Synthesis and Characterization of a Diazirine-Based Photolabel of the Nonanesthetic Fropofol

Article abstract of DOI:10.1021/acschemneuro.0c00667

The mechanisms of general anesthetics have been debated in the literature for many years and continue to be of great interest. As anesthetic molecules are notoriously difficult to study due to their low binding affinities and multitude of binding partners, it is advantageous to have additional tools to study these interactions. Fropofol is a hydroxyl to fluorine-substituted propofol analogue that is able to antagonize the actions of propofol. Understanding fropofol's ability to antagonize propofol would facilitate further characterization of the binding interactions of propofol that may contribute to its anesthetic actions. However, the study of fropofol's molecular interactions has many of the same difficulties as its parent compound. Here, we present the synthesis and characterization of ortho-azi-fropofol (AziFo) as a suitable photoaffinity label (PAL) of fropofol that can be used to covalently label proteins of interest to characterize fropofol's binding interactions and their contribution to general anesthetic antagonism.

Full text of DOI:10.1021/acschemneuro.0c00667

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Research Article
Synthesis and Characterization of a Diazirine-Based Photolabel of
the Nonanesthetic Fropofol
E. Railey White, David M. Leace, Victoria M. Bedell, Natarajan V. Bhanu, Benjamin A. Garcia,
William P. Dailey, and Roderic G. Eckenhoff*
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ABSTRACT: The mechanisms of general anesthetics have been debated in
the literature for many years and continue to be of great interest. As anesthetic
molecules are notoriously difficult to study due to their low binding affinities
and multitude of binding partners, it is advantageous to have additional tools
to study these interactions. Fropofol is a hydroxyl to fluorine-substituted
propofol analogue that is able to antagonize the actions of propofol.
Understanding fropofol’s ability to antagonize propofol would facilitate further characterization of the binding interactions of
propofol that may contribute to its anesthetic actions. However, the study of fropofol’s molecular interactions has many of the same
difficulties as its parent compound. Here, we present the synthesis and characterization of ortho-azi-fropofol (AziFo) as a suitable
photoaffinity label (PAL) of fropofol that can be used to covalently label proteins of interest to characterize fropofol’s binding
interactions and their contribution to general anesthetic antagonism.
KEYWORDS: Anesthetic antagonist, propofol, molecular probe, photoaffinity label, hydrogen bonding, fluorine
excitation, fropofol is able to antagonize the sedative effect of
propofol.7 The mechanism of this antagonism is not fully
understood, but there are data to support that fropofol does
not modulate the α1β2γ2L isoform of GABAA, making a
GABAergic mechanism less likely than another as yet
unidentified molecular target.7
INTRODUCTION
■
The introduction of propofol (2,6-diisopropylphenol) in the
late 1980s marked the first alkylphenol anesthetic agent to be
used in anesthetic practice.1 This molecule remains structurally
unique compared to other general anesthetic agents and has
become one of the most frequently used medications in the
practice of anesthesia.2 Many studies have explored the
medicinal chemistry of alkylphenol anesthetics and have
worked to define the chemical space that they inhabit.3−6
Seemingly subtle structural changes occasionally result in the
complete elimination of anesthetic activity, but the loss of
hypnotic or sedating activity does not necessarily render these
compounds biologically inactive. One such molecule that was
initially thought to be “inactive” is a fluorinated analogue of
propofol (fropofol) where a fluorine has been substituted for
the hydroxyl group (Figure 1).7 This loss of hydrogen bonding
character abrogates any sedative/hypnotic activity and exhibits
an excitatory phenotype at high doses (200 mg/kg in mice).7
At concentrations much lower than necessary to generate this
Due to the structural similarity of fropofol to propofol and
their seemingly opposite pharmacologic effects, fropofol could
prove a useful tool in probing the anesthetic mechanism of
propofol, which remains an area of significant interest.8,9 As
small hydrophobic molecules, anesthetics have been notori-
ously difficult to study due to their relatively low binding
affinities at a large number of target sites. These hurdles have
previously been overcome in part by developing diazirine-
based photoaffinity labels (PALs) of the molecules of
interest.10 The incorporation of a diazirine moiety allows the
formation of nonspecific covalent bonds through a reactive
carbene intermediate formed in the presence of UV light.11,12
By irreversibly linking the PAL to the macromolecule of
interest, a snapshot of a low-affinity, transient interaction can
be studied in detail.10 Fropofol, like propofol and most other
anesthetics, is a small molecule (MW = 246.21 amu), and it is
well established that making seemingly minor changes to these
Received: October 16, 2020
Accepted: December 8, 2020
Published: December 23, 2020
Figure 1. Chemical structures. Shown are the structures of the
anesthetic propofol, its fluorine-substituted derivative, fropofol, and
the diazirine-based photolabel of fropofol, ortho-azi-fropofol (AziFo,
1).
