Design and synthesis of a new class of membrane-permeable triazaborolopyridinium fluorescent probes

Article abstract of DOI:10.1021/ja2005175

A new class of fluorescent triazaborolopyridinium compounds was synthesized from hydrazones of 2-hydrazinylpyridine (HPY) and evaluated as potential dyes for live-cell imaging applications. The HPY dyes are small, their absorption/emission properties are

Full text of DOI:10.1021/ja2005175

ARTICLE
pubs.acs.org/JACS
Design and Synthesis of a New Class of Membrane-Permeable
Triazaborolopyridinium Fluorescent Probes
Sudath Hapuarachchige,† Gilbert Monta~no,‡ Chinnasamy Ramesh,† Delany Rodriguez,‡ Lauren H. Henson,‡
Casey C. Williams,‡ Samuel Kadavakkollu,† Dennis L. Johnson,† Charles B. Shuster,‡ and
Jeffrey B. Arterburn*,†
†Department of Chemistry and Biochemistry and ‡Department of Biology, New Mexico State University, MSC 3C, Las Cruces,
New Mexico 88003, United States
S Supporting Information
b
ABSTRACT: A new class of fluorescent triazaborolopyridi-
nium compounds was synthesized from hydrazones of 2-
hydrazinylpyridine (HPY) and evaluated as potential dyes for
live-cell imaging applications. The HPY dyes are small, their
absorption/emission properties are tunable through variation of
pyridyl or hydrazone substituents, and they offer favorable
photophysical characteristics featuring large Stokes shifts and
general insensitivity to solvent or pH. The stability, neutral
charge, cell membrane permeability, and favorable relative
influences on the water solubility of HPY conjugates are
complementary to existing fluorescent dyes and offer advantages for the development of receptor-targeted small-molecule probes.
This potential was assessed through the development of a new class of cysteine-derived HPY-conjugate imaging agents for the
kinesin spindle protein (KSP) that is expressed in the cytoplasm during mitosis and is a promising chemotherapeutic target.
Conjugates possessing the neutral HPY or charged Alexa Fluor dyes that function as potent, selective allosteric inhibitors of the KSP
motor were compared using biochemical and cell-based phenotypic assays and live-cell imaging. These results demonstrate the
effectiveness of the HPY dye moiety as a component of probes for an intracellular protein target and highlight the importance of dye
structure in determining the pathway of cell entry and the overall performance of small-molecule conjugates as imaging agents.
’ INTRODUCTION
mechanisms. The large size, polycyclic aromatic structures, and
presence of charged functional groups in many fluorescent dyes
present major challenges when applied to the development of
imaging agents based on small molecules, where the physicochem-
ical properties of the dye may dramatically alter the solubility,
biodistribution, and binding properties of the conjugate.4 The
ideal reporter dye should have characteristics that include ease and
versatility of methods for attachment to small-molecule targeting
agents, efficient cellular uptake, and lack of inherent biological
activity or toxicity. The propensity of existing dyes to localize in
specific sites or organelles is an additional factor that must be
recognized and considered when designing dye-conjugates. There-
fore, a significant need exists for the development of new small,
neutral biocompatible fluorescent cores that exhibit good aqueous
solubility, membrane permeability, favorable photophysical prop-
erties, and versatile coupling chemistries that yield minimal
perturbation of targeting properties. The spectroscopic character-
istics must be compatible with the instrumentation used for
detection, and the fluorescent output of the probe must be
Fluorescent compounds serve as versatile tools for molecular
and cellular imaging, flow cytometry, and a wide variety of
applications in biology and biotechnology.1 The development
of modular approaches, whereby different reporter groups can be
conjugated to the targeting agent, is well-suited for specific
applications and benefits from the availability of structurally
diverse fluorescent dyes with different spectroscopic properties.
Biomolecules such as DNA, proteins, and antibodies are routinely
labeled with fluorescent dyes that match the required spectro-
scopic properties of the application, frequently involving deriva-
tives such as fluorescein, rhodamine, and boron dipyrromethene
(BODIPY) (Figure 1).2 Advances in chemical biology and
molecular library screening approaches provide exceptional
opportunities for the rapid identification of novel small-molecule
ligands with high affinity and selectivity for biological targets of
interest and the discovery of new fluorescent scaffolds.3 The
structure and physicochemical properties of the dye-conjugate
are key considerations for preservation of the targeting character-
istics of the small-molecule ligand conjugates. The development of
agents for intracellular targets is faced with additional challenges,
since access to the corresponding intracellular compartments must
be achieved by simple permeation, active transport, or endocytotic
Received: January 18, 2011
Published: April 07, 2011
r
2011 American Chemical Society
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Figure 1. Structures of representative fluorescent dyes.
Figure 2. Structures of 5-substituted HPY dyes 9, 10, 12, and 14.
’ RESULTS AND DISCUSSION
Scheme 1. Synthesis of Triazaborolopyridinium HPY Dyes
Synthesis of the Fluorescent Triazaborolopyridinium Core.
Hydrazin-2-ylpyridine reacts rapidly and efficiently with alde-
hydes and ketones to form the corresponding hydrazones. Our
initial studies included the representative hydrazones 1, 3, 5,
and 7 derived from 4-(thiophen-2-yl)benzaldehyde (Scheme 1).
These compounds exhibited strong absorption maxima in the
range of 354ꢀ360 nm with molar absorptivity constants ε ≈
38000 Mꢀ1 cmꢀ1 (Supporting Information). However, these
hydrazones exhibited weak fluorescence emission spectra λemi
when excited at the λmax values with very low quantum yields
(Φf = 6 ꢁ 10ꢀ3). The emission was associated with relatively large
Stokes shifts (∼60 nm), but the low intensity precludes their use
for most biological imaging applications. The simple benzalde-
hyde-derived hydrazone 9 (Figure 2) exhibits shifts in the
absorption and emission spectra to shorter wavelength (λabs/λemi
328/397 nm) and reduced quantum yield (Φf = 2 ꢁ 10ꢀ3),
consistent with expectations for a small blue shift due to loss of the
extended conjugation of the 2-thiophenyl substituent.
We next considered the possible formation of cyclic systems
incorporating the distal nitrogen atoms into a rigid scaffold with
enforced planarity of the extended π system, anticipating that this
would enhance the resulting fluorescence emission properties.
Considering the basicity of the pyridin-2-yl-hydrazone moiety, we
investigated the formation of Lewis acid complexes with boron
trifluoride diethyl etherate. The reaction conditions for cyclization
to produce the triazaborolopyridinium dyes from the hydrazone
sufficienttopermitthe detection of the targetatnatural abundance
levels.
We have previously used metal-mediated coupling strategies
to incorporate chelates derived from the 2-hydrazinylpyridine
core into estrogen derivatives for the development of 99mTc-
imaging agents and became interested in the possibility of
developing fluorescent dyes based on this heterocyclic scaffold.5
Hydrazines are versatile reagents in organic and aqueous media,
with rapid kinetics and favorable thermodynamics of hydrazone
formation that are advantageous for bioorthogonal coupling
strategies.6 SoluLink offers proprietary technologies for biocon-
jugation using hydrazone formation with hydrazinylnicotinamide
groups to connect proteins, DNA, antibodies, and solid surfaces.7
The UV-traceable bis-aryl hydrazone chromophore provides a
basis for quantitative determination of protein labeling using
absorbance spectroscopy. Considering the versatile coupling
chemistries associated with 2-hydrazinylpyridine and the promis-
ing photophysical properties associated with the extended
π systems that result from hydrazone formation, we explored
strategies to construct fluorescent derivatives based on this
scaffold. Herein, we report the synthesis of a new class of hydra-
zinylpyridine-derived hydrazones (HPY) that incorporate a rigid
triazaborolopyridinium core structure. The photophysical prop-
erties and initial assessment of cellular permeability suggest the
suitability of HPY dyes for use as imaging probes. This potential
was used to develop a new class of cysteine-derived HPY-
conjugate imaging agents that function as potent, selective
inhibitors of the promising chemotherapeutic target kinesin
spindle protein (KSP). Comparison of fluorescent HPY and
charged Alexa Fluor conjugate probes in biochemical and cell-
based phenotypic assays and live-cell imaging demonstrates the
importance of dye structure in determining the pathway of cell
entry and the overall performance of targeted imaging agents.
precursors used BF3 OEt2 and 1,8-diazabicyclo[5.4.0]undec-7-ene
3
(DBU) as shown in Scheme 1 and provided yields ranging from 78
to 90%. The six-membered-ring system of the well-known 4,4-
difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) dyes are typically
prepared from dipyrromethene intermediates under similar con-
ditions,2 and structural elaboration of this scaffold has yielded new
analogues with modified photophysical properties.8
The photophysical properties of a series of HPY dyes are
summarized in Table 1. The HPY compounds derived from
4-(thiophen-2-yl)benzaldehyde exhibit absorption spectra with
λmax 472ꢀ490 nm and molar absorptivities ε = 26 600ꢀ32 900
M
ꢀ1 cmꢀ1 in CH3OH and emission spectra λemi 551ꢀ563 nm
with characteristically large Stokes shifts (72ꢀ80 nm). These
compounds exhibited high quantum yields with Φf = 0.69ꢀ0.95.
