Discovery of new A- and B-type laxaphycins with synergistic anticancer activity

Article abstract of DOI:10.1016/j.bmc.2018.03.022

Two new cyclic lipopeptides termed laxaphycins B4 (1) and A2 (2) were discovered from a collection of the marine cyanobacterium Hormothamnion enteromorphoides, along with the known compound laxaphycin A. The planar structures were solved based on a combined interpretation of 1D and 2D NMR data and mass spectral data. The absolute configurations of the subunits were determined by chiral LC-MS analysis of the hydrolysates, advanced Marfey's analysis and 1D and 2D ROESY experiments. Consistent with similar findings on other laxaphycin A- and B-type peptides, laxaphycin B4 (1) showed antiproliferative effects against human colon cancer HCT116 cells with IC₅₀ of 1.7 μM, while laxaphycins A and A2 (2) exhibited weak activities. The two major compounds isolated from the sample, laxaphycins A and B4, were shown to act synergistically to inhibit the growth of HCT116 colorectal cancer cells.

Full text of DOI:10.1016/j.bmc.2018.03.022

Accepted Manuscript
Discovery of New A- and B-Type Laxaphycins with Synergistic Anticancer
Activity
Weijing Cai, Susan Matthew, Qi-Yin Chen, Valerie J. Paul, Hendrik Luesch
PII:
S0968-0896(18)30232-3
https://doi.org/10.1016/j.bmc.2018.03.022
BMC 14260
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
4 February 2018
3 March 2018
12 March 2018
Please cite this article as: Cai, W., Matthew, S., Chen, Q-Y., Paul, V.J., Luesch, H., Discovery of New A- and B-
Type Laxaphycins with Synergistic Anticancer Activity, Bioorganic & Medicinal Chemistry (2018), doi: https://
doi.org/10.1016/j.bmc.2018.03.022
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Discovery of New A- and B-Type
Laxaphycins with Synergistic Anticancer
Activity
Leave this area blank for abstract info.
Weijing Caia,b, Susan Matthewa, Qi-Yin Chena,b, Valerie J. Paulc and Hendrik Luescha, b,

aDepartment of Medicinal Chemistry and bCenter for Natural Products, Drug Discovery and Development
(CNPD3), University of Florida, 1345 Center Drive, Gainesville, Florida 32610, United States.
cSmithsonian Marine Station, Fort Pierce, Florida 34949, United States.

Bioorganic & Medicinal Chemistry
Discovery of New A- and B-Type Laxaphycins with Synergistic Anticancer Activity
Weijing Caia,b, Susan Matthewa, Qi-Yin Chena,b, Valerie J. Paulc and Hendrik Luescha,b,
aDepartment of Medicinal Chemistry and bCenter for Natural Products, Drug Discovery and Development (CNPD3), University of Florida, 1345 Center Drive,
Gainesville, Florida 32610, United States.
cSmithsonian Marine Station, Fort Pierce, Florida 34949, United States.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Two new cyclic lipopeptides termed laxaphycins B4 (1) and A2 (2) were discovered from a
collection of the marine cyanobacterium Hormothamnion enteromorphoides, along with the
known compound laxaphycin A. The planar structures were solved based on a combined
interpretation of 1D and 2D NMR data and mass spectral data. The absolute configurations of
the subunits were determined by chiral LC-MS analysis of the hydrolysates, advanced Marfey’s
analysis and 1D and 2D ROESY experiments. Consistent with similar findings on other
laxaphycin A- and B- type peptides, laxaphycin B4 (1) showed antiproliferative effects against
human colon cancer HCT116 cells with IC50 of 1.7 µM, while laxaphycins A and A2 (2)
exhibited weak activities. The two major compounds isolated from the sample, laxaphycins A
and B4, were shown to act synergistically to inhibit the growth of HCT116 colorectal cancer
cells.
Available online
Keywords:
Marine natural products
Cyanobacteria
Laxaphycin
Anticancer activity
Synergy
2018 Elsevier Ltd. All rights reserved.
———
 Corresponding author. Tel.: +1-(352) 273-7738; fax: +1-(352) 273-7741; e-mail: luesch@cop.ufl.edu

1. Introduction
Supporting Information Figures S1 and S2). Laxaphycins A and
B were isolated from the freshwater cyanobacterium Anabaena
laxa in 1992.6,7 The absolute configurations of these two
compounds were not elucidated until 1997, when laxaphycins A
and B were identified again from a collection of a tropical marine
cyanobacterium identified as Lyngbya majuscula.8 Within the last
two decades, laxaphycin A and B groups have been expanded,
with several laxaphycin analogues isolated from marine
cyanobacteria (Supporting Information Figures S1 and S2),
Cyanobacteria represent
a prolific source of bioactive
secondary metabolites with wide pharmaceutical importance.1,2
A
majority of these bioactive molecules are peptides, polyketides or
hybrid polyketide-polypeptides being biosynthesized by multi-
modular enzymatic systems integrating nonribosomal peptide
synthetases (NRPS) and polyketide synthases (PKS) biosynthetic
pathways.1,3 The intriguing structural features of cyanobacterial
compounds allow them to interact with a variety of cellular
targets, which results in a broad spectrum of biological activities,
including anticancer, antifungal, antimicrobial, protease
including
hormothamnin
A
from
Hormothamnion
enteromorphoides,9,10 laxaphycins B2 and B3 from Lyngbya
majuscula,11 lobocyclamides A–C from Lyngbya confervoides,12
and lyngbyacyclamides A and B from Lyngbya sp.13 Related
inhibitory,
immunomodulatory
and
neuromodulatory
properties.4,5
compounds
from
A
freshwater
cyanobacteria
include
trichormamides
and
B
from Trichormus sp.,14 and
The laxaphycins are a large family of cyclic lipopeptides
found in cyanobacteria, which are characterized by a rare fatty β-
amino acid with a linear chain of up to 12 carbons. All
laxaphycins can be separated into cyclic undeca- and
dodecapeptides, the major representative of each class being
laxaphycin A and laxaphycin B, respectively (Figure 1,
trichormamides C and D from cf. Oscillatoria sp.15 Laxaphycin
A-type peptides are characterized by
a segregation of
hydrophobic and hydrophilic residues, while laxaphycin B- type
peptides have alternating hydrophobic and hydrophilic residues
(Figure 1).8

