Depsipeptides from the Actinomycete Kutzneria
Journal of Natural Products, 2006, Vol. 69, No. 1 101
The plates were incubated at 20 ( 1 °C for 30 days, and the outcome
was studied by ocular observations.
NH), 3.10 (1H, d, J ) 13 Hz, H-5a), 2.80 (1H, q, J ) 12 Hz, H-5b),
2.34 (1H, d, J ) 13.2 Hz, H-3a), 2.08 (1H, m, H-4a), 1.61 (1H, m,
H-3b), 1.56 (1H, m, H-4b); OHdiMeBu 5.85 (1H, s, H-2), 1.12 (9H, s,
H-4); 13C NMR (CDCl3, 100 MHz) δ MecPGly 173.4 (CO, C-1), 60.2
(CH, C-2), 19.3 (C, C-3), 18.8 (CH3, CH3), 14.4 (CH2, C-4), 12.0 (CH2,
C-5); diClPIC 172.0 (CO, CHCONH), 147.0 (C, C-7a), 133.4 (C, C-6),
131.2 (C, C-3b), 122.3 (CH, C-5), 122.0 (CH, C-4), 115.8 (C, C-7),
91.7 (C, C-3a), 85.5 (CH, C-8a), 60.7 (CH, C-2), 39.3 (CH2, C-3);
OHGlu 174.7 (CO, C-5), 171.4 (CO, C-1), 68.0 (CH, C-3), 52.6 (CH,
C-2), 36.7 (CH2, C-4); MeSer 172.6 (CO, C-1), 71.5 (CH2, C-3), 59.9
(CH3, CH3), 55.9 (CH, C-2); Pip 171.4 (CO, C-1), 50.8 (CH, C-2),
46.6 (CH2, C-5), 23.7 (CH2, C-3), 20.8 (CH2, C-4); OHdiMeBu 172.8
(CO, C-1), 78.6 (CH, C-2), 33.9 (C, C-3), 26.6 (CH3, C-4); HRFABMS
m/z 854.2888 (M + H)+ (calcd for C37H50N7O12Cl2 854.2895).
Conidiospores of the root pathogen F. aVenaceum were used to guide
the isolation of antifungal compounds. Conidiospores were produced
on MMN agar media in 9 cm diameter Petri dishes at 20 ( 1 °C in the
dark, and the spores were harvested from 15- to 30-day-old cultures
by flooding mycelium with liquid MMN medium and gently rubbing
the culture colony with a glass rod. The resulting suspensions were
filtered through a 50 µm mesh to separate mycelial fragments. A
haemacytometer was used to determine the conidial concentration.
Bioassays were performed as previously described.28 Aliquots of SPE
or HPLC fractions were transferred to microtiter plates and the solvents
evaporated overnight in a fume hood. Subsequently, 100 µL of MMN
media containing 105 conidiospores/mL of F. aVenaceum was added
to each well, and the microtiter plates were incubated at 20 ( 1 °C for
48 h. Control wells comprised conidiospores suspended in liquid MMN
medium. The extent of germination of conidiospores was estimated by
ocular observation of the microtiter plates with a stereomicroscope as
well as by an automated plate reader (Labsystems Multiscan RC,
Helsinki, Finland) operated at 620 nm.
The growth inhibition caused by the isolated compounds was tested
for conidiospores of F. aVenaceum as well as for the root-rotting fungi
Cylindrocladium canadense strain aurim1149, F. oxysporum strain
aurim1101-10, and Nectria radicicola strain aurim1148-1. Solutions
of the peptides were transferred to wells in microtiter plates, and the
solvent (CH3OH) was allowed to evaporate overnight in a fume hood.
To the dried wells were added suspensions of conidiospores of the
different fungal species (all conidiospores prepared as described above),
in liquid MMN media (total volume 100 µL per well). The growth
inhibition studies were performed in triplicate and covered the
concentration range 1.0 mg/mL to 0.5 µg/mL. The extent of germination
of conidiospores was estimated by ocular observation of the microtiter
plates with a stereomicroscope as well as by an automated plate reader
(Labsystems Multiscan RC) at 620 nm.