Large Stokes shifts facilitate signal detection, improve resolution
by separating absorption and emission spectra, and are advanta-
geous for imaging applications in which self-quenching must be
avoided.8d Substituents on the 5-position of the pyridine ring
were varied to identify related effects on the spectroscopic
properties, since we anticipated that we would eventually use
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Table 1. Photophysical Properties of HPY Dyesa
Scheme 2. Synthesis of HPY Dyes 16 and 18
dyea λmax, nm ε, Mꢀ1 cmꢀ1 λemi,b nm
Φf
solvent
2
472
491
482
490
471
491
481
403
404
408
403
406
400
403
404
406
407
410
400
28 800
26 800
32 900
26 600
22 600
33 800
36 700
8 500
551
563
562
562
539
572
572
493
491
497
504
500
496
492
492
497
504
501
476
0.75 MeOH
0.69 MeOH
0.95 MeOH
0.71 MeOH
0.31 MeOH
0.37 MeOH
0.38 MeOH
0.21 MeOH
0.17 1% DMSO/PBSc
0.22 CH2Cl2
0.17 acetone
0.07 toluene
0.20 MeOH
0.21 MeOH
0.16 1% DMSO/PBSc
0.20 CH2Cl2
0.23 acetone
0.51 toluene
0.21 MeOH
4
6
8
10
12
14
16
16
16
16
16
18
21
21
21
21
21
22
Scheme 3. Synthesis of HPY Dyes 21 and 22
8 600
9 300
8 600
9 800
8 300
12 000
12 300
11 200
10 800
10 600
9 400
and preferentially interact with lipophilic components. While the
relatively narrow emission bandwidth of the BODIPY dyes
provides an intense signal, the small associated Stokes shift
may require that they be excited or detected at suboptimal
wavelengths, which limits the capacity to fully utilize their intense
absorption/emission properties. In contrast, the HPY dyes
exhibit much larger Stokes shifts and increased resolution of
absorption/emission spectra (see Figure 2.3.12 in the Supporting
Information). The FITC and Alexa Fluor dyes present larger
relative steric profiles and consist of a mixture of two regio-
isomers, although single-isomer products are now commercially
available. The Alexa Fluor dyes are charged at physiological pH,
due to the multiple anionic sulfate and carboxylate groups. The
ionic charge increases water solubility but reduces membrane
permeability.
We were interested in optimizing the core structure of the
HPY dyes for potential biological applications, intending to
further reduce the steric profile and molecular weight while also
increasing water solubility without having to introduce charged
groups that would compromise membrane permeability. We
elected to eliminate the hydrophobic aryl hydrazone group while
retaining the 5-halogen substituent as a potential site for con-
necting linkages to biological targeting entities. The 2-hydrazi-
nylpyridines react rapidly with acetone to form the corre-
sponding hydrazone derivatives 15 and 17, which underwent
cyclization to form the 5-bromo- and 5-iodo-substituted triaza-
borolopyridinium compounds 16 and 18 under similar condi-
tions (Scheme 2). To provide an additional parameter for
structural modification, we investigated the possibility of replac-
ing the fluoride substituents attached to boron with alkoxide
ligands and found that this transformation occurred efficiently
using mildly basic conditions in methanol to produce the stable
bis-methoxide compounds 21 and 22, respectively (Scheme 3).
A related example of this type of substitution has been reported
for the six-membered BODIPY dyes, although more forcing
conditions involving a stepwise procedure for fluoride activation
with AlCl3 followed by alcohol substitution were required.10 The
acetone-derived HPY compounds 16, 18, 21, and 22 were
soluble in a variety of solvents, and comparison of the photo-
physical properties of derivatives 16 and 21 demonstrated only
a 20 μM samples were prepared. b Sample was excited at λmax. c pH 7.4.
this position for attaching targeting agents. The 5-brominated
compound 6 exhibited the highest absorptivity and quantum
yield in comparison with the parent scaffold 2 and the chlorinated
and iodinated halogen congeners 4 and 8, respectively (Scheme 1).
The 1,2,3-triazole and ethyne derivatives 12 and 14 were selected
to represent possible functional group substitutions on the
pyridine backbone that could be elaborated to provide connect-
ing linkages in small-molecule probes and used for bioorthogonal
labeling strategies (Figure 2).9 While these carbon- and nitrogen-
linked derivatives produced minimal perturbation of the absorp-
tion and emission maxima, the resulting quantum yields were
reduced nearly 2-fold relative to the parent compound 2. Com-
parison of 6 with the simple benzaldehyde hydrazone derivative
10 reveals that the conjugated 2-thiophene group confers both
longer wavelength absorption and increased quantum yield.
Computational analysis of the structural effects exhibited by this
series may help to identify the basis for the observed electronic
trends and would provide valuable insight for further optimiza-
tion of photophysical properties.
A comparison of the structure and photophysical properties of
the HPY dyes with the commercially available amine-reactive
fluorescent dyes fluorescein-isothiocyanate (FITC), BODIPY
493/503, and Alexa Fluor 488 provides a basis for considering the
structural effects associated with small-molecule conjugates and
their utility in live-cell imaging applications (Figure 1). These
commercial dyes exhibit strong absorption and emission proper-
ties and are available with reactive isothiocyanate or N-hydro-
xysuccinimidyl ester (NHS) groups to facilitate their conjugation
to amines. The BODIPY and HPY dyes each incorporate a
heterocyclic boron-containing ring system; however, they differ
in their respective six- or five-membered-ring sizes and the
inclusion of additional polar nitrogen atoms in the HPY dyes.
Aza-dipyrromethene analogues incorporating a nitrogen in the
backbone have been described recently.8e,f The BODIPY and
HPY dyes are electronically neutral, and BODIPY dyes have
demonstrated their ability to effectively cross cell membranes
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Scheme 4. Synthesis of Oxazolidine-2,5-dione 20
Figure 3. Membrane permeability of 16 and 21 in living HeLa cells. HeLa
cells were incubated in 20 μM 16 (AꢀC) or 21 (DꢀF) for 1 h, washed free
of the dye, and imaged live. Additionally, 100 μg/mL Alexa Fluor 546-
labeled bovine serum albumin (BSA) was included as a marker for fluid
phase endocytosis (B and E). The bar denotes 20 μm.
minor solvent-dependent variations in their absorption and
emission properties in methanol, dichloromethane, acetone,
toluene, and aqueous 1% DMSO in PBS buffer solution at the
physiological pH 7.4 (Table 1). The acetone-derived HPY dyes
required higher energy excitation (λmax ∼405 nm) in compar-
ison with the aryl hydrazone derivatives, attributed to the
truncated π system in these simple aliphatic derivatives. The
smaller, acetone-derived hydrazone group was expected to be
advantageous for increasing the relative water solubility of HPY-
conjugates designed for biological applications. The absorbance/
emission properties and chemical stability of 16 were unaffected
over a wide range of pH (4ꢀ9) in aqueous buffer solution, such
that no decomposition in solution was evident after several days
at ambient temperature.
