Pembamide, a N-methylated linear peptide from a sponge Cribrochalina sp.

Article abstract of DOI:10.1016/j.tetlet.2016.05.054

A new highly N-methylated linear peptide, pembamide (1), has been isolated from the marine sponge Cribrochalina sp. (family Niphatidae) collected off the coast of Pemba (Tanzania). The planar structure of 1 was assigned on the basis of extensive 1D and 2D NMR spectroscopy and mass spectrometry. The absolute configuration of the amino acid residues in 1 was determined by application of the Advanced Marfey's method. Compound 1 displayed significant cytotoxicity against three human tumor cell lines with GI₅₀values in the micromolar range.

Full text of DOI:10.1016/j.tetlet.2016.05.054

Accepted Manuscript
Pembamide, a N-Methylated Linear Peptide from a Sponge Cribrochalina sp
Carlos Urda, Marta Pérez, Jaime Rodríguez, Carlos Jiménez, Carmen Cuevas,
Rogelio Fernández
PII:
S0040-4039(16)30577-9
DOI:
http://dx.doi.org/10.1016/j.tetlet.2016.05.054
TETL 47673
Reference:
To appear in:
Tetrahedron Letters
Received Date:
Revised Date:
Accepted Date:
11 April 2016
11 May 2016
13 May 2016
Please cite this article as: Urda, C., Pérez, M., Rodríguez, J., Jiménez, C., Cuevas, C., Fernández, R., Pembamide,
a N-Methylated Linear Peptide from a Sponge Cribrochalina sp, Tetrahedron Letters (2016), doi: http://dx.doi.org/
1
0.1016/j.tetlet.2016.05.054
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Essential title page information
Title
Pembamide, a N-Methylated Linear Peptide from a Sponge Cribrochalina sp.
Author names and affiliations
a
a
b
b
a,
Carlos Urda, Marta Pérez, Jaime Rodríguez, Carlos Jiménez, Carmen Cuevas * and Rogelio
a,
Fernández *
a
Medicinal Chemistry Department, PharmaMar S. A., Pol. Ind. La Mina Norte, Avenida de los
Reyes 1, 28770, Colmenar Viejo (Madrid), Spain
b
Departamento de Química Fundamental, Facultade de Ciencias e Centro de Investigacións
Científicas Avanzadas (CICA) Universidade da Coruña. 15071 A Coruña, Spain.
Corresponding autor
Rogelio Fernández (rfernandez@pharmamar.com) Tel: +34 91 823 4664. Fax: +34 91 846 6001
1

Abstract
A new highly N-methylated linear peptide, pembamide (1), has been isolated from the marine
sponge Cribrochalina sp. (family Niphatidae) collected off the coast of Pemba (Tanzania). The
planar structure of 1 was assigned on the basis of extensive 1D and 2D NMR spectroscopy and
mass spectrometry. The absolute configuration of the amino acid residues in 1 was determined by
application of the Advanced Marfey´s method. Compound 1 displayed significant cytotoxicity
against three human tumor cell lines with GI values in the micromolar range.
5
0
Keywords: Cribrochalina; isolation; peptide; advanced Marfey’s method; pembamide.
1
Numerous studies on N-methylamino acids (NMA) containing peptides reveal that replacing
natural amino acids with N-methylated analogs produce changes in the pharmacokinetic properties
2
,3
by blocking the proteolytic cleavage sites, decreasing the number of hydrogen bonds, increasing
4
,5,6
lipophilicity and, in many cases, enhancing membrane permeability,
as well as altering the
7
,8,9
conformational characteristics or properties of the amide bonds.
Thus, cyclic N-methylamino
acids such as vancomycin, cyclosporine and actinomycin D isolated from microorganisms have
found clinical use in part due to the physical properties and chemical stability conferred by the
1
0,11,12
NMA present in their structures.
Highly N-methylated linear peptides have also been isolated from different marine sources. Thus,
1
3
koshikamides are peptides with four or more N-methylamino acids isolated from the marine
1
4
sponge Theonella sp. Dictyonamide A, was isolated from the fungus K063 separated from the red
1
5
alga Ceratodictyon spongiosum, while the linear octapeptides RHM-1 and RHM-2 were obtained
2

