Unique polyhalogenated peptides from the marine sponge Ircinia sp.

Article abstract of DOI:10.3390/MD18080396

Two new bromopyrrole peptides, haloirciniamide A (1) and seribunamide A (2), have been isolated from an Indonesian marine sponge of the genus Ircinia collected in the Thousand Islands (Indonesia). The planar structure of both compounds 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 and 2 was determined by the application of Marfey's method. Compound 1 is the first dibromopyrrole cyclopeptide having a chlorohistidine ring, while compound 2 is a rare peptide possessing a tribromopyrrole ring. Both compounds failed to show significant cytotoxicity against four human tumor cell lines, and neither compound was able to inhibit the enzyme topoisomerase I or impair the interaction between programmed cell death protein PD1 and its ligand, PDL1.

Full text of DOI:10.3390/MD18080396

marine drugs
Article
Unique Polyhalogenated Peptides from the Marine
Sponge Ircinia sp.
Rogelio Fernández 1, Asep Bayu 2, Tri Aryono Hadi 3, Santiago Bueno 1, Marta Pérez 1,
Carmen Cuevas 1 and Masteria Yunovilsa Putra 2
* ,
1
Natural Products Department, PharmaMar S.A., Pol. Ind. La Mina Norte, Avda. de los Reyes 1,
28770 Colmenar Viejo (Madrid), Spain; rfernandez@pharmamar.com (R.F.); sbueno@pharmamar.com (S.B.);
ccuevas@pharmamar.com (C.C.)
Research Center for Biotechnology, Indonesian Institute of Sciences, Jl. Raya Jakarta-Bogor No. Km46,
Cibinong, Bogor, Jawa Barat 16911, Indonesia; asepbayu86@gmail.com (A.B.);
masteria.yunovilsa@gmail.com (M.Y.P.)
2
3
Research Center for Oceanography, Indonesian Institute of Sciences, Jl. Pasir Putih I, Ancol Timur,
Jakarta 14430, Indonesia; tri.aryono.hadi@lipi.go.id
*
Correspondence: mperez@pharmamar.com; Tel.: +34-91-823-4527
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 7 July 2020; Accepted: 27 July 2020; Published: 28 July 2020
Abstract: Two new bromopyrrole peptides, haloirciniamide A (
1
) and seribunamide A (2), have been
isolated from an Indonesian marine sponge of the genus Ircinia collected in the Thousand Islands
(Indonesia). The planar structure of both compounds 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
and
2
was determined by the application of Marfey’s method. Compound
1 is the first
dibromopyrrole cyclopeptide having a chlorohistidine ring, while compound
2
is a rare peptide
possessing a tribromopyrrole ring. Both compounds failed to show significant cytotoxicity against
four human tumor cell lines, and neither compound was able to inhibit the enzyme topoisomerase I
or impair the interaction between programmed cell death protein PD1 and its ligand, PDL1.
Keywords: marine sponge; Ircinia sp.; polyhalogenated peptides; Marfey’s analysis
1. Introduction
Indonesia is located at the center of a biodiversity hotspot, and around 750 structures from
Indonesian waters have been published in the last 50 years [1]. The structural diversity and bioactive
properties of the compounds isolated from this region encouraged us to continue to investigate this
area, which still remains largely unexplored. Thus, one of the recent PharmaMar expeditions was
carried out in the Thousand Islands, in collaboration with Indonesian Institute of Sciences (LIPI).
The Thousand Islands archipelago is located about 25 miles from the coast to the northeast of Jakarta;
the collection site is an area full of gentle rock and coral slopes, and it is potentially a highly productive
area, both in terms of the quantity and the nature of the biodiversity. In this paper, we describe the
isolation of two new peptides isolated from an Ircinia specimen from this area.
