Kutznerides 1-4, depsipeptides from the actinomycete Kutzneria sp. 744 inhabiting mycorrhizal roots of Picea abies seedlings

Article abstract of DOI:10.1021/np050378g

Bioassay-guided fractionation of culture supernatants of the actinomycete Kutzneria sp. 744 resulted in the isolation of four new depsipeptides (1-4). Structure analysis revealed the general structure: cyclo[2-(1-methylcyclopropyl) -D-glycine-(2S,3aR,8aS)

Full text of DOI:10.1021/np050378g

J. Nat. Prod. 2006, 69, 97-102
97
Kutznerides 1-4, Depsipeptides from the Actinomycete Kutzneria sp. 744 Inhabiting
Mycorrhizal Roots of Picea abies Seedlings
Anders Broberg,*,† Audrius Menkis,‡,§ and Rimvydas Vasiliauskas‡
Department of Chemistry, Swedish UniVersity of Agricultural Sciences, P.O. Box 7015, SE-750 07 Uppsala, Sweden, Department of Forest
Mycology and Pathology, Swedish UniVersity of Agricultural Sciences, P.O. Box 7026, SE-750 07 Uppsala, Sweden, and Lithuanian Forest
Research Institute, Liepø 1, Girionys, LT-53101 Kaunas Region, Lithuania
ReceiVed September 29, 2005
Bioassay-guided fractionation of culture supernatants of the actinomycete Kutzneria sp. 744 resulted in the isolation of
four new depsipeptides (1-4). Structure analysis revealed the general structure: cyclo[2-(1-methylcyclopropyl)-D-
glycine-(2S,3aR,8aS)-6,7-dichloro-3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid-3-hydroxy-
D-glutamic acid-O-methyl-L-serine-L-piperazic acid-(S)-2-hydroxy-3,3-dimethylbutanoic acid]. The 3-hydroxy-D-glutamic
acid was present as its threo-isomer in 1 and 2 and as its erythro-isomer in 3 and 4. The piperazic acid was modified
to its (R)-4-chloro analogue in 2 and to its C-5/N unsaturated analogue in 4. Compounds 1-4 displayed moderate spore
germination inhibiting activity against several common root-rotting fungi.
Pathogenic fungi cause significant economic losses annually in
agriculture and forestry. During recent decades, various chemical
fungicides have been extensively used to control these fungi, a
method that, however, may be hazardous in the long run, since the
pathogenic fungi may evolve resistance to the chemicals in use or
the fungicides may be accumulated in biological systems. An
alternate approach is to use antagonistic microorganisms to control
pathogenic fungi.1 The mechanisms behind this type of microbial
antagonism may be of many different types, e.g., competition for
nutrients or ecological niche, or the production of biologically active
metabolites.1-3 At present, there are a few examples of commercial
products, based on living fungi or bacteria, designed to control
pathogenic fungi in forestry and agriculture.1 There is, however,
still a need for research leading to the isolation and characterization
of new microorganisms suitable for control of pathogenic fungi.
The present report is part of a larger study where microorganisms
antagonistic to fungi that cause root-rot in north-temperate and
boreal forests have been isolated and characterized. An important
part of this characterization is the investigation of the biologically
active metabolites produced by the organisms, since these metabo-
lites may be crucial to the biological activity of the antagonistic
organisms. Cyclic depsipeptides are a frequently occurring type of
Results and Discussion
secondary metabolites displaying antimicrobial properties. Depsi-
peptides have been isolated from many types of organisms,
including mollusks, sponges, fungi, and bacteria, and the biological
activity ascribed to different depsipeptides has been quite varied,
e.g., anticancer, antibacterial, antiviral, antifungal, anti-inflamma-
tory, and cardiovascular activity.4,5 Examples include the dolast-
atins,6 the luzopeptins,7 and globomycin.8
The actinomycete Kutzneria sp. 744 inhibited the growth of the
root pathogens Pythium undulatum, Ceratobasidium bicorne, and
Fusarium aVenaceum in dual cultures on agar. In addition, the
conidial germination of F. aVenaceum was completely inhibited in
a culture filtrate of Kutzneria sp. 744. These findings indicated that
Kutzneria sp. 744 would make an interesting candidate for studies
of antifungal compounds.
The aim of this study was to investigate the secondary metabo-
lites produced by the actinomycete Kutzneria sp. 744, demonstrated
to have antagonistic effects on the growth of several common root-
rotting fungi. In this paper, the isolation and characterization of
novel depsipeptides, kutznerides 1, 2, 3, and 4 (1-4), are described.
These depsipeptides display moderate antifungal effects against
several root-rotting fungi.
