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column chromatography using 40–60 petroleum ether/ethyl ace-
tate (10:1) as eluent to afford the title compound 6a (2.69 g,
5.7 mmol, 92%) as a colourless oil. Rf =0.34 [40–60 petroleum
ether/diethyl ether=10:1]; [a2D3:7]=À30.9 (c 1.00, CHCl3); 1H NMR
(400 MHz, [D6]-DMSO, T=403 K): d=7.47–7.13 (m, 10H, d-Hiv-
C1’cH–d-Hiv-C1’eH and Phe-C5H–Phe-C7H), 5.20 (s, 2H, d-Hiv-
C1’aH2), 4.99 (dd, J=10.4, 5.4 Hz, 1H, Phe-C2H), 4.92 (d, J=4.6 Hz,
1H, d-Hiv-C2H), 3.23 (dd, J=14.6, 5.4 Hz, 1H, Phe-C3H), 3.01 (dd,
J=14.6, 10.4 Hz, 1H, Phe-C3H), 2.70 (s, 3H, Phe-NCH3), 2.28–2.13
(m, 1H, d-Hiv-C3H), 1.33 (s, 9H, Phe-C2’cH3), 0.96 (d, J=6.9 Hz, 3H,
d-Hiv-C4H3), 0.93 ppm (d, J=6.9 Hz, 3H, d-Hiv-C4H3); 13C NMR
(101 MHz, [D6]-DMSO, T=403 K): d=169.6 (quat., Phe-C1), 167.8
(quat., d-Hiv-C1), 155.5 (quat., Phe-C2’a), 136.8 (quat., Phe-C4),
135.0 (quat., d-Hiv-C1’b), 128.0, 127.7, 127.4, 127.3, 125.6 (10CH,
d-Hiv-C1’c–d-Hiv-C1’e, Phe-C5–Phe-C7), 78.7 (quat., Phe-C2’b), 76.4
(CH, d-Hiv-C2), 65.7 (CH2, d-Hiv-C1’a), 58.8 (CH, Phe-C2), 33.8 (CH2,
Phe-C3), 30.2 (CH3, Phe-NC), 29.1 (CH, d-Hiv-C3), 27.3 (3CH3, Phe-
4.3 Hz, 6H, 2d-HivB-C4H3), 0.78 (d, J=6.8 Hz, 3H, d-HivA-C4H3),
0.71–0.55 ppm (m, 3H, d-HivA-C4H3); 13C NMR (101 MHz, [D6]-
DMSO, T=403 K): d=169.3 (quat., PheA-C1), 169.1 (quat., PheB-C1),
168.3 (quat., d-HivA-C1), 167.8 (quat., d-HivB-C1), 154.1 (quat., PheA-
C2’a), 136.9 (quat., PheA-C4), 136.4 (quat., PheB-C4), 135.0 (quat., d-
HivB-C1’b), 128.1, 128.0, 127.7, 127.5, 127.4, 127.4, 125.7, 125.6 (15
CH, PheA+B-C5–PheA+B-C7 and d-HivB-C1’c–d-HivB-C1’e), 78.5 (quat.,
PheA-C2’b), 76.7 (CH, d-HivB-C2), 74.4 (CH, d-HivA-C2), 65.7 (CH2, d-
HivB-C1’a), 58.7 (CH, PheA-C2), 57.5 (CH, PheB-C2), 33.9 (CH2, PheB-
C3), 33.6 (CH2, PheA-C3), 31.0 (CH3, PheB-NC), 30.0 (CH3, Phe A-NC),
29.0 (CH, d-HivB-C3), 28.6 (CH, d-HivA-C3), 27.3 (3CH3, PheA-C2’c),
17.8 (CH3, d-HivA-C4), 17.4 (CH3, d-HivB-C4), 16.3 (CH3, d-HivB-C4),
A
~
15.7 ppm (CH3, d-Hiv -C4); IR (neat): n=2962, 2937, 1761, 1737,
1683, 1662, 1454, 1394, 1290, 1167, 1128, 1029, 745, 696 cmÀ1
HRMS (ESI): found 731.3878 [M+H]+ C42H55N2O9 requires 731.3907
D=3.9 ppm.