Of all the marine organisms investigated, sponges (Porifera) are the most primitive multicellular
animals with ample time to evolve into more complex living organisms. In fact, marine sponges are
recognized as the richest sources of MNP, contributing to nearly 30% of all marine natural products
discovered so far [
a rich source of varied bioactive natural products, including fatty acids [
macrolides [ ], and peptides [10], many of which have biological activities. This structural diversity
could be due to the fact that sponges harbor diverse microorganisms and in numerous cases, bacteria
2]. Previous reports revealed that Marine sponges of the genus Ircinia are known as
3
], steroids [ ], terpenes [ ],
4
,
5
6,7
8,9
Mar. Drugs 2020, 18, 396; doi:10.3390/md18080396
www.mdpi.com/journal/marinedrugs

Mar. Drugs 2020, 18, 396
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isolated from sponges or symbiotic bacteria are the true producers of the compounds found in their
extracts [11]. Specifically, an intriguing group of Ircinia-derived peptides are assumed to be of microbial
origin due to the presence of both d-amino acids and unusual amino acids, as illustrated by the cyclic
hexapeptide waiakeamide from Ircinia dendroides [12] and the cyclotheonamides E4 and E5, which are
cyclic pentapeptides also from Ircinia species [13].
Undeniably, among the compounds isolated from marine sources, linear and cyclic peptides are
recognized as an important class with great structural diversity and a wide range of bioactivities,
and these include the antimalarial carmabin A [14], the antiproliferative jaspamides [15], and the
cytotoxic patellamides [16]. Furthermore, two marine peptide-derived products have reached the
market: ziconotide [17] for analgesic use and a synthetic derivative of dolastatin 10 [18] linked to an
antibody for the treatment of Hodgkin’s lymphoma. PharmaMar has also developed a marine natural
peptide Aplidin, which was originally found in the Ascidian Aplidium albicans and has recently been
approved for commercialization in Australia for the treatment of multiple myeloma. Recent studies
suggest that Aplidin may also have antiviral properties, and a clinical trial to treat patients with
COVID-19 has been initiated.
In the course of our screening program to isolate novel compounds with antitumor properties from
marine sources, the organic extract of an Ircinia specimen collected off the coast of the Thousand Islands
showed hints of activity, and although the fractions did not confirm cytotoxicity, the chromatographic
profiles along with the mass spectra showed peaks that were interesting enough for us to purify them.
Thus, we have isolated two unique peptides haloirciniamide A (1) and seribunamide A (2). It is worth
mentioning that the number of known peptides with a halogenated pyrrole ring is limited, with only
cyclocinamides and corticiamide A [19] as well as gunungamide A [20] having been described as
possessing chlorinated pyrrole rings. Although there are dozens of dibromopyrrolecarboxamide
derivatives from porifera such as nagelamide [21] and carteramine [22], mainly from Agela and
Stylissa species, compound 1 is the first example of a peptide containing a halogenated pyrrole ring
in its structure. Indeed, haloirciniamide A represents a structurally unique cyclopeptide, since it
also has an unprecedented chlorohistidine moiety. Furthermore, there are only two examples of
tribromopyrrole rings derived from natural sources: 2,3,4-tribromopyrrole itself, which was isolated
from the marine Poychaete Polyphysia crassa [23], and tribromopyrrol-2-methylphenol isolated from a
coralline algal-associated Pseudoalteromone [24], with compound 2 being the first of its class.
Details of the isolation and structural elucidation of the new halogenated peptides
1 and 2 are
provided. The results of cytotoxicity and other related antitumor programmed cell death protein (PD1)
and TOPO-I screenings are also described.
2. Results and Discussion
Isolation and Structure Elucidation
The sponge Ircinia sp. was collected by hand while diving in the Thousand Islands (Indonesia).
The specimen was repeatedly extracted using CH2Cl2:MeOH (1:1 v/v). The combined concentrated
extracts, after vacuum liquid chromatography (VLC) and semipreparative reverse-phase HPLC
separations, led to the isolation of the two pure compounds shown in Figure 1.