Consequently, cell-free supernatants from liquid cultures of
Kutzneria sp. 744 were extracted by reversed-phase SPE. The
resulting 95% CH3CN fraction displayed conidiospore germination
inhibiting activity against F. aVenaceum, whereas the H2O fraction
did not show any activity. Gradient HPLC fractionation of the 95%
CH3CN fraction, followed by spore germination inhibition bioassay,
resulted in the isolation of several fractions with antifungal activity
1
(Figure 1). H NMR data indicated that two antifungal fractions
* To whom correspondence should be addressed. Tel: +46 18 672217.
Fax: +46 18 673392. E-mail: Anders.Broberg@kemi.slu.se.
† Department of Chemistry, Swedish University of Agricultural Sciences.
‡ Department of Forest Mycology and Pathology, Swedish University
of Agricultural Sciences.
contained single compounds (1 and 2), whereas two further
compounds (3 and 4) were isolated after one additional chromato-
graphic step (at 61% aqueous CH3CN). The production of 1-4 in
cultures of Kutzneria sp. 744 varied significantly between batches,
from several milligrams to less than a milligram of each compound.
§ Lithuanian Forest Research Institute
10.1021/np050378g CCC: $33.50
© 2006 American Chemical Society and American Society of Pharmacognosy
Published on Web 01/05/2006

98 Journal of Natural Products, 2006, Vol. 69, No. 1
Broberg et al.
Table 1. Data from GC-MS Analysis for Determination of
Absolute Configuration of the Subunits Constituting 1-4a
elution time (min) for samples
reference
1
2
3
4
2OHdiMeBu
tert-Leuc
R
S
D
L
24.87
25.21
39.47
40.81
39.77
40.16
57.14
57.29
56.33
56.41
57.89
57.99
b
b
b
b
25.23
39.50
b
b
40.12
b
57.26
56.31
25.22
39.50
b
b
40.20
f
f
25.23
39.51
b
b
40.23
b
57.33
b
25.23
39.50
b
b
40.20
f
f
b
Serd
D
L
Orne
D
L
Figure 1. Chromatogram from isolation of compounds 1-4 with
gradient reversed-phase HPLC. Compounds 3 and 4 were further
purified by isocratic reversed-phase HPLC.
threo-3OHGlu
erythro-3OHGlu
D
L
56.36
b
b
b
b
b
b
b
b
D
L
57.90
b
57.92
b
Compounds 1-4 were determined by NMR, ESIMS, and HR-
FABMS analysis to be four new depsipeptides (1-4), named
kutzneride 1, kutzneride 2, kutzneride 3, and kutzneride 4,
respectively. The absolute configuration was investigated by acidic
hydrolysis followed by GC-MS after chemical modification, as well
as by molecular modeling.
a Following acidic hydrolysis, the samples were hydrogenated and
treated with (S)-2-BuOH/AcCl (10:1) followed by perfluoropropanoic
anhydride, and finally analyzed by GC-MS b The corresponding
diastereomeric compounds were detected at approximately 10% of the
main peaks. These compounds were formed during the hydrolysis and
esterification steps. c Formed from 2-(1-methylcyclopropyl)glycine dur-
ing hydrogenation. d Formed from O-methylserine during acidic hy-
drolysis. e Formed from piperazic acid during hydrogenation. f Not
detected.
Analysis of compound 1 by ESIMS in negative mode (m/z 852.4
[M - H]-) and positive mode (m/z 854.3 [M + H]+ and 876.7 [M
+ Na]+) indicated a molecular mass of 853 Da. The isotope
distribution pattern of the pseudomolecular ions, 100/67/13 for
M/M+2/M+4, pointed to the presence of two chlorine atoms in
the molecule. This was corroborated by HRFABMS, which resulted
in a calculated molecular formula of C37H49N7O12Cl2. Compound
1 gave 1H NMR signals evenly dispersed over a wide range. Data
1
1
1
from H-1H COSY, H-13C HSQC, H-13C HSQC-DEPT, and
1H-13C HMBC experiments enabled the reconstruction of the
skeletons of the subunits of the peptide, as described below.