https://dx.doi.org/10.1021/acschemneuro.0c00667
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Scheme 1. Synthesis of AziFo (1)
drugs can have a significant impact on their function.3−6 Thus,
before a photolabel can be deployed to study the molecular
actions of its parent compound, it is critical to establish that
the substitution of the isopropyl group for a diazirine results in
comparable activity.13,14 Without assuring a similar function, a
PAL will fail to be an adequate surrogate of its parent
compound. Here, we describe the synthesis of ortho-azi-
fropofol (AziFo) and its characterization as a suitable PAL of
fropofol.
for fropofol and AziFo of 11 and 17 μM, respectively (Figure
2). Utilizing these values and an experimentally determined
RESULTS AND DISCUSSION
■
Synthesis of Azi-fropofol (1). The proposed PAL analog
of fropofol presented here is 3-(2-fluoro-3-isopropylphenyl)-3-
(trifluoromethyl)-3H-diazirine (ortho-azi-fropofol, AziFo 1,
Figure 1). Compared with fropofol, AziFo replaces one of
the isopropyl groups on the aromatic ring with the photo-
reactive trifluoromethyl diazirine groups. The synthesis of
AziFo (Scheme 1) involved the conversion of 1-fluoro-2-
isopropylbenzene to the known trifluoromethyl ketone 3.15
The conversion of ketone 3 to the corresponding diazirine 1
followed well-established protocols.16 Azi-fropofol (1) was
obtained as a liquid in >98% purity as measured by capillary
GC.
Figure 2. 1-AMA competition assay. HSAF binding affinities of AziFo
and fropofol were calculated from their displacement of the known
binding partner 1-AMA, which exhibits increased fluorescence when
bound to HSAF.
binding affinity of 1-AMA and HSAF (9 μM) (Figure S9), the
Cheng-Prusoff equation was used to calculate KD values, which
were 4.3 μM (95% CI of 3.1−5.7) for fropofol and 6.6 μM
(95% CI of 4.7−11.7) for AziFo.18
Physiochemical Characterization. The first step in
establishing the suitability of AziFo as a PAL of fropofol was
to compare physiochemical properties, which are shown in
Table 1. When compared to fropofol, AziFo has a 36% increase
Activity of AziFo in Zebrafish. Zebrafish (Danio rerio)
larvae at 5 days postfertilization (dpf) were initially exposed to
concentrations as high as 100 μM AziFo or fropofol for 30 min.
No decrease in spontaneous movement was observed, and no
increases in activity or alterations in swimming pattern that
may be consistent with an excitatory or seizure phenotype were
observed. After these responses were noted, larvae were then
transferred to fresh zebrafish embryo water (E3) and observed
for 24 h after which 3 of 12 larvae in the 100 μM fropofol
exposure were found dead. No toxicity was seen during tests of
up to 100 μM AziFo, and concentrations no higher than 25 μM
were used for either compound in the subsequent experiments.
Because of the seemingly absent pharmacologic effect of both
ligands, AziFo and fropofol (at 5 or 25 μM) were
coadministered with propofol (0.03−10 μM) to look for
pharmacologic additivity. Instead of additivity, both AziFo and
fropofol showed a dose-dependent antagonism of propofol-
induced hypnosis as measured by a decrease in spontaneous
movement (Figure 3).19
AziFo Diazirine Half-Life. The UV−vis absorption
spectrum of AziFo showed a local maximum from the diazirine
at 317 nm with additional aromatic absorbance maxima at 273
and 267 nm (Figure S7). An experimentally determined
extinction coefficient of 1600 M−1 cm−1 at 273 nm was used
for all determinations of concentration based on the
absorption. The signal at 317 nm was of insufficient intensity
to be useful at working concentrations in aqueous solutions;
thus, the half-life of the AziFo diazirine was determined in
methanol to overcome the limitation in solubility. Upon
exposure to 356 nm light, the half-life (t1/2) of the diazirine
peak (317 nm) was 11.0 min (95% CI of 6.6−26.5) (Figure 4).
Degradation after a 300 nm exposure was faster with a t1/2 of
Table 1. Physiochemical Properties of Fropofol and AziFo
a
physiochemical properties
molecular weight (amu)
fropofol
azi-fropofol (AziFo)
180.26
189
0.9
246.21
195
1.19 0.01
4.14
van der Waals volume (Å3)
density (g/cm3; mean SD)
b
cLogP
3.96
solubility in water (μM; mean SD)
116
4
95
4
a
All fropofol data are values that have been previously reported.18
b
cLogP = octanol/water partition coefficient.
in molecular weight but does not have a proportional increase
in van der Waals volume. Therefore, it is no surprise that AziFo
has a higher density of 1.2 g/cm3 compared to fropofol’s
density of 0.9 g/cm3. This increase corresponds to general
trends in the fluorination of hydrocarbons.17 The calculation of
the octanol/water partition coefficient (cLogP) indicates a
marginal increase in the hydrophobicity of AziFo (cLogP of
4.14) compared to fropofol (cLogP of 3.96), which correlates
to their relative maximal water solubility (95 and 116 μM,
respectively).
