Nocardiopsins C and D and nocardiopyrone A: New polyketides from an Australian marine-derived Nocardiopsis sp.

Article abstract of DOI:10.1016/j.tet.2012.10.104

A Nocardiopsis sp. (CMB-M0232) recovered from marine sediment collected off the coast of South Molle Island, Queensland, Australia, yielded two new examples of rare prolinyl-macrolactam polyketides, nocardiopsins C (1) and D (2), a new highly substituted α-pyrone polyketide, nocardiopyrone A (3), and the previously reported macrolide polyketides nocardiopsins A (4) and B (5). Structures were assigned on the basis of detailed spectroscopic analysis, degradation, and chemical derivatization. PCR amplification of CMB-M0232 genomic DNA revealed the presence of type I and type II polyketide synthase and nonribosomal peptide synthase domains. 2012 Elsevier Ltd. All rights reserved.

Full text of DOI:10.1016/j.tet.2012.10.104

Tetrahedron 69 (2013) 692e698
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Nocardiopsins C and D and nocardiopyrone A: new polyketides
from an Australian marine-derived Nocardiopsis sp.
*
Ritesh Raju, Andrew M. Piggott, Michelle Quezada, Robert J. Capon
Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD 4072, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 August 2012
Received in revised form 10 October 2012
Accepted 29 October 2012
Available online 15 November 2012
A Nocardiopsis sp. (CMB-M0232) recovered from marine sediment collected off the coast of South Molle
Island, Queensland, Australia, yielded two new examples of rare prolinyl-macrolactam polyketides, no-
cardiopsins C (1) and D (2), a new highly substituted a-pyrone polyketide, nocardiopyrone A (3), and the
previously reported macrolide polyketides nocardiopsins A (4) and B (5). Structures were assigned on the
basis of detailed spectroscopic analysis, degradation, and chemical derivatization. PCR amplification of
CMB-M0232 genomic DNA revealed the presence of type I and type II polyketide synthase and non-
ribosomal peptide synthase domains.
Keywords:
Nocardiopsis
Marine-derived actinomycetes
Prolinyl-macrolactam polyketides
Ó 2012 Elsevier Ltd. All rights reserved.
a
-Pyrone polyketides
Microbial natural products chemistry
1. Introduction
2. Results and discussion
As part of our ongoing research program into the chemical di-
versity of Australian marine-derived microorganisms, we recently
reported on our investigations into a Nocardiopsis sp. (CMB-M0232)
recovered from marine sediment collected at a depth of 55 m off
the coast of South Molle Island, Queensland, Australia. When cul-
tivated under saline conditions, CMB-M0232 produced nocardioa-
zines A and B1dthe first reported diketopiperazines to incorporate
a normal C-3 prenyl substituent. By contrast, when cultivated under
non-saline conditions, CMB-M0232 ceased production of nocar-
dioazines and instead produced nocardiopsins A and B2dthe first
new natural examples of the FK506/rapamycin class of FKBP-
binding polyketide macrolides to be described in over a decade.
In the course of these earlier studies, we also noted that non-
saline cultivation of CMB-M0232 resulted in low yields of three
minor metabolites, including a pair of putative nocardiopsin ana-
logs and an unrelated small molecular weight polyketide. In this
present study, we revisit these minor metabolites, performing
a larger-scale non-saline cultivation to successfully isolate, char-
acterize, and identify two new polyketide macrolides, nocardiop-
2.1. Cultivation and isolation
The EtOAc extract of a seven-day 3-L non-saline broth cultiva-
tion of Nocardiopsis sp. CMB-M0232 was concentrated in vacuo and
the residue was triturated with hexane, CH2Cl2, and MeOH. The
CH2Cl2-soluble material was subsequently fractionated by re-
versed-phase HPLC to yield small quantities of nocardiopsins C (1;
0.5 mg) and D (2; 0.4 mg), nocardiopyrone A (3; 3.7 mg), and
previously reported2 nocardiopsins A (4; 8.7 mg) and B (5; 8.1 mg)
(Fig. 1). The spectroscopic analyses leading to the structure eluci-
dation of compounds 1e3 are detailed below.
