Design, Synthesis, and Evaluation of a Luminescent Cholesterol Mimic

Article abstract of DOI:10.1021/acs.joc.0c02460

The development of new chemical tools with improved properties is essential to chemical and cell biology. Of particular interest is the development of mimics of small molecules with important cellular function that allow the direct observation of their trafficking in a cell. To this end, a novel 15-azasterol has been designed and synthesized as a luminescent cholesterol mimic for the monitoring of cholesterol trafficking. The brightness of this probe, which is ～32-times greater than the widely used dehydroergosterol probe, is combined with resistance to photobleaching in solution and in human fibroblasts and an exceptionally large Stokes-like shift of ～150-200 nm. The photophysical properties of the probe have been studied experimentally and computationally, suggesting an intersystem crossing to the triplet excited state with subsequent phosphorescent decay. Molecular dynamics simulations show a similar binding mode of cholesterol and the azasterol probe to NPC proteins, demonstrating the structural similarity of the probe to cholesterol.

Full text of DOI:10.1021/acs.joc.0c02460

pubs.acs.org/joc
Article
Design, Synthesis, and Evaluation of a Luminescent Cholesterol
Mimic
Emily M. Work, Guillermo Ferraudi, Luke Kiefer, Gang Liu, Michael Grigalunas, Atul Bhardwaj,
̈
Rasmin Kaur, Janel M. Dempsey, Daniel Wustner, Paul Helquist,* and Olaf Wiest*
Cite This: J. Org. Chem. 2021, 86, 1612−1621
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: The development of new chemical tools with improved properties is essential to
chemical and cell biology. Of particular interest is the development of mimics of small molecules
with important cellular function that allow the direct observation of their trafficking in a cell. To
this end, a novel 15-azasterol has been designed and synthesized as a luminescent cholesterol
mimic for the monitoring of cholesterol trafficking. The brightness of this probe, which is ∼32-
times greater than the widely used dehydroergosterol probe, is combined with resistance to
photobleaching in solution and in human fibroblasts and an exceptionally large Stokes-like shift
of ∼150−200 nm. The photophysical properties of the probe have been studied experimentally
and computationally, suggesting an intersystem crossing to the triplet excited state with
subsequent phosphorescent decay. Molecular dynamics simulations show a similar binding mode
of cholesterol and the azasterol probe to NPC proteins, demonstrating the structural similarity of
the probe to cholesterol.
cholestatrienol (CTL) (Figure 1) are structurally similar to
cholesterol and intrinsically fluorescent, but their photo-
INTRODUCTION
■
Cholesterol plays a vital role in the cell processes of eukaryotic
organisms. It is important in cell signaling, regulating
membrane fluidity, and biosynthesis of steroid hormones.1
Furthermore, the disruption of cholesterol trafficking results in
its accumulation, which can lead to diseases such as Niemann-
Pick type C (NPC) disease and other lipid-associated
disorders.2 The understanding of cholesterol processing and
transport in cells and organisms is therefore very important
from both the basic chemical biology standpoint and the study
of numerous disease states. The need for a method of
monitoring cholesterol trafficking on a cellular level has
resulted in the development of luminescent molecules that
can be used as biological tools for the tracking of cholesterol.
Recent reports of new probes confirm that this is an area of
intense interest,3 with dozens of studies published every year.
A major problem in the field is that strongly luminescent
probes incorporate large fluorophores and thus fail to preserve
key structural features of cholesterol, which can result in
dissimilar interactions with proteins or in lipid bilayers.4 On
the other hand, the photophysical properties of commonly
used structural analogs of sterols leave much to be desired,
especially under the conditions of long time-scale microscopy
on living cells,4 during which the brightness of a probe fades
significantly due to photodecomposition and photobleaching.
To serve as an effective cholesterol mimic, a chemical probe
must be strongly luminescent at excitation wavelengths >300
nm and have high structural similarity to cholesterol.
Dehydrosterols are a widely used type of probe for cholesterol
tracking. Compounds such as dehydroergosterol (DHE) and
Figure 1. Structures of common dehydrosterols.
physical properties are not optimal.5−8 The low extinction
coefficient (10500 M−1 cm−1 in methanol) and low
fluorescence quantum yield (0.024 in methanol) of DHE
combine to give low molecular brightness,9 which is even
further diminished by rapid photobleaching. DHE is known to
rapidly decompose upon exposure to ultraviolet (UV) light,
even in deaerated solutions.10 These pitfalls can lead to poor
imaging or require the use of large quantities of the probes.
Consequently, there is an urgent need for a better analog that
incorporates a strong fluorophore while avoiding photo-
bleaching pathways.
Received: October 16, 2020
Published: December 28, 2020
https://dx.doi.org/10.1021/acs.joc.0c02460
© 2020 American Chemical Society
J. Org. Chem. 2021, 86, 1612−1621
1612

The Journal of Organic Chemistry
pubs.acs.org/joc
Article
A variety of azasteroids have been reported to have
biological activities, suggesting that they can interact with
cholesterol binding sites. Common azasteroid scaffolds include
functionalization of the aliphatic side chain as well as
incorporation of amide,11−13 amine,11−14 imine,14−17 or
pyrazole18 moieties in the carbocyclic steroid core (Figure
2a). These azasteroids have mainly been studied for their
Figure 3. Structure of cholesterol and proposed azasterol (1).
system would undergo rapid intersystem crossing (ISC) to the
triplet excited state, resulting in a lower energy emission that
would be desirable to separate the excitation and detection
wavelengths, thus increasing the signal/noise ratio in
fluorescence microscopy. Rapid ISC is well-known for
aromatic aldehydes and ketones, such as benzophenone,31
and has been more recently reported for an aromatic donor−
acceptor imine.32
This design prompted the development of a convergent
synthesis for 1 that is based on a Michael addition of an
enolate to a nitroalkene to give nitroketone 7 (Scheme 1).33
Nitroalkene 5 could be obtained in 67% yield over four steps
on a gram scale without intermediate chromatographic
purification starting from readily available 1-bromo-3-methyl-
butane and tert-butyl acrylate. Copper-catalyzed conjugate
addition of isopentylmagnesium bromide to tert-butyl acrylate
yielded ester 2, which was subjected to reduction with DIBAL
to give aldehyde 3 in very good yield. Henry reaction of 3 with
nitromethane followed by dehydration of nitrol 4 afforded
nitroalkene 5 in excellent yield. The other building block,
ketone 6, was obtained by a modified literature procedure from
6-methoxy-1-tetralone and methyl 4-bromocrotonate.16,34 The
conjugate addition of the potassium enolate of 6 to 5 gave
nitroketone 7 in 93% yield as a 4:1 mixture of diastereomers.
Following Raney nickel-catalyzed hydrogenation of the nitro
group and spontaneous cyclization to form the conjugated
imine, the diastereomers could be separated via chromatog-
raphy or crystallization. The relative stereochemistry of the
major diastereomer was confirmed via X-ray crystallography
(Figure S7). Deprotection of the methyl ether moiety of 8 with
boron tribromide yielded 1 as a crystalline racemate in 22%
overall yield over seven linear steps. The final three steps could
be conducted on millimole scales without significant reduction
in yield, allowing access to hundreds of milligrams of ( )-1,
which is readily available for biological applications. While this
racemate is useful for certain applications, the enantiomers
may be separated by chiral HPLC (see Supporting
Information) for cases in which the properties are dependent
on stereochemistry. Absolute configuration assignments were
made by X-ray crystallography (Figure S8) and circular
dichroism (CD) spectroscopy (Figure S9).