Fluorescence Competition with Horse Spleen Apo-
ferritin. 1-Aminoanthracene (1-AMA) exhibits enhanced
fluorescence when bound to horse spleen apoferritin
(HSAF). Fropofol and AziFo are both able to displace 1-
AMA from its known binding pocket in HSAF. This
competition assay with fropofol and AziFo yielded IC50 values
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that identified its peptide sequence (for the best spectra, see
Figures S14 and S15). This low frequency of labeling,
combined with the overall poor-quality MS/MS spectra,
makes these more likely to be nonspecific labeling sites. On
the other hand, the labeling of Arg-59 with AziFo was seen in
over half of the MS/MS sample spectra that identified the
peptide sequence that contained this site (ELAEEKR59-
EGAER) and had overall higher quality MS/MS spectra
(Figure S16), which makes it much more likely to be a
photoadduction site representative of a meaningful interaction
of HSAF and AziFo.
Given that no photoadduction sites were identified at 10 μM
AziFo and there were some possible nonspecific labeling sites
identified at 100 μM AziFo, an additional round of photo-
labeling was conducted with an intermediate concentration of
AziFo (50 μM). This intermediate concentration only
supported one photolabeled site, again showing strong
evidence for the same Arg-59 site identified in the 100 μM
photolabeling experiment (Figure 5). There was also some
weak evidence for a second possible biding site between Asn-
17 and Leu-31 in the middle of the same 34 amino acid length
peptide previously identified (Figure S17). This peptide is the
product of two missed trypsin cleavages, and the “blind spot”
peptide was identified this time and included a photolableded
site. The spectrum of this peptide (LVNLYLR) (Figure S18)
identified Leu2-4 as the labeled site; however, there was only a
single spectrum at low intensity.
Both Leu-24 and Arg-59 of HSAF have been shown to be
among the amino acids to demonstrate specific noncovalent
interactions with propofol and fropofol,20,21 and Leu-24 was
the photolabeled site previously identified with the propofol
analogue AziPm.13 The evidence presented here only strongly
supports an Arg-59 site, despite many other residues that line
the known “anesthetic” pocket of HSAF. These residues of this
pocket can be seen in a CASTp analysis (Figure S19). Each of
these residues have been previously identified via computa-
tional modeling and the propofol bound crystal structure of
HSAF.20,21
Figure 3. Activity of AziFo in vivo. No change (neither decrease nor
increase) in spontaneous movement was observed upon exposure of 5
dpf zebrafish to maximal nontoxic doses of AziFo or fropofol. The
administration of propofol (Prop) alone demonstrates a dose-
dependent decrease in spontaneous movement. Shown here is the
effect of coadministration of AziFo or fropofol (Frop) with propofol,
and the resultant dose-dependent antagonism of propofol’s effects (5
and 25 μM doses shown). Data for propofol alone has been previously
published.19
3.8 min (95% CI of 2.9−4.8) (Figure 4). The exposure to a
300 nm light was used for all photolabeling experiments.
Photoadduction to HSAF. After irradiating a solution of
10 μM AziFo and HSAF with a 300 nm UV light for 25 min,
trypsinization, and analysis by LC/MS/MS, 98.3% sequence
coverage of the HSAF light chain sequence was detected, and
MaxQuant was used to search for AziFo adducts (+218.0719
m/z). At a 10 μM AziFo concentration, there was only one
potential photolabeled site detected. This site was in the
middle of a peptide 34 amino acids in length (Figures S12 and
S13), so it is not surprising that there was insufficient spectral
data to identify the labeling of a specific amino acid within this
sequence. However, it was somewhat promising that portions
of this suspected labeling site fall within the known ligand
binding site of HSAF, which was previously photolabeled with
AziPm (meta-azi-propofol), a propofol PAL.13 An additional
photolabeling reaction was conducted with 100 μM AziFo.
From this reaction, protein sequence coverage was 78.7%
(Figure S11) and there were 3 possible photoadduction sites
identified (Arg-59, Met-144, and Gln-82). The labeled Gln-82
was only seen in <2% of spectra that identified its peptide
sequence, and the labeled Met-144 was seen in 12% of spectra
In providing an answer to which amino acids are
photolabeled, it is important to consider the questions we
posed by performing a photolabeling experiment with AziFo vs
AziPm. It is reasonable to think that the preference for AziFo to
label Arg-59, rather than Leu-24, is due to differences in the
pose that AziPm and AziFo assume in the HSAF site. For
example, a significant determinant of adduct formation is the
position of the reactive carbene derived from the diazirine arm
relative to the lining residues. For AziFo and AziPm, this arm is
Figure 4. Determination of diazirine half-life. The disappearance of the diazirine upon exposure to UV light was monitored via UV−vis absorption.
(A) UV−vis spectra during exposure to 356 nm light. (B) UV−vis spectra during exposure to 300 nm light. (C) Graphical representation of
absorption vs time showing the relative difference in diazirine degradation kinetics between the two exposure wavelengths. Absorbance was
measured at 317 nm.
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Figure 5. MS/MS spectrum of AziFo adduction to R59. MS/MS fragmentation spectrum of the peptide ELAEEKREGAER (amino acids 53 to 64),
which contains the Arg identified as a site photolabeled by AziFo (50 μM). Fragments identified on the spectrum are shown on the peptide
sequence (b ions in blue; y ions in red). A detailed ion table can be found in Table S6.