2.2. Nocardiopsins C and D
HRESI(þ)MS analysis of nocardiopsin C (1) returned an adduct
ion [(MþNa)þ] indicative of a molecular formula (C32H49NO7,
D
mmu ꢀ0.3) consistent with a lower homolog (eCH2) of the pre-
viously reported2 nocardiopsin A (4). Comparison of the NMR
spectroscopic data (methanol-d4) for 1 (Table 1) with those pre-
viously reported for 4 revealed a high degree of similarity for the C-
1 to C-23 backbone, with the differences clearly centered on the
pipecolic acid residue. Variation in the 1H and 13C chemical shifts at
C-24 (1: dH 4.41/dC 61.4; 4: dH 5.23/dC 54.5) and C-29 (1: dH 3.67/dC
48.0; 4: dH 3.91, 3.21/dC 45.5), together with one less COSY corre-
lation in the pipecolic acid spin system, suggested incorporation of
a proline residue in 1, in place of the pipecolic acid residue in 4. A
sins
C (1) and D (2), and a new a-pyrone polyketide,
nocardiopyrone A (3).
* Corresponding author. Tel.: þ61 7 3346 2979; fax: þ61 7 3346 2090; e-mail
address: r.capon@uq.edu.au (R.J. Capon).
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2012.10.104

R. Raju et al. / Tetrahedron 69 (2013) 692e698
693
Table 1
1H (600 MHz) and 13C (150 MHz) NMR spectroscopic data for nocardiopsins C (1)
and D (2) in methanol-d4
Position Nocardiopsin C (1)
dH, mult (J in Hz)
Nocardiopsin D (2)
dH, mult (J in Hz)
a
a
dC
dC
1
1.04, t, (7.2)
7.8 0.97, t, (7.2)
9.8
2a
2b
3
4a
4b
5
6
7a
7b
8
9
2.60, m
37.2 1.58, m
1.47, m
213.0 3.83, m
45.8 1.76, mg
1.42, mh
30.5
73.5
38.5
2.66, dd, (15.6, 2.4)
2.49, dd, (15.6, 10.2)
4.01, dd, (10.2, 2.4)
74.7 3.70, dd, (10.8, 1.8)
85.5
77.9
86.0
41.6
1.79, md
42.4 1.77, mg
1.74, m
1.72, me
2.54, m
37.0 2.56, m
78.5 4.06, ddd, (10.8, 7.2, 3.0)
36.3 1.76, mg
1.47, m
36.7
78.3
35.9
4.05, ddd, (10.6, 7.2, 3.0)
10a
10b
11
12
13a
13b
14a
14b
15
16
17
18
19a
19b
20a
20b
21
22
23
24
25
26a
26b
27a
27b
29b
30
31
32
33
1.73, me
1.50, m
1.39, m
25.7 1.43, mh
25.5
74.8
35.9
4.92c
74.9 4.90c
1.81, md
33.5 1.75, m
1.55, m
1.54, m
2.29, m
2.17, m
6.70, dd, (9.6, 4.8)
29.7 2.31, mi
2.18, m
29.5
145.3 6.70, dd, (9.6, 4.2)
137.2
208.2
37.3 3.50, m
34.8 1.83, m
1.62, m
30.2 2.37, m
2.11, m
145.0
137.0
208.3
37.1
3.49, m
1.81, md
1.61, m
2.34, m
2.12, m
34.5
30.0
6.89, ddd, (15.0, 10.2, 4.2) 147.8 6.90, ddd, (15.0, 10.8, 4.2) 147.6
6.17, dd, (15.0, 1.2)
4.40, dd, (8.4, 5.1)
123.7 6.18, dd, (15.0, 1.2)
166.6
123.5
166.7
61.4
170.7
29.5
61.6 4.42, dd, (8.4, 5.4)
173.1
2.29, m
29.7 2.30, mi
1.98, mf
2.00, mj
2.13, m
26.0 2.14, m
2.02, mj
25.7
2.00, mf
3.67, dd (7.0, 6.5)
0.96, d, (7.4)
1.12, s
1.73, s
1.05, d, (7.2)
48.2 3.67, m
14.6 0.97, d, (6.6)
22.2 1.13, s
11.3 1.74, s
18.4 1.06, d, (6.6)
48.0
14.2
22.1
11.1
18.2
Fig. 1. Structure of Nocardiopsis sp. (CMB-M0232) polyketides 1e5.
Marfey’s analysis performed on 1 confirmed the presence of L-
a
proline. The absence of doubling of NMR resonances suggested the
proline residue in 1 existed as a single solution conformer, which
a ROESY correlation between H-22 and H-29 (Fig. 2) confirmed as
trans. Key HMBC correlations (Fig. 2) from H-29 to carbonyl C-23
Assignments supported by HSQC and HMBC experiments.
C-28 has been omitted from the numbering scheme to facilitate comparison
b
with nocardiopsins A and B.
c
Obscured by H2O signal.
