Trichormamides A and B with antiproliferative activity from the cultured freshwater cyanobacterium Trichormus sp. UIC 10339

Article abstract of DOI:10.1021/np5003548

Two new cyclic lipopeptides, trichormamides A (1) and B (2), were isolated from the cultured freshwater cyanobacterium Trichormus sp. UIC 10339. The strain was obtained from a sample collected in Raven Lake in Northern Wisconsin. The planar structures of trichormamides A (1) and B (2) were determined using a combination of spectroscopic analyses including HRESIMS and 1D and 2D NMR experiments. The absolute configurations of the amino acid residues were assigned by the advanced Marfey's method after acid hydrolysis. Trichormamide A (1) is a cyclic undecapeptide containing two d-amino acid residues (d-Tyr and d-Leu) and one β-amino acid residue (β-aminodecanoic acid). Trichormamide B (2) is a cyclic dodecapeptide characterized by the presence of four nonstandard α-amino acid residues (homoserine, N-methylisoleucine, and two 3-hydroxyleucines) and one β-amino acid residue (β-aminodecanoic acid). Trichormamide B (2) was cytotoxic against MDA-MB-435 and HT-29 cancer cell lines with IC₅₀ values of 0.8 and 1.5 μM, respectively.

Full text of DOI:10.1021/np5003548

Article
pubs.acs.org/jnp
Open Access on 08/04/2015
Trichormamides A and B with Antiproliferative Activity from the
Cultured Freshwater Cyanobacterium Trichormus sp. UIC 10339
Shangwen Luo,† Aleksej Krunic,† Hahk-Soo Kang,† Wei-Lun Chen,† John L. Woodard,‡ James R. Fuchs,‡
Steven M. Swanson,† and Jimmy Orjala*,†
†Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, 833 S. Wood
Street, Chicago, Illinois 60612, United States
‡Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, Ohio 43210,
United States
S
* Supporting Information
ABSTRACT: Two new cyclic lipopeptides, trichormamides A
(1) and B (2), were isolated from the cultured freshwater
cyanobacterium Trichormus sp. UIC 10339. The strain was
obtained from a sample collected in Raven Lake in Northern
Wisconsin. The planar structures of trichormamides A (1) and
B (2) were determined using a combination of spectroscopic
analyses including HRESIMS and 1D and 2D NMR
experiments. The absolute configurations of the amino acid
residues were assigned by the advanced Marfey’s method after
acid hydrolysis. Trichormamide A (1) is a cyclic undecapep-
tide containing two D-amino acid residues (D-Tyr and D-Leu)
and one β-amino acid residue (β-aminodecanoic acid). Trichormamide B (2) is a cyclic dodecapeptide characterized by the
presence of four nonstandard α-amino acid residues (homoserine, N-methylisoleucine, and two 3-hydroxyleucines) and one β-
amino acid residue (β-aminodecanoic acid). Trichormamide B (2) was cytotoxic against MDA-MB-435 and HT-29 cancer cell
lines with IC50 values of 0.8 and 1.5 μM, respectively.
any structurally diverse lipopeptides, characterized by
peptide backbone. The α position of the lipid unit is either
Mthe presence of a lipid tail connected to a linear or cyclic hydroxylated, methylated, or left as a methylene. The chain of
the lipid unit often shows modifications such as methylation,
hydroxylation, carbonylation, and chlorination.
oligopeptide, have been discovered from bacteria and fungi to
date.1 As a class, lipopeptides are known to possess a wide
range of biological activities including antimicrobial, antiproli-
ferative, immunosuppressant, and surfactant properties.2−4
Studies into the modes of action of lipopeptides such as
daptomycin, syringomycin, and syringopeptin revealed that the
lipid tail plays a key role in their interactions with plasma
membranes, which leads to ion pump activation, causing
membrane depolarization and eventually gives rise to cell
death.5−7 The cyclic forms of lipopeptides are thought to
possess restricted conformational freedom and improved
stability compared to their linearized forms. These character-
istics are important in the interactions with relevant biological
targets.1,8 There has been a considerable interest in the
discovery, biosynthesis, and mechanisms of action studies of
cyclic lipopeptides for their potential pharmaceutical applica-
tions.9
Cyanobacteria are prolific producers of cyclic lipopeptides,
and many structurally diverse cyclic lipopeptides have been
reported. Their activities include antimicrobial, antiproliferative,
antitumor, 20S proteasome inhibitory, and allelopathic
activities.10−18 A common feature of the lipid portion of
cyanobacterial cyclic lipopeptides is the presence of a β-amino
or a β-hydroxy group, which is incorporated into the cyclic
In our continuing search for antiproliferative natural products
from cultured freshwater cyanobacteria, we evaluated the
antiproliferative extract from Trichormus sp. UIC 10339.
Morphological characterization and phylogenetic analysis
using a partial 16S rDNA gene sequence identified this strain
to be a Trichormus sp. (Supporting Information S1−S3).
Herein we report the isolation, structure elucidation, and
biological activity of two new cyclic lipopeptides, named
trichormamides A (1) and B (2), each containing a β-
aminodecanoic acid unit as part of their cyclic backbones.
RESULTS AND DISCUSSION
■
Trichormus sp. UIC 10339 was obtained from a sample
collected from Raven Lake in Northern Wisconsin in August
2010 and cultured in Z media. It was harvested after 8 weeks of
growth.19 The lyophilized cells were extracted with CH2Cl2 and
MeOH (1:1) and dried in vacuo. This extract showed
antiproliferative activity against the human colon cancer cell
line HT-29 and was subjected to a bioassay-guided
Received: April 23, 2014
Published: August 4, 2014
© 2014 American Chemical Society and
American Society of Pharmacognosy
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peptides with molecular weights of 1184 and 1446 Da.
Subsequent reversed-phase HPLC separations yielded trichor-
mamides A (1) and B (2).
