Synthesis and characterization of BODIPY-α-tocopherol: A fluorescent form of vitamin e

Article abstract of DOI:10.1021/jo100095n

Fluorescent nitrobenzoxadiazole analogues of α-tocopherol (NBD-α-Tocs; λ_ex = 468 nm, λ_em = 527 nm) have been made previously to aid study of the intracellular location and transfer of vitamin E. However, these analogues are susceptible to photobleaching while under illumination for confocal microscopy as well as in in vitro FRET transfer assays. Here we report the synthesis of three fluorescent analogues of α-tocopherol incorporating the more robust dipyrrometheneboron difluoride (BODIPY) fluorophore. A BODIPY-linked chromanol should have no intervening polar functional groups that might interfere with binding to the hydrophobic binding site of the tocopherol transfer protein (α-TTP). A key step in bringing the two ring systems together was a metathesis reaction of vinyl chromanol and an alkenyl BODIPY. An o-tolyl containing second generation Grubbs catalyst was identified as the best catalyst for effecting the metathesis without detectable alkene isomerization, which when it occurred produced a mixture of chain lengths in the alkyl linker. C8-BODIPY-α-Toc 10c (λ_ex = 507 nm, λ_em = 511 nm, ε₅₀₇ = 83,000 M^-1 cm^-1) having an eight-carbon chain between the chromanol and fluorophore, had the highest affinity for α-TTP (K_d = 94 ' 3 nM) and bound specifically as it could not be displaced with cholesterol.

Full text of DOI:10.1021/jo100095n

pubs.acs.org/joc
Synthesis and Characterization of BODIPY-r-Tocopherol: A
Fluorescent Form of Vitamin E
Ryan West, Candace Panagabko, and Jeffrey Atkinson*
Department of Chemistry and Centre for Biotechnology, Brock University, St. Catharines,
Ontario, Canada L2S 3A1
jatkin@brocku.ca
Received January 22, 2010
Fluorescent nitrobenzoxadiazole analogues of R-tocopherol (NBD-R-Tocs; λex = 468 nm, λem = 527 nm)
have been made previously to aid study of the intracellular location and transfer of vitamin E. However,
these analogues are susceptible to photobleaching while under illumination for confocal microscopy
as well as in in vitro FRET transfer assays. Here we report the synthesis of three fluorescent analogues
of R-tocopherol incorporating the more robust dipyrrometheneboron difluoride (BODIPY) fluoro-
phore. A BODIPY-linked chromanol should have no intervening polar functional groups that might
interfere with binding to the hydrophobic binding site of the tocopherol transfer protein
(R-TTP). A key step in bringing the two ring systems together was a metathesis reaction of vinyl
chromanol and an alkenyl BODIPY. An o-tolyl containing second generation Grubbs catalyst was
identified as the best catalyst for effecting the metathesis without detectable alkene isomerization,
which when it occurred produced a mixture of chain lengths in the alkyl linker. C8-BODIPY-R-Toc
10c (λex = 507 nm, λem = 511 nm, ε507 = 83,000 M-1 cm-1) having an eight-carbon chain between
the chromanol and fluorophore, had the highest affinity for R-TTP (Kd = 94 ( 3 nM) and bound
specifically as it could not be displaced with cholesterol.
Introduction
Vitamin E is a family of eight hydrophobic chromanols
including four tocopherols and four tocotrienols. One of
these compounds, (RRR)-R-tocopherol (Figure 1; hereafter
referred to as R-tocopherol), is selectively retained by mam-
mals in plasma and tissues following ingestion as a result of
the actions of a specific binding and transfer protein called
the tocopherol transfer protein (TTP).1-3 TTP facilitates the
secretion of R-tocopherol from hepatocytes to the plasma,
where it is carried to other tissues by very-low-density
lipoproteins (VLDL).1,3
FIGURE 1. Structure of naturally occurring R-tocopherol.
antioxidant of free radical lipid peroxidation.6 The kinetics
and physical chemistry of this action have been well des-
cribed for in vitro conditions,7-13 and there is evidence for
Following the discovery of biological activities4 that
would later be ascribed to R-tocopherol,5 research on this
vitamin was dominated by its activity as a chain-terminating
(6) Wolf, R.; Wolf, D.; Ruocco, V. J. Eur. Acad. Dermatol. Venereol.
1998, 10, 103.
(7) Burton, G. W.; Ingold, K. U. Acc. Chem. Res. 1986, 19, 194.
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Antioxidants in Biomedicine; Miquel, J., Quintanilha, A. T., Weber, H., Eds.;
CRC Press, Inc.: Boca Raton, FL, 1989; Vol. 2, p 29.
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(1) Manor, D.; Morley, S. Vitam. Horm. 2007, 76, 45.
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(3) Traber, M. G.; Arai, H. Ann. Rev. Nutr. 1999, 19, 343.
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DOI: 10.1021/jo100095n
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Published on Web 04/13/2010
J. Org. Chem. 2010, 75, 2883–2892 2883
2010 American Chemical Society

West et al.
JOCArticle
similar actions in vivo12,14,15 and in cells.16,17 Recently
described biological actions of R-tocopherol and other
forms of vitamin E may be unrelated to radical scavenging
activity.18-20
high quantum yield, and low polarity.35,36 The lipophilicity
of BODIPY readily allows entry into cell membranes and
other hydrophobic environments such as plasma lipo-
proteins.37,38 Unlike NBD and other common fluorophores
such as dansyl and anthroyloxy that are linked to molecules
by heteroatom-containing functional groups such as esters,
BODIPY analogues can be prepared with an alkyl linker that
maximizes lipophilicity. The fluorescence quantum yield of
BODIPY is two to three times greater than that of NBD, and
its fluorescence intensity is approximately four to five times
greater.39 Seeking to incorporate the advantages of a BOD-
IPY fluorphore in our work with vitamin E, we describe here
the synthesis of three BODIPY-containing R-tocopherol
analogues (C6-, C7-, and C8-BODIPY-R-Toc, 10a-c) as
new probes to assist monitoring the location of tocopherol
within cells. The binding affinities of these analogues to
R-TTP are also reported.
Most investigations of the nature of vitamin E absorption
and distribution have used deuterium-labeled samples to
enable detection of tocopherols by GC-MS following or-
ganic solvent extraction of plasma and tissue samples.21-26
More recently, we prepared a series of fluorescent ana-
logues of vitamin E27 that have expanded our ability to
follow the localization and trafficking of R-tocopherol at
the cellular level.28-31 The most useful of these fluores-
cent analogues contains a 7-nitrobenz-2-oxa-1,3-diazole
(NBD) fluorophore separated from a 6-hydroxychromanol
similar to R-tocopherol by a C9-n-alkyl linker. This so-
called C9-NBD-R-Toc bound specifically and reversibly to
TTP with a dissociation constant, Kd, of approximately
60 nM.27
The NBD fluorophore has been used extensively in lipid
probes,32 but its significant polarity is known to accelerate
spontaneous intermembrane transfer of phospholipid ana-
logues33 and to perturb the behavior of NBD-modified acyl
chains,34 which is a drawback for molecules designed to
probe protein-catalyzed transfer of hydrophobic ligands to
and from membranes. The tendency of the NBD chromo-
phore to photobleach during prolonged observation in
fluorescence confocal microscopy also detracts from its use
as an intracellular probe of lipid trafficking.
Results and Discussion
Reagents are available commercially (BODIPY-FL, 4,4-
difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pro-
pionic acid, is available from Molecular Probes) that include
an intact fluorophore with a short alkanoic side chain
(Figure 2). This allows functionalization of amines and
alcohols with a BODIPY fluorophore. However, for a
desired BODIPY-tocopherol analogue this would install a
polar functional group between the fluorophore and the
chromanol nucleus. The X-ray crystal structure of TTP with
bound R-tocopherol40,41 shows that the tocopherol is held in
a deep hydrophobic site of the protein, suggesting that polar
functional groups might reduce binding affinity. Conse-
quently, we wished to prepare a dipyrromethene structure
that would contain a hydrophobic linkage between fluoro-
phore and chromanol.
Dipyrrometheneboron difluoride (BODIPY) is a fluoro-
phore that has been widely incorporated into lipid probes
because of its high photostability, large extinction coefficient,
(14) Roberts, L. J.; Oates, J. A.; Linton, M. F.; Fazio, S.; Meador, B. P.;
Gross, M. D.; Shyr, Y.; Morrow, J. D. Free Radical Biol. Med. 2007, 43, 1388.
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Traber, M. G. Free Radical Biol. Med. 2004, 36, 1329.
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4449.
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D. A.; Webb, A. Lipids 1987, 22, 163.
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Radical Res. Commun. 1990, 11, 99.
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K. U.; Kayden, H. J. J. Lipid Res. 1990, 31, 687.
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H.; Hughes, L.; Ingold, K. U. Am. J. Clin. Nutr. 1998, 67, 669.
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Cross, C. E.; Traber, M. G. Free Radical Biol. Med. 2005, 38, 857.
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D. Biochemistry 2006, 45, 1075.
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Atkinson, J.; Manor, D. J. Biol. Chem. 2008, 283, 17797.
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2009, 44, 631.