Figure 2. (a) Representative examples of reported azasteroids and (b)
luminescent 8-azasteroid scaffold.
antibacterial and antifungal activities. Although some analogs
incorporate fluorophores, their luminescent properties have to
the best of our knowledge not been examined or exploited. A
common type of luminescent azasteroid is based on an 8-
azasteroid scaffold (Figure 2b), the fluorescent and phosphor-
escent properties of which have been extensively studied.19−25
However, their photophysical properties have not been
optimized for use as chemical probes. In addition, their
structures are not well-suited as probes in the study of
cholesterol trafficking due to the lack of an aliphatic side chain
at C17. This side chain plays a notable role in binding of
cholesterol to protein targets26,27 and in membrane inter-
actions.28 We thus designed a new luminescent azasterol probe
that would possess the key structural features of cholesterol as
well as superior photophysical properties. Desirable photo-
physical properties include strong emission upon irradiation
with wavelengths above 300 nm to prevent interfering
absorption by other cell components and UV damage to the
cells under study, and stability for extended periods of time
under irradiation to prevent photobleaching or undesired side
reactions.
Studies were performed to confirm that ( )-1 possessed
desired luminescent properties. To give insight into the
photophysical behavior of 1, we compared the luminescent
properties in methanol and water as examples for hydrophilic
environments and 1-octanol as a model for lipophilic
environments such as membranes. The absorbance of 1 was
similar in each solvent (Figure 4), and the absorbance
maximum (λabs) was above 300 nm, which is important to
reduce interfering absorption, autofluorescence, and UV
damage in cell studies. While the longest wavelength
absorption is similar to the known probes DHE9 and CTL,35
the molar extinction coefficient (ε) is 40−50% higher (Table
1). The long wavelength absorption in aryl imines is generally
RESULTS AND DISCUSSION
■
Our probe design is based on the replacement of the A and B
rings of cholesterol by a naphthalene conjugated π-system as a
means of introducing a fluorophore. We envisioned an
improvement of the photophysical properties through this
incorporation of an aromatic system with extended con-
jugation. A second design element to enhance the luminescent
properties was to introduce a push−pull effect29,30 by replacing
C15 with an imine nitrogen. These structural changes in analog
1 are highlighted in color in Figure 3. As discussed in the
computational studies, we predicted that a conjugated imine
1613
https://dx.doi.org/10.1021/acs.joc.0c02460
J. Org. Chem. 2021, 86, 1612−1621

The Journal of Organic Chemistry
pubs.acs.org/joc
Article
Scheme 1. Synthesis of Azasterol 1
Table 1. Photophysical Properties of 1 and Other
Luminescent Sterols
probe
λabs (nm)
ε (M−1 cm−1
)
λem (nm)
Φ
a
1
321
308
316
324.4
15 000
15 000
15 100
10 500
11 250
504
514
526
372.5
374
0.03
0.54
0.10
0.024
-
b
c
1
1
a
9
,
DHE
CTL
d
35
,
324
a
b
c
Water as solvent. Methanol as solvent. Octanol as solvent.
Ethanol as solvent.
d
Figure 5. Irradiation with hand-held UV lamp (254 nm) of 10 μM
solutions of (left to right) 1 in water, 1 in methanol, 1 in octanol,
DHE in methanol, and 1 in methanol after 30 min of UV irradiation.
DHE. The quantum yield of 1 in octanol remains higher than
that of DHE in various solvents,9 suggesting that the improved
brightness holds in lipophilic environments. Resistance to
photobleaching was demonstrated by the ability of 1 to
luminesce after 30 min of UV irradiation (Figure 5). Although
the probe was designed to have a large shift in emission relative
to excitation, the observed shift of ∼200 nm was much larger
than anticipated, exceeding the criteria of mega-Stokes shifts
(>100 nm) desired for bioimaging.37,38 Mega-Stokes shifts of
this magnitude are unusual for organic fluorophores,
prompting further investigation of the photophysics of 1,
which suggested that the observed shift in emission was not a
true Stokes shift due to participation of a triplet excited state.
Given the remarkable properties of 1, flash photolysis
experiments were conducted to give more detailed information
Figure 4. Absorbance (top) and emission (bottom, λex = 310 nm)
spectra of 10 μM solutions of 1 in water (blue), methanol (red), and
octanol (green), measured at 22 °C.
thought to derive from a n → π* excitation,36 which is
confirmed by computational studies (Figure S11).
Significant differences in emission were observed under
continuous irradiation in the different solvents (Figures 4 and
5). While emission was brightest in methanol, we were pleased
to find that it was readily detectable in both water and octanol.
Furthermore, the luminescence quantum yield (Φ) of 1 was
0.54 in methanol, giving a brightness that is 32-times that of
1614
https://dx.doi.org/10.1021/acs.joc.0c02460
J. Org. Chem. 2021, 86, 1612−1621

The Journal of Organic Chemistry
pubs.acs.org/joc
Article
about the photophysical behavior. A deaerated solution of
( )-1 in methanol was irradiated at 308 nm in a stationary cell
to produce the transient absorption spectrum shown in Figure
6. A trace was recorded at λob = 360 nm to show the decay of
excited states using time-dependent density functional theory
(TDDFT) (Table S3). These results were used to simulate
absorption spectra using Gaussian functions with a full width at
half-maximum (fwhm) of 0.3 eV to convolute the peaks. The
calculated absorption spectrum shows good agreement with
experiment (Figure 7). As expected, the first excited state
corresponds to the n → π* transition (Figure S6).
Figure 7. Experimental (green) and calculated (black) absorption
spectra of 1 in 1-octanol. Gaussian functions with a fwhm of 0.3 eV
were used to convolute the calculated stick spectrum.
The structures of the S1 and T1 states in 1-octanol were
optimized using TDDFT/PCM/B3LYP-D3/6-311++G** and
ΔSCF/PCM/B3LYP-D3/6-311++G**, respectively. Vertical
emission energies were calculated and compared to experiment
(Table 2). On the basis of these results, the higher energy
Figure 6. Transient absorption of ( )-1 in methanol upon irradiation
at 308 nm.
this signal with second order kinetics (Figure S3). The decay
of the emission spectrum at λob = 360 nm approximately
follows second-order kinetics, with small deviations due to
some contribution of decay with first-order kinetics (Figure
S4). The second-order kinetics observed for the decay of the
transient absorption and emission spectra can be attributed to
a triplet−triplet annihilation reaction. The accessibility of the
triplet state for aryl imines is well-known in the literature36 and
confirms our original design of a system undergoing rapid ISC.
It should also be noted that prior to deaeration, the
luminescence was slightly quenched by triplet oxygen, giving
further evidence for the presence of a triplet excited state. It is
important to note that the stability of the baseline absorbance
in the stationary cell confirmed that 1 was resistant to
photobleaching. This is observed even in the presence of
oxygen (Figure 5) and is another important quality for the use
of 1 as a biological probe and a significant improvement over
the DHE and CTL probes.
In an effort to gain a better understanding of the observed
photophysical properties of 1, specifically the structural
elements responsible for the large Stokes-like shift in emission
and the unusual stability of the triplet state even in the
presence of oxygen, a computational study was completed
using Gaussian 16.39 After benchmarking a number of
functionals and basis sets (see Tables S1 and S2 and Figure
S5 in the Supporting Information) against the experimentally
observed spectra, the structure of 1 in the S0 state was
optimized using B3LYP-D3/6-311++G** and solvent effects
of n-octanol were included using the polarizable continuum
model (PCM). Vertical excitation energies and oscillator
strengths were calculated for the 15 lowest singlet and triplet
Table 2. Comparison of Experimental and Calculated
Vertical Emission Wavelengths in 1-Octanol
transition
λexp (nm)
λcalc (nm)
S0 ← S1
S0 ← T1
365
527
374
581
emission at λ = 365 nm in octanol is assigned to the emission
from the S1 state. This emission is not observed experimentally
in methanol or water, both of which show only emission from
the T1 state with wavelength longer than 500 nm. In analogy to
the photochemistry of benzophenone,40 the T1 (n → π*) state
is generated through rapid ISC from the S1 (n → π*) and then
undergoes radiative decay (phosphorescence) to the S0 state.
