Letter reSeArCH
MꢁꢂꢃOꢄS
2 2 4
CH Cl . The organic phase was washed with brine, dried over MgSO and concen-
No statistical methods were used to predetermine sample size. However, for trated in vacuo. Purification by flash column chromatography on silica gel (50–80%
mutants in the cAMP accumulation assays, after an n=3 was obtained, an addi- diethyl ether in hexanes) gave 124mg of 15 as a white solid in 84% yield. Melting
3
tional power analysis was performed (alpha=0.05; power=80%) to determine point=59–61°C; IR (neat) 3343 (br, OH), 2931, 2860, 2093 (s, N ), 1713, 1621,
-1 1
3
the n required to have confidence in the values produced; additional curves were 1537, 1413, 1331, 1268, 1138, 1011, 967, 839cm ; H NMR (500MHz, CDCl )
added as indicated.
δ6.35 (d, J=1.0Hz, 1H, Ar-H), 6.19 (d, J=1.5Hz, 1H, Ar-H), 4.81 (br s, 1H, ArOH),
2
Synthesis of AM11542 and AM841: experimental procedures and spectroscopic 3.57–3.47 (m, 2H, -CH OH), 3.23–3.17 (m and t overlapping 3H, C-ring, 7′-H,
data. Experimental procedures for steps a–m (Extended Data Fig. 1) are similar to especially, 3.21, t, J=6.5Hz, 2H, 7′-H), 2.52–2.44 (m as td, J=11.0Hz, J=2.5Hz,
9,21,22
those we reported earlier for closely related systems
.
1H, C-ring), 2.02–1.91 (m, 2H, C-ring), 1.82–1.74 (m, 1H, C-ring), 1.56–1.46
- of the
- of the side
(
6aR,9R,10aR)-3-(8-bromo-2-methyloctan-2-yl)-1-((tert-butyldimethylsilyl)oxy)- (m 5H, 6′-H, 2′-H, C-ring), 1.38 (s, 3H, 6-Me), 1.35–1.26 (m, 2H, -CH
2
6
,6-dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromene-9-carbaldehyde (12). side chain), 1.25–1.11 (s and m overlapping, 10H, -C(CH
3
)
2
-, -CH
2
1
Colourless oil. H NMR (500MHz, CDCl3) δ 9.63 (d, J=1.5Hz, 1H, 9β-CHO), chain, C-ring, especially, 1.20, s, 6H, -C(CH
)
3 2
-), 1.10–1.02 (s and m overlapping,
6
.38 (d, J=2.0Hz, 1H, Ar-H), 6.32 (d, J=2.0Hz, 1H, Ar-H), 3.52–3.46 (t and 5H, 6-Me, -CH
m as br d overlapping, t, J = 6.5 Hz, 2H, -CH2Br, m as br d, J = 13.5 Hz, 1H, q, J=12Hz, 1H, C-ring). C NMR (100MHz, CDCl
C-ring), 2.46–2.33 (m, 2H, C-ring), 2.14–2.06 (m, 1H, C-ring), 2.02–1.96 107.8, 105.4, 68.5, 51.5, 49.3, 44.2, 40.5, 37.2, 34.9, 33.1, 29.7, 29.6, 28.8, 28.7, 27.7,
2
- of the side chain, especially, 1.09, s, 3H, 6-Me), 0.87–0.78 (m as
13
3
) δ 154.6, 154.5, 149.7, 109.6,
+
(
40 3 3
m, 1H, C-ring), 1.69 (sextet, J= 6.7 Hz, 2H, 6′-H), 1.52–1.42 (m, 4H, 2′-H, 27.4, 26.5, 24.4, 19.0. HRMS (m/z): [M] calculated for C25H N O , 430.3070;
C-ring), 1.42–130 (m and s, overlapping, 5H, -CH
2
- of the side chain and 1.39, s, found, 430.3065.