Trichormamide A (1) was obtained as a white, amorphous
powder. The molecular formula of 1 was deduced as
C58H93N11O15 based on HRESIMS analysis (m/z 1184.6978
[M + H]+). The 1H NMR spectrum of 1 in DMSO-d6 showed
1
broad signals. The H NMR spectra of 1 in CD3OH with
suppression at 4.9 ppm exhibited sharper peaks, but the
resonances close to 4.9 ppm were also suppressed. Therefore,
1H NMR, COSY, and TOCSY experiments were conducted in
both CD3OD (without signal suppression, but NH signals
absent) and CD3OH (suppressed at 4.9 ppm, but NH signals
present), respectively, to obtain both NH signals and clear
signals close to 4.9 ppm (Supporting Information S4−S12).
The values of chemical shifts were reported using 1D and 2D
1
NMR spectra obtained in CD3OH. The H NMR spectrum of
1 exhibited a signal pattern characteristic of a lipopeptide,
including exchangeable amide signals (δH 6.7−8.9), signals
typical of α-protons (δH 4.1−5.0), aliphatic methylene signals
(δH 1.1−1.4), and several doublet and triplet methyl signals (δH
0.7−1.0). The two downfield doublets (δH 7.25, J = 8.7 Hz; δH
6.71, J = 8.7) were attributed to a para-substituted phenyl
moiety. Analysis of COSY and TOCSY correlations of 1
revealed the presence of 11 amino acid residues, including 10
standard α-amino acid residues: three serines (Ser1, Ser2,
Ser3), two prolines (Pro1, Pro2), two isoleucines (Ile1, Ile2),
tyrosine (Tyr), leucine (Leu), and glycine (Gly). Sequential
COSY correlations between NH (δH 7.44), methine H-3 (δH
4.36), and methylene H2-2 (δH 1.59; 2.06), as well as COSY
correlations between methine H-3 (δH 4.36) and methylene
H2-4 (δH 1.50; 1.58), suggested the presence of a β-amino acid
residue. Sequential COSY correlations from H2-4 (δH 1.50;
1.58) to the region of highly overlapped methylene signals (H2-
5−9, δH 1.28−1.33), which corresponded to five carbons in the
HSQC spectrum (δC 32.9, 30.3, 30.2, 27.2, and 23.6), and
finally to the methyl triplet H-10 (δH 0.86) identified this β-
amino acid residue as β-aminodecanoic acid (Ada).
fractionation scheme using Diaion vacuum liquid chromatog-
raphy. The fraction eluting with 60% isopropyl alcohol (IPA)
displayed antiproliferative activity. LC/MS and 1H NMR
dereplication indicated the presence of two potentially new
Assignment of the amino acid sequence was carried out by a
combination of HMBC, ROESY, and ESIMS/MS analysis
(Figure 1). HMBC correlations from Ile2-NH (δH 8.84) to Ile1
C-1 (δC 174.1), from Ile1-NH (δH 6.95) to Leu C-1 (δC 173.6),
Figure 1. Key 2D NMR correlations of trichormamide A (1) and trichormamide B (2).
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Figure 2. ESIMS/MS fragmentation of trichormamide A (1).
from Leu-NH (δH 7.71) to Tyr C-1 (δC 175.1), from Tyr-NH
(δH 7.30) to Ser3 C-1 (δC 173.0), and from Ser3-NH (δH 7.57)
to Pro1 C-1 (δC 172.2) suggested a partial sequence of Ile2-
Ile1-Leu-Tyr-Ser3-Pro1. HMBC correlation from Pro1 H2-5
(δH 3.60; 3.88) to Ser2 C-1 (δC 174.3) extended this sequence
to Ile2-Ile1-Leu-Tyr-Ser3-Pro1-Ser2. Similarly, the HMBC
correlations from Ser1-NH (δH 6.79) to Ada C-1 (δC 172.2),
from Ada-NH (δH 7.44) to Gly C-1 (δC 170.4), and from Gly-
NH (δH 8.75) to Pro2 C-1 (δC 176.1) established the partial
sequence of Ser1-Ada-Gly-Pro2. This partial sequence was
confirmed by ROESY correlations between Ser1-NH (δH 6.79)
and Ada H2-2 (δH 1.59; 2.06) and between Ada-NH (δH 7.44)
and Gly H2-2 (δH 3.42; 3.97). A ROESY correlation between
Ser2-NH (δH 7.88) and Ser1 H2-3 (δH 4.00; 4.27) linked the
two partial sequences together into Ile2-Ile1-Leu-Tyr-Ser3-
Pro1-Ser2-Ser1-Ada-Gly-Pro2. The 18 degrees of unsaturation
and the molecular formula suggested that 1 was a cyclic
peptide. The chemical shifts of Ile2 C-1 (δC 173.8), C-2 (δC
52.1) and Pro2 C-2 (δC 64.5), C-5 (δC 49.5), H2-5 (δH 4.07;
4.13) suggested the presence of an amide moiety at Ile2 C-1
and Pro2 N.16,17 Therefore, the cyclic undecapeptide ring was
closed between Ile2 and Pro2. Mass fragmentation analysis
using a quadrupole-time-of-flight (qTOF) tandem mass
spectrometer confirmed the sequence of amino acid residues
determined by NMR analysis. The parent ion [M + H]+ 1184.6
was fragmented using a 40 eV collision energy. As expected,
protonation and breakage of the proline amide bond is a highly
favored process, and the macro ring of 1 opened between Ile2
and Pro2, forming a linear acylium ion.20 The acylium ion was
continuously fragmented to generate fragment ions as
illustrated in Figure 2. The ESIMS/MS fragments were in
complete agreement with the structure determined by NMR
studies, and the planar structure of 1 was determined as
cyclo[Ile2-Ile1-Leu-Tyr-Ser3-Pro1-Ser2-Ser1-Ada-Gly-Pro2].
