New amino acid derivatives from the marine ascidian Leptoclinides dubius

Article abstract of DOI:10.1021/np9603535

From the cytotoxic extracts of the marine ascidian Leptoclinides dubius, N-(p-hydroxybenzoyl)L-arginine (1), N-(1H-indolyl-3-carbonyl)-D-arginine (2), and N-(6-bromo-1H-indolyl-3-carbonyl)-R, where R is L-Arg (4), L-His (5), and the very rare amino acid L-enduracididine (3), have been isolated and identified by spectroscopic data, hydrolysis, and comparison with authentic samples.

Full text of DOI:10.1021/np9603535

782
J . Nat. Prod. 1996, 59, 782-785
New Am in o Acid Der iva tives fr om th e Ma r in e Ascid ia n Leptoclin id es d u biu s
Angel Garc´ıa,† Mar´ıa J esu´s Va´zquez,† Emilio Quin˜oa´,† Ricardo Riguera,*,† and Ce´cile Debitus‡
Departamento de Qu´ımica Orga´nica, Facultad de Quı´mica, and Instituto de Acuicultura, Universidad de Santiago,
15706 Santiago de Compostela, Spain, and Centre ORSTOM, B.P. A5, Noumea´ Cedex, New Caledonia
Received March 12, 1996X
From the cytotoxic extracts of the marine ascidian Leptoclinides dubius, N-(p-hydroxybenzoyl)-
L-arginine (1), N-(1H-indolyl-3-carbonyl)-D-arginine (2), and N-(6-bromo-1H-indolyl-3-carbonyl)-
R, where R is L-Arg (4), L-His (5), and the very rare amino acid L-enduracididine (3), have
been isolated and identified by spectroscopic data, hydrolysis, and comparison with authentic
samples.
Marine ascidians have been shown to be a very rich
source of unique and biologically active secondary
metabolites that have attracted the interest of both
chemists and pharmacologists.1 A major group of those
metabolites are nitrogen-containing compounds, par-
ticularly aromatic heterocycles.2 As part of our ongoing
investigation on bioactive compounds from marine
organisms, we describe in this paper the isolation of
simple peptides 1-5 (Chart 1) from the polar cytotoxic
extract (100% inhibition of KB cells at 10 µg/mL and
80% inhibition of P-388 cells at 10 µg/mL) of the ascidian
Leptoclinides dubius3 (Sluiter, 1909) (order Enterogona,
family Didemnidae), collected in New Caledonia.
The methanolic extracts of L. dubius (340 g dry wt)
were sequentially submitted to solvent partition be-
tween aqueous MeOH and hexanes, then CH2Cl2, and
then n-BuOH. The n-BuOH-soluble material was first
chromatographed on Amberlite XAD-2 and then on
Sephadex LH-20 and finally subjected to reversed-phase
HPLC to afford pure 1 (11 mg), 2 (3 mg), 3 (5 mg), 4 (15
mg), and 5 (5 mg).
Compound 1 was obtained as a pale yellow solid. The
UV spectra indicated the presence of a phenol chro-
mophore with bands at λmax 210 nm and 256 nm that
shifted to longer wavelenghts on addition of base
[MeOH/NaOH, λmax 216 nm and 294 nm]. Its (+)-
FABMS (in glycerol) showed the molecular ion at 295
([M + H]+), confirmed by the ion at m/ z 317 ([M +
Na]+), which was obtained when the (+)-FABMS was
taken in glycerol + NaCl. These ions are consistent
with the molecular formula C13H18N4O4 for 1, requiring
seven sites of unsaturation. For its part, HREIMS
showed the highest mass ion at m/ z ) 234.0768 ([M -
CH6N3]+; C12H12NO4, ∆ ) 0.2 ppm) in accordance with
the facile â cleavage of the guanidine moiety and the
proposed structure.