Considering the favorable characteristics of 16 and 21, we
selected these dyes to evaluate their respective cell membrane
permeability and imaging properties in HeLa cells (Figure 3). To
visualize the staining by conventional epifluorescence micro-
scopy, a custom filter set was assembled that closely matched the
optimal excitation/emission spectra (395/500 nm) for the two
dyes in physiological buffers. Examination of cells incubated for
60 min in the presence of 20 μM 16 and 21 revealed that both
dyes could be visualized throughout the cytoplasmic compart-
ment and were particularly enriched in intracellular membranes
(Figure 3A,D). To distinguish between membrane permeability
and uptake by endocytosis, 100 μg/mL Alexa Fluor546 con-
jugated bovine serum albumin (BSA) was included to mark
membrane compartments involved in endocytic trafficking
(Figure 3B,E). As shown in the merged images (Figure 3C,F),
the HPY dyes were distributed throughout membranous com-
partments that were distinct from those endosomal vesicles that
contained the fluid-phase marker. The dyes 16 and 21 did not
exhibit any obvious localization in specific organelles or other
sites and could be removed by washing free with buffered saline.
Cells treated for up to 6 h with dye concentrations of 100 μM
exhibited no toxicity or other obvious effects on cell viability or
phenotypic changes. Coumarin-based dyes also undergo uptake
into cells without differential localization; however, sequestration
in internal membranes is a common feature of many dyes such as
coumarin 6 and can be washed away.4 In contrast, rhodamine
dyes 123 and B are taken up into the mitochondria, cyanine dyes
often localize in the nucleus, and BODIPY dyes typically localize
in the endoplasmic reticulum. These imaging results from live
cells demonstrate that the triazaborolopyridinium dyes were
capable of entering mammalian cells in a pathway independent
of endocytosis, suggesting that these dyes were membrane-
permeable and were suitable candidates for elaboration as probes
for intracellular targets.
Development of Inhibitory KSP Imaging Probes. S-Trityl-
L-cysteine is an allosteric small-molecule inhibitor of the class
5 kinesin motor kinesin spindle protein (KSP).11 The tetrameric
microtubule motor KSP plays a vital role in separating the spindle
poles during mitosis, and when KSP is antagonized with small-
molecule inhibitors, cells form a characteristic monopolar spin-
dle, resulting in mitotic arrest and eventual cell death by
apoptosis.12 The development of small-molecule inhibitors as
probes has played an invaluable role in the study of the
cytoskeleton.13 Fluorescent ATP-substrate analogues have been
used to investigate kinesin motor function,14 but no examples of
fluorescent inhibitors have been reported. Because KSP func-
tions only in the cytoplasm during mitosis, an inhibitory dye
conjugate would not only serve as a potential imaging probe but
would also provide an independent measure of the membrane
permeability and performance of the synthetic triazaborolopyr-
idinium probes.
Structureꢀactivity analyses have identified the triphenyl-
methyl-mercapatoethanamine moiety as the primary pharmaca-
phore in this class of KSP inhibitor, and the 40-methoxy derivative
19 was found to exhibit increased potency in biochemical and
cellular assays.11 Consideration of possible sites for the attach-
ment of a dye to 19 requires that the resulting fluorescent
conjugates retain the high-affinity allosteric binding interaction.
Docking studies have suggested that the carboxylic acid group
projects outside of the hydrophobic binding pocket and is not
required for binding, and amide derivatives were shown to retain
inhibitory activity; therefore, we selected this position for
derivatization.11b The R-amino acid functionality of cysteine
derivative 19 can be easily converted to the amine-reactive
oxazolidine-2,5-dione group 20, which provides a viable syn-
thetic route for developing carboxamide-linked selective fluor-
escent inhibitors of KSP (Scheme 4).
The iodide substituted difluoro- and bis-methoxy-HPY dyes
18 and 22 were converted to the amine-functionalized alkyne
products 23 and 24 in excellent yields (g95%) by Sonogashira
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coupling of tert-butylbut-3-ynylcarbamate. Deprotection of the
tBoc group using TFA/CH2Cl2 followed by neutralization of the
acidic reaction mixture with triethylamine provided the corre-
sponding primary amines, which were immediately coupled with
oxazolidine-2,5-dione 20 to give the desired conjugates 25 and
26 (Scheme 5). No significant changes in the spectroscopic
properties of conjugates 25 and 26 were evident in comparison to
the representative HPY dyes 16 and 21 measured in 1% DMSO/
PBS at pH 7.4 (Figure 4). These results demonstrate that the
attachment of the triphenylmethyl substituent does not affect the
photophysical properties or induce self-quenching and suggest
that the distance of separation and absence of extended conjuga-
tion in the aliphatic carboxamide linkage allow the HPY dyes to
function effectively as a fluorescent reporter group in conjugates
25 and 26.
In order to compare the performance characteristics of the
KSP-targeted HPY-conjugates with a representative analogue
containing a commercially available fluorescent dye, we synthe-
sized the Alexa Fluor 488 analogue 28 (Scheme 6). The amine-
reactive oxazolidine-2,5-dione 20 was treated with excess 2,20-
oxybis(ethylamine) to give the monocarboxamide 27 in moder-
ate yield. The activated N-hydroxysuccinimide ester of the Alexa
Fluor 488 dye was coupled with the free amine of 27 to yield the
desired conjugate 28. This product was purified by reverse-phase
chromatography followed by ion-exchange chromatography to
provide the Naþ salt form. The observed regioselective
Scheme 6. Synthesis of Alexa Fluor 488-KSP Inhibitor
Conjugate 28
Scheme 5. Synthesis of HPY-KSP Inhibitor Conjugates 25
and 26
Figure 4. Absorption and emission spectra of (a) 25 (λmax 400 nm, ε = 9 900 Mꢀ1 cmꢀ1, λemi 489 nm, Φf = 0.31) and (b) 26 (λmax 400 nm,
ε = 12 900 Mꢀ1 cmꢀ1, λemi = 486 nm, Φf = 0.29). Samples (20 μM) were prepared in 1% DMSO/PBS at pH 7.4 and 25 °C and excited at 405 nm.
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Table 2. IC50 Values for Inhibition of Microtubule-
Stimulated KSP ATPase Activity and EC50 Values for Mitotic
Arrest in Cell-Based Assays for Compounds 19 and 25ꢀ29a
compd
IC50
EC50
19
25
26
27
28
29
0.27
0.85
0.60
0.75
0.73
7.8
0.23
1.13
1.03
0.58
5.40
49.00
a Inhibitory concentrations are expressed in μM.
Figure 6. Imaging of fluorescent cysteine conjugates in living cells.
HeLa cells were treated with fluorescent conjugates of 10 μM 25, 26, and
28, coincubated with fluid-phase endocytosis marker Alexa Fluor 546-
conjugated BSA for 4 h, and then imaged using wide-field epifluores-
cence. Fluorescence was confined to punctate structures colocalizing
with the endocytosis marker (BSA) in cells treated with 28, whereas cells
treated with conjugates 25 and 26 displayed diffuse fluorescence
throughout the cytoplasm. The bar denotes 20 μm.
To determine the performance of the dye-conjugated KSP
inhibitors in living cells, HeLa cells were incubated in the absence
or presence of 10 μM compound (19, 25ꢀ28) for 4 h and then
fixed and probed for spindle organization using antitubulin
antibodies. Whereas carrier control (DMSO)-treated cells ex-
hibited normal bipolar spindle morphology (Figure 5), cells
treated with 19, 25, 26, and 28 all exhibited dose-dependent
formation of monopolar spindles consistent with compromised
KSP activity. Comparison of the half-maximal effective concen-
trations (EC50) for each conjugate revealed that while the HPY
conjugates exhibited a 5-fold reduction in activity relative to the
inhibitor 19 (Table 2), the Alexa Fluor conjugate 28 displayed a
23-fold decrease in inhibitory activity in the cell-based assay.