Compound
1 was isolated as an amorphous white solid. The isotopic distribution observed in the
(+)-LRESIMS mass spectrum with four protonated ion [M+Na]+ peaks at m/z 830, 832, 834, and 836
in the ratio 3:6:3:1 respectively, showed the presence of two bromine atoms and a chlorine atom in
the molecule. The presence of these halogens in the structure was confirmed by (+)-HRESI-TOFMS
analysis, with the ion peak observed at m/z 830.0400 [M+Na]+ corresponding to the molecular
formula C25H3279Br235ClN11O8Na (calcd. 830.0383). Interpretation of the mono NMR data (1H, 13
C
and 1D-TOCSY) compiled in Table 1 and two-dimensional NMR spectra (gHSQC, gCOSY, gHMBC,
and 2D-TOCSY) in CD3OD led to identification of 6 spin systems. Taking into consideration the
seven carbonyl carbon resonances (δC 161.4–175.8) and the number of α-amino acid proton signals

Mar. Drugs 2020, 18, 396
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(δH 4.06–4.84), the peptide nature of compound
1
was expected. This hypothesis was confirmed by the
NH signals observed in the 1H NMR spectrum in CD3OH (δH 7.13–12.68) and DMSO-d6 (δH 7.37–12.03),
with the latter solvent being chosen for full structural elucidation. The COSY correlations observed
between methines at δH 4.23/δC 50.5 and δH 4.49/δC 51.5 with the diastereotopic methylenes at δH 2.87,
3.13/δC 49.2 and δH 3.22, 4.04/δC 40.5 respectively, indicated the presence of two 2,3-diaminopropionic
acid units (Figure 2). Both amino acids were directly connected based on the HMBC correlation of
the NH signals at δH 9.11 for Dap1 and δH 7.72 for Dap2 with the same carbonyl carbon at δC 169.5.
The next amino acid present in the peptide core was an isoserine with a methylene group at δH 2.75,
3.47/δC 42.8, a methine carbon at δH 4.13/δC 67.8, and an NH signal at δH 8.23. Isoserine was linked
to the Dap2 by the correlation between NH at δH 8.23 and the carbonyl carbon of Dap2 at δC 170.6.
In addition, an HMBC correlation of the methine carbon at δH 4.13 and the NH signal at δH 7.13, which
belongs to a unit of isoasparagine (δH 4.58/δC 48.4, δH 2.80, 3.09/δC 35.0), with the carbonyl group at
δC 170.6 allowed the sequence of amino acids to be continued. Finally, the peptide ring was closed
by an NMe-histidine (δH 3.99/δC 65.6, δH 3.05/δC 25.0), whose N-methyl group showed an HMBC
correlation with carbons belonging to the carbonyl group of iAsn at δC 173.5 and its own methine at δC
65.6, with an additional HMBC correlation between the α-aminoacid proton signal of NMe-histidine
and the NH signal at δH 9.11 of Dap1 with the carbonyl carbon at δC 169.0 (Figure 2). NMe-histidine
ring shifts at δC 110.5, δC 128.2, and δH 6.76/δC 109.5 revealed that the non-protonated carbon at δC
128.2 bore one of the three halogen atoms.
Figure 1. Chemical structures of the compounds 1 and 2 isolated from Ircinia sp.
Figure 2. Selected key COSY (bold), HMBC (red), and ROESY (blue) correlations for 1 and 2.

Mar. Drugs 2020, 18, 396
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Table 1. NMR spectroscopy data for 1 (1H NMR MHz, 13C NMR 125 MHz).
δC, Mult b
δ , Mult (J in Hz) b
Pos
δC, Mult a
δ , Mult (J in Hz) a
H
H
Dap1
1
2
3
169.5, C
50.3, CH
49.2, CH2
172.3, C
52.2, CH
50.5, CH2
4.23 ddd (6.2, 6.2, 2.8)
2.87 d (13.9, 6.2)
3.13 d (13.9, 2.8)
9.11 d (6.2)
4.54 dd (5.6, 2.9)
3.17 dd (14.6, 5.6)
3.54 d (14.2)
NH
8.97 d (6.6)*
NH2
NMeClHis
1
2
3
169.0, CO
65.6, CH
24.9, CH2