This observation that an emission from the S1 state is observed
in octanol but not in water or methanol may be explained by a
narrowing of the singlet−triplet energy gap in more polar
solvents, which is known to increase the rate of intersystem
crossing.41
We considered two hypotheses to explain the large Stokes-
like shift in emission wavelength observed for the decay from
T1 to S0: (i) significant changes in the electron distribution
could lead to a solvent reorganization that further lowers the
energy of the T1 state relative to the S1 state and (ii) changes in
the geometry of the molecule could lead to a vibronic
relaxation upon ISC. Comparisons of Hirshfeld charges and
geometries of S0, S1, and T1 showed no notable changes in
charges. In contrast, significant differences in the geometries
were calculated for the three states, especially in the length of
1615
https://dx.doi.org/10.1021/acs.joc.0c02460
J. Org. Chem. 2021, 86, 1612−1621

The Journal of Organic Chemistry
pubs.acs.org/joc
Article
the C8−C9 bond, as shown in Figure 8 for the S0 and T1
states. This bond is elongated upon excitation from the S0 to
Figure 9. Photobleaching kinetics of azasterol and cholestatrienol
(CTL) in cells acquired in the UV channel are shown in A. A
corresponding bleach profile for a bleach stack of azasterol acquired
without emission filter is shown in B.
Figure 8. Highest occupied molecular orbitals of 1 in the optimized
geometries of the S0 and T1 states. The most notable change is in the
length of the C8−C9 bond (denoted by arrow). We rationalize this
change in geometry with contribution from the resonance structure
shown.
Having demonstrated the superior photophysical properties
of 1 over DHE and CTL, we sought to investigate whether our
probe is sufficiently similar to cholesterol for use in protein
binding studies. To study the similarity of 1 to cholesterol and
their interaction to biomedically relevant cholesterol binding
proteins, we compared the binding of 1 and cholesterol to
NPC1 and NPC2, two key lysosomal proteins essential for
maintenance of proper cholesterol homeostasis.42 The crystal
structures of cholesterol bound to the N-terminal domain of
NPC1 (PDB ID: 3GKI, 1.80 Å)43 and to NPC2 (PDB ID:
2HKA, 1.81 Å)42 have been determined. It is known from
these structures that the hydroxyl group and the aliphatic side
chain of cholesterol are key in binding to NPC1 and NPC2,
respectively, suggesting that these moieties need to be
preserved in a successful cholesterol mimic. Docking followed
by 100 ns molecular dynamics (MD) simulations of 1 and
cholesterol to NPC1 and NPC2 proteins showed that 1
overlays well with cholesterol in the binding sites (Figure 10
and Figures S11−S15 in the Supporting Information). Like the
H-bonding interactions observed for cholesterol with ASN 41
of NPC1, the hydroxyl group of 1 also indicated H-bonding
with the side chain amide of the ASN 41 residue (2.06 Å).
Moreover, one of the aromatic rings of 1 also indicated π−π
interactions with the PHE 203 residue of NPC1 (Figure S11).
The free energies of binding for 1 and cholesterol in NPC
proteins were estimated using MM-GBSA rescoring of
snapshots from a 100 ns MD simulation44 and were found
to be similar (Table S5), further supporting our hypothesis
the S1 state and is longer yet in the optimized geometry of the
T1 state. Meanwhile, the C8−C14 bond shortens and the
C14−N15 bond lengthens. These changes can be represented
by the resonance structure shown in Figure 8, which is also
supported by our calculation that the greatest spin density is
on C9 and N15. The aromaticity in the ring is decreased, but
conjugation is extended through participation of the imine
moiety, which has adopted a more enamine-like geometry.
These results are in line with the low energy of the T1 state
leading to the large Stokes shift as well as its relative stability in
the presence of oxygen.
Live cell imaging and bleaching analysis studies were
performed to demonstrate the improved photophysical
properties of 1 in a cellular environment compared to CTL.
In human fibroblasts, 1 showed weak emission in the UV but
very strong emission over the whole spectral range when
excited between 320 and 340 nm. Furthermore, 1 was shown
to bleach very slowly compared to CTL (Figure 9). Pixel-wise
bleaching analysis using a stretched exponential fitting function
showed that CTL bleaches slightly faster than exponential and
fades after a few seconds, whereas bleaching of 1 has a strongly
compressed exponential with a much longer time constant
(Figure S10). Because of a combination of the strong signal in
the absence of a UV emission filter and the long delay before
significant photobleaching takes place, we conclude that 1 can
be imaged for a much longer time than probes such as CTL.
1616
https://dx.doi.org/10.1021/acs.joc.0c02460
J. Org. Chem. 2021, 86, 1612−1621

The Journal of Organic Chemistry
pubs.acs.org/joc
Article
Figure 10. Overlay of cholesterol (gray) and azasterol (1, green) docked in (a) NPC1 and (b) NPC2.
MHz, respectively, or a Bruker AVANCE II 800 instrument operating
that 1 is an effective mimic of cholesterol. These results
strongly suggest that modification of the polycyclic core and
removal of the C21 methyl group do not significantly affect
binding.
1
at 800 and 201 MHz, respectively. All H and 13C spectral data are
reported in ppm (δ) relative to the residual solvent peak for CDCl3 at
7.26 and 77.23 ppm, respectively. Coupling constants (J) are reported
in Hz. High-resolution mass spectrometry (HRMS) was performed
on a Bruker micrOTOF-Q II instrument. Infrared spectra were
recorded on a Thermo Nicolet IR 200 spectrometer. X-ray diffraction
collections were obtained on a Bruker APEX-II diffractometer or a
Bruker PHOTON-II diffractometer. Absorbance spectra were
collected using an Evolution 201 UV−vis spectrometer with
ThermoInsight software. Luminescence spectra were collected using
a Horiba Fluoromax-4 fluorometer with FluorEssence software. Chiral
HPLC was performed using a Waters 1525 Binary HPLC pump and
Waters 2487 dual λ absorbance detector with Breeze software and a
Chiralcel OZ-H column purchased from Chiral Technologies, Inc.
Circular dichroism (CD) spectra were collected using a Jasco J-815
spectrometer. 7-Methoxy-3,4-dihydrophenanthren-1(2H)-one was
prepared from 6-methoxy-1-tetralone and methyl 4-bromocrotonate
according to a literature procedure.34
tert-Butyl 6-methylheptanoate (2). A 100 mL round-bottom
flask with a stir bar was charged with freshly ground turnings of
magnesium (365 mg, 15 mmol) and a crystal of iodine. The flask was
capped with a septum, evacuated, and backfilled with argon (3×).
THF (15 mL) was added, and 1-bromo-3-methylbutane (1.8 mL, 15
mmol) was added dropwise via syringe with stirring. The yellow
solution turned colorless and then dark and cloudy with heat
generation upon initiation. The reaction was stirred for 2 h at 22 °C.
