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1H NMR (400 MHz, CDCl3): δ 5.91–5.82 (m, 1H), 5.53–5.46 (m, 1H), 5.37 (dd,
To a solution of 1h (37 mg, 0.092 mmol), and imidazole (12.5 mg, 0.184 mmol)
in DCM (0.6 ml) at 0 °C was added a solution of tert-butyldimethylsilyl chloride
(15.4 mg, 0.1 mmol) in DCM (0.3 ml) dropwise. The mixture was stirred for 1 h,
and warmed to RT, and stirred for 0.5 h. The mixture was concentrated in vacuo,
and the crude material was purified by flash chromatography (5–15% ethyl acetate
in hexanes) to yield 3a (39 mg, 0.076 mmol) as a white solid.
J = 15.3, 6.9 Hz, 1H), 3.95–3.87 (m, 1H), 3.83–3.71 (m, 1H), 3.48 (br s, 1H),
2.11–2.00 (m, 5H), 1.39–1.19 (m, 22H), 0.88 (t, J = 7.0, 3H).
13C NMR (100 MHz, CDCl3): δ 170.93, 139.50, 121.36, 71.82, 58.05, 56.05,
32.23, 31.93, 29.71, 29.68, 29.67, 29.67, 29.59, 29.41, 29.36, 29.24, 28.54, 22.69,
20.76, 14.11.
1H NMR (400 MHz, CDCl3): δ 5.80–5.72 (m, 1H), 5.50 (dd, J = 15.4, 5.9 Hz,
1H), 5.22 (br d, J = 7.8 Hz, 1H), 4.22–4.16 (m, 1H), 3.94 (dd, J = 10.3, 3.0 Hz, 1H),
3.78–3.71 (m, 1H), 3.57 (br s, 1H), 3.30 (br d, J = 8.1 Hz, 1H), 2.09–2.02 (m, 2H),
1.45 (s, 9H), 1.40–1.24 (m, 22H), 0.91–0.86 (m, 12H), 0.07 (d, J = 1.1 Hz, 6H).
HRMS (DART, M+H+): m/z calcd. for C20H40NO3+ 342.3003, found 342.3002.
N-((2S,3R,E)-1,3-Dihydroxyoctadec-4-en-2-yl)acetamide (N-AS)
OH
13C NMR (100 MHz, CDCl3): δ 155.74, 133.06, 129.42, 79.39, 74.62, 63.45,
54.49, 32.30, 31.91, 29.68, 29.64, 29.62, 29.50, 29.35, 29.19, 28.38, 25.81, 22.68,
18.12, 14.10, −5.62, −5.65.
HO
(CH2)12
MS (APCI, M+H+): m/z found 514.4.
HN
(2S,3R,E)-2-((tert-Butoxycarbonyl)amino)-1-((tert-butyldimethylsilyl)oxy)
octadec-4-en-3-yl acetate (3b)
Ac
Synthesis of N-AS from 1-O-AS
OAc
To a solution of 1-O-AS (13.5 mg, 0.029 mmol) in DCM (0.53 ml) at RT. was
added triethylamine (90 μl, 0.64 mmol), and was stirred for 3 h. The mixture was
concentrated in vacuo, and purified by flash chromatography (1–5% methanol in
DCM) to yield N-AS (9 mg, 0.026 mmol) as a white solid.
TBDMSO
(CH2)12
HN
Synthesis of N-AS from 3-O-AS
Boc
To a solution of 3-O-AS (17 mg, 0.037 mmol) in DCM (0.6 ml) at RT was
added triethylamine (115 μl, 0.82 mmol) and was stirred for 3 h. The mixture was
concentrated in vacuo, and purified by flash chromatography (1–5% methanol in
DCM) to yield N-AS (10 mg, 0.029 mmol) as a white solid.
1H NMR (400 MHz, CDCl3): δ 6.33 (br d, J = 5.5 Hz, 1H), 5.83–5.75 (m, 1H),
5.53 (dd, J = 15.4, 6.4 Hz, 1H), 4.35–4.31 (m, 1H), 3.99–3.89 (m, 2H), 3.71 (dd, J =
11.0, 3.1 Hz, 1H), 2.09–2.02 (m, 5H), 1.40–1.22 (m, 22H), 0.88 (t, J = 7.0, 3H).