Interestingly, laxaphycin A- and B-type compounds have been

frequently co-isolated from the same cyanobacterium.8,12,14,15 This

phenomenon might be explained from the perspective of their

biological activities. Laxaphycin B-type compounds exhibited

strong to moderate antifungal activities against C. albicans and

C. glabrata12 and anticancer activity towards various cancer cells

(IC50 < 2 µM)11,13–15 as well as antibacterial activities against M.

tuberculosis.15 In contrast, most of the laxaphycin A-type

compounds show weak cytotoxicity (IC50 >10 µM) with the

cytotoxic hormothamnin
A
as an exception.7,9–11,14,15

Hormothamnin A is a (Z)-α,β-didehydro-α-aminobutyric acid (Z-

Dhb) analog of laxaphycin A. It is possible that the geometry of

the Dhb unit in laxaphycin A-type compounds is one of the

contributing factors to its cytotoxicity. Importantly, laxaphycin

A-type and B-type compounds have been reported to act

Figure 1. Structures of laxaphycins B and A and characteristic features of B- and A-type compounds. Laxaphycin B- type peptides have
alternating non-polar and polar amino acids. Laxaphycin A-type peptides are characterized by a segregation of non-polar and polar amino acids.
synergistically with each other to inhibit fungus and cancer

cells.11,12 In other words, in order to achieve maximum biological
potency, a member of each class of peptide must be present.
Although the exact mechanism of action of the synergism remain
unknown, the cooperativity provides a possible explanation for
the frequently observed coproduction of laxaphycin A-type and
B-type peptides from the same cyanobacteria.
detailed NMR interpretation of 1H NMR, 13C NMR, HSQC,
HMBC, COSY and ROESY spectra (Table 1, Figure 2,
Supporting Information Figures S3–S8). The H NMR spectrum
1
of 1 exhibited a signal pattern characteristic of a lipopeptide: a
group of signals for exchangeable amide protons (δH 6.9–8.2),
signals of α -protons (δH 4.0–5.0), aliphatic methylene signals (δH
1.1–1.4) and methyl signals (δH 0.7–1.0). Eleven α-amino acid
units were characterized by interpretation of COSY, HSQC and
HMBC spectrum: two threonines (Thr1/2), two 3-
hydroxyleucines (3OH-Leu 1/2), valine (Val), leucine (Leu), 4-
hydroxyproline (4-OHPro), N-methyl isoleucine (N-Me-Ile),
homoserine (Hse), glutamine (Gln), 3-hydroxyasparagine (3-
OHAsn). The presence of a lipophilic β-amino acid, β-
aminodecanoic acid (Ada), was evident by sequential COSY
correlations between NH (δH 7.64)/ H-3 (δH 4.07)/H2-2(δH 2.50;
2.31) as well as sequential COSY correlations from H2-4 (δH
1.38; 1.31) to the region of highly overlapping methylene signals
(H2-5 to H2-9), which correspond to five carbons in the HSQC
spectrum (δC 28.7, 28.3, 25.0, 30.9, 21.8) and then to a methyl
triplet (δH 0.84) (Table 1). The amino acid sequence was assigned
based on HMBC and ROESY correlations as 4-OHPro-Leu-
Thr2-Ada-Val-3-OHLeu1-HSe-3-OHLeu2-Gln-N-Me-Ile-3-
OHAsn-Thr1 (Table 1, Figure 2). The 16 degrees of unsaturation
and the molecular formula suggested that 1 was a cyclic peptide.
In the present study, a field collection of the marine
cyanobacterium Hormothamnion enteromorphoides has afforded
two new laxaphycin analogues: laxaphycin B4 (1) and
laxaphycin A2 (2) (Figure 2). Here, we describe the isolation,
total structure determination, and evaluation of their
antiproliferative effects in a colon cancer cell line, HCT116, as
well as their synergistic effects.
2. Results and Discussion
The cyanobacterium was collected from Garden Key in the
Dry Tortugas National Park and extracted with CH2Cl2 and
MeOH (1:1) to provide the nonpolar extract and EtOH and H2O
(1:1) to provide a polar extract. The nonpolar extract (2.6 g) was
subjected to silica chromatography and two rounds of reversed-
phase HPLC to yield laxaphycin B4 (1) (20 mg), laxaphycin A2
(2) (0.4 mg) and laxaphycin A (20 mg).
Figure 2. Structures and key NMR correlations of laxaphycin B4 (1) and laxaphycin A2 (2).
The HR-ESIMS spectrum of compound 1 showed a [M + Na]+
peak at m/z 1463.8334, consistent with the molecular formula
C66H116N14O21. The structure of 1 was established based on a
The chemical shifts of C1 (δC 168.6) in Thr1 indicated the
presence of an amide or ester moiety, and therefore the cyclic
dodecapeptide ring was closed between Thr1 and 4-OHPro.