Then the septum was removed, and DMAP (2.443 g, 20 mmol) and
copper(I) bromide-dimethyl sulfide complex (206 mg, 1 mmol) were
quickly added. The reaction turned dark blue immediately. The flask
was fitted with an addition funnel, which was capped with a septum
and placed under an argon atmosphere. The flask was cooled to −78
°C, and the addition funnel was charged with tert-butylacrylate (1.47
mL, 10 mmol), TMSCl (2.54 mL, 20 mmol), and THF (2 mL). This
solution was added dropwise with vigorous stirring, and then the
addition funnel was rinsed with additional THF (2 mL). The reaction
mixture was allowed to warm to 22 °C and stirred for 14 h. The
reaction was quenched with 1 M HCl (70 mL) and extracted with
diethyl ether (3 × 50 mL). The combined organic layers were dried
over anhyd Na2SO4 and concentrated on a rotary evaporator to give a
slightly yellow oil. The crude material was passed through a silica
plug, eluting with 20% diethyl ether in hexanes, and then concentrated
to give 1.67 g (83%) of the title compound as a colorless oil. 1H NMR
(400 MHz, CDCl3) δ 2.20 (t, J = 7.5 Hz, 2H), 1.59−1.49 (m, 3H),
1.44 (s, 9H), 1.33−1.25 (m, 2H), 1.20−1.14 (m, 2H), 0.86 (d, J = 6.6
Hz, 6H); 13C{1H} NMR (201 MHz, CDCl3) δ 173.5, 80.0, 38.8, 35.9,
28.3, 28.0, 27.0, 25.6, 22.8; HRMS (ESI) calcd for C12H24NaO2 [M +
Na] 223.1669, found 223.1661; IR (neat) 2955, 2933, 2870, 1731,
1366, 1151 cm−1.
CONCLUSIONS
■
A novel 15-azasterol 1 was designed as a biochemical tool for
the study of cholesterol trafficking and synthesized in 22%
overall yield in seven linear steps in high purity and on a scale
of 100s of mg. Our probe incorporates a strong fluorophore as
well as key structural properties to serve as an effective
luminescent cholesterol mimic without issues of photo-
bleaching. The probe was shown to have desirable photo-
physical properties including absorption at ∼310−320 nm, a
large Stokes-like shift of ∼150−200 nm due to ISC and
vibronic relaxation, emission in the visible region at ∼450−525
nm with a quantum yield of 0.54 (methanol), and resistance to
photobleaching upon UV irradiation in a stationary cell and in
human fibroblasts. This new probe has the potential to be used
in a variety of applications related to lysosomal storage
disorders and other cholesterol-related diseases, including
time-lapse microscopy, for which use of the DHE probe is very
limited.4,45 Our probe is applicable to the study of intracellular
cholesterol trafficking but may also prove useful in other realms
in which luminescent sterols are used such as the study of
atherosclerosis and Alzheimer’s disease.46 Efforts toward an
asymmetric synthesis of 1 are currently underway to increase
synthetic efficiency for applications where only the native
sterol stereochemistry is desired. Future work will also include
experimental investigation of the binding of 1 in NPC proteins.
Further studies in biological systems will be published in the
future.
EXPERIMENTAL SECTION
■
General Methods. All reactions were conducted in oven-dried
glassware under an atmosphere of argon unless otherwise specified.
Tetrahydrofuran, toluene, and dichloromethane were purified by
passage through an Inert PureSolv Micro solvent purification system.
Other solvents were purified by literature procedures or used as
received. All other reagents were used as received from commercial
sources unless otherwise stated. MPLC was performed to obtain
analytically pure material using a Biotage Isolera Prime (Version
1.5.2) system with Silicycle, Inc. SiliaSep cartridges or Biotage SNAP
KP-SIL cartridges. Corrected melting points were obtained using a
Mel-Temp II device. 1H and 13C spectra were obtained using either a
Bruker AVANCE III HD 400 instrument operating at 400 and 101
1617
https://dx.doi.org/10.1021/acs.joc.0c02460
J. Org. Chem. 2021, 86, 1612−1621

The Journal of Organic Chemistry
pubs.acs.org/joc
Article
6-Methylheptanal (3). A 100 mL round-bottom flask with a stir
bar was charged with ester 2 (2.145g, 10.7 mmol). The flask was
capped with a septum, evacuated, and backfilled with argon (3×).
DCM (27 mL) was added and the solution cooled to −78 °C.
DIBALH (10.7 mL of 1 M solution in DCM, 10.7 mmol) was added
dropwise via syringe. The reaction was stirred at −78 °C for 15 min,
then warmed to 22 °C and stirred for an additional 5 min. Next, 1 M
HCl was added carefully until all aluminum salts were dissolved. The
layers were separated, and the aq layer was extracted with DCM (3 ×
25 mL). The combined organic layers were dried over anhyd Na2SO4
and concentrated to give a clear fragrant liquid, which was partially
purified by passing through a silica plug with DCM and carefully
concentrated to give the title compound (1.213 g, 88%) as a clear,
colorless liquid. Because of volatility issues, this compound was used
over anhyd Na2SO4, and concentrated to a give an orange oil that
solidified upon standing. The crude material was purified by MPLC
(7−15% ethyl acetate in hexane) to give 80 mg (83%) of the title
compound as a white crystalline solid, which was recrystallized from
1
hexane to give colorless needles. Mp = 108−109 °C; H NMR (400
MHz, CDCl3) δ 8.07 (d, J = 8.7 Hz, 1H), 7.99 (d, J = 9.2 Hz, 1H),
7.61 (d, J = 8.6 Hz, 1H), 7.20 (dd, J = 9.2, 2.2 Hz, 1H), 7.13 (d, J =
2.3 Hz, 1H), 3.93 (s, 3H), 3.47 (dt, J = 17.2, 4.1 Hz, 1H), 3.20 (ddd, J
= 17.3, 10.9, 4.8 Hz, 1H), 2.65 (m, 1H), 2.34 (dq, J = 13.1, 4.4 Hz,
1H), 1.93 (qd, J = 20.2, 4.5 Hz, 1H), 1.31 (d, J = 6.7 Hz, 3H);
13C{1H} NMR (101 MHz, CDCl3) δ 200.9, 159.6, 142.6, 137.6,
128.2, 126.5, 126.0, 124.1, 119.1, 107.1, 55.6, 41.7, 30.9, 25.2, 15.6.
(lit.16 1H NMR)
7-Methoxy-2-methyl-2-(7-methyl-1-nitrooctan-2-yl)-3,4-di-
hydrophenanthren-1(2H)-one (7). A 25 mL round-bottom flask
with a stir bar was charged with ketone 6 (240 mg, 1 mmol). The flask
was capped with a septum, evacuated, and backfilled with argon (3×).
THF (13.5 mL) was added, and the solution was cooled to −78 °C.
