Tauramamide, a lipopeptide antibiotic produced in culture by Brevibacillus laterosporus isolated from a marine habitat: Structure elucidation and synthesis

Article abstract of DOI:10.1021/np070209r

Tauramamide (1), a new lipopeptide antibiotic, is produced by cultures of the marine bacterial isolate Brevibacillus laterosporus PNG276 obtained from Papua New Guinea. Tauramamide was isolated as its methyl and ethyl esters 2 and 3, whose structures were elucidated by analysis of NMR, MS, and chemical degradation data. A total synthesis of tauramamide (1) and tauramamide ethyl ester (3) confirmed the structure proposed from spectroscopic analysis and provided the natural product for antimicrobial testing. Tauramamide (1) and ethyl ester 3 show potent and relatively selective inhibition of pathogenic Enterococcus sp.

Full text of DOI:10.1021/np070209r

1850
J. Nat. Prod. 2007, 70, 1850–1853
Tauramamide, a Lipopeptide Antibiotic Produced in Culture by BreWibacillus laterosporus
Isolated from a Marine Habitat: Structure Elucidation and Synthesis
Kelsey Desjardine,† Alban Pereira,† Helen Wright,† Teatulohi Matainaho,‡ Michael Kelly,§ and Raymond J. Andersen*,†
Departments of Chemistry and Earth & Ocean Sciences, UniVersity of British Columbia, 2036 Main Mall, VancouVer, British Columbia,
Canada V6T 1Z1, Discipline of Pharmacology, UniVersity of Papua New Guinea, N.C.D., Papua New Guinea, and Ocean Pharmaceuticals,
Inc., 4156 H Street Road, Blaine, Washington 98230
ReceiVed May 4, 2007
Tauramamide (1), a new lipopeptide antibiotic, is produced by cultures of the marine bacterial isolate BreVibacillus
laterosporus PNG276 obtained from Papua New Guinea. Tauramamide was isolated as its methyl and ethyl esters 2
and 3, whose structures were elucidated by analysis of NMR, MS, and chemical degradation data. A total synthesis of
tauramamide (1) and tauramamide ethyl ester (3) confirmed the structure proposed from spectroscopic analysis and
provided the natural product for antimicrobial testing. Tauramamide (1) and ethyl ester 3 show potent and relatively
selective inhibition of pathogenic Enterococcus sp.
Microorganisms isolated from marine habitats are proving to be
an extremely rich source of novel bioactive secondary metabolites.1
As part of an ongoing program aimed at discovering new antibiot-
ics,2 we have been exploring crude extracts obtained from laboratory
cultures of a marine isolate of BreVibacillus laterosporus (PNG-
276). To date, we have reported three structurally unrelated families
of antibiotics, the loloatins,3 the bogorols,4 and the basiliskamides,5
that are produced by PNG-276. Several fractions from the chro-
matographic separation of the PNG-276 cell extract, which did not
contain members of the known families, showed strong antibacterial
activity. Investigation of one of these sets of fractions has resulted
in the identification of tauramamide (1), a new lipopeptide
antibiotic.6 The details of the isolation and structure elucidation of
tauramamide methyl ester (2), the total synthesis of tauramamide
(1), and the antibacterial activities of the free acid 1 and its ethyl
ester 3 are presented below.
HPLC led to the isolation of pure tauramamide methyl ester (2) as
a pale yellow oil (7.4 mg, 0.02% dry cell weight).
Methyl ester 2 gave a [M + H]+ ion at m/z 878.5181 in the
HRESI-TOFMS, consistent with
a molecular formula of
C45H67N9O9 (calcd for C45H68N9O9, 878.5140), requiring 17 sites
1
of unsaturation. Preliminary examination of the H and 13C NMR
data recorded for 2 (Supporting Information) revealed that the
molecule was a peptide. Detailed analysis of the COSY, HSQC,
HMBC, and TOCSY data identified arginine, tryptophan, leucine,
tyrosine, and serine residues (Table 1). Five aliphatic methylene
resonances between δ 1.07 and 1.36, a methine resonance at δ 1.44,
a pair of isochronous methyl doublets at δ 0.83, and a methylene
1
resonance at δ 2.01 in the H NMR spectrum were tentatively
assigned to a 7-methyloctanoyl fragment on the basis of COSY,
HSQC, and HMBC correlations (Table 1). A methyl resonance at
δ 3.62, which showed a HMBC correlation to a carbon resonance
at δ 172.1, was assigned to a methyl ester.