170.9, CO
67.0, CH
25.9, CH2
3.99 dd (10.6, 3.9)
3.05 m
4.06 dd (10.1, 4.5)
3.19 dd (15.2, 10.1)
3.30 dd (15.2, 4.5)
4
5
110.5, C
109.5, CH
128.2, C
39.6, CH3
173.5, C
135.6, C
120.8, CH
131.0, C
40.4, CH3
175.8, C
6.76 s
2.84 s
6.96 s
3.02 s
6
NMe
1
iAsn
2
35.0, CH2
2.80 dd (16.6, 2.7)
3.09 dd (16.6, 5.8)
4.58 ddd (8.0, 5.8, 2.7)
36.2, CH2
3.02 m
3.27 m
4.84 m
3
4
48.4, CH
172.2, CO
50.3, CH
173.8, CO
NH
NH2
1
2
3
7.13 d (8.0)
7.61 d (6.3) *
iSer
171.9, C
67.8, CH
42.8, CH2
173.3, C
69.5, CH
44.2, CH2
4.13 dd (9.0, 4.2)
2.75 ddd (9.0, 9.4, 5.4)
3.47 m
4.40 dd (9.5, 4.0)
3.00 m
3.75 dd (13.2, 4.0)
8.25 s *
NH
1
2
8.23 t (5.4)
Dap2
170.5, C
51.5, CH
40.1, CH2
172.8, C
53.4, CH
41.6, CH2
4.49 ddd (9.2, 9.2, 6.3)
3.22 m
4.84 m
3.57 dd (13.8, 8.6)
3
4.04 ddd (12.9, 6.3,
6.3)
7.72 d (9.2)
7.20 t (6.3, 6.3)
4.22 dd (13.8, 5.4)
NH-1
NH-2
7.98 d (9.5) *
7.37 t (6.0) *
Br2Py
1
2
3
158.6, CO
118.0, C
110.4, CH
96.9, C
161.4, CO
120.2, C
112.0, CH
99.4, C
6.30 d (2.7)
6.15 s
4
5
123.2, C
124.2, C
NH
12.68 d (2.7)
12.03 s
a
b
In DMSO-d6. In CD3OD (* CD3OH).
To complete the structure elucidation, the two remaining doublets with a small coupling constant
value of 2.7 Hz were assigned to an sp2 methine at δH 6.30/δC 110.4 and a significant downfield NH
signal at δH 12.68. An HMBC correlation of these two protons with three non-protonated sp2 carbons
(δC 96.9, 118.1, and 123.2), demonstrated the existence of a trisubstituted pyrrol moiety, with two
of these three positions bearing halogens. The placement of this heterocycle was established by the
HMBC correlation of the methylene group of Dap2 with a carbonyl group at δC 158.6. To confirm
the direct connection between the pyrrol moiety and this carbonyl group, a new gHMBC experiment
with J = 3 Hz was conducted. The position of the sp2 methine in the pyrrol unit and the bond to
the ring with the cyclopeptide was settled by the HMBC correlation between the methine proton
at δH 6.30 and the carbonyl group at δC 158.6. Although the chlorine and two bromine atoms were
undoubtedly located on the three free positions of the heterocycle ring and the carbon shifts (δC 96.9
and 123.2) suggested that both bromine atoms were on the pyrrol moiety, this evidence was insufficient
to fully confirm this proposal. Fortunately, this could be resolved by detailed study of the peptide

Mar. Drugs 2020, 18, 396
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structure by (+)-HRESI-TOFMS and QTOFMS (Figure 3), which showed significant cluster ions at
m/z 158.0487/160.0460 in a 3:1 ratio corresponding to the iAsn moiety. These m/z values, the mass
error observed, and the isotopic distribution clearly confirmed the presence of a chlorine atom on the
NMeHis amino acid (Figure S30).
Figure 3. Fragment found for 1 by QTOF.
The absolute stereochemistry of compound
with the 1-fluoro-2,4-di-nitrophenyl-5-l-alanine amide (l-FDAA) [25]. Compound
strong acid conditions and derivatization of the free amino acids with l-FDAA allowed an exhaustive
analysis by HPLC-MS. A comparison of the retention times of the derivatized amino acids present in
1
was established on the basis of Marfey’s analysis
1
was hydrolyzed in
1
and the suitably derivatized pure amino acid standards unambiguously demonstrated the absolute
configuration as l-Dap, l-iSer, and d-Asn. The absolute configuration of NMeClHis could not be
determined due to the absence of the standard amino acid.
