatography on neutral alumina
(CH2Cl2 to methanol/CH2Cl2 1:9) yielded 13 as a brown oil (76 mg, 72%). A 30 mg aliquot of 13
was purified by preparative HPLC according to the conditions below to obtain a sample with sufꢀ
ficient purity for spectroscopic characterization. Method started at 30% solvent B (0.1% triꢀ
fluoroacetic acid in acetonitrile) and 70% solvent A (0.1% trifluoroacetic acid in water), then 30ꢀ
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60% solvent B over 15 min, then 60ꢀ100% solvent B over 1 min, then 100% solvent B for 5 min.
HPLC absorbance detector was set at 254 nm. The product eluted at 15.7 min. HPLC fractions
were combined and lyophilized to yield 13 as a brown oil. Rf = 0.32 (neutral alumiꢀ
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na/methanol/CH2Cl2 1:49). H NMR (CDCl3, 400 MHz) δ: 7.12 (dd, J = Hz, J = Hz, 1H), (m,
7H), 6.74 (m, 2H), 6.20 (dd, J = 2.8 Hz, J = 8.4 Hz, 1H), 6.03 (d, J = 2.8 Hz, 1H), 4.23 – 4.20
(m, 4H), 4.12 – 4.09 (m, 2H), 3.79 – 3.61 (m, 26H), 3.43 – 3.40 (m, 4H), 3.35 (s, 3H), 3.29 –
3.26 (m, 8H), 2.69 (t, J = 6.4 Hz, 2H), 2.36 (s, 6H), 1.94 – 1.84 (m, 4H), 1.49 – 1.45 (m, 2H). 13C
NMR (CDCl3, 100 MHz): δ 162.7, 152.7, 146.5, 139.5, 134.2, 127.7, 121.3, 115.3, 113.6, 113.2,
71.2, 67.5 67.0, 55.1, 58.9, 52.4, 50.1, 36.4, 31.3, 21.1. HRMS (ESI): m/z calculated for
C51H70N4O9Na ([M+Na]+): 905.5040; found: 905.5042.
KNIRꢀ1. The aryl azobenzene 3 (28 mg, 0.095 mmol, 1.0 eq) and TACꢀalkane 13 (40 mg, 0.095
mmol, 1.0 eq) were combined in acetic acid (0.5 mL) and stirred at 80 °C for 18 h. The resulting
dark green reaction was concentrated under vacuum, and purified by preparative HPLC accordꢀ
ing to the conditions. Method started at 30% solvent B (0.1% trifluoroacetic acid in acetonitrile)
and 70% solvent A (0.1% trifluoroacetic acid in water), then 30ꢀ60% solvent B over 15 min, then
60ꢀ100% solvent B over 1 min, then 100% solvent B for 5 min. HPLC absorbance detector was
set at 254 nm. The product eluted at 15.7 min. HPLC fractions were combined and lyophilized to
yield the trifluoroacetate salt of KNIR-1 as a blue solid (mg, 22%), which was first converted to
the hydroxide salt and then to the acetate salt by passing through DOWEX® 1X8 200ꢀ400 hyꢀ
1
droxide and acetate anion exchange columns. Rf = 0.20 (silica/methanol/CH2Cl2 1:9). H NMR
(CD3OD, 400 MHz) δ: 7.58 (d, J = 9.6 Hz, 2H), 7.45 (d, J = 14.4 Hz, 2H), 7.22 (d, 8.0 Hz, 1H),
7.03 – 6.98 (m, 5H), 6.92 (s, 2H), 6.85 (d, J = 8.0 Hz, 1H), 6.60 (s, 1H), 4.35 – 4.20 (m, 6H),
4.25 – 3.85 (m, 12H), 3.85 – 3.75 (m, 5H), 3.75 – 3.50 (m, 17H), 3.50 – 3.35 (m, 5H), 3.31 (s,
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3H), 3.85 – 3.75 (m, 4H), 2.70 (t, J = 7.2 Hz, 2H), 2.30 (s, 6H), 2.20 – 1.80 (m, 6H), 1.38 (t, J =
7.2 Hz, 3H). 13C NMR (DMSOꢀd6, 100 MHz): δ 172.5, 163.5, 156.0, 152.9, 152.8, 151.3, 151.1,
147.4, 131.9, 123.9, 122.2, 121.6, 121.5, 121.4, 116.7, 114.5, 114.4, 96.9, 95.3, 70.7, 70.5, 70.3,
67.9, 58.5, 52.6, 52.5, 52.5, 35.5, 32.5, 27.1, 21.5, 14.5, 11.8. HRMS (ESI): m/z calculated for
C62H81N6O9Na ([M+Na]2+): 527.3069; found: 527.3068.
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ASSOCIATED CONTENT
Supporting Information
Experimental procedures, compound characterization, and supplementary figures are available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
* william.kobertz@umassmed.edu
Present Addresses
§Department of Pure and Applied Chemistry, University of Strathclyde, 295 Cathedral Street,
Glasgow, G1 1XL, United Kingdom
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENTS
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This work was supported by a grant to W.R.K. from the National Institutes of Health (GMꢀ
070650). S.C.M. acknowledges support from the NIH (GMꢀ087460), and S.M.P. received felꢀ
lowship support from the American Heart Association.
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