the presence of an amino acid moiety, and that at δ
156.9 (s, C15) was in good agreement with the expected
value for the terminal guanidine group. Acid hydrolysis
of 1 (6 N HCl/120 °C/12 h) afforded p-hydroxybenzoic
acid and L-Arg [CD (+) (c ) 1.49 × 10-3 M, HCl 2 N),
λmax ) 215 nm (∆ꢀ ) 0.06)] whose stereochemistry was
determined by Marfey’s method.4
Compound 2 also contained arginine as the amino
acid component, but its aromatic chromophore had UV
bands at λmax 222, 250, and 280 nm, typical of an indole
system. The (+)-FABMS showed the molecular ion at
m/ z 318 ([M + H]+; molecular formula C15H19N5O3),
1
that together with the H NMR spectral data suggested
structure 2, with an amide bond between the indole
carboxylic acid and the Arg group. Similar analyses
were carried out with compound 4, which showed a
molecular mass 78 amu higher than 2 in the (+)-
FABMS. It also showed a typical cluster due to the
presence of one bromine atom, and this atom was
located at C6 on the basis of 2D NMR data.5,6
Acid
hydrolysis of 2 afforded the expected 1H-indole-3-
carboxylic acid and D-Arg [CD (-) (c ) 2.87 × 10-3 M,
HCl 2 N), λmax ) 207.5 nm (∆ꢀ ) -0.09)], while
hydrolysis of 4 produced 6-bromo-1H-indole-3-carboxylic
acid and L-Arg [CD (+) (c ) 2.42 × 10-3 M, HCl 2 N),
λmax ) 213.0 nm (∆ꢀ ) 0.63)].
Spectroscopic data and hydrolysis indicated that
compounds 3 and 5 contained the 6-bromo-1H-indole-
3-carboxylic acid moiety and that the amino acid
component of 5 was L-His. For its part, comparison of
the 13C and DEPT NMR data of 3 with those of 4 showed
differences mainly at C12, C13 (a CH2 in 4; a CH in 3),
and C14. All these signals were shifted to lower field
in 3, suggesting that C13 was bonded to a nitrogen
1
atom. These and other significant data from H NMR
and FABMS indicated that the amino acid of 3 was a
cyclic derivative of Arg, with the guanidine moiety
forming a partially saturated imidazole ring. In addi-
tion, the amino acid isolated by hydrolysis of 3 showed
a positive CD band indicative of an L configuration at
C11 and a positive optical rotation.7
Two natural amino acids with a partially saturated
imidazole structure were reported 30 years ago and
their absolute stereochemistry determined by X-ray
diffraction and ORD. These amino acids were named
enduracididine [6; L-series; (2S,4R)] and its diastereo-
isomer alloenduracididine [D-series; (2R,4R)] isolated
from the fungal peptide antibiotic enduracidin.8 En-
duracididine has since been found as a component of
The 1H NMR spectrum (D2O; 500 MHz) of 1 contained
the resonances of an AA'BB' system at δ 6.68, d (H4/
H6) and δ 7.46, d (H3/H7), due to a p-hydroxyphenyl
fragment. The 13C and DEPT NMR spectra showed 13
resonances that were correlated with the corresponding
protons by standard 2D NMR experiments (COSY,
HMQC, and HMBC). Thus, the carbon resonance at
55.2 ppm and its proton at 4.13 ppm (dd, H10) indicated
* Author to whom correspondence should be addressed. Tel./Fax:
34-81-591091.
† Departamento de Qu´ımica Orga´nica.
‡ Centre ORSTOM.
X Abstract published in Advance ACS Abstracts, J uly 15, 1996.
S0163-3864(96)00353-9 CCC: $12.00
© 1996 American Chemical Society and American Society of Pharmacognosy

Notes
J ournal of Natural Products, 1996, Vol. 59, No. 8 783
Ch a r t 1
the seeds of a leguminoseae.9 Comparison with the
reported data for those isomers indicates that the amino
acid component of 3 is enduracididine, and therefore the
absolute stereochemistry of 3 is (11S,13R). This is the
first time the very rare amino acid enduracididine has
been found in a marine organism, albeit in very small
amounts.
In summary, L. dubius is the source, as other ascid-
ians, of amino acid-derived metabolites.2 In this par-
ticular case, they are modified dipeptides where one of
the amino acid constituents has lost two carbon atoms
and the amino group.
matrix with NaCl as additive for positive ion mode.
HREIMS were obtained on a Kratos MS50 spectrometer
and LREIMS on a Hewlett-Packard 5988A mass spec-
trometer operating at 70 eV.