The amino acid sequence of tauramamide methyl ester (2) was
determined by analysis of ROESY data. ROESY correlations
observed between the Arg NH (δ 8.39) and the Trp RH (δ 4.53),
between the Trp NH (δ 8.14) and the Leu RH (δ 4.20), between
the Leu NH (δ 7.75) and the Ser RH (δ 4.22), between the Ser NH
(δ 8.03) and the Tyr RH (δ 4.41), and between the Tyr NH (δ
7.96) and the C-2 methylene protons of the 7-methyloctanoyl
residue (δ 4.22) demonstrated that the Tyr residue was the
N-terminus of the pentapeptide Tyr-Ser-Leu-Trp-Arg, and it was
N-acylated with a 7-methyloctanoyl fragment. The methyl ester
functionality indicated by the NMR data had to be on the C-terminal
Arg residue, leading to the proposed linear lipopeptide structure 2
for tauramamide methyl ester, which accounted for all of the atoms
and the 17 sites of unsaturation indicated by the HRMS analysis.
Hydrolysis of 2 with aqueous HCl followed by Marfey’s analysis7
of the amino acids revealed that Tyr and Leu had the D configu-
ration, while Arg, Trp, and Ser were L as shown.
Results and Discussion
The marine bacterial isolate PNG-276, identified as BreVibacillus
laterosporus by the analysis of 16S RNA, was obtained from tissues
of an unidentified tube worm collected off the coast of Loloata
Island, Papua New Guinea. Crude MeOH extracts of B. laterosporus
cells harvested from cultures grown as lawns on solid agar showed
broad-spectrum antibiotic activity against the human pathogens
MRSA, VRE, Mycobacterium tuberculosis, Candida albicans, and
Escherichia coli. Bioassay-guided fractionation of the cell extracts
employing sequential application of Sephadex LH20 chromatog-
raphy, reversed-phase flash chromatography, and reversed-phase
It was possible that the methyl ester in 2 was an artifact of the
isolation procedure because MeOH had been used to extract the B.
laterosporus cells. In order to determine if the methyl ester was
natural or an isolation artifact, a second batch of B. laterosporus
cells was extracted with EtOH, and MeOH was not used at any
point in the purification. In this case, tauramamide ethyl ester 3
was isolated from the cell extract, and there was no detectable trace
of the methyl ester 2, demonstrating that the free carboxylic acid
1 must be the natural product. All attempts to convert the small
amounts of the ester 2 obtained from the B. laterosporus cultures
to the natural product 1 failed to generate sufficient amounts of
* To whom correspondence should be addressed. Tel: 604 822 4511.
Fax: 604 822 6091. E-mail: randersn@interchange.ubc.ca.
† Chemistry and EOS, UBC.
‡ UPNG.
§ Ocean Pharmaceuticals.
10.1021/np070209r CCC: $37.00
2007 American Chemical Society and American Society of Pharmacognosy
Published on Web 11/29/2007

Lipopeptide Antibiotic Produced by BreVibacillus laterosporus
Journal of Natural Products, 2007, Vol. 70, No. 12 1851
Table 1. NMR Data for Tauramamide Methyl Ester (2), Recorded at 500 MHz (1H) and 100 MHz (13C) in DMSO-d6
δC
δH (J in Hz)
δC
δH (J in Hz)
OMe
Ser
1
51.8
3.62 s, 3H
Arg
1
2
3
55.1
61.7
4.22 m
172.1
H2 3.47 m
H2′ 3.52 m
4.83 bs
2
3
51.3
27.5
4.29 m
3-OH
NH
H2 1.67 m
H2′ 1.77 m
1.52 m, 2H
3.11 m, 2H
8.03 bs
4
5
25.0
40.6
Tyr
1
6
156.7
2
3
54.5
36.5
4.41 bs
H2 2.64 m
H2′ 2.85 m
2-NH
8.39 bd (7.0)
7.50 bs
5-NH
Trp
1
2
3
1′
127.8
130.0
114.7
155.7
2′, 6′
3′, 5′
4′
7.02 d (7.8), 2H
6.62 d (7.8), 2H
53.1
27.8
4.53 bs
H2 2.89 m
H2′ 3.15 m
4′-OH
9.13 s
3′
3a′
4′
5′
6′
7′
7a′
2′
2-NH
1′-NH
Leu
1
109.8
127.1
118.4
118.0
120.7
111.1
136.0
123.9
NH
7.96 bs
methyloctanoyl
7.60 d (7.7)
1
2
3
4
5
6
7
8
8′
6.96 dd (7.7, 7.2)
7.04 bd (7.2)
7.30 d (7.8)
35.1
25.1
28.7
26.5
38.0
27.0
22.5
22.5
2.01 m, 2H
1.36 m, 2H
1.07 m, 2H
1.14 m, 2H
7.11 bs
8.14 bs
10.76 bs
1.07 m, 2H
1.44 bm (6.7)
0.83 d (6.7), 3H
0.83 d (6.7), 3H
2
3
4
5
5′
NH
51.7
40.5
23.7
22.8
21.5
4.20 m
1.08 m, 2H
1.25
0.71 d (6.3), 3H
0.68 d (6.3), 3H
7.75 bs
Scheme 1. Synthesis of Tauramamide (1)
the natural product for biological testing. Therefore, we undertook
a total synthesis of tauramamide (1) in order to confirm the proposed
structure and to generate sufficient quantities of compound for
antimicrobial evaluation.