The observed differences in inhibitory properties of the probes
in the cell-based assays can be attributed to structural differences
in the attached dyes. The Alexa Fluor 488 dye is significantly
larger than the HPY structure; however, the in vitro results
demonstrated that there were no significant differences asso-
ciated with this increased steric requirement (Table 2). The ionic
charge associated with the sulfate and carboxylate groups of the
Alexa Fluor 488 dye would, however, be expected to decrease the
permeability across the membrane bilayer and would result in
increased apparent EC50 values.
In order to further investigate the differences in KSP inhibition
observed between the two classes of dye conjugates in the cell-
based assay, the fluorescence imaging capabilities of the dye
conjugates were employed to determine their subcellular distribu-
tion. HeLa cells were incubated with the dye conjugates 25, 26,
and 28 for 4 h and then washed free of unbound dye and imaged
live (Figure 6). Examination of both interphase (left panels) and
mitotic cells (right panels) revealed differences in the localization
patterns for the HPY and Alexa Fluor conjugates. The HPY-
conjugates displayed a cytoplasmic localization that mirrored that
of the free dye (Figure 3). The presence of fluoride or alkoxide
substituents on boron in 25 and 26 did not affect the imaging
patterns and suggests that both probes exhibit similar cellular
properties. However, the Alexa Fluor derivative 28 colocalized
with the fluid-phase endocytosis marker (BSA) in both interphase
and mitotic cells, as shown in Figure 6. The observation that 28
retained KSP inhibitory activity in the cytoplasm was inconsistent
Figure 5. Fluorescent cysteine conjugates retain KSP inhibitory activ-
ity. HeLa cells were incubated in the absence or presence of 10 μM 19 or
conjugates 25, 26, and 28 for 4 h and then fixed and probed for tubulin
(red) and DNA (blue) localization. The bar denotes 10 μm.
carboxamide formation involving the sterically accessible terminal
amine group was consistent with expectations for increased reactiv-
ity relative to the R-amine in 27. Acetylation of the R-amine group of
19, which is part of the pharmacophore, inactivates KSP inhibition.11
Our attempts to synthesize the analogous BODIPY-493/503 con-
jugates following a parallel synthetic route were unsuccessful, due to
major difficulties encountered in purification of the final products.
These derivatives were very hydrophobic, amphiphilic compounds
and tended to form intractable aggregates that were incompatible
with normal or reverse-phase chromatography purification meth-
ods, and this effort was abandoned.
Characterization of KSP Inhibition. The synthetic dye con-
jugates 25, 26, and 28 were evaluated for their ability to inhibit
KSP using both an in vitro biochemical assay for microtubule-
stimulated ATPase activity and a cell-based assay for bipolar
spindle assembly in HeLa cells (Table 2). Results of the biochem-
ical assay using purified human KSP motor domain revealed that
the original KSP inhibitor 19 exhibited a half-maximal inhibitory
concentration (IC50) of 0.27 μM, while the fluorescent HPY dye
conjugates 25 and 26 and Alexa Fluor derivative 28 had similar
IC50 values of 0.85, 0.60, and 0.73μM, respectively. The IC50 value
of the monocarboxamide derivative 27 was nearly identical with
that of 28, demonstrating that the 2,20-oxybis(ethylamine) linkage
itself had no distinct effect on KSP inhibition. These results show
that each of the conjugates elicit similar inhibitory properties
against the purified KSP motor, irrespective of the length of the
carboxamide linkages used or the differences in size and ionic
charge of the dye structures.
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Scheme 7. Acid Stability Study of Alexa Fluor 488-KSP
Inhibitor Conjugate 28
that fluorescence was observed from the cytoplasm in all cases
and excluded from the nucleus in ER-positive MCF7 cell lines.
Attempts to identify possible cleavage products were unsuccess-
ful but were not ruled out, due to the high probe concentrations
and long incubation times involved, combined with the difficul-
ties faced in quantifying subnanomolar concentrations of active
probes. The results obtained in our investigation with Alexa
Fluor conjugate 28 illustrate an important caveat for the inter-
pretation of fluorescence localization and activity data and
suggest that endocytotic uptake can serve as a route for delivering
intracellular probes possessing an acid-cleavable trityl linkage.
with the imaging data that suggested the Alexa Fluor dye remained
sequestered within endocytic compartments and raised the pos-
sible scenario that the Alexa Fluor conjugate 28 was not stable
under the acidic conditions found in the endosomal compartment.
Molecules taken up by endocytosis encounter an increasingly
acidic environment as they progress through the endosomal and
lysosomal compartments. The stability of Alexa Fluor conjugate
28 under conditions that resemble the acidic luminal pH of the
midlate endosome (pH 4.5) was evaluated by HPLC-MS
(Supporting Information, Figure 5.1). The S-trityl and carbox-
amide linkages in 28 are potential hydrolytic cleavage sites.
Compound 28 underwent a time-dependent acid-catalyzed
hydrolysis of the 40-methoxytrityl group to produce the alcohol
29, which was characterized by HPLC retention time and
identified by mass spectrometry in comparison with authentic
material (Scheme 7). Compound 29 was evaluated individually
in the biochemical and cell-based assays, revealing that this
simple trityl alcohol moiety itself exhibited KSP inhibitory
properties, although it was markedly less potent than any of
the corresponding thioethanamine analogues. The poor water
solubility of 29 contributes to the observation of higher EC50
values in the cell-based assay; however, the efficiency of endo-
cytic uptake and delivery of the charged, acid-sensitive hydro-
lyzable probe 28 is evident from the higher relative potency.
These results, in combination with the observed subcellular
localization of the Alexa Fluor fluorescence that was retained in
the endocytic compartments, suggest the observed apparent
KSP-inhibitory activity of conjugate 28 includes a contribution
from the hydrolytic release of 29 in the acidic endosomes, which
then diffuses into the cytoplasm where it inhibits KSP. In
comparison, the HPY conjugates 25 and 26 are able to enter
the cells via simple permeation and exhibit potent inhibitory
properties in both in vitro and cell-based assays.
Consideration of dye structure is important for the design and
performance of probes.4,15 The fluorescein group is often pre-
sumed to be innocuous and permeable; however, associated
effects on both transport pathway and subcellular localization
have been observed in conjugates of cell-penetrating peptides.16
A BODIPY-androgen conjugate derived from dehydroepian-
drosterone mimicked the steroid in functional assays and was
effective for live-cell imaging but exhibited weak uniform staining
of the cytoplasm that was not displaced by competition with
excess steroids and was metabolized differently.17 A recent
investigation of a series of hydrophobic tamoxifen conjugates
containing Alexa Fluor, carboxyfluorescein, and BODIPY dyes
revealed differences in binding affinities to estrogen receptors R
and β, such that the large Alexa Fluor conjugate exhibited the
weakest binding, but all three antagonized ER-mediated tran-
scription in the nucleus with similar inhibitory IC50 values.18 The
Alexa Fluor conjugate exhibited slower uptake than the neutral
derivatives, which is consistent with reduced permeability at
short time intervals due to the charged dye. However, expecta-
tions for nuclear localization of the dyes were not realized, such
’ CONCLUSIONS
The triazaborolopyridinium HPY core represents a new class
of fluorescent dye that is readily synthesized from hydrazone
precursors and provides complementary structural and physico-
chemical properties that are well suited for use in the construc-
tion of small-molecule probes. These neutral compounds are
structurally related to the widely used BODIPY dyes and share
favorable characteristics such as membrane permeability, and
although they are not as bright, the HPY dyes exhibit larger
Stokes shifts for better resolved absorption and emission spectra.
The small overall size and presence of three nitrogen atoms in the
conjugated heterocyclic backbone of the HPY dyes are structural
factors that contribute toward favorable water solubility. The
HPY dyes demonstrated tunable absorption/emission properties
with variation of the hydrazone substituent, but the individual
photophysical properties were relatively insensitive to varying
solvents from nonpolar organic to aqueous media. The size and
polarity of the hydrazone group can be easily varied, and the
ability to exchange fluoride and alkoxide substituents on boron
provides additional routes for structural optimization of HPY
dyes for specific applications. The versatile chemistry associated
with the HPY dye core structure should facilitate the construc-
tion of new fluorescent probe conjugates using a variety of
synthetic strategies such as modification of the hydrazone, direct
substitutions on the pyridine scaffold, or incorporation of
linkages with appropriately matched reactive functional groups.