The absolute configurations of the amino acids were
determined using acid hydrolysis followed by the advanced
Marfey’s method (Supporting Information S14).21−23 The
chromatographic comparison between Marfey’s derivatives of
the hydrolysate of 1 and appropriate amino acid standards
assigned the L configurations for Ser1/2/3 and Pro1/2 and the
D configuration for Tyr. The absolute configuration of Ada was
determined as 3R by comparison of the elution order of
hydrolysate Ada L- and D-FDLA derivatives with those reported
in the literature.24 The stereoisomers of leucine and isoleucine
standards were analyzed by Marfey’s method, and retention
times were compared with Marfey’s derivatives of hydrolysate
of 1. As leucine and isoleucine isomers have identical molecular
weight and very similar retention behavior in HPLC, a rigorous
co-injection scheme was utilized to assign Leu as D and Ile1/2
as L (2S, 3S) (Supporting Information S14). The final structure
of 1 was determined as cyclo[L-Ile−L-Ile−D-Leu−D-Tyr−L-
Ser−L-Pro−L-Ser−L-Ser−(3R)-Ada−Gly−L-Pro].
Trichormamide B (2) was obtained as a white, amorphous
powder. The molecular formula was determined as
C69H115N13O210 (m/z 1446.8508 [M + H]+) by HRESIMS
analysis. The H NMR spectrum of 2 in DMSO-d6 (Table 2)
also exhibited signals characteristic of a lipopeptide. Signal
1
doubling was observed for some of the signals in both the H
NMR and DEPTQ spectra, indicating the presence of two
conformers of 2 in solution (integration ratio 2:1 in DMSO-d6,
according to integration of the phenyl protons from tyrosine).
This phenomenon has been reported for cyclic peptides with
one or more N-methylated amino acid residues.24−27 An
elevated-temperature NMR experiment was attempted to
generate one average set of signals. However, the coalescence
temperature of 2 was higher than the highest operating
temperature permitted by a cryogenically cooled probe (T =
328 K), and no signal coalescence was observed. Nevertheless,
the elevated-temperature NMR experiment showed improved
peak shapes and better resolved coupling patterns as compared
to the room-temperature experiments. Thus, the structure
elucidation of 2 was performed using the signals from the major
conformer at elevated temperature (T = 328 K). Combined
analysis of the COSY and TOCSY spectra identified the
structures of seven standard amino acids: isoleucine (Ile),
glutamine (Glu), serine (Ser), proline (Pro), tyrosine (Tyr),
and two threonines (Thr1 and Thr2) (Figure 1 and Table 2).
An N-methylated isoleucine (NMeIle) was deduced by TOCSY
and the HMBC correlation observed from N-Me (δH 3.00) to
NMeIle C-2 (δC 59.8). The presence of homoserine (Hse) was
evident by sequential COSY correlations from NH (δH 8.01) to
Hse H2-4 (δH 3.42; 3.50). The downfield chemical shifts of the
Hse C-4 methine (δC 57.3) and H2-4 (δH 3.42; 3.50) indicated
the attachment of a hydroxy group to C-4. A broad hydroxy
signal (δH 5.05) with a COSY correlation to a downfield
methine (δH 3.52), as well as sequential COSY correlations
between NH (δH 8.16)/H-2 (δH 4.39)/H-3 (δH 3.52)/H-4 (δH
1.61)/H3-5 (δH 0.93), combined with HMBC correlations from
4-Me (δH 0.81) and H3-5 (δH 0.93) to both C-3 (δC 76.3) and
C-4 (δC 30.5), indicated the presence of a 3-hydroxyleucine (3-
OHLeu1). An additional 3-hydroxyleucine (3-OHLeu2) was
identified using a similar approach (Table 2). The presence of a
β-aminodecanoic acid was suggested from COSY, TOCSY, and
HSQC correlations as described for trichormamide A (1).
The sequence of the 12 amino acid residues was established
by analysis of the HMBC and ROESY spectra (Figure 1 and
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Table 1. NMR Spectroscopic Data of Trichormamide A (1) in CD3OH, T = 300 K
a
b
position
δC, mult
δH, mult. (J in Hz)
COSY
HMBC
ROESY
NHSer
Ada
1
2
172.2, C
42.8, CH2
1.59, m
3
1
2.06, m
3
4
47.7, CH
35.9, CH2
4.36, m
2, 4, NH
3
4, 5
1, 3
1.50, m
1.58, m
c
5
32.9, CH2
1.28, m
overlapped
c
6
30.3, CH2
1.29, m
overlapped
c
7
30.2, CH2
1.32, m
overlapped
c
8
27.2, CH2
1.33, m
overlapped
9
23.6, CH2
12.1, CH3
1.31, m
10
9
10
NH
1
0.86, t (7.4)
7.44, d (9.9)
9
3
3, 1Gly
2Gly
Ser1
175.5, C
53.5, CH
63.1, CH2
2
4.94, dd (6.2, 3.0)
4.00, t (9.7)
4.27, m
NH, 3
2
3
1, 2
NH, NHSer2
d
OH
NH
1
nd
6.79, d (7.6)
2
2, 3, 1Ada
3, 2Ada
Ser2
Pro1
174.3, C
59.4, CH
61.9, CH2
2
4.34, m
3.91, m
NH, 3
2
1