Subunit A of 1 contained two spin-systems. One signal at δΗ
7.66 (NH) was coupled to a CH signal (δΗ 4.02), and there was a
spin-system consisting of upfield-shifted signals from two CH2
groups (δH 1.02/0.67 and 0.71/0.39). The CH group was assigned
by HMBC to be linked to a carbonyl at δC 173.4, as well as to the
two CH2 groups through a quaternary carbon with a signal at δC
19.3. Furthermore, a CH3 group (δH 1.04) was linked to the
quaternary carbon. These findings indicated this residue to be 2-(1-
methylcyclopropyl)glycine (MecPGly), in accordance with the
upfield-shifted CH2 signals. This was later corroborated by acidic
hydrolysis of the peptide followed by catalytic hydrogenation (H2/
PtO2/HOAc), which afforded tert-leucine (Table 1), a compound
previously demonstrated to be formed from MecPGly by catalytic
hydrogenation under these conditions.9
Figure 2. Left: ROESY correlations indicating the relative
configuration of diClPIC in 1-4. Right: ROESY correlations
indicating the relative configuration of the chlorinated Pip in 2.
Solid arrows indicate strong ROESY correlations, whereas dashed
arrows indicate weak ones.
Subunit B was found to contain several spin-systems. A signal
at δH 5.29 (CH) was coupled to signals from two CH2 protons at
δΗ 2.79 and 2.17, and a signal at δΗ 5.25 (CH) was coupled to a
signal at δΗ 6.42 (NH). Additionally, there were signals from two
vicinal aromatic protons at δΗ 6.97 and 7.16, as well as a singlet
signal at δΗ 5.88 (OH). HMBC experiments afforded data enabling
the identification of this residue as a tryptophan derivative similar
to 3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-car-
boxylic acid (PIC, Figure 2), and the NMR data for subunit B were
in good agreement with literature data for PIC residues in
peptides.10,11 Subunit B, however, displayed signals from only two
aromatic protons; that is, two positions on the aromatic ring were
carrying unknown substituents. An HMBC correlation between H-4
(δΗ 7.16) and C-3a (δC 91.7), together with ROESY correlations
between H-4 and the H-3 signals (δΗ 2.79 and 2.17, respectively),
indicated the vicinal aromatic protons to be on positions 4 and 5.
HRFABMS data indicated the presence of two chlorine atoms in
1, and the chemical shifts of C-6 and C-7 (δC 133.4 and 115.8,
respectively) were compatible with chlorine substituents on these
positions. Thus, the aromatic ring was assumed to be substituted
with chlorine atoms on C-6 and C-7. This was later verified by
hydrolysis experiments analyzed by LCMS (Figure 3). During acidic
Figure 3. Fragments obtained from compound 1 by partial
hydrolysis (6 M HCl, 110 °C, 0.5 to 5 h). The fragments were
identified by LCMS.
hydrolysis, PIC is almost quantitatively transformed to 2-hydroxy-
tryptophan,12 and most likely, the PIC derivative constituting subunit
B of compound 1 reacts in the same way. The LCMS data showed
the presence of a compound with m/z 289 [M + H]+, displaying
the expected isotope distribution pattern for a compound containing
two chlorine atoms. The molecular mass 288 Da is the mass of a
PIC molecule carrying two chlorine substituents, as well as the mass
of dichloro-2-hydroxytryptophan. Thus, regardless of the stability
of the subunit during acidic hydrolysis, the LCMS data verified
that subunit B carried two chlorine substituents. Consequently,

Depsipeptides from the Actinomycete Kutzneria
Journal of Natural Products, 2006, Vol. 69, No. 1 99
subunit B was identified as 6,7-dichloro-3a-hydroxy-1,2,3,3a,8,-
8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid (diClPIC). The
relative configuration of this residue was investigated by ROESY
experiments, which afforded cross-peaks between the signals of
H-8a and OH, OH and H-3â, OH and H-3R (weak), H-3R and
H-2, and H-3â and H-2 (weak) (Figure 2). No correlation was
observed between H-2 and H-8a. The ROESY data indicated that
the OH group was on the same side of the ring system as the
carbonyl group and H-8a (Figure 2). The same relative configuration
has been reported for a PIC residue in a peptide,10 with NMR data
in good agreement with the data obtained for the diClPIC residue
in 1. A different relative configuration, i.e., H-2, H-8a, and OH on
the same face of the ring system, was reported for a PIC residue in
a peptide.11 This finding was based on ROESY data including a
cross-peak between H-2 and H-8a, a correlation absent in the
ROESY data for 1.
the depsipeptide. However, correlations were found between both
H-2 and NH of residue A and the carbonyl group of residue B, as
well as between NH of residue C and the carbonyl group of residue
D. These findings were in agreement with the sequence proposed
by ROESY experiments. This sequence was further corroborated
by partial hydrolysis experiments, by treatment of 1 with 6 M HCl
at 110 °C, between 0.5 and 5 h, and subsequent analysis by LCMS.
These experiments showed fragments all in agreement with the
suggested sequence (Figure 3).