Compound 1 (0.3 mg) was hydrolyzed with 6 N HCl (110 °C,
20 h) and the hydrolysate was subjected to chiral HPLC-MS,
revealing the presence of D-Leu, L-Thr/ L-allo-Thr, L-Gln, L-Val,
N-Me-L-Ile and trans-4OH-L-Pro/cis-4OH-D-Pro in the molecule
(Figure 2, Table 2). The exact assignment for Thr and 4-OHPro
and all the other amino acids in 1 was elucidated using advanced
Marfey’s analysis (Figure 2, Table 3).16–18 The L-FDLA
derivative of the acid hydrolysate of 1 was compared with FDLA
derivatives of authentic standards of 3-OHLeu [(2S,3S)-3-
allowed for assignment of 2R,3S configurations for 3-OHLeu and
L configurations for HSe and Thr and the assignment of trans-
4OH-L-Pro and D-threo-3-OHAsn (Table 3). A comparison of the
elution orders of L-FDLA and D-FDLA derivatives of the acid
hydrolysate with those reported in the literature assigned 3R
configuration for Ada in 1 (Table 3).12,14,16,17,19
Compound 1 is a laxaphycin B-type peptide featuring a
dodecapeptide core and a fatty β-amino acid. It is closely related
to laxaphycin B3 with the alanine at position 4 in laxaphycin B3
being replaced by homoserine (Supporting Information, Figure
S2). Thus, compound 1 was termed laxaphycin B4 (1). Although
the configuration of 3-OH-Leu1 in laxaphycin B3 was first
deduced as 2S,3S based on the remarkable NMR spectra
similarities between laxaphycins B3 and B, a later study revised
the configuration of 3-OH-Leu1 to (2R,3S)-3-OH-Leu1 in
OHLeu-L-FDLA,
(2S,3S)-3-OHLeu-DL-FDLA,
(2S,3R)-3-
OHLeu-L-FDLA, (2S,3R)-3-OHLeu-DL-FDLA], HSe (L-HSe-L-
FDLA, L-HSe-DL-FDLA), Thr (L-Thr-L-FDLA, L-allo-Thr-L-
FDLA) and 4-OHPro (trans-4OH-L-Pro-L-FDLA, cis-4OH-D-
Pro-L-FDLA), 3-OHAsp [L-threo-3-OHAsp-L-FDLA, L-threo-3-
OHAsp-DL-FDLA,
D/L-erythro-3-OHAsp-L-FDLA],
which
Table 1. NMR data for 1 in DMSO-d6 (600 MHz for 1H and 150 MHz for 13C NMR data)
C/H no
δC
δH (J in Hz)
COSY
HMBC
ROESY
β-Ada
1
171.5, C
39.6, CH2
2a
2b
3
2.50, m
2.31, m
4.07, m
1.38, m
1.31, m
1.19, m
1.21, m
1.20, m
1.20, m
1.23, m
0.84, t (7.0)
7.64, d (8.9)
3, 2b
1, 3, 4
NH (Val)
NH (Val)
NH (Val)
3, 2a
1, 3, 4
45.7, CH
33.4, CH2
2, 4, NH
3, 4b, 5
3, 4a, 5
1
2
5
4a
4b
5
28.7, CH
28.3, CH
25.0, CH2
30.9, CH2
21.8, CH2
13.7, CH3
6
7
8
9
10
9
10
NH
1
3
3, 1 (Thr2)
2 (Thr2), 3 (Thr2)
NH (3-OHLeu1)
Val
171.4, C
58.4, CH
29.4, CH
18.1, CH3
18.8, CH3
2
4.16, t (7.2)
1.98, m
3, NH
2, 4
3
1, 3, 4
3
2, 5
4
0.88, d (6.8)
0.85, m
3, 5
5
2, 3, 4
NH
8.17, d (7.6)
2
2, 3, 1 (β-Ada)
2 (β-Ada), 3 (β-Ada)
3-OHLeu1
1
171.6, C
55.0, CH
76.6, CH
30.5, CH
19.0, CH3
18.5, CH3
2
4.43, m
3, NH
1, 3
NH (Hse)
NH (Hse)
3
3.50 m
2, 4, OH
4, 6
4
1.59, m
3, 5, 6
3, 5
5
0.92, m
4
4
2
3
3, 4, 6
3, 4, 5
2, 1 (Val)
2, 3, 4
6
0.78, d (6.7)
7.91, d (8.3)
4.83, d (7.1)
NH
OH
1
2 (Val)
Hse
171.8, C
50.8, CH
34.3, CH2
2
4.36, m
NH
3, 4
NH (3-OHLeu2)
3a
3b
4a
4b
NH
1.86, m
2, 3b, 4a, 4b
2, 3a, 4a, 4b
3a, 3b, 4b, OH
3a, 3b, 4a, OH
2
1, 2, 4
1.82, m
1, 2, 4
57.4, CH2
3.49, m
2, 3
3.42, m
2, 3
7.96, br d (5.6)
1, 2, 3, 1 (3-OHLeu1)
2 (3-OHLeu1), 3 (3-OHLeu1)

OH
4.50, t (5.2)
4a, 4b
3, 4
3-OHleu2
1
171.0, C
55.5, CH
76.0, CH
29.8, CH
18.5, CH3
18.7, CH3
2
4.31, m
3, NH
2, OH
3, 5, 6
4, 6
4, 5
2
1
NH (Gln)
3
3.44, m
4
1.57, m
2, 3, 5
3, 4, 6
3, 4, 5
2, 1 (Hse)
3, 4
5
0.90, m
6
0.76, m
NH
OH
1
7.79, d (8.9)
4.91, d (5.1)
2 (Hse)
3
Gln
173.0, C
49.2, CH
25.4, CH2
2
4.56, m
2.00, m
1.67, m
2.29, m
2.17, m
3a, 3b, NH
2, 3b, 4a, 4b
2, 3a, 4a, 4b
3a, 3b, 4b
1
3a
3b
4a
4b
5
4, 5
4, 5
30.4, CH2
174.8, C
2, 3, 5
2, 3, 5
3a, 3b, 4a
NH
NH2a
NH2b
7.84, d (7.3)
7.37, br s
2
2, 1 (3-OHLeu2)
2 (3-OHLeu2)
NH2b
NH2a
5
6.96, br s
4, 5
N-Me-Ile
1
170.2, C
59.7, CH
31.5, CH
23.5, CH2
2
4.74, d (10.8)
1.92 m
3, 6
2, 6
3, 4b, 5
4a, 5
4a
1, 3, 4, 6, N-Me
NH (3-OHAsn)
3
4a
4b
5
1.29, m
0.91, m
0.77, m
0.75, m
3.02, s
3, 6
3, 6
10.0, CH3
14.8, CH3
30.0, CH3
169.1, C
55.3, CH
70.0 CH
173.6, C
6
3
1, 2, 3, 4
N-Me
1
2, 1 (Gln)
3-OHAsn
2
4.64, dd (8.3, 1.4)
4.37, m
H-3, NH
H-2, OH
1, 3, 4
1, 2, 4
NH (Thr1)
NH (Thr1)
3
4
NH
NHa
NHb
OH
1
7.67, d (8.3)
7.30, NH2
2
3
2, 3, 1 (N-Me-Ile)
2 (N-Me-Ile)
4
7.27, NH2
3, 4
2, 3, 4
5.82, d (6.4)
Thr1
168.6, C
55.7, CH
66.6, CH
18.6, CH3
2
4.46, m
3, NH
2, 4, OH,
3, OH
3
1, 3, 4
1, 2, 4
2, 3
3
3.88, dq (11.5, 6.1)
1.05, d (6.1)
4.93, d (4.6)
7.25, d (8.0)
4
OH
NH
2, 3, 4
2
2
2 (3-OHAsn), 3 (3-OHAsn)
NH (Leu)
4-OHPro
1
171.3, C
58.5, CH
37.6, CH2
2
4.44, m
1.99, m
1.85, m
3a, 3b
1, 3, 4
3a
3b
2, 4, 3b, 5b
2, 4, 3a
1, 2, 4, 5
1, 2, 4, 5