3
1, 2
d
OH
NH
1
nd
7.88, br
2
3Ser
172.2, C
62.4, CH
30.1, CH2
2
4.13, m
1.95, m
2.14, m
2.21, m
2.28, m
3.60, m
3.88, m
3
3
4
NHSer3
3
2, 4
4
5
26.2, CH2
48.6, CH2
3, 5
4
3
3, 1Ser2
Ser3
Tyr
1
2
3
173.0, C
55.5, CH
62.4, CH2
4.51, m
3.78, m
4.13, m
NH, 3
2
1
NHTyr
NH
1, 2
d
OH
NH
1
nd
7.57, d (9.8)
2
2, 1Pro
3, 2Pro
175.1, C
59.9, CH
37.7, CH2
2
4.25, m
NH, 3
2
1, 3
1, 2
NHLeu
3
3.06, dd (13.9, 3.2)
2.98, dd (13.9, 10.8)
NH, NHLeu
4
129.2, C
5/9
6/8
7
131.2, CH
116.4, CH
157.4, C
7.25, d (8.7)
6.71, d (8.7)
6/8
5/9
3, 4, 6/8, 7
4, 5/9, 7
d
OH
NH
1
nd
7.30, d (5.7)
2
2, 1Ser3
3, 2Ser3
NHIle
Leu
173.6, C
52.9, CH
40.1, CH2
2
4.53, m
NH, 3
2, 4
1, 3, 4
3
1.12, m
1, 2, 4, 4-Me, 5
1.45, m
4
26.0, CH
23.6, CH3
20.6, CH3
1.73, m
3, 4-Me, 5
4-Me, 5
3, 4, 5
4-Me
1.00, d (6.5)
0.84, d (6.3)
7.71, d (8.7)
4
4
2
5
3, 4, 4-Me
2, 3, 1Tyr
NH
2Tyr, 3Tyr
NHIle2
Ile1
1
174.1, C
2
58.1, CH
41.6, CH
16.2, CH3
24.1, CH2
4.70, d (3.7)
1.92, m
NH, 3
2, 3-Me, 4
3
1
3
3-Me
4
0.91, d (6.3)
1.19, m
2, 3, 4
5
3, 5
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Table 1. continued
position
Article
a
b
δC, mult
δH, mult. (J in Hz)
COSY
HMBC
ROESY
1.28, m
5
14.3, CH3
0.89, t (10.2)
6.95, d (9.8)
4
2
3, 4
1Leu
NH
2Leu
Ile2
Pro2
Gly
1
173.8, C
2
52.1, CH
26.0, CH
20.5, CH3
40.5, CH2
4.65, dd (12.2, 2.4)
1.44, m
NH, 3
2, 3-Me, 4
3
1, 3-Me, 4
5
3
3-Me
4
0.96, d (6.5)
1.43, m
3, 5
1.73, m
5
23.6, CH3
0.85, t m
4
2
3, 4
NH
1
8.84, d (7.0)
2, 1Ile
2Ile
176.1, C
64.5, CH
30.6, CH2
2
4.31, m
2.11, m
2.46, m
2.00, m
2.06, m
4.07, m
4.13, m
3
1
3
2, 4
1, 4
4
5
26.2, CH2
49.5, CH2
3, 5
4
3
3
1
2
170.4, C
43.9, CH2
3.42, d (17.3)
3.97, d (17.3)
8.75, br
NH
1, 1Pro2
NHAda
NH
2
a
b
c
Carbon chemical shifts were assigned from the DEPTQ spectrum recorded at 226 MHz. Recorded at 600 MHz. Carbon chemical shifts are
d
interchangeable. nd: not detected.
Table 2). HMBC correlations from 3-OHLeu1-NH (δH 8.01)
to Ile C-1 (δC 170.8), from Ile-NH (δH 8.16) to Ada C-1 (δC
171.2), from Ada-NH (δH 7.52) and Ada H-3 (δH 4.08) to
Thr2 C-1 (δC 169.6), from Thr2-NH (δH 7.60) to Tyr C-1 (δC
170.8), and from Tyr-NH (δH 7.97) to Pro C-1 (δC 171.5)
indicated a partial sequence of 3-OHLeu1-Ile-Ada-Thr2-Tyr-
Pro. ROESY correlations between 3-OHLeu1-NH (δH 8.01)
and Ile H-2 (δH 4.19), between Ile-NH (δH 8.16) and Ada H2-2
(δH 2.31; 2.49), between Ada-NH (δH 7.52) and Thr2 H-2 (δH
4.05), between Thr2-NH (δH 7.60) and Tyr H-2 (δH 4.45), and
between Tyr-NH (δH 7.97) and Pro H-2 (δH 4.33) confirmed
this partial sequence. Similarly, a partial sequence of Ser-
NMeIle-Gln-3-OHLeu2-Hse was established by the HMBC
correlations from Ser-NH (δH 7.83) to NMeIle C-1 (δC 169.8),
from NMeIle N-Me (δH 3.00) to Gln C-1 (δC 172.3), from
Gln-NH (δH 7.93) to 3-OHLeu2 C-1 (δC 171.3), and from 3-
OHLeu2-NH (δH 7.76) to Hse C-1 (δC 171.3). This partial
sequence was also confirmed by the ROESY correlations
between Ser-NH (δH 7.83) and NMeIle H-2 (δH 4.73),
between Gln-NH (δH 7.93) and 3-OHLeu2 H-2 (δH 4.32), and
between 3-OHLeu2-NH (δH 7.76) to Hse H-2 (δH 4.35). The
ROESY correlations between Thr1-NH (δH 7.51) and Ser H-2
(δH 4.33) and between Thr1-NH (δH 7.51) and Ser H2-3 (δH
3.60) further expanded this partial sequence to Thr1-Ser-
NMeIle-Gln-3-OHLeu2-Hse. An HMBC correlation from Hse-
NH (δH 7.95) to 3-OHLeu1 C-1 (δC 172.3), together with a
ROESY correlation between Hse-NH (δH 7.95) and 3-OHLeu1
H-2 (δH 4.39), connected those two partial sequences together
into Thr1-Ser-NMeIle-Gln-3-OHLeu2-Hse-3-OHLeu1-Ile-
Ada-Thr2-Tyr-Pro. The molecular formula and degree of
unsaturation indicated the cyclic nature of 2. The chemical
shifts of Thr1 C-1 (δC 168.7) and C-2 (δC 55.6) suggested the
presence of an amide moiety at Thr1 C-1.16 Similarly, the
chemical shifts of Pro C-2 (δC 59.5), C-5 (δC 47.0), and H2-5
(δH 3.61; 3.67) also indicated that Pro N was part of an amide
bond.17 Therefore, the cyclic dodecapeptide ring was closed
between Thr1 and Pro. Mass fragmentation analysis confirmed
the sequence assignment of 2 (Figure 3). Protonation on the
amide nitrogen at Pro generated amide bond cleavage to form a
linear acylium ion, which produced series of fragments (Figure
3). These observed fragments were consistent with the amino
acid sequence determined by NMR analysis, and the planar
structure of 2 was assigned as cyclo[Thr1-Ser-NMeIle-Gln-3-
OHLeu2-Hse-3-OHLeu1-Ile-Ada-Thr2-Tyr-Pro].