In this study, we observed improved performance of the HPY-
conjugated inhibitors in comparison with Alexa Fluor conjugates.
Because KSP inhibitors can be used to synchronize cell cultures
at G2/M, conjugates such as 25 and 26 may have broad utility as
reversible probes in cell cycle research. Synthetic and computa-
tional efforts designed to further optimize the photophysical
properties of the HPY scaffold, expanded comparison of addi-
tional synthetic dye conjugates, and exploration of other targeted
biological imaging applications are currently in progress.
’ EXPERIMENTAL PROCEDURES
All compounds were synthesized in an efficient fume hood. Commer-
cially available solvents and reagents were purchased and used without
further purification. Alexa Fluor488-NHS was purchased from Invitro-
gen. Preparative chromatography was performed by medium-pressure
column chromatography using AnaLogix SuperFlash or Sorbent Tech-
nologies RediSep C18 prepacked columns. 1H NMR spectra were
acquired at 300 or 400 MHz, and 13C NMR spectra were acquired at
75 or 100 MHz at ambient temperature (20 ( 2 °C). 1H NMR spectra in
CDCl3 were referred to TMS, and C6F6 was used as an internal reference
in 19F NMR spectra. The purities and molecular weights of compounds
were determined by a Waters 2695 Alliance HPLC/ESI-MS instrument
with a C18 reverse-phase column system and 20ꢀ70% acetonitrile in
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water as eluent. High-resolution mass spectra were obtained at the
University of California at Riverside.
General Procedure A for Triazaborolopyridinium Cycliza-
was obtained as a red solid. 1H NMR (300 MHz, CDCl3): δ 8.42
(m, 1H), 8.39 (m, 1H), 7.77 (m, 2H), 7.74 (m, 1H), 7.56 (ddd, J = 9.12,
1.77, 1.66 Hz, 1H), 7.50ꢀ7.47 (m, 2H), 7.39 (d, J= 4.98 Hz, 1H), 7.14 (ddd,
J = 6.77, 3.74, 1.17 Hz, 1H), 6.68 (d, J = 9.39 Hz, 1H). 13C NMR
(75 MHz, CDCl3): δ 148.2, 143.0, 141.1, 140.5, 138.1, 133.6, 133.5,
128.8, 128.5, 126.8, 126.0, 125.8, 124.9, 115.0. 19F NMR (CDCl3):
δ ꢀ149.4 (q, J = 28 Hz, 2F). FT-IR (KBr): 1634 (s), 1606 (m), 1493
(s), 1403 (m), 1147 (m) cmꢀ1. HPLC/ESI-MS (m/z): calcd for C16H12-
BF2IN3S [M þ H]þ 453.99, found 454.03.
tion. Boron trifluoride diethyl etherate (BF3 OEt2, 5.0 equiv) was
3
added dropwise to a mixture of the hydrazone (1.0 equiv) and DBU
(3.0 equiv) in anhydrous toluene (0.02 M) at room temperature and
heated at 90 °C for 1ꢀ6 h under an argon atmosphere. The reaction
mixture was quenched with water (5.0 mL), and the product was
extracted with CH2Cl2 (2 ꢁ 50 mL). The combined organic layers
were washed with water (3 ꢁ 50 mL), dried over anhydrous Na2SO4,
and evaporated under reduced pressure. The solid residue was purified
by silica gel column chromatography using CH3OH/CH2Cl2 (0ꢀ1%)
as the eluent.
2-Benzylidene-6-bromo-3,3-difluoro-2,3-dihydro[1,2,4,3]-
triazaborolo[4,5-a]pyridin-2-ium-3-uide (10). The reaction was
performed following the general procedure A for 4 h starting with 9
(0.138 g, 0.5 mmol). The product 10 (0.133 g, 82%) was obtained as
an orange solid. 1H NMR (400 MHz, DMSO-d6): δ 8.57 (d, J =
7.28 Hz, 2H), 8.22 (s, 1H), 8.05 (s, 1H), 7.77 (dd, J = 9.14, 1.78 Hz, 1H),
7.62ꢀ7.57 (m, 3H), 6.94 (d, J = 9.48 Hz, 1H). 13C NMR (100 MHz,
DMSO-d6): δ 160.1, 144.6, 141.9, 136.5, 133.0, 132.8, 130.0, 128.9,
114.4, 102.8. 19F NMR (DMSO-d6): δ ꢀ145.62 (q, J = 28 Hz, 2F).
General Procedure B for Methoxide Substitution. The
fluorinated HPY dye (1.0 equiv) was dissolved in methanol (2.0 M)
and treated with K2CO3 (2.2 equiv). The mixture was stirred for 2 h and
diluted with CH2Cl2 (100 mL). The organic layer was washed with
water (3 ꢁ 50 mL) and dried over anhydrous Na2SO4. Volatiles were
removed under reduced pressure, and the residue was dried in vacuo.
The solid residue was purified by reverse-phase C18 column chroma-
tography using CH3CN/H2O (20ꢀ50%) as the eluent.
FT-IR (KBr): 1643 (m), 1608 (w), 1489 (s), 1405 (m), 1143 (m) cmꢀ1
.
HPLC/ESI-MS (m/z): calcd for C12H10BBrF2N3 [M þ H]þ 324.01,
found 324.07.
3,3-Difluoro-6-(4-phenyl-1H-1,2,3-triazol-1-yl)-2-(4-
(thiophen-2-yl)benzylidene)-2,3-dihydro[1,2,4,3]triazaborolo-
[4,5-a]pyridin-2-ium-3-uide (12). The reaction was performed
following the general procedure A for 3 h starting with 11 (0.018 g,
0.043 mmol). The product 12 (0.018 g, 87%) was obtained as a red
solid. 1H NMR (300 MHz, DMSO-d6): δ 9.20 (s, 1H), 8.63 (s, 1H),
8.60 (s, 1H), 8.58 (d, J = 1.89 Hz, 1H), 8.21 (dd, J = 9.69, 2.34 Hz,
1H), 8.14 (s, 1H), 7.93ꢀ7.88 (m, 4H), 7.77 (dd, J = 3.66, 1.02 Hz,
1H), 7.71 (dd, J = 5.06, 1.17 Hz, 1H), 7.53ꢀ7.48 (m, 2H), 7.41ꢀ
7.35 (m, 1H), 7.23ꢀ7.18 (m, 2H). 13C NMR (75 MHz, DMSO-d6):
δ 147.2, 142.3, 142.1, 137.8, 136.0, 134.1, 130.2, 129.2, 129.1, 128.9,
128.7, 128.5, 128.4, 128.2, 126.2, 125.5, 125.4, 123.5, 120.1, 114.1.
19F NMR (CDCl3): δ ꢀ148.4 (q, J = 28 Hz, 2F). FT-IR (KBr): 1655
(w), 1614 (m), 1519 (m), 1137 (w), 1025 (m) cmꢀ1. HPLC/ESI-MS
(m/z): calcd for C24H18BF2N6S [M þ H]þ 471.14, found 471.41.
6-Ethynyl-3,3-difluoro-2-(4-(thiophen-2-yl)benzylidene)-
2,3-dihydro[1,2,4,3]triazaborolo[4,5-a]pyridin-2-ium-3-uide (14).
The reaction was performed following the general procedure A for
1 h starting with 9 (0.112 g, 0.37 mmol). The product 14 (0.107 g, 83%)
was obtained as a red solid. 1H NMR (400 MHz, CDCl3): δ 8.35 (s. 1H),
7.98ꢀ7.96 (m, 2H), 7.75ꢀ7.73 (m, 2H), 7.64 (m, 1H), 7.47 (dd,
J = 3.72, 1.08 Hz, 1H), 7.40 (dd, J = 5.02, 1.07 Hz, 1H), 7.34 (dd, J =
9.38, 1.85 Hz, 1H), 7.14 (dd, J = 5.07, 3.62 Hz, 1H), 6.58 (d, J = 9.37 Hz,
1H), 3.05 (s, 1H). 13C NMR (75 MHz, CDCl3): δ 153.4, 143.2, 142.9,
140.0, 138.2, 137.0, 133.6, 128.5, 126.9, 125,8, 125.0, 117.2, 117.2, 113.1,
79.3, 78.5. 19F NMR (CDCl3): δ ꢀ150.01 (q, J = 28 Hz, 2F). FT-IR
(KBr): 2112 (m), 1644 (s), 1609 (w), 1509 (s), 1044 (m) cmꢀ1. HPLC/
ESI-MS (m/z): calcd for C18H13BF2N3S [M þ H]þ 352.09, found
352.12.