Figure 6. Visualization of AziFo and photolabeled residues. Utilizing a previously reported crystal structure (PDB ID 3f32), docking calculations
using AutoDock Vina were conducted to analyze the relationship between possible ligand confirmations and the photolabeled Arg-59. (A) HSAF
dimer (gray) with the location of possible photolabeled adducts (red = Arg-59, blue = Leu-24, orange = Gln-82, and yellow = Met-144) and the
location of the amino acids known to line the ligand binding pocket (light green, Ser-27, Tyr-28, Glu-63, and Leu-81). (B) Demonstration of lowest
energy AziFo docking pose within the ligand binding pocket (dark green = carbon, light green = fluorine, and blue = diazirine nitrogens). (C)
Lowest energy docking pose for 1-AMA (bright pink), fropofol (yellow), and AziFo (teal) within the ligand binding pocket. (D) All docking poses
of AziFo. The closest distance of any atom of any AziFo confirmation to the nearest Arg-59 atom was 2.9 Å. (E) The chemical structure of meta-
Azi-Propofol (AziPm). Note the meta position of the diazirine arm compared to the ortho position of AziFo (Figure 1). (F) Docking poses 2, 3, 5, 7,
and 9 of AziPm with the diazirine group oriented toward one of the two Leu-24. Poses 1, 4, 6, and 8 were of approximately the opposite symmetry
with the diazirine arm oriented toward the other Leu-24. (G) A single AziPm pose indicating the difference in position between the meta diazirine
arm of AziPm and the ortho position of AziFo. Arrow and dotted line indicate the theoretical movement of the arm to the ortho position. (H) A
single pose of AziFo showing a closer proximity of the diazirine carbon to Arg-59 (red) than Leu-24 (light green). Measurements are in angstroms.
in different positions on the aromatic ring (ortho vs meta with
respect to the fluorine/hydroxyl). Because of these differences
in geometry, it is not entirely surprising that these PALs result
in different binding sites. In fact, in the crystal structure of
propofol bound to HSAF, the isopropyl arms are packed
against the hydrophobic portions of both Arg-59 side chains.21
Given the fact that AziFo has a direct substitution of an
isopropyl arm for a diazirine arm, it is not surprising that this
close interaction results in selective photolabeling of this
residue. This change in orientation can be more easily seen by
docking AziFo within the HSAF pocket (Figure 6).
Molecular Docking Calculations. To demonstrate the
occupation of AziFo in the known ligand binding site of HSAF,
molecular docking calculations were conducted with Auto-
Dock Vina.22 This model helps to provide a visual
representation and approximate measurement of atomic
distances between the docked PAL and protein crystal
structure. The ligand binding pocket is formed at the interface
of HSAF homodimers and is lined by 6 amino acids (L24, S27,
Y28, R59, E63, and L81; see Figures 6A and S19). The
symmetry of this dimerization puts the Arg-59 from both
proteins in close proximity within the ligand binding pocket
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(Figure 6A). The lowest energy poses of AziFo, fropofol, and
1-AMA can be seen in Figure 6B,C. Even with this simple
docking model, AziFo shows a preference of position within
the pocket. It is easiest to see the clustering of the diazirine
nitrogens, which are oriented in proximity to Arg-59 and Leu-
24 (Figure 6D).
not ruled out the possibility of entirely different binding sites
and different molecular targets.
Like many approaches, AziFo is only one of many tools in
the toolbox needed to understand anesthetic mechanisms. It
can provide a starting point for subsequent, complementary
experimental and computational methods.
To compare how AziPm and AziFo might bind in the HSAF
pocket, docking simulations were also conducted with AziPm
(Figure 6E). Consistent with AziFo docking, AziPm showed a
clustering of the diazirine groups in proximity to Leu-24 and
Arg-59 (Figure 6F), but there is an important difference in the
position of the diazirine arm. It is known from the crystal
structure of ligands bound to HSAF that the propofol hydroxl
prefers to face the opening of the pocket (toward the two
arginines). If this orientation is preserved, then the position of
the diazirine arm becomes an important predictor of what
residues are photolabeled by each ligand. Figure 6G shows
AziPm with the diazirine arm oriented toward Leu-24 (shown
in blue). If one imagines the theoretical repositioning of that
arm to an ortho position (dotted line in Figure 6G), then the
arm moves into closer proximity of Arg-59. Figure 6H shows a
single pose of AziFo and the relative closeness of Arg-59 (red)
and Leu-24 (blue). It should be noted that the measurements
shown in Figure 6 are the closest distances that were found;
there is no way of knowing exactly to which atom on the
arginine (including the backbone) the PAL adducted.
During docking calculations, the backbone is rigid and the
side chains are allowed to move. Therefore, comparisons made
between a previously determined crystal structure and a
docking calculation with a novel ligand may not be a reliable
representation of binding conditions. Despite these limitations,
the structural relationships support the photoadduction site
identified by MS/MS sequencing as a realistic target of the
AziFo PAL and may help to explain the difference in selectivity
between AziFo and AziPm.