from a marine sponge-derived fungus Acremonium sp. Marine cyanobacteria are also sources of
1
6
17,18,19,20,21
this type of compounds. For instance, apramides A-G, dragonamides A-E,
almiramides
2
2
23
23
24
A-C, carmabin A, dragomabin, and kurahyne have been isolated from the marine
2
5
Cyanobacterium Lyngbya majuscule whilst micromide was found from a species belonging to the
genus Symploca.
On the other hand, marine sponges belonging to the genus Cribrochalina sp. have been found to
contain a variety of structurally unique metabolites with interesting biological activities. These
2
6
include the polyacetylenic alcohol duryne that inhibits the growth of several human tumor cell
2
7
lines including leukemia, colon, lung, gastric, and breast cancers, and the kapakahines , a family
of cyclic peptides. Other studies have been related with marine alkaloids such as the manzadin
2
8
29
family of bromopyrrole alkaloids, pyrinadine A which was the first pyridine alkaloid isolated
from natural sources with an azoxy moiety and showed cytotoxic activity against L1210 murine
leukemia and KB human epidermoid carcinoma cells in vitro. Also the tetrahydroisoquinoline
3
0
cribrostatins 1-5, displaying some of them cytotoxic, antibacterial and antifungal activities, were
found in Cribochalina species.
2. Results and discussion.
3
1
As part of our ongoing efforts to find novel antitumor agents from marine organisms, a detailed
biological investigation of a specimen of the Tanzanian sponge Cribrochalina sp. was undertaken.
Preliminary data from the extracts of this sponge displayed cytotoxic activity against the human
tumor cell lines A-549 (lung), HT-29 (colon), and MDA-MB-231 (breast). Bioassay-guided
fractionation of the active organic extract of this sample resulted in the isolation of a new highly N-
methylated linear peptide which was named as pembamide (1). This compound shows significant
cytotoxicity towards different human cancer cells.
3

A sample of the marine sponge Cribrochalina sp. collected by hand off the coast of Pemba Island,
north of Tanzania, was extracted several times using CH Cl /MeOH (1:1). The crude extract was
2
2
subsequently fractionated by vacuum flash chromatography (VFC) on Lichoprep RP-18 using a
gradient mixture of H O, MeOH and CH Cl with decreasing polarity. Bioguide fractionation
2
2
2
against the former human tumor cell lines afforded an active fraction eluted with 100% MeOH that
was subjected to reversed-phase HPLC to yield 1.
Pembamide (1) was obtained as a colourless amorphous solid. The molecular formula of
+
compound 1 was determined to be C H N O (8 degrees of unsaturation) based on the [M + H]
4
6
86
8
9
ion peak at m/z 895.6596 observed in the (+)-HRESI-TOFMS and on NMR data. A peptidic
1
structure was evident from the NMR data in CD OD (Table 1). The H NMR spectrum of 1
3
revealed the presence of five tertiary N-methyl amide singlets at δ 3.16, 3.15, 3.09, 3.08 and 3.05,
H
as well as a characteristic secondary amide NH doublet at δ 8.07 (deuterium exchangeable). Also,
H
the presence of eight low-fielded signals (δ 5.32-4.30) assigned to the -protons of amino acids
H
and 15 methyl groups between 1.45 and 0.77 ppm were representative of alkyl amino acids
1
3
residues. Moreover, the C NMR spectrum of 1 exhibits eight carbonyl carbons at δ 174.6, 174.3,
C
172.9, 172.2, 172.1, 172.0, 171.8 and 171.2 attributable to ester/amide functionalities and five N-
1
3
methyl groups at δ 31.8, 31.4, 31.3, 31.2 and 31.0. Further examination of the C NMR data
C
4

showed the presence of eight carbons resonating in the δ 64.0-49.0 ppm range, and 25 signals in
C
the high-field region, supporting the alkyl nature of the side chains of the amino acid residues.
2D NMR experiments of 1, including COSY, TOCSY and edited-HSQC, established the presence
of eight spin systems (Figure 1), three of which belong to the common amino acids alanine, valine
and isoleucine. The identification of the remaining fragments was based on the long range HMBC
correlations detected between N-methyl groups and the corresponding -protons of the amino acid
residues. Thus, two N-methylated isoleucines were deduced by the presence of two HMBC cross
peaks between the N-Me at δ 3.05 and C-2 at δ 58.0 ppm (N-MeIle-1) and between the N-Me at δ
H
C
H
3
.09 and C-2 at δ 58.1 (N-MeIle-2). In the same way, long range correlations from three N-Me
C
groups at 3.08, 3.16 and 3.15 ppm to the corresponding C-2 at δ 64.1, 59.7, 63.8 respectively,
C
allowed us to identify the remaining amino acids as N-methyl valines (N-MeVal-1, N-MeVal-2 and
N-MeVal-3), thereby accounting for all 8 degrees of unsaturation and signifying an overall linear
arrangement.
5