An im a l Ma t er ia l. Speciments of the ascidian L.
dubius (voucher ref. UA-284) were collected by hand
using SCUBA (-5 to -10 m) in Woodin Canal, New
Caledonia and stored at -5°C. They were identified by
Mrs. F. Monniot of the Muse´um National d′Histoire
Naturelle (Paris, France).3
Extr a ction a n d Isola tion . The freeze-dried ascid-
ians (340 g) were homogeneized in methanol (six times)
and the solvent evaporated to dryness under reduced
pressure. The extract was partitioned between 10%
aqueous MeOH (200 mL) and hexane (2 × 200 mL). The
aqueous portion was made 40% MeOH and extracted
sequentially with CH2Cl2 (3 × 200 mL) and n-BuOH (5
× 200 mL). The n-BuOH portion was passed through
an Amberlite XAD-2 column, eluting first with water
(3 L) to retain the organic compounds that were then
eluted with MeOH (4 L). The methanol eluates were
concentrated under reduced pressure to give 1.2 g of
material, which was chromatographed on Sephadex LH-
20 (flow rate 50 mL/h) with MeOH/H2O (2:1) as eluant
and TLC monitoring (silicagel, 6:4:1 CHCl3/MeOH/H2O)
giving seven main fractions. Fractions 4 and 6 con-
tained compounds 1-5. The fraction four (127 mg) was
submitted to HPLC on a 300 × 7.8 mm µBondapak NH2
column, eluting with MeOH, to afford pure compound
1 (11 mg, retention time 22 min, flow rate 2 mL/min).
Identical separation conditions applied to fraction 6 (214
mg) afforded 5 mg of pure compound 5 (retention time
30 min, flow rate 3 mL/min).
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Optical rota-
tions were measured in MeOH using a J ASCO DIP-370
polarimeter with a sodium lamp operating at 598 nm.
The UV spectra were recorded on a Hewlett-Packard
8452A spectrometer. The IR spectra on a MINAC
Prospect FTI spectrometer. CD data were obtained in
2 N HCl on a J ASCO J -720 spectropolarimeter. The
NMR spectra were recorded on a Bruker AMX-500 at
500.13 MHz for 1H and 125.77 for 13C. The two-
dimensional 1H-13C heteronuclear shift correlation
(HMQC) spectra (512 × 2K) for directly bonded protons
and carbons were obtained by accumulating 48 scans
per t1, with a relaxation delay of 1 s and J CH ) 130 Hz.
The two-dimensional 1H-13C heteronuclear shift cor-
relation (HMBC) spectrum (1024 × 2K) was obtained
by accumulating 128 scans per t1; the relaxation delay
was 1.5 s, and the value of J CH selected was 9 Hz. The
data were zero-filled to 1024 in F1 and subjected to a
QSINE transformation.
HPLC was performed with a Waters Model 590
apparatus equipped with an R401 differential refrac-
tometer and a 440 UV detector working at 254 nm.
Semipreparative HPLC was carried out using 300 × 7.8
mm µBondapak NH2 and µBondapak C18 reversed-
phase columns and a 500 × 7.8 mm Partisil M9 10/50
ODS-3 reversed-phase column; for analytical HPLC a
330 × 3.9 mm µBondapak C18 column was used. Fast
atom bombardment mass (FABMS) spectra were ob-
tained on a Kratos MS50 mass spectrometer in glycerol
The rest of the compounds were also isolated from
fraction 6, using different HPLC conditions as follows:
HPLC on a 300 × 7.8 mm µBondapak C18 column,
eluting with 30:70 MeOH/H2O, afforded 3 mg of pure
compound 2 (retention time 21 min, flow rate 1.5 mL/
min) and HPLC on a 500 × 7.8 mm Partisil M9 10/50
ODS-3 column eluting with 45:55 MeOH/10-2 M NH4-
AcO gave pure compounds 4 (15 mg, retention time 46
min, flow rate 2 mL/min) and 3 (5 mg, retention time
48 min, flow rate 2 mL/min).