Compound 2
was isolated as an amorphous white solid. Its (+)-LRESI showed an m/z = 825 [M + H]+
with a characteristic cluster corresponding to the presence of three bromine atoms. The molecular
formula C29H4379Br3N6O7 was established by (+)-HRESI-TOFMS analysis of the [M + H]+ at m/z
825.0806 (calcd. 825. 0816). The peptide nature of
2
was evident from its 1H and 13C NMR spectra
1
(Table 2). H NMR in DMSO showed the characteristic α-proton resonances of four α-amino acids in
the range δH 5.35 to 4.10 ppm, five interchangeable protons at δH 12.53, 8.56, 8.08, 7.19, and 6.77 ppm,
and two NMe signals at δH 3.61 and 3.04 ppm. 13C NMR data displayed six carbonyl signals between δC
173.5 and 159.1 ppm, four adjacent methine carbons in the range δC 59.1–51.5 ppm, and two NMe signals
at δC 35.7 and 30.5 ppm. Extensive 2D NMR analysis, including COSY, TOCSY, HSQC, and HMBC
was used to determine the identity of the four amino acids and to assign the NMR signals. As a result
of these studies, the amino acids were found to be one Ile, one NMe-Leu, one Pro, and one Asn unit.
A long-range correlation between protons at δH 7.19/6.77 and 2.15/2.11 ppm with the carbonyl group at
δC 173.5 ppm and the observation of ROESY cross-peaks between protons at δH 7.19/6.77 ppm and the
CH2 of position 4 at δH 2.15/2.11 ppm established the presence of a Gln. A N-methyl-2,3,4-bromopyrrol
unit was inferred by the presence of four aromatic non-protonated carbons at δC 128.5, 107.9, 100.7 and
93.4 ppm with chemical shifts similar to those described for bromopseudoceratines [23].
The sequencing for compound
data. Long-range correlations from
acids plus ROESY correlations between
(see Figure 2) allowed us to establish the sequence as Br3Py-Ile-NMeLeu-Pro-Gln.
2
α
was carried out using a combination of HMBC and ROESY
-protons, NH, and NMe to carbonyl carbons of adjacent amino
-protons, NH, and NMe protons of adjacent amino acids
α

Mar. Drugs 2020, 18, 396
6 of 10
Table 2. NMR spectroscopy data for 2 (1H NMR 500 MHz, 13C NMR 125 MHz).
δ , Mult (J in Hz) b
δC, Mult b
Pos
δ , Mult (J in Hz) a
δC, Mult a
H
H
Br3Py
1
2
3
4
-
-
-
-
-
161.8, CO
128.6, C
102.7, C
101.3, C
110.8, C
36.7, CH3
174.3, CO
55.7, CH
38.0, CH
25.9, CH2
11.2, CH3
15.7, CH3
-
171.8, CO
54.3, CH
38.0, CH2
25.7, CH
23.6, CH3
22.3, CH3
31.8, CH3
174.5, CO
61.6, CH
30.5, CH2
26.0, CH2
48.8, CH2
174.7, CO2H
52.8, CH
30.5, CH2
32.6, CH2
177.9,
-
-
-
-
-
159.1, CO
128.5, C
100.7, C
93.4, C
107.9, C
35.7, CH3
171.4, CO
53.7, CH
35.8, CH
24.2, CH2
10.7, CH3
15.1, CH3
-
168.8, CO
51.7, CH
36.7, CH2
23.9, CH
23.1, CH3
21.8, CH3
30.5, CH3
171.4, CO
59.1, CH
28.9, CH2
24.4, CH2
46.7, CH2
173.3, CO2H
51.5, CH
27.0, CH2
31.3, CH2
173.5,
5
NMe
1
2
3
4
5
6
NH
1
2
3
4
5
6
3.76, s
3.61, s
Ile
-
-
4.84, m
1.96, m
4.65, dd, 8.35, 8.5
1.87, m
1.73, m, 1.22, m
0.95, t, 7.4
0.99, d, 6.8
8.24, d, 8.1
-
5.51, dd, 10.3, 4.7
1.77, m; 1.61, m
1.56, m
1.57, m; 1.21, m
0.83, t, 7.4
0.85, d, 6.9
8.56, d, 8.2
-
5.35, dd, 10.1, 4.3
1.59, m; 1.42, m
1.43, m
NMeLeu
0.97, d, 6.2
0.93, d, 6.1
3.21, s
0.87, d, 6.2
0.83, d, 6.2
3.04, s
NMe
Pro
Gln
1
2
3
4
5
1
2
3
4
-
-
4.41, m
4.31, dd, 8.3, 4.1
2.02, m; 1.80, m
1.90, m; 1.77, m
3.53, m; 3.50; m
12.53, brs
4.10, ddd, 8.6, 8.5, 5.2
1.93, m; 1.74, m
2.15, m; 2.11, m
2.23, m; 2.00, m
2.08, m; 1.92, m
3.75, m; 3.69, m
-
4.41, m
2.27, m; 1.92, m
2.41, m; 2.32, m
5
-
7.19, s; 6.77, s
8.08, brs
CONH2
-
CONH2
-
NH
-
a
In CD3OD. b In DMSO-d6.
