An efficient and cost-effective approach to kahalalide F N-terminal modifications using a nuisance algal bloom of Bryopsis pennata

Article abstract of DOI:10.1016/j.bbagen.2015.05.004

Background: Kahalalide F (KF) and its isomer iso-kahalalide F (isoKF), both of which can be isolated from the mollusk Elysia rufescens and its diet alga Bryopsis pennata, are potent cytotoxic agents that have advanced through five clinical trials. Due to a short half-life, narrow spectrum of activity, and a modest response in patients, further efforts to modify the molecule are required to address its limitations. In addition, due to the high cost in producing KF analogues using solid phase peptide synthesis (SPPS), a degradation and reconstruction approach was employed using natural KF from a seasonal algal bloom to generate KF analogues. Methods: N-protected KF was carefully hydrolyzed at the amide linkage between L-Thr12 and D-Val13 using dilute HCl. The synthesis of the C-terminal fragment began with the formation of hexanoic succinimide ester, followed by a reaction with dipeptides. The final coupling reaction was performed between the semisynthesized Fmoc-KF hydrolysis product and the C-terminal fragment, followed by the deprotection of the Fmoc group. Results: Six KF analogues with an addition of an amino acid residue on the N-terminal chain, D-Val14-isoKF (2), Val13-Val14-isoKF (3), D-Leu14-isoKF (4), D-Pro14-isoKF (5), D-Phe14-isoKF (6), and 3,4-2F-D-Phe14-isoKF (7) were prepared using semisynthesis at the exposed N-terminal chain. Conclusions: The overall yield of the medication was 45%. This approach is economical, efficient and amendable to large-scale production while eliminated a nuisance algal bloom. General significance: B. pennata blooms are capable of producing KF in good yields. The semisynthesis from the natural product produced N-terminal modifications for the construction of inexpensive semisynthetic KF libraries.

Full text of DOI:10.1016/j.bbagen.2015.05.004

Biochimica et Biophysica Acta 1850 (2015) 1849–1854
Contents lists available at ScienceDirect
Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbagen
An efficient and cost-effective approach to kahalalide F N-terminal
modifications using a nuisance algal bloom of Bryopsis pennata
a
a
d
a,b,c,
Bin Wang , Amanda L. Waters , Frederick A. Valeriote , Mark T. Hamann
⁎
a
Division of Pharmacognosy, Department of BioMolecular Sciences, The University of Mississippi, University, MS 38677, USA
Division of Pharmacology, Department of BioMolecular Sciences, The University of Mississippi, University, MS 38677, USA
Department of Chemistry and Biochemistry and National Center for Natural Products Research, The University of Mississippi, University, MS 38677, USA
Henry Ford Health System, Department of Internal Medicine, Division of Hematology and Oncology, Detroit, MI 48202, USA
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Background: Kahalalide F (KF) and its isomer iso-kahalalide F (isoKF), both of which can be isolated from the
mollusk Elysia rufescens and its diet alga Bryopsis pennata, are potent cytotoxic agents that have advanced
through five clinical trials. Due to a short half-life, narrow spectrum of activity, and a modest response in patients,
further efforts to modify the molecule are required to address its limitations. In addition, due to the high cost in
producing KF analogues using solid phase peptide synthesis (SPPS), a degradation and reconstruction approach
was employed using natural KF from a seasonal algal bloom to generate KF analogues.
Received 25 January 2015
Received in revised form 27 April 2015
Accepted 4 May 2015
Available online 9 May 2015
Keywords:
Methods: N-protected KF was carefully hydrolyzed at the amide linkage between L-Thr12 and D-Val13 using di-
lute HCl. The synthesis of the C-terminal fragment began with the formation of hexanoic succinimide ester,
followed by a reaction with dipeptides. The final coupling reaction was performed between the semisynthesized
Fmoc–KF hydrolysis product and the C-terminal fragment, followed by the deprotection of the Fmoc group.