784 J ournal of Natural Products, 1996, Vol. 59, No. 8
Ta ble 1. 1H NMR Data of 1-5
Notes
1a
2b
3a
4a
5a
δH, mult, J (Hz)
δH, mult, J (Hz)
δH, mult, J (Hz)
δH, mult, J (Hz)
δH, mult, J (Hz)
H2
7.81, s
8.04, s
7.99, s
7.96, s
H3
H4
H5
7.46, d, 8.7
6.68, d, 8.7
7.55, d, 1.2
7.90, d, 8.5
7.20, dd, 8.5, 1.2
7.82, dd, 8.8, 3.4
7.10, m
8.06, d, 7.7
7.28, dd, 7.6, 1.7
7.90, d, 8.5
7.20, dd, 8.5, 1.5
H6
H7
H10
H11
6.68, d, 8.7
7.46, d, 8.7
4.13, dd, 8.3, 5.0
a ) 1.58, m
b ) 1.70, m
1.42, q
7.10, m
7.39, dd, 6.0, 3.0
7.66, d, 1.7
4.61, m
7.38, d, 1.5
7.55, d, 1.2
4.27, dd, 7.7, 4.9
4.50, dd, 8.7, 4.3
4.70, dd, 7.5, 5.1
H12
a ) 1.66, m
b ) 1.78, m
1.52, q
a ) 2.20, m
b ) 2.30, m
4.27, m
a ) 1.80, m
b ) 1.90, m
1.64, q
a ) 3.2, dd, 15.0, 5.1
b ) 3.4, dd, 15.0, 5.1
H13
H14
2.90, t, 6.9
3.00, t, 6.9
a ) 3.56, t, 9.6
b ) 3.94, t, 9.5
a ) 3.10, m
b ) 3.20, m
7.20, s
8.50, s
H16
a
b
In CD3OD. In D2O.
Ta ble 2. 13C NMR Data of 1-5a
(100), 197 (10), 195 (11), 143 (15), 115 (21), 114 (40);
HREIMS 335.0032 (C14H12N2O379Br, ∆ ) 0.2 ppm).
δC (1)b
δC (2)c
δC (3)b
δC (4)b
δC (5)b
Com p ou n d 4, C15H18N5O3Br. [R]20 +3.6° (c 0.55 ,
D
C1
C2
C3
169.9
125.4
129.6
MeOH); UV (MeOH) λmax (log ꢀ) 222 (3.78), 250 (3.43),
280 (3.18) nm; IR (dry film) ν max 1670 (CdN, guani-
dine), 1623 (CdO, amide), 1592 (CdC), 1576 (CdO,
128.4
110.5
137.6
123.0
121.1
120.9
113.5
128.2
168.4
130.8
112.3
139.3
123.3
125.6
117.2
116.2
126.2
167.9
130.1
111.8
138.6
122.9
124.9
116.7
115.6
125.9
167.4
130.5
112.1
133.0
123.1
125.4
117.0
116.0
126.4
167.3
C3a
C4
C5
C6
C7
115.2
159.4
115.2
129.6
1
carboxylic acid) cm-1; H NMR see Table 1; 13C NMR
see Table 2; FABMS (positive ion mode) m/ z 398 (55),
396 (53), 224 (23), 222 (22), 144 (10), 115 (100); EIMS
(70 eV) m/ z 337 (16), 335 (16), 240 (23), 238 (24), 224
(97), 222 (100), 197 (25), 195 (27), 115 (30), 114 (40);
HREIMS 335.0029 (C14H12N2O379Br, ∆ ) 0.3 ppm).
C7a
C8
C9
178.9
55.2
29.0,
24.7
40.8
C10
C11
C12
C13
C14
C15
C16
180.5
56.5
31.6
27.0
42.2
179.0
55.3
40.5
55.3
50.1
180.1
55.5
31.0
26.3
42.1
176.8
54.9
29.9
139.1
118.5
Com p ou n d 5, C15H13N4O3Br. [R]20D +26.6° (c 0.55 ,
MeOH); UV (MeOH) λmax (log ꢀ) 222 (3.53), 250 (3.17),
282 (2.92) nm; IR (dry film) ν max 3400 (N-H), 1595
(CdO, amide), 1538 (CdO, carboxylic acid) cm-1 1H
;
156.9
NMR: see Table 1; 13C NMR see Table 2; FABMS
(positive ion mode) m/ z 379 (17), 377 (16), 224 (9), 222
(8), 115 (100); EIMS (70 eV) m/ z 241 (48), 240 (28), 239
(41), 224 (96), 222 (100), 197 (97),196 (35), 195 (99), 194
(20), 143 (21), 116(64), 115 (39), 82(48), 81(42); HREIMS
376.0169 (C15H13N4O379Br, ∆ ) 0.3 ppm).
160.0
161.5
158.9
134.5
a
b
Assignments based on DEPT experiments. In CD3OD. c In
D2O.