4
68.4, CH
55.3, CH2
4.32, m
3a, 3b, 5a, 5b, OH
22
5a
5b
OH
1
3.73, dd (10.6, 3.9)
3.58, d (10.7)
5.08, d (3.3)
4, 5b
3a, 4, 5a
4
3
4
3, 4, 5
Leu
171.9, C
51.3, CH
41.0, CH2
24.0, CH
22.6, CH3
21.6, CH3
2
4.33, m
3, NH
1, 3, 4
NH (Thr2)
3
1.47, t (7.2)
1.53, m
2, 4
1, 2, 4, 5, 6
2, 3, 5, 6
3, 4, 6
4
3, 5, 6
5
0.88, m
4
4
2
6
0.82, d (7.0)
7.95, br d (5.5)
3, 4, 5
NH
1
1 (4-OHPro)
2 (4-OHPro)
Thr2
168.8, C
58.2, CH
66.1, CH
19.4, CH3
2
4.09, m
3, NH
1, 3, 4, 1 (Leu)
NH (β-Ada)
NH (β-Ada)
3
3.99, m
2, 4, OH
4
1.02, d (6.3)
7.78, d (9.2)
4.78, d (5.4)
3
2
3
3, 2
NH
OH
1 (Leu)
2, 3, 4
2 (Leu)
Table 2. Chiral amino acid analysis of 1
Compound parameters for amino acidsa
Retention times (min) b
Amino
acid
M
W
Q1
m/z
Q3
m/z
D
P
E
P
C
E
CX
P
CE
P
Measure
d
L
D
L-allo
NA
D-allo
NA
Assignment
Val
Glu
117
147
118
146
72
31
8
15
4
12
8.4
6.1
14.1
7.5
8.5
L
L
-
30
-
20
102
-2
-4
-14
NA
NA
6.0
Thr
119
131
145
120
132
146
74
21
31
35
7
8
7
13
13
17
4
4
2
10
10
10
7.7
8.9
7.8
11.0
NA
7.8
L or L-allo
Leu
86
9.3
17.4
49.7
NA
15.1
18.1
12.4
D
L
N-Me-Ile
100
12.5
49.3
9.1 (cis-
L)
10.6 (cis-
D)
10.9 (trans-
L)
28.6 (trans-
D)
trans-L or cis-
D
4-OHPro
131
132
68
31
5
27
4
12
10.8
a MS parameters: Q1 (parent ion); Q3 (product ion); DP (declustering potential); EP (entrance potential) CE (collision energy); CXP (collision cell exit potential);
CEP (collision cell entrance potential); Positive and negative values indicate positive and negative ionization modes. b Measured by LC-MS selected ion
chromatogram on a chiral column (Chirobiotic TAG (250x4.6 mm), Supelco; solvent: MeOH:10mM NH4OAc (40:60, pH 5.12)); flow rate 0.5 mL/min
Table 3. Advanced Marfey’s analysis of 1
Compound parameters for FDLA derivatives of amino acids
Retention times (min)
Amino
acid
M
W
Q1
m/z
Q3
m/z
CX
P
CE
P
Measure Assignme
DP EP
CE
L
D
L-allo
D-allo
d
nt
N
A
N
A
N
A
Adaa
482
442
414
426
414
444
480
440
412
426
412
442
32.0 (S)
14.1 (2S,3S)
11.0 (S)
38.8 (R)
38.4
R
NA
162
NA
-2
NA
-22
NA
NA
3-OH-
Leua
-
30
-
9.5
-
48
19.3
(2R,3R)
21.8
(2R,3S)
13.9
(2S,3R)
22.2
11.0
8.0
2R,3S
L
-
45
-
5.5
-
38
Hsea
176
381
192
398
-4
8
-20
24
12.0 (R)
NA
NA
NA
NA
7.9 (trans-
L)
4-OHProa
Thrb
66
4.5
-8
25
9.4 (cis-D)
trans-L
L
NA
-
40
-
26
19.0
(2S,3R)
-4
-4
-20
-22
19.9 (2S,3S)
18.9
37.4
NA
3-OH-
Aspc
-
45
-
24
38.1 (L-
threo)
37.8 (D-
threo)
-3
41.0/43.5 (L/D-erythro)
D-threod
aMeasured by LC-MS selected ion chromatogram on a reversed-phase column (Phenomenex Kinetex C18, 100 x 2.10 mm, 2.6 μm, flow rate 0.2 mL/min) with a
b
linear gradient from 25% to 65% aqueous acetonitrile containing 0.1% formic acid over 50 min. Measured by LC-MS selected ion chromatogram on a reversed-
phase column (Alltech Alltima C18, 5 μm, 250 × 4.6 mm, 5 μm, flow rate 1.0 mL/min) with a linear gradient from 25% to 65% aqueous acetonitrile containing
c
0.1% formic acid over 50 min. Measured by LC-MS selected ion chromatogram on a a reversed-phase column (Alltech Altima C18, 5 μm, 250 × 4.6 mm; flow
d
rate, 1.0 mL/min ) using a linear gradient of MeOH in 0.1% formic acid (20–50% in 60 min). Confirmed by co-injection.