Results: Six KF analogues with an addition of an amino acid residue on the N-terminal chain, D-Val14–isoKF (2),
Val13–Val14–isoKF (3), D-Leu14–isoKF (4), D-Pro14–isoKF (5), D-Phe14–isoKF (6), and 3,4-2F-D-Phe14-isoKF (7)
were prepared using semisynthesis at the exposed N-terminal chain.
Kahalalide F
Partial hydrolysis
N-terminal modification
Conclusions: The overall yield of the medication was 45%. This approach is economical, efficient and amendable to
large-scale production while eliminated a nuisance algal bloom.
General significance: B. pennata blooms are capable of producing KF in good yields. The semisynthesis from the
natural product produced N-terminal modifications for the construction of inexpensive semisynthetic KF libraries.
©
2015 Elsevier B.V. All rights reserved.
1
. Introduction
their potential in the treatment of multiple drug resistant cancers [5].
Additionally, in the NCI-60 DTP human tumor cell line screen, the
biological response pattern of KF and isoKF was not similar to any of
compounds included in the library, suggesting that they have a unique
mode of action (MOA) [6]. Although the mechanism of these molecules
is not completely elucidated, they were shown to effectively inhibit the
receptor ErbB3, thus down-regulating PI3K/Akt signaling pathway [7].
The ErbB family including ErbB1 (EGFR, HER1), ErbB2 (HER2, Neu),
ErbB3 (HER3), and ErbB4 (HER4), is part of the epidermal growth factor
receptor (EGFR) family which are tyrosine kinase receptors that
mediate cell interactions in human cells [8,9]. Inhibitors targeting the
ErbB family exert potent anti-tumor activity. Several ErbB1/2 inhibitors,
such as gefitinib, erlotinib, lapatinib, and vandetanib have been ap-
proved by the FDA as anticancer drugs. An ErbB4 inhibitor, dacomitinib,
developed by Pfizer is currently in phase III clinical trials [10]. However,
there are no drugs which target ErbB3 over-expressing tumor cells.
Therefore KF and analogues derived using the methods reported here
could yield promising ErbB3 inhibitors with clinical applications.
KF has completed its safety evaluation in phase I clinical trials in
patients with various advanced solid tumors. Unfortunately, the lead
The Sacoglossan sea slug, Elysia rufescens, is an herbivorous marine
mollusk. These and many other mollusks protect themselves from
predatory attacks by accumulating defensive chemicals. In this case
E. rufescens accumulates kahalalide F (KF) in high quantities which is
also found in their dietary algae Bryopsis spp. [1] KF occurs naturally
as a mixture with its isomer, isoKF, and is a depsipeptide isolated from
both E. rufescens and Bryopsis pennata (Fig. 1) [2,3]. KF and isoKF exhibit
significant activity against human prostate, breast, non-small cell lung,
and colon solid tumors, in particular prostate and breast cancer cell
lines are highly sensitive with IC50 values ranging from 0.07 to
0
.28 μM [2–5]. The cytotoxicity of KF and isoKF did not correlate with
the expression level of MDR1 in prostate and breast cancer, indicating
⁎
Corresponding author at: Divisions of Pharmacognosy and Pharmacology,
Department of BioMolecular Sciences; Department of Chemistry and Biochemistry and
National Center for Natural Products Research, The University of Mississippi, University,
MS 38677, USA. Tel.: +1 662 915 5730; fax: +1 662 915 6975.
E-mail address: mthamann@olemiss.edu (M.T. Hamann).
http://dx.doi.org/10.1016/j.bbagen.2015.05.004
0304-4165/© 2015 Elsevier B.V. All rights reserved.