3,3-Difluoro-2-(4-(thiophen-2-yl)benzylidene)-2,3-dihydro-
[1,2,4,3]triazaborolo[4,5-a]pyridin-2-ium-3-uide (2). The reac-
tion was performed following the general procedure A for 5 h starting
with 1 (0.056 g, 0.2 mmol). The product 2 (0.051 g, 78%) was obtained
as a red solid. 1H NMR (400 MHz, DMSO-d6): δ 8.67 (s, 1H), 8.05 (d,
J = 8.48 Hz, 2H), 7.86 (d, J = 8.48 Hz, 2H), 7.74 (dd, J = 3.32, 1.06 Hz,
1H), 7.69 (dd, J = 5.02, 1.06 Hz, 2H), 7.52 (ddd, J = 8.25, 7.67, 1.68 Hz,
1H), 7.20 (dd, J = 5.02, 3.66 Hz, 1H), 6.70 (d, J = 8.88 Hz, 1H), 6.40
(dt, J = 6.49, 6.24, 0.86 Hz, 1H). 13C NMR (75 MHz, CDCl3):
δ 141.2, 138.9, 135.7, 133.2, 128.4, 127.2, 126.6, 126.0, 125.8, 125.3,
124.7, 123.5, 113.2, 110.2. 19F NMR (CDCl3): δ ꢀ150.33 (q, J = 28 Hz,
2F). FT-IR(KBr):1641(s), 1609(w), 1491(s), 1105(m), 1023(w) cmꢀ1
.
HPLC/ESI-MS (m/z): calcd for C16H13BF2N3S [M þ H]þ 328.09,
found 328.19.
6-Chloro-3,3-difluoro-2-(4-(thiophen-2-yl)benzylidene)-
2,3-dihydro[1,2,4,3]triazaborolo[4,5-a]pyridin-2-ium-3-uide (4).
The reaction was performed following the general procedure A for 3 h
starting with 3 (0.063 g, 0.2 mmol). The product 4 (0.057 g, 78%) was
obtained as a red solid. 1H NMR (300 MHz, CDCl3): δ 8.33 (s, 1H),
7.98ꢀ7.94 (m, 2H), 7.75ꢀ7.71 (m, 2H), 7.46 (dd, J = 3.74, 1.14 Hz,
1H), 7.45 (s, 1H), 7.39 (dd, J = 5.06, 1.02 Hz, 1H), 7.29 (d, J = 2.31 Hz,
1H), 7.13 (dd, J = 5.10, 4.23 Hz, 1H), 6.60 (d, J = 9.64 Hz, 1H).
13C NMR (100 MHz, DMSO-d6): δ 154.5, 144.2, 142.8, 140.6, 138.8,
134.2, 133.9, 128.6, 127.1, 126.1, 125.6, 124.2, 120.4, 108.6. 19F NMR
(CDCl3): δ ꢀ150.4 (q, J = 28 Hz, 2F). FT-IR (KBr): 1645 (w), 1597 (w),
1491 (s), 1133 (m), 1059 (m) cmꢀ1. HPLC/ESI-MS (m/z): calcd for
C16H12BClF2N3S [M þ H]þ 362.05, found 362.32.
6-Bromo-3,3-difluoro-2-(4-(thiophen-2-yl)benzylidene)-
2,3-dihydro[1,2,4,3]triazaborolo[4,5-a]pyridin-2-ium-3-uide (6).
The reaction was performed following the general procedure A for 3 h
starting with 5 (0.085 g, 0.24 mmol). The product 6 (0.090 g, 90%) was
6-Bromo-3,3-difluoro-2-(propan-2-ylidene)-2,3-dihydro-
[1,2,4,3]triazaborolo[4,5-a]pyridin-2-ium-3-uide (16). The re-
action was performed following the general procedure A for 6 h using 15
(0.146 g, 0.64 mmol). The product 16 (0.107 g, 60%) was obtained as a
greenish yellow solid. 1H NMR (300 MHz, DMSO-d6): δ 7.82 (d, J =
1.32 Hz, 1H), 7.46 (dd, J = 9.69, 2.20 Hz, 1H), 6.69 (dd, J = 9.53 Hz,
1H), 2.36 (s, 3H), 2.32 (s, 3H). 13C NMR (75 MHz, DMSO-d6):
δ 166.3, 157.8, 142.8, 135.3, 113.6, 99.7, 21.2, 21.0. 19F NMR (DMSO-
d6): δ ꢀ145.81 (q, J = 28 Hz, 2F). FT-IR (KBr): 1658 (w), 1625 (m),
1498 (s), 1154 (s), 1018 (m) cmꢀ1. HRMS/ESI-TOF (m/z): calcd for
C8H10BBrF2N3 [M þ H]þ 276.0119, found 276.0114.
1
obtained as a red solid. H NMR (300 MHz, CDCl3): δ 8.42ꢀ8.39
(m, 2H), 7.78ꢀ7.75 (m, 2H), 7.67 (s, 1H), 7.50ꢀ7.45 (m, 3H), 7.40
(d, J = 5.12 Hz, 1H), 7.14 (dd, J = 4.90, 3.81 Hz, 1H), 6.77 (d, J =
9.51 Hz, 1H). 13C NMR (75 MHz, CDCl3): δ 143.8, 143.0, 140.6, 138.1,
136.1, 133.5, 128.8, 128.5, 126.8, 126.0, 125.8, 124.9, 114.6, 102.7.
19F NMR (CDCl3): δ ꢀ149.4 (q, J = 28 Hz, 2F). FT-IR(KBr):1641(m),
1605 (w), 1526 (m), 1498 (s), 1048 (m) cmꢀ1. HPLC/ESI-MS (m/z):
calcd for C16H12BBrF2N3S [M þ H]þ 406.00, found 406.04.
6-Iodo-3,3-difluoro-2-(4-(thiophen-2-yl)benzylidene)-
2,3-dihydro[1,2,4,3]triazaborolo[4,5-a]pyridin-2-ium-3-uide (8).
The reaction was performed following the general procedure A for
3 h starting with 7 (0.061 g, 0.15 mmol). The product 8 (0.054 g, 80%)
3,3-Difluoro-6-iodo-2-(propan-2-ylidene)-2,3-dihydro-
[1,2,4,3]triazaborolo[4,5-a]pyridin-2-ium-3-uide (18). The re-
action was performed following the general procedure A for 4 h starting
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with 17 (0.235 g, 0.85 mmol). The product 18 (0.212 g, 77%) was
obtained as a greenish yellow solid. 1H NMR (300 MHz, CDCl3): δ 7.54
(b s, 1H), 7.35 (dd, J = 9.40, 2.01, 1H), 6.41 (d, J = 9.54, 1H), 2.41
(s, 3H), 2.37 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 163.4, 158.6,
147.0, 140.3, 114.0, 66.6, 21.6, 21.5. 19F NMR (CDCl3): δ ꢀ149.38
(q, J = 28 Hz, 2F). FT-IR (KBr): 1658 (w), 1619 (m), 1501 (s), 1152 (s),
1014 (m) cmꢀ1. HRMS/ESI-TOF (m/z): calcd for C8H10BF2IN3
[M þ H]þ 323.9975, found 323.9979.
CDCl3): δ 163.1, 158.7, 155.7, 142.3, 138.2, 111.8, 104.3, 87.3, 79.5, 77.8,
39.5, 28.4, 21.5, 21.4, 21.0. 19FNMR(CDCl3):δꢀ149.94 (q, J=28Hz, 2F).
FT-IR (neat): 3300 (b), 2171 (w), 1650 (m), 1510 (s), 1110 (m) cmꢀ1
.