METHODS
■
General Synthetic Procedures. Proton and 13C NMR spectra
were obtained on a Bruker DMX 500 MHz nuclear magnetic
resonance spectrometer, and 19F NMR spectra were obtained on a
Bruker DMX 360 MHz nuclear magnetic resonance spectrometer.
Spectra for compounds 1 and 3 are reported in the Supporting
Information. Accurate mass measurement analyses were conducted on
either a Waters GCT Premier, time-of-flight GCMS with electron
ionization (EI) or an LCT Premier XE, time-of-flight LCMS with
electrospray ionization (ESI). Samples were taken up in a suitable
solvent for analysis. The signals were mass measured against an
internal lock mass reference of perfluorotributylamine (PFTBA) for
EI-GCMS and leucine enkephalin for ESI-LCMS. Waters Masslynx
software calibrates the instruments and reports measurements by the
use of neutral atomic masses. The mass of the electron is not
included.
Preparation of 2, 2, 2-Trifluoro-1-(2-fluoro-3-
isopropylphenyl)ethan-1-one (3). A 250 mL round-bottom flask
equipped with a magnetic stirring bar was filled with 100 mL of dry
THF and 2.76 g (20 mmol) of 1-fluoro-2-isopropylbenzene. Under a
dry nitrogen atmosphere, the clear, colorless solution was cooled in a
dry ice/acetone bath with stirring for 30 min. A solution of 1.3 M sec-
BuLi in cyclohexane (18.0 mL, 23 mmol) was added dropwise over
the course of 10 min. The solution was stirred for an additional 10
min at dry ice temperature. Ethyl trifluoroacetate (4.0 mL, 4.8 g, 34
mmol) was added dropwise over 10 min to the cooled, stirred
solution. After stirring an additional 10 min in the cold bath, the
solution was allowed to warm to room temperature and was quenched
with 100 mL of 10% hydrochloric acid (HCl) solution. The mixture
was extracted with methylene chloride (3 × 100 mL). The combined
organic extracts were washed with water, and the dried organic layer
was evaporated. Short-path distillation of the residue under reduced
pressure produced 3.43 g (73%) of clear, colorless oil, bp 108−109 °C
CONCLUSION
1
■
at 25 mmHg. H NMR (500 MHz, CDCl3) δ 7.69 (t, J = 7.1, 1H),
In order to determine if AziFo closely mimics its parent
compound fropofol and is viable as a PAL, we compared
physiochemical properties, interactions with a model protein,
and pharmacologic activity in zebrafish. When combined with
the ability of AziFo to successfully adduct HSAF, this data
serves to support the use of AziFo as a surrogate molecule for
further study of the mechanism of action of fropofol. A single
amino acid (Arg-59) in HSAF was identified as a photolabeled
site within the known binding pocket. In addition to the MS/
MS data, AziFo’s ability to displace 1-AMA provides additional
evidence that this site is specific and not the result of random
off-site labeling. Specificity is a necessary trait for deploying
AziFo into more complex biological systems of interest. The
functional similarity of fropofol and AziFo combined with the
ability of AziFo to successfully adduct a known fropofol
binding site of HSAF serves to support the use of AziFo as a
surrogate molecule for further study of the mechanism of
action of fropofol.
7.60 (td, J = 7.1, 1.8 Hz, 1H), 7.25 (t, J = 7.8 Hz, 1H), 3.33 (p, J = 6.9
Hz, 1H), 1.29 (dd, J = 7.0, 2.4 Hz, 6H). 19F-NMR (340 MHz,
CDCl3) −74.43 (d, J = 15.7 Hz, 3F), −114.97 (td, J = 15.7, 6.8 Hz,
1F) ppm. 13C NMR (90 MHz, CDCl3) 179.80 (q, J = 38 Hz), 159.8
(d, J = 261 Hz), 137.3 (d, J = 15 Hz), 134.3 (d, J = 7 Hz), 128.8,
124.5 (d, J = 4 Hz), 119.7 (d, J = 11 Hz), 116.0 (q, J = 291 Hz), 27.0
(d, J = 3 Hz), 22.4 ppm. HRMS (EI+) calculated for C11H11F4O [M +
H]+: 235.0746, found: 235.0753.
Preparation of 3-(2-Fluoro-3-isopropylphenyl)-3-(trifluoro-
methyl)-3H-diazirine (Azi-fropofol) (1). A 100 mL round-bottom
flask equipped with a magnetic stirrer was filled with 3.00 g (12.8
mmol) of ketone 3, 1.00 g (14.5 mmol) of hydroxylamine
hydrochloride, and 50 mL of pyridine. A water-cooled reflux
condenser was attached, and the mixture was heated to reflux for 1
h. After cooling to room temperature, pyridine was evaporated under
reduced pressure. The semisolid residue was dissolved in a mixture of
50 mL of water and 50 mL of methylene chloride with vigorous
mixing. The organic layer was separated and washed with water. The
evaporation of the solvent left 3.0 g of the crude oxime as a thick
colorless oil. A portion (2.5 g, 10 mmol) of the thick oil was dissolved
in 50 mL of pyridine, and 2.7 g (14 mmol) of tosyl chloride was
added in one portion. The mixture was heated to reflux for 1 h and
then cooled to room temperature. Pyridine was evaporated at reduced
pressure. A mixture of 50 mL of water and 50 mL of methylene
chloride was added to the semisolid residue and mixed well. The
organic layer was separated and washed with water, 1 N aqueous HCl,
and water. The solvent was evaporated under reduced pressure to
leave 4.38 g of colorless semisolid crude oxime-tosylate. This was
One plausible explanation of fropofol’s inhibition of
propofol’s action is simply that it is binding in the same
“anesthetic” site(s). This would support the notion that the
mere occupancy of a site is insufficient to generate anesthesia.