laxaphycin B by conducting total synthesis.11,20 Therefore, the
configuration of 3-OH-Leu1 in laxaphycin B3 is likely to be
2R,3S as well, which is the same as laxaphycin B4 (1).
aminobutyric acid (Dhb) and one β amino acid unit β-
aminooctanoic acid (Aoa) (Table 4, Figure 2). The presence of β-
Aoa was deduced by the COSY, TOCSY and HSQC correlations
as described for 1. The amino acid sequence was assigned based
on interpretation of HMBC and ROESY correlations as Dhb-4-
The molecular formula of 2 was deduced as C59H95N11O14
based on a [M + Na]+ peak at m/z 1204.6930 in HR-ESIMS
spectrum and the NMR spectra. Due to the broad NMR signals
observed in DMSO-d6, 1D and 2D NMR experiments of 2 were
conducted in CH3CN-d3 (Table 4). The structure of 2 was
elucidated based on combined analysis of 1H NMR, HSQC,
HMBC, COSY, TOCSY and ROESY experiments (Table 4,
Figure 2, Supporting Information Figures S9-S15). COSY and
TOCSY correlations of 2 revealed the presence of ten α-amino
acid residues, including glycine (Gly), phenylalanine (Phe), two
leucines (Leu1/2), valine (Val), 4-hydroxyproline (4-OHPro),
isoleucine (Ile), two homoserines (HSe1/2), α,β-didehydro-α-
OHPro-HSe2-Phe-Leu1-Val-Ile-Leu2-Gly-Aoa-HSe1.
The
molecular formula and degree of unsaturation indicated the cyclic
nature of 2. Thus, the cyclic undecapeptide ring was closed
between Dhb and HSe1.
Compound 2 was hydrolyzed with 6 N HCl (110 °C, 20 h) and
the hydrolysate of 2 was subjected to chiral HPLC-MS to reveal
the presence of D-Phe, L-Val, D-allo-Ile and trans-4OH-L-
Pro/cis-4OH-D-Pro (Figure 2, Table 5). Peaks for both L- and D-
Leu were detected by chiral HPLC-MS, preventing unambiguous
Table 4. NMR data for 2 in CH3CN-d3 (600 MHz for 1H and 150 MHz for 13C NMR data)
a
δC
δH (J in Hz)
COSY
HMBC
ROESY
β-Aoa
1
171.1, C
41.4, CH2
41.4, CH2
45.9, CH2
36.1, CH
28.9, CH2
32.2, CH2
32.5, CH2
14.4, CH3
2a
2b
3
1.65, m
1.98, m
4.34, m
1.41, m
1.25, m
1.32, m
1.25, m
0.87, m
6.92, m
3, 2b
1
NH (HSe1)
3, 2a
NH, 2a, 2b, 4
4
2, 3
6
5
6
7
8
NH
3
2a (Gly)
Hse1
1
2
50.0, CH
34.3, CH2
57.8, CH2
4.69, m
1.80, m
3.58, m
4.22, m
6.81, m
3, NH
2, 4
3, OH
4
3
4
OH
NH
2
2a (β-Aoa)
Dhb
1
2
3
4
169.3, C
131.9, C
122.6, CH
11.0, CH3
5.66, q (7.2)
1.76, d (7.2)
4
3
1
3, 2
2 (4-OHPro), 5a (4-OHPro), 5b (4-OHPro)
4 (Dhb), NH (HSe2)
4-OHPro
1
172.1, C
60.7, CH
38.9, CH2
38.9, CH2
68.4, CH
2
4.65, dd (8.0, 10.0)
1.95, m
3a, 3b
1
1
4
3a
3b
4
2
2.39, d (13.8, 7.9)
4.36, br s
2, 4
5a, 5b, 3a, 3b
OH
5a
5b
58.5, CH2
58.5, CH2
3.46, d (11.0)
4, 5b
4, 5a
4, 1 (Dhb)
3.62, dd (11.0, 2.9)
Hse2
1
2
174.2, C
50.3, CH
4.30, m
NH, 3, 2
NH (Phe)

3
33.4, CH2
59.1, CH2
59.1, CH2
2.14, m
3.42, m
3.51, m
2.74, br s
7.15, m
2
1
4a
OH, 3, 4b
OH, 3, 4a
3a, 3b
2
4b
OH
NH
2
1 (4-OHPro)
2 (4-OHPro)
NH (Leu1)
Phe
1
174.2, C
2
58.5, CH
38.2, CH2
38.2, CH2
139.4, C
4.31, m
3a, 3b
1
3a
3b
4
3.13, dd (13.5, 12.0)
3.05, dd (13.5, 3.0)
2
2
2, 5/9
4, 5/9
5, 9
6, 8
7
130.8, CH
129.5, CH
127.8, CH
7.45, d (7.7)
7.29, t (7.5)
7.22, t (7.3)
7.91, d (7.4)
3a, 3b, 6/8
3, 6/8, 7
7
5/9, 7
6/8
2
6/8
NH
1 (HSe2)
2 (HSe2)
NH (Val)
Leu1
1
173.5, C
53.2, CH
40.3, CH2
40.3, CH2
25.2, CH
23.7, CH3
20.6, CH3
2
4.30, m
NH, 3a, 3b
1
3a
3b
4
1.08, m
2
1.33, m
2
1.58, m
3a, 3b, 5, 6
5
0.82, d (6.9)
0.75, d (6.6)
6.98, d (8.0)
4
4
2
3, 4, 6
3, 5, 6
1 (Phe)
6
NH
2 (Phe)
Val
1
174.5, C
56.7, CH
35.0, CH
19.8, CH3
16.0, CH3
2
4.80, m
NH, 3
1
NH (Ile)
3
2.14, m
2, 4, 5
4
0.84, d (7.0)
0.73, d (6.9)
6.47, d (9.9)
3
3
2
2, 3, 5
5
2, 3, 4
NH
1 (Leu1)
2 (Leu1)
Ile
1
174.9, C
53.1, CH
37.6, CH
27.7, CH2
27.7, CH2
12.7, CH3
14.8, CH3
2
4.79, m
1.94. m
0.91, m
1.23, m
0.90, m
0.83, m
7.09, d (9.5)
NH, 3
1
NH (Leu2)
3
2, 4b, 6
4a
4b
5
3
4b
6
NH
2
2 (Val), 3 (Val)
NH (Gly)
Leu2
1
175.2, C
55.3, CH
39.9, CH2
39.9, CH2
25.3, CH
22.1, CH3
22.8, CH3
2
3.92, m
NH, 3a, 3b
1
3a
3b
4
1.56, m
2, 3b
2
1.46, m
2, 3a
2, 4, 5
1.63, m
3a, 3b, 5, 6
5
0.88, d (6.5)
0.94, d (6.5)
4
4
3, 4, 6
3, 5, 6
6