KHMDS (2.2 mL of 0.5 M solution in toluene, 1.1 mmol) was added
dropwise via syringe, and the solution turned dark red. The solution
was stirred at −78 °C for 30 min, and then a solution of nitroalkene 5
(188 mg, 1.1 mmol in 1 mL THF) was added dropwise to the enolate
solution. The solution turned from dark red to yellow and was
allowed to stir at −78 °C for an additional 30 min. The reaction
mixture was warmed to 22 °C, quenched with satd aq NH4Cl, and
extracted 3 times with DCM. The combined organic layers were
washed with brine, dried over anhyd Na2SO4, and concentrated to a
give a pale yellow oil. The crude material was purified by MPLC (2−
7% ethyl acetate in hexane) to give 381 mg (93%) of the title
compound in 4:1 d.r. as a thick colorless oil. The diastereomers were
characterized as a mixture. Major diastereomer: 1H NMR (400 MHz,
CDCl3) δ 8.05 (d, J = 8.7 Hz, 1H), 8.02 (d, J = 9.2 Hz, 1H), 7.65 (d, J
= 8.7 Hz, 1H), 7.23 (dd, J = 9.2, 2.4 Hz, 1H), 7.15 (d, J = 2.4 Hz,
1H), 4.55 (dd, J = 13.3, 4.8 Hz, 1H), 4.30 (dd, J = 13.3, 6.5 Hz, 1H),
3.95 (s, 3H), 3.42−3.25 (m, 1H), 2.94−2.89 (m, 1H), 2.33 (ddd, J =
13.9, 8.2, 5.6 Hz, 1H), 2.05−1.99 (m, 1H), 1.56−1.43 (m, 2H),
1.42−1.26 (m, 4H), 1.20 (s, 3H), 1.16−1.11 (m, 3H), 0.85 (d, J = 6.6
Hz, 6H); 13C{1H} NMR (101 MHz, CDCl3) δ 201.1, 159.9, 141.2,
137.8, 126.52, 126.47, 126.3, 124.6, 119.4, 107.2, 77.3, 55.6, 47.1,
41.0, 38.9, 30.6, 30.4, 28.5, 28.1, 27.8, 22.8, 21.9, 19.5; Minor
1
directly in the next step without further purification. H NMR (400
MHz, CDCl3) δ 9.76 (t, J = 1.9, 1H), 2.42 (td, J = 7.4, 1.9 Hz, 2H),
1.61 (quint, J = 7.5 Hz, 2H), 1.53 (nonet, J = 6.6 Hz, 1H), 1.36−1.28
(m, 2H), 1.21−1.15 (m, 2H), 0.86 (d, J = 6.6 Hz, 6H); 13C{1H}
NMR (201 MHz, CDCl3) δ 203.1, 44.2, 38.9, 28.0, 27.2, 22.8, 22.5;
IR (neat) 2954, 2931, 2869, 2716, 1726, 1467, 1366, 1155 cm−1.
(lit.47 1H NMR, 13C NMR)
(E)-7-Methyl-1-nitrooct-1-ene (5). A 100 mL round-bottom
flask with a stir bar was charged with aldehyde 3 (1.201 g, 9.4 mmol)
and methanol (3.7 mL) and cooled to 0 °C in an ice bath.
Nitromethane (502 μL, 9.4 mmol) was added dropwise with stirring,
and then a solution of sodium hydroxide (588 mg, 14.7 mmol) in
water (7.3 mL) was added dropwise. The reaction was stirred at 0 °C
for 1 h and then diluted with water (9.2 mL). Conc hydrochloric acid
(2.1 mL) was added slowly, and the mixture was extracted with
diethyl ether. The combined organic layers were washed with brine,
dried over anhyd Na2SO4, and concentrated to give a pale yellow oil
(1.725 g, 97%), which was used directly in the next step without
further purification. An analytically pure sample was obtained by
1
MPLC (7−10% ethyl acetate in hexane) as a colorless oil. H NMR
(400 MHz, CDCl3) δ 4.43 (dd, J = 12.7, 2.7 Hz, 1H), 4.37 (dd, J =
12.7, 8.3 Hz, 1H), 4.33−4.27 (m, 1H), 2.65 (s, 1H), 1.58−1.41 (m,
4H), 1.38−1.24 (m, 3H), 1.17 (q, J = 7.1 Hz, 2H), 0.86 (d, J = 6.6
Hz, 6H); 13C{1H} NMR (101 MHz, CDCl3) δ 80.9, 68.9, 38.9, 34.0,
28.1, 27.3, 25.6, 22.8, 22.7; IR (neat) 3411, 2952, 2930, 2867, 1551,
1384 cm−1.
1
A 100 mL round-bottom flask with a stir bar was charged with
nitrol 4 (1.725 g, 9.1 mmol) and DCM (5.4 mL). Trifluoroacetic
anhydride (1.27 mL, 9.1 mmol) was added dropwise with stirring.
Then the reaction was cooled to −10 °C in an ice/brine bath and
stirred for 2 min. Triethylamine (2.54 mL, 18.2 mmol) was added
slowly dropwise over several min. The reaction was stirred at −10 °C
for an additional 30 min and then was diluted with DCM (6.1 mL)
and washed with satd aq ammonium chloride. The organic layer was
dried over anhyd Na2SO4 and concentrated to give a crude yellow−
orange oil. The crude material was purified by MPLC (2−5% DCM in
hexane) to give the title compound (1.482 g, 95%) as a colorless oil.
diastereomer: H NMR (400 MHz, CDCl3) δ 8.05 (d, J = 8.7 Hz,
1H), 8.02 (d, J = 9.2 Hz, 1H), 7.65 (d, J = 8.7 Hz, 1H), 7.23 (dd, J =
9.2, 2.4 Hz, 1H), 7.15 (d, J = 2.4 Hz, 1H), 4.68 (dd, J = 13.0, 4.1 Hz,
1H), 4.43 (dd, J = 13.0, 7.4 Hz, 1H), 3.95 (s, 3H), 3.52−3.44 (m,
1H), 2.70 (sept, J = 3.9 Hz, 1H), 2.26 (ddd, J = 13.9, 8.2, 5.6 Hz, 1H),
2.12−2.07 (m, 1H), 1.56−1.43 (m, 2H), 1.42−1.26 (m, 4H), 1.24 (s,
3H), 1.07−1.05 (m, 3H), 0.79 (d, J = 6.6 Hz, 6H); 13C{1H} NMR
(101 MHz, CDCl3) δ 201.4, 141.1, 137.7, 137.3, 127.4, 127.1, 126.6,
119.5, 78.5, 42.3, 38.8, 31.8, 29.6, 28.6, 28.0, 27.6, 22.73, 22.67, 22.2,
19.7, other resonances may be indistinguishable from those
corresponding to the major diastereomer; HRMS (ESI) calcd for
C25H34NO4 [M + H] 412.2482, found 412.2491; IR (neat) 2932,
2866, 1666, 1619, 1550, 1223, 1033, 857, 821, 780 cm−1.
7-Methoxy-11a-methyl-1-(5-methylhexyl)-2,10,11,11a-tet-
rahydro-1H-naphtho[1,2-g]indole (8). A 250 mL round-bottom
flask with a stir bar was charged with nitroketone 7 (380 mg, 0.92
mmol) and ethanol (10 mL), evacuated, and backfilled with argon.
Raney nickel (880 mg of ∼50% slurry in water) was washed with
water (3 × 2 mL) and then ethanol (3 × 2 mL). The washed Raney
nickel was suspended in ethanol (4 mL) and transferred via pipet into
the reaction flask with rapid stirring. The flask was evacuated and
backfilled with a balloon of hydrogen gas (3×). The reaction was
allowed to stir under H2 (1 atm) at 22 °C for 7 h. The flask was
evacuated and backfilled with argon, and the reaction mixture was
diluted with DCM, filtered through Celite, and concentrated to give a
pale yellow oil. The crude material was purified by MPLC (15% ethyl
acetate in hexane) to give 310 mg (92%) combined yield of the
separated diastereomers of the title compound as a white solid. The
major diastereomer was recrystallized from hexane to yield colorless
crystals for characterization. Single crystals suitable for X-ray
1
The yield over three steps was 81% based on the starting ester. H
NMR (400 MHz, CDCl3) δ 7.28 (dt, J = 13.7, 7.2 Hz, 1H), 6.98 (d, J
= 13.4 Hz, 1H), 2.27 (qd, J = 7.2, 1.3 Hz, 2H), 1.58−1.46 (m, 3H),
1.37−1.30 (m, 2H), 1.21−1.15 (m, 2H), 0.87 (d, J = 6.6 Hz, 6H);
13C{1H} NMR (101 MHz, CDCl3) δ 143.0, 139.8, 38.7, 28.7, 28.2,
28.0, 27.1, 22.7; HRMS (ESI) calcd for C9H17NO2 [M + H]
172.1332, found 172.1337; IR (neat) 3105, 2954, 2930, 2867, 1650,
1524, 1467, 1350 cm−1.