The synthesis of tauramamide (1) started with the commercially
available N-protected arginine derivative 4, which was converted
to its benzyl ester 5 by reaction with benzyl alcohol, DCC, and
DMAP (Scheme 1). Deprotection of the R-amino nitrogen by
treatment of 5 with TFA, followed by three cycles of standard
PyBOP-activated peptide coupling with Boc-L-Trp, Boc-D-Leu, and
Boc-L-Ser(Bn) in sequence, generated the protected tetrapeptide 8.
The phenolic hydroxyl in the Tyr methyl ester 9 was protected
by treatment with benzyl bromide and potassium carbonate in
acetone to give 10. Exposure of the amino group in 10 by reaction
with TFA, followed by DCC-mediated amide formation with 7-meth-
yloctanoic acid, and subsequent LiOH-catalyzed ester hydrolysis
gave acid 12. Treatment of the protected tetrapeptide 8 with TFA
followed by PyBOP-mediated amide coupling with the acid 12 gave

1852 Journal of Natural Products, 2007, Vol. 70, No. 12
Desjardine et al.
Acid Hydrolysis of Tauramamide Methyl Ester (2). Ester 2 (0.8
mg, 0.9 mol) was added to 0.5 mL of 6 N HCl and sealed in a screw-
top vial, and the mixture was heated to 108 °C with stirring for 16 h.
After cooling, the mixture was evaporated under N2 to remove HCl. A
second hydrolysis for Trp analysis was performed using ester 2 (0.2
mg, 0.2 umol) under identical conditions, but heating for 1 h instead
of 16 h.
Preparation of FDAA Derivative Standards. Amino acid stan-
dards (2.0 mmol) were dissolved in H2O (40 mL), and to this solution
was added Marfey’s reagent (N-(2,4-dinitro-5-fluorophenyl)-L-alani-
namide, 62.5 mM in 60 mL of Me2CO), followed by NaHCO3 (1 M,
20 mL). The mixture was heated for 1 h at 43 °C, after which HCl (2
N, 10 mL) was added. An additional aliquot of Me2CO (50 mL) was
added to solubilize the samples.
Table 2. Antimicrobial Activity (MICs in µg/mL) of Synthetic
Tauramamide (1) and Tauramamide Ethyl Ester (3)
tauramamide
bioasay
pathogen
MRSA
C. albicans
Enterococcus sp.
MRSA
tauramamide (1) ethyl ester (3)
Alamar Blue
ppta
ppta
0.1
25
200
0.1
9.4
75
agar inclusion
200
50
C. albicans
a The compound precipitated from solution at higher concentrations
and was not active at lower concentrations where it was soluble.
Marfey’s Analysis of Tauramamide Methyl Ester (2). The acid
hydrolysate of ester 2 was treated in the same fashion as the amino
acid standards described above. HPLC analyses of derivatized amino
acids were performed using an Alltech Econosil C18 HPLC column
eluted with the following solutions. Solution A: prepared by adding
Et3N (28 mL, 0.2 mol) to H2O (3.57 L) while stirring; to this mixture
was added H3PO4 dropwise until the mixture was pH 3.0; MeCN (400
mL) was then added, resulting in a mixture of 9:1 triethylammonium
phosphate (50 mM, ph 3.0)/MeCN. Solution B: HPLC grade MeCN.
The HPLC analysis employed a linear gradient from 100% solution A
to 60% solution A/40% solution B over 40 min at a flow rate of 1
mL/min, with UV detection at 210 nm.
protected tauramamide. Hydrogenolysis using Pd/C in EtOH
removed the Bn and Cbz protecting groups to liberate tauramamide
(1), which was purified by reversed-phase HPLC to give a pure
sample. Esterification of 1 with EtOH and HCl gave ethyl ester 3,
which was identical by HPLC, MS, and NMR comparison with
the material extracted from B. laterosporus cells with EtOH,
confirming the structure proposed for the natural product 1.
Table 2 shows the antimicrobial activity of tauramamide (1) and
tauramamide ethyl ester (3). Both compounds show potent (MICs
0.1 µg/mL) and relatively selective activity against the important
Gram-positive human pathogen Enterococcus sp. The ethyl ester
3 shows weaker activity against MRSA, but neither compound is
appreciably active against C. albicans. Tauramamide (1) is a new
lipopeptide antibiotic that contains two D amino acids and is acylated
at the N-terminus, which are both hallmarks of a non-ribosomal
peptide synthase biosynthetic origin.