HPLC/ESI-MS (m/z): calcd for C17H24BF2N4O2 [Mþ H]þ 365.20, found
365.30.
6-(4-(tert-Butoxycarbonylamino)but-1-ynyl)-3,3-dimethoxy-
2-(propan-2-ylidene)-2,3-dihydro[1,2,4,3]triazaborolo[4,5-a]-
pyridin-2-ium-3-uide (24). The mixture of 22 (0.069 g, 0.2 mmol),
Boc-but-3-yn-1-amine (0.068 g, 0.4 mmol), PdCl2(PPh3)2 (7.0 mg,
1.0 ꢁ 10ꢀ2 mmol), and CuI (1.9 mg, 1.0 ꢁ 10ꢀ2 mmol) in Et3N (4 mL)
was stirred for 2 h at ambient temperature under an argon atmosphere.
Volatiles were evaporated under reduced pressure. The solid residue was
dissolved in CH2Cl2 (10 mL) and this solution filtered through a plug
of Celite. The filtrate was concentrated, and the residue was purified by
silica gel column chromatography using CH3OH/CH2Cl2 (0ꢀ1.0%) to
isolate the product 24 (0.074 g, 95%) as a greenish yellow solid. 1H NMR
(300 MHz, CDCl3): δ 7.47 (d, J = 1.00 Hz, 1H), 7.21 (dd, J = 9.39,
1.91 Hz, 1H), 6.51 (dd, J = 9.39, 0.90 Hz, 1H), 4.85 (bs, 1H), 3.33 (dt, J =
6.30, 6.17 Hz, 2H), 3.06, (s, 6H), 2.57 (t, J = 6.60 Hz, 2H), 2.39 (s, 3H),
2.36 (s, 3H), 1.46 (s, 9H). 13C NMR (75 MHz, CDCl3): δ 160.3, 158.9,
155.7, 141.6, 139.0, 111.5, 103.8, 87.2, 79.5, 78.2, 49.9, 39.5, 28.4, 21.3,
21.0, 20.5. FT-IR (neat): 3259 (b), 2167 (w), 1630 (m), 1510 (s), 1141
(m) cmꢀ1. HPLC/ESI-MS (m/z): calcd for C19H30BN4O2 [M þ H]þ
389.24, found 389.16.
4-(((4-Methoxyphenyl)diphenylmethylthio)methyl)-
oxazolidine-2,5-dione (20). Triphosgene (0.932 g, 3.15 mmol) was
added to a mixture of 19 (0.589 g, 1.5 mmol) and cyclohexene (1.23 g,
15 mmol) in ethyl acetate (10 mL) at 0 °C. The reaction mixture was
heated at 85 °C for 3 h. The volatiles were removed in vacuo. The
residue was dissolved in CH2Cl2 (20 mL) and washed with water
(35 mL). The organic layer was dried over anhydrous Na2SO4 and
concentrated under reduced pressure to isolate the product 20 (0.525 g,
84%) as a colorless solid. This compound was used directly in sub-
sequent reactions or could be stored at 5 °C under anhydrous conditions
for up to 2 days. 1H NMR (300 MHz, CDCl3): δ 7.53ꢀ7.08 (m, 12H),
6.93ꢀ6.73 (m, 2H), 5.15 (s, 1H), 3.80 (s, 3H), 3.58ꢀ3.51 (dd, J = 8.51,
3.96 Hz, 1H), 2.87ꢀ2.68 (m, 2H); 13C NMR (75 MHz, CDCl3):
δ 167.85, 158.43, 151.17, 144.00, 135.65, 130.58, 129.22, 128.24, 127.15,
113.50, 67.28, 56.53, 55.26, 33.27. FT-IR (KBr, cmꢀ1): 3399 (s),
1860 (s), 1785 (m), 1601 (w), 1508 (w).
6-(4-(2-Amino-3-((4-methoxyphenyl)diphenylmethylthio)-
propanamido)but-1-ynyl)-3,3-difluoro-2-(propan-2-ylidene)-
2,3-dihydro[1,2,4,3]triazaborolo[4,5-a]pyridin-2-ium-3-uide (25).
Trifluoroacetic acid (TFA, 50 μL) was added dropwise to compound 23
(0.026 g, 0.07 mmol) in methylene chloride (1.0 mL) and stirred for 3 h
at ambient temperature under an argon atmosphere. The mixture was
diluted with CH2Cl2 (2 mL), cooled in an ice bath, and neutralized by
dropwise addition of Et3N (90 μL). The cyclic anhydride oxazolidine-
2,5-dione 20 (0.036 g, 0.089 mmol) in CH2Cl2 (2 mL) was added to the
neutralized reaction mixture dropwise over 15 min, and this mixture was
stirred for 3 h at 0 °C to room temperature under an argon atmosphere.
The reaction mixture was diluted with CH2Cl2 (100 mL). The organic
layer was washed with saturated NH4Cl (2 ꢁ 50 mL), saturated
NaHCO3 (2 ꢁ 50 mL), and H2O (3 ꢁ 50 mL) and dried over
anhydrous Na2SO4. Volatiles were evaporated under reduced pressure,
and the solid residue was purified by silica gel column chromatography
using CH3OH/CH2Cl2 (0ꢀ1%) to isolate the product 25 (0.039 g,
86%) as a greenish yellow solid. 1H NMR (300 MHz, CDCl3):
δ 7.45ꢀ7.28 (m, 10H), 7.23ꢀ7.14 (m, 4H), 6.81 (d, J = 9.10 Hz,
2H), 6.48 (d, J = 9.39 Hz, 1H), 3.78 (s, 3H), 3.76 (m, 1H), 3.43ꢀ3.28
(m, 2H), 3.06 (dd, J = 8.37, 3.96 Hz, 1H), 2.74 (dd, J = 12.60,
4.02 Hz, 1H), 2.62ꢀ2.50 (m, 3H), 2.42 (s, 3H), 2.37 (s, 3H), 1.60
(b s, 2H). 13C NMR (75 MHz, CDCl3): δ 172.9, 163.1, 158.7, 158.2,
144.9, 144.7, 142.2, 138.2, 136.6, 130.7, 129.5, 127.9, 126.7, 113.2,
111.8, 104.3, 87.2, 77.9, 66.5, 55.2, 54.0, 37.9, 37.3, 21.5, 20.4.
19F NMR (CDCl3): ꢀ149.88 (q, J = 28 Hz, 2F). FT-IR (neat): 3360 (b),
2120 (w), 1680 (m), 1510 (m), 1110 (m) cmꢀ1. HRMS/ESI-TOF
(m/z): calcd for C35H36BF2N5NaO2S [M þ Na]þ 662.2543, found
662.2537.
6-Bromo-3,3-dimethoxy-2-(propan-2-ylidene)-2,3-dihydro-
[1,2,4,3]triazaborolo[4,5-a]pyridin-2-ium-3-uide (21). The
reaction was performed following the general procedure B starting
with 16 (0.028 g, 0.1 mmol) and a reaction time of 3.5 h. The solid
residue was purified by reverse-phase column chromatography with
CH3CN/H2O (50/50) as the eluent to isolate the product 21 (0.023 g,
77%) as a greenish yellow solid. 1H NMR (300 MHz, CDCl3): δ 7.37
(dd, J = 2.06, 0.74 Hz, 1H), 7.25 (dd, J = 9.53, 1.76 Hz, 1H), 6.48 (dd,
J = 9.53, 0.73 Hz, 1H), 3.07 (s, 6H), 2.38 (s, 3H), 2.36 (s, 3H). 13C NMR
(75 MHz, CDCl3): δ 160.4, 159.0, 142.0, 135.9, 113.2, 100.1, 49.9,
21.3, 20.5. FT-IR (KBr): 1650 (m), 1619 (m), 1528 (m), 1500 (s),
1222 (m) cmꢀ1. HRMS/ESI-TOF (m/z): calcd for C10H16BBrN3
[M þ H]þ 300.0519, found 300.0513.