The character of the molecule and the interactions it makes
within the site may have important and even opposing effects
on pharmacodynamics. However, at this point in time, we have
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dissolved in 20 mL of methylene chloride in a 100 mL round-bottom
flask equipped with a magnetic stir bar and a dry ice/acetone cold
bath. A dry ice cooled gas condenser was attached, and 20 mL of
liquid ammonia was condensed into the flask. The cold bath was
removed, and the mixture was stirred under the dry ice/acetone filled
gas condenser for several hours. Then, the condenser was allowed to
warm, and the ammonia was allowed to evaporate from the solution
overnight. In the morning, a mixture of 50 mL of water and 50 mL of
methylene chloride was added. The organic layer was removed and
was washed with water and then evaporated under reduced pressure.
The semisolid residue was triturated with 50 mL of hexane.
Evaporation of the hexane left 2.5 g of semisolid crude diaziridine.
A 100 mL round-bottom flask with magnetic stir bar was filled with
1.40 g (5.65 mmol) of the crude diaziridine, 60 mL of methylene
chloride, and 3.5 mL of triethylamine. The stirred solution was cooled
in an ice bath. Iodine (1.45 g, 5.70 mmol) was added in one portion.
The mixture was stirred in the ice bath until the solid iodine
dissolved; then, it was allowed to warm to room temperature and to
stir for 1 h. A solution of 1 N potassium hydroxide (30 mL) was
added, and the mixture was vigorously stirred for 30 min. Water (100
mL) was added, and the mixture was extracted with methylene
chloride (3 × 30 mL). The combined organic extracts were washed
with 100 mL of 1 M aqueous HCl solution and 100 mL of water and
then dried. The evaporation of the solvent left 1.3 g of brown oil. The
residue was dissolved in hexane and flushed through a short plug of
silica gel using additional hexane as eluent. The evaporation left a
clear colorless oil that was dynamically transferred under high vacuum
to a liquid nitrogen cooled U-trap to give 1.0 g (72%) of a very pale
pink liquid. The product was greater than 98% pure when analyzed by
capillary GC (30 m dimethylsilicone column, 150 °C injector, 100 °C
column temp). 1H NMR (500 MHz, CDCl3) 7.35 (t, J = 7.0 Hz, 1H),
7.30 (t, J = 7.0 Hz, 1H), 7.13 (t, J = 7.8 Hz), 3.27 (septet, J = 7 Hz,
1H), 1.25 ppm (d, J = 7 Hz, 6H). 19F-NMR (340 MHz, CDCl3)
−68.65 (d, J = 7.8 Hz, 3F), −119.40 ppm (td, J = 7.8, 7.0 Hz, 1F).
13C NMR (90 MHz, CDCl3) 160.2 (d, J = 252 Hz), 136.8 (d, J = 15
Hz), 130.0 (d, J = 5 Hz), 127.9 (d, J = 2 Hz), 124.7 (d, J = 5 Hz),
121.8 (q, J = 275 Hz), 115.5 (d, J = 16 Hz), 27.0 (d, J = 2 Hz), 25.6
(q, J = 43 Hz), 22.5 ppm. HRMS(EI+) calculated for C11H11F4N2 [M
+ H]+: 247.0858, found: 247.0869.
Physicochemical Properties of AziFo. Octanol/water partition
coefficients were calculated using XLOGP3.23 Molecular volume was
calculated using the Molinspiration property calculation toolkit
(Molinspiration Cheminformatics). The density of fropofol was
determined from replicate measurements of the volume/mass
relationship. The measurement of the UV−vis absorbance (Varian
Cary 300 Bio UV−vis spectrophotometer) of AziFo showed a
maximum diazirine absorbance at 317 nm with additional aromatic
absorption maxima at 267 and 273 nm. The extinction coefficient
(Σ273 = 1600 M−1 cm−1) was calculated from UV absorption
measurements from the aromatic absorption at 273 nm in methanolic
solutions of known concentrations. The extinction coefficient was
used to calculate the maximal water solubility of AziFo after 24 h of
sonication in double distilled water (ddH2O) and filtration with a 0.22
μm polyvinylidene difluoride (PVDF) syringe (MidSci, St. Louis,
MO).