Isolation of Tauramamide Ethyl Ester (3). PNG-276 was cultured
and the cells were collected as described above. Extraction of the cells
and fractionation of the crude extract proceeded as described above
for the isolation of 2 except that EtOH was substituted for MeOH.
This extraction yielded tauramamide A ethyl ester (3; 4.1 mg, 0.015%
dry cell weight).
Tauramamide ethyl ester (3): pale yellow glass; [R]25 -31.9 (c
D
Experimental Section
0.8, MeOH); for 1D and 2D NMR data, see the Supporting Information;
HRESIMS [M + H]+ m/z 892.5294 (calcd for C46H70N9O9, 892.5297).
Boc-Arg(Cbz)2-OBn (5). To a solution of Boc-Arg(Cbz)2-OH (4)
(2.5 g, 4.6 mmol) and benzyl alcohol (0.71 mL, 6.9 mmol) in CH2Cl2
(50 mL) at 0 °C were added DCC (1.04 g, 5.06 mmol) and DMAP
(0.06 g, 0.46 mmol), and the resulting mixture was stirred overnight.
The solution was poured into water and extracted with CH2Cl2 (30 mL
× 3). The organic extracts were dried (Na2SO4) and concentrated in
Vacuo. Purification by Sephadex LH20 column chromatography (eluent:
100% MeOH) afforded 5 as a colorless solid (2.91 g, 97%). 5: 1H NMR
(CDCl3, 600 MHz) δ 9.36 (1H, s), 9.12 (1H, s), 7.30–7.10 (15H, m),
5.30 (1H, d, J ) 8.3 Hz), 5.10–4.90 (6H, m), 4.24 (1H, m), 3.83 (2H,
m), 1.85–1.70 (4H, m), 1.31 (9H, s); HRESIMS m/z 655.2743 (calcd
for C34H40N4O8Na, 655.2744).
General Experimental Procedures. Optical rotations were recorded
with a JASCO J-1010 polarimeter equipped with a halogen lamp (589
nm) and a 10 mm microcell. NMR spectra were recorded on Bruker
Avance 400, Bruker AMX 500, and Bruker Avance 600 (equipped with
a cryoprobe) spectrometers at 400, 500, and 600 MHz, respectively, in
CDCl3 and DMSO-d6. Chemical shifts are given in δ (ppm) with the
residual CDCl3 solvent peak referenced to δH 7.24 and δC 77.0 and
the residual DMSO solvent peak referenced to δH 2.49 and δC 39.5 as
the internal reference. ESIMS spectra were obtained with Kratos MS-
50, Micromass LCT, and Bruker Esquire-LC mass spectrometers. Si
gel (Silicycle, 230–400 mesh) and Sephadex LH20 were used for
column chromatography; precoated Si gel plates (Merck, Kieselgel 60
F
254, 0.25 mm and Whatman, MKC18F 60 A) were used for TLC
analysis. A Waters 1500 Series pump system equipped with a Waters
2487 dual λ absorbance detector and a CSC-Inertsil 150A/ODS2 column
was used for HPLC. All reagents were purchased in analytical or higher
grade. Dry CH2Cl2 was prepared by distillation from CaH.
Culture Conditions for PNG 276. PNG-276 was grown as lawns
on 21 pans of solid tryptic soy agar (24 × 37 × 0.5 cm) supplemented
with NaCl to a final concentration of 1%. Cultures were grown at room
temperature for 5 days before live cells were scraped from the solid
agar surface and lyophilized.
Isolation of Tauramamide Methyl Ester (2). Lyophilized cells
(∼30 g) were exhaustively extracted with MeOH (3 × 250 mL). The
combined MeOH extract was concentrated in Vacuo to yield a brown
gum (3.8 g), which was partitioned between H2O and EtOAc. The
EtOAc-soluble portion (1.0 g) was fractionated via sequential Sephadex
LH-20 chromatographies, the first eluted with MeOH and the second
eluted with EtOAc/MeOH/H2O (20:5:2) yielding fractions that were
pooled according to bioactivity, NMR, and TLC characteristics [2: Rf
) 0.7 on normal-phase Si gel (eluent: 4:1:1 n-BuOH/MeOH/H2O,
visualized with ceric sulfate/phosphomolybdic acid dip)].