1
13
Table1. NMR Data of 1 in CD OD (500 MHz for H and 125 MHz for C)
3
Amino acid
Pos.
NH
1
2
3
C, mult.
H, mult., J in Hz
Amino acid Pos.
C, mult.
31.3, CH
172.1, C
58.0, CH
34.5, CH
25.3, CH2
19.6, CH3
11.0, CH3
H, mult., J in Hz
3.05, s
Ala
2
-
-
-
N-MeIle-1
N-MeIle-2
N-MeVal-3
Val
NMe
3
171.8, C
49.0, CH
16.4, CH3
1
2
3
4
5
6
-
4.42, q, 7.0
1.45, d, 7.0
5.32, d, 11.5
2.09, m
1.23, m; 0.96 ,m
a
0.85
0.85
a
N-MeVal-1
NMe 31.0, CH
3
3.08, s
NMe
31.4, CH
3
3.09, s
-
5.30, d, 12.0
2.13, m
1
2
3
4
5
171.2, C
-
1
2
3
4
5
6
172.9, C
58.1, CH
34.8, CH
25.2, CH2
19.0, CH3
11.0, CH3
64.1, CH
27.4, CH
15.8, CH3
18.9, CH3
4.65, d, 12.5
2.26, m
0.89, d, 6.5
0.87, d, 6.5
1.23, m; 0.96, m
a
0.85
0.86
a
Ile
NH
1
2
3
4
-
-
-
NMe
31.8, CH
3
3.15, s
-
4.70, d, 9.0
2.24, m
0.96, d, 6.5
0.77, d, 6.5
174.6, C
54.6, CH
37.8, CH
25.5, CH2
18.2, CH3
10.9, CH3
1
2
3
4
5
174.3, C
63.8, CH
27.8, CH
19.7, CH3
19.0, CH3
4.70, d, 8.5
1.86, m
1.52, m; 1.17, m
0.92, d, 7.0
0.89, t, 7.5
5
6
b
N-MeVal-2
NMe 31.2, CH
3
3.16, s
NH
1
-
8.07, d, 7.0
1
2
3
4
5
172.2, C
-
172.0, C
59.0, CH
31.7, CH
19.6, CH3
19.8, CH3
-
59.7, CH
28.6, CH
15.8, CH3
18.6, CH3
5.22, d, 11.0
2.33, m
0.88, d, 6.0
0.80, d, 6.5
2
3
4
5
4.30, d, 5.5
2.16, m
0.92, d, 6.5
0.92, d, 6.5
a
b
Signal overlapped. Multiplicity and coupling constant could not be determined. Signal deuterium exchangeable.
6

A combination of HMBC and ROESY data allowed us to determine the sequence of these units.
Thus, long range correlations from -protons or N-Me groups to the carbonyl carbons of adjacent
amino acids, plus ROESY correlations between the -protons and the N-Me protons allowed us to
establish the sequence as Ala-N-MeVal-1-Ile-N-MeVal-2-N-MeIle-1-N-MeIle-2-N-MeVal-3-Val
as shown as in Figure 1.
Figure 1. Selected ROESY (red) and HMBC (blue) and COSY (bold bonds) correlations for 1.
The mass fragmentation pathways observed in ESIMS/MS of 1 confirmed the proposed
sequence (Figure 2). Thus, the peaks at m/z 298 (Ala/N-MeVal-1/Ile), 411 (Ala/N-MeVal-1/Ile/N-
MeVal-2), 538 (Ala/N-MeVal-1/Ile/N-MeVal-2/N-MeIle-1), 665 (Ala/N-MeVal-1/Ile/N-MeVal-
2
3
/N-MeIle-1/N-MeIle-2) and 778 (Ala/N-MeVal-1/Ile/N-MeVal-2/N-MeIle-1/N-MeIle-2/N-MeVal-
) displayed in the MS/MS experiment of 1 were used to confirm the former proposed sequence.
The molecular formula of those fragments was confirmed by HRESIMS/TOF (Figure 2).
7

A
B
Figure 2. (A) Fragmentation pathway of pembamide (1) along with the (+)-HRESIMS/TOF of
the fragments. (B) MS/MS ESI fragmentation at (m/z)= 778.6.
8