1850
B. Wang et al. / Biochimica et Biophysica Acta 1850 (2015) 1849–1854
B
A
Fig. 1. (A) Structures of KF and isoKF; (B) Sources of KF: E. rufescens (Photo taken by Megan Huggett) and B. pennata.
was dropped from phase II clinical trials due to a lack of efficacy despite
results indicating a limited number of patients achieved a positive
response [11]. The pharmacokinetic studies suggested that KF has a
short half-life (approximately 30 min) [11], which could in part be the
reason for its poor efficacy. However, no further studies have provided
evidence of KF degradation products in vivo. Considering the high
potential for a cytotoxic drug candidate from this class, several attempts
have been made to modify the molecule to produce analogues with a
higher potency, better delivery, or longer half-life. The structure-activity
relationship (SAR) analysis based on the analogues prepared using solid
phase peptide synthesis (SPPS) revealed that the cyclic peptide system
(amino acid residues 1–6) and the configurations of amino acid residues
7–13 are crucial to the activity. The substitutions with aliphatic residues,
especially the modifications of the N-terminal lipid chain, can increase
Scheme 1. Semisynthesis of KF analogues.

B. Wang et al. / Biochimica et Biophysica Acta 1850 (2015) 1849–1854
1851
Table 1
Structure of KF analogues
isoKF (1)
D-Val14–isoKF (2)
Val13–Val14–isoKF (3)
D-Leu14–isoKF (4)
D-Pro14–isoKF (5)
D-Phe14–isoKF (6)
3,4-2F-D-Phe14–isoKF (7)
Structure
AA13
AA14
D-Val
–
D-Val
D-Val
L-Val
L-Val
D-Val
D-Leu
D-Val
trans-D-Pro
D-Val
D-Phe
D-Val
3,4-2F-D-Phe
the activity [12]. Although the SPPS technique is well developed, more
than 29 steps were involved [12,13]. Challenges such as the peptide
cyclization by coupling L-Phe3 with D-Val4 and the formation of Z-
didehydroaminobutyric acid residue [(Z)-Dhb2] decreased the overall
yield. Additionally, it is costly to synthesize 1 kg of KF due in part to the
high price of Fmoc-D-allo-Ile and Fmoc-D-allo-Thr. Semisynthetic ap-
proaches based on natural KF isolated from B. pennata blown on shore
after an algal bloom could provide a less expensive method to produce
KF congeners. Our group has modified natural KF at the Orn-residue
which yielded two molecules with a similar or higher potency in selected
cancer cell lines [14,15]. In this report we provide an approach to produce
modified KF analogues with additional amino acid residues on the N-
terminal side chain after removal of the terminal fatty acid by selective
hydrolysis between threonine and valine.
2. Materials and methods
2.1. General experiment procedures
NMR spectra were recorded on a Bruker Avance DRX-400 spectrom-
1
13
eter. The H and C NMR chemical shifts were reported in ppm. IR spec-
tra were measured on a PerkinElmer Spectrum 100 FT-IR spectrometer.
HPLC–ESI-TOF was performed on Bruker MicroTOF spectrometer in line
with an Agilent 1100 Series HPLC system and G1316A DAD detector.
Table 2
1
13
a
H and C NMR data of additional amino acid residues of 2–7 (δ in ppm, DMSO-d
6
).