Boc-Ser(Bn)-D-Leu-Trp-Arg(Cbz)2-OBn (8). A solution of Boc-
Arg(Cbz)2-OBn (5) (1.87 g, 2.9 mmol, 1 equiv) in CH2Cl2 (25 mL)
was treated with TFA (3 mL, 40.3 mmol, 14 equiv) and stirred for 4 h,
whereupon it was concentrated in Vacuo to a pale red oil. This oil was
dissolved in CH2Cl2 (20 mL) and added to a solution of Boc-Trp-OH
(0.82 g, 2.7 mmol, 0.90 equiv) and PyBOP (1.69 g, 3.24 mmol, 1.10
equiv) in CH2Cl2 (10 mL), followed by addition of DIEA (1.55 mL,
8.90 mmol, 3 equiv). After stirring overnight at 25 °C, saturated NH4Cl
was added, and the resulting solution was extracted with CH2Cl2 (3 ×
20 mL). The organic extracts were combined, dried (Na2SO4), and
concentrated in Vacuo. Column chromatography on Si gel (80% EtOAc/
hexanes) afforded 6 as a white, amorphous solid (1.87 g, 85%). The
procedure above was repeated successively with Boc-D-Leu-OH (0.48
g, 2.07 mmol) and Boc-Ser(Bn)-OH (0.46 g, 1.57 mmol) to afford Boc-
D-Leu-Trp-Arg(Cbz)2-OBn (7) (1.93 g, 76%) and Boc-Ser(Bn)-D-Leu-
Trp-Arg(Cbz)2-OBn (8) (1.75 g, 81%), respectively.
Tetrapeptide 8: 1H NMR (CDCl3, 400 MHz) δ 9.40 (1H, s), 9.28
(1H, s), 9.00 (1H, s), 7.26–6.90 (30H, m), 5.65 (1H, m), 5.20–4.90
(6H, m), 4.80 (1H, dd, J ) 6.3, 7.4 Hz), 4.55–4.40 (2H, m), 4.40–4.25
(2H, m), 3.95–3.80 (2H, m), 3.67 (1H, m), 3.50 (1H, dd, J ) 6.1, 2.8
Hz), 3.31 (1H, dd, J ) 6.3, 8.7 Hz), 3.15 (1H, dd, J ) 5.7, 8.7 Hz),
1.85–1.75 (1H, m), 1.75–1.60 (2H, m), 1.60–1.35 (4H, m), 1.41 (9H,
s), 0.80 (6H, m); HRESIMS m/z 1131.5162 (calcd for C61H72N8O12Na,
1131.5167).
Boc-D-Tyr(Bn)-OMe (10). A solution of Boc-D-Tyr-OMe (9) (2.0
g, 6.8 mmol), K2CO3 (1.4 g, 10.1 equiv), and BnBr (1 mL, 8.1 mmol)
in acetone was refluxed for 3 h and then stirred overnight at 25 °C.
After filtration and solvent evaporation, column chromatography on
The most active fraction (140.9 mg) was subjected to reversed-phase
C-18 chromatography (Waters 2 g Sep-Pak) using step gradient elution
from H2O to MeOH to CH2Cl2 to give 92.8 mg of active material.
This material was fractionated by isocratic reversed-phase HPLC (40%
MeCN/H2O + 0.1% TFA) to give pure tauramamide methyl ester (2;
7.4 mg, 0.02% dry cell weight).
Tauramamide methyl ester (2): pale yellow glass; [R]25 -14.6
D
(c 0.6, MeOH); for NMR data, see Table 1; HRESIMS [M + H]+ m/z
878.5181 (calcd for C45H67N9O9, 878.5185).

Lipopeptide Antibiotic Produced by BreVibacillus laterosporus
Journal of Natural Products, 2007, Vol. 70, No. 12 1853
Si gel (30% EtOAc/hexanes) afforded 10 as an amorphous, white solid
(2.60 g, 99%). 10: 1H NMR (CDCl3, 400 MHz) δ 7.35–7.20 (5H, m),
6.94 (2H, d, J ) 5.6), 6.81 (2H, d, J ) 5.7), 4.94 (2H, s), 4.89 (1H, d,
J ) 5.0 Hz), 4.45 (1H, dd, J ) 4.4 Hz), 3.61 (3H, s), 2.95 (1H, dd, J
) 3.64, 9.2 Hz), 2.91 (1H, dd, J ) 3.6, 9.0 Hz), 1.33 (9H, s); HRESIMS
m/z 408.1790 (calcd for C22H27NO5Na, 408.1787).
(1H, m), 1.45 (1H, m), 1.35 (2H, m), 1.35 (1H, m), 1.24 (1H, m), 1.17
(1H, m), 1.15 (1H, m), 1.14 (1H, m), 1.09 (2H, m), 1.08 (1H, m), 1.07
(1H, m), 1.06 (1H, m), 0.826 (3H, d, J ) 6.8 Hz), 0.823 (3H, d, J )
6.8 Hz), 0.68 (3H, d, J ) 6.8 Hz), 0.66 (3H, d, J ) 6.5 Hz); HRESIMS
m/z 864.4981 (calcd for C44H66N9O9, 864.4984).
