Photolabile Diazirine as Inhaled Anesthetic
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9 1885
quenched with 3 mL of methanol. The mixture was added to
tryptophan-containing regions of bovine serum albumin, in-
creasing concentrations of these compounds (from stock buffer
solutions) were added to 4 mL fluorescence cuvettes containing
∼2 µM protein and examined in a Shimadzu RF 5301 PC
spectrofluorophotometer (Shimadzu Scientific Instruments,
MD) using 295 nm excitation and emission scanning. Care was
taken to eliminate all air from the cuvette with the last
addition, and spectra were collected at room temperature.
Fluorescence values were corrected for inner filter effects due
to these compounds as previously published.2
2
00 mL of 0.5 N HCl, and the solution was extracted with three
portions of ether. The combined ether layers were washed with
aqueous 0.5 N HCl solution, saturated NaHCO , and brine.
The organic layer was dried over Na SO and evaporated.
3
2
4
Toluene (40 mL) was added to the oil, and the resulting
solution was brought to reflux for 2 h. After the solution had
cooled, the water layer was removed. tert-butyl amine (22 mL,
0
.20 mol) was added, and the resulting solution was again
brought to reflux 1.5 h. After cooling, the solution was dried
over Na SO and evaporated. Short-path distillation under
2
4
Hydr ogen -Tr itiu m Exch an ge. We have previously shown
aspirator pressure yielded 15.1 g (0.057 mol, 67% yield) of
that stabilization of serum albumin correlates well with
1
clear, colorless product. BP25 ) 90 °C. H NMR: δ 7.6 (t, 1H,
27
anesthetic potency in a series of volatile compounds. Amide
J
H-F ) 3.5 Hz), 6.0 (t, 1H, J H-F ) 9.9 Hz), 1.2 (s, 9H). 13
C
)
hydrogen-tritium exchange was used to measure the effect
of these compounds on bovine serum albumin stability. For
NMR: δ 29 (s), 55 (t, J C-F ) 33.1 Hz), 59 (s), 114 (t, J C-F
2
J
1
9
49 Hz), 149 (t, J C-F ) 33.3 Hz); F NMR: δ -106 (qAB, 2F,
these measurements, protein solutions (10 mg/mL) were incu-
F-F ) 258 Hz, J F-H ) 9.8 Hz, J F-H ) 3.4 Hz). Anal. (C
N): C, H, N.
7
H
11
-
3
bated with ∼5 mCi HOH in 1 M GdnCl, 0.1 M NaH
2 4
PO pH
BrClF
2
8
.5 buffer for at least 18 h at room temperature. The GdnCl
P r ep a r a tion of (2-Br om o,2-ch lor o-1,1-d iflu or oeth yl)-
-ter t-bu tyl-d ia zir id in e (16). A 25 mL rb flask containing a
stir bar was filled with 1.0 g (0.0038 mol) of 3-bromo-3-chloro-
,2-difluoropropylidene)-tert-butyl amine, 4 mL of anhydrous
ethanol, and 2 mL of triethylamine. The solution was cooled
to 0 °C and 0.88 g (0.007 mol) of hydroxylamine-O-sulfonic acid
was added. After the mixture had stirred for 2 h at 0 °C, 50
mL of ether was added. The resulting solution was washed
with three 10 mL portions of water. The organic layer was
increased exposure of protected hydrogens to solvent, and the
elevated pH increased the rate of chemical hydrogen exchanges
both conditions intended to ensure equilibration of all ex-
changeable hydrogens in the protein prior to initiation of
exchange-out. Free 3HOH was removed and the buffer ex-
changed with a PD-10 gel filtration column (Sigma, MO), and
exchange-out was thereby initiated. After recovery from the
column, the protein solution was immediately transferred to
pre-filled Hamilton (Reno, NV) gas-tight syringes containing
the photolabels and equipped with repeaters (see figure leg-
ends). Aliquots were precipitated with 2 mL of ice-cold 10%
trichloroacetic acid at timed intervals over at least 6 h. The
precipitated protein was rapidly vacuum filtered through
Whatman GF/B filters and washed with 8 mL of ice-cold 2%
TCA. 3H retained by the protein was determined by liquid
scintillation counting as above. Protection factor ratios (PFRs)
were determined by dividing the time required for a given
hydrogen to exchange under the different conditions for the
last three to five hydrogens in common for the two conditions,
and ∆∆G was determined using the equation ∆∆G ) RT ln-
1
2
dried (Na
2 4
SO ) and evaporated to leave 0.58 g (0.0021 mol,
5
5% yield) of product that was solid below room temperature
1
but turned to an oil at room temperature. H NMR: δ 5.9 (t,
1
1
J
H, J H-F ) 3.6 Hz), 5.8 (t, 1H, J H-F ) 3.4 Hz), 3.4 (multiplet,
13
H), 2.3 (broad s, 1H), 1.1 (ds, 9H). C NMR: δ 26 (s), 55 (t,
C-F ) 35 Hz), 51 (q, J C-F ) 32.4 Hz), 60, 115 (t, J C-F ) 246
1
9
Hz). F NMR: δ -115, -116 (dqAB, 2F, J F-F ) 245 Hz, J F-H
)
5.2 Hz). Anal. (C
P r ep a r a tion of 3-(2-Br om o-2-ch lor o-1,1-d iflu or oeth yl)-
H-d ia zir in e (4). A 50 mL rb flask with a stir bar was filled
with 2.0 g (0.0073 mol) of 2-bromo,2-chloro-1,1-difluoroethyl)-
-tert-butyl-diaziridine, 40 mL of methylene chloride, and 1.56
7 2 2
H12BrClF N ): C, H, N.