FIGURE 2. Structures of the commercially available BODIPY-FL,
a hypothetical ester-linked BODIPY tocopherol, and an n-alkyl-
linked BODIPY-R-tocopherol prepared in this work.
(37) Drummen, G. P.; van Liebergen, L. C.; Op den Kamp, J. A.; Post,
J. A. Free Radical Biol. Med. 2002, 33, 473.
(32) Chattopadhyay, A. Chem. Phys. Lipids 1990, 53, 1.
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502.
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SCHEME 1. Successful43 and Unsuccessful Coupling Reactions
for Halogenated BODIPY Frameworks
SCHEME 2. Preparation of Terminal Alkenyl 1-Ketopyrroles
SCHEME 3. Preparation of Alkenyl BODIPYs
The BODIPY nucleus can be prepared with 3,5-dihalogen
substituents,42 and these groups used as substrates for
Suzuki and Heck cross-couping reactions.43 We attempted
to couple BODIPY halides with an appropriate alkene, but
with no success (Scheme 1). TLC showed several products
that weakly fluoresced, but after chromatography none
could be confirmed as the expected product. The BODIPY
halides were never recovered from these reactions, suggest-
ing that lack of a substituent at the meso-position introduces
alternate reactivities. There has been one other reported
failure of a cross-coupling in the presence of a BODIPY
halide fluorophore, in this case using the Sonogashira reac-
tion.44 Interestingly, this fluorophore also lacked a substi-
tuent at the meso-position, and multiple uncharacterizable
products were formed.
We then considered whether a BODIPY system function-
alized with a suitable alkene could be used in a metathesis
reaction. The successful olefin metathesis of a BODIPY dye
functionalized with a terminal olefin was reported in the
synthesis of sphingosine analogues.45 The BODIPY used
lacked a meso-substituent and yet remained intact after this
reaction. The alkenyl BODIPYs that are required for this
metathesis can be prepared from ω-alkenoic acids. If the
ω-alkenoic acids are not commercially available, they can be
prepared by Grignard reaction, as was required for obtaining
7-octenoic acid 1 (Scheme 2).
2-Ketopyrroles can be produced by the acylation of
pyrrylmagnesium chloride with acyl halides or esters.46
Acylations with esters are more favorable because they react
less vigorously than other acylating agents and are less likely
to form tars.47 2-Ketopyrroles are stable species that can be
purified by chromatography.
Alkenylpyrroles 4a-c were synthesized in yields near 50%
after treating the 2-ketopyrroles with sodium borohydride48
(Scheme 3). Compounds 4a-c tended to decompose on silica
during attempted chromatography, and therefore the pro-
ducts were best obtained by simply passing them through a
short alumina plug prior to using them in the next step.
Despite hydrobromic acid being used to condense the
alkenylpyrroles with substituted pyrrole carboxaldehydes
in the methods described by Peters et al.,45 multiple attempts
by us did not yield the expected precipitate. The use of
phosphorus oxychloride,44 however, successfully generated
dipyrromethenes 5a-c in situ as the PO2Cl2- salts.49 Treat-
ing solutions of 5a-c with boron trifluoride diethyl etherate
and base yielded alkenyl BODIPYs 6a-c.
The olefin metathesis between 1 equiv of vinyl Trolox 7,
prepared from Trolox aldehyde,50 and 2 equiv of alkenyl
BODIPY 6a-c produced 8a-c with yields that varied from
48 to 65% (Scheme 4). Two equivalents of alkenyl BODIPY
are required to obtain the best yields of the cross-coupled
product. A dimerization byproduct arising from coupling of
the alkenyl BODIPY (a bis-BODIPY) can be easily removed
by silica gel chromatography. We never observed any dimer-
ized Trolox byproduct from this metathesis.
We did not explore the geometry of the alkenes as they
were to be reduced to prepare the target molecules. However,
it has been noted45,51 that the cross-metathesis products are
formed almost exclusively in the E geometry. The double
bonds in 8a-c need to be reduced to obtain the saturated
alkyl linker of the target molecules. There are also multiple
reports of functional groups on molecules that possess a
BODIPY fluorophore being reduced using standard hydro-
gen conditions (10% Pd/C, H2) without decomposition.52-56
(42) Rohand, T.; Baruah, M.; Qin, W. W.; Boens, N.; Dehaen, W. Chem.
Commun. 2006, 266.
(49) van Koeveringe, J. A.; Lugtenburg, J. Recl. Trav. Chim. Pay-Bas
1977, 96, 55.
(43) Rohand, T.; Qin, W. W.; Boens, N.; Dehaen, W. Eur. J. Org. Chem.
2006, 4658.
(50) Lei, H. S.; Atkinson, J. J. Org. Chem. 2000, 65, 2560.
(51) Nussbaumer, P.; Ettmayer, P.; Peters, C.; Rosenbeiger, D.; Hogenauer,
K. Chem. Commun. 2005, 5086.
(52) Azov, V. A.; Diederich, F.; Lill, Y.; Hecht, B. Helv. Chim. Acta 2003,
86, 2149.
(44) Li, Z.; Bittman, R. J. Org. Chem. 2007, 72, 8376.
(45) Peters, C.; Billich, A.; Ghobrial, M.; Hogenauer, K.; Ullrich, T.;
Nussbaumer, P. J. Org. Chem. 2007, 72, 1842.
(46) Castro, A. J.; Lowell, J. R., Jr; Marsh, J. P., Jr J. Hetercycl. Chem.
1964, 1, 207.
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Lett. 2007, 9, 3375.
(47) Bean, G. P. J. Org. Chem. 1967, 32, 228.
€
(48) Furstner, A.; Grabowski, J.; Lehmann, C. W. J. Org. Chem. 1999,64, 8275.
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2007, 129, 5597.
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SCHEME 4. Preparation of BODIPY-R-Tocopherols of Varying
Linker Chain Length via Cross-Coupling Olefin Metathesis
FIGURE 3. HPLC chromatogram of the BODIPY-R-tocopherols
produced from a metathesis reaction of C7-alkenyl-BODIPY 6b
with vinyl chromanol 7 using Hoveyda-Grubbs second generation
catalyst.
only if run with extreme care. In the end, we removed the
protecting group in HCl/methanol/THF45,59to provide
BODIPY-R-Tocs 10a-c in yields of 66-77%.
However, when 8a-c was treated with hydrogen gas and
Pd/C, TLC showed rapid decomposition of the starting
material within minutes and an uncharacterizable transpa-
rent oil that gave a fluorescent blue spot on the TLC baseline
was formed. Again, the dyes that were successfully trans-
formed using Pd/C52-56 possessed meso-aryl groups, which
must increase the stability of the backbone during noble
metal reductions. A report of the synthesis of the bile
pigment nonylprodigiosin (also a pyrrole) reported similar
difficulties in reducing a double bond using hydrogen gas
and Pd/C,48 but good yields were obtained when Wilkinson’s
catalyst was used instead.48 This was also noted in the
reduction of the BODIPY-ceramide analogues reported by
Boath et al.57 When Wilkinson’s catalyst was applied to the
reduction of 8a-c, selective reduction of the alkene only
began at pressures of 40 psi or greater and was sensitive to the
concentration of alkene. Decomposition of the fluorophore
was still observed, but there was enough chemoselectivity to
obtain 9a-c in decent yields. The best conditions (70% yield)
for the catalytic hydrogenation of 8a-c with Wilkinson’s
catalyst were in ethanol, 80 psi H2, for 28 h with an alkene
concentration of ∼20 mM.
The NMR spectra of 10a-c appeared straightforward,
but the HPLC chromatograms showed multiple peaks
(Figure 3) when detection was at the BODIPY absorbance
maximum of 511 nm. LC-MS analysis of the expected C7-
BODIPY-R-Toc product 10b revealed that the main peak at
13.38 min was the desired material but that peaks with
shorter and longer retention times gave molecular ions
differing by mass 14, suggesting a group of analogues with
differing numbers of methylenes in the alkyl linker. H NMR
cannot easily detect the relatively minor abundance of short-
er and longer chain-length materials. Inspection of synthetic
intermediates prior to 8a-c by HPLC showed them to be free
of such homologues, suggesting that they arose during the
metathesis reactions.
Second generation olefin metathesis catalysts are known
to facilitate olefin isomerization by 1,3-hydride shifts during
the intended metathesis.60 As a result, chain-length homo-
logues can be generated. Unfortunately, these side reactions
are not always reported in the many papers that perform
metatheses. It is not clear if the isomerization is caused by the
ruthenium catalyst itself, by some decomposition product of
the catalyst, or by some impurity made during the synthesis
of the catalyst.60 The suggested mechanism for this isomer-
ization has been reported.61,62 To obtain homologues that
are one or two methylenes less, the isomerization of the
terminal alkene must occur more than once. To obtain a
homologue with an extra methylene the alkene can isomerize
after the first successful metathesis and then undergo a
second metathesis, as depicted in Figure 4.60 An isolated
bis-BODIPY 11 (from reaction of 6a), which we expected to
have an alkyl spacer length of 10 carbons, in fact showed a
range of chain lengths from 9 to 11 carbons, suggesting that
the sterically encumbered vinyl chroman 7 reacts only
slowly, allowing time for isomerization (and coupling) of the
more accessible alkenyl BODIPY (Scheme 5). Bis-BODIPY
Deprotection of the TBS group from 9a-c using tetra-
butylammonium fluoride also resulted in decomposition and
loss of the characteristic BODIPY fluorescence. The desily-
lation of an intermediate for the synthesis of an anthracene-
BODIPY analogue, also lacking a substituent at the meso-
position, was reported to occur with great difficulty.58 This
step was specifically stated as being the most troublesome in
the entire synthesis and could be successfully transformed
(55) Ziessel, R.; Bonardi, L.; Retailleau, P.; Ulrich, G. J. Org. Chem.