7-Methoxy-2-methyl-3,4-dihydrophenanthren-1(2H)-one
(6). A 10 mL crimp cap vial with a stir bar was capped, evacuated, and
backfilled with argon (3×). LiHMDS (0.42 mL of 1 M solution in
THF, 0.42 mmol) and THF (2.5 mL) were added, and the solution
was cooled to 0 °C. A solution of 7-methoxy-3,4-dihydrophenanthren-
1(2H)-one34 (91 mg, 0.40 mmol in 1.5 mL THF) was added
dropwise. The solution was stirred at 0 °C for 15 min, and then
iodomethane (27.4 μL, 0.44 mmol) was added dropwise to the
enolate solution. The reaction mixture was warmed to 22 °C, stirred
for 14 h, quenched with satd aq NH4Cl, and extracted 3 times with
DCM. The combined organic layers were washed with brine, dried
1618
https://dx.doi.org/10.1021/acs.joc.0c02460
J. Org. Chem. 2021, 86, 1612−1621

The Journal of Organic Chemistry
pubs.acs.org/joc
Article
diffraction analysis were obtained as colorless plates via cooling of a
concentrated solution in diethyl ether. mp = 97−98 °C; H NMR
Paul Helquist − Department of Chemistry and Biochemistry,
University of Notre Dame, Notre Dame, Indiana 46566,
United States; Email: phelquis@nd.edu
1
(400 MHz, CDCl3) δ 8.12 (d, J = 8.6 Hz, 1H), 7.98 (d, J = 9.2 Hz,
1H), 7.63 (d, J = 8.6 Hz, 1H), 7.19 (dd, J = 9.2, 2.1 Hz, 1H), 7.15 (d,
J = 2.1 Hz, 1H), 4.17 (dd, J = 15.2, 7.4 Hz, 1H), 3.93 (s, 3H), 3.47
(dd, J = 15.1, 10.8 Hz, 1H), 3.39 (dd, J = 17.8, 5.7 Hz, 1H), 3.24
(ddd, J = 17.7, 12.5, 5.3 Hz, 1H), 2.25 (dd, J = 12.8, 4.8 Hz, 1H), 2.05
(quint, J = 8.1 Hz, 1H), 1.84 (td, J = 12.7, 5.8 Hz, 1H), 1.56 (sept, J =
6.6 Hz, 1H), 1.49−1.43 (m, 1H), 1.40−1.32 (m, 4H), 1.24−1.19 (m,
2H), 0.97 (s, 3H), 0.89 (d, J = 6.6 Hz, 6H); 13C{1H} NMR (201
MHz, CDCl3) δ 179.3, 158.6, 136.6, 136.3, 127.2, 126.1, 126.0, 125.2,
124.1, 118.8, 107.3, 63.9, 55.6, 50.7, 47.9, 39.2, 35.3, 29.3, 28.2, 28.11,
28.07, 23.6, 22.89, 22.85, 14.2; HRMS (ESI) calcd for C25H34NO [M
+ H] 364.2635, found 364.2646; IR (solid) 2924, 2843, 1615, 1248,
1033, 855 cm−1.
Authors
Emily M. Work − Department of Chemistry and Biochemistry,
University of Notre Dame, Notre Dame, Indiana 46566,
United States
Guillermo Ferraudi − Radiation Research Laboratory,
University of Notre Dame, Notre Dame, Indiana 46556,
United States
Luke Kiefer − Department of Chemistry and Biochemistry,
University of Notre Dame, Notre Dame, Indiana 46566,
United States
11a-Methyl-1-(5-methylhexyl)-2,10,11,11a-tetrahydro-1H-
naphtho[1,2-g]indol-7-ol (1). A 25 mL round-bottom flask with a
stir bar was charged with methyl ether 8 (271 mg, 0.75 mmol). The
flask was capped, evacuated, and backfilled with argon (3×). DCM
(7.5 mL) was added, and the solution was stirred at 0 °C in an ice
bath. Boron tribromide (1.5 mL of 1 M solution in DCM, 0.198
mmol) was added dropwise over 5 min via syringe, the yellow
solution was allowed to warm to 22 °C, and then it was heated to 30
°C in a sand bath for 6 h. The reaction mixture was quenched with
water and extracted 3 times with DCM. The organic layers were
combined, dried over anhyd Na2SO4, and concentrated on a rotary
evaporator to give a crude yellow solid. The crude material was
purified by MPLC (3−10% methanol in DCM) to give the title
compound as a powdery solid (252 mg, 96%), which was
recrystallized from methanol/DCM to give colorless crystals of a
Gang Liu − Department of Chemistry and Biochemistry,
University of Notre Dame, Notre Dame, Indiana 46566,
United States
Michael Grigalunas − Department of Chemistry and
Biochemistry, University of Notre Dame, Notre Dame,
Indiana 46566, United States
Atul Bhardwaj − Department of Chemistry and Biochemistry,
University of Notre Dame, Notre Dame, Indiana 46566,
United States
Rasmin Kaur − Department of Biochemistry and Molecular
Biology, University of Southern Denmark, DK-5230 Odense
M, Denmark
Janel M. Dempsey − Department of Chemistry and
Biochemistry, University of Notre Dame, Notre Dame,
Indiana 46566, United States
1
single diastereomer. Mp = 206−207 °C (decomp.); H NMR (800
MHz, CDCl3) δ 8.03 (d, J = 8.7 Hz, 1H), 7.95 (d, J = 9.2 Hz, 1H),
7.41 (d, J = 8.7 Hz, 1H), 7.16 (dd, J = 9.1, 2.6 Hz, 1H), 7.09 (d, J =
2.6 Hz, 1H), 4.19 (dd, J = 14.8, 7.4 Hz, 1H), 3.50 (dd, J = 14.4, 10.8
Hz, 1H), 3.40 (dd, J = 17.6, 4.8 Hz, 1H), 3.25 (ddd, J = 17.8, 12.4, 5.5
Hz, 1H), 2.27 (ddd, J = 12.9, 5.6, 1.5 Hz, 1H), 2.08 (dddd, J = 10.5,
9.2, 7.4, 5.9 Hz, 1H), 1.87 (td, J = 12.9, 5.8 Hz, 1H), 1.58−1.51 (m,
2H), 1.47−1.43 (m, 1H), 1.39−1.31 (m, 4H), 1.22−1.18 (m, 2H),
0.99 (s, 3H), 0.88 (d, J = 6.6 Hz, 6H); 13C{1H} NMR (201 MHz,
CDCl3) δ 179.8, 155.4, 137.0, 136.5, 126.9, 126.4, 125.9, 124.6, 124.1,
118.4, 111.0, 63.5, 51.1, 50.6, 48.1, 39.2, 35.2, 29.3, 28.2, 28.1, 23.6,
22.89, 22.86, 14.2; HRMS (ESI) calcd for C24H32NO [M + H]
350.2478, found 350.2481; IR (solid) 3100, 2955, 2927, 2857, 1613,
1391, 1231, 1028, 857 cm−1.
Daniel Wustner − Department of Biochemistry and Molecular
̈
Biology, University of Southern Denmark, DK-5230 Odense
M, Denmark; orcid.org/0000-0003-4995-9709
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.joc.0c02460
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. A. Oliver for crystallographic assistance, Dr. B.
Smith for access to a fluorometer, J. Burton for assistance with
the synthesis, and the Biophysics Instrumentation Core Facility
for use of the CD spectrometer. E.M.W. thanks the Arthur J.