3
(
PFR).
Oocyte Electr op h ysiology. (i) P r ep a r a tion of cRNA
fr om cDNA. GABA receptor cDNA sequences cloned into
1
g (0.009 mol) of NBS (N-bromosuccinimide). The flask was
covered with tin foil and was stirred for 1.5-2.0 h at room
temperature. A flow of nitrogen gas was used to evaporate
most of the solvent. When approximately 5 mL remained, the
volatiles were fractioned under vacuum (0.01 Torr) through a
series of three U-traps cooled to -10 °C, -45 °C, and -78 °C.
The contents of the -45 °C U-trap were further purified by
preparative gas chromatography (10 ft, 20% SF-96) to give
A
plasmid vectors were used to synthesize capped RNA tran-
scripts for expression in oocytes. Phage polymerases (T7, SP6,
or T3) were used to make full-length capped RNA transcripts
from linearized template DNA using a commercially available
kit (mMessage mMachine (Ambion)) according to manufac-
turer’s protocol.
0
.355 g (0.0016 mol, 23% yield) of 5 as a colorless oil. BP: >110
(ii) In jection of Xen op u s Oocytes a n d Volta ge-Cla m p
1
°
C (decomposition). H NMR: δ 5.8 (t, 1H, J H-F ) 7.3 Hz), 1.7
Recor d in g. Oocytes were obtained from adult female Xenopus
1
3
28
(t, 1H, J H-F ) 7.4 Hz). C NMR: δ 19.6 (t, J C-F ) 32.6 Hz),
as described before. Ovarian lobes were opened with forceps,
1
9
5
4.2 (t, J C-F ) 36.4 Hz), 114.4 (t, J C-F ) 251.5 Hz). F NMR:
and the follicular layer was softened by incubation with
collagenase in OR2 solution (82.5 mM NaCl, 2 mM KCl, 1 mM
δ -106.8 (dt, 2F, J F-F ) 24 Hz, J F-H ) 7.3 Hz).
P r ep a r a tion of 1-Azid o-2-br om o-2-ch lor o-1,1-d iflu or o-
eth a n e (6). To a mixture of 10.0 g (0.154 mol) of sodium azide
in 200 mL of methylene chloride was added 10 mL of
concentrated HCl and 3 mL of water. The mixture was
vigorously stirred for 10 min, then 6.0 g (0.038 mol) of bromine
was added in one portion. After the mixture was stirred for
an additional 30 min, the organic layer was separated and was
added to a 500 mL rb flask equipped with a dry ice cooled gas
condenser and magnetic stir bar. 1-Bromo-1-chloro-2,2-difluo-
roethene (4.8 g, 0.027 mol) was condensed into the flask, and
the flask was irradiated with a 200 W sodium lamp for 4 h
under a dry ice gas condenser. The methylene chloride was
removed by distillation through a Vigreux column and the
MgCl
manually removed, and the oocytes were washed and placed
in ND96 medium (96 mM NaCl, 2 mM KCl, 1 mM MgCl , 1.8
mM CaCl , 5 mM HEPES, 5 mM pyruvate, pH 7.5). Stage
V-VI oocytes were injected in the vegetal pole with RNA (50
nL; 1-2 ng of each cRNA transcript). For expression of these
heteromeric receptors, subunit cRNAs were injected in 1:1
ratio. Oocytes were maintained at 18 °C in ND96 with
antibiotic (50 µg/mL gentamicin) for 3-5 days before use in
experiments.
For recording, oocytes were positioned in a small Perspex
chamber and continuously superfused (5 mL/min) with ND96
solution. Oocytes were impaled with borosilicate glass micro-
electrodes filled with 3 M KCl (resistance 0.5-3 MΩ), and
currents were recorded from oocytes using a two-electrode
voltage-clamp amplifier (GeneClamp 500, Axon Instruments).
To record currents, signals were low-pass filtered and digitized,
using an A/D interface (MacLab 4/S with chart and scope
software, A.D. Instruments, Mountain View, CA), and stored
on the hard disk of a computer for offline analysis.
2
, 5 mM HEPES, pH 7.5). Remaining follicular cells were
2
2
product was distilled to yield 80% of a clear, colorless oil, bp
1
23 °C. IR: 2250 cm-1. H NMR: δ 5.71 (t, J H-F ) 5.9 Hz).
1
1
3
C NMR: δ 55.0 (t, J C-F ) 39.6 Hz), 119.0 (t, J C-F ) 70.0
Hz). F NMR: δ -81.5 (m).
1
9
F lu or escen ce Sp ectr oscop y. Several of the designed
compounds contain heavy atoms or delocalized electrons that
can quench tryptophan fluorescence if bound in the immediate
2
vicinity (<5 Å). Thus, to determine if the various inhaled
All compounds were solubilized in GABA-containing oocyte
buffer as above, loaded into gas-tight Hamilton syringes, and
anesthetics can gain access to and exhibit selectivity for the