2006, 71, 3093.
(56) Ueno, T.; Urano, Y.; Kojima, H.; Nagano, T. J. Am. Chem. Soc.
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Bornancin, F. J. Biol. Chem. 2008, 283, 8517.
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L. B. A.; Wolford, M. F.; Kim, T. G.; Topp, M. R.; Hochstrasser, R. M.;
Burgess, K. Chem.;Eur. J. 2003, 9, 4430.
(59) Ye, Y. Q.; Koshino, H.; Onose, J. I.; Yoshikawa, K.; Abe, N.;
Takahashi, S. Org. Lett. 2007, 9, 4131.
(60) Lehman, S. E.; Schwendeman, J. E.; O’Donnell, P. M.; Wagener,
K. B. Inorg. Chim. Acta 2003, 345, 190.
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TABLE 1. Summary of Metathesis Reactions with Various Ruthenium Catalysts
SCHEME 5. Generation of a Cross-Coupled Bis-BODIPY
during Attempted Coupling of Vinyl Trolox 7 and Alkenyl
BODIPY 6a
using different catalysts is provided in Table 1. Grubbs
generation I catalyst was used in attempts to metathesize
vinyl Trolox 7 with heptenyl-BODIPY 6b but was presum-
ably not active enough to complex to the bulky chromanol;
only the bis-BODIPY was produced from metathesis of the
alkenyl BODIPY. In other attempts to eliminate olefin
isomerization,63 acids (e.g., trifluoroacetic acid, phenylphos-
phoric acid) or benzoquinones have been added to titrate the
species responsible for the isomerization. Removal of impu-
rities from the catalyst by silica gel chromatography has been
reported to eliminate isomerization activity as well.64 Both of
these methods were applied to this metathesis using Grubbs
generation II catalyst. The composition of products drama-
tically improved (93-95% C7 and 5-7% C6), but these
product mixtures would still need purification by preparative
HPLC. When Hoveyda-Grubbs generation II catalyst was
used, the composition and yield improved as well, but
purification would again be required. The o-tolyl deriva-
tive of Hoveyda-Grubbs generation II catalyst has been
FIGURE 4. Proposed pathway for the isomerization and produc-
tion of a C8-linker from a C7-alkenyl BODIPY following metathesis
with a vinyl chromanol.
mixture 11 was also subjected to cross-coupling conditions
with 7 and produced BODIPY chromans of an even more
complex mixture of chain lengths, showing that bis-BODI-
PY was an intermediate in the original reactions, capable of
continued rounds of metathesis, and is the likely source for
homologues of chain lengths higher than the sum of carbons
in the starting alkenes 7 and 6a-c.
We did manage to isolate milligram amounts of pure
10a-c using preparative HPLC but also wished to find a
more efficient means of synthesis that avoided olefin iso-
(63) Fokou, P. A.; Meier, M. A. R. J. Am. Chem. Soc. 2009, 131, 1664.
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Chem. Soc. 2002, 124, 13390.
merization. A summary of metathesis reactions performed
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TABLE 2. Dissociation Constants for NBD-R-Tocs and BODIPY-R-
Tocs
NBD-R-Toc
Kd (nM)
BDP-R-Toc
Kd (nM)
C6
C7
C8
C9
299 ( 37
106 ( 21
142 ( 35
56 ( 15
C6
C7
C8
232 ( 10
130 ( 4
94 ( 3
FIGURE 5. Titration curves showing the increase in fluorescence
intensity at 514 nm (λex = 506 nm) following sequential additions of
C6-, C7-, and C8-BODIPY-R-Toc, 10a-c, to a 0.2 μM solution of
R-TTP in SET buffer. The resulting saturation curves were fitted to
a one-site binding model. Averages of triplicate data sets are
graphed with the associated standard errors.
FIGURE 6. Competitive displacement of 1.0 μM C8-BODIPY-R-
Toc, 10c, bound to 0.2 μM R-TTP in SET buffer by addition of
increasing amounts of (RRR)-R-tocopherol (Δ) or cholesterol (0).
Fluorescence was monitored at 514 nm after excitation at 506 nm.
The average of triplicate data sets are reported for tocopherol and
standard errors. Data for R-tocopherol was fit to a one-site compe-
tition model using GraphPad Prism.
developed to facilitate easy interaction with bulkier sub-
strates.65 When this catalyst was used, the crude product
showed only one peak by analytical HPLC that corre-
sponded to the desired homologue. While the yield was
significantly lower, it saved considerable effort in obtaining
pure material.
For BODIPY-R-Tocs 10a-c to be of any utility, they must
show some affinity toward R-TTP. Binding should be both
specific and reversible for these fluorescent probes to be
suitable analogues of vitamin E. The dissociation constants
were assessed from fluorescence titrations of R-TTP with
BODIPY-R-Tocs 10a-c (Figure 5) and calculated using a
one-site binding model with Prism software. The average
results of the multiple titrations on a range of ligand con-
centrations (3-640 nM) are listed in Table 2. C8-BODIPY-
R-Toc 10c appears to have the best affinity to R-TTP (Kd=
94 nM). The dissociation constant for R-tocopherol, the
natural ligand for R-TTP, is 25 nM.66
ethanol as the carrier solvent, which is known to interfere
with the binding of hydrophobic ligands to lipid transfer
proteins.67 Together, Figures 5 and 6 show that there is
specific competitive binding of C8-BDP-R-Toc to R-TTP.
Conclusions
Three BODIPY analogues of R-tocopherol 10a-c were
successfully prepared. They all bind specifically and rever-
sibly to R-TTP with high affinity and thus can serve as
fluorescent probes in the study of the location and the
intracellular transfer of vitamin E. We will use these probes
in cultured hepatocytes to explore the mechanism of R-TTP-
mediated transfer and secretion of tocopherol.
Experimental Section
To illustrate the specificity of binding, R-TTP was first
equilibrated with 10c and then titrated with R-tocopherol or
cholesterol, the latter of which is known not to bind.66 C8-
BODIPY-R-Toc exhibited a dose-dependent decrease in
fluorescence upon the addition of R-tocopherol, suggesting
that the environmentally sensitive fluorophore is displaced
from the hydrophobic-binding pocket of R-TTP by the
native ligand (Figure 6). After the addition of 5 μM
R-tocopherol to the assay, there is near total displacement
of C8-BODIPY-R-Toc by the native ligand. After 5 μM
cholesterol had been added, fluorescence had been reduced
by ∼15%, which is in large part due to the presence of
Fluorescent Binding Assays of BODIPY-r-Tocopherols 10a-c.
Human R-TTP was expressed and purified following pub-
lished methods as were fluorescence titrations.27 Briefly,
0.2 μM R-TTP was incubated with each of the BODIPY
analogues at concentrations ranging from 0 to 5 μM in SET
buffer (250 mM sucrose, 100 mM KCl, 1 mM EDTA, 50 mM
Tris-HCl, pH 7.4). The ligands were added from ethanolic
stock solutions such that the total volume of ethanol was only
0.1% of the total sample. After brief vortexing, samples were
agitated on a rocker for 25-40 min at room temperature.
Subsequently, the fluorescence of each sample was measured at
514 nm on a QuantaMaster2000 fluorometer (Photon Technologies
International, London, Ontario) using an excitation wavelength
of 506 and 5 nm slit widths. Fluorescence values were plotted in
(65) Stewart, I. C.; Douglas, C. J.; Grubbs, R. H. Org. Lett. 2008, 10, 441.
(66) Panagabko, C.; Morley, S.; Hernandez, M.; Cassolato, P.; Gordon,
H.; Parsons, R.; Manor, D.; Atkinson, J. Biochemistry 2003, 42, 6467.
(67) Avdulov, N. A.; Chochina, S. V.; Igbavboa, U.; Warden, C. S.;
Schroeder, F.; Wood, W. G. Biochim. Biophys. Acta 1999, 1437, 37.
2888 J. Org. Chem. Vol. 75, No. 9, 2010

West et al.
JOCArticle
Prism Graphpad 4.0 and fit to a nonlinear regression one-site
binding model.68
mmol, 96%). Light yellow syrup, Rf = (0.35, hexanes/diethyl
ether 3:2). 1H NMR (300 MHz, CDCl3) δ 8.40-8.38 (dd, J =
4.8 Hz, J = 0.9 Hz, 1H), 7.52-7.47 (m, 1H), 7.42-7.39 (d, J =
8.1 Hz, 1H), 7.06-7.02 (m, 1H), 5.61-5.52 (ddt, J = 16.8 Hz,
J = 10.4 Hz, J = 6.6 Hz, 1H), 4.88-4.79 (m, 2H), 2.53-2.48 (t,
J = 7.5 Hz, 2H), 1.96-1.89 (m, 2H), 1.67-1.57 (m, 2H). 13C
NMR (75 MHz, CDCl3) δ 195.4, 150.9, 149.7, 136.6, 136.4,
129.4, 122.8, 115.1, 42.7, 32.1, 23.7. MS (þEI) m/z 207 (Mþ,
0.4%), 179 (1.2%), 166 (3.1%), 125 (4.3%), 111 (100%), 78
(12.2%), 69 (19.4%), 55 (32.5%), 41 (38.2%). HRMS (EI)
calculated for C11H13NOS: 207.07179, found 207.07210.