6
-Me), 1.26–1.10 (m, 10H, -CH2- of the side chain, C-ring and -C(CH3)2-), 1.09- (6aR,9R,10aR)-9-(Hydroxymethyl)-3-(8-isothiocyanato-2-methyloctan-2-yl)-6,6-
.00 (m, s and s, overlapping, 14H as follows: 2H, -CH2- of the side chain, 1.08, dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol (AM841). To a
1
s, 3H, 6-Me, 1.01, s, 9H, -Si(Me)
2
CMe
3
), 0.26 (s, 3H, Si(Me)
2
CMe
3
), 0.15 (s, 3H, solution of 15 (120mg, 0.28mmol), in anhydrous THF (5.6ml) at room tem-
1
3
Si(Me)2CMe3). C NMR (100MHz, CDCl3) δ 203.5, 154.5, 154.2, 149.5, 112.5, perature, was added triphenyl phosphine (365mg, 1.4mmol). Carbon disulfide
1
2
09.5, 108.4, 50.5, 49.0, 45.0, 44.4, 37.3, 35.4, 32.6, 30.2, 29.5, 28.8, 28.6, 27.6, (0.55ml, 8.4mmol) was then added dropwise and the reaction mixture was stirred
6.9, 26.8, 25.9, 24.5, 18.8, 18.2, −3.6− -4.2. HRMS (m/z): [M+H]+ calculated for an additional 10h at the same temperature. Upon completion, the reaction
7
9
81
for C31
H
52
O
3
BrSi, 579.2869; found, 579.2862; calculated for C31
H O
52 3
BrSi, mixture was concentrated under reduced pressure and purified by flash column
chromatography on silica gel (50–80% diethyl ether in hexanes) to give 95mg of
5
81.2849; found, 581.2850.
{
(6aR,9R,10aR)-3-(8-Bromo-2-methyloctan-2-yl)-1-[(tert-butyldimethylsilyl)oxy]- AM841 as white solid in 76% yield. Melting point=63–65°C. IR (neat) 3332 (br,
6
,6-dimethyl-6a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-9-yl}methanol (13). OH), 2931, 2860, 2093 (s, NCS), 1620, 1537, 1451, 1413, 1331, 1269, 1137, 1037,
1
−1 1
Colourless viscous oil. H NMR (500MHz, CDCl
.30 (d, J=2.0Hz, 1H, Ar-H), 3.54 (dd, J=10.5Hz, J=5.5Hz, half of an AB sys- (d, J=2.0Hz, 1H, Ar-H), 4.76 (br s, 1H, ArOH), 3.52 (m, 2H, -CH2OH), 3.46
tem, 1H, -CH2OH), 3.50–3.43 (dd and t overlapping, especially, 3.48, t, J=6.5, (t, J=6.5Hz, 2H, 7′-H), 3.22-3.16 (m as d, J=13Hz, 1H, C-ring), 2.51–2.44 (m
′-H, dd, J=10.0Hz, J=6.5Hz, half of an AB system, 1H, -CH OH), 3.18-3.16 as td, J=11.0Hz, J=2.5Hz, 1H, C-ring), 2.02-1.91 (m, 2H, C-ring), 1.82-1.74
m as br d, J=13.0Hz, 1H, C-ring), 2.40-2.32 (m as td, J=11.0Hz, J=3.0Hz, 1H, (m, 1H, C-ring), 1.65-1.56 (m, 2H, 6′-H), 1.54–1.46 (m, 3H, 2′-H, C-ring), 1.39
C-ring), 2.04–1.97 (m, 1H, C-ring), 1.94–1.88 (m, 1H, C-ring), 1.8–1.64 (m, 1H, (s, 3H, 6-Me), 1.37–1.29 (m, 2H, -CH2- of the side chain group), 1.26–1.11 (s and
C-ring, 2H, 6′-H), 1.52–1.44 (m, 3H, 2′-H, C-ring), 1.4–1.3 (m and s overlapping, m overlapping, 10H, -C(CH -, -CH - of the side chain, C-ring, especially, 1.20,
H, -CH - of the side chain, 6-Me, especially 1.25, s, 3H, 6-Me), 1.24–1.1 (m, s, s, 6H, -C(CH -), 1.10–1.03 (s and m overlapping, 5H, 6-Me, -CH - of the side
and s overlapping, 10H, -C(CH -, -CH - of the side chain, C-ring, especially, chain, especially, 1.09, s, 3H, 6-Me), 0.87–0.7 (m as q, J=12Hz, 1H, C-ring);