Schmitt Foundation for fellowship support. D.W. acknowl-
edges technical assistance from Maria Szomek and thanks Dr.
Douglas Covey (Washington University, St. Louis) for kindly
providing CTL. We gratefully acknowledge financial support
from the National Institutes of Health (NS092653), the Ara
Parseghian Medical Research Foundation, and the Danish
Council for Independent Research (Grant No. DFF-7014-
00054).
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.joc.0c02460.
■
sı
Analytical data and NMR spectra for all new
compounds; photophysical studies; live cell imaging
and bleaching analysis; computational details (PDF)
Accession Codes
CCDC 2016041 and 2022200 contain the supplementary
crystallographic data for this paper. These data can be obtained
free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by
emailing data_request@ccdc.cam.ac.uk, or by contacting The
Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
DEDICATION
■
We dedicate this paper to Professor Robert M. Carlson on the
occasion of his 80th birthday in 2020.
AUTHOR INFORMATION
Corresponding Authors
Olaf Wiest − Department of Chemistry and Biochemistry,
University of Notre Dame, Notre Dame, Indiana 46566,
United States; orcid.org/0000-0001-9316-7720;
Email: owiest@nd.edu
■
REFERENCES
■
(1) Hu, J.; Zhang, Z.; Shen, W. J.; Azhar, S. Cellular Cholesterol
Delivery, Intracellular Processing and Utilization for Biosynthesis of
Steroid Hormones. Nutr. Metab. 2010, 7, 7−9.
(2) Maxfield, F. R.; Tabas, I. Role of Cholesterol and Lipid
Organization in Disease. Nature 2005, 438, 612−621.
1619
https://dx.doi.org/10.1021/acs.joc.0c02460
J. Org. Chem. 2021, 86, 1612−1621

The Journal of Organic Chemistry
pubs.acs.org/joc
Article
(3) Gupta, A.; Rivera-Molina, F.; Xi, Z.; Toomre, D.; Schepartz, A.
Endosome Motility Defects Revealed at Super-Resolution in Live
Cells Using HIDE Probes. Nat. Chem. Biol. 2020, 16, 408−414.
Luminescent Properties of 2,3-Dimethoxy-8-Aza-D-Homogona-
1,3,5(10),13-Tetraene-12,17a-Dione and Its Delta9,11 Derivative.
Opt. Spectrosc. 2006, 101, 882−888.
(23) Bazyl, O. K.; Artyukhov, V. Y.; Mayer, G. V. A Quantum-
Chemical Study of the Spectral and Luminescent Properties of
Photoproducts of Some 8-Azasteroids. Opt. Spectrosc. 2008, 104,
172−179.
(4) Maxfield, F. R.; Wustner, D. Analysis of Cholesterol Trafficking
̈
with Fluorescent Probes. Methods Cell Biol. 2012, 108, 367−393.
(5) Mukherjee, S.; Zha, X.; Tabas, I.; Maxfield, F. R. Cholesterol
Distribution in Living Cells: Fluorescence Imaging Using Dehy-
droergosterol as a Fluorescent Cholesterol Analog. Biophys. J. 1998,
75, 1915−1925.
(24) Basyl, O. K.; Artykhov, V. Y.; Mayer, G. V. Proton-Acceptor
Properties of 8-Azasteroids. Russ. Phys. J. 2005, 48, 196−199.
(25) Bagnich, S. A.; Khropik, N. N.; Knyukshto, V. N.; Mikhalchuk,
A. L.; Rubinov, D. B. Phosphorescence of 8-Azasteroids and Related
Compounds. Spectrochim. Acta, Part A 2005, 61, 2161−2167.
(26) Fantini, J.; Barrantes, F. J. How Cholesterol Interacts with
Membrane Proteins: An Exploration of Cholesterol-Binding Sites
Including CRAC, CARC, and Tilted Domains. Front. Physiol. 2013, 4,
1−9.
(6) Scheidt, H. A.; Muller, P.; Herrmann, A.; Huster, D. The
̈
Potential of Fluorescent and Spin-Labeled Steroid Analogs to Mimic
Natural Cholesterol. J. Biol. Chem. 2003, 278, 45563−45569.
(7) Solanko, K. A.; Modzel, M.; Solanko, L. M.; Wustner, D.
̈
Fluorescent Sterols and Cholesteryl Esters as Probes for Intracellular
Cholesterol Transport. Lipid Insights 2015, 2015, 95−114.
(8) Sezgin, E.; Can, F. B.; Schneider, F.; Clausen, M. P.; Galiani, S.;
Stanly, T. A.; Waithe, D.; Colaco, A.; Honigmann, A.; Wustner, D.;
̈
(27) Bukiya, A. N.; Dopico, A. M. Common Structural Features of
Cholesterol Binding Sites in Crystallized Soluble Proteins. J. Lipid Res.
2017, 58, 1044−1054.
Platt, F.; Eggeling, C. A Comparative Study on Fluorescent
Cholesterol Analogs as Versatile Cellular Reporters. J. Lipid Res.
2016, 57, 299−309.
(28) Scheidt, H. A.; Meyer, T.; Nikolaus, J.; Baek, D. J.;
(9) Smutzer, G.; Crawford, B. F.; Yeagle, P. L. Physical Properties of
the Fluorescent Sterol Probe Dehydroergosterol. Biochim. Biophys.
Acta, Biomembr. 1986, 862, 361−371.
Haralampiev, I.; Thomas, L.; Bittman, R.; Muller, P.; Herrmann, A.;
̈
Huster, D. Cholesterol’s Aliphatic Side Chain Modulates Membrane
Properties. Angew. Chem., Int. Ed. 2013, 52, 12848−12851.
(10) Morton, R. A.; Heilbron, I. M.; Spring, F. S. Absorption Spectra
in Relation to Vitamin A. Biochem. J. 1930, 24, 136−140.
(29) Abbotto, A.; Beverina, L.; Bozio, R.; Bradamante, S.; Ferrante,
C.; Pagani, G. A.; Signorini, R. Push-Pull Organic Chromophores for
Frequency-Upconverted Lasing. Adv. Mater. 2000, 12, 1963−1967.
́
(11) Martins-Duarte, E. S.; Lemgruber, L.; Lorente, S. O.; Gros, L.;
Magaraci, F.; Gilbert, I. H.; De Souza, W.; Vommaro, R. C. Evaluation
of Three Novel Azasterols against Toxoplasma Gondii. Vet. Parasitol.
2011, 177, 157−161.
(12) Gigante, F.; Kaiser, M.; Brun, R.; Gilbert, I. H. SAR Studies on
Azasterols as Potential Anti-Trypanosomal and Anti-Leishmanial
Agents. Bioorg. Med. Chem. 2009, 17, 5950−5961.
(13) Gros, L.; Lorente, S. O.; Jimenez, C. J.; Yardley, V.; Rattray, L.;
Wharton, H.; Little, S.; Croft, S. L.; Ruiz-Perez, L. M.; Gonzalez-
Pacanowska, D.; Gilbert, I. H. Evaluation of Azasterols as Anti-
Parasitics. J. Med. Chem. 2006, 49, 6094−6103.
(14) Alonso, F.; Cirigliano, A. M.; Cabrera, G. M.; Ramírez, J. A.
Synthesis and Preliminary Biological Screening of Sterol Analogues as
New Antifungal Agents against Plant Pathogens. Steroids 2010, 75,
659−664.
(15) Woloshuk, C. P.; Sisler, H. D.; Dutky, S. R. Mode of Action of
the Azasteroid Antibiotic 15-Aza-24-Methylene-D-Homocholesta-
8,14-Dien-3B-Ol in Ustilago Maydis. Antimicrob. Agents Chemother.