;
Synthesis of Boc-Me-Phe-d-Hiv-Me-Phe-d-Hiv-OBn (8a) [flow]: Di-
peptidol 6a (0.024 g, 0.05 mmol) was taken up in dichloromethane
and this solution was hydrogenated using an H-Cubeꢁ Pro (Thales-
Nano) with a 10% Pd/C CatCartꢁ. The pump was run at 1 mLminÀ1
using dichloromethane with the temperature set to 608C and the
pressure to 6 bar. The solvent was then removed in vacuo and the
crude acid was used directly in the coupling.
~
C2’c), 17.5 (CH3, d-Hiv-C4), 16.2 ppm (CH3, d-Hiv-C4); IR (neat): n=
2972, 2933, 2880, 1741, 1694, 1455, 1390, 1366, 1167, 1127, 1029,
750, 697 cmÀ1; HRMS (ESI): found 470.2531 [M+H]+ C27H36NO6 re-
quires 470.2543 D=2.4 ppm.
Synthesis of Boc-Me-Phe-d-Hiv-Me-Phe-d-Hiv-OBn (8a) [batch]:
Anhydrous HCl (2.5 mL, 4m in dioxane) was added to a solution of
Boc-Me-Phe-d-Hiv-OBn (6a; 0.31 g, 0.66 mmol) in dioxane (2.5 mL)
at room temperature and the resulting mixture was stirred for 3 h.
The solvent was removed in vacuo and the residue was taken up
sequentially in ethanol (25 mL) and methanol (5 mL) with the sol-
vent removed in vacuo after each addition to afford the amine HCl
salt, which was used in the next step without further purification.
Dipeptidol 6a (0.024 g, 0.05 mmol) was taken up in dioxane (1 mL)
and anhydrous HCl (1 mL, 4m in dioxane) was added and the reac-
tion mixture stirred at room temperature for 3 h. The solvent was
then removed in vacuo and the crude oil was taken up in ethanol
(22 mL) and methanol (2 mL) successively, removing the solvent
in vacuo after each addition to afford the amine as the HCl salt,
which was used directly in the coupling.
Hydrogen gas was bubbled through a solution of Boc-Me-Phe-d-
Hiv-OBn (6a; 0.31 g, 0.66 mmol) and palladium (70 mg, 10% on
charcoal, 0.07 mmol) in tetrahydrofuran (5 mL) for 3 min at room
temperature before the resulting mixture was stirred under a hy-
drogen atmosphere for 2.5 h. The reaction mixture was filtered
through Celite, eluting with ethyl acetate (20 mL). The solvent was
removed in vacuo from the filtrate to afford the acid which was
used in the next step without further purification.
The flow equipment was set up according to Table 3. The crude
acid was taken up in dichloromethane (5 mL) and filled into loop 1.
Ghosez reagent (0.013 mL, 0.1 mmol) was taken up in dichlorome-
thane (5 mL) and filled into loop 2. The crude amine HCl salt and
diisopropylethylamine (0.032 mL, 0.18 mmol) were taken up in di-
chloromethane (4.968 mL) and filled into loop 3. Pumps 1–3 were
run a 1 mLminÀ1 with dichloromethane and injection loops 1–3 in-
jected at the appropriate time to ensure coordinated meeting of
streams at the T-pieces (determined by runs using Sudan red dye):
loop 1 at t=0, loop 2 at t=23 s and loop 3 at t=4 min 53 s. From
t=15 min to t=26 min the reaction outflow was collected in stir-
ring aqueous HCl (10 mL, 1m) at 08C. The reaction mixture was ex-
tracted with dichloromethane (320 mL) and the combined organ-
ic extracts were washed with water (10 mL), saturated NaHCO3
(10 mL) and saturated NaCl (10 mL) before being dried over MgSO4
and the solvent being removed in vacuo. The resultant crude prod-
uct was then purified using preparative TLC using 40–60 petrole-
um ether/ethyl acetate (2:1) as eluent to afford the title compound
8a (0.031 g, 0.042 mmol, 82%) as a colourless solid, which was
spectroscopically identical to that reported using method A.