Overlapping resonances.
dej
and from H-24 and H-26 to carbonyl C-25 positioned the L-proline
as indicated. A relative 9,12-cis configuration was tentatively
assigned to 1 by comparison of NMR data with 4. Thus the structure
for nocardiopsin C (1) was assigned as shown.
demonstrate that this racemization occurs for a range of N-acyl
substituted pipecolic acids, including FK506 and rapamycin, and
that the nature of the N-acyl substituent can influence the extent to
which racemization occurs. With these previous findings in mind,
we were interested to determine whether a similar racemization
would occur for N-acyl substituted proline derivatives. To this end,
HRESI(þ)MS analysis of nocardiopsin D (2) returned an adduct
ion [(MþNa)þ] indicative of a molecular formula (C32H51NO7,
D
mmu ꢀ0.1) consistent with a lower homolog (eCH2) of the pre-
viously reported2 nocardiopsin B (5). Comparison of the NMR data
(methanol-d4) for 2 (Table 1) with those previously reported for 5
revealed a high degree of similarity for the C-1 to C-23 backbone,
we synthesized N-crotonyl-L-proline and N-butanoyl-L-proline and
with the main differences again centered on the
residue. A Marfey’s analysis performed on 2 confirmed the pres-
ence of -proline, while a ROESY correlation between H-22 and H-
L-pipecolic acid
L
29 (Fig. 2) confirmed a single trans amide conformation. Key HMBC
correlations (Fig. 2) from H-29 to carbonyl C-23 and from H-24 and
H-26 to carbonyl C-25 positioned the L-proline as indicated. Rela-
tive configurations of 3,5-syn and 9,12-cis were tentatively assigned
to 2 by comparison of NMR data with 5. Thus the structure for
nocardiopsin D (2) was assigned as shown.
In our previous study,2 we discovered that the
L-pipecolic acid
residue of nocardiopsins A (4) and B (5) underwent acid-catalyzed
partial racemization during Marfey’s analysis. We went on to
Fig. 2. Key 2D NMR correlations for nocardiopsins C (1) and D (2).

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R. Raju et al. / Tetrahedron 69 (2013) 692e698
subjected the compounds to Marfey’s analysis along with nocar-
diopsins C (1) and D (2). No racemization was observed for any of
the N-acyl substituted proline derivatives (Supplementary data),
confirming the acid-catalyzed racemization process is unique to N-
acyl substituted pipecolic acids. At this time the complete relative
and absolute configurations for 1 and 2 remain unassigned.
Isothermal titration calorimetry (ITC) was performed to assess
the binding affinities of nocardiopsins C (1) and D (2) with FKBP12.
Unlike nocardiopsins A (4) and B (5), which were previously shown
neurons.6 It is noteworthy that nocardiopsins C (1) and D (2) and
the previously reported prolinyl-macrolactams, were all isolated as
relatively minor co-metabolites of their pipecolic acid-containing
homologs. This pattern is indicative of relaxed substrate specific-
ity by the pipecolate-incorporating enzyme (PIE), which catalyzes
incorporation of
L-pipecolic acid into the polyketide chain imme-
diately prior to macrolactonization.7 Previous studies have shown
that the rapamycin PIE, encoded by the rapP gene, can accept L-
proline8 and other non-natural substrates9,10 to generate a range of
unique rapamycin analogs.
to bind with Kd values of 2.9 and 5.0 m
M, respectively,2 1 and 2
showed no appreciable affinity for FKBP12. In addition, none of the
nocardiopsins were able to induce dimerization of EGFP-FKBP12
and Cherry-FRB and hence are unlikely to display immunosup-
pressive properties.