NH
7.16, s
2
2 (Ile), 3 (Ile)
Gly
1
168.6, C
43.6, CH2
43.6, CH2
2a
2b
NH
3.91, m
3.37, m
7.43, m
NH, 2b
NH, 2a
2a, 2b
1
NH (β-Aoa)
1, 1 (Leu)
2 (Leu2)
aDeduced from HSQC and HMBC spectra.
Table 5. Chiral amino acid analysis of 2
Compound parameters for amino acids
Retention times (min)
Amino
acid
M
W
Q1
m/z
Q3
m/z
D
P
E
P
C
E
CX
P
CE
P
L
D
L-allo
D-allo
Measured
Assignment
Vala
Phea
Ileb
117 117
165 166
131 132
72
31
31
31
8
8
8
15
19
13
4
4
4
12
12
10
8.4
14.1
18.2
95.0
NA
NA
19.8
NA
NA
75.1
8.5
L
120
86
13.0
17.3
18.6
75.7
D
D-allo
19.1 and
65.8
Leub
131 132
86
31
8
13
4
10
19.0
65.7
NA
NA
L and D
9.1 (cis-
L)
10.6 (cis-
D)
10.9 (trans-
L)
28.6 (trans-
D)
trans-L or
cis-D
4-OHProa
131 132
68
31
5
27
4
12
10.8
aMeasured by LC-MS selected ion chromatogram on a chiral column (Chirobiotic TAG (250 × 4.6 mm), Supelco; solvent: MeOH:10mM NH4OAc (40:60, pH
b
5.12)); flow rate 0.5 mL/min. Measured by LC-MS selected ion chromatogram on the same chiral column (Chirobiotic TAG (250 × 4.6 mm), Supelco; solvent:
MeOH:10mM NH4OAc (90:10, pH 6.0)); flow rate 0.5 mL/min.
Table 6. Advanced Marfey’s analysis of 2
Compound parameters for FDLA derivatives of amino acids
Retention times (min) a
Amino acid MW
Q1 m/z Q3 m/z DP
EP
NA NA NA NA
-45 -6 -38 -4
66 25
CE
CXP CEP
L
D
L-allo
NA
D-allo Measured
Assignment
Aoa
454
414
426
452
412
426
NA
176
381
NA
-20
24
26.2 (S) 32.0 (R)
NA
NA
32.0
11.0
8.0
R
Hse
11.0
12.0
NA
L
4-OHPro
5
8
NA
9.4 (cis-D) 7.9 (trans-L) NA
trans-L
aMeasured by LC-MS selected ion chromatogram on a reversed-phase column (Phenomenex Kinetex C18, 100 × 2.10 mm, 2.6 μm, flow rate 0.2 mL/min) with a
linear gradient from 25% to 65% aqueous acetonitrile containing 0.1% formic acid over 50 min.
configurational assignment for the two leucines in 2 (Table 5).
Partial hydrolysis was not able to be conducted due to limited
supply of 2 (0.4 mg), and thus we proposed that the sequence of
the nonpolar tetrapeptide residue is the same as that found in all
the other laxaphycin A-type compounds (Supporting Information
Figure S1). The L-FDLA derivative of the acid hydrolyzates of 2
was compared with FDLA derivatives of authentic standards of
HSe (L-HSe-L-FDLA, L-HSe-DL-FDLA) and 4-OHPro (trans-
4OH-L-Pro-L-FDLA, cis-4OH-D-Pro-L-FDLA), which allowed
for assignment of L-HSe and trans-4OH-L-Pro (Table 6). LC-MS
comparison between L-FDLA and DL-FDLA derivatives of the
acid hydrolysate of 2 assigned 3R configuration for Aoa, similar
to the assignment of Ada in 1 (Table 6). The geometric
configuration of Dhb was determined to be E based on 1D and
2D ROESY correlations between Dhb H3-4 (δH 1.76) and 4-
OHPro H2-5 (δH 3.62; 3.46) as well as between Dhb H3-4 (δH
1.76) and 4-OHPro H-2 (δH 4.65) (Table 4, Supporting
Information Figures S13, S14).12,15
respectively. These results are consistent with the trend observed
in other laxaphycins in that laxapycin B-type compounds are
more potent than laxaphycin A-type compounds.
To further elucidate the synergistic effect between laxaphycin
A- and B-type compounds, we aimed to test the combinatorial
effect of laxaphycin B4 (1) and laxaphycin A in HCT116 cells.
We selected these two compounds for this study based on the fact
that they are the two major compounds produced by this
collection as well as due to the limited supply of laxaphycin A2
(2). To conduct the combination study, HCT116 cells were
treated concomitantly with both compounds at fixed ratios (A:B4
=32:1; 10:1) for 48 h. These are at equipotency ratios according
to their IC50 values (1.7 µM and 23 µM) so that the contribution
to the effect of each compound would be about equal.21 Cell
viability was measured using the MTT assay, and the
combination index (CI) was calculated as an indicator of the
mode of drug-drug interactions using CompuSyn software, a
software designed based on the principles of Chou–Talalay
method.21 As shown in Figure 3A,B, strong synergism was
observed in HCT116 at both combination ratios. Only one
combination at low doses (laxaphycin A = 1 µM, laxaphycin B4
= 0.032 µM) showed an antagonistic effect (Figure 3A), which
was further explained by a correlation trend between CI and
percentage cell viability generated by computer simulation
(Figure 3C). In general, the combination index decreases as the
cell viability decreases (Figure 3C). When cell viability is lower
than 70 %, synergism is predicted between laxaphycin A and B4
(1) under the tested combination ratios. A very strong synergistic
Compound 2 is structurally related to laxaphycin A with the
Ile at position 8 in laxaphycin A being replaced by a valine
(Figure 1, Supporting Information Figure S1). Therefore,
compound 2 was named laxaphycin A2 (2).
Due to the known anticancer activities of the laxaphycin
familiy,11 we evaluated the antiproliferative activities of these
two new laxaphycins in colon cancer HCT116 cells using the
MTT assay. We observed antiproliferative activity of laxaphycin
B4 (1) in HCT116 cells with an IC50 value of 1.7 µM, while
laxaphycin A2 (2) and laxaphycin A only exhibited weak
antiproliferative activity with IC50 values of 29 µM and 23 µM,