Acidic hydrolysis, followed by catalytic hydrogenation, esteri-
fication with (S)-2-BuOH, treatment with perfluoropropanoic
anhydride, and subsequent analysis with GC-MS, identified (S)-2-
hydroxy-3,3-dimethylbutanoic acid, threo-3-hydroxy-D-glutamic
acid, and O-methyl-L-serine (identified as L-serine formed during
the acidic hydrolysis), as well as 2-(1-methylcyclopropyl)-D-glycine
(identified as D-tert-leucine formed by catalytic hydrogenation9)
and L-piperazic acid (identified as L-ornithine formed by catalytic
hydrogenation13) (Table 1). To obtain information about the absolute
configuration of the diClPIC residue, molecular modeling using
the MM2 force field was employed. Compound 1 was constructed
of the above configurationally determined subunits, together with
the D- or L-isomer of diClPIC with the relative configuration as
indicated by ROESY experiments (Figure 2). Subsequently, starting
from different initial conformations, energetically favorable con-
formers of 1 were calculated, and selected interatomic distances
were measured. In 1, containing the L-isomer of diClPIC, the
average MecPGly NH/diClPIC H-2 and MecPGly NH/diClPIC
H-8a distances were 2.5 and 4.6 Å, respectively, whereas the
corresponding distances were 3.4 and 2.6 Å, respectively, for 1
containing the D-isomer. In ROESY experiments on 1, using mixing
times between 100 and 300 ms, strong correlations were observed
between MecPGly NH and diClPIC H-2, whereas no cross-peaks
were detected between MecPGly NH and diClPIC H-8a. Thus, the
data from ROESY experiments were in accordance with the
Subunit C showed two separate spin-systems. A signal at δΗ
7.20 (NH) was coupled to a signal at δΗ 5.31 (CH), whereas a
signal at δH 4.76 (CH) was coupled to the signals of a CH2 group
(δH 2.62 and 2.54), as well as to a broad signal at δH 3.96 (OH,
coupling only observable in COSY experiments). HMBC experi-
ments gave correlations between the CH2 protons and the carbons
of both CH groups, as well as to a carbonyl group (δC 174.7). The
presence of this carbonyl was also indicated by the large geminal
coupling constant of the CH2 group (15.6 Hz). Furthermore, HMBC
gave a cross-peak between the signal of the CH group at δH 5.31
and another carbonyl group (δC 171.4). The data pointed to the
existence of a 3-hydroxyglutamic acid or 3-hydroxyglutamine
1
residue in 1, and since no H NMR signal was detected from a
NH2 group linked to the side chain carbonyl, subunit C was
identified as 3-hydroxyglutamic acid (OHGlu).
Subunit D contained one spin-system. One signal at δΗ 7.62 (NH)
was connected via a CH group (δH 4.36) to a CH2 group (δH 3.78
and 3.41). HMBC experiments showed the presence of a carbonyl
linked to the CH group, as well as a CH3O group (δH 3.25, δC
59.9) bound to the CH2 group, all in accordance with an O-
methylserine (MeSer) residue.
molecular modeling data for 1 containing
L-diClPIC. Taken together
with the relative configuration of the diClPIC residue (Figure 2),
this suggests the residue to be (2S,3aR,8aS)-6,7-dichloro-3a-
hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxyl-
ic acid. According to these findings, the structure of 1 was cyclo(D-
MecPGly-(2S,3aR,8aS)-diClPIC-threo-D-OHGlu-L-MeSer-L-Pip-(S)-
OHdiMeBu), which is a novel depsipeptide.
Subunit E also contained one single spin-system. A CH group
(δH 5.01) was linked to three consecutive CH2 groups (δH 2.34/
1.61, δH 2.08/1.56, and δH 3.10/2.80) and finally to a NH (δH 4.70).
The marked differences between the chemical shifts within the
geminal proton pairs indicated a ring-closed structure, possibly a
six-membered ring in a chairlike conformation. Comparison with
literature data showed good agreement with piperazic acid (Pip) in
peptides, e.g., ref 13. This finding was later confirmed by catalytic
hydrogenation (H2/PtO2/HOAc), which afforded the amino acid
ornithine13 as identified by GC-MS (Table 1).
Subunit F gave only two singlet signals in the 1H NMR spectrum
(δH 5.85 and 1.12), corresponding to one CH and three CH3 groups,
respectively. HMBC experiments proved the CH group to be linked
to a carbonyl (δC 172.8) as well as to a quaternary carbon (δC 33.9),
which also carried the three CH3 groups. The downfield-shifted
CH signal (δC 78.6) indicated that an oxygen atom was linked to
this group and that the oxygen was part of an ester linkage to one
of the amino acid residues in the peptide. Thus, this subunit was
assigned as 2-hydroxy-3,3-dimethylbutanoic acid (OHdiMeBu).