Tauramamide ethyl ester (3). A solution of tauramamide (1) (0.063
g, 0.073 mmol) and HCl (1 mL of 2 M HCl in diethyl ether, 2 mmol)
in EtOH was stirred at 25 °C until TLC spots for starting material (RP,
90% CH3CN/H2O) disappeared (around 5 days). Solvent evaporation,
followed by reversed-phase HPLC (50% CH3CN/H2O, 0.1% TFA),
yielded pure tauramamide ethyl ester (3) (0.0559 g, 86%): [R]25D –4.7
(c 1.85, MeOH); 1H NMR (DMSO, 600 MHz) δ 10.77 (1H, d, J ) 1.6
Hz), 9.14 (1H, s), 8.43 (1H, d, J ) 7.4 Hz), 8.16 (1H, d, J ) 8.3 Hz),
8.04 (1H, d, J ) 7.7 Hz), 7.98 (1H, d, J ) 7.7 Hz), 7.75 (1H, d, J )
7.7 Hz), 7.62 (1H, d, J ) 7.7 Hz), 7.48 (1H, dd, J ) 5.5, 5.6 Hz), 7.30
(1H, d, J ) 8.0 Hz), 7.10 (1H, d, J ) 2.2 Hz), 7.04 (1H, dd, J ) 7.2,
7.5 Hz), 7.02 (1H, d, J ) 8.6 Hz), 7.02 (1H, d, J ) 8.6 Hz), 6.96 (1H,
dd, J ) 7.2, 7.5 Hz), 6.61 (1H, d, J ) 8.6 Hz), 6.61 (1H, d, J ) 8.6
Hz), 4.84 (1H, dd, J ) 5.2, 5.5 Hz), 4.54 (1H, m), 4.42 (1H, m), 4.25
(1H, m), 4.24 (1H, m), 4.23 (1H, m), 4.09 (2H, m), 3.51 (1H, m), 3.45
(1H, m), 3.15 (1H, dd, J ) 3.8, 14.6 Hz), 3.11 (2H, m), 2.88 (1H, m),
2.84 (1H, m), 2.62 (1H, dd, J ) 10.0, 13.8 Hz), 2.00 (2H, m), 1.77
(1H, m), 1.66 (1H, m), 1.53 (2H, m), 1.45 (1H, m), 1.35 (2H, m), 1.26
(1H, m), 1.18 (3H, t, J ) 7.2 Hz), 1.14 (2H, m), 1.08 (2H, m), 1.08
(2H, m), 1.06 (2H, m), 0.824 (3H, d, J ) 6.7 Hz), 0.822 (3H, d, J )
6.6 Hz), 0.70 (3H, d, J ) 6.6 Hz), 0.67 (3H, d, J ) 6.6 Hz); HRESIMS
m/z 892.5295 (calcd for C46H70N9O9, 892.5297).
(CH3)2CH2(CH2)5CONH-D-Tyr(Bn)-OMe (11). A solution of Boc-
D-Tyr(Bn)-OMe (10) (1.50 g, 3.9 mmol) in CH2Cl2 (35 mL) was treated
with TFA (2 mL, 27 mmol) and stirred for 4 h, whereupon it was
concentrated in Vacuo to a colorless oil. This oil was dissolved in
CH2Cl2 (10 mL) and added to a solution of 7-methyloctanoic acid (0.62
g, 3.9 mmol) and PyBOP (2.0 g, 3.8 mmol) in CH2Cl2 (40 mL),
followed by addition of DIEA (2.0 mL, 12 mmol). After stirring
overnight at 25 °C, saturated NH4Cl was added and the resulting
solution was extracted with CH2Cl2 (3 × 20 mL). The combined organic
extracts were dried (Na2SO4) and concentrated in Vacuo. Column
chromatography on Si gel (80% EtOAc/hexanes) afforded 11 as a
1
yellowish powder (1.67 g, 88%). 11: H NMR (CDCl3, 400 MHz) δ
7.35–7.15 (5H, m), 6.98 (2H, d, J ) 8.5), 6.80 (2H, d, J ) 8.5), 6.71
(1H, d, J ) 8.0), 4.87 (2H, s), 4.79 (1H, dd, J ) 6.8, 7.2 Hz), 3.57
(3H, s), 3.01 (1H, dd, J ) 5.6, 14.0 Hz), 2.89 (1H, dd, J ) 7.0, 14.0
Hz), 2.10 (2H, t, J ) 7.6 Hz), 1.51 (2H, quintet, J ) 7.96 Hz), 1.43
(1H, septet, J ) 6.5 Hz), 1.18 (4H, m), 1.08 (2H, quintet, J ) 6.8 Hz),
0.80 (6H, d, J ) 6.5 Hz); HRESIMS m/z 426.2645 (calcd for
C26H36NO4, 426.2644).