Competition assays were performed under similar conditions.
To a 0.2 μM solution of R-TTP in 3 mL of SET buffer was added
2 μL of a stock solution of fluorescent analogue in absolute
ethanol (1.5 mM) such that the final concentration of fluorophore
was 1 μM. The solution was then mixed until the fluorescence
signal reached a constant value. To this solution were added 2 μL
aliquots of R-tocopherol (or cholesterol) in absolute ethanol (1.5
to 15 mM stock solutions) to yield final concentrations of toco-
pherol ranging from 1 to 5 μM. After each addition of tocopherol,
the samples were equilibrated for 15 min, and the final fluorescence
was recorded. Control experiments included an identical competi-
tion substituting cholesterol as the competitor.
S-Pyridin-2-yl Hept-6-enethioate (2b, n = 2). (1.42 g, 6.43
mmol, 95%). Light yellow syrup, Rf = (0.35, hexanes/diethyl
1
ether 3:2). H NMR (300 MHz, CDCl3) δ 8.29-8.26 (m, 1H),
Synthesis. (S)-tert-Butyldimethyl(2,5,7,8-tetramethyl-2-vinyl-
chroman-6-yloxy)silane (7).50 Methyltriphenylphosphonium
bromide (169 mg, 0.473 mmol) was dissolved in tetrahydrofuran
(10 mL) under nitrogen, and to this was added dropwise a 1.0 M
solution of LHMDS in tetrahydrofuran (753 μL, 0.753 mmol).
After stirring for 1 h, a solution of (S)-Trolox aldehyde (150 mg,
0.430 mmol) in tetrahydrofuran (5 mL) was added and stirred for
3 h. The reaction was then quenched with saturated ammonium
chloride solution and extracted with dichloromethane. The crude
material was subjected to chromatography (hexanes/dichloro-
methane 3:1) to afford product 7 (132 mg, 0.380 mmol, 88%).
White solid, Rf = (0.30, hexanes/dichloromethane 3:1), mp
54 °C. 1H NMR (300 MHz, CDCl3) δ 5.94-5.85 (dd, J = 17.1
Hz, J = 10.8 Hz, 1H), 5.20-5.13 (dd, J = 17.1 Hz, J = 1.1 Hz,
1H), 5.07-5.03 (dd, J = 10.8 Hz, J = 1.1 Hz, 1H), 2.66-2.48 (m,
2H), 2.19 (s, 3H), 2.17 (s, 3H), 2.09 (s, 3H), 2.01-1.81 (m, 2H),
1.44 (s, 3H), 1.10 (s, 9H), 0.17 (s, 6H). 13C NMR (75 MHz,
CDCl3) δ 146.0, 144.2, 141.9, 125.8, 123.4, 122.2, 117.5, 113.1,
75.2, 31.9, 27.0, 26.1, 21.1, 18.6, 14.3, 13.4, 12.0, -3.3. MS (þEI)
m/z 346 (Mþ, 100%), 331 (8.6%), 289 (12.4%), 278 (27.3%), 234
(10.9%), 221 (49.5%), 163 (9.0%), 129 (6.7%), 73 (53.8%), 59
(13.6%). HRMS (EI) calculated for C21H34O2Si: 346.23281,
found 346.23288.
7.37-7.30 (m, 2H), 6.93-6.89 (m, 1H), 5.48-5.42 (m, 1H),
4.74-4.64 (m, 2H), 2.42-2.37 (t, J=7.5 Hz, 2H), 1.79-1.71
(m, 2H), 1.47-1.37 (m, 2H), 1.20-1.10 (m, 2H). 13C NMR (75
MHz, CDCl3) δ 194.8, 150.8, 149.3, 137.1, 136.0, 128.9, 122.4,
113.9, 42.9, 32.3, 27.0, 23.8. MS (þEI) m/z 221 (Mþ, 0.2%), 188
(1.1%), 160 (3.1%), 125 (2.7%), 111 (100%), 78 (10.0%), 67
(13.9%), 55 (31.1%), 41 (23.8%). HRMS (EI) calculated for
C12H15NOS: 221.08744, found 221.08726.
S-Pyridin-2-yl Oct-7-enethioate (2c, n = 3). (733 mg, 3.12
mmol, 98%). Light yellow syrup, Rf = (0.35, hexanes/diethyl
ether 3:2). 1H NMR (300 MHz, CDCl3) δ 8.36-8.34 (dd, J =
4.8 Hz, J = 1.2 Hz, 1H), 7.47-7.36 (m, 2H), 7.01-6.96 (m, 1H),
5.59-5.50 (ddt, J = 16.8 Hz, J = 10.2 Hz, J = 6.6 Hz, 1H),
4.80-4.69 (m, 2H), 2.47-2.43 (t, J = 7.5 Hz, 2H), 1.84-1.77
(m, 2H), 1.53-1.43 (m, 2H), 1.22-1.09 (m, 4H). 13C NMR (75
MHz, CDCl3) δ 195.2, 151.0, 149.5, 137.8, 136.3, 129.2, 122.7,
113.8, 43.3, 32.7, 27.7, 27.5, 24.4. MS (þEI) m/z 235 (Mþ, 0.8%),
174 (2.2%), 125 (2.7%), 111 (100%), 78 (10.3%), 67 (13.0%), 55
(43.7%), 41 (15.1%). HRMS (EI) calculated for C13H17NOS:
235.10309, found 235.10277.
1-(1H-Pyrrol-2-yl)hex-5-en-1-one (3a, n=1).48 Pyrrole (669 μL,
9.65 mmol) was dissolved in tetrahydrofuran (4 mL) and cooled to
0 °C under nitrogen. To this was slowly added a 3.0 M solution of
methylmagnesium chloride in tetrahydrofuran (2.41 mL, 7.24
mmol), and the resulting solution was stirred for 15 min. Next,
the solution was cooled further to -78 °C, and a solution of thioate
2a (500 mg, 2.41 mmol) in tetrahydrofuran (18 mL) was added.
The reaction was warmed to 0 °C and stirred for 1 h. Afterward,
the reaction mixture was condensed in vacuo and then extracted
with dichloromethane. This crude material was purified via
column chromatography (hexanes/diethyl ether 3:2) to yield
pure ketopyrrole 3a (356 mg, 2.17 mmol, 90%). Clear and colorless
oil, Rf = (0.40, hexanes/diethyl ether 3:2). 1H NMR (300 MHz,
CDCl3) δ 10.29 (br s, 1H), 7.06 (m, 1H), 6.93 (m, 1H), 6.27-6.26
(m, 1H), 5.89-5.76 (ddt, J = 17.0 Hz, J = 10.1 Hz, J = 6.9 Hz,
1H), 5.07-4.98 (m, 2H), 2.82-2.77 (t, J = 7.2 Hz, 2H), 2.18-2.11
(m, 2H), 1.89-1.80 (m, 2H). 13C NMR (75 MHz, CDCl3) δ 191.0,
138.0, 131.9, 124.9, 116.4, 115.1, 110.4, 37.1, 33.2, 24.3. MS
(þEI) m/z 163 (Mþ, 25.3%), 146 (1.1%), 122 (1.6%), 109
(100%), 94 (63.0%), 80 (6.7%), 66 (14.9%), 55 (5.1%), 44
(15.4%). HRMS (EI) calculated for C10H13NO: 163.09971, found
163.09972.
Oct-7-enoic Acid (1).48 To a stirring solution of 7-bromo-1-
heptene (2.00 g, 11.3 mmol) in dry diethyl ether (10 mL) were
added magnesium turnings (302 mg, 12.4 mmol) and a catalytic
amount of iodine. The mixture was heated to 34 °C for 3.5 h and
then cooled to -20 °C upon which carbon dioxide (dry ice
subliming through a Drierite drying tube) was bubbled into the
solution. After 2 h, the reaction was quenched with 19%
hydrochloric acid (10 mL) and extracted with diethyl ether to
obtain acid 1 (452 mg, 2.94 mmol, 26%). Clear and colorless
liquid, Rf = (0.25, hexanes/ethyl acetate 5:1, visualized by
H2SO4/MeOH). 1H NMR (300 MHz, CDCl3) δ 11.79 (br s,
1H), 5.82-5.73 (ddt, J = 17.0 Hz, J = 10.3 Hz, J = 6.7 Hz, 1H),
5.02-4.90 (m, 2H), 2.36-2.31 (t, J = 1.5 Hz, 2H), 2.08-2.01
(m, 2H), 1.66-1.61 (m, 2H), 1.43-1.28 (m, 4H). 13C NMR (75
MHz, CDCl3) δ 180.3, 138.4, 114.3, 33.9, 33.4, 28.4, 24.4. MS
(þFAB) m/z 143 ([MH]þ, 20.4%), 137 (15.2%), 125 (28.2%),
107 (9.3%), 97 (34.0%), 81 (16.1%), 67 (13.4%), 55 (100%), 41
(39.2%). HRMS (FAB) calculated for C8H15O2þ: 143.10720,
found 143.10647.