The absolute configurations of the amino acids were determined by comparing the
hydrolysis products of
(6 N HCl, 110 ◦C, 18 h) after derivatization with Marfey’s reagent
2
(N-(3-fluoro-4,6-dinitrophenyl)-l-alaninamide, l-FDAA), with appropriate amino acid standards
using HPLC-MS chromatography. As a result, all the amino acids were determined to be L.
3. Materials and Methods
3.1. General Experimental Procedures
Optical rotations were determined using a Jasco P-1020 polarimeter. UV spectra were performed
using an Agilent 8453 UV
FT-IR spectrometer with ATR sampling. NMR spectra were recorded on a Varian “Unity 500” spectrometer
at 500/125 MHz (1H/13C). Chemical shifts were reported in ppm using residual CD3OH (
3.31 ppm for
1H and 49.0 ppm for 13C) and DMSO-d6 ( 2.50 ppm for 1H and 39.5 ppm for 13C) as an internal reference.
−vis spectrometer. IR spectra were obtained with a Perkin-Elmer Spectrum 100
δ
δ
HRESI-TOFMS was performed on an Agilent 6230 TOF LC/MS chromatograph spectrometer. (+)-ESIMS
were recorded using an Agilent 1100 Series LC/MSD spectrometer. HRESI-TOFMS was performed on an
Agilent 6230 TOF LC/MS chromatograph spectrometer. ESI(+) and MSe were performed on an Waters
UHPLC-QTOF Acquity I-Class + Xevo G2-XS.

Mar. Drugs 2020, 18, 396
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3.2. Biological Material
The sponge Ircinia sp. (158 g) was collected by hand using a diving rebreather system in the
Thousand Islands (Indonesia). The sponge was immediately frozen and kept under these conditions
until extraction. The specimen was identified by María Jesús Uriz at CEAB, Blanes, Spain. A voucher
specimen (ORMA155272) was deposited at PharmaMar facilities (Madrid, Spain).
3.3. Extraction and Isolation
The sponge Ircinia sp. (158 g) was triturated and exhaustively extracted with MeOH:DCM (1:1,
3
×
500 mL). The combined extracts were concentrated to yield a crude mass of 7.9 g. The crude
product was subjected to VLC on a Lichroprep RP-18 with a stepped gradient from H2O to MeOH
to CH2Cl2. The fractions eluting with H2O:MeOH (3:1, 606 mg) and H2O:MeOH (1:1, 89.6 mg) were
subjected to semipreparative HPLC (Symmetry Prep C18
0.04% TFA: CH3CN + 0.04% TFA 95:5 and then gradient from 5% to 68% CH3CN + 0.04% TFA in 25 min,
flow 3 mL/min, UV detection) to obtain 4.3 mg of compound . The fraction eluting with H2O:MeOH
(1:3, 43.9 mg) was subjected to semipreparative HPLC (Symmetry Prep C18 m, 10 150 mm; 3 min.
5
µm, 10
×
150 mm; 3 min isocratic H2O +
1
5
µ
×
isocratic H2O + 0.04% TFA: CH3CN + 0.04% TFA 90:10 and then gradient from 10% to 75% CH3CN +
0.04% TFA in 25 min, flow 3 mL/min, UV detection) to obtain 3.0 mg of compound 2.
25
Haloirciniamide A (
1
): amorphous white solid; [
α
]
–62.7
º
(c 0.1, MeOH); IR
υ
max 3314,
D
2920, 2850, 1644, 1523, 1416, 1311, 1239, 1199, 1041 cm−1; UV (MeOH)
(500 MHz) and 13C NMR (125 MHz) see Table 1; (+)-HREI-TOFMS m/z 830.0400 [M+Na]+ (calcd for
λ
max
198, 268 nm. H NMR
1
C25H3279Br2N11O8Na m/z 830.0383).