(CH3)2CH2(CH2)5CONH-D-Tyr(Bn)-Ser(Bn)-D-Leu-Trp-Arg(Cbz)2-
OBn (13). A solution of (CH3)2CH2(CH2)5CONH-D-Tyr(Bn)-OMe (11)
(0.717 g, 1.68 mmol) and LiOH ·H2O (0.705 g, 16.8 mmol) in 1,4-dioxane/
H2O (150 mL, 2:1) was stirred at 25 °C. After 1 h, TLC (80% EtOAc/
hexanes) showed an absence of starting material. Solvents were evaporated,
and the resulting residue was dissolved in H2O, acidified, and extracted
with EtOAc (3 × 30 mL). The combined organic extracts were dried
(Na2SO4) and concentrated in Vacuo to give a white solid (acid 12, 0.68 g,
98%). In a separate reaction, Boc-Ser(Bn)-D-Leu-Trp-Arg(Cbz)2-OBn (8)
(1.42 g, 1.3 mmol) in CH2Cl2 (50 mL) was treated with TFA (3 mL, 40.3
mmol) and stirred for 5 h, whereupon it was concentrated in Vacuo to a
red oil. This oil was dissolved in CH2Cl2 (20 mL) and added to a solution
of acid 12 (0.58 g, 1.41 mmol) and PyBOP (0.73 g, 1.41 mmol) in CH2Cl2
(30 mL), followed by addition of DIEA (0.66 mL, 3.84 mmol). After
stirring 16 h at 25 °C, the reaction was quenched with saturated NH4Cl,
and 1 M HCl was used to neutralize the aqueous layer. CH2Cl2 (3 × 30
mL) extractions were performed; the organic extracts were combined, dried
(Na2SO4), and concentrated in Vacuo. Column chromatography on Si gel
(80% EtOAc/hexanes) afforded 13 as a pale yellow solid (1.45 g, 81%).
Antibacterial Assays. Culture media for bacterial strains: Tryptic
soya agar (TSA+) or broth (TSB+) with 5 g/L NaCl; for C. albicans
Sabouraud agar (Sab) or TSB+.
Alamar Blue Bioassays. This assay was run as a 1:2 serial dilution
bioassay. The compound to be tested was dissolved in solvent (DMSO
or EtOH). Alamar Blue (AB), an oxidation–reduction indicator,
undergoes colorimetric change in response to cellular metabolic
reduction. If the compound has antimicrobial activity (i.e., pathogen is
killed and/or inhibited), then the AB remains blue/purple. If the
pathogen is not killed and is able to grow, then the AB is oxidized and
changes to bright pink.
Agar Extract Inclusion Bioassay. The compounds were dissolved
in solvent (DMSO or EtOH) and incorporated into Mueller Hinton agar
cooled to 50 °C. Agar was then serially diluted 1:2 with additional cooled
agar and aliquoted into Petri plates. Several pathogens (each ∼2.5 × 105
cells/mL inoculum concentration) were spotted onto the agar surface. Plates
were incubated overnight at 37 °C, and then the plates were checked for
inhibition of pathogen growth to determine MIC.
1
13: H NMR (DMSO, 600 MHz) δ 10.74 (1H, s), 9.16 (1H, s, broad),
8.52, (1H, d, J ) 6.9 Hz), 8.15, (1H, d, J ) 8.0 Hz), 8.13, (1H, d, J ) 8.6
Hz), 8.00 (2H, d, J ) 6.9 Hz), 7.58 (1H, d, J ) 7.7 Hz), 7.60–6.80 (25H,
m), 7.13 (3H, m), 7.09 (1H, d, J ) 2.0 Hz), 7.03 (1H, dd, J ) 7.1, 7.1
Hz), 6.93 (1H, dd, J ) 7.4, 7.4), 6.85 (3H, m), 5.20 (2H, m), 5.05–5.00
(6H, m), 4.55 (3H, m), 4.29 (2H, m), 3.90 (1H, m), 3.85 (1H, m), 3.61–3.45
(4H, m), 3.07 (1H, m), 2.85 (1H, m), 2.65 (1H, m), 1.98 (2H, m), 1.70
(1H, m), 1.43 (3H, m), 1.32 (4H, m), 1.25–1.00 (10H, m), 0.79 (3H, d, J
) 6.2 Hz), 0.78 (3H, d, J ) 6.3 Hz), 0.62 (6H, d, J ) 5.5 Hz); HRESIMS
m/z 1402.7125 (calcd for C81H96N9O13, 1402.7128).