1979, 16, 98−103.
(16) Berlin, K. D.; Morgan, J. G.; Durham, N. N.; Chestnut, R. W.
Synthesis of 1,10,11,11a-Tetrahydro-11a-Methyl-2H-Naphth[1,2-g]-
Indol-7-Ol, an Equilenin-Like 15-Aza Steroid. J. Org. Chem. 1971, 36,
1599−1602.
(17) Chesnut, R. W.; Durham, N. N.; Brown, R. A.; Mawdsley, E. A.;
Berlin, K. D. Antibacterial Activity of 15-Azasteroids Alone and in
Combination with Antibiotics. Steroids 1976, 27, 525−541.
(18) Mawdsley, E. A.; Berlin, K. D.; Chesnut, R. W.; Durham, N. N.
Synthesis and Structure-Activity Relations of Heterocyclic Com-
pounds Containing a Trimethoxyarene Function. J. Med. Chem. 1976,
19, 239−243.
̌
(30) Bures, F. Fundamental Aspects of Property Tuning in Push-Pull
Molecules. RSC Adv. 2014, 4, 58826−58851.
́
(31) Dorman, G.; Nakamura, H.; Pulsipher, A.; Prestwich, G. D. The
Life of Pi Star: Exploring the Exciting and Forbidden Worlds of the
Benzophenone Photophore. Chem. Rev. 2016, 116, 15284−15398.
(32) Uraguchi, D.; Tsuchiya, Y.; Ohtani, T.; Enomoto, T.; Masaoka,
S.; Yokogawa, D.; Ooi, T. Unveiling Latent Photoreactivity of Imines.
Angew. Chem., Int. Ed. 2020, 59, 3665−3670.
́
(33) Seebach, D.; Golinski, J. Synthesis of Open-Chain 2,3-
Disubstituted 4-nitroketones by Diastereoselective Michael-addition
of (E)-Enamines to (E)-Nitroolefins. A Topological Rule for C, C-
bond Forming Processes between Prochiral Centres. Preliminary
Communication. Helv. Chim. Acta 1981, 64, 1413−1423.
(34) Morgan, J. G.; Berlin, K. D.; Durham, N. N.; Chestnut, R. W.
Syntheses of Some Indazoles Structurally Related to Equilenin. J.
Heterocycl. Chem. 1971, 8, 61−63.
(35) Fischer, R. T.; Stephenson, F. A.; Shafiee, A.; Schroeder, F.
Δ5,7,9(10)-Cholestatrien-3β-Ol: A Fluorescent Cholesterol Ana-
logue. Chem. Phys. Lipids 1984, 36, 1−14.
(36) Padwa, A. Photochemistry of the Carbon−Nitrogen Double
Bond. Chem. Rev. 1977, 77, 37−68.
(37) Martin, A.; Long, C.; Forster, R. J.; Keyes, T. E. Near IR
Emitting BODIPY Fluorophores with Mega-Stokes Shifts. Chem.
Commun. 2012, 48, 5617−5619.
́
(38) Mas-Montoya, M.; Montenegro, M. F.; Espinosa Ferao, A.;
́
́
Tarraga, A.; Rodríguez-Lopez, J. N.; Curiel, D. Rigid π-Extended
Boron Difluoride Complex with Mega-Stokes Shift for Bioimaging.
Org. Lett. 2020, 22 (9), 3356−3360.
(39) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci,
B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H.
P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.;
Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.;
Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.;
Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.;
Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi,
R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar,
S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox,
J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.;
Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.;
Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.;
Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A.
(19) Borisevich, N. A.; Raichyonok, T. F.; Sukhodola, A. A.;
Tolstorozhev, G. B.; Shashilov, V. A. Absorption and Fluorescence of
8-Azasteroids in the Gas Phase. J. Appl. Spectrosc. 2005, 72, 49−58.
(20) Dubovskii, V. L.; Gulyakevich, O. V.; Mikhal’chuk, A. L.;
Raichenok, T. F.; Tikhomirov, S. A.; Tolstorozhev, G. B. Spectral and
Kinetic Properties of an Immunoactive 8-Azasteroid and a Product of
Its Photochemical Transformation in Aqueous Solutions. Opt.
Spectrosc. 2008, 104, 875−885.
(21) Borisevich, N. A.; Raichyonok, T. F.; Sukhodola, A. A.;
Tolstorozhev, G. B. Delayed Fluorescence and Phosphorescence of 8-
Aza-D-Homogonane in the Gas and Condensed Phases. J. Fluoresc.
2006, 16, 649−653.
(22) Dubouski, V. L.; Gulyakevich, O. V.; Mikhal’chuk, A. L.;
Raichenok, T. F.; Tikhomirov, S. A.; Tolstorozhev, G. B. Spectral and
1620
https://dx.doi.org/10.1021/acs.joc.0c02460
J. Org. Chem. 2021, 86, 1612−1621

The Journal of Organic Chemistry
pubs.acs.org/joc
Article
D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J.
Gaussian 16, revision B.01; Gaussian, Inc.: Wallingford, CT, 2016.
(40) Wilkinson, F. Luminescence in Chemistry; Bowen, E. J., Ed.; Van
Nostrand: London, 1968; pp 155−182.
(41) Yguerabide, J. Nanosecond Fluorescence Spectroscopy of
Biological Macromolecules and Membranes. In Fluorescence Techni-
ques in Cell Biology; Thaer, A. A., Sernetz, M., Eds.; Springer: Berlin,
Heidelberg, 1973; pp 311−331.
(42) Xu, S.; Benoff, B.; Liou, H.; Lobel, P.; Stock, A. M. Structural
Basis of Sterol Binding by NPC2, a Lysosomal Protein Deficient in
Niemann-Pick Type C2 Disease. J. Biol. Chem. 2007, 282, 23525−
23531.
(43) Kwon, H. J.; Abi-Mosleh, L.; Wang, M. L.; Deisenhofer, J.;
Goldstein, J. L.; Brown, M. S.; Infante, R. E. Structure of N-Terminal
Domain of NPC1 Reveals Distinct Subdomains for Binding and
Transfer of Cholesterol. Cell 2009, 137, 1213−1224.
́
(44) Shen, Y.; Liu, J.; Estiu, G.; Isin, B.; Lee, D.-S.; Barabasi, A.-L.;
Kapatral, V.; Wiest, O.; Oltvai, Z. N.; et al. A Blueprint for
Antimicrobial Hit Discovery Targeting Metabolic Networks. Proc.
Natl. Acad. Sci. U. S. A. 2010, 107, 1082−1087.
(45) Berzina, Z.; Solanko, L. M.; Mehadi, A. S.; Jensen, M. L. V.;
Lund, F. W.; Modzel, M.; Szomek, M.; Solanko, K. A.; Dupont, A.;
Nielsen, G. K.; Heegaard, C. W.; Ejsing, C. S.; Wustner, D. Niemann-
̈
Pick C2 Protein Regulates Sterol Transport between Plasma
Membrane and Late Endosomes in Human Fibroblasts. Chem. Phys.
Lipids 2018, 213, 48−61.
(46) Chaudhuri, A.; Anand, D. Cholesterol: Revisiting Its
Fluorescent Journey on 200th Anniversary of Chevruel’s “Choles-
terine. Biomed. Spectrosc. Imaging 2017, 6, 1−24.
(47) Steinhaus, M. Characterization of the Major Odor-Active
Compounds in the Leaves of the Curry Tree Bergera Koenigii L. by
Aroma Extract Dilution Analysis. J. Agric. Food Chem. 2015, 63,
4060−4067.
1621
https://dx.doi.org/10.1021/acs.joc.0c02460
J. Org. Chem. 2021, 86, 1612−1621