S-Pyridin-2-yl hex-5-enethioate (2a, n = 1).48 To a solution of
5-hexenoic acid (1.12 g, 9.81 mmol) in dry toluene (10 mL) were
added 2,20-dipyridyl disulfide (2.81 g, 12.8 mmol) and triphe-
nylphosphine (3.34 g, 12.8 mmol). This mixture was stirred
overnight. After washing the toluene with water and then
washing the combined water fractions with diethyl ether, the
organic phases were combined and condensed in vacuo, and the
crude material was loaded onto a silica gel column eluting with
hexanes/ethyl acetate 6:1 to afford pure thioate 2a (1.95 g, 9.42
1-(1H-Pyrrol-2-yl)hept-6-en-1-one (3b, n = 2). (1.11 g, 6.23
mmol, 97%). Clear and colorless oil, Rf = (0.45, hexanes/
diethyl ether 3:2). H NMR (300 MHz, CDCl3) δ 11.21 (br s,
1
1H), 7.12-7.10 (m, 1H), 6.99-6.98 (m, 1H), 6.31-6.28 (m, 1H),
5.91-5.80 (ddt, J = 17.0 Hz, J = 10.3 Hz, J = 6.7 Hz, 1H),
5.12-5.01 (m, 2H), 2.87-2.82 (t, J = 7.2 Hz, 2H), 2.18-2.11
(m, 2H), 1.88-1.77 (m, 2H), 1.58-1.48 (m, 2H). 13C NMR (75
MHz, CDCl3) δ 191.0, 138.1, 131.6, 125.2, 116.6, 114.2, 109.9,
37.3, 33.2, 28.2, 24.5. MS (þEI) m/z 177 (Mþ, 21.8%), 149
(2.1%), 134 (2.2%), 122 (25.9%), 109 (91.7%), 94 (100%), 80
(6.8%), 67 (36.2%), 55 (11.9%), 41 (15.4%). HRMS (EI)
calculated for C11H15NO: 177.11536, found 177.11487.
(68) Norris, A. W.; Cheng, L.; Giguere, V.; Rosenberger, M.; Li, E.
Biochim. Biophys. Acta 1994, 1209, 10.
J. Org. Chem. Vol. 75, No. 9, 2010 2889

West et al.
JOCArticle
1-(1H-Pyrrol-2-yl)oct-7-en-1-one (3c, n = 3). (551 mg, 2.88
mmol, 93%). Clear and colorless oil, Rf = (0.45, hexanes/
diethyl ether 3:2). H NMR (300 MHz, CDCl3) δ 10.81 (br s,
1.48-1.43 (m, 4H). 13C NMR (75 MHz, CDCl3) δ 160.3, 158.6,
142.7, 138.7, 134.4, 133.2, 128.4, 123.3, 119.6, 116.5, 114.1, 33.5, 28.9,
28.5, 28.4, 28.3, 14.6, 10.9. 11BNMR(96MHz,CDCl3) 1.29-0.60 (t,
J = 32.7 Hz, BF2). 19F NMR (282 MHz, CDCl3) -144.7 to -145.1
(overlapping q and sextet, J = 33.3 Hz, BF2). MS (þEI) m/z 316
(Mþ, 49.0%), 277 (2.1%), 248 (61.2%), 233 (100%), 213 (23.0%),
194 (4.3%), 149 (3.9%), 116 (9.9%), 81 (4.2%), 69 (7.6%), 55
(7.5%), 43 (13.2%). HRMS (EI) calculated for C18H23BF2N2:
316.19224, found 316.19279.
7-(Oct-7-enyl)-5,5-difluoro-1,3-dimethyl-5H-dipyrrolo[1,2-c:10,20-
f][1,3,2]diazaborinin-4-ium-5-uide (6c, n = 3). (370 mg, 1.12 mmol,
39% over two steps). Dark red oil, Rf = (0.50, hexanes/diethyl ether
3:2). λmax excitation in ethanol = 507 nm (ε507 = 85 000 M-1
cm-1), λmax emission in ethanol = 511 nm. 1H NMR (300 MHz,
CDCl3) δ7.02 (s, 1H), 6.87-6.85 (d, J= 3.9 Hz, 1H), 6.26-6.25 (d,
J= 3.9 Hz, 1H), 6.04 (s, 1H), 5.89-5.75 (ddt, J=17.0Hz,J= 10.3
Hz, J = 6.7 Hz, 1H), 5.02-4.92 (m, 2H), 3.00-2.95 (t, J = 7.8 Hz,
2H), 2.54 (s, 3H), 2.18 (s, 3H), 2.07-2.02 (m, 2H), 1.78-1.69 (m,
2H), 1.52-1.40 (m, 6H). 13C NMR (75 MHz, CDCl3) δ 160.6,
158.7, 142.7, 139.0, 134.5, 133.2, 128.4, 123.4, 119.7, 116.6, 114.1,
33.7, 29.3, 28.8, 28.7, 28.6, 28.5, 14.7, 11.0. 11B NMR (96 MHz,
CDCl3) 1.29-0.60 (t, J = 32.7 Hz, BF2). 19F NMR (282 MHz,
CDCl3) -144.9 to -145.3 (overlapping q and sextet, J = 33.3 Hz,
BF2). MS (þEI) m/z 330 (Mþ, 10.2%), 248 (100%), 233 (40.5%),
218 (15.4%), 149 (10.6%), 116 (15.3%), 94 (10.9%), 83 (7.9%), 69
(14.3%), 57 (16.0%), 44 (40.8%). HRMS (EI) calculated for
C19H25BF2N2: 330.20789, found 330.20818.
1
1H), 7.09-7.06 (m, 1H), 6.97-6.94 (m, 1H), 6.28-6.26 (m, 1H),
5.87-5.78 (ddt, J = 17.0 Hz, J0 = 10.3 Hz, J00 = 6.7 Hz, 1H),
5.05-4.95 (m, 2H), 2.83-2.78 (t, J = 7.2 Hz, 2H), 2.11-2.04
(m, 2H), 1.80-1.75 (m, 2H), 1.48-1.39 (m, 4H). 13C NMR (75
MHz, CDCl3) δ 191.3, 138.6, 131.8, 125.2, 116.5, 114.2, 110.1,
37.7, 33.4, 28.7, 28.5, 25.1. MS (þEI) m/z 191 (Mþ, 20.8%), 148
(2.2%), 136 (1.7%), 122 (18.5%), 109 (100%), 94 (77.5%), 80
(8.1%), 67 (19.6%), 55 (8.9%), 41 (10.1%). HRMS (EI) calcu-
lated for C12H17NO: 191.13101, found 191.13106.
2-(Hex-5-enyl)-1H-pyrrole (4a, n = 1).48 Ketopyrrole 3a (194
mg, 1.19 mmol) was dissolved in isopropyl alcohol (6 mL) and
heated to reflux. To this boiling solution was added a suspension
of sodium borohydride (126 mg, 3.33 mmol) in isopropyl
alcohol (4 mL). The reaction refluxed overnight. Afterward,
the crude material was condensed in vacuo and passed through a
neutral alumina column (hexanes/ethyl acetate 10:1) to afford
crude 4a that was reacted immediately in the next step. Note:
extreme care must be used when handling these alkenyl pyrroles
because they readily decompose, especially in the presence of an
acid, and must be used immediately. Clear and colorless liquid,
fruity odor, Rf = (0.50, hexanes/ethyl acetate 6:1, alumina).
2-(Hept-6-enyl)-1H-pyrrole (4b, n = 2). Clear and colorless
liquid, fruity odor, Rf = (0.60, hexanes/ethyl acetate 6:1, alumina).
2-(Oct-7-enyl)-1H-pyrrole (4c, n = 3). Clear and colorless
liquid, fruity odor, Rf = (0.60, hexanes/ethyl acetate 6:1, alumina).
7-(Hex-5-enyl)-5,5-difluoro-1,3-dimethyl-5H-dipyrrolo[1,2-c:10,20-
f][1,3,2]diazaborinin-4-ium-5-uide (6a, n = 1).44 Crude 4a and 3,5-
dimethylpyrrole-2-carboxaldehyde (70.0 mg, 0.568 mmol) were
dissolved in dry dichloromethane (8.5 mL) and cooled to 0 °C
under nitrogen. A solution of phosphorus oxychloride (52.0 μL,
0.568 mmol) in dichloromethane (0.5 mL) was added via syringe,
and the resulting solution was kept on ice for 1 h. Afterward, the
reaction was brought to room temperature and stirred overnight.