2.3. Nocardiopyrone A
HRESI(þ)MS analysis of nocardiopyrone A (3) revealed an ad-
duct ion [(MþNa)þ] consistent with a molecular formula (C15H24O3,
Prolinyl-macrolactam polyketides are surprisingly rare, with
only four reported naturally occurring examples (Fig. 3). FR-900525
(prolylFK506), reported in 1989 as a minor metabolite from the
original FK506-producing strain of Streptomyces tsukubaensis,
exhibited similar immunosuppressive activity to FK506.3 Prolylra-
pamycin, reported in 1994 from Streptomyces hygroscopicus, was
patented as an immunosuppressive, anti-inflammatory, antifungal,
and antitumor agent.4 Antascomycin D, reported in 1996 from a soil
Micromonospora sp., exhibited five-fold lower immunosuppressive
activity than FK506.5 Finally, 3-normeridamycin, reported in 2006
from a soil Streptomyces sp., exhibited no immunosuppressive ac-
tivity, but was found to be neuroprotective in dopaminergic
Dmmu ꢀ0.5) requiring four double bond equivalents. The NMR
spectroscopic data (methanol-d4) for 3 (Table 2) revealed reso-
nances for five quaternary sp2 carbons, consistent with one ester/
lactone carbonyl (dC 169.0) and two fully substituted mono-
oxygenated double bonds (dC 98.6, 113.7, 161.2, 168.9), requiring 3
to be monocyclic. Examination of the 1D and COSY spectra revealed
two isolated spin systems (H2-6 to H3-9 and H2-10 to H3-14),
consistent with isobutyl and isopentyl groups, respectively, and one
olefinic methyl (dH 1.90, s; dC 8.8). Reacquisition of the 1H NMR
spectrum in CDCl3 revealed a broad exchangeable OH singlet at dH
6.16, accounting for all atoms in 3. These observations suggested
that 3 was most likely a 2,3,4,5-tetrasubstituted
port of this proposal, the UVevis spectrum of 3 showed an absor-
bance maximum at 295 nm, typical for
-pyrones.11 Diagnostic
a-pyrone. In sup-
a
HMBC correlations (Fig. 4) from H3-15 to C-1, C-2 and C-3 posi-
tioned the olefinic methyl between the lactone carbonyl and oxy-
genated olefinic carbon. Additional HMBC correlations from H2-6 to
C-3, C-4 and C-5 and from H2-10 to C-4 and C-5 established the
regiochemistry of the isobutyl and isopentyl groups as indicated.
Table 2
1H (600 MHz) and 13C (150 MHz) NMR spectroscopic data for nocardiopyrone A (3)
in methanol-d4
a
Position
dH, mult (J in Hz)
dC
1
2
3
4
5
6
7
169.0
98.5
169.8
113.7
161.2
34.1
29.7
22.5
29.5
37.4
28.8
22.5
8.8
2.31, d (7.4)
1.86, m
0.93, d (6.5)
2.53, m
1.54, m
1.60, m
8/9
10
11
12
13/14
15
0.95, d (6.5)
1.90, s
a
Assignments supported by HSQC and HMBC experiments.
Fig. 4. Key 2D NMR correlations for nocardiopyrone A (3).
Nocardiopyrone A (3) displayed no antimicrobial activity against
Escherichia coli (ATCC 11775), Staphylococcus aureus (ATCC 25923),
Bacillus subtilis (ATCC 6051) or Candida albicans (ATCC 10231) in
Fig. 3. Previously reported prolinyl-macrolactam polyketides.
disc diffusion assays up to 150 mg per disc, nor any cytotoxicity

R. Raju et al. / Tetrahedron 69 (2013) 692e698
695
against gastric adenocarcinoma (AGS), neuroblastoma (SH-SY5Y),
and colorectal adenocarcinoma (HT-29) cell lines in MTT assays up
to 30 M.
Polyketide
m
a-pyrones have been widely reported from plants,
marine invertebrates, and microorganisms, although 2,3,4,5-
tetrasubstituted examples such as nocardiopyrone A (3) are less
common. Noteworthy examples of
a-pyrones isolated from acti-
nomycetes include the germicidins,11e13 first reported in 1993 from
a Streptomyces viridochromogenes as autoregulative germination
inhibitors, the elijopyrones,14 reported in 1995 from a marine-
derived actinomycete, the lagunapyrones,15 reported in 1996 from
a marine-derived actinomycete as cytotoxic agents, and the 2,3,4,5-
tetrasubstituted salinipyrones,16 reported in 2007 from a marine-
derived Salinispora pacifica (Fig. 5).
Fig. 6. A plausible biosynthesis of nocardiopyrone A (3). ACP¼acyl carrier protein;
KS¼ketosynthase; KR¼ketoreductase.
and 800 bp, respectively (Fig. 7). The presence of NRPS modules is
consistent with our previous isolation of the nocardioazines1 from
a saline cultivation of CMB-M0232.
Fig. 5. Selected examples of a-pyrones reported from actinomycetes.
Fig. 7. Gel electrophoresis of DNA products obtained from PCR amplification of No-
cardiopsis sp. CMB-M0232 genomic DNA with degenerate primers for conserved re-
gions of type I and II polyketides and non-ribosomal peptide synthase genes.