25
Seribunamide A (
2
): amorphous white solid; [
α
]
–38.4
º
(c 0.2, MeOH); IR
υ
max 3352, 2932,
D
2850, 1658, 1515, 1320, 1236, and 1035 cm−1; UV (MeOH)
λ
max 197, 266 nm. H NMR (500 MHz) and
1
13C NMR (125 MHz) see Table 2; (+)-HREI-TOFMS m/z 825.0806 [M + H]+ (calcd for C29H4479Br3N6O7
m/z 825.0816).
3.4. Absolute Configuration
Absolute Configuration of
HCl at 110 ◦C for 15 h. The excess aqueous HCl was removed under a N2 stream, and a solution of
700 g of l-FDAA (N-(3-fluoro-4,6-dinitrophenyl)-l-alanine-amide) in acetone (160 L), H2O (100 L),
and NaHCO3 1N (50 L) was added to the dry hydrolysate. The resulting mixture was heated at 40
for 1 h, before being cooled to 23 C, quenched by addition of 2N HCl (20 L), dried, and dissolved in
H2O (800 L). The resultant aqueous solution was subjected to reversed-phase LC/MS (column: Waters
1. First, 0.5 mg of haloirciniamide A was hydrolyzed in 0.5 mL of 6 N
µ
µ
µ
µ
ºC
º
µ
µ
Symmetry 4.6 × 150 mm, 3.5 µm, flow rate 0.8 mL/min) in three different gradients.
Gradient 1 for iSer (mobile phase CH3CN + 0.04% formic acid /H2O + 0.04% formic acid, using
a linear gradient from 5% to 20% CH3CN in 10 min and then from 20% to 35% CH3CN in 25 min):
the retention time was 23.9 min for l-iSer.
Gradient 2 for Asp (mobile phase CH3CN + 0.04% formic acid/H2O + 0.04% formic acid, using
a linear gradient from 5% to 30% CH3CN in 10 min and then from 30% to 50% CH3CN in 30 min):
the retention time was 17.0 min for d-Asp.
Gradient 3 for Dap: mobile phase CH3CN + 0.04% formic acid/H2O + 0.04% formic acid, using
a linear gradient from 5% to 10% CH3CN in 5 min and then from 10% to 35% CH3CN in 25 min):
the retention times was 15.5 min for l-Dap.
Retention times for the derivatized amino acids standards were as follows: gradient 1 (23.2 min for
d-iSer and 23.8 min for the l-iSer); gradient 2 (16.2 min for l-Asp and 17.0 min for d-Asp), and gradient
3 (15.5 min for l-Dap and 16.6 min for the d-Dap).
Absolute Configuration of
6 N HCl, 110 ◦C for 15 h. The excess aqueous HCl was removed under a N2 stream, and a solution of
400 g of l-FDAA (N-(3-fluoro-4,6-dinitrophenyl)-l-alanine-amide) in acetone (160 L), H2O (100 L),
2. First, 0.3 mg of seribunamide A was hydrolyzed in 0.4 mL of
µ
µ
µ

Mar. Drugs 2020, 18, 396
8 of 10
and NaHCO3 1N (50
µL) was added. The vial was heated at 40
ºC for 1 h, and the contents were
neutralized with 2N HCl (20 µL) after cooling to room temperature. The resulting solution was
dried in vacuum and reconstituted in H2O (600
µL) before being analyzed by HPLC-MS using two
different methods.
Ile was analyzed using Lux Cellulose-4, 5
µm, flow 1 mL/min, H2O/AcN + 0.04%TFA isocratic
65:35 in 60 min. The retention time of the l-FDAA amino acid in the hydrolysate of 2 was established
as l-Ile 36.1 min. Retention times for the derivatized amino acids standards were as follows: l-allo-Ile
31.7 min, l-Ile 36.1 min, d-allo-Ile 38.1 min, and d-Ile 51.4 min.