Compound
Formula
Amino acid
Position
δC, mult
δC, mult
2
C
C
C
80
80
81
H
H
H
134
134
136
N
N
N
15
15
15
O
O
O
17
17
17
D-Val 14
Val 14
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
6
1
2
3
4
5
1
2
3
4
171.7
59.0, CH
31.4, CH
18.3, CH
19.8, CH
171.6
59.0, CH
31.5, CH
18.3, CH
19.7, CH
172.9, qC
51.5, CH
41.3, CH
25.0, CH
23.2, CH
22.2, CH
172.2, qC
59.7, CH
31.9, CH
26.5, CH
46.5, CH
171.4, qC
54.1, CH
37.5, CH
4.30, m
2.02, m
0.86, m
0.79, m
3
3
3
4
4.28, m
2.01, m
0.87, m
0.80, m
3
3
D-Leu 14
4.36, m
1.41, 1.48, m
1.58, m
0.86, m
0.83, m
2
3
3
5
6
C
C
80
84
H
131
134
N
N
15NaO17
D-Pro 14
D-Phe 14
4.49, m
2
2
2
2.00, 2.26, m
1.36, 1.51, m
3.38, 3.52, m
H
15
O
17
4.60, m
2.75, 3.05, m
2
138.7, qC
129.6, CH
128.3, CH
126.5, CH
171.5, qC
54.1, CH
5
6
7
1
2
3
4
5
6
7
8
9
,5′
,6′
7.15, m
7.17, m
7.20, m
7
C
81
H
132
F
2
N
15
O
17
3,4-2F-D-Phe 14
4.62, m
2.72, 2.98, m
37.1, CH
2
136.2, qC
117.3, CH
149.5, qC
148.5, qC
118.6, CH
126.5, CH
7.26, m
7.30, m
7.07, m
a
Assignments based on 1H, 13C, and HSQC NMR (13C 100/ H 400 MHz) experiments at room temperature.
1

1852
B. Wang et al. / Biochimica et Biophysica Acta 1850 (2015) 1849–1854
Preparative HPLC was carried out on a Waters PrepLC system with a
single wavelength detector. Phenomenex Luna C18 5 μm column
Table 3
Differential cytotoxicity of KF and isoKF against human tumor cell lines.
a
(
250 mm × 20.2 mm) was used. Optical rotations were measured on a
H125
MCF-7
LNCAP
OVC-5
MDA
HepG2
JASCO DIP-370 polarimeter.
KF
Δ CEM
Δ CFU
Δ CEM
Δ CFU
300
450
350
500
450
600
400
600
250
450
150
300
450
600
500
650
250
500
350
500
450
650
450
600
2
.2. Isolation of KF and isoKF mixture from B. pennata
isoKF
a
B. pennata (30 kg, wet weight) was collected on the beaches at Black
Values shown are differential cell killing against either leukemia, CEM (Δ CEM) or
normal (Δ CFU) cells.
Point, O'ahu, during the algal bloom season. An EtOH extract of
B. pennata was fractionated on a silica flash column with a gradient sol-
vent system of hexanes–EtOAc (100:0, 50:50, 0:100) and EtOAc–MeOH
(s), 1389 (s), 1344 (s), 1230 (s), 1024 (s) cm−1; HRESIMS m/z calc. for
+
(
50:50, 0:100). Two EtOAc–MeOH fractions were further eluted on an
HP-20 column with MeOH–H O (50:50, 60:40, 80:20, 90:10, 100:0).
The MeOH–H O (90:10) and 100% MeOH fractions were subjected to
18 glass open-tubular column with an isocratic solvent system of
C
84
H
134
N
15
O
17 [M + H] 1625.0077, detected 1625.0131.
3,4-2F-D-Phe14–isoKF (7, 6.2 mg), white amorphous powder.
2
2
5
D
[α] −18 (c 0.1, MeOH); UV (DAD) λmax (MeOH) 208, 228 nm; IR
2
C
neat (KBr) 3288 (s, br), 2965 (s), 2932 (s), 2877 (s), 1736 (s), 1641
(s), 1521 (s), 1455 (s), 1389 (s), 1343 (s), 1280 (s), 1233 (s), 1026
2
MeOH–H O (90:10), followed by preparative HPLC (Phenomenex C18
−
1
132 2 15
(s) cm ; HRESIMS m/z calc. for C84H F N O
17 [M + H]+
column 250 mm × 21.2 mm) with a gradient elution of MeCN–H
70:30–100:0) at a flow rate of 8 mL/min to afford a KF and isoKF
mixture (0.95 g, 0.0032% yield).
2
O
(
1660.9888, detected 1660.9999.