A plausible biosynthesis for nocardiopyrone A (3) draws on the
recent characterization of germicidin Type III polyketide synthase
(PKS) with substrate flexibility inclusive of both acyl-ACP (polyke-
tide) and acyl-CoA (fatty acid) modules.17 This hypothesis relies on
condensation of malonyl-ACP with an isobutyryl-CoA residue to
deliver a pre-extended PKS starter unit, 3-oxo-4-methylpentyl-ACP.
Transfer of this starter unit to the ketosynthase (KS) module fol-
lowed by chain elongation with methylmalonyl-ACP delivers 2,6-
dimethyl-3,5-dioxoheptanyl-ACP. At this stage, the assembled
polyketide substrate acts as the elongation module for a second
unit of 3-oxo-4-methylpentyl-KS, delivering the complete carbon
skeleton for 3 pendant to ACP. Reduction of one and enolization of
the remaining two ketone moieties followed by ring closure, re-
leases nocardiopyrone A (3). Fig. 6 is illustrative of this biosynthetic
concept, but is not intended to be proscriptive of the precise nature
of the mixed polyketide/fatty acid synthase system.
2.4. Conclusion
In summary, this current study extends knowledge of the sec-
ondary metabolism of a Queensland marine-derived Nocardiopsis
sp. (CMB-M0232), leading to the isolation, characterization, and
structure elucidation of two new examples of a very rare class of
prolinyl-macrolactam polyketide, nocardiopsins C (1) and D (2),
together with a new unusually substituted
nocardiopyrone A (3). All these Nocardiopsis sp. polyketides were
only produced under non-saline broth cultivation.
a-pyrone polyketide,
3. Experimental section
3.1. General experimental details
PCR amplification of genomic DNA from Nocardiopsis sp. CMB-
M0232 was performed using previously reported18,19 degenerate
primers targeting conserved regions of actinomycete PKS-I keto-
synthase (KS) and methylmalonyl-CoA transferase domains, PKS-II
Chiroptical measurements ([a]D) were obtained on a JASCO P-
1010 polarimeter in a 100ꢁ2 mm cell. UVevisible spectra were
obtained on a Cary 50 spectrophotometer in 1 cm quartz cells. NMR
spectra were obtained on a Bruker Avance DRX600 spectrometer, in
the solvents indicated and referenced to residual 1H and 13C signals
KS
a and KSb domains and non-ribosomal peptide synthase (NRPS)
adenylation domains, yielding major PCR products of w1270, 100,

696
R. Raju et al. / Tetrahedron 69 (2013) 692e698
in deuterated solvents. Electrospray ionization mass spectra
(ESIMS) were acquired using an Agilent 1100 Series separations
module equipped with an Agilent 1100 Series LC/MSD mass de-
tector in both positive and negative ion modes. High-resolution
ESIMS measurements were obtained on a Bruker micrOTOF mass
a single colony of CMB-M0232 followed by shaking at 200 rpm for
7 d at 26.5 ꢂC. Aliquots (2.8 mL) of the seed culture were transferred
to six 2-L Fernbach flasks, each containing the same liquid medium
(500 mL), and the flasks were shaken at 200 rpm for 7 d at 26.5 ꢂC.
The resulting cultures were each extracted with EtOAc (400 mL)
and the combined organic phases concentrated in vacuo to yield
a crude extract (197 mg).
spectrometer by direct infusion in MeCN at 3 mL/min using sodium
formate clusters as an internal calibrant. HPLC was performed using
an Agilent 1100 Series separations module equipped with Agilent
1100 Series diode array and/or multiple wavelength detectors and
Agilent 1100 Series fraction collector, controlled using ChemStation
Rev.B.02.01.
3.6. Isolation and purification of natural products
The crude EtOAc extract of CMB-M0232 was sequentially tritu-
rated with 25 mL each of hexane, CH2Cl2, and MeOH to afford, after
concentration in vacuo, 39.8, 120.6, and 25.6 mg fractions, re-
spectively. The CH2Cl2 fraction was further fractionated by
3.2. Microbial isolation
Strain CMB-M0232 was isolated from a sediment sample ob-
tained from South Molle Island at a depth of 55 m. The fresh sed-
iment sample was promptly (16 h) transferred to the laboratory in
a sealed 50 mL Falcon tube at ambient temperature, where it was
stored in the dark at ꢀ30 ꢂC for one week. Approximately 1 g of the
thawed sediment sample was suspended in 4 mL of Ocean Nature
seawater (Aquasonic, Australia) and then subjected to heat-shock at
reversed-phase HPLC (Zorbax SB-C8 250ꢁ9.4 mm, 5
mm column,
4 mL/min gradient elution from 10 to 100% MeCN/H2O over
20 min), to yield nocardiopsin C (1) (tR¼16.2 min; 0.5 mg, 0.3%),
nocardiopsin D (2) (tR¼16.0 min; 0.4 mg, 0.3%) and nocardiopyrone
A (3) (tR¼18.6 min; 3.7 mg, 1.9%), as well as the previously reported
nocardiopsin A (4) (tR¼17.6 min; 8.7 mg, 4.4%) and nocardiopsin B
(5) (tR¼17.2 min; 8.1 mg, 4.1%). Note: percent yields are relative to
the crude EtOAc extract (197 mg).