Compounds 2, 3, and 4 were also subjected to extensive
spectroscopic analysis. NMR data indicated that these compounds
all were similar to 1. The differences between the compounds were
confined to two of the amino acids: the Pip and the OHGlu
residues. The Pip residues of 2 and 4 were modified relative to 1
and 3, whereas the relative configuration of the OHGlu residues
of 3 and 4 was different compared to 1 and 2. All compounds were
found to contain L-MeSer, D-MecPGly, and (S)-OHdiMeBu (Table
1), and the absolute configuration of the diClPIC residues was
assumed to be (2S, 3aR, 8aS) for all compounds, as supported by
molecular modeling (data not shown) and ROESY experiments.
Moreover, the general sequence of subunits was determined by
ROESY and HMBC experiments to be the same for 2-4 as for 1.
The structure analysis of these compounds will not be described in
full detail, since it was performed in very similar manners as for 1.
Thus, only important differences will be highlighted.
The molecular formula C37H49N7O12Cl2, obtained by HRFABMS,
and the identification of the subunits made it apparent that the
peptide was ring-closed by five amide bonds and one ester linkage.
The sequence of amino and hydroxy acids was determined by
ROESY, HMBC, and partial hydrolysis experiments. Diagnostic
ROESY cross-peaks were obtained between the signals of A-NH
and B-2, B-NH and C-2, B-8a and C-NH, C-NH and D-2, C-NH
and D-NH, D-NH and E-2, E-2 and F-4, E-NH and F-2, E-5 and
F-4, and F-2 and A-2, suggesting the sequence of 1 to be cyclo-
(MecPGly-diClPIC-OHGlu-MeSer-Pip-OHdiMeBu). HMBC ex-
periments failed to yield data determining the whole sequence of
Compound 2 was identical to 1 except for a modification of the
Pip residue, which was found to carry a chlorine substituent at C-4.
This was manifested as changes in the corresponding NMR spectra,
as this Pip residue contained only two CH2 groups with signals at
δΗ 2.77/1.81 and δΗ 3.36/2.85 and one additional CH group at δH
4.66 (δC 50.5). COSY experiments showed this CH group to be
positioned between the two CH2 groups and thus that an unknown
substituent was linked to C-4 of the Pip residue. HRFABMS pointed
to the molecular formula C37H48N7O12Cl3 for 2, which taken
together with the NMR data was in good agreement with the

100 Journal of Natural Products, 2006, Vol. 69, No. 1
Broberg et al.
presence of a chlorine substituent at C-4 in the Pip residue. The
relative configuration of the chlorinated Pip residue was investigated
by ROESY experiments, which yielded diagnostic cross-peaks
between the signals of H-2 and H-3a (weak), H-2 and H-3b, H-3a
and H-4, H-3b and H-5b, H-4 and H-5a, and H-4 and NH (Figure
2). These data indicated a trans-relation between the carbonyl group
and the chlorine atom and an axial position for the H-4, assuming
a chairlike conformation of the residue. The axial position for H-4
was further supported by the coupling patterns of the H-3b and
H-5b signals, which both displayed large couplings to H-4,
indicating a trans-diaxial relation to H-4 for both protons. The Pip
residues of 1 and 3 were both determined to be of L-configuration
(Table 1), making it likely that the chlorinated Pip residue of 2
also was of L-configuration. Assuming this, the chirality of C-4
was deduced to be R; that is, the residue was (R)-4-chloro-L-
piperazic acid.
Compounds 1-4 were tested for growth inhibition of four
common root-rotting fungi, Cylindrocladium canadense, F. aVe-
naceum, F. oxysporum, and Nectria radicicola. The growth
inhibition was studied in the concentration range 0.5 µg/mL to 1.0
mg/mL, and in no case did the growth inhibition at the highest
concentration, 1.0 mg/mL, exceed 60-99%. At this concentration
of 1-4, C. canadense displayed the highest sensitivity with 60-
99% inhibition for all tested compounds, whereas F. oxysporum
was the least sensitive, with observable inhibition only for 2 (30-
59% inhibition). N. radicicola and F. aVenaceum were inhibited
60-99% by 1-3 and 1-2, respectively, and 30-59% by 4. F.
aVenaceum was not tested with 3 at this concentration. Due to the
low water solubility of the compounds, the growth inhibition was
not tested at higher concentrations.