The absolute configuration of all units in 1 was established as using the Advanced Marfey
L
3
2,33
Method.
Acid hydrolysis of 1 was subjected to Marfey´s derivatization with 1-fluoro-2-4-
dinitrophenyl-5- -alanine amide ( -FDAA) and racemic -FDAA separately, and then analyzed by
L
L
LD
LC/MS. After comparing both chromatograms, we could assign the configurations to the valine,
L
alanine, and the three N-Me-valines. Since N-Me- -Val and -Ile have the same molecular weight,
L
L
3
4
these two amino acids were distinguished by their UV spectrum. Although Marfey’s analysis
clearly suggested the -configuration of the N-MeIle moieties, we were unable to distinguish N-
L
Me- -Ile and N-Me- -allo-Ile by this method due to the lack of a standard of N-Me- -allo-Ile. In
L
L
L
order to distinguish between the and -allo configurations in 1, racemization of the N-Me- -Ile
D
L
L
standard was carried out, and the reaction mixture, containing both N-Me- -Ile and N-Me- -allo-
L
D
Ile, was subjected to advanced Marfey´s analysis using LC-MS. The retention times of -Marfey
L
derivatives of the N-Me- -Ile standard and the N-Me- -Ile units, in the hydrolysate of 1, were
L
L
identical. In the same way, we determined that the configuration for the remaining isoleucine was
L-Ile instead of -allo-Ile.
L
Cell proliferation assays against the human tumor cell lines A-549 (lung), HT-29 (colon), and
MDA-MB-231 (breast) displayed that pembamide exhibited significant cytotoxic activity with a
GI range at micromolar level (Table 2). As a positive standard antitumour compound,
5
0
doxorubicin is usually included in our assays and it is tested in parallel to the compounds,
following identical procedure. The results obtained with doxorubicin are included in the table 2
9

Table 2. Cytotoxic Activity Data ( M) of 1.
Tumor cell lines
Compound
Breast, MDA-MB-
31
Colon, HT-
29
Lung, NSCLC
A-549
2
GI50
3.35
.15
3.80
3.91
4.13
0.27
0.86
>17.2
2.46
> 11.2
> 11.2
0.21
Pembamide
Doxorubicin
TGI
8
LC50
> 11.2
0.15
GI50
TGI
0
.50
0.85
LC50
2.41
>17.2
GI , compound concentration that produces 50% inhibition on cell growth as compared to control cells, TGI,
5
0
compound concentration that produces total growth inhibition as compared to control cells, and LC , compound
5
0
concentration that produces 50% cell death as compared to control cells.
In summary, a new N-methylated linear peptide, pembamide (1), with a structure of Ala-N-
MeVal-1-Ile-N-MeVal-2-N-MeIle-1-N-MeIle-2-N-MeVal-3-Val, was isolated from an extract of a
sponge Cribrochalina sp., being the first time that this type of compounds is isolated from
specimens belonging to this genus. Application of the advanced Marfey's method has allowed us to
determine its absolute configuration. This compound showed significant cytotoxic activity against
three human tumor cell lines. As in the case of other linear N-methylated peptides that have been
isolated from marine sources, such as the octapeptides RHM1 and RHM2, the true origin of the
3
5,36
compounds maybe from microorganisms living within the marine invertebrates.
actual source of pembamide remains to be determined.
As such, the
5. Acknowledgments
1
0

We gratefully acknowledge the help of our PharmaMar colleagues, C. de Eguilior and S. Bueno for
collecting the marine samples, Dr. L. F. García for the design of the biological assays, Dr. S. Munt
for revision of the manuscript, and Dr. S. González for performance of the NMR experiments. We
also thank Dr. M. J. Uriz (CEAB-Centro de Estudios Avanzados de Blanes, Spain) for determining
the sponge taxonomy. We also acknowledge Mr. P. J. Ruysenaars, The Pemba Channel Fishing
Club, Shimoni (Kenya). Ministry of Livestock and Fisheries Development, Fisheries Department
(Tanzania).
6. Supplementary data
1
13
Experimental procedure and H, C, HSQC, COSY, HMBC and ROESY NMR spectra of
pembabide (1) along with its analysis by advance Marfey´s method. Supplementary data
associated with this article can be found, in the online version, at xx.
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1
2

Highlights




A new linear peptide, named Pembamide, was isolated from the marine sponge
Cribrochalina sp.
Structural elucidation was determined by a combination of NMR and MS-MS
experiment.
The absolute configuration of the amino acid residues was determined by
application of the Advanced Marfey´s method.
Pembamide has shown interesting cytotoxic properties against several tumour
cell lines.

Graphical Abstract
Pembamide