effect (CI < 0.1) is predicted when percentage cell viability is
lower than 25 %.
Figure 3. Antiproliferative effect of laxaphycins A and B4 in HCT116 cells.
A) Combinatorial effect at a fix ratio of laxaphycin A: laxaphycin B4 = 32:1.
B) Combinatorial effect at a fix ratio of laxaphycin A: laxaphycin B4 = 10:1.
Cell viability (fraction survival, Fs) values were obtained using MTT assay,
48 h. These data were then converted to the level of cell growth inhibition, or
affected fraction (Fa) using the equation of: Fa = 1- Fs. CI values were
calculated using CompuSyn software as described in the Experimental
Section. A CI value of <1 (in green), 1 and >1 (in red) indicates synergism,
additivity and antagonism, respectively. Data are represented by the mean of
three independent experiments +SD. C) Effect of the combinatorial ratios of
laxaphycins A and B4 (32:1; 10:1) on the Combination Index (CI). CI is
plotted against levels of cell growth inhibition or affected fraction (Fa) by
computer simulation using CompuSyn software. The vertical bars indicate
95% confidence intervals based on Sequential Deletion Analysis (SDA) using
CompuSyn software (see Experimental Section for details).
In summary, laxaphycins are cyclic lipopeptides featuring a
fatty β-amino acid moiety with a linear chain of up to 12 carbons.
In the present study, we have discovered two new laxaphycins
from a collection of the marine cyanobacterium Hormothamnion
enteromorphoides. The total structures were determined using a
combined analysis of NMR spectra, mass spectral data, chiral
LC-MS analysis and advanced Marfey’s analysis. Due to their
structural similarity to the known laxaphycin B3 and laxaphycin
A, these two new compounds were named laxaphycin B4 (1) and
laxaphycin A2 (2), respectively. Laxaphycin B4 (1) exhibited
antiproliferative effect against human colon cancer HCT116 cells
with IC50 value of 1.7 µM, while laxaphycin A2 (2) had a much
weaker effect. Using the same cancer cell model, we have also
elucidated that laxaphycins A and B4 (1), the two major
compounds co-produced in this collection, work synergistically
to inhibit cancer cell growth.
with a 5 mm TXI cryogenic probe using residual solvent signals
(δH 2.50; δC 39.51 ppm, DMSO-d6; δH 1.94; δC 118.69 ppm,
CH3CN-d3) as internal standards. HSQC experiments were
optimized for 145 Hz, and HMBC experiments were optimized
for 7 Hz. LC-MS data were obtained using an API 3200 (Applied
Biosystems) equipped with a Shimadzu LC system. Optical
rotations were measured on a Perkin-Elmer 341 polarimeter. UV
was measured on a SpectraMax M5 (Molecular Devices). HRMS
data was obtained using an Agilent LC-TOF mass spectrometer
equipped with an APCI/ESI multimode ion source detector.
3. Experimental Section
3.1. General Experimental Procedures
1H and 2D NMR spectra in DMSO-d6 and CH3CN-d3 were
recorded on a Bruker Avance II 600 MHz spectrometer equipped
3.2. Chemicals
Standard amino acids, four isomers of 4-hydroxyproline and
L-homoserine were obtained from Sigma-Aldrich, (St. Louis,
MO).
L-threo-3-hydroxyaspartic
acid
and (2S,3R)-3-
hydroxyleucine was obtained from Santa Cruz Biotechnology
(Santa Cruz, CA). (2S,3S)-3-hydroxyleucine was obtained from
AstaTech, Inc (Bristol, PA).
3.3. Biological Material
The cyanobacterium was collected on May 8 and May 9 in
2009 at Garden Key, Dry Tortugas National Park. Voucher
specimens (DRTO0000033) are maintained at the Smithsonian
Marine Station at Fort Pierce, Florida, and at South Florida
Collections Management Center, Everglades National Park. The
cyanobacterium corresponds morphologically and chemically
with Hormothamnion enteromorphoides, which is broadly
distributed in tropical waters in Florida, the Caribbean and
Pacific and is known to produce hormothamnin A9 and
laxaphycins22. It has been suggested that this name should be
regarded as a synonym of Hydrocoryne enteromorphoides.23
3.4. Extraction and Isolation
The freeze-dried sample (95 g) was extracted with CH2Cl2 and
MeOH (1:1) to provide the nonpolar extract and EtOH and H2O
(1:1) to provide a polar extract. The nonpolar extract (2.6 g) was
subjected to silica column using a gradient system of increasing
i-PrOH in CH2Cl2. The fraction eluting with 20% i-PrOH was
further purified by reversed-phase HPLC (Phenomenex Luna
C18, 250 × 10 mm, 5µm, 2.0 mL/min; PDA detection) using a
MeOH–H2O linear gradient (50–100% MeOH for 30 min and
100% MeOH for 10 min). Fractions were pooled on the basis of
retention times, 1H NMR analysis and low-resolution MS
measurements to afford laxaphycin B4 (1) (tR 29.5 min),
laxaphycin A2 (2) (tR 29.8 min,) and laxaphycin A (tR 30.5 min).
The obtained semi-pure laxaphycins were further purified by the
same HPLC column (Phenomenex Luna C18, 250 × 10 mm,