Acid hydrolysis of trichormamide B (2) followed by Marfey’s
derivatization with L- and D-FDLA and LC/MS analysis
assigned the L configurations for Gln, Pro, Ile (2S, 3S),
Thr1/2 (2S, 3R), and the nonproteinogenic amino acid Hse, D
configurations for Tyr and Ser, and 3R configuration for Ada.
The four stereoisomers of NMeIle were synthesized according
to the literature.28,29 On the basis of these standards, the
absolute configuration of NMeIle was determined to be L (2S,
3S). The 2S, 3R and 2S, 3S isomers of 3-hydroxyleucine were
synthesized following Bonnard’s procedure16 and were
derivatized with L- and D-FDLA respectively. Because
enantiomers exhibit identical retention behavior under non-
chiral HPLC conditions, the (2S,3R)-D-FDLA derivative shows
the same retention time as (2R,3S)-L-FDLA, and the (2S,3S)-D-
FDLA derivative shows the same retention time as (2R,3R)-L-
FDLA (detailed explanations can be found in Supporting
Information S26). By comparing the above four retention times
with retention times of 3-OHLeu1 and 3-OHLeu2 L-FDLA
derivatives, both 3-OHLeu1 and 3-OHLeu2 were assigned as
2R, 3S. The complete structure of 2 was determined as cyclo[L-
Thr−D-Ser−L-NMeIle−L-Gln−(2R,3S)-3-OHLeu−L-Hse−
(2R,3S)-3-OHLeu−L-Ile−(3R)-Ada−L-Thr−D-Tyr−L-Pro].
Trichormamide A (1) is a cyclic undecapeptide characterized
by the presence of β-aminodecanoic acid and two D-amino acid
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Table 2. NMR Spectroscopic Data of Trichormamide B (2) in DMSO-d6, T = 328 K
a
b
position
δC, mult
δH, mult. (J in Hz)
COSY
HMBC
ROESY
3, NHIle
Ada
1
2
171.2, C
40.2, CH2
2.31, m
3
1
1
2.49, m
3
4
46.0, CH
33.4, CH2
4.08, m
2, 4, NH
3
2, 4
3
1.36, m
1.44, m
c
5
28.8, CH2
1.22, m
overlapped
c
6
28.4, CH2
1.22, m
overlapped
c
7
25.1, CH2
1.21, m
overlapped
8
31.0, CH2
21.8, CH2
13.6, CH3
1.22, m
overlapped
9
1.24, m
10
9
10
NH
1
0.85, t (7.2)
7.52, d (7.2)
8, 9
3
1Thr2
2Thr2
Ile
170.8, C
2
57.6, CH
35.7, CH
15.1, CH3
24.4, CH2
4.19, m
NH, 3
2, 3-Me, 4
3
1, 3, 4
2, 3, 4
3
2
3
1.80, m
3-Me
4
0.92, d (7.0)
1.16, m
3, 5
1.47, m
5
10.2, CH3
0.91, m
4
2
3, 4
1Ada
NH
8.16, d (7.4)
2Ada
3-OHLeu1
1
172.3, C
55.3, CH
76.3, CH
30.5, CH
18.9, CH3
18.5, CH3
2
4.39, m
NH, 3
1, 4
3
3
3.52, m
2, 3-OH, 4
2, 4-Me, 5
4
1.61, m
3, 4-Me, 5
4-Me
0.81, d (6.5)
0.93, d (6.2)
5.05, s
4
4
3
2
3, 4, 5
3
3
5
3, 4, 4-Me
3-OH
NH
1
8.01, d (7.4)
1Ile
1
2Ile
Hse
171.3, C
50.9, CH
34.3, CH2
2
4.35, m
1.83, m
1.89, m
3.42, m
3.50, m
NH, 3
2, 4
3
2
3
4
57.3, CH2
3
2
d
OH
nd
NH
7.95, d (9.6)
1OHLeu
1
2OHLeu
3-OHLeu2
1
171.3, C
55.6, CH
75.9, CH
29.7, CH
18.8, CH3
18.5, CH3
2
4.32, m
NH, 3
3
3
3.47, m
2, 3-OH, 4
2, 4-Me, 5
4
1.58, m
3, 4-Me, 5
4-Me, 5
3, 4, 5
4-Me
0.90, d (6.4)
0.78, d (6.8)
4.93, s
4
4
3
2
3
3
5
3, 4, 4-Me
3-OH
NH
1
7.76, d (9.1)
1Hse
Gln
172.3, C
49.3, CH
26.1, CH2
2
4.59, m
1.75, m
1.92, m
2.12, m
2.15, m
NH, 3
2, 4
3, 4, N-MeNMeIle
2
3
4
30.6, CH2
174.4, C
3
2
5
2
5
NH
NH2
7.93, d (9.1)
6.76, s
1OHLeu2
2OHLeu2
7.17, s
NMeIle
1
169.8, C
2
59.8, CH
31.5, CH
15.1, CH3
23.8, CH2
10.6, CH3
4.73, d (11.0)
1.95, m
3
1, 3
N-Me
N-Me
3
2, 3-Me, 4
3-Me
0.83, m
3
2, 3, 4
3, 4
4
5
1.31, m
3, 5
4
0.84, m
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Table 2. continued
a
b
position
N-Me
δC, mult
δH, mult. (J in Hz)
COSY
HMBC
2, 1Gln
ROESY
2, 3, 2Gln
30.0, CH3
170.6, C
3.00, s
Ser
1
2
54.9, CH
60.8, CH2
4.33, m
3.60, m
NH, 3
1
1
3
2
d
OH
nd
NH
7.83, d (6.6)
2
1NMeIle
2, 3
1
2NMeIle
Thr1
1
168.7, C
55.6, CH
66.2, CH
18.8, CH3
2
4.42, dd (8.5, 3.4)
3.93, m
NH, 3
2, 4
3
3
4
1.08, d (5.9)
d
3-OH
nd
NH
1
7.51, d (7.2)
2
2Ser, 3Ser
Pro
Tyr
171.5, C
59.5, CH
28.6, CH2
2
4.33, m
1.78, m
1.93, m
1.78, m
3.61, m
3.67, m
3
3
2, 4
4
5
24.0, CH2
47.0, CH2
3, 5
4
1
2
3
170.8, C
54.5, CH
36.6, CH2
4.45, m
2.72, m
2.92, m
NH, 3
2
3
2
1, 2, 5/9
4
127.4, C
5/9
6/8
7
129.8, CH
114.7, CH
155.6, C
6.99, d (8.3)
6.63, d (8.3)
6/8
5/9
3, 5/9, 7