Pro, NMeLeu, and Glu were analyzed using Symmetry 4.6
×
150 mm, 3.5 µm, flow 0.8 mL/min,
H2O+ 0.04%TFA/CH3CN + 0.04%TFA from 20% to 50% in 30 min. The retention time of the l-FDAA
amino acids in the hydrolysate of 2 were established as l-Glu 14.0 min, l-Pro 16.8 min, and NMe-l-Leu
28.5 min. Retention times for the derivatized amino acids standards were as follows: l-Glu 14.1 min,
d-Glu 15.2 min, l-Pro 16.8 min, d-Pro 14.01 min, NMe-l-Leu 28.5 min, and NMe-d-Leu 30.1 min.
3.5. Biological Activity
The cytotoxic activity of
colon (HT-29), breast (MDA-MB-231), and pancreas PSN-1, and both compounds displayed a GI50
1.2
10−5 M in all cell lines. Since these compounds did not show cytotoxicity, we further evaluated
an anticancer response through other targets. Thus, compound was further tested for the capacity
to inhibit the enzyme topoisomerase I, but it showed no inhibition of the enzyme at 1.0
10−5 M,
and therefore, it was not considered active in inhibiting this enzyme. Likewise, compounds and
1 and 2 was tested against four human tumor cell lines, lung (A-549),
>
×
1
×
1
2
were unable to impair the interaction between the programmed cell death protein PD-1 and its natural
ligand PDL1 as demonstrated by their lack of effect in a cell-based assay whose final readout was
dependent on the interaction between the two proteins (Table 3).
Table 3. % PD-1 and TOPO-I inhibition for compounds
1 and 2. PD-1: programmed cell death protein.
Compound
Target %Inhibition at 1 × 10−5
M
1
1
2
Top-I
PD-1
PD-1
3
0.3
−3.5
4. Conclusions
In summary, two new peptides bearing unprecedented halogenated moieties, haloirciniamide A (
and seribunamide A ( ), were isolated from a marine sponge belonging to the Irnicia genus, which was
1)
2
selected for further studies. The sample was collected around the Thousand Islands (Indonesia) by the
Pharmamar expedition team in collaboration with the Research Center for Oceanography, Indonesian
Institute of Sciences (RCO-LIPI). The planar structures of the novel compounds were determined by a
combination of extensive NMR and HPLC-MS experiments. The absolute configuration was achieved
by Marfey’s analysis after acid hydrolysis. Cytotoxic activity in the four cancer cell lines tested was not
observed for
immune response was also evaluated, and neither compound was able to impair PD1–PDL1 interaction.
Moreover, compound failed to inhibit the enzyme topoisomerase I. Unfortunately, the amount of the
1 and 2, as in the case of gunungamide A and cyclocinamide B. In addition, an anticancer
1
compounds isolated was not enough for further assays. This work is the first example of the isolation
and structural elucidation of novel compounds with unique structural features from an Ircinia sponge,
which highlights this gender and its microbiota as a distinctive source of novel structures.
Supplementary Materials: The following are available online at http://www.mdpi.com/1660-3397/18/8/396/s1,
Figure S1: Picture of the fresh sponge; Figures S2–S16: NMR spectra of compound 1. Figures S17–S29: NMR
spectra of compound 2. Figures S30: QTOFMS spectrum of compound 1 and fragments found. Figures S31–S38:
Analysis by Marfey’s method.

Mar. Drugs 2020, 18, 396
9 of 10
Author Contributions: Sample collection: T.A.H., A.B. and S.B.; Conceptualization and supervision: C.C. and
M.Y.P.; Methodology and writing: R.F.; Investigation: A.B.; Original draft preparation and writing: M.P.; Review
and editing: C.C., M.P. and M.Y.P. All the authors read, reviewed, and agreed with the structure and content of the
manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: The present research was financed in part by Grants from Ministerio de Ciencia, Innovación y
Universidades of Spain (AGL2015-63740-C2-2-R and RTC-2016 4611-1, Inmunotop project), cofunded by the
FEDER Programme from the European Union.
Acknowledgments: We gratefully acknowledge the help of our PharmaMar colleagues C. Crespo for her excellent
technical assistance, E. Millá
n for performing the ESI(+) and MSe experiments, J. M. Dominguez for the biological
assays and S. Munt for revision of the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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