2.4. Disk diffusion assay
2
.3. Semisynthesis of KF analogues from an isomeric mixture
The synthetic procedures are displayed in Scheme 1. The experimental
The cytotoxicity of natural KF and its analogues was tested using a
soft-agar based disk diffusion assay [16,17]. The screen included 11
cell types: human leukemia CCRF-CEM, nine solid tumors (human
colon HCT116, human lung H125, human ovarian OVCAR5, human
breast cancers MCF7 and MDA-231, human prostate LNCaP, human
pancreatic cancer PANC1, human liver cancer HePG2, and human
brain U251N) as well as a human normal cell (hematopoietic progenitor
cell, CFU-GM).
The human cancer cell lines were maintained in cell culture. They
were removed from their cultures by a trypsin–collagenase–DNAase
cocktail. For plating of all of the cell types other than the normal CFU,
the 60 mm plates were first prepared with a hard agar bottom layer
(0.6% agar in RPMI-1640 plus 15% BCS). A soft agar top layer (0.3%
agar with the serum and media as above) plus 30,000 cells in 3 mL
were poured into the plates and allowed to solidify.
details are described in the Supporting Information.
25
IsoKF (1, 6.2 mg), white amorphous powder. [α]
D
−14 (c 0.1,
MeOH); UV λmax (MeOH) 205, 224 nm; IR neat (KBr) 3280 (s, br),
2
1
C
964 (s), 2932 (s), 2877 (s), 1736 (s), 1632 (s), 1514 (s), 1465 (s),
−
1
383 (s), 1342 (s), 1227 (s), 1025 (s) cm ; HRESIMS m/z calc. for
+
75
H
125
N
14
O
16 [M + H] 1477.9393, detected 1477.9453.
D-Val14–isoKF (2, 6.1 mg), white amorphous powder. [α]
.1, MeOH); UV (DAD) λmax (MeOH) 205, 224 nm; IR neat (KBr) 3280
2
5
D
−16 (c
0
(s, br), 2964 (s), 2932 (s), 2876 (s), 1736 (s), 1633 (s), 1515 (s), 1464
−
1
(
s), 1383 (s), 1342 (s), 1227 (s), 1025 (s) cm ; HRESIMS m/z calc. for
+
C
80 134 15
H N O
17 [M + H] 1577.0077, detected 1577.0082.
Val13–Val14–isoKF (3, 6.4 mg), white amorphous powder. [α]
c 0.1, MeOH); UV (DAD) λmax (MeOH) 205, 224 nm; IR neat (KBr) 3280
2
5
D
−14
(
For human CFU-GM, the cells were obtained from Poietic Technolo-
gies, Inc. (Gaithersburg, MD) overnight and washed twice with PBS. A
(s, br), 2964 (s), 2932 (s), 2876 (s), 1736 (s), 1633 (s), 1515 (s), 1464 (s),
−
1
1
C
383 (s), 1342 (s), 1227 (s), 1025 (s) cm ; HRESIMS m/z calc. for
6
+
total of 1.5 × 10 cells were plated in 3 ml of 0.3% agar with the addition
80
H
134
N
15
O
17 [M + H] 1577.0077, detected 1577.0137.
D-Leu14–isoKF (4, 5.8 mg), white amorphous powder. [α]
.1, MeOH); UV (DAD) λmax (MeOH) 205, 224 nm; IR neat (KBr) 3281
of 10% L-cell conditioned media (which provides colony stimulating
factor) in MEM-alpha plus 10% BCS.