4, 6/8, 7
d
OH
NH
1
nd
7.97, d (8.6)
2
1Pro
1, 3
2, 3
1Tyr
2Pro
Thr2
169.6, C
58.0, CH
66.0, CH
19.2, CH3
2
4.05, m
NH, 3
2, 4
3
3
3.97, m
4
0.87, d (6.6)
d
3-OH
NH
nd
7.60, d (8.1)
2
2Tyr
a
b
c
Carbon chemical shifts were assigned from the DEPTQ spectrum recorded at 226 MHz. Recorded at 600 MHz. Carbon chemical shifts are
d
interchangeable. nd: not detected.
Figure 3. ESIMS/MS fragmentation of trichormamide B (2).
residues (D-Tyr and D-Leu). Trichormamide A (1) is a lipid-
containing compound and displays amphiphilic character with
half of the amino acid sequence being hydrophobic (Ada-Gly-
Pro2-Ile2-Ile1-Leu) and the other half of the sequence
hydrophilic (Tyr-Ser3-Pro1-Ser2-Ser1). The amphiphilic nature
of lipopeptides has been found to be strongly correlated with
their plasma membrane interactive potential.5−7 Trichorma-
mide A (1) is structurally related to hormothamnin A,
lobocyclamide A, and laxaphycin A with conserved amino
acid residues of Leu, Ile1, Ile2, and Gly.14−16 The amino acid
residues homoserine, 4-hydroxyproline, α,β-didehydro-α-ami-
nobutyric acid, and β-aminooctanoic acid in the east portion of
the three related compounds were replaced by structurally
related residues of Ser, Pro, Ser, and Ada in trichormamide A
(1) (Supporting Information S27). Trichormamide B (2) is a
cyclic dodecapeptide characterized by the presence of a β-
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with an 18/6 h light/dark cycle. The temperature of the culture room
was maintained at 22 °C. The biomass of UIC 10339 was harvested
after 8 weeks of growth by centrifugation and freeze-dried.
amino acid residue (β-aminodecanoic acid), two D-amino acid
residues (D-Tyr and D-Ser), and four nonstandard amino acid
residues (3-OHLeu1, Hse, 3-OHLeu2, NMeIle). Trichorma-
mide B (2) is also an amphiphilic molecule containing a
hydrophobic Ada residue and seven hydroxy groups.
Trichormamide B (2) is structurally related to the cyclic
dodecapeptides lobocyclamides B and C, laxaphycins B, B2, and
B3, and lyngbyacyclamides A and B.15−17 A comparison of 2 to
these structures revealed that the presence and location of 3-
OHLeu1, Gln, NMeIle, Thr1, and Thr2 are conserved in all
these peptides, indicating these amino acid residues may be
crucial for the biological functions of these molecules.
Trichormamides A (1) and B (2) were evaluated for their
antiproliferative activities against the human melanoma cell line
MDA-MB-435 and the human colon cancer cell line HT-29.
Trichormamide B (2) displayed significant cytotoxic activity
against MDA-MB-435 and HT-29 cell lines with IC50 values of
0.8 and 1.5 μM, respectively. Trichormamide A (1) was less
active than 2 and showed IC50 values of 9.9 and 16.9 μM
against MDA-MB-435 and HT-29, respectively. Compounds
structurally related to 1 and 2 were reported to have 2−3-fold
increased synergistic activities in antimicrobial and cytotoxicity
assays.15,16,26,30 However, the limited supply of 1 stopped us
from obtaining statistically significant data in synergistic assays.
In summary, we have obtained two new cyclic lipopeptides,
trichormamides A (1) and B (2), each containing the β-amino
acid residue β-aminodecanoic acid, from the cultured fresh-
water cyanobacterium Trichormus sp. UIC 10339. Their planar
structures were determined by analysis of the HRESIMS, MS/
MS, and 1D and 2D NMR experiments. The stereoconfigura-
tions of the amino acid residues were assigned by the advanced
Marfey’s method using synthesized amino acid standards.
Compounds 1 and 2 are not analogues, but both exhibited
amphiphilic properties. Trichormamide B (2) was found to be
more active against MDA-MB-435 and HT-29 human cancer
cell lines than trichormamide A (1), with IC50 values of 0.8 and
1.5 μM, respectively.
Morphological Identification. Initial taxonomic identification
was performed using a cultured UIC 10339 cyanobacterium.