55 ꢂC for 8 min after which 50
mL was transferred on to agar plates
(comprising 25 mL of 1% starch, 0.4% yeast extract, 0.2% peptone,
1.8% agar, and 0.0005% rifampicin). The resulting agar plate was
incubated at 27 ꢂC for four weeks. Pure strains of individual colo-
nies were obtained by standard microbiological techniques, and
were grown to dense colonies on a single agar plate.
3.7. Characterization of natural products
22
3.7.1. Nocardiopsin C (1). Colorless oil; [
UV (MeOH) lmax (log
a]
þ10 (c 0.04, MeOH);
D
3 ) 218 (3.36) nm; NMR (600 MHz, methanol-
d4) see Table 1; HRESI(þ)MS m/z 582.3415 (calcd for C32H49NNaO7,
3.3. Microbial taxonomy
582.3412).
22
Genomic DNA extraction was performed using the DNeasy
blood and tissue kit (Qiagen) as per the manufacturers protocol.
The 16S rRNA genes were amplified from gDNA by PCR using
primers FC 27 (50-AGAGTTTGATCCTGGCTCAG-30) and RC 1492 (50-
3.7.2. Nocardiopsin D (2). Colorless oil; [
UV (MeOH) lmax (log
a
]
D
þ13 (c 0.04, MeOH);
3
) 217 (3.35) nm; NMR (600 MHz, methanol-
d4) see Table 1; HRESI(þ)MS m/z 584.3559 (calcd for C32H51NNaO7,
584.3558).
TACGGCTACCTTGTTACGACTT-30). The 50
m
L PCR mixture contained
20e40 ng gDNA, 250 pmol of each primer, 2.5 U Taq DNA poly-
merase and 100 mol of each dNTP. The 16S rRNA gene sequence
3.7.3. Nocardiopyrone A (3). White powder; nmax (thin film) 3370,
m
2956, 2871, 1698, 1670, 1610, 1467, 1385, 1367, 1226, 1169, 1136,
(GenBank accession no. JX441867) showed 99% homology with
1048 cmꢀ1. UV (MeOH) lmax (log
) 293 (3.76) nm; NMR (600 MHz,
3
other members of the genus Nocardiopsis.
methanol-d4) see Table 2; HRESI(þ)MS m/z 275.1625 (calcd for
C15H24NaO3, 275.1618).
3.4. PCR amplification of conserved PKS and NRPS genes
3.8. Marfey’s analysis
PCR amplifications of gDNA were performed using pairs of de-
generate primers18,19 targeting conserved regions of type I polyke-
tide synthase KS and methylmalonyl-CoA transferase domains (K1F
50-TSAAGTCSAACATCGGBCA-30 and M6R 50-CGCAGGTTSCSGTAC-
A sealed vial containing the nocardiopsin to be hydrolyzed
(100
16 h and then reduced to dryness under a stream of nitrogen. The
residue was redissolved in H2O (50 L) and aliquots of -1-fluoro-
2,4-dinitrophenyl-5-alanine amide ( -FDAA; 1% w/v in acetone;
