The Journal of Organic Chemistry
ARTICLE
106.8, 103.7, 83.4, 61.0, 56.4, 55.6, 39.3, 31.3, 27.9, 14.4, 14.2; Enriched
13C-peak: 175.6; IR (neat) 3388, 2973, 1730, 1469, 1238, 1149,
888 cmꢀ1; HRMS (ESI/APCI) calcd for C22[13C]H33N2O5 (M þ H)
418.2418, found 418.2422.
from the Department of Agriculture in Peoria, IL. This culture was
transferred to malt extract agar slants and allowed to incubate for
fourteen days. Potato dextrose broth was prepared by dissolving 48 g
of the medium and 6 g tryptose in 2 L of doubly distilled H2O
(DDH2O). The solution was heated to aid in dissolving the medium,
which was then transferred to Fernbach flasks (4 ꢁ 500 mL) and
autoclaved. Spores of A. versicolor were added to the broth from the agar
slants. The Fernbach flasks were covered and gently placed in the
incubator for fourteen days. [13C]2-[15N]-6-Hydroxy-deoxybreviana-
mide E (8) (59 mg, 0.159 mmol) was dissolved in 0.5 mL DMSO and
added to 75 mL of hot DDH2O containing 10 mL of 1% TWEEN 80.
The solution was allowed to cool to room temperature and was added to
350 mL of a sterile trace element solution (35 mM NaNO3, 5.7 mM
K2HPO4, 4.2 mM MgSO4 7H2O, 1.3 mM KCl, 36 μM FeSO4 7H2O,
[
13C]2-[15N]-(2R)-Tert-butyl 2-((3-(6-((Tert-butoxycarbonyl)oxy)-
2-(2-methylbut-3-en-2-yl)-1H-indol-3-yl)-1-ethoxy-1-oxopropan-2-
yl)carbamoyl)pyrrolidine-1-carboxylate (12). 13C-Amine 11 (610 mg,
1.46 mmol) and [13C]-[15N]-N-Boc-L-proline (317 mg, 1.46 mmol)
were stirred in acetonitrile (15 mL) at 0 °C. HATU (832 mg, 2.19
mmol) and iPr2NEt (1.0 mL, 5.84 mmol) were added and the reaction
stirred for 3 h at room temperature. The reaction was quenched with 1 M
HCl (30 mL) and extracted with CH2Cl2 (3 ꢁ 50 mL). The combined
organic layer was washed with brine (50 mL), dried over Na2SO4, and
concentrated under reduced pressure. The crude residue was purified by
flash column chromatography and eluted with 1:1 hexanes/EtOAc to
3
3
25 μM MnSO4 H2O, 7 μM ZnSO4 7H2O, and 1.5 μM CuCl2 2H2O).
3
3
3
1
The fungus broth was decanted and the fungal cells were washed with
100 mL sterile DDH2O. The precursor/trace element solution
(110 mL) was added to each flask using a syringe and needle. The
fungal cells were incubated at 25 °C for fourteen days and each flask was
swirled daily to ensure even distribution of the labeled compound.
Isolation and Purification. The trace element solution was
decanted, and the fungus was pureed in a blender with 1:1 MeOHꢀ
CHCl3. The puree was transferred to a 2 L Erlenmeyer flask, diluted to
1.2 L with 1:1 MeOHꢀCHCl3, and placed in the shaker for
24 h. Celite (30 g) was added to the flask and allowed to shake for an
extra 10 min. The suspension was filtered through Whatman #2 paper
and the filtrate was stored at 4 °C. The mycelia “cake” was diluted with
600 mL 1:1 MeOHꢀCHCl3 and placed on the shaker for an additional
48 h. The suspension was filtered through Whatman #2 paper, and the
combined filtrates were concentrated under vacuum. The residue was
dissolved in 250 mL H2O and extracted with EtOAc (3 ꢁ 300 mL). The
organic layer was concentrated and partitioned between MeCN and
hexanes. The layers were separated, and the MeCN layer was concen-
trated under vacuum.4c,e,5b The crude material was purified via silica gel
flash column chromatography (1% MeOH in DCM ꢀ 3% MeOH in
DCM) to afford three fractions that were each analyzed by 13C NMR
spectroscopy. Further purification was carried out on those fractions
containing 13C-labeled material via preparative thin layer chromatogra-
phy (1000 μm, 3% MeOH in DCM x5) to afford triply labeled
6-hydroxydeoxybrevianamide E (40 mg, 0.108 mmol) and notoamide
J (1.4 mg, 0.003 mmol).
afford 860 mg (95%) of 12 as a yellow amorphous solid. H NMR
(300 MHz, CDCl3) δ 8.40 (bs, 1H), 7.42ꢀ7.34 (m, 1H), 7.02 (d, J = 1.2
Hz, 1H) 6.83ꢀ6.81 (m, 1H), 6.09ꢀ5.98 (m, 1H), 5.13ꢀ5.06 (m, 2H),
4.80ꢀ4.69 (m, 1H), 4.22ꢀ3.74 (m, 3H), 3.47ꢀ2.95 (m, 4H),
2.00ꢀ1.67 (m, 2H) 1.66ꢀ1.16 (m, 28H), 1.12ꢀ0.92 (m, 2H); 13C
NMR (75 MHz, CDCl3) δ 172.6, 172.4, 171.9, 170.7, 169.5, 169.2,
154.9, 152.8, 146.6, 145.7, 141.6, 134.1, 128.1, 118.5, 118.2, 113.5, 112.4,
105.4, 105.2, 103.7, 83.3, 80.6, 77.0, 61.4, 53.8, 53.0, 47.3, 39.3, 28.4,
27.9, 27.6, 14.1, 13.8; 13C-enriched peaks: 172.6, 172.4; IR (neat) 3342,
2984, 1750, 1644, 1481, 1447, 1140 cmꢀ1; HRMS (ESI/APCI) calcd for
C31[13C]2H47N2[15N] NaO8 (M þ Na) 639.3293, found 639.3293.