Com p ou n d 1, C13H18N4O4: [R]20 +19.5° (c 2.05 ,
D
MeOH); UV (MeOH) λmax (log ꢀ) 210 (3.46), 256 (3.49)
nm; IR (dry film) ν max 3200-3600 (OH), 1630 (CdN,
guanidine), 1614 (CdO, amide), 1576 (CdO, carboxylic
Acid Hyd r olysis of 1-5. A solution of the com-
pound (2-4 mg) in 6 N HCl (1 mL) was heated at 120
°C overnight in a stoppered reaction vial. The solution
was then evaporated under reduced pressure and the
residue partitioned between water (3 mL) and CHCl3
(3 × 2 mL). The combined organic extracts were washed
with water and evaporated to dryness under reduced
pressure to give p-hydroxybenzoic acid from 1, 1H-
indole-3-carboxylic acid from 2, and 6-bromo-1H-indole-
3-carboxylic acid from 3-5, identified by MS and
comparison with authentic samples.
The aqueous layer was lyophilized and the residue
derivatized with Marfey’s reagent and compared by
HPLC on 330 × 3.9 mm µBondapak C18 column, with
the corresponding derivatives of L- and D-Arg and L- and
D-His. In this way, L-Arg was identified in 1 and 4,
D-Arg in 2, and L-His in 5. Hydrolysis of 3 gave
enduracididine (6), identified by comparison of its
spectroscopic properties (NMR, CD, R) with reported
data.9
acid), 1264 (OH, phenol moeity) cm-1 1H NMR see
;
Table 1; 13C NMR see Table 2; FABMS (positive ion
mode) m/ z 295 (100), 121 (14), 93 (11); EIMS (70 eV)
m/ z 234 (9), 137 (5), 121 (100), 93 (16); HREIMS
234.0768 (C12H12NO4, ∆ ) 0.2 ppm).
Com p ou n d 2, C15H19N5O3: [R]20 -133.3° (c 0.15 ,
D
MeOH); UV (MeOH) λmax (log ꢀ) 214 (3.65), 246 (3.13),
282 (3.11) 286 (3.10) nm; IR (dry film) ν max 1651 (CdN,
guanidine), 1623 (CdO, amide), 1592 (CdC), 1539
(CdO, carboxylic acid) cm-1 1H NMR see Table 1;
;
FABMS (positive ion mode) m/ z 318 (22), 259 (19), 115
(100); EIMS (70 eV) m/ z 257 (19), 160 (11), 145 (12),
144 (100), 116 (15); HREIMS 258.1212 (C14H14N2O3, ∆
) 0.3 ppm).
Com p ou n d 3. C15H16N5O3Br, [R]20D +16.5° (c 0.85 ,
MeOH); UV (MeOH) λmax (log ꢀ) 222 (2.98), 250 (2.63),
282 (2.39) nm; IR (dry film) ν max 1682 (CdN, guani-
dine), 1616 (CdO, amide), 1592 (CdC), 1535 (CdO,
1
carboxylic acid) cm-1; H NMR see Table 1; 13C NMR:
Ack n ow led gm en t. This work was supported by
grants from Xunta de Galicia (XUGA-20907B94 and
20901B95), CICYT (MAR95-1933-CO2-O2) and the OR-
STOM-CNRS SMIB Program.
see Table 2; FABMS (positive ion mode) m/ z 396(42),
394 (40), 224 (12), 222 (11), 115 (100); EIMS (70 eV)
m/ z 337 (12), 335 (12), 240 (16), 238 (17), 224 (97), 222

Notes
J ournal of Natural Products, 1996, Vol. 59, No. 8 785
(6) Kobayashi, J .; Cheng, J .; Ohta, T.; Nozoe, S.; Ohizumi, Y.;
Sasaki, T. J . Org. Chem., 1990, 55, 3666-3670.
(7) J ennings, J . P.; Klyne, W.; Scopes, P. M. J . Chem. Soc. 1965,
294-296.
Refer en ces a n d Notes
(1) Faulkner, D. J . Nat. Prod. Rep. 1995, 12, 223-269.
(2) Davidson, B. S. Chem. Rev. 1993, 93, 1771-1791.
(3) Monniot, F. Bull. Mus. Nat. His. Nat., Sect. A 1989, 11, 673-
691.
(4) Marfey, P. Carlsberg. Res. Commun. 1984, 49, 591-596.
(5) Lake, R. J .; Blunt, J .; Munro, M. Aust. J . Chem. 1989, 42, 1201-
1206.
(8) Horii, S.; Kameda, Y. J . Antibiot. 1968, 21, 665-667.
(9) Fellows, L. E.; Hider, R. C.; Bell, E. A. Phytochemistry 1977,
16, 1957-1959.
NP9603535