100 L) and aqueous sodium bicarbonate (1 M; 20 L) were added.
The reaction mixture was heated at 40 ꢂC for 1 h and then quenched
by the addition of aqueous HCl (1 M; 20 L). Finally, the solution
was diluted with acetonitrile (500 L) and an aliquot (5 L) was
injected onto an Agilent Zorbax SB-C3 HPLC column (150ꢁ4.6 mm,
m) maintained at 50 ꢂC, which was developed with a linear
mg) and aqueous HCl (6 M; 200 m
L) was heated at 105 ꢂC for
CAGTA-30), type II polyketide synthase KS
TSGRCTACRTCAACGGSCACGG-30 and KS
a
b
/b
domains (KS
a
50-
m
L
50-TACSAGTCSWT-
L
CGCCTGGTTC-30), and non-ribosomal peptide synthase adenylation
domains (A3F 50-GCSTACSYSATSTACACSTCSGG-30 and A7R 50-
m
m
SASGTCVCCSGTSCGGTAS-30). Each 50
mL PCR mixture contained
m
60 ng gDNA, 400 pmol of each primer, 200 mol dNTPs, 1.25 U Taq
m
m
m
DNA polymerase (Fisher Biotec),1.25 mM MgCl2, and 5% DMSO. PCR
was performed with an Eppendorf Mastercycler ep gradient using
the following conditions: 5 min at 95 ꢂC, then 35 cycles of 30 s at
5
m
gradient of 15e60% MeOH/water (þisocratic 5% acetonitrile and 1%
formic acid) at 1 mL/min over 55 min. Peaks were observed using
diode array (340 nm) and ESIMS detectors. The procedure was re-
95 ꢂC, 2 min at either 55 ꢂC (for K1F/M6R), 58 ꢂC (for KS
a/KSb) or
59 ꢂC (for A3F/A7R), and 4 min at 72 ꢂC, followed by 10 min at 72 ꢂC.
PCR products were analyzed by electrophoresis on a 1% agarose gel
stained with SybrSafe and compared to a 1 kb Plus Ladder
(Invitrogen).
peated on analytical standards of
L-proline and also the N-crotonyl
and N-butanoyl amides of
L
-proline, using both -FDAA and -FDAA.
L
D
3.9. Bioassays
3.5. Microbial cultivation and extraction
3.9.1. Antimicrobial assay. Cultures of E. coli (ATCC 11775), S. aureus
(ATCC 25923), and B. subtilis (ATCC 6051) in nutrient broth (Bacto)
and C. albicans (ATCC 10231) in potato dextrose broth (Bacto) were
A seed culture was prepared by inoculating a 100 mL flask
containing non-saline liquid medium (15 mL deionized water
containing 1% starch, 0.4% yeast extract, and 0.2% peptone) with
incubated overnight at 37 ꢂC and an aliquot (100
mL) of each broth

R. Raju et al. / Tetrahedron 69 (2013) 692e698
697
was spread over the surface of sterile nutrient agar plates. Pure
compounds were dissolved in MeOH (5 mg/mL) and an aliquot
(30 mL) of each solution was added to sterile paper discs. The discs
were air dried for 1 h and then placed onto the surface of the in-
oculated agar plates. The plates were then incubated overnight at
37 ꢂC, after which the zones of inhibition were recorded. Rifampicin
and cycloheximide were used as positive controls against bacteria
and fungi, respectively, while MeOH was used as a negative control.
(1H, m, 1H, NCH2), 2.15e2.10 (1H, m, NCHCH2), 2.08e2.00 (1H, m,
NCH2CH2), 2.06e2.01 (1H, m, NCHCH2), 1.94e1.90 (1H, m,
NCH2CH2), 1.88 (3H, dd, J 6.8, 1.7 Hz, CH3); 13C NMR (150 MHz,
methanol-d4) dC trans rotamer 179.8, 167.5, 141.5, 124.3, 63.6, 47.7,
32.8, 23.7, 17.9. dC cis rotamer 180.3, 167.5, 141.5, 124.5, 62.9, 48.3,
31.0, 25.4, 17.9. HRESI(þ)MS m/z 206.0788 (calcd for C9H13NNaO3,
206.0793).
3.10.4. N-Butanoyl-
L
-proline.22 Prepared as described for N-cro-
3.9.2. Cytotoxicity assay. MTT cytotoxicity assays were performed
using gastric adenocarcinoma (AGS), neuroblastoma (SH-SY5Y),
and colorectal adenocarcinoma (HT-29) cell lines. Cells were
seeded in a 96-well microtiter plate at 1ꢁ105 to 4ꢁ105 cells/mL in
tonyl-L-proline. Colorless oil (tR¼9.7 min; 4.0 mg, 25%); [
a
]
D
22 ꢀ45 (c
0.10, MeOH); 1H NMR (600 MHz, methanol-d4) dH trans rotamer 4.24
(1H, dd, J 8.7, 3.3 Hz, NCHCO2H), 3.64e3.60 (1H, m, NCH2), 3.51e3.45
(1H, m, NCH2), 2.36e2.31 (1H, m, COCH2), 2.29e2.20 (1H, m,
NCHCH2), 2.27e2.22 (1H, m, COCH2), 2.15e2.10 (1H, m, NCHCH2),
1.97e1.89 (1H, m, NCHCH2), 1.86e1.80 (1H, m, NCH2CH2), 1.69e1.58
(2H, m, CH3CH2), 0.94 (3H, t, J 7.6 Hz, CH3). dH cis rotamer 4.32 (1H,
dd, J 8.7, 3.8 Hz, NCHCO2H), 3.69e3.65 (1H, m, NCH2), 3.54e3.50 (1H,
m, NCH2), 2.36e2.32 (1H, m, NCH2CH2), 2.27e2.22 (1H, m, COCH2),
2.16e2.11 (1H, m, NCHCH2), 2.05e2.00 (1H, m, CH2CH2), 2.02e1.98
(1H, m, NCHCH2), 1.93e1.89 (1H, m, NCH2CH2), 1.69e1.58 (2H, m,
CH3CH2), 0.97 (3H, t, J 7.6 Hz, CH3); 13C NMR (150 MHz, methanol-d4)
dC trans rotamer 180.0, 174.7, 63.8, 47.5, 37.3, 32.7, 23.9, 19.3, 14.2. dC
cis rotamer 179.6, 173.8, 62.7, 48.4, 37.3, 31.1, 25.5, 19.3, 14.2.