.20, s, 3H, -C(CH -, and 1.19, s, 3H, -C(CH -), 1.09-1.02 (s and m overlap- NMR (100MHz, CDCl ) δ 154.6 (ArC-1 or ArC-5), 154.5 (ArC-5 or ArC-1), 130.1
ping, 5H, 6-Me, -CH - of the side chain, especially, 1.06, s, 3H, 6-Me), 1.0 (s, 9H, (NCS), 149.6 (tertiary aromatic), 109.7 (tertiary aromatic), 107.8 (ArC-2 or ArC-4),
Si(Me) CMe ), 0.82-0.7 (m, 1H, C-ring), 0.23 (s, 3H, Si(Me) CMe ), 0.12 (s, 3H, 105.3 (ArC-4 or ArC-2), 68.5 (-CH OH), 49.3, 45.0, 44.1, 40.5, 37.2, 35.0, 33.1, 29.8,
3 3
) δ 6.37 (d, J=2.0Hz, 1H, Ar-H), 966, 838cm ; H NMR (500MHz, CDCl ) δ 6.35 (d, J=1.5Hz, 1H, Ar-H), 6.19
6
7
(
2
)
3 2
2
5
2
)
3 2
2
13
)
3 2
2
C
1
)
3 2
)
3 2
3
2
2
3
2
3
2
1
3
+
Si(Me)2CMe3). C NMR (100MHz, CDCl3) δ 154.5, 154.3, 149.0, 113.5, 109.7, 29.7, 29.3, 28.7, 28.6, 27.7, 27.4, 26.3, 24.3, 19.0. HRMS (m/z): [M] calculated for
1
08.4, 68.5, 49.6, 45.1, 44.4, 40.5, 37.2, 35.5, 33.2, 32.6, 29.8, 29.5, 28.8, 28.6, 27.6, C26H40NO3S, 446.2729; found, 446.2726.8
+
27.5, 26.8, 25.9, 24.5, 18,8, 18.2, −3.6, −4.3. HRMS (m/z): [M+H] calculated (-)-7′-Bromo-1′,1′-dimethylheptyl-Δ -tetrahydrocannabinol (AM11542).
79
81
for C31
H O
54 3
BrSi, 581.3026; found, 581.3018; calculated for C31
H O
54 3
BrSi, Experimental procedures for the synthesis and purification, along with spectro-
8
5
83.3005; found, 583.3007.
scopic and analytical data were reported earlier from our laboratory .
(
6aR,9R,10aR)-3-(8-Bromo-2-methyloctan-2-yl)-9-(hydroxymethyl)-6,6-dimethyl- Purification of CB1–flavodoxin protein and crystallization in lipidic cubic
6
a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol (14). To a solution of 13 phase. CB
1
–flavodoxin construction, expression and membrane preparation were
2
(210mg, 0.36mmol) in anhydrous THF (9ml) at −40°C, under an argon atmos- performed using the same procedure as descried before . In brief, the construct has
phere, was added tetra-n-butylammonium fluoride (0.72 ml, 0.72 mmol, 1 M truncations of residues 1–98, 307–331 and 415–472, the flavodoxin (PDB accession
solution in anhydrous THF). The reaction mixture was stirred for 30min at the 1I1O, molecular mass 14.9kDa, with Y98W mutation) fusion protein was fused
4
same temperature, and then quenched using a saturated aqueous NH Cl solution. to the truncated third intracellular loop of the human CNR1 (also known as CB1)
1
Extractive isolation with diethyl ether, and purification by flash column chro- gene. The resulting CB –flavodoxin chimaera sequence was subcloned into a mod-
matography on silica gel (20–50% ethyl acetate in hexane) gave 14 (164mg, 96% ified mammalian expression vector pTT5 that contains a haemagglutinin (HA)
1
yield) as a white solid. Melting point=68–70°C. H NMR (500MHz, CDCl3) signal sequence, a Flag tag and 10× His tag, followed by a tobacco etch virus (TEV)
δ6.35 (d, J=1.0Hz, 1H, Ar-H), 6.18 (d, J=1.5Hz, 1H, Ar-H), 4.75 (br s, 1H, ArOH), protease cleavage site, before the N terminus of the chimaera sequence. The CNR1
23
3
.61–3.42 (m and t overlapping, 4H, -CH2OH, 7′-H, especially, 3.49, t, J=6.5Hz, gene was further modified by introducing four rationally designed mutations ,
3.46
5.37
5.47
6.32
2
H, -CH2OH), 3.23–3.16 (m as br d, J=13.0Hz, 1H, C-ring), 2.52–2.44 (m as td, Thr210 Ala, Glu273 Lys, Thr283 Val and Arg340 Glu, using standard
J=11.0Hz, J=2.5Hz, 1H, C-ring), 2.02–1.91 (m, 2H, C-ring), 1.82-1.74 (m, 1H, QuickChange PCR. The protein was expressed using the FreeStyle 293 Expression
C-ring), 1.72–1.64 (m, 1H, 6′-H), 1.54–1.46 (m, 3H, 2′-H, C-ring), 1.44–1.31 system (Invitrogen) in HEK293F cells for 48h, and the membrane was washed
(
s and m overlapping, 5H, 6-Me, -CH
2
- of the side chain, especially, 1.39, s, 3H, repeatedly using hypotonic buffer with low and high salt. Notably, the receptor used
3
6
-Me), 1.29–1.15 (s, and m overlapping, 10H, -C(CH
C-ring, especially, 1.20, s, 6H, -C(CH
-Me, -CH - of the side chain, especially, 1.09, s, 3H, 6-Me), 0.89–0.78 (m as q, 0.80)nM) as the wild-type receptor, and cold CP55,940 (K
J=11.5Hz, 1H, C-ring). C NMR (100MHz, CDCl
3 2
) -, -CH
2
- of the side chain, for crystallization was capable of binding to [ H]CP55,940 and this binding could
)
3 2
-), 1.10–1.01 (s and m overlapping, 5H, be replaced by AM11542 (K
i
=0.29 (0.17–0.50)nM); AM841 (K
=2.0 (1.2–3.5)nM).
construct yielded no signalling in signalling assays (not shown), which is
07.9, 105.4, 68.5, 49.3, 45.2, 44.2, 40.5, 37.2, 34.9, 33.1, 32.6, 29.7, 29.5, 28.7, 28.6, probably due to the flavodoxin insert that prevents coupling secondary effectors.
i
=0.53 (0.36–
6
2
i
13
3
) δ 154.7, 154.4, 149.7, 109.6, This CB
1
1
2
4
+
79
40 3
7.7, 27.4, 26.7, 24.4, 19.0. HRMS (m/z): [M+H] calculated for C25H O Br, However, the individual point mutations did not interfere with agonist activity
81
3.46
23
40 3
67.2161; found, 467.2162; calculated for C25H O Br, 469.2140; found, 469.2144. except for Thr210 Ala, which has been previously reported . These controls are
(
6aR,9R,10aR)-3-(8-Azido-2-methyloctan-2-yl)-9-(hydroxymethyl)-6,6-dimethyl- summarized in Extended Data Fig. 4c and Extended Data Table 2.
6
a,7,8,9,10,10a-hexahydro-6H-benzo[c]chromen-1-ol (15). To a stirred solution
Purified membranes were thawed at room temperature and then incubated with
of 14 (160 mg, 0.34 mmol) in anhydrous CH3Cl/CH3NO2 (1:1 mixture, 6ml) 20μM corresponding ligand (AM11542 or AM841) in the presence of 1.0mg ml−
at room temperature, under an argon atmosphere was added N,N,N′,N′- iodoacetamide, and EDTA-free protease inhibitor cocktail (Roche) for 30min at
tetramethylguanidinium azide (1.6g, 10.2mmol) and stirring was continued for room temperature, and then further incubated at 4°C for 3h. The membranes
1
1
day. On completion, the reaction was quenched with water and diluted with were then solubilized with 50 mM HEPES (pH 7.5), 500 mM NaCl, 1% (w/v)
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