2
5
D
−30 (c
0
Natural KF and its analogues were provided as a lyophilized powder
and were dissolved in DMSO at 2–3 mg/mL and 15 μL (30–45 ug) was
dropped onto a 6.5 mm filter disk. The disk was allowed to dry
overnight and then placed close to the edge of the petri dish. The plates
were incubated for 7–10 days (depending upon the cell type) and
examined by an inverted stereo-microscope (10×) for measurement
of the zone of inhibition measured from the edge of the filter disk to
the beginning of normal-sized colony formation. The diameter of the
filter disk, 6.5 mm, was arbitrarily taken as 200 units. A difference in
zones between solid tumor cells and either normal or leukemia cells
of 250 units defines solid tumor selective compounds. If the test
material was excessively toxic at the first dosage, a range of dilutions
(
(
C
s, br), 2964 (s), 2932 (s), 2874 (s), 1736 (s), 1633 (s), 1516 (s), 1462
−
1
s), 1384 (s), 1343 (s), 1227 (s), 1024 (s) cm ; HRESIMS m/z calc. for
+
81
H
136
N
15
O
17 [M + H] 1591.0233, detected 1591.0335.
D-Pro14–isoKF (5, 5.6 mg), white amorphous powder. [α]
.1, MeOH); UV (DAD) λmax (MeOH) 205, 224 nm; IR neat (KBr) 3281
2
5
D
−16 (c
0
(s, br), 2965 (s), 2932 (s), 2877 (s), 1736 (s), 1633 (s), 1515 (s), 1464
−
1
(
C
s), 1383 (s), 1342 (s), 1227 (s), 1025 (s) cm ; HRESIMS m/z calc. for
+
80 131
H N15NaO17 [M + Na] 1596.9745, detected 1596.9690.
25
D-Phe14–isoKF (6, 6.4 mg), white amorphous powder. [α]
D
−22 (c
0
.1, MeOH); UV (DAD) λmax (MeOH) 205, 224 nm; IR neat (KBr) 3284
(s, br), 2965 (s), 2932 (s), 2873 (s), 1736 (s), 1633 (s), 1525 (s), 1454
Scheme 2. Proposed mechanism of partial hydrolysis of Fmoc-KF.

B. Wang et al. / Biochimica et Biophysica Acta 1850 (2015) 1849–1854
1853
of the agents (at either 1:4 or 1:10 decrements) against the same
tumors were retested. At some dilution, quantifiable cytotoxicity was
invariably obtained for KF.
similar or higher potency in selected cancer cell lines [14,15]. Perhaps it
will be necessary to reconsider the KF modification approach focusing
on improving the short half-life instead of increasing its potency.
Much has been learned about the optimization of half-life, metabolism
and bioavailability from the 15 year journey leading to the development
for caspofungin (Cancidas®), the first in class antifungal derived from
the echinocandins. These lipopeptides inhibit enzyme (1 → 3)-β-D-glu-
can synthase ultimately disrupting the fungal cell wall. This drug was
based on the original natural product pneumocandin which exhibited
good antifungal activity but poor water solubility, stability and pharma-
cokinetic profile [20]. Years of medicinal chemistry studies addressed
these problems and it was found that simply adding cationic groups to
the structure greatly improved not only the potency and selectivity of
the molecule but also increased water solubility, stability and the phar-
macokinetic profile [20]. The same challenges regarding water solubility
and stability associated with the kahalalides may be addressed with the
assistance of the approach presented here. Alternatively, simply
exchanging the hydrogen(s) for deuterium(s) in the KF molecule
could have a positive impact on prolonging half-life. The C–D bond is
stronger than C–H bond due to the deuterium isotope effect, making it
a more stable bond to chemical and enzymatic bond cleavages [21,22].
Several deuterated analogues, such as tramadol, linezolid, and indiplon,
have higher biological half-lives than their non-deuterated drugs [22].
The next step in the KF modifications will include either the addition
of more cationic groups or exchange of selective hydrogen(s) in N-
terminal residues to deuterium(s) on the basis of our semisynthetic
approach in this paper to investigate the possibility of more stable and
active analogues.