Morphological observation was performed using a Zeiss Axiostar
Plus light microscope equipped with a Canon PowerShot A620
camera. The following characters were utilized for identification:
structure of colony, morphology of thallus, size, shape, and motility of
trichomes, shape of vegetative cells, presence or absence of mucilage or
sheath, presence and arrangement of heterocytes, presence and relative
size of akinetes, and morphology of the end cell (Supporting
Information S1, S2). The morphological characterization and initial
taxonomical designation of UIC 10339 were made according to the
system by Komar
́
ek et al.32
DNA Extraction, 16S rDNA PCR Amplification, and Sequenc-
ing. Prior to DNA extraction, 8 mL of a static culture of Trichormus
sp. UIC 10339 was combined with 1.5 mL of lysozyme buffer and 0.5
mL of 20 mg/mL lysozyme stock solution and incubated at 35 °C for
1 h. After incubation, the cell mass was centrifuged and transferred to a
2 mL microcentrifuge tube. A Wizard Genomic DNA purification kit
(Promega) was used to extract the genomic DNA. A partial sequence
of the 16S rDNA gene was PCR-amplified using the cyanobacteria-
specific primers 106F and 1509R.33 For a total volume of 25 μL, the
reaction mixture contained DNA (1 μL, approximately 300 ng),
GoTaq reaction buffer (5 μL, 5×), dNTP mix (0.5 μL, 10 mM),
upstream and downstream primer (1 μL each, 10 μM), GoTaq DNA
polymerase (0.25 μL, 2.5 u/μL), and H2O (16.25 μL). The reaction
was performed in a Bio-Rad C1000 thermal cycler with the following
reaction program: initial denaturation for 30 s at 98 °C, 35
amplification cycles of 10 s at 98 °C, 30 s at 53 °C, 30 s at 72 °C,
and a final extension for 10 min at 72 °C. PCR products were purified
using a MinElute PCR purification kit (Qiagen) and sequenced using
the cyanobacteria-specific primers 106F, 359F, and 1509R.33 The
resulting 16S rDNA gene sequence was deposited in the NCBI
GenBank under the accession no. KF444210.
Extraction and Isolation. Lyophilized biomass (6.14 g) from
eight 2 L cultures of UIC 10339 was extracted with 1:1 CH2Cl2/
MeOH three times and concentrated in vacuo to yield 650.9 mg of
extract, which was fractionated using Diaion HP-20SS resin with an
H2O/IPA step gradient (1:0, 4:1, 3:2, 2:3, 3:7, 1:4, 1:9, 0:1 v/v) to
yield eight fractions. The fraction eluting with H2O/IPA (2:3 v/v)
(58.8 mg) displayed significant antiproliferative activity against the
human melanoma cell line MDA-MB-435 with 100% growth
inhibition at a concentration of 25 μg/mL. LC-MS combined with
1H NMR dereplication of this fraction revealed the presence of new
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured on a PerkinElmer 241 polarimeter at 22 °C in MeOH.
UV spectra were recorded on a Shimadzu UV spectrophotometer
UV2401. IR spectra were acquired on a PerkinElmer 577 IR
spectrophotometer. 1D and 2D NMR spectra including 1H NMR,
COSY, TOCSY, HSQC, HMBC, and ROESY were obtained on a
Bruker Avance DRX 600 MHz spectrometer. A Bruker AVII 900 MHz
lipopeptides. The fraction was subjected to semipreparative reversed-
phase HPLC (Varian C8 semipreparative column, 10 × 250 mm, 3
mL/min) with a linear gradient from 80% to 100% MeOH with H2O
over 20 min, and the subfraction eluting from 9 to 10 min was
1
collected and dried in vacuo. H NMR and MS flow injection analysis
1
NMR spectrometer was used to acquire the DEPTQ spectra. H and
revealed that this subfraction consisted of two compounds. The
subfraction was subjected to semipreparative reversed-phase HPLC
(Varian C18 semipreparative column, 10 × 250 mm, 3 mL/min) using
isocratic 50% aqueous CH3CN. Trichormamide A (1, 0.8 mg, 0.01% of
dry weight) was eluted at 8 min, and trichormamide B (2, 3.7 mg,
0.06% of dry weight) was eluted at 5 min.
13C NMR chemical shifts were referenced to the DMSO-d6 (δH 2.50
and δC 39.51, respectively) and CD3OD/CD3OH (δH 3.31 and δC
49.0, respectively) residual solvent signals. The TOCSY experiments
were conducted using a 60 ms mixing time. The ROESY spectra were
acquired with a 200 ms mixing time. The HMBC spectrum was
3
Trichormamide A (1): white, amorphous powder; [α]2D2 +20 (c 0.06,
MeOH); UV (MeOH) λmax (log ε) 240 (3.07), 278 (2.95) nm; IR
(neat) νmax 3312 (br), 2984, 2942, 2831, 1449 cm−1; 1H and 13C NMR
see Table 1; HR-ESI-TOF-MS (+) m/z 1184.6978 [M + H]+ (calcd
for C58H94N11O15, 1184.6931).
recorded with an average JCH of 8 Hz, and the HSQC spectrum was
1
recorded with an average JCH of 145 Hz. High-resolution ESI mass
spectra and LC-MS data were obtained using a Shimadzu HPLC-IT-
TOF spectrometer. The HPLC experiments were performed using an
Agilent 1100 series instrument equipped with a diode array detector,
and a Waters instrument equipped with a Waters 600 pump and a
Waters 2487 detector.