This solution was then cooled back to 0 °C, and boron trifluoride
diethyl etherate (288 μL, 2.27 mmol) and DIPEA (396 μL, 2.27
mmol) were added. After warming back to room temperature and
stirring for 6 h, the crude product was extracted in dichloromethane
and condensed in vacuo. The residue was loaded onto a silica
column (hexanes/diethyl ether 3:1) to afford pure 6a (143 mg,
0.476 mmol, 40% over two steps). Dark red oil, Rf = (0.30,
hexanes/diethyl ether 3:2). λmax excitation in ethanol = 507 nm
(ε507 = 87 000 M-1 cm-1), λmax emission in ethanol = 511 nm. 1H
NMR (300 MHz, CDCl3) δ 7.04 (s, 1H), 6.89-6.87 (d, J = 3.9 Hz,
1H), 6.28-6.26 (d, J = 3.9 Hz, 1H), 6.07 (s, 1H), 5.89-5.76 (ddt,
J = 17.0 Hz, J = 10.3 Hz, J = 6.7 Hz, 1H), 5.05-4.94 (m, 2H),
3.01-2.96 (t, J = 7.8 Hz, 2H), 2.55 (s, 3H), 2.21 (s, 3H), 2.16-2.09
(m, 2H), 1.81-1.70 (m, 2H), 1.58-1.48 (m, 2H). 13C NMR (75
MHz, CDCl3) δ160.5, 142.9, 138.7, 134.6, 133.3, 128.4, 123.4, 119.8,
118.9, 116.7, 114.5, 33.5, 28.7, 28.4, 28.0, 14.8, 11.2. 11B NMR (193
MHz, CDCl3) 1.11-0.77 (t, J = 32.7 Hz, BF2). 19F NMR (282
MHz, CDCl3) -145.2 to -145.5 (overlapping q and sextet, J=33.3
Hz, BF2).MS(þEI) m/z302 (Mþ,50.3%),282([M- HF]þ,3.5%),
248 (11.6%), 233 (100%), 213 (24.1%), 195 (7.5%), 149 (19.3%),
111 (12.9%), 97 (8.6%), 83 (9.6%), 71 (12.9%), 57 (22.7%), 43
(17.7%). HRMS (EI) calculated for C17H21BF2N2: 302.17659,
found 302.17684.
(S,E)-7-(6-(6-(tert-Butyldimethylsilyloxy)-2,5,7,8-tetramethyl-
chroman-2-yl)hex-5-enyl)-5,5-difluoro-1,3-dimethyl-5H-dipyrrolo-
[1,2-c:10,20-f][1,3,2]diazaborinin-4-ium-5-uide (8a, n = 1).45 Vinyl
Trolox 7 (61.9 mg, 0.179 mmol) and hexenylBODIPY 6a (108 mg,
0.357 mmol) were dissolved in dry dichloromethane (1.5 mL).
Grubbs second generation catalyst (15.2 mg, 0.018 mmol) was
then added, and the reaction refluxed for 6 h. The mixture was
condensed in vacuo, and the crude residue was loaded onto a silica
column (hexanes/diethyl ether 6:1) to obtain 8a (72.3 mg, 0.117
mmol, 65%). Dark red oil, Rf = (0.50, hexanes/diethyl ether 3:2).
1H NMR (300 MHz, CDCl3) δ 7.05 (s, 1H), 6.89-6.88 (d, J = 3.9
Hz, 1H), 6.21-6.19 (d, J = 3.9 Hz, 1H), 6.08 (s, 1H), 5.54-5.50
(m, 2H), 2.96-2.91 (t, J = 7.8 Hz, 2H), 2.56-2.50 (m, 5H), 2.24
(s, 3H), 2.14 (s, 3H), 2.12 (s, 3H), 2.03 (s, 3H), 2.07-2.00 (m, 1H),
1.94-1.71 (m, 3H), 1.68-1.60 (m, 2H), 1.46-1.40 (m, 2H), 1.38
(s, 3H), 1.07 (s, 9H), 0.14 (s, 6H). 13C NMR (75 MHz, CDCl3) δ
160.7, 158.9, 146.1, 144.1, 142.8, 134.6, 134.2, 133.3, 128.6, 128.5,
128.3, 125.7, 123.4, 122.2, 119.8, 117.7, 116.8, 74.8, 32.3, 31.9, 29.0,
28.4, 27.8, 27.3, 26.1, 21.2, 18.6, 14.8, 14.3, 13.4, 12.0, 11.2, -3.4.
11B NMR (96 MHz, CDCl3) 1.27-0.58 (t, J = 32.7 Hz, BF2). 19
F
NMR (282 MHz, CDCl3) -145.0 to -145.5 (overlapping q and
sextet, J = 33.3 Hz, BF2). MS (þEI) m/z 620 (Mþ, 0.6%), 378
(1.3%), 346 (13.2%), 316 (26.8%), 278 (6.1%), 233 (100%), 213
(25.4%), 149 (25.3%), 129 (6.5%), 97 (11.0%), 83 (20.2%), 71
(32.7%), 57 (54.0%), 43 (78.6%). HRMS (EI) calculated for
C36H51BF2N2O2Si: 620.37810, found 620.37428.
(S,E)-7-(7-(6-(tert-Butyldimethylsilyloxy)-2,5,7,8-tetramethyl-
chroman-2-yl)hept-6-enyl)-5,5-difluoro-1,3-dimethyl-5H-dipyrrolo-
[1,2-c:10,20-f][1,3,2]diazaborinin-4-ium-5-uide (8b, n = 2). (61.8 mg,
0.097 mmol, 53%). Dark red oil, Rf = (0.55, hexanes/diethyl ether
1
3:2). H NMR (300 MHz, CDCl3) δ 7.08 (s, 1H), 6.92-6.90 (d,
J = 3.9 Hz, 1H), 6.29-6.28 (d, J = 3.9 Hz, 1H), 6.10 (s, 1H),
5.54-5.51 (m, 2H), 2.99-2.94 (t, J = 7.8 Hz, 2H), 2.58 (s, 3H),
2.56-2.49 (m, 2H), 2.26 (s, 3H), 2.16 (s, 3H), 2.14 (s, 3H), 2.00 (s,
3H), 1.96-1.54 (m, 6H), 1.51-1.39 (m, 4H), 1.39 (s, 3H), 1.07 (s,
9H), 0.13 (s, 6H). 13C NMR (75 MHz, CDCl3) δ 160.8, 158.9,
146.1, 144.1, 142.8, 134.6, 134.2, 133.3, 128.8, 128.6, 128.5, 125.7,
123.4, 122.2, 119.8, 117.7, 116.8, 74.7, 32.4, 32.0, 29.0, 28.8, 28.6,
28.4, 27.2, 26.1, 21.2, 18.6, 14.8, 14.3, 13.4, 12.0, 11.2, -3.4. 11B
NMR (96 MHz, CDCl3) 1.30-0.61 (t, J = 32.7 Hz, BF2). 19F
NMR (282 MHz, CDCl3) -144.9 to -145.5 (overlapping q and
7-(Hept-6-enyl)-5,5-difluoro-1,3-dimethyl-5H-dipyrrolo[1,2-c:10,20-
f][1,3,2]diazaborinin-4-ium-5-uide (6b, n = 2). (313 mg, 0.988 mmol,
34% over two steps). Dark red oil, Rf = (0.45, hexanes/diethyl ether
3:2). λmax excitation in ethanol = 507 nm (ε507 =86000M-1 cm-1),
λ
max emission in ethanol = 511 nm. 1H NMR (300 MHz, CDCl3) δ
7.00 (s, 1H), 6.84-6.83 (d, J = 3.9 Hz, 1H), 6.25-6.23 (d, J = 3.9
Hz, 1H), 6.02 (s, 1H), 5.88-5.75 (ddt, J= 17.0 Hz, J=10.3Hz,J=
6.7 Hz, 1H), 5.05-4.92 (m, 2H), 3.00-2.95 (t, J = 7.8 Hz, 2H), 2.53
(s, 3H), 2.15 (s, 3H), 2.08-2.04 (m, 2H), 1.79-1.69 (m, 2H),
2890 J. Org. Chem. Vol. 75, No. 9, 2010

West et al.
JOCArticle
sextet, J = 33.3 Hz, BF2). MS (þEI) m/z 634 (Mþ, 0.1%), 422
(1.2%), 346 (20.7%), 278 (11.6%), 233 (100%), 213 (24.6%),
195 (3.8%), 149 (11.4%), 116 (2.6%), 73 (11.5%), 41 (7.9%).
HRMS (EI) calculated for C37H53BF2N2O2Si: 634.39375, found
634.39555.