HRESI(þ)MS m/z 208.0944 (calcd for C9H15NNaO3, 208.0950).
either 100 mL of DNEM supplemented with 10% FBS (for AGS and
HT-29 cells) or 100 mL of RPMI supplemented with 15% FBS (for SH-
SY5Y cells), and the plate was incubated for 3e5 h (37 ꢂC; 5% CO2).
Compounds to be tested were dissolved in DMSO (to 1 mg/mL) and
diluted from 300
mM to 300 nM with 10% aqueous DMSO. Aliquots
(10 L) of each dilution (or 10% aqueous DMSO for controls) were
m
added to the plate in duplicate and the plate was incubated for 24 h
(37 ꢂC; 5% CO2). A solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT; Sigma, USA) in PBS was added
to each well to a final concentration of 0.5 mg/mL and the plate was
incubated for a further 4 h (37 ꢂC; 5% CO2). The medium was then
carefully aspirated from each well and precipitated formazan
crystals were dissolved in DMSO (100 mL). Finally, the absorbance of
Acknowledgements
each well at 600 nm was measured spectrophotometrically.
We thank A. Grinham (UQ) for acquiring the sediment samples,
F. Lafi and L. Sly (UQ) for taxonomic analysis, Z. Tnimov and K.
Alexandrov (UQ) for FKBP-binding studies and M. Conte (UQ) for
cytotoxicity testing. R.R acknowledges the provision of an Austra-
lian Postgraduate Research Award. This research was funded in part
by the Institute for Molecular Bioscience, The University of
Queensland and the Australian Research Council (LP0989954).
3.9.3. Isothermal titration calorimetry with FKBP12. Performed as
described previously.2
3.10. Synthetic procedures
3.10.1. Crotonic acid NHS ester. A solution of crotonic acid (200 mg,
2.32 mmol), DCC (479 mg, 2.32 mmol), and N-hydroxysuccinimide
(NHS) (267 mg, 2.32 mmol) in MeCN (25 mL) was stirred at room
temperature for 18 h, during which time a white precipitate
formed. The reaction mixture was then cooled to ꢀ20 ꢂC, filtered,
and the filtrate reduced to dryness in vacuo to give crotonic acid
NHS ester as a white solid (130 mg, 31%). 1H NMR (600 MHz,
methanol-d4) dH 7.24 (1H, dq, J 15.8, 7.0 Hz, CH3CH]), 6.00 (1H, d, J
15.8 Hz, ]CHCO), 2.80 (4H, s, 2ꢁNCOCH2),1.95 (3H, d, J 7.0 Hz, CH3).
Supplementary data
NMR spectra and tabulated 2D NMR data for all compounds, and
chromatograms from Marfey’s analyses. Supplementary data as-
sociated with this article can be found in the online version, at
http://dx.doi.org/10.1016/j.tet.2012.10.104. These data include MOL
files and InChiKeys of the most important compounds described in
this article.
3.10.2. Butyric acid NHS ester.20 Prepared as described for crotonic
acid NHS ester. Viscous yellow oil (125 mg, 30%). 1H NMR (600 MHz,
methanol-d4) dH 2.83 (4H, s, 2ꢁNCOCH2), 2.60 (2H, t, J 7.5 Hz,
CH2CO), 1.78e1.72 (2H, m, CH3CH2), 1.04 (3H, t, J 7.5 Hz, CH3).
References and notes
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