5µm, 2.0 mL/min; PDA detection) using a different solvent
system: an CH3CN–H2O isocratic method (50% CH3CN for 30
min) to yield laxaphycin B4 (1) (tR 10.5 min, 20 mg), laxaphycin
A2 (2) (tR 20.5 min, 0.4 mg), and laxaphycin A (tR 26.7 min, 20
mg).
3 and Table 6. The source and gas-dependent MS parameters
were as follows: CUR 40, CAD medium, IS 4500, TEM 450,
GS1 40, GS2 40.
3.8. Cell Viability Assay (MTT)
20
HCT116 cells were cultured in Dulbecco’s modified Eagle
medium (DMEM, Invitrogen) supplemented with 10% fetal
bovine serum (FBS, Hyclone) under a humidified environment
with 5% CO2 at 37 °C. HCT116 (10,000) cells were seeded in
96-well plates. Cells were treated with a series of concentrations
of laxaphycins in DMSO (0.5% for both single compound and
combinatory studies), 24 h post seeding. Cells were incubated for
an additional 48 h before the addition of the MTT reagent. Cell
viability (fraction survival, Fs) was measured according to the
manufacturer’s instructions (Promega, Madison, WI, USA).
Treatments were done in triplicate. Nonlinear regression analysis
was carried out using GraphPad Prism software for IC50 value
calculations.
Laxaphycin B4 (1). White amorphous solid; [α]D
15.6 (c
0.09, MeOH); UV (MeOH) λmax 200 nm (ε 20893), 230 nm (ε
2882), 270 nm (ε 362); NMR data, H NMR,13C NMR, COSY,
1
HSQC, HMBC and ROESY in DMSO-d6, see Table 1;
HRESI/APCIMS m/z [M
+
Na]+ 1463.8334 (calcd for
C66H116N14NaO21 1463.8337).
20
Laxaphycin A2 (2). White amorphous solid; [α]D +6.9(c
0.032, MeOH); UV (MeOH) λmax 200 nm (ε 26788), 230 nm (ε
11346); NMR data, H NMR, COSY, HSQC, HMQC, HMBC
1
and ROESY in CH3CN-d3, see Table 4; HRESI/APCIMS m/z [M
+ Na]+ 1204.6930 (calcd for C59H95N11NaO14 1204.6958).
20
Laxaphycin A. White amorphous solid. [α]D +25.0 (c 0.5,
MeOH); UV (MeOH) λmax 202 nm (ε 29346), 230 nm (ε 23604 )
(hormothamnin A from literature [α]D25 + 47.2 (c 1.22, MeOH);
no data of laxaphycin A available)10; NMR data match literature
values.6
3.9. Combination Index (CI) Calculation
The cell viability (fraction survival, Fs) data was obtained
from the MTT assay. For combinatory studies, compound
effects were calculated as levels of cell growth inhibition or
affected fraction (Fa) of treated verses control cells. Affected
fraction (Fa) was obtained using the equation of: Fa = 1- Fs.
Dose–effect analyses and calculation of combination index (CI)
were performed using CompuSyn software (ComboSyn Inc,
Paramus, NJ, USA).21 CI reflects the extent of synergy or
antagonism for two drugs: CI<1, synergy; CI =1, additive effect;
CI>1, antagonism. CI is plotted against levels of cell growth
inhibition or affected fraction (Fa) by computer simulation. The
vertical bars indicate 95% confidence intervals based on
Sequential Deletion Analysis (SDA).
3.5. Synthesis of erythro-3-hydroxy-L/D-aspartic acid.24
Ammonia aqueous solution (28–30%) (2 mL) was added to
the solid (2R,3R)-epoxysuccinic acid (100 mg, 0.76 mmol) at 0
oC. The mixture was stirred 48 h at 45–48 oC, then cooled down
and concentrated under reduced pressure. Water (3 × 15 mL) was
added to the concentrated residue and evaporated again three
times to remove traces of ammonia. The crude product was
subjected to advanced Marfey’s analysis.
Acknowledgments
The research was supported by the National Institutes of
Health grant R01CA172310. We thank the Florida Institute of
Oceanography for supporting use of the R/V Bellows, and the
National Park Service for granting permission to collect within
Dry Tortugas National Park. We thank the crew of R/V Bellows,
J. Kwan, L. A. Salvador, and L. Angermeier for help with the
collection.
3.6. Acid Hydrolysis and Chiral Amino Acid Analysis
Samples of 1 (0.3 mg) and 2 (~0.1 mg) were heated with 6 N
HCl (110 °C, 20 h) and the hydrolysates subjected to chiral
HPLC-MS [column, Chirobiotic TAG (4.6 × 250 mm), Supelco;
solvent, MeOH-10mM NH4OAc (40:60, pH 5.12): flow rate, 0.5
mL/min; detection by ESIMS in positive or negative ion modes
(MRM scan)]. The retention times (tR, min; MRM ion pair,
parent→product) of the authentic amino acids and compound-
dependent MS parameters were listed in Table 2 and Table 5.
The source and gas-dependent MS parameters were as follows:
CUR 50, CAD medium, IS 5500, TEM 750, GS1 65, GS2 65.
Supplementary Data
Structures of laxaphycins and analogues (Figures S1 and S2),
NMR spectra (1H NMR, 13C NMR, COSY, HSQC, HMBC,
ROESY) for compound 1, NMR spectra (1H NMR, COSY,
TOCSY, HSQC, HMBC, ROESY) for compound 2 and NMR
spectra (1H NMR, 13C NMR) for laxaphycin A (Figures S3–S17).
Notes
3.7. Advanced Marfey’s Analysis
Samples of 1 and 2 (30 μg) were subjected to acid hydrolysis
and reconstituted in water. Then, 10 μL of 1 M NaHCO3 and 50
μL of 1-fluoro-2,4-dinitrophenyl-5-L-leucinamide (L-FDLA, 1%
w/v in acetone) or 1-fluoro-2,4-dinitrophenyl-5-DL-leucinamide
(DL-FDLA, 1% w/v in acetone) were added to 25 μL of these
solutions. After heating at 40 °C for 1 h, with frequent mixing,
the reaction mixtures were acidified with 5 μL 2 N HCl,
concentrated to dryness and then reconstituted with 250 μL
MeCN–H2O (1:1). Amino acid standards were made into 50 mM
stock solutions in water, derivatized with L-FDLA or DL-FDLA
in a similar method. Standards and hydrolysates were subjected
to reversed-phase HPLC-MS analysis. Details are listed in Table
The authors declare no competing financial interest.
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