3
. Results and discussion
3
.1. KF analogues
Seven semisynthetic analogues, isoKF (1), D-Val14–isoKF (2), Val13–
Val14–isoKF (3), D-Leu14–isoKF (4), D-Pro14–isoKF (5), D-Phe14–isoKF
(
6), and 3,4-2F-D-Phe14–isoKF (7) were synthesized (Table 1). The
structures were confirmed using HRMS, H, C, and 1H- C HSQC
1
13
13
NMR overlay experiments (Table 2, Figs. S1–S19). The overall yield
(
synthesis and purification) for each analogue was nearly 45%.
3
.2. Proposed mechanism of partial hydrolysis of KF
N-protected KF was hydrolyzed at the amide linkage between
L-Thr12 and D-Val13 under acidic condition at a relative high yield
70%). It is hypothesized that the release of the MeHex–D-Val13
(
fragment is kinetically faster than any other residues, which results
from the contribution of the hydroxy group of L-Thr12. The proposed
mechanism of the partial hydrolysis is based on N → O acyl rearrange-
ment [18]. The oxygens on the amide carbonyl groups would be proton-
ated under acidic conditions. The nucleophilic β-hydroxy group interacts
with the carbonyl of D-Val13, thus forming a hydroxyoxazolidine as an in-
termediate, followed by acyl rearrangement to produce an ester which is
more easily hydrolyzed than the amide linkage (Scheme 2).
Transparency document
3
.3. Cytotoxicity of KF analogues
The Transparency document associated with this article can be
found, in the online version.
Natural KF and isoKF demonstrated cytotoxicity based on the zone
assay although the activity was minimal for HCT-116, U251N and
PANC-1. Unfortunately, the other 6 analogues tested were inactive in
the assay against all 8 solid tumor cell lines tested. Further, KF and
isoKF both demonstrated selective cytotoxicity to H125, MCF-7,
LNCaP, MDA-235 and HepG2 when compared to either leukemia,
CEM, or normal cell, CFU-GM. The zone differential (selectivity) is pre-
sented in Table 3 and it is noted that the zone differentials were larger
when compared to normal cells than when compared to the leukemia
cells. Indeed, there was little or no cell killing noted against the normal
cells, as has been previously commented upon Natural KF and synthetic
isoKF all display selectivity against MCF-7 and OVC-5 cell lines [6].
Acknowledgement
This investigation was supported by the NCCIH RO1AT007318 and
conducted in a facility constructed from a Research Facilities Improve-
ment Program NIH Grant Number C06 RR-14503-01. For A.W., this
investigation was supported in part by a National Science Foundation
Graduate Research Fellowship under Grant No. 1144250. For B.W., this
investigation was supported in part by PharmaMar, Madrid, Spain.
Appendix A. Supplementary data
4
. Conclusions
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.bbagen.2015.05.004.
B. pennata is a potentially invasive green algae native to Hawaii, but
is also widely distributed off the coast of Australia, the Atlantic Ocean,
the Mediterranean, the Caribbean, the Indian and the Pacific Oceans
References
[
1] M.A. Becerro, G. Goetz, V.J. Paul, P.J. Scheuer, Chemical defenses of the sacoglossan
[19]. When conditions are favorable, these algae can grow quickly and
mollusk Elysia rufescens and its host alga Bryopsis sp, J. Chem. Ecol. 27 (2001)
become toxic to most herbivorous organisms [19]. During the algal
bloom season, considerable quantities of B. pennata are washed onto
the shore. A large quantity of algae producing kilograms of KF (0.003%
yield) could be recovered easily. The route presented here could
produce N-terminal modifications for the construction of improved and
inexpensive KF libraries from the naturally occurring blooms and rational
drug design. This approach is economical, efficient and amendable to
large-scale production.
It was reported that the substitutions with aliphatic residues of the
N-terminal lipid chain could increase the activity [12]. In this paper,
we proposed a modification method of N-terminal elongation using
one or two more low-polar amino acid residues on KF. However, the
products, except for isoKF, did not display activity against selected
cancer cell lines. Of the hundreds of KF analogues produced by different
research groups, only two modifications on the L-Orn residue showed a
2
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