Experimental Section
General Experimental Procedures. Optical rotation was measured
on a Model 341 polarimeter (Perkin-Elmer) using CH3OH as solvent,
in a 1 mL cell (path 10.0 cm, λ 589 nm, 20 °C). UV data were acquired
at 20 °C with a U-2100 spectrophotometer (Hitachi) using CH3OH as
solvent. NMR data were obtained using Bruker DRX400 and DRX600
NMR spectrometers operated at 400 and 600 MHz, respectively, for
1H, and 100 and 150 MHz, respectively, for 13C. The Bruker DRX400
was equipped with 5 mm QNP or BBO probe heads, whereas the
DRX600 was equipped with a 5 mm QXI probe head. NMR experi-
ments were performed at 30 or 20 °C, with CDCl3 as solvent, using
pulse programs for one-dimensional 1H and 13C, and two-dimensional
When analyzing compound 3 by HRFABMS the data pointed
to the same molecular formula (C37H49N7O12Cl2) for 3 as for 1.
NMR data indicated that the only difference between 1 and 3 was
the configuration of the OHGlu residues, which was manifested
by the coupling pattern of the corresponding H-2 signals. In 1, no
coupling was observed between the H-2 and H-3 signals of the
OHGlu residue, whereas in 3 the H-2 signal was a triplet (10 Hz)
due to vicinal couplings to the NH signal as well as to the H-3
signal. Subsequent component analysis proved the OHGlu residue
of 3 to be erythro-3-hydroxy-D-glutamic acid (Table 1).
1H-1H COSY, H-13C HSQC-DEPT, H-13C HMBC (65 ms), and
1H-1H ROESY (100, 200, or 300 ms) experiments, supplied by Bruker.
Chemical shifts were determined relative to CHCl3 (δH 7.27) and CDCl3
(δC 77.23). ESIMS data were obtained using a Bruker Esquire mass
spectrometer in positive and negative mode, using CH3OH as solvent.
HRFABMS data were obtained using a four-sector instrument (JEOL
SX102/SX102) using glycerol as matrix and PEG as internal standard.
LCMS was performed on a HP1100 LC system (Hewlett-Packard)
connected to a Bruker Esquire mass spectrometer, and GCMS on a
HP5890/5970 GCMS (Hewlett-Packard).
Isolation and Identification of Microorganisms. The actino-
mycetous strain 744 was isolated on Melin Norkrans medium (MMN)22
from surface-sterilized mycorrhizal root tips of bare root cultivated
Picea abies seedlings in a forest nursery (54°58′ N, 23°38′ E) in
Lithuania.23 The isolated culture was maintained on MMN media in 9
cm diameter Petri dishes at 20 ( 1 °C. For long-term storage it was
maintained at 4 ( 1 °C.
Morphological studies (the Central Bureau of Fungal Cultures in
Utrecht, The Netherlands) determined the isolate to be Streptomyces-
like aerobic actinomycete. Molecular studies, involving extraction of
DNA, amplification of 16S rDNA with primers 27F and 1492R,24 and
sequencing, as previously described,25 resulted in a nucleotide sequence,
1425 bp in length, that was searched against the 16S rDNA sequences
available at the GenBank database.26 The total length of the sequence
was analyzed by nucleotide-nucleotide BLAST (blastn) and pairwise
two sequence alignments (bl2seq). Identification-based comparison
showed that the sequence was the most similar (98%) to the sequence
of the actinomycete Kutzneria kofuensis, differing by 18 nucleotides
as another two nucleotides in the reference sequence were provided as
unknown. Interspecific 16S rDNA sequence comparison of the type
species K. Viridogrisea and K. kofuensis originating from a phylogenetic
study27 showed that nucleotide homology within the genus Kutzneria
is at least 97%; therefore it was concluded that the actinomycetous
strain 744 was eligible for assignment to this particular genus. The
isolate, defined as Kutzneria sp. 744, has been deposited at the culture
collection of the Department of Forest Mycology and Pathology,
Swedish University of Agricultural Sciences (SLU), Uppsala, and the
16S rDNA sequence at the GenBank under the accession number
DQ181633.
1
1
The difference between compounds 3 and 4 was shown by NMR
to be in the Pip residues. This was demonstrated by the absence of
the C-5 CH2 group as well as the NH, in the Pip residue of 4.
Instead, a signal at δΗ 7.17 (δC 146.9) was present, which was
coupled to the signals of the C-4 CH2 group, suggesting a double
bond between C-5 and the neighboring nitrogen atom. The NMR
data were in good agreement with literature data describing this
modified Pip residue (Pip*) in aurantimycins.14 Additionally, data
from HRFABMS supported this conclusion, as the measured
molecular mass was two mass units lower than for 1 and 3, giving
a calculated molecular formula of C37H47N7O12Cl2. The absolute
configuration of the Pip* residue was assumed to be the same as
for Pip residues in 1 and 3, i.e., the L-configuration.