Acknowledgment. Financial support was provided by the Natural
Sciences and Engineering Research Council of Canada (to R.J.A.).
Supporting Information Available: NMR spectra for 1, 2, and 3;
tables of NMR data for 1 and 3, 13C NMR data for synthetic
intermediates. This material is available free of charge via the Internet
at http://pubs.acs.org.
References and Notes
Tauramamide (1). A solution of (CH3)2CH2(CH2)5CONH-D-
Tyr(Bn)-Ser(Bn)-D-Leu-Trp-Arg(Cbz)2-OBn (13) (0.21 g, 0.15 mmol)
and 10% Pd/C, wet Degussa type E101NE/W, was stirred in the
presence of H2 at 25 °C and 20 atm, until TLC (RP, 90% CH3CN/
H2O) showed the absence of starting material (around 1 week). Catalyst
filtration, followed by solvent evaporation and reversed-phase column
chromatography (90% CH3CN/H2O), afforded a colorless solid residue.
Further purification by reversed-phase HPLC (50% CH3CN/H2O, 0.1%
(1) (a) Bernan, V. S.; Greenstein, M.; Carter, G. T. Curr. Med. Chem.:
Anti-InfectiVe Agents 2004, 3, 181–195. (b) Fenical, W.; Jensen, P. R.
Nat. Chem. Biol. 2006, 2, 666–673. (c) Jensen, P. R.; Mincer, T. J.;
Williams, P. G.; Fenical, W. Antonie Van Leeuwenhoek 2005, 87, 43–
48. (d) Blunt, J. W.; Copp, B. R.; Munro, M. H. G.; Northcote, P. T.;
Prinsep, M. R. Nat. Prod. Rep. 2006, 23, 26–78.
(2) Davies, J.; Wang, H.; Taylor, T.; Warabi, K.; Huang, X.-H.; Andersen,
R. J. Org. Lett. 2005, 7, 5233–5236.
(3) (a) Gerard, J. M.; Haden, P.; Kelly, M. T.; Andersen, R. J. Tetrahedron
Lett. 1996, 37, 7201–7204. (b) Gerard, J. M.; Haden, P.; Kelly, M. T.;
Andersen, R. J. J. Nat. Prod. 1999, 62, 80–85.
(4) (a) Barsby, T.; Kelly, M. T.; Gagne, S.; Andersen, R. J. Org. Lett. 2001,
3, 437–440. (b) Barsby, T.; Warabi, K.; Sorenson, D.; Zimmerman,
W.; Kelly, M. T.; Andersen, R. J. J. Org. Chem. 2006, 71, 6031–6037.
(5) Barsby, T.; Kelly, M. T.; Andersen, R. J. J. Nat. Prod. 2002, 65, 1447–
1451.
(6) This compound is named after Taurama Point, which is adjacent to
Loloata Island, Papua New Guinea.
TFA) yielded pure tauramamide (1) (0.132 g, 81%): [R]25 –51.8 (c
D
1
0.9, MeOH); H NMR (DMSO, 600 MHz) δ 12.69 (1H, s, br), 10.76
(1H, d, J ) 1.5 Hz), 9.14 (1H, s), 8.32 (1H, d, J ) 7.8 Hz), 8.14 (1H,
d, J ) 8.3 Hz), 8.03 (1H, d, J ) 7.8 Hz), 7.99 (1H, d, J ) 7.7 Hz),
7.75 (1H, d, J ) 7.8 Hz), 7.62 (1H, d, J ) 7.8 Hz), 7.48 (1H, dd, J )
5.8, 5.8 Hz), 7.30 (1H, d, J ) 8.0 Hz), 7.10 (1H, d, J ) 1.9 Hz), 7.03
(1H, dd, J ) 7.1, 7.1 Hz), 7.02 (1H, d, J ) 8.3 Hz), 7.02 (1H, d, J )
8.3 Hz), 6.95 (1H, dd, J ) 7.4, 7.4 Hz), 6.61 (1H, d, J ) 8.4 Hz), 6.61
(1H, d, J ) 8.4 Hz), 4.82 (1H, s, broad), 4.54 (1H, m), 4.42 (1H, m),
4.23 (1H, m), 4.22 (1H, m), 4.21 (1H, m), 3.51 (1H, m), 3.44 (1H, m),
3.16 (1H, m), 3.12 (2H, m), 2.88 (1H, m), 2.86 (1H, m), 2.62 (1H, dd,
J ) 10.2, 14.0 Hz), 2.00 (2H, m), 1.79 (1H, m), 1.64 (1H, m), 1.54
(7) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591–596.
NP070209R