[
13C]2-[15N]-(3S,8aS)-3-((6-Hydroxy-2-(2-methylbut-3-en-2-yl)-1H-
indol-3-yl)methyl) Hexahydropyrrolo[1,2-a]pyrazine-1,4-dione (8).
TFA (0.3 M) was slowly added to a solution of 12 (400 mg, 0.65
mmol) in CH2Cl2 (0.3 M) at 0 °C. The reaction was stirred for 3 h at
room temperature. The mixture was quenched with saturated NaHCO3
to a pH 9 and extracted with EtOAc (3 ꢁ 20 mL). The combined organic
layers were dried over Na2SO4 and concentrated under reduced
pressure. The residue was dissolved in toluene (0.2 M) and 2-hydro-
xypyridine (8.6 mg, 0.09 mmol) was added. The reaction refluxed for 14
h under Ar atmosphere, cooled to room temperature, and concentrated
under reduced pressure. The residue was rediluted with CH2Cl2
(10 mL) and washed with 1 M HCl (20 mL). The organic layer was
dried over Na2SO4 and concentrated under reduced pressure. The crude
residue was purified via flash column chromatography eluting with 3:97
MeOH/CH2Cl2 to afford 68 mg (40%) of the desired cis-diastereomer
as cream foam. The trans-diastereomer was isolated in 38% yield (64
Notoamide J (4). HRMS (ESI/APCI) calcd for C19[13C]2H26N2-
22
[15N]O4 (M þ H) 387.1955, found 387.1951. [R]D ꢀ141 (c 0.0014,
1
mg) as yellow foam (overall: 52% for two steps). Cis: H NMR (300
19
MeOH) lit. [R]D ꢀ156 (c 0.067, MeOH)4b
MHz, 20:1 CDCl3/CD3OD) δ 8.05 (bs, 1H), 7.22 (d, J = 8.7 Hz, 1H),
6.74 (d, J = 2.1 Hz, 1H), 6.61 (dd, J = 8.4, 2.1 Hz, 1H), 6.08 (dd, J = 17.4,
10.5, 1H), 5.15ꢀ5.09 (m, 2H), 4.38 (d, J = 11.4 Hz, 1H), 4.09ꢀ4.01 (m,
1H), 3.72ꢀ3.54 (m, 3H), 3.16ꢀ3.06 (m, 1H), 2.38ꢀ1.81 (m, 6H),
1.49ꢀ1.44 (m, 6H); 13C NMR (75 MHz, CDCl3) δ 169.7, 166.3, 153.0,
146.1, 140.3, 135.7, 123.2, 118.5, 112.5, 110.2, 104.1, 97.1, 60.7, 45.6,
39.1, 28.5, 28.1, 28.0, 26.2, 26.1, 22.7; 13C-enriched peaks: 169.7, 166.3;
IR (neat) 3353, 2925, 1664, 1457 cmꢀ1; HRMS (ESI/APCI) calcd for
C19[13C]2H26N2[15N]O3 (MþH) 371.2006, found 371.2004. Trans:
1H NMR (300 MHz, (CD3)2CO) δ 7.91 (bs, 1H), 7.32 (d, J = 8.4 Hz,
1H), 6.81 (d, J = 2.4 Hz, 1H), 6.64 (dd, J = 8.4, 2.1 Hz, 1H), 6.22 (dd, J =
17.4, 10.5 Hz, 1H), 5.67ꢀ5.63 (m, 1H), 5.14ꢀ5.03 (m, 2H), 4.42ꢀ4.38
(m, 1H), 4.20ꢀ4.17 (m, 1H), 3.64ꢀ3.42 (m, 3H), 3.07ꢀ2.98 (m, 1H),
2.00ꢀ1.82 (m, 3H), 1.56 (s, 3H), 1.54 (s, 3H); 13C NMR (75 MHz,
CDCl3) δ 169.7, 166.3, 166.1, 152.8, 146.0, 140.2, 135.7, 123.2, 118.4,
112.3, 110.2, 104.1, 97.1, 65.1, 64.9, 60.5, 45.7, 39.1, 29.9, 28.1, 26.2,
22.7, 14.4; 13C-enriched peaks: 169.7, 166.3; IR (neat) 3358, 2920, 1662,
1456 cmꢀ1; HRMS (ESI/APCI) calcd for C19[13C]2H25N2[15N]O3Na
(M þ Na) 393.1826, found 393.1826.
Calculation of Percent Incorporation from Mass Spectra.
The percentage of 13C-enrichment in notoamide J from isotopically
labeled 6-hydroxydeoxybrevianamide E was calculated according to the
method by Lambert et al.11 These calculations are based on the
comparison of the mass spectrum of the labeled material (fixed 15N)
to the mass spectrum of the unlabeled material. For these experiments,
electrospray mass spectroscopy was used, thus the base peak was the
M(15N) þ H (see Supporting Information).
’ ASSOCIATED CONTENT
S
Supporting Information. Spectroscopic data and experi-
b
mental details for the preparation of all new compounds, as well
as copies of H NMR and 13C NMR spectra. This material is
1
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: rmw@lamar.colostate.edu.
General Procedure for the Precursor Incorporation Ex-
periment. A culture of Aspergillus versicolor NRRL 35600 was obtained
5957
dx.doi.org/10.1021/jo200218a |J. Org. Chem. 2011, 76, 5954–5958