Compounds 1-4 were all constructed of several unusual building
blocks, of which diClPIC and the chlorine-substituted Pip have not
been described before. The non-chlorinated analogue of diClPIC,
i.e., PIC, has been shown to occur in peptides and proteins as a
product of oxidation of tryptophan residues12 as well as a product
of oxidation of free tryptophan,15 but also as a constituent of
peptides produced by bacteria and fungi.10,11 The other subunits of
these depsipeptides have previously all been described, either as
free molecules or as parts of peptides or other natural products.
The synthesis and optical resolution of the enantiomers of OHdiMe-
Bu has been described,16 but to our knowledge, this is the first
report of this R-hydroxy acid as part of a natural product. MecPGly
has been isolated from Micromonospora miyakonensis9 as a free
amino acid, whereas OHGlu has been reported from different
sources, e.g., in the peptidoglycan of Microbacterium lacticum.17
MeSer, as well as Pip, has been found to occur in several
depsipeptides, isolated from, for example, sponges18 or strains of
Streptomyces.13
On the basis of phenotypic and genomic differences, three species
of the genus Streptosporangium were previously transferred to the
new genus Kutzneria,19 and these three species have been described
with respect to secondary metabolites only in a few reports. K.
albida and K. Viridogrisea (formerly S. albidum and S. Viridogri-
seum19) have both been found to produce the antibiotic macrocyclic
lactone sporaviridin,20 whereas aculeximycin,21 also a macrocyclic
lactone, has been isolated from K. albida. However, nothing similar
to compounds 1-4 has been described from this genus earlier, or
from any other source.
Assay of Antifungal Activity. To estimate the antifungal activity
of the isolate, the actinomycete Kutzneria sp. 744 was confronted on
MMN agar media in 9 cm diameter Petri dishes with the root pathogens
Pythium undulatum strain SN1, Ceratobasidium bicorne strain 248,
and Fusarium aVenaceum strain D2, originating from the culture
collection of the Department of Forest Mycology and Pathology, SLU.

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

102 Journal of Natural Products, 2006, Vol. 69, No. 1
Broberg et al.
Hz, H-5), 6.36 (1H, s, NH), 5.76 (1H, s, OH), 5.31 (1H, d, J ) 7.9 Hz,
H-2), 5.28 (1H, d, J ) 5.3 Hz, H-8a), 2.80 (1H, d, J ) 14.2 Hz, H-3â),
2.16 (1H, dd, J ) 14.2, 7.9 Hz, H-3R); OHGlu 6.72 (1H, d, J ) 10.5
Hz, NH), 5.20 (1H, dd, J ) 10, 9 Hz, H-2), 4.58 (1H, q, J ) 8 Hz,
H-3), 3.62 (1H, s, OH), 2.62 (2H, m, H-4ab); MeSer 7.91 (1H, d, J )
7.4 Hz, NH), 4.51 (1H, m, H-2), 3.84 (1H, d, J ) 7.2 Hz, H-3a), 3.32
(1H, dd, J ) 7.2, 2.6 Hz, H-3b), 3.27 (3H, s, CH3); Pip 7.17 (1H, d, J
) 4 Hz, H-5), 5.23 (1H, d, J ) 3.7 Hz, H-2), 2.78 (1H, m, H-4a), 2.46
(1H, dd, J ) 13.4, 6.6 Hz, H-3a), 2.21 (1H, m, H-4b), 1.57 (1H, m,
H-3b); OHdiMeBu 5.91 (1H, s, H-2), 1.11 (9H, s, H-4); 13C NMR
(CDCl3, 150 MHz) δ MecPGly 172.8 (CO, C-1), 60.0 (CH, C-2), 19.9
(C, C-3), 19.0 (CH3, CH3), 14.9 (CH2, C-4), 12.1 (CH2, C-5); diClPIC
172.2 (CO, CHCONH), 147.3 (C, C-7a), 133.3 (C, C-6), 131.1 (C,
C-3b), 122.2 (CH, C-5), 122.0 (CH, C-4), 115.7 (C, C-7), 91.8 (C,
C-3a), 85.3 (CH, C-8a), 60.8 (CH, C-2), 39.1 (CH2, C-3); OHGlu 173.2
(CO, C-5), 171.4 (CO, C-1), 69.1 (CH, C-3), 54.9 (CH, C-2), 38.0
(CH2, C-4); MeSer 172.1 (CO, C-1), 72.6 (CH2, C-3), 60.1 (CH3, CH3),
55.7 (CH, C-2); Pip 172.4 (CO, C-1), 146.9 (CH, C-5), 50.5 (CH, C-2),
21.0 (CH2, C-4), 17.3 (CH2, C-3); OHdiMeBu 172.5 (CO, C-1), 79.0
(CH, C-2), 35.2 (C, C-3), 26.7 (CH3, C-4); HRFABMS m/z 852.2688
(M + H)+ (calcd for C37H48N7O12Cl2 852.2738).
