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Organic & Biomolecular Chemistry
Page 7 of 9
DOI: 10.1039/C6OB00938G
Journal Name
ARTICLE
CH2Cl2 ( 2 x 15 mL). The combined organic extracts were washed 60% MeCN: H2O/TFA (0.1%) to 80% MeCN: H2O/TFA (0.1%). See S14
with brine (20 mL), dried over Na2SO4 and concentrated in vacuo for detailed HPLC experimental. Tautomeric ratio
~ 4:1. 1H NMR
The crude product was purified by column chromatography on (500 MHz, CDCl3) δ 7.53 (d, = 15.5 Hz, 1H, H-8), 7.28 (d, = 15.5 Hz,
Sephadex using 50% CH2Cl2:MeOH to yield the target compound as 1H, H-7), 6.28 (dq, = 16.0, 7.0 Hz, 1H, H-14), 6.06 (s, 1H, H-10),
an orange oil (74 mg, 52%). Tautomeric ratio = ~2:1 in CDCl3. For 5.96 (br s, 1H, NH), 5.76 (d, = 16.0 Hz, 1H, H-13), 3.94 (s, 2H, H-5
1H NMR spectrum the major tautomer is assigned, for the 13C NMR minor), 3.84 (s, 2H, H-5 major), 2.14 (s, 3H, C-9 Me), 1.87 (d,
= 7.0
spectrum, both tautomers are assigned. 1H NMR (400 MHz, CDCl3) δ Hz, 3H, H-15). 13C (125 MHz, CDCl3) δ 192.4 (C-4), 176.2 (C-2), 174.2
7.59 (d, = 15.6 Hz, 1H, H-8), 7.31 (d, = 15.6 Hz, 1H, H-7), 6.29 (dq, (C-6), 146.8 (C-8), 144.9 (C-9), 141.7 (C-14), 121.0 (C-10), 119.0 (C-
= 15.6, 6.8 Hz, 1H, H-14), 6.10 (s, 1H, H-10), 5.74 (dq, = 15.6, 2.0 7), 110.9 (C-13), 101.9 (C-12), 100.5 (C-3), 86.7 (C-11), 51.6 (C-5),
Hz, 1H, H-13), 4.06 (s, 2H, H-5), 2.11 (s, 3H, C-9 Me), 1.86 (dd,
19.0 (C-15), 14.9 (C-9 Me). EI-MS m/z (%) 257 (100, M•+), 242 (10),
.
Z:E
J
J
J
Z
:E
J
J
J
J
J
J
J
=
6.8, 2.0 Hz, 3H, H-15), 1.56 (s, 9H, C(CH3)3). 13C (100 MHz, CDCl3) δ 126 (48), 84 (56), 69 (67). HREI-MS: calc 257.1052 (C15H15NO3)
197.7 (C-4 minor), 189.3 (C-4 major), 178.0 (C-6 minor), 176.2 (C-6 found 257.1051. ESI-MS (−ve) m/z (%) 513 (3, [2M − H]−), 256 (19,
major), 173.6 (C-2 major), 164.8 (C-2 minor), 149.8 (NCO2tBu [M − H]−), 140 (8), 124 (12), 98 (38), 69 (100), 42 (67). HRESI-MS:
minor), 149.4 (NCO2tBu major), 148.7 (C-8), 144.8 (C-9), 142.1 (C- calc 256.0974 (for C15H14NO3, [M − H]−) found 256.0974.
14), 122.4 (C-10), 118.6 (C-7 major), 118.0 (C-7 minor), 110.9 (C-13),
(Z)-3-((2E,4E,6E)-1-Hydroxy-4-methyldeca-2,4,6-trien-8-yn-1-
103.5 (C-3 minor), 102.9 (C-12), 101.5 (C-3 major), 86.7 (C-11), 84.0
(C(CH3)3 major), 83.4 (C(CH3)3 minor), 54.7 (C-5 major), 52.3 (C-5
minor), 28.0 (C(CH3)3), 19.0 (C-15), 14.8 (C-9 Me). IR: ν (cm-1) 1743
(m), 1613 (s), 1551 (m), 1343 (s), 1310 (s), 1156 (s). EI-MS m/z (%)
357 (3, M•+), 301 (7), 277 (52), 2257 (43), 126 (20), 71 (48), 56 (100).
HREI-MS: calc 357.1576 (C20H23NO5) found 357.1563.