13C NMR (100 MHz, CDCl3): δ 171.09, 134.18, 128.59, 74.24, 62.12, 54.56,
32.28, 31.90, 29.66, 29.63, 29.61, 29.48, 29.33, 29.22, 29.13, 23.31, 22.66, 14.09.
HRMS (DART, M+H+): m/z calcd. for C20H40NO3+ 342.3003, found 342.3031.
To a solution of 3a (38 mg, 0.074 mmol), and 4-dimethylaminopyridine (18 mg,
0.15 mmol) in DCM (0.7 ml) at RT, was added acetic anhydride (10.5 μl,
0.11 mmol). The mixture was stirred for 1 h, and concentrated in vacuo. The crude
material was purified by flash chromatography (5–10% ethyl acetate in hexanes) to
yield 3b (38 mg, 0.068 mmol) as a clear oil.
1H NMR (400 MHz, CDCl3): δ 5.81–5.73 (m, 1H), 5.41 (dd, J = 15.4, 7.6, 1H),
5.27 (t, J = 7.2 Hz, 1H), 4.71 (br d, J = 8.9 Hz, 1H), 3.85 (br s, 1H), 3.70 (dd, J =
10.1, 3.5 Hz, 1H), 3.60 (dd, J = 10.2, 4.5 Hz, 1H), 2.05–1.98 (m, 5H), 1.44 (s, 9H),
1.38–1.22 (m, 22H), 0.90–0.86 (m, 12H), 0.04 (s, 6H).
13C NMR (100 MHz, CDCl3): δ 169.71, 155.41, 136.77, 124.74, 79.29, 76.68,
76.68, 73.78, 61.79, 53.70, 32.34, 31.91, 29.67, 29.65, 29.59, 29.47, 29.35, 29.22,
28.89, 28.36, 25.82, 22.68, 21.25, 18.21, 14.11, −5.57, −5.59.
MS (APCI, M+H+): m/z found 556.4.
(2S,3R,E)-2-((tert-Butoxycarbonyl)amino)-1-hydroxyoctadec-4-en-3-yl acetate
(3c)
LC-ELSD analysis. To a solution of 1-O-AS (0.25 mg) or 3-O-AS (0.25 mg) in
water (400 μl) and DMSO (50 μl) at RT was added sodium phosphate buffer (pH
7.4, 0.1 M, 50 μl). The solution was mixed homogeneously with micropipette and
was incubated at 25 °C for 10 min. Reverse-phase HPLC was performed using
Agilent 1220 coupled with Agilent 1260 Infinity ELSD. Phenomenex Kinetex
1.7 μm C8 100 A (50 × 21 mm LC column) at a flow rate of 0.5 ml min−1 was used.
A gradient of acetonitrile with 0.1% formic acid was used as an eluent.
OAc
LC-MS/MS analysis for structure elucidation of N-AS. To determine whether
N-AS was synthesized by acetyl-CoA and sphingosine within SphK1, 10 mM
acetyl-CoA (Sigma-Aldrich, A2056) and 100 μM sphingosine (Sigma-Aldrich,
S7049) were incubated with purified SphK1 (Cayman, 10348) at 37 °C for 24 h in
acetylation buffer (50 mM Tris-HCl pH 7.6, 1 mM DTT, 1 mM EDTA, and 10%
glycerol, all from Sigma-Aldrich). Also, SphK1 WT, SphK1 D178A, and SphK1
F192A were incubated with 100 μM sphingosine and acetyl-CoA ranging from 0 to
10 mM. Reactions were quenched by adding equal volume of cold methanol. A 100
μl aliquot of the reaction mixture was mixed with 30 μl of distilled water containing
5 ng ml−1 of furosemide (internal standard) for 1 min and vigorously mixed with
0.7 ml−1 of ethyl acetate for 20 min. After centrifugation at 16,000×g for 10 min,
the mixtures were kept for 2 h for freezing aqueous phase. The upper ethyl acetate
layer was transferred to a clean tube and evaporated to dryness under a gentle
stream of nitrogen. Then, the dried extract was reconstituted in 100 µl of mobile
phase and 10 µl aliquot of the solution was injected into an Agilent 6470 Triple
Quad LC–MS/MS system (Agilent, Wilmington, DE) coupled with an Agilent 1260
HPLC system. Separation was performed on a Synergy Polar RP column (4 μm