(R)-7-(8-(6-(tert-Butyldimethylsilyloxy)-2,5,7,8-tetramethyl-
chroman-2-yl)octyl)-5,5-difluoro-1,3-dimethyl-5H-dipyrrolo[1,2-
c:10,20-f][1,3,2]diazaborinin-4-ium-5-uide (9c, n = 3). (122.8 mg,
0.189 mmol, 70%). Dark red oil, Rf = (0.55, hexanes/diethyl ether
3:2). 1H NMR (300 MHz, CDCl3) δ 7.06 (s, 1H), 6.91-6.89 (d, J =
3.9 Hz, 1H), 6.30-6.29 (d, J = 3.9 Hz, 1H), 6.08 (s, 1H), 3.02-2.97
(t, J = 7.8 Hz, 2H), 2.58-2.55 (m, 5H), 2.24 (s, 3H), 2.13 (s, 3H),
2.11 (s, 3H), 2.08 (s, 3H), 1.89-1.30 (m, 16H), 1.22 (s, 3H), 1.05 (s,
9H), 0.14(s, 6H). 13C NMR (75 MHz, CDCl3) δ160.9, 158.8, 145.9,
144.0, 142.7, 134.5, 133.3, 128.5, 125.7, 123.5, 123.4, 122.6, 119.8,
117.4, 116.8, 74.4, 39.6, 31.5, 29.6, 29.5, 29.4, 29.3, 28.7, 28.6, 26.1,
23.7, 23.6, 20.9, 18.6, 14.8, 14.3, 13.4, 11.9, 11.2, -3.4. 11B NMR (96
MHz, CDCl3) 1.32-0.63 (t, J = 32.7 Hz, BF2). 19F NMR (282
MHz, CDCl3) -145.0 to -145.4 (overlapping q and sextet, J=33.0
Hz, BF2). MS (þEI) m/z 650 (Mþ, 4.1%), 360 (14.2%), 346
(42.9%), 332 (26.2%), 233 (100%), 213 (21.7%), 195 (6.3%), 149
(3.5%), 55 (2.1%), 43 (4.2%). HRMS (EI) calculated for
C38H57BF2N2O2Si: 650.42505, found 650.42546.
(R)-5,5-Difluoro-7-(6-(6-hydroxy-2,5,7,8-tetramethylchroman-
2-yl)hexyl)-1,3-dimethyl-5H-dipyrrolo[1,2-c:10,20-f][1,3,2]diaza-
borinin-4-ium-5-uide (10a, n = 1).59 Compound 9a (30.0 mg,
0.048 mmol) was dissolved in tetrahydrofuran (1.2 mL), and a
10% solution of hydrochloric acid in methanol (1.2 mL) was
added dropwise. The reaction stirred for 4.5 h and then was
extracted in dichloromethane. After condensation, the crude
residue was loaded onto a column (hexanes/diethyl ether 3:1) to
afford final compound 10a (18.8 mg, 0.039 mmol, 77%). Dark
red oil, Rf = (0.25, hexanes/diethyl ether 3:2), tR = (25.8 min,
acetonitrile, 1.00 mL/min, pHPLC). λmax excitation in ethanol =
507 nm (ε507 = 81000 M-1 cm-1), λmax emission in ethanol =
511 nm. 1H NMR (600 MHz, CDCl3) δ 7.08 (s, 1H), 6.93-6.92 (d,
J = 3.9 Hz, 1H), 6.30-6.29 (d, J = 3.9 Hz, 1H), 6.11 (s, 1H),
2.99-2.96 (t, J = 7.8 Hz, 2H), 2.63-2.61 (t, J = 7.2 Hz, 2H), 2.58
(s, 3H), 2.27 (s, 3H), 2.18 (s, 3H), 2.13 (s, 6H), 1.84-1.71 (m, 4H),
1.63-1.50 (m, 2H), 1.47-1.41 (m, 4H), 1.39-1.35 (m, 2H), 1.22 (s,
3H). 13C NMR (151 MHz, CDCl3) δ 160.9, 159.0, 145.5, 144.5,
142.8, 134.6, 133.3, 128.5, 123.5, 122.6, 121.0, 119.9, 118.5, 117.4,
116.8, 74.5, 39.4, 31.5, 29.9, 29.6, 28.7, 28.6, 23.8, 23.5, 20.8, 14.9,
12.2, 11.8, 11.3. 11B NMR (96 MHz, CDCl3) 1.28-0.60 (t, J = 32.7
Hz, BF2). 19F NMR (282 MHz, CDCl3) -145.1 - -145.5 (over-
lapping q and sextet, J = 33.3 Hz, BF2). MS (þEI) m/z 508 (Mþ,
2.4%), 488 ([M - HF]þ, 44.4%), 368 (2.8%), 325 (16.8%), 277
(3.9%), 227 (6.9%), 213 (14.2%), 149 (10.4%), 129 (19.1%),
112 (10.7%), 83 (15.2%), 71 (16.2%), 57 (30.4%), 43 (100%).
HRMS (EI) calculated for C30H38BFN2O2: 488.30104, found
488.30050.
(R)-5,5-Difluoro-7-(7-(6-hydroxy-2,5,7,8-tetramethylchro-
man-2-yl)heptyl)-1,3-dimethyl-5H-dipyrrolo[1,2-c:10,20-f][1,3,2]-
diazaborinin-4-ium-5-uide (10b, n = 2). (14.8 mg, 0.029 mmol,
66%). Dark red oil, Rf = (0.10, hexanes/diethyl ether 3:1), tR =
(29.1 min, acetonitrile, 1.00 mL/min, pHPLC). λmax excitation
in ethanol = 507 nm (ε507 = 85 000 M-1 cm-1), λmax emission in
ethanol = 511 nm. 1H NMR (600 MHz, CDCl3) δ 7.08 (s, 1H),
6.93-6.92 (d, J = 3.9 Hz, 1H), 6.30-6.29 (d, J = 3.9 Hz, 1H),
6.11 (s, 1H), 2.99-2.98 (t, J = 7.8 Hz, 2H), 2.63-2.61 (t, J = 7.2
Hz, 2H), 2.58 (s, 3H), 2.26 (s, 3H), 2.18 (s, 3H), 2.13 (s, 6H),
1.82-1.72 (m, 4H), 1.62-1.53 (m, 2H), 1.45-1.28 (m, 8H), 1.22
(s, 3H). 13C NMR (151 MHz, CDCl3) δ 161.0, 159.0, 145.5,
144.5, 142.8, 134.6, 133.3, 128.5, 123.5, 122.6, 121.0, 119.9,
118.5, 117.4, 116.8, 74.5, 39.5, 31.5, 30.0, 29.5, 29.4, 28.7, 28.6,
23.8, 23.6, 20.8, 14.9, 12.2, 11.8, 11.3. 11B NMR (96 MHz,
CDCl3) 1.29-0.60 (t, J = 32.7 Hz, BF2). 19F NMR (282 MHz,
CDCl3) -145.1 to -145.5 (overlapping q and sextet, J = 33.0
Hz, BF2). MS (þEI) m/z 522 (Mþ, 12.1%), 502 ([M - HF]þ,
100%), 368 (4.8%), 302 (26.1%), 227 (14.1%), 213 (25.1%), 205
(12.5%), 165 (12.8%), 149 (22.8%), 129 (32.7%), 112 (19.5%),
86 (31.3%), 73 (49.7%), 57 (53.5%), 45 (26.1%). HRMS (EI)
calculated for C31H40BFN2O2: 502.31669, found 502.31872.
(S,E)-7-(8-(6-(tert-Butyldimethylsilyloxy)-2,5,7,8-tetramethyl-
chroman-2-yl)oct-7-enyl)-5,5-difluoro-1,3-dimethyl-5H-dipyrrolo-
[1,2-c:10,20-f][1,3,2]diazaborinin-4-ium-5-uide (8c, n = 3). (174 mg,
0.268 mmol, 48%). Dark red oil, Rf = (0.55, hexanes/diethyl
ether 3:2). 1H NMR (300 MHz, CDCl3) δ 7.05 (s, 1H), 6.90-6.89
(d, J = 3.9 Hz, 1H), 6.29-6.28 (d, J = 3.9 Hz, 1H), 6.08 (s, 1H),
5.53-5.50 (m, 2H), 3.00-2.95 (t, J = 7.8 Hz, 2H), 2.57-2.49 (m,
5H), 2.23 (s, 3H), 2.16 (s, 3H), 2.14 (s, 3H), 2.06(s, 3H), 2.02-1.67
(m, 6H), 1.46-1.32 (m, 6H), 1.39 (s, 3H), 1.07 (s, 9H), 0.14 (s,
6H). 13C NMR (75 MHz, CDCl3) δ 160.8, 158.8, 146.1, 144.0,
142.7, 134.5, 133.9, 133.3, 128.8, 128.5, 128.4, 125.6, 123.4, 122.1,
119.7, 117.6, 116.7, 74.7, 32.3, 32.1, 29.1, 28.9, 28.7, 28.6, 28.5,
27.2, 26.1, 21.1, 18.5, 14.7, 14.3, 13.4, 12.0, 11.1, -3.4. 11B NMR
(96 MHz, CDCl3) 1.31-0.62 (t, J = 32.7 Hz, BF2). 19F NMR
(282 MHz, CDCl3) -145.0 to -145.4 (overlapping q and sextet,
J = 33.0 Hz, BF2). MS (þEI) m/z 648 (Mþ, 0.8%), 406 (11.8%),
344 (23.8%), 233 (100%), 213 (26.2%), 194 (3.8%), 117 (2.5%),
55 (4.0%), 41 (5.4%). HRMS (EI) calculated for C38H55-
BF2N2O2Si: 648.40940, found 648.41062.