ranging from 1 to 125 μM. Upon addition of the ligand, the samples
were mixed and immediately analyzed with a spectrofluorometer
(Shimadzu RF-5301 PC) with an excitation wavelength of 380 nm
and emission detection from 400 to 700 nm. The fluorescence curves
were corrected by subtracting contributions from 1-AMA alone and
HSAF alone. There was no significant fluorescent signal from
unbound ligands. Fluorescence intensities at 515 nm were plotted
as a percentage of the control (1-AMA and HSAF bound with no
competing ligand present) and were fitted to a logarithmic four
parameter variable slope using GraphPad Prism (version 8.4.0,
GraphPad Software, La Jolla California, USA). This experimentally
determined IC50 was used to calculate the dissociation constant via an
experimentally determined KD of 1-AMA and the Cheng-Prusoff
equation.18
Activity in Zebrafish. All zebrafish were treated in strict
accordance with NIH and institutional guidelines, and procedures
were approved by the University of Pennsylvania Animal Care and
Use Committee and conducted in accordance with the Guide for Care
and Use of Laboratory Animals. Adult Tubingen long fin wild-type
zebrafish (Danio rerio) were maintained at the University of
Pennsylvania’s aquatic facility and overseen by the University
Laboratory Animal Resources using standard husbandry conditions.
In vivo behavioral activity studies were performed on zebrafish at 5
days postfertilization (dpf). Zebrafish embryos were raised in E3
zebrafish embryo water (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2,
0.33 mM MgSO4, pH 7.2) at 28.5 °C in a 14:10 h light/dark cycle.
Each replicate consisted of 12 larvae. At least three independent
biological replicates for dosage were derived from different clutches of
embryos and were recorded on different days. Because no decrease in
spontaneous movement was observed with any AziFo or fropofol
concentration, these ligands were coadministered with propofol to
evaluate pharmacological additivity. Each sample well contained 2 mL
of E3 with various concentrations of AziFo or fropofol (0, 5, or 25
μM) and propofol (0.003−30 μM, Aldrich, 97%). Stock solutions of
drug were made in DMSO and diluted in E3. The final concentration
of DMSO (Sigma, sterile-filtered, BioReagent) was always below 2%
v/v, which showed no signs of toxicity or change in the measured
movement parameters. Larvae were acclimatized to the 24-well plates
(1 fish/well) for approximately 20 min in E3 at 25 °C prior to ligand
exposure. The solution was removed from each well and replaced with
2 mL of E3 with drug for a total of 30 min. Infrared video recordings
were made using a Daniovision Observation Chamber (Noldus). The
recordings were analyzed for spontaneous propulsive movements
(total distance moved) for the final 10 min of the 30 min exposure.
After drug exposure, the drug solution in each well was replaced with
fresh E3, and the zebrafish were kept for observation over the next 24
h and monitored for signs of toxicity and spontaneous recovery in
anesthetized groups. At 24 h, all zebrafish were euthanized by
submersion in ice water for at least 20 min.
̈
AziFo Diazirine Half-Life. The rate of photolysis of the AziFo
diazirine was determined in methanol in a 1 cm path length quartz
cuvette exposed to 300 and 350 nm UV light (Rayonet RPR-3500
lamp) at a distance from the light source of approximately 1 cm. The
half-life was unable to be determined in aqueous solution due to
limitations of solubility and the ability to detect the absorption of the
diazirine peak. The disappearance of the diazirine absorption peak was
monitored via serial UV−vis measurements.
Photolabeling HSAF with AziFo. Solutions containing 50 μg (1
mg/mL, 25 μM dimer) of HSAF (Sigma-Aldrich, 0.2 m filtered) were
equilibrated with 10 or 100 μM AziFo in DPBS (pH = 7.4) for 25 min
on ice in the dark. AziFo stock solutions were prepared in DMSO for
a final concentration of 1% DMSO (v/v). The samples were
transferred to 1 mm path length quartz cuvettes and exposed to a 300
nm light (Rayonet RPR-3500 lamp) for a total of 25 min. Proteins
were then precipitated with acetone, resuspended in buffer, reduced
with dithiothreitol, and alkylated with iodoacetamide and underwent
in-solution protease digestion with trypsin. Samples were desalted
with C18 stage tips in preparation for LC/MS/MS analysis.25
Digested protein preparations were analyzed by an Orbitrap Elite
Hybrid Ion Trap-Orbitrap Mass Spectrometer (MS) coupled to an
1-Aminoanthracene Competition Fluorescence Assay. This
assay was performed in a similar manner to that previously described
with a few modifications.7,13,24 A saturated solution of 1-AMA (1-
aminoanthracene, Sigma-Aldrich, technical grade) was prepared by
sonication in Dulbecco’s phosphate buffered saline (DPBS, pH = 7.4)
followed by filtration with a 1.2 μm glass microfiber filter (Whatman).
The concentration was determined by UV−vis spectroscopy utilizing
an experimentally determined extinction coefficient of 1-AMA (Σ368
=
4073 M−1 cm−1; see Figure S8). Stock solutions of ligand (AziFo or
fropofol) were prepared in DMSO due to limited aqueous solubility.