Biological Material. Trichormus sp. UIC 10339 was isolated from
a sample collected at Raven Lake, Wisconsin, in 2010 (N 45°58.7′, W
89°51.5′°). The unialgal (not axenic) strain UIC 10339 was obtained
through micropipette isolation techniques.31 The strain was cultured in
8 × 2 L of Z medium in 2.8 L Fernbach flasks with sterile air
aeration.19 Cultures were illuminated with fluorescent lamps at 1.03 klx
Trichormamide B (2): white, amorphous powder; [α]2D2 −25.9 (c
0.32, MeOH); UV (MeOH) λmax (log ε) 240 (3.16), 278 (2.97) nm;
IR (neat) νmax 3317 (br), 2943, 2831, 1449, 1414, 1114 cm−1; 1H and
13C NMR see Table 2; HR-ESI-TOF-MS (+) m/z 1446.8508 [M +
H]+ (calcd for C69H116N13O20, 1446.8460).
Synthesis of L-N-Methylisoleucine, D-N-Methylisoleucine, L-
allo-N-Methylisoleucine, and D-allo-N-Methylisoleucine (Based
on Shendage’s and Malkov’s Methods28,29). The amino group of
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L-Ile was Boc-protected using a previously published protocol.28 The
methylation of NBoc-L-Ile was carried out using Malkov’s method.29
The Boc-protected NMeIle was subjected to acid hydrolysis using the
general peptide hydrolysis scheme as described below, to yield L-N-
methylisoleucine (73.11 mg), which was analyzed by the advanced
Marfey’s method. Although citric acid from the workup was observed
as a minor impurity in the 1H NMR spectrum of L-N-methylisoleucine,
it did not interfere with the advanced Marfey’s analysis. D-N-
Methylisoleucine, L-allo-N-methylisoleucine, and D-allo-N-methyliso-
leucine were synthesized using the same protocol with D-Ile, L-allo-Ile,
and D-allo-Ile as starting compounds. All were analyzed using the
advanced Marfey’s method as described for the peptide hydrolysate. L-
N-Methylisoleucine: [α]2D2 +17.6 (c 0.39, MeOH); 1H NMR data
(DMSO-d6, 400 MHz) δ 3.84 (1H, d, J = 3.0 Hz), 2.59 (3H, s), 1.95
(1H, m), 1.50 (1H, m), 1.32 (1H, m), 0.91 (3H, d, J = 8.5 Hz), 0.90
(3H, t, J = 7.1 Hz); DEPTQ data (DMSO-d6, 400 MHz) δ 169.5, 64.6,
35.3, 32.5, 26.4, 14.5, 12.0. NMR spectra of D-Ile, L-allo-Ile, and D-allo-
Ile can be found in Supporting Information S24.
ASSOCIATED CONTENT
* Supporting Information
Supplementary data (morphological and phylogenetic analysis
■
S
1
of Trichormus sp. UIC 10339; H NMR, DEPTQ, COSY,
TOCSY, HSQC, HMBC, and ROESY spectra of 1 and 2;
Marfey’s and advanced Marfey’s analysis of 1 and 2; MS/MS
spectra of 1 and 2) associated with this article are available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: +1-312-996-5583. Fax: +1-312-996-7107. E-mail: orjala@
uic.edu.
■
Notes
The authors declare no competing financial interest.
Synthesis of (2S,3R)-3-Hydroxyleucine and (2S,3S)-3-Hy-
droxyleucine. These compounds were synthesized using the
procedures by Bonnard et al.16 We obtained 148.0 mg of major
precursor (R)-1-((2S,5R)-5-isopropyl-3,6-dimethoxy-2,5-dihydropyra-
zin-2-yl)-2-methylpropan-1-ol and 54.5 mg of minor precursor (S)-1-
((2S,5R)-5-isopropyl-3,6-dimethoxy-2,5-dihydropyrazin-2-yl)-2-meth-
ylpropan-1-ol. The hydroxyleucine precursors were hydrolyzed
following the general peptide acid hydrolysis process as described
below. The major precursor gave a mixture of (2S,3R)-3-
hydroxyleucine and D-valine, and the minor precursor gave a mixture
of (2S,3S)-3-hydroxyleucine and D-valine. These two mixtures were
derivatized with L-FDLA and D-FDLA, respectively, and the resulting
four derivatization mixtures were analyzed using the general LC-MS
method as described for peptide hydrolysate analysis. 1H NMR data of
major precursor (CDCl3, 300 MHz): δ 4.08 (1H, dd, J = 3.2 Hz, 2.9
Hz), 3.98 (1H, dd, J = 3.6 Hz, 3.4 Hz), 3.71 (3H, s), 3.67 (3H, s), 3.61
(1H, br), 2.24 (1H, m), 1.99, (1H, m), 1.69 (1H, br), 1.03 (3H, d, J =
6.9 Hz), 1.02 (3H, d, J = 6.9 Hz), 0.97 (3H, d, J = 6.9 Hz), 0.70 (3H,
d, J = 6.9 Hz). 1H NMR data of minor precursor (CDCl3, 300 MHz):
δ 4.17 (1H, dd, J = 4.3 Hz, 4.0 Hz), 3.98 (1H, dd, J = 3.7 Hz, 3.6 Hz),
3.71 (3H, s), 3.70 (3H, s), 3.65 (1H, br), 2.27 (1H, m), 1.82, (1H, m),
1.68 (1H, br), 1.04 (3H, d, J = 6.9 Hz), 0.90 (3H, d, J = 6.9 Hz), 0.89
(3H, d, J = 6.9 Hz), 0.69 (3H, d, J = 6.9 Hz).
ACKNOWLEDGMENTS
■
This research was supported by grant PO1 CA125066 from
NCI/NIH. The 900 MHz NMR spectrometer was purchased
by NIH grant GM068944 to Dr. Peter G. W. Gettins. We thank
Dr. B. E. Ramirez for providing access to NMR spectrometers
at the UIC Center for Structural Biology (CSB). We thank Dr.
G. E. Chlipala for assisting in the taxonomic identification and
phylogenetic analysis. We thank Dr. D. Nikolic and R. Davis for
their aid in qTOF instrument operating. We thank Dr. G. R.
Thacher and R. Xiong for assisting with NMeIle synthesis.
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