(R)-7-(6-(6-(tert-Butyldimethylsilyloxy)-2,5,7,8-tetramethyl-
chroman-2-yl)hexyl)-5,5-difluoro-1,3-dimethyl-5H-dipyrrolo[1,2-
c:10,20-f][1,3,2]diazaborinin-4-ium-5-uide (9a, n = 1).48 A mixture
of 8a (93.6 mg, 0.151 mmol) and Wilkinson’s catalyst (69.8 mg,
0.075 mmol) in absolute ethanol (25 mL) was shaken under an
atmosphere of hydrogen gas (80 psi) for 52 h. Afterward, the
crude product was condensed in vacuo and purified via column
chromatography (hexanes/diethyl ether 6:1) to afford pure 9a
(32.6 mg, 0.052 mmol, 35%). Dark red oil, Rf = (0.50, hexanes/
diethyl ether 3:2). 1H NMR (300 MHz, CDCl3) δ 7.06 (s, 1H),
6.91-6.89 (d, J = 3.9 Hz, 1H), 6.28-6.27 (d, J = 3.9 Hz, 1H),
6.08 (s, 1H), 3.00-2.94 (t, J = 7.8 Hz, 2H), 2.56-2.53 (m, 5H),
2.24 (s, 3H), 2.10 (s, 3H), 2.08 (s, 3H), 2.06 (s, 3H), 1.87-1.27 (m,
12H), 1.22 (s, 3H), 1.05 (s, 9H), 0.14 (s, 6H). 13C NMR (75 MHz,
CDCl3) δ 160.9, 158.9, 145.9, 144.0, 142.8, 134.6, 133.3, 128.5,
125.8, 123.5, 123.1, 122.7, 119.8, 117.5, 116.7, 74.4, 39.6, 31.5,
29.9, 29.7, 28.7, 28.6, 26.1, 23.8, 23.5, 20.9, 18.6, 14.8, 14.3, 13.4,
11.9, 11.2, -3.4. 11B NMR (96 MHz, CDCl3) 1.29-0.60 (t, J =
32.7 Hz, BF2). 19F NMR (282 MHz, CDCl3) -145.1 to -145.5
(overlapping q and sextet, J = 33.3 Hz, BF2). MS (þEI) m/z 622
(Mþ, 2.6%), 433 (100%), 302 (2.8%), 233 (16.5%), 208 (7.7%),
168 (53.1%), 149 (26.8%), 69 (12.2%), 55 (18.4%), 43 (30.8%).
HRMS (EI) calculated for C36H53BF2N2O2Si: 622.39375, found
622.39439.
(R)-7-(7-(6-(tert-Butyldimethylsilyloxy)-2,5,7,8-tetramethyl-
chroman-2-yl)heptyl)-5,5-difluoro-1,3-dimethyl-5H-dipyrrolo[1,2-
c:10,20-f][1,3,2]diazaborinin-4-ium-5-uide (9b, n = 2). (27.3 mg,
0.043 mmol, 58%). Dark red oil, Rf = (0.55, hexanes/diethyl
ether 3:2). 1H NMR (300 MHz, CDCl3) δ 7.06 (s, 1H), 6.90-6.89
(d, J = 3.9 Hz, 1H), 6.29-6.27 (d, J = 3.9 Hz, 1H), 6.08 (s, 1H),
2.99-2.94 (t, J = 7.8 Hz, 2H), 2.56-2.53 (m, 5H), 2.24 (s, 3H),
2.10 (s, 3H), 2.08 (s, 3H), 2.06 (s, 3H), 1.83-1.68 (m, 4H),
1.46-1.42 (m, 4H), 1.41-1.27 (m, 6H), 1.22 (s, 3H), 1.05 (s,
9H), 0.14 (s, 6H). 13C NMR (151 MHz, CDCl3) δ 161.0, 158.9,
145.9, 144.0, 142.8, 134.6, 133.3, 128.5, 125.6, 123.5, 123.4, 122.7,
119.9, 117.5, 116.9, 74.5, 39.6, 31.5, 30.0, 29.7, 29.5, 28.7, 28.6,
26.1, 23.8, 23.5, 20.9, 18.6, 14.7, 14.2, 13.4, 12.0, 11.3, -3.3. 11B
NMR (96 MHz, CDCl3) 1.29-0.60 (t, J = 32.7 Hz, BF2). 19F
NMR (282 MHz, CDCl3) -145.1 to -145.6 (overlapping q and
sextet, J = 33.3 Hz, BF2). MS (þEI) m/z 636 (Mþ, 1.1%), 394
(46.0%), 380 (31.0%), 318 (5.4%), 248 (13.0%), 233 (100%), 213
(38.3%), 149 (6.1%), 115 (2.4%), 91 (14.8%), 57 (4.5%), 43
(14.5%). HRMS (EI) calculated for C37H55BF2N2O2Si: 636.40940,
found 636.40915.
J. Org. Chem. Vol. 75, No. 9, 2010 2891

West et al.
JOCArticle
(R)-5,5-Difluoro-7-(8-(6-hydroxy-2,5,7,8-tetramethylchroman-
2-yl)octyl)-1,3-dimethyl-5H-dipyrrolo[1,2-c:10,20-f][1,3,2]diaza-
borinin-4-ium-5-uide (10c, n = 3). (17.0 mg, 0.033 mmol, 74%).
Dark red oil, Rf = 0.25 (hexanes/diethyl ether 3:2), tR = (33.4 min,
acetonitrile, 1.00 mL/min, pHPLC). λmax excitation in ethanol =
507 nm (ε507 = 83000 M-1 cm-1), λmax emission in ethanol = 511
nm. 1H NMR (600 MHz, CDCl3) δ 7.08 (s, 1H), 6.93-6.92 (d, J =
3.9 Hz, 1H), 6.31-6.30 (d, J = 3.9 Hz, 1H), 6.11 (s, 1H), 2.99-2.97
(t, J = 7.8 Hz, 2H), 2.63-2.61 (t, J = 7.2 Hz, 2H), 2.58 (s, 3H), 2.26
(s, 3H), 2.18 (s, 3H), 2.13 (s, 6H), 1.84-1.71 (m, 4H), 1.62-1.54
13C NMR (151 MHz, CDCl3) δ 160.9, 158.9, 142.7, 134.6, 133.3,
130.7, 130.3, 129.9, 129.8, 128.5, 123.4, 119.8, 116.9, 32.5,
32.4, 31.0, 29.6, 29.43, 29.39, 28.6, 28.5, 28.3, 28.09, 28.06,
27.1, 14.9, 11.3. MS (þEI) m/z 548 (4.0%), 562 (5.6%), and
576 (5.2%) corresponding to chain lengths of 9-11 carbons in
the linking alkyl chain. HRMS (EI) calculated for C30H34-
B2F4N4: 548.2906, found 548.2911; calculated for C31H36-
B2F4N4: 562.3062, found 562.3063; calculated for C32H38B2-
F4N4: 576.3219, found 576.3212.
(m, 2H), 1.44-1.41 (m, 4H), 1.35-1.28 (m, 6H), 1.23 (s, 3H). 13
C
Acknowledgment. This work was supported by an
NSERC Discovery Grant (J.A.) and an NSERC Post-
Graduate Scholarship (R.W.), and award DK067494 from
the National Institutes of Health. Metathesis catalysts were
provided by Richard Pederson, Director Fine Chemicals
R&D, Materia Inc. Pasadena, CA.
NMR (151 MHz, CDCl3) δ 161.0, 159.0, 145.5, 144.5, 142.8, 134.6,
133.3, 128.5, 123.5, 122.6, 121.0, 119.9, 118.5, 117.4, 116.9, 74.5,
39.5, 31.5, 30.1, 29.6, 29.5, 29.4, 28.7, 28.6, 23.8, 23.6, 20.8, 14.9, 12.2,
11.8, 11.3. 11B NMR (96 MHz, CDCl3) 1.29-0.60 (t, J = 32.7 Hz,
BF2). 19F NMR (282 MHz, CDCl3) -145.2 to -145.5 (overlapping
q and sextet, J = 33.0 Hz, BF2). MS (þEI) m/z 536 (Mþ, 1.4%),
516 ([M - HF]þ, 36.8%), 408 (3.1%), 353 (10.1%), 298 (33.3%),
278 (3.6%), 233 (17.6%), 213 (18.4%), 191 (3.8%), 171 (100%),
129 (14.1%), 97 (17.0%), 69 (22.3%), 55 (33.3%), 43 (47.1%).
HRMS (EI) calculated for C32H42BFN2O2: 516.33234, found
516.33308.
Note Added after ASAP Publication. Acknowledgement
for a shared NIH grant was omitted in the version published
ASAP on April 13, 2010; the corrected version was reposted on
April 30, 2010.
Bis-BODIPY Mixture (11). (21.0 mg, 0.037 mmol, 17% based
on starting material 6a). Copper red oil, Rf = 0.20 (hexanes/
diethyl ether 3:2). 1H NMR (600 MHz, CDCl3) δ 7.07 (s, 2H),
6.91 (d, J = 4.0 Hz, 2H), 6.30 (d, J = 3.6 Hz, 2H), 5.43-5.52 (m,
2H), 2.99 (t, J = 7.6 Hz, 4H), 2.57 (s, 6H), 2.26 (s, 6H), 2.19 (m,
4H), 2.06-2.14 (m, 4H), 1.72-1.82 (m, 4H), 1.51-1.60 (m, 4H).
Supporting Information Available: Proton and carbon
NMR spectra for compounds 2-4, 6, 8-10 and mixture 11;
fluorescence spectra of 10a-c; and LC-MS spectra for mixture
11 and products from cross coupling metathesis of 11 with 7.
This material is available free of charge via the Internet at http://
pubs.acs.org.
2892 J. Org. Chem. Vol. 75, No. 9, 2010