Horse spleen apoferritin (HSAF, Sigma-Aldrich, 0.2 μm filtered) was
used as received. 1-AMA (15 μM final concentration) was pre-
equilibrated with HSAF (15 μM dimer final concentration), and 5 μL
of ligand stock in DMSO was then added for a final volume of 500 μL
(1% DMSO v/v in DPBS) with the final ligand concentrations
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pubs.acs.org/chemneuro
Research Article
Easy-nanoLC 1000 system. Spectral analysis was conducted using
MaxQuant (Max Planck Institute of Biochemistry).26 For more
detailed methods, see the Supporting Information.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acschemneuro.0c00667
Molecular Docking Calculations. The calculations of ligand
docking poses were generated using a previously published crystal
structure of HSAF (PDB ID 3f32, 1.7 Å resolution).21 From the
molecular assembly, a homodimer was generated, and small molecules
(water and sulfate and cadmium ions) were removed with PyMOL
Author Contributions
R.G.E. and W.P.D. conceived the original idea. E.R.W. directed
the project with supervision from R.G.E. W.P.D. performed all
chemical syntheses and characterizations. N.V.B. analyzed the
MS/MS samples under the supervision of B.A.G. V.M.B.
performed the zebrafish experiments. E.R.W. and D.M.L.
performed all the remaining experiments and took the lead in
writing the manuscript with input from all the authors who
provided critical feedback and helped shape the research,
analysis, and manuscript.
̈
(The PyMOL Molecular Graphics System, Version 2.3.5 Schrodinger,
LLC.). AutoDock Tools was used to add Gasteiger charges and merge
nonpolar hydrogens.27 The 2D drawing and 3D optimization of the
ligand structures were performed in ChemDoodle (version 9.1.0,
iChemLabs) followed by conversion to .pdbqt files with PyMOL and
AutoDock Tools. Maximum torsions were added to each ligand to
allow full flexibility. Ligand docking was conducted with AutoDock
Vina with an 18 × 20 × 20 Å grid box centered at the homodimer
interface.22,27 All default algorithmic parameters were used including
side chain flexibility and exhaustiveness. Images and atomic
measurements were generated with PyMOL. The identification of
atoms lining the solvent accessible pocket was conducted with
CASTp.28
Funding
This work was funded by the following sources: Research
Fellowship Grant from the Foundation for Anesthesia
Education and Research, National Institutes of Health
National Institute of General Medical Sciences grants P01-
GM-055876, R01-GM-110174, and T32-GM-112596.
Notes
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acschemneuro.0c00667.
■
The authors declare no competing financial interest.
sı
ACKNOWLEDGMENTS
■
The authors would like to thank Dr. Michael A. Pack
(University of Pennsylvania, Perelman School of Medicine,
Department of Medicine) for graciously providing wild-type
zebrafish larvae.
NMR, absorbance, fluorescence, and MS/MS spectra
with associated ion fragmentation tables, HSAF
sequence coverage, CASTp analysis, an additional table
of docking simulation data, and supplemental methods
(PDF)
ABBREVIATIONS
■
1-AMA, 1-aminoanthracene; AziFo, ortho-azi-fropofol; AziPm,
meta-Azi-propofol; CDCl3, deuterated chloroform; CI, con-
fidence interval; cLogP, octanol/water partition coefficient; E3,
E3 zebrafish embryo water; HCl, hydrochloric acid; HCM,
hypertrophic cardiomyopathy; HSAF, horse spleen apoferritin;
dpf, days postfertilization; DPBS, Dubelco’s phosphate
buffered saline; HRMS, high resolution mass spectrometry;
PAL, photoaffinity label; PFTBA, perfluorotributylamine;
AUTHOR INFORMATION
Corresponding Author
Roderic G. Eckenhoff − Perelman School of Medicine,
Department of Anesthesiology and Critical Care, University of
Pennsylvania, Philadelphia, Pennsylvania 19104, United
States; Phone: 215-662-3705; Email: roderic.eckenhoff@
pennmedicine.upenn.edu; Fax: 215-349-5078
■
PVDF, polyvinyl difluoride; sec-BuLi, sec-butyllithium; t1/2
half-life
,
Authors
E. Railey White − Perelman School of Medicine, Department
of Anesthesiology and Critical Care, University of
Pennsylvania, Philadelphia, Pennsylvania 19104, United
States; orcid.org/0000-0001-9729-6394
David M. Leace − Perelman School of Medicine, Department
of Anesthesiology and Critical Care, University of
Pennsylvania, Philadelphia, Pennsylvania 19104, United
States
Victoria M. Bedell − Perelman School of Medicine,
Department of Anesthesiology and Critical Care, University of
Pennsylvania, Philadelphia, Pennsylvania 19104, United
States
Natarajan V. Bhanu − Perelman School of Medicine,
Department of Biochemistry and Biophysics, Smilow Center
for Translational Research, University of Pennsylvania,
Philadelphia, Pennsylvania 19104, United States
Benjamin A. Garcia − Perelman School of Medicine,
Department of Biochemistry and Biophysics, Smilow Center
for Translational Research, University of Pennsylvania,
Philadelphia, Pennsylvania 19104, United States;
orcid.org/0000-0002-2306-1207
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