particle size, 2.0 mm × 150 mm, Phenomenex) using a mobile phase that consisted
of methanol and water (95:5, v/v) with 0.1% formic acid at a flow rate of 0.2 ml
min−1. Quantification was carried out using multiple reaction monitoring (MRM)
at m/z 340.3→263.3 for N-AS and m/z 328.9→284.8 for furosemide (internal
standard) in negative ionization mode and collision energy of 20 eV. The lower
limit of quantification was determined to be 0.5 ng ml−1 and linearity was observed
in the standard range of 0.5–200 ng ml−1. Also, to confirm N-AS generation in
neurons, microglia, and astrocytes, these cells were treated with Aβ, and sonicated
in neuronal and glial culture medium. The supernatants were supplemented with
acetyl-CoA (100 μM, Sigma-Aldrich, A2056), SphK1 (20 ng, Cayman, 10348), or
sphingosine (1 μM, Sigma-Aldrich, S7049), and incubated at 37 °C for 2 h. To
evaluate N-AS in microglia and neurons in vivo, the cells were isolated from WT
mice, APP/PS1 mice and APP/PS1 mice treated with N-AS at 9 months of age. The
cells were sonicated and resuspended in glial and neuronal culture medium,
respectively. A 100 μl aliquot of each cell lysate were used for the determination of
N-AS using a method previously described.
HO
(CH2)12
HN
Boc
A solution of HF-pyridine was prepared by addition of ca. 70% HF-pyridine
(6.6 mg) to a solution of pyridine (10 μl) in THF (0.13 ml). This solution was added
to a solution of 3b (19 mg, 0.034 mmol) in THF (0.55 ml) at 0 °C. The mixture was
stirred for 1 h, and slowly warmed to 50 °C over 1 h, and stirred for 18 h. The
reaction was quenched by addition of saturated NaHCO3 solution (30 μl), and
diluted with water. The aqueous layer was extracted with ethyl acetate, and
combined organic layers were dried over Na2SO4 and filtered, and concentrated in
vacuo. The crude material was purified by flash chromatography (10–30% ethyl
acetate in hexanes) to yield 3c (13 mg, 0.029 mmol) as a white solid.
1H NMR (400 MHz, CDCl3): δ 5.81–5.74 (m, 1H), 5.46 (dd, J = 15.4, 7.8 Hz,
1H), 5.27 (t, J = 7.3 Hz, 1H), 4.96 (br d, J = 8.5 Hz, 1H), 3.82–3.73 (m, 1H), 3.65 (d,
J = 3.5 Hz, 2H), 2.09 (s, 3H), 2.06–2.00 (m, 2H), 1.44 (s, 9H), 1.39–1.23 (m, 22H),
0.88 (t, J = 7.0, 3H).
13C NMR (100 MHz, CDCl3): δ 170.79, 155.80, 137.18, 124.50, 79.68, 74.48,
61.86, 54.36, 32.28, 31.90, 29.66, 29.65, 29.63, 29.56, 29.43, 29.33, 29.19, 28.82,
28.31, 22.66, 21.21, 14.09.
MS (APCI, M+H+): m/z found 442.4.
(2S,3R,E)-3-Acetoxy-1-hydroxyoctadec-4-en-2-aminium trifluoroacetate (3-O-
AS)
To a solution of 3c (18 mg, 0.04 mmol) in DCM (0.56 ml) at 0 °C was added
trifluoroacetic acid (240 μl, 3.14 mmol), and triethylsilane (19.3 μl, 0.12 mmol). The
mixture was stirred for 2 h, and concentrated in vacuo to yield 3-O-AS (18 mg,
0.039 mmol) as a pale-yellow oil.
LC-MS/MS analysis of the acetylated site in COX2. To identify the acetylation
site of COX2, N-AS (Sigma-Aldrich, 01912)-treated COX2 enzymes (LSBio, LS-
G21094) were immediately precipitated with trichroloacetic acid (Merck) and
dried. The dried extract was resuspended in 10 μl of 5 M Urea solution and
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