ylidene)pyrrolidine-2,4-dione (2) Prepared in a similar manner to
tetramic acid 3, from tetramic acid 18 (6 mg, 17 µmole, 1 eq). Yield
(2 mg, 46%). This compound was markedly more unstable than the
natural product 3 and decomposed rapidly in our hands. 1H NMR
(300 MHz, CDCl3) δ 7.55 (d,
J
= 15.3 Hz, 1H, H-8), 7.25 (d,
J
= 15.3 Hz,
1H, H-7), 6.90 (dd, = 14.7, 11.1 Hz, 1H, H-11), 6.53 (d,
J
J = 11.1 Hz,
(Z
)-tert-Butyl-3-((2E,4E,6E)-1-hydroxy-4-methyldeca-2,4,6-trien-8-
1H, H-12), 5.80-5.90 (m, 2H, NH and H-10), 3.82 (s, 2H, H-5), 2.02-
yn-1-ylidene)-2,4-dioxopyrrolidine-1-carboxylate
(18)
Same 2.04 (m, 6H, H-15 and C-9 Me). ESI-MS (−ve) m/z (%) 535 (3, [2M –
procedure as for tetramic acid 19, between tetramic acid 4 (251 mg, 2H + Na]−), 256 (12, [M – H]−), 140 (8), 125 (13), 113 (100), 98 (52),
0.50 mmol, 1.0 eq) and aldehyde 5 (0.50 mmol, 1.0 eq). Red oil (111 42 (37). HRESI-MS: calc 256.0974 (C15H14NO3, [M – H]−) found
mg, 62%). Tautomeric ratio
Z:E
= ~2:1 in CDCl3. For 1H NMR 256.0976.
spectrum the major tautomer is assigned, for the 13C NMR
spectrum, both tautomers are assigned. 1H NMR (400 MHz, CDCl3) δ
Isolation of Fungus (MINAP-9902).2
A fruiting body of the myxomycete Lycogala epidendrum was
surface sterilised by immersion in a 10% aqueous NaOCl solution for
90 seconds. The fruiting body was then washed twice with sterile
water and ruptured onto a 110 mm potato dextrose agar (PDA)
plate (an initially white culture developed which turned green upon
sporulation). Yellow globules developed after 4 days. A sterile
inoculating loop was used to transfer fungal material from the
radius of the loop to a new PDA plate which grew to give a pure
culture of the fungus sp. MINAP-9902, which was identified as a
Penicillium species.
7.62 (d,
J
= 15.6 Hz, 1H, H-8), 7.32 (d,
= 15.2, 12.0 Hz, 1H, H-11), 6.51 (d,
= 15.2, 2.4 Hz, 1H, H-12), 4.06 (s, 2H, H-5), 2.04 (d,
J
= 15.6 Hz, 1H, H-7), 6.90 (dd,
= 12.0 Hz, 1H, H-10), 5.88 (dq,
= 2.4 Hz, 3H,
J
J
J
J
H-15), 2.00 (s, 3H, C-9 Me), 1.56 (s, 9H, C(CH3)3). 13C (100 MHz,
CDCl3) δ 197.7 (C-4 minor), 189.2 (C-4 major), 178.1 (C-6 minor),
176.5 (C-6 major), 173.2 (C-2 major), 165.0 (C-2 minor), 151.9
(NCO2tBu minor), 151.2 (NCO2tBu major), 150.6 (C-8), 141.9 (C-9, C-
10 and C-11), 136.1, 133.9, 118.7 (C-7 and C-12), 117.5, 103.0 (C-3
minor), 101.1 (C-3 major), 94.4 (C-14), 83.9 (OC(CH3)3 major), 83.3
(OC(CH3)3 minor), 79.7 (C-13), 54.6 (C-5 major), 52.3 (C-5 minor),
28.2 (C(CH3)3), 12.6 (C-9 Me), 4.9 (C-15). IR: ν (cm-1) 1706 (m), 1615
(s), 1573 (s), 1310 (s), 1154 (s). EI-MS m/z (%) 357 (21, M•+), 301
(22), 257 (97), 126 (54), 91 (100), 84 (53), 57 (100). HREI-MS: calc
357.1576 (C20H23NO5) found 357.1566.
Culturing of MINAP-9902
The fungus was grown on 110 mm malt extract agar plates at 25 °C
for 4 days. Eight 250 mL conical flasks were prepared, four with 50
mL potato dextrose broth (PDB) and four with 50 mL of malt extract
broth (MEB). Each flask was inoculated with two 1 cm2 blocks of
fungal culture of the malt extract plate. These flasks were
maintained at 25 °C, and agitated on an orbital shaker at 120 rpm
for 4, 6, 8 and 10 days (two conical flasks, one each of PDB and
MEB, were removed periodically every two days from the four day
mark). For each of the conical flasks, the mycelia were mechanically
separated from the broth by filtration and the mycelia were
homogenised in a solution of CH2Cl2/EtOH (1:4). The crude extracts
were concentrated, then suspended in 50 mL of deionised water,
and extracted successively with hexane (2 x 50 mL), CH2Cl2 (2 x
50mL) and EtOAc (2 x 50 mL). Previous studies1 indicated that the
(Z)-3-((2E,4E,8E)-1-Hydroxy-4-methyldeca-2,4,8-trien-6-yn-1-
ylidene)pyrrolidine-2,4-dione (3) TFA (100 μL) was added dropwise
to a solution of tetramic acid 19 (13 mg, 36 µmole, 1 eq) in CH2Cl2 (1
mL) at room temperature. After stirring for 20 min, hexane (10 mL)
was added and the solvent removed under reduced pressure. The
addition of hexane and removal of solvent was repeated two times.
The crude product was purified by column chromatography using
LH-20 Sephadex, with 50% CH2Cl2:MeOH as eluent to afford the title
compound as an orange solid (6 mg, 65%). Further purification was
achieved using semi-preparative HPLC using a gradient eluent of
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