Partial Hydrolysis of 1. Compound 1 (approximately 50 µg for
each experiment) was hydrolyzed for 0.5, 1, 2, and 5 h, in 0.5 mL of
6 M HCl at 110 °C, in evacuated glass ampules. The samples were
dried under a stream of N2, redissolved in 1 mL of H2O, and dried
under vacuum. Each sample was dissolved in 1 mL of H2O for analysis
by LCMS. Samples (10 µL) were injected on a reversed-phase column
(C-18, 2 × 150 mm, 5 µm, Dr. A. Maisch High Performance LC
GmbH), eluted with a gradient of CH3OH in H2O (45-60% in 10 min
at 0.2 mL/min, followed by 60% CH3OH for 10 min). MS data were
acquired in the positive ion mode.
Absolute Configuration of Subunits A, C, D, E, and F of 1-4.
Compounds 1-4 (25-50 µg of each) were hydrolyzed overnight in
0.5 mL of 6 M HCl at 110 °C, in evacuated glass ampules. The samples
were dried under a stream of N2 and hydrogenated overnight, H2 (1
atm)/PtO2/AcOH, at room temperature, followed by filtration and
evaporation under a stream of nitrogen. (S)-2-Butyl esters were formed
by treatment with 200 µL of (S)-2-BuOH/AcCl (10:1) at 100 °C for
40 min in sealed test tubes. Following evaporation under a stream of
nitrogen, 200 µL of perfluoropropanoic anhydride was added and the
mixtures were kept at 100 °C for 40 min in sealed test tubes. Each
reaction mixture was dried under a stream of N2 and dissolved in EtOAc
(100 µL). Analysis by GC-MS was performed on a fused silica column
(HP-5MS, 0.25 mm × 30 m, Agilent Technologies) using He as carrier
gas at 1 mL/min. The injector was held at 240 °C and the oven at 60
°C for 5 min, followed by a gradient to 100 °C at 1 °C/min, to 150 °C
at 5 °C/min, and finally to 240 °C at 3 °C/min. The transfer line to the
MS was kept at 260 °C. Samples of D- and L-serine, (S)-2-hydroxy-
3,3-dimethylbutanoic acid (Sigma-Aldrich), D- and L-tert-leucine
(Sigma-Aldrich), D- and L-ornithine, and threo-3-hydroxy-D-glutamic
acid, threo-3-hydroxy-L-glutamic acid, erythro-3-hydroxy-D-glutamic
acid, and erythro-3-hydroxy-L-glutamic acid (synthesized as described
in Supporting Information) were derivatized as above (or with racemic
2-BuOH) and used as reference compounds.
help with the bioassays. This work was supported by the Foundation
for Strategic Environmental Research (MISTRA).
Supporting Information Available: 1H and 13C NMR spectra and
UV spectrum for 1, as well as NMR data for 1, 2, 3, and 4, together
with experimental procedures for the synthesis of 3-hydroxyglutamic
acid, and associated NMR and MS data. This material is available free
of charge via the Internet at http://pubs.acs.org.
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Absolute Configuration of Subunit B (diClPIC) of 1-4. Starting
from several different conformations, the energetically most favorable
conformers of 1-4, constructed of the configurationally determined
subunits A, C, D, E, and F (as above), together with the D- or L-isomer
of diClPIC, were calculated by molecular modeling employing the MM2
force field using the Chem3D Ultra ver. 8.0.3 software (CambridgeSoft
Corp.). Subsequently, the relevant interatomic distances were measured
using the same software.
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Acknowledgment. We are grateful to Mr. S. Gohil at the Depart-
ment of Chemistry, SLU, for help in acquiring the HRFABMS data,
Prof. T. Norberg at the Department of Chemistry, SLU, for helpful
discussions regarding the synthesis of 3-hydroxyglutamic acid, and Dr.
K. Capieau, Department of Forest Mycology and Pathology, SLU, for
2000, 50, 331-336.
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Kenne, L. J. Nat. Prod. 2004, 67, 851-857.
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