6-Iodo-3,3-dimethoxy-2-(propan-2-ylidene)-2,3-dihydro-
[1,2,4,3]triazaborolo[4,5-a]pyridin-2-ium-3-uide (22). The
reaction was performed following the general procedure B starting with
18 (0.097 g, 0.3 mmol) and a reaction time of 3.5 h. The solid residue
was purified by reverse-phase flash chromatography with CH3CN/H2O
(20/80) as eluent to isolate the product 22 (0.096 g, 90%) as a greenish
yellow solid. 1H NMR (300 MHz, CDCl3): δ 7.47 (dd, J = 2.00, 0.80 Hz,
1H), 7.35 (dd, J = 9.39, 2.06 Hz, 1H), 6.41 (dd, J = 9.39, 0.88 Hz, 1H),
3.07 (s, 6H), 2.38 (s, 3H), 2.36 (s, 3H). 13C NMR (75 MHz, CDCl3):
δ 160.3, 158.8, 146.3, 141.0, 113.7, 66.4, 50.0, 21.3, 20.5. FT-IR (KBr):
1651 (m), 1615 (m), 1523 (m), 1498 (s), 1219 (s) cmꢀ1. HRMS/
ESI-TOF (m/z): calcd for C10H16BBrN3O2 [M þ H]þ 348.0375, found
348.0372.
6-(4-(tert-Butoxycarbonylamino)but-1-ynyl)-3,3-difluoro-
2-(propan-2-ylidene)-2,3-dihydro[1,2,4,3]triazaborolo[4,5-a]-
pyridin-2-ium-3-uide (23). A mixture of 18 (0.134 g, 0.42 mmol),
Boc-but-3-yn-1-amine (0.141 g, 0.83 mmol), PdCl2(PPh3)2 (14.6 mg,
2.07 ꢁ 10ꢀ2 mmol), and CuI (4.0 mg, 2.07 ꢁ 10ꢀ2 mmol) in Et3N
(4 mL) was stirred for 2 h at ambient temperature under an argon
atmosphere. Volatiles were removed in vacuo. The solid residue was
dissolved in CH2Cl2 (10 mL) and filtered through a plug of Celite. The
filtrate was concentrated, and the residue was purified by silica gel column
chromatographyusing CH3OH/CH2Cl2 (0ꢀ0.5%) toisolate theproduct
6-(4-(2-Amino-3-((4-methoxyphenyl)diphenylmethylthio)-
propanamido)but-1-ynyl)-3,3-dimethoxy-2-(propan-2-ylidene)-
2,3-dihydro[1,2,4,3]triazaborolo[4,5-a]pyridin-2-ium-3-uide (26).
Trifluoroacetic acid (TFA, 50 μL) was added dropwise to compound 24
(0.027 g, 0.07 mmol) in CH2Cl2 (1.0 mL), and the mixture was
stirred for 3 h at room temperature under an argon atmosphere. The
mixture was diluted with CH2Cl2 (2 mL), cooled in an ice bath, and
neutralized by dropwise addition of Et3N (90 μL). The oxazolidine-2,
5-dione 20 (0.038 g, 0.09 mmol) in CH2Cl2 (2 mL) was added to the
neutralized reaction mixture dropwise over 15 min and stirred for 3 h at
0 °C to room temperature under an argon atmosphere. The reaction
1
23 (0.147 g, 96%) as a greenish yellow solid. H NMR (300 MHz,
CDCl3): δ 7.47 (s, 1H), 7.21 (dd, J = 9.30, 1.80 Hz, 1H), 6.51 (d, J = 9.39
Hz, 1H), 4.83 (bs, 1H), 3.32 (dt, J = 6.44, 6.31 Hz, 2H), 2.55 (t, J = 6.46,
2H), 2.42 (s, 3H), 2.37 (s, 3H), 1.46 (s, 9H). 13C NMR (75 MHz,
6788
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Journal of the American Chemical Society
ARTICLE
mixture was diluted with CH2Cl2 (100 mL). The organic layer was
washed with saturated NH4Cl (2 ꢁ 50 mL), saturated NaHCO3 (2 ꢁ
50 mL), and H2O (3 ꢁ 50 mL) and dried over anhydrous Na2SO4.
Volatiles were evaporated under reduced pressure, and the solid residue
was purified by silica gel column chromatography using CH3OH/
CH2Cl2 (0ꢀ1%) to isolate the product 26 (0.035 g, 76%) as a greenish
’ ACKNOWLEDGMENT
We thank Dr. Lori Wilmeth for her assistance in the cell
culture experiments and acknowledge funding from NM-INBRE
P20 RR16480, R25 GM61222, and the Cowboys for Cancer
Research Foundation.
1
yellow solid. H NMR (300 MHz, CDCl3): δ 7.45ꢀ7.28 (m, 10H),
7.25ꢀ7.15 (m, 4H), 6.81 (d, J = 9.09 Hz, 2H), 6.49 (dd, J = 9.39, 0.90 Hz,
1H), 3.79 (s, 3H), 3.77ꢀ3.25 (m, 1H), 3.42ꢀ3.29 (m, 2H), 3.08ꢀ3.02 (m,
7H), 2.73 (dd, J = 12.75, 3.96 Hz, 1H), 2.61ꢀ2.51 (m, 3H), 2.39
(s, 3H), 2.39 (s, 3H), 1.60 (b s, 2H). 13C NMR (75 MHz, CDCl3):
δ 173.0, 160.4, 158.9, 158.2, 144.9, 144.7, 141.6, 139.0, 136.5, 130.8, 129.5,
127.9, 126.7, 113.2, 111.5, 103.8, 86.9, 78.4, 66.5, 55.2, 54.0, 49.9, 37.9, 37.4,
21.3, 20.4. FT-IR (KBr): 3270 (b), 2110 (w), 1689 (m), 1510 (s), 1140
(m) cmꢀ1. HRMS/ESI-TOF (m/z): calcd for C37H43BN5O4S [M þ H]þ
664.3123, found 664.3121.
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2-Amino-N-(2-(2-aminoethoxy)ethyl)-3-((4-methoxy-
phenyl)diphenylmethylthio)propanamide (27). The oxazolidine-
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chromatography using CH3OH/CH2Cl2 1(20/80) to provide the product
27 (0.067 g, 56%) as a colorless solid. H NMR (300 MHz, CDCl3):
δ 7.45ꢀ7.17 (m, 12H), 6.85 ꢀ6.75 (m, 2H), 3.78 (s, 3H), 3.55ꢀ3.30 (m,
6H), 3.10ꢀ3.00 (dd, J = 8.37, 3.97 Hz, 1H), 2.85ꢀ2.78 (t, J= 5.06 Hz, 2H),
2.76ꢀ2.67 (dd, J = 12.62, 4.11 Hz, 1H), 2.60ꢀ2.50 (dd, J = 12.62, 8.36 Hz,
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136.6, 130.7, 129.5, 127.9, 126.7, 113.2, 72.7, 69.5, 66.4, 55.2, 54.0, 41.5,
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single peak at 4.43 min. HPLC/ESI-MS (m/z): calcd for C27H34N3O3S
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Synthesis of Alexa Fluor488 Conjugate (28). To a mixture of
27 (1.0 mg, 0.002 mmol) and Et3N (5 μL) in dry DMF (0.25 mL) was
added Alexa Fluor488-NHS (1.0 mg, 0.0016 mmol) in dry DMF
(0.20 mL) at 0 °C over 15 min, and this mixture was stirred at room
temperature for 12 h. Volatiles were removed in vacuo, and the crude
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concentrated in vacuo. A pipet column containing Dowex 50WX2-400
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(1 mL), and H2O (2 mL), respectively. The concentrate was dissolved in
water (0.5 mL) and eluted through the column; fractions containing the
Naþ salt of the product were combined and concentrated in vacuo to
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C48H46N5O13S3 [M þ H]þ 996.22, found 996.13.
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’ ASSOCIATED CONTENT
S
Supporting Information. Text and figures giving experi-
b
mental details for synthetic and biological studies, compound
identity and purity characterization spectra, and the complete ref
4. This material is available free of charge via the Internet at
http://pubs.acs.org.
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’ AUTHOR INFORMATION
Corresponding Author
jarterbu@nmsu.edu
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