Compound 2: 3.7 mg; white powder; [R]20 -19.4 (c 0.29, CH3-
D
1
OH); H NMR (CDCl3, 600 MHz) δ MecPGly 7.61 (1H, d, J ) 9.4
Hz, NH), 4.03 (1H, d, J ) 9.4 Hz, H-2), 1.05 (3H, s, CH3), 1.03 (1H,
m, H-4a), 0.71 (1H, m, H-5a), 0.69 (1H, m, H-4b), 0.41 (1H, m, H-5b);
diClPIC 7.17 (1H, d, J ) 8.1 Hz, H-4), 6.99 (1H, d, J ) 8.1 Hz, H-5),
6.37 (1H, d, J ) 5.3 Hz, NH), 5.84 (1H, s, OH), 5.28 (1H, d, J ) 7.9
Hz, H-2), 5.25 (1H, d, J ) 5.3 Hz, H-8a), 2.79 (1H, d, J ) 14.1 Hz,
H-3â), 2.17 (1H, dd, J ) 14.1, 7.9 Hz, H-3R); OHGlu 7.13 (1H, d, J
) 11.0 Hz, NH), 5.30 (1H, d, J ) 11.0 Hz, H-2), 4.75 (1H, dd, J )
9.2, 4.6 Hz, H-3), 4.0 (1H, s, OH), 2.65 (1H, dd, J ) 14.8, 9.2 Hz,
H-4a), 2.55 (1H, dd, J ) 14.8, 4.6 Hz, H-4b); MeSer 7.52 (1H, d, J )
4.6 Hz, NH), 4.35 (1H, m, H-2), 3.79 (1H, dd, J ) 8.6 Hz, H-3a), 3.41
(1H, dd, J ) 8.6, 2.2 Hz, H-3b), 3.26 (3H, s, CH3); Pip 5.21 (1H, d, J
) 13.0 Hz, NH), 5.18 (1H, d, J ) 5.1 Hz, H-2), 4.66 (1H, m, H-4),
3.36 (1H, d, J ) 12.6 Hz, H-5a), 2.85 (1H, q, J ) 12.0 Hz, H-5b),
2.77 (1H, m, H-3a), 1.81 (1H, ddd, J ) 13, 12, 5.9 Hz, H-3b);
OHdiMeBu 5.75 (1H, s, H-2), 1.13 (9H, s, H-4); 13C NMR (CDCl3,
100 MHz) δ MecPGly 173.8 (CO, C-1), 60.2 (CH, C-2), 19.2 (C, C-3),
18.8 (CH3, CH3), 14.5 (CH2, C-4), 12.0 (CH2, C-5); diClPIC 171.9
(CO, CHCONH), 147.0 (C, C-7a), 133.5 (C, C-6), 131.1 (C, C-3b),
122.3 (CH, C-5), 122.0 (CH, C-4), 115.9 (C, C-7), 91.7 (C, C-3a),
85.6 (CH, C-8a), 60.7 (CH, C-2), 39.3 (CH2, C-3); OHGlu 173.6 (CO,
C-5), 171.4 (CO, C-1), 68.2 (CH, C-3), 52.6 (CH, C-2), 36.5 (CH2,
C-4); MeSer 172.3 (CO, C-1), 71.3 (CH2, C-3), 59.9 (CH3, CH3), 56.1
(CH, C-2); Pip 170.5 (CO, C-1), 53.5 (CH2, C-5), 53.4 (CH, C-2), 50.5
(CH, C-4), 34.2 (CH2, C-3); OHdiMeBu 172.6 (CO, C-1), 78.5 (CH,
C-2), 33.9 (C, C-3), 26.5 (CH3, C-4); HRFABMS m/z 888.2534 (M +
H)+ (calcd for C37H49N7O12Cl3 888.2505).
Isolation of Compounds 1, 2, 3, and 4. Twenty Erlenmeyer flasks
(500 mL) with 300 mL of liquid MMN medium were each inoculated
with three MMN agar plugs (diameter ca. 3 mm) colonized by Kutzneria
sp. 744. Inoculated flasks were incubated for 14 days at 21 ( 2 °C on
a Unitwist 400 rotary shaker (Uniequip, Martinsried, Germany) at 120
rpm. The liquid cultures were filtered (50 µm), and the pooled filtrates
were subsequently extracted by SPE. For each batch a 150 g SPE
column (C-18, International Sorbent Technology, UK) was packed and
activated in CH3CN. Following equilibration with H2O, the culture
filtrate was passed through the column. Nonbound substances were
eluted from the column with H2O (1 L), whereas compounds adsorbed
to the SPE column were eluted with aqueous 95% CH3CN (1 L). The
H2O phase as well as the CH3CN phase were analyzed for antifungal
activity as described above. The 95% CH3CN fraction from SPE was
dried under reduced pressure (yield typically 450 mg) and the resulting
material subsequently dissolved in 6 mL of aqueous 40% CH3CN. This
sample was filtered (0.45 µm) and further fractionated (6 × 1 mL
injected) by preparative reversed-phase HPLC (C-18, 3 µm, 20 × 100
mm and 20 × 30 mm guard column, Dr. A. Maisch High Performance
LC GmbH, Germany) using a gradient of CH3CN in H2O (10-100%
in 10 min at 10 mL/min, followed by a 10 min hold at 100% CH3CN).
The eluate was monitored by UV detection at 210 nm, and fractions
(12 s) were collected in 96-well plates (2.2 mL wells) by a fraction
collector. Aliquots (200 µL) were transferred to microtiter plates for
bioassay as described above. Isocratic elution at 61% aqueous CH3-
CN, 10 mL/min, using the same column, was used to further purify
compounds 3 and 4.
Compound 3: 3.0 mg; white powder; [R]20 -30.9 (c 0.16, CH3-
D
1
OH); H NMR (CDCl3, 600 MHz) δ MecPGly 7.88 (1H, d, J ) 9.4
Hz, NH), 4.11 (1H, d, J ) 9.4 Hz, H-2), 1.08 (1H, m, H-4a), 1.07
(3H, s, CH3), 0.71 (1H, m, H-5a), 0.69 (1H, m, H-4b), 0.38 (1H, m,
H-5b); diClPIC 7.15 (1H, d, J ) 8.0 Hz, H-4), 6.97 (1H, d, J ) 8.0
Hz, H-5), 6.38 (1H, d, J ) 5.5 Hz, NH), 5.68 (1H, s, OH), 5.33 (1H,
d, J ) 7.7 Hz, H-2), 5.28 (1H, d, J ) 5.5 Hz, H-8a), 2.82 (1H, d, J )
14.2 Hz, H-3â), 2.16 (1H, dd, J ) 14.2, 8.1 Hz, H-3R); OHGlu 6.81
(1H, d, J ) 10.5 Hz, NH), 5.24 (1H, t, J ) ∼10 Hz, H-2), 4.74 (1H,
m, H-3), 3.25 (1H, s, OH), 2.74 (1H, dd, J ) 14.5, 3.7 Hz, H-4a), 2.69
(1H, dd, J ) 14.5, 10.5 Hz, H-4b); MeSer 7.53 (1H, d, J ) 7.0 Hz,
NH), 4.60 (1H, d, J ) 7.0 Hz, H-2), 3.88 (1H, d, J ) 8.9 Hz, H-3a),
3.27 (1H, dd, J ) 8.9, 3.1 Hz, H-3b), 3.24 (3H, s, CH3); Pip 5.10 (1H,
m, NH), 5.08 (1H, d, J ) 5.0 Hz, H-2), 3.11 (1H, d, J ) 12.7 Hz,
H-5a), 2.87 (1H, q, J ) 12.7 Hz, H-5b), 2.40 (1H, d, J ) 13.6 Hz,
H-3a), 2.12 (1H, m, H-4a), 1.64 (1H, m, H-3b), 1.54 (1H, m, H-4b);
OHdiMeBu 5.85 (1H, s, H-2), 1.15 (9H, s, H-4); 13C NMR (CDCl3,
100 MHz) δ MecPGly 172.7 (CO, C-1), 59.9 (CH, C-2), 19.9 (C, C-3),
18.8 (CH3, CH3), 15.0 (CH2, C-4), 11.9 (CH2, C-5); diClPIC 172.0
(CO, CHCONH), 147.0 (C, C-7a), 133.4 (C, C-6), 131.0 (C, C-3b),
122.1 (CH, C-5), 121.9 (CH, C-4), 115.7 (C, C-7), 91.8 (C, C-3a),
85.2 (CH, C-8a), 60.7 (CH, C-2), 39.0 (CH2, C-3); OHGlu 174.2 (CO,
C-5), 171.2 (CO, C-1), 69.3 (CH, C-3), 55.8 (CH, C-2), 38.6 (CH2,
C-4); MeSer 172.2 (CO, C-1), 73.1 (CH2, C-3), 60.0 (CH3, CH3), 55.8
(CH, C-2); Pip 175.1 (CO, C-1), 51.1 (CH, C-2), 46.5 (CH2, C-5), 23.5
(CH2, C-3), 20.1 (CH2, C-4); OHdiMeBu 172.8 (CO, C-1), 78.3 (CH,
C-2), 34.2 (C, C-3), 26.8 (CH3, C-4); HRFABMS m/z 854.2889 (M +
H)+ (calcd for C37H50N7O12Cl2 854.2895).
Compound 1: 6.8 mg; white powder; [R]20 -16.7 (c 0.33, CH3-
D
1
OH); H NMR (CDCl3, 600 MHz) δ MecPGly 7.66 (1H, d, J ) 9.4
Hz, NH), 4.02 (1H, d, J ) 9.4 Hz, H-2), 1.04 (3H, s, CH3), 1.02 (1H,
m, H-4a), 0.71 (1H, m, H-5a), 0.67 (1H, m, H-4b), 0.39 (1H, m, H-5b);
diClPIC 7.16 (1H, d, J ) 8.1 Hz, H-4), 6.97 (1H, d, J ) 8.1 Hz, H-5),
6.42 (1H, d, J ) 5.5 Hz, NH), 5.88 (1H, s, OH), 5.29 (1H, d, J ) 7.7
Hz, H-2), 5.25 (1H, d, J ) 5.5 Hz, H-8a), 2.79 (1H, d, J ) 14.3 Hz,
H-3â), 2.17 (1H, dd, J ) 14.3, 7.7 Hz, H-3R); OHGlu 7.20 (1H, d, J
) 10.8 Hz, NH), 5.31 (1H, d, J ) 10.8 Hz, H-2), 4.76 (1H, dd, J )
8.8, 5.0 Hz, H-3), 3.96 (1H, s, OH), 2.62 (1H, dd, J ) 15.6, 8.8 Hz,
H-4a), 2.54 (1H, dd, J ) 15.6, 5.0 Hz, H-4b); MeSer 7.62 (1H, d, J )
5.9 Hz, NH), 4.36 (1H, dt, J ) 5.8, 2.8 Hz, H-2), 3.78 (1H, dd, J )
9.5, 2.2 Hz, H-3a), 3.41 (1H, dd, J ) 9.5, 2.9 Hz, H-3b), 3.25 (3H, s,
CH3); Pip 5.01 (1H, d, J ) 5.5 Hz, H-2), 4.70 (1H, d, J ) 12.3 Hz,
Compound 4: 1.1 mg; white powder; [R]20D -17.9 (c 0.067, CH3-
1
OH); H NMR (CDCl3, 600 MHz) δ MecPGly 7.84 (1H, d, J ) 9.4
Hz, NH), 4.12 (1H, d, J ) 9.4 Hz, H-2), 1.08 (1H, m, H-4a), 1.07
(3H, s, CH3), 0.71 (1H, m, H-5a), 0.71 (1H, m, H-4b), 0.40 (1H, m,
H-5b); diClPIC 7.15 (1H, d, J ) 8.1 Hz, H-4), 6.97 (1H, d, J ) 8.1