Ghosez reagent (0.096 mL, 0.72 mmol) was added to a solution of
the crude acid in dichloromethane (2.5 mL) at 08C and the result-
ing mixture was stirred for 20 min at this temperature. A solution
of the crude amine HCl salt and N,N-diisopropylethylamine
(0.40 mL, 2.36 mmol) in dichloromethane (2.5 mL) was added and
the reaction mixture was allowed to warm to room temperature
and stirred for 17 h. Aqueous HCl (20 mL, 1m) was added and the
phases were separated. The aqueous layer was extracted with di-
chloromethane (320 mL) and the combined organic layers were
washed with water (40 mL), saturated NaHCO3 solution (40 mL)
and saturated NaCl solution (40 mL) before drying over MgSO4 and
removing the solvent in vacuo. The crude product was purified by
flash column chromatography using 40–60 petroleum ether/ethyl
acetate (10:1) as eluent to afford the title compound 8a (0.33 g,
0.45 mmol, 68%) as a colourless solid. Rf =0.06 [40–60 petroleum
ether/diethyl ether=10:1]; m.p.=120–1228C; [a2D3:7]=À47.4 (c
1.00, CHCl3); 1H NMR (400 MHz, [D6]-DMSO, T=403 K): d=7.51–
7.31 (m, 5H, d-HivB-C1’cH–d-HivB-C1’eH), 7.31–7.08 (m, 10H, PheA+
Synthesis of beauvericin (1a) [batch]: Hydrogen gas was bubbled
through a solution of dipeptidol trimer 10a (83 mg, 84 mmol) and
palladium (9 mg, 10% on charcoal, 8.4 mmol) in tetrahydrofuran
(2 mL) for 3 min at room temperature before the mixture was
stirred under hydrogen for 6 h. The reaction mixture was filtered
through Celite and washed with ethyl acetate (30 mL). The solvent
was removed in vacuo from the filtrate to afford the acid, which
was then taken up in dioxane (2 mL). Anhydrous HCl (2 mL, 4m in
dioxane) was added and the resulting mixture was stirred for 6 h.
The solvent was removed in vacuo and the residue was taken up
sequentially in ethanol (25 mL) and methanol (5 mL) removing
the solvent in vacuo after each addition to afford the deprotected
linear precursor. This was taken up in dichloromethane (20 mL)
B-C5H–PheA+B-C7H), 5.39 (brs, 1H, PheB-C2H), 5.21 (d, J=1.4 Hz,
2H, d-HivB-C1’aH2), 5.11 (d, J=4.8 Hz, 1H, d-HivA-C2H), 4.99–4.90
(m, 2H, PheA-C2H and d-HivB-C2H), 3.32 (dd, J=14.6, 5.4 Hz, 1H,
PheB-C3HH), 3.21 (dd, J=14.6, 5.2 Hz, 1H, PheA-C3HH), 3.07–2.96
(m, 2H PheA-C3HH and PheB-C3HH), 2.94 (s, 3H, PheB-NCH3), 2.72 (s,
3H, PheA-NCH3), 2.22 (pd, J=6.9, 4.8 Hz, 1H, d-HivB-C3H), 1.81 (br.
s, 1H, d-HivA-C3H), 1.31 (s, 9H, 3PheA-C2’cH3), 0.95 (dd, J=6.9,
Chem. Eur. J. 2016, 22, 4206 – 4217
4215 ꢀ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim