Isolation and structural elucidation of callipeltins J-M: antifungal peptides from the marine sponge Latrunculia sp.

Article abstract of DOI:10.1016/j.tet.2006.10.032

Continued investigation of the polar extracts of the marine sponge Latrunculia sp. has resulted in the discovery of callipeltins J-M. The new structures were determined by interpretation of their NMR and MS data. The stereochemistry of the unusual 3-methy

Full text of DOI:10.1016/j.tet.2006.10.032

Tetrahedron 63 (2007) 131–140
Isolation and structural elucidation of callipeltins
J–M: antifungal peptides from the marine
sponge Latrunculia sp.
Maria Valeria D’Auria,a Valentina Sepe,a Rosa D’Orsi,a Filomena Bellotta,a
b
Cecile Debitus and Angela Zampella
a,
*
ꢀ
aDipartimento di Chimica delle Sostanze Naturali, Universitaꢁ di Napoli ‘Federico II’, via D. Montesano 49, 80131 Napoli, Italy
b
´
´
IRD (ex ORSTOM), Centre de Noumea, B.P. A5 Noumea Cedex, New Caledonia
Received 27 July 2006; revised 18 September 2006; accepted 12 October 2006
Available online 1 November 2006
Abstract—Continued investigation of the polar extracts of the marine sponge Latrunculia sp. has resulted in the discovery of callipeltins J–M.
The new structures were determined by interpretation of their NMR and MS data. The stereochemistry of the unusual 3-methylglutamine
residue in callipeltins J and K was secured by stereoselective synthesis and Marfey’s LC–MS analysis.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
stereochemistry of b-OMeTyr residue,20 left unassigned in
the original paper due to the decomposition of the above
residue under standard acidic hydrolysis of the parent
peptide, the revision of the stereochemistry of 3-hydroxy-
2,4,6-trimethylheptanoic acid16 and confirmation of the ste-
reochemistry of diMeGln and AGDHE residues. In addition,
by employing quantum molecular calculation of coupling
constants and LC–MS Marfey’s analysis, we found that
both of the threonine residues in callipeltin A have the
D-allo configuration, consistent with what was indepen-
dently found in the structurally related cyclic depsipeptide
neamphamide A.21 All these structural and synthetic
studies have led to a complete stereochemical assignment
of callipeltin A and its congeners that recently received
indirect support by the total synthesis of callipeltins D
and E.22,23
Callipeltins are a group of marine peptides with unusual
structural features and remarkable biological properties, iso-
lated from the sponges Callipelta sp. and Latrunculia sp.1–4
From a structural point of view the most distinctive feature
of callipeltins is the presence of several non-proteinogenic
units; from a biological point of view, callipeltin A displays
a broad range of biological activities, ranging from antiviral
activity, cytotoxic activity against several human tumour cell
lines and regulatory activity of the myocardial force of con-
traction.5,6 The unusual structural features of these peptide
metabolites and the interesting biological activities have
aroused considerable interest among the synthetic chemistry
community. As a result, all the non-proteinogenic units
in callipeltins, namely (3S,4R)-3,4-dimethyl-L-glutamine
(diMeGln),7–9 (3S,4R)-3,4-dimethyl-L-pyroglutamic acid
(the N-terminus unit in callipeltin B),9 (2R,3R,4S)-4-amino-
7-guanidino-2,3-dihydroxyheptanoic acid (AGDHE),10–13
(2R,3R,4R)-3-hydroxy-2,4,6-trimethylheptanoic acid14–17
linked to the N-terminus of callipeltins A, C, D and
F–I; and (R)-b-methoxy-D-tyrosine (b-OMeTyr)18–20 were
obtained in a stereoselective manner. The availability of
synthetic standards allowed the assignment of the
Apart from synthetic studies, further knowledge on the
structure–activity relationship studies could arise from the
chemical and biological evaluations of new natural ana-
logues of the parent callipeltin A. In this respect the sponge
Latrunculia sp., collected at the Vanuatu Island, was proved
to be a rich source of new linear callipeltin derivatives
structurally related to callipeltin C (1),2 which in turn
represents the acyclic counterpart of callipeltin A. Continu-
ing investigation of the polar extracts of this sponge afforded
four new callipeltins, named callipeltins J–M (2–5), the
structural elucidation of which is the subject of the present
paper.
Keywords: Callipeltin; Peptides; Antifungal.
* Corresponding author. Tel.: +39 081678525; fax: +39 081678552; e-mail:
azampell@unina.it
0040–4020/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.10.032

132
M. V. D’Auria et al. / Tetrahedron 63 (2007) 131–140
D-erythro- OMeTyr
L-DiMeGln
NH2
OH
L-Leu
D-alloThr-2
O
AGDHE
O
MeO
O
OH
NH
OH
O
O
O
H
N
H
Me
N
Me
N
N
COOH
H2N
N
N
H
N
N
H
N
H
H
H
NH OH
O
O
O
O
HO
L-NMeAla
D-alloThr-1
HN
O
NH
O
NH2
L-NMeGln
H2N
NH
D-Arg
OH
Callipeltin C (1)
2. Results and discussion
d 1.04 and to an allylic methylene at d 2.39 and 2.17.
HMBC data confirmed the assignment, whereas the identity
of glutamine rather than glutamic was deduced from MS
data.
The lyophilised sponge was extracted with methanol and
the crude methanolic extract was subjected to a modified
Kupchan’s partitioning procedure. Fractionation of the buta-
nol-soluble material (ca. 4 g) by DCCC (Droplet Counter
Current Chromatography, CHCl3/MeOH/H2O, 7:13:8 as-
cending mode) followed by repeated reversed phase HPLC
afforded pure callipeltins J–M (2–5).
The amino acid sequence of 2 and placement of acyl substit-
uent were assigned from the analysis of the fragmentation
pattern in the ESIMS/MS mass spectrum (Fig. 1). The align-
ment of four amino acid residues in 2 was the same as that of
callipeltin D, with 3-methylglutamine in 2 replacing the
3,4-dimethylglutamine in callipeltin D.
The molecular formula of callipeltin J (2) was established as
C31H58N8O11 by HR ESIMS calculation of the [M+H]+
molecular ion at m/z 719.4297 (calcd for C31H59N8O11:
719.4303). Extensive interpretation of 2D NMR data ob-
tained by the gradient-enhanced versions of COSY, TOCSY,
HSQC, HMBC spectra (see Table 1), by comparison with
those of callipeltin D, disclosed the presence of one residue
each of alanine (Ala), 4-amino-7-guanidino-2,3-dihydroxy-
heptanoic acid (AGDHE), threonine (Thr) and 3-hydroxy-
2,4,6-trimethylheptanoic acid. In addition, the presence of
a 3-methylglutamine residue, not previously found in other
callipeltin derivatives, was easily inferred from the analysis
of COSY, TOCSY, HMBC and MS data. Starting from the
a-proton at d 4.46, this was found to be coupled to a methine
proton at d 2.21 in turn coupled with a methyl group at
The absolute stereochemistry of Ala, Thr and AGDHE resi-
dues in callipeltin J was determined by complete acid hy-
drolysis of 2 and Marfey’s analysis of the resulting amino
acids. The acid hydrolysate was derivatised with (1-fluoro-
2,4-dinitrophenyl)-5-L-alaninamide (L-FDAA),24 and then
the LC–MS comparison of the derivatives from 2 with the
FDAA derivatives of appropriate standards established the
presence of D-Ala, D-alloThr and (2R,3R,4S)-4-amino-7-
guanidino-2,3-dihydroxyheptanoic acid.
The molecular formula of callipeltin K (3) was established
as C67H116N18O21 by HR ESIMS. The NMR and MS data
for 3 were consistent with those of callipeltin C, except for
Table 1. 1H and 13C NMR data (500 MHz, CD3OD) for compounds 2–5
aa
2
3
aa
4
aa
5
a
a
dH
a
a
dH
dC
dC
dH
dC
dH
dC
TMHEA
TMHEA
1
2
3
4
5
6
7
8
9
10
177.8
45.6 2.61 m
79.9 3.51 dd (8.5, 2.4)
33.8 1.79 m
39.8 1.20 m
177.8
44.4
79.4
33.1
39.0
25.9
24.7
14.6
17.5
1
2
3
4
5
6
7
8
9
179.4
44.5
79.4
33.8
39.3
25.5
23.9
14.6
17.5
21.6
2.61 m
3.50 m
1.79 m
1.20 m
1.67 m
0.95 d (6.4)
1.07 d (7.1)
0.98 d (6.4)
0.89 d (6.4)
2.61 m
3.51 dd (9.3, 3.1)
1.76 m
1.20 m
26.5 1.67 m
1.67 m
24.7 0.95 d (6.2)
14.4 1.07 d (6.5)
17.5 0.98 d (6.9)
22.0 0.88 d (6.9)
0.95 d (6.2)
1.10 d (6.0)
0.99 d (6.7)
0.89 d (6.4)
22.0 10
Ala
a
b
CONH
Ala
a
b
4.31 q (7.1)
1.39 d (7.1)
50.9 4.32 ovl
17.6 1.42 d (7.5)
175.9 8.27b br s
51.2
17.7
4.32 ovl
1.42 d (7.2)
—
51.6
17.5
175.1
175.9 CONH
AGDHE
AGDHE
a
b
3.98 d (6.4)
3.75 dd (6.4, 2.6)
72.7 3.99 d (6.6)
75.3 3.69 dd (6.6, 2.2)
72.7
75.3
a
b
3.98 d (7.6)
3.73 dd (7.6, 3.5)
72.8
75.0
(continued)

M. V. D’Auria et al. / Tetrahedron 63 (2007) 131–140
133
Table 1. (continued)
aa
2
3
aa
4
aa
5
a
a
a
a
dH
dC
dH
dC
50.9
26.4
29.5
41.7
175.9 CO
157.8 Guan
NH-4
dH
dC
50.6
25.9
29.5
41.7
175.4
na
dH
dC
g
d
3
z
CO
Guan
NH-4
4.15 m
50.7 4.15 m
26.4 1.63, 1.30 m
29.5 1.68 m
42.0 3.18 m
175.9
g
d
3
4.15 m
1.66, 1.30 m
1.66 m
3.18 m
1.63, 1.41 m
1.68 m
3.21 m
z
—
—
157.8 7.26b br s
7.74b br s
—
—
3-MeGln
a
b
bMe
g
diMeGln
a
b
diMePyroGlu
4.46 ovl
2.21 m
1.04 d (6.8)
2.39, 2.17 m
58.1 4.46 ovl
39.7 2.21 m
17.2 1.04 d (6.8)
40.2 2.39, 2.17 m
58.2
39.7
4.43 d (8.5)
2.25 m
1.02 d (7.0)
2.66 m
1.18 d (6.7)
57.0
39.3
a
b
3.80 d (3.3)
2.59 m
63.4
39.7
14.7
39.7
10.0
17.2 bMe
39.4
13.7 bMe
41.3
15.6 gMe
1.12 d (7.2)
2.68 m
1.07 d (7.3)
g
gMe
g
CONH
CONH2 7.61 br s
173.1
173.1 CONH
180.8 CONH2
172.6 CONH
179.3 CONH
182.0
174.5
180.8 7.61b br s
—
—
Thr
a
b
g
CONH
Thr
Thr
4.47 d (6.3)
4.15 m
1.24 d (7.1)
—
60.0 4.36 d (8.1)
69.0 4.13 m
20.1 1.26 d (6.5)
60.1
68.4
20.4
a
b
g
4.30 d (7.0)
4.11 m
1.26 d (6.1)
—
60.0
68.0
20.2
a
b
g
4.33 d (6.7)
4.08 m
1.26 d ovl
—
60.4
68.0
20.1
172.1
172.7 8.43b br s
172.7 CONH
172.6 CONH
Thr
a
b
g
CONH
Thr
Thr
4.22 d (6.4)
4.06 m
1.31 d (6.5)
60.9
68.4
20.6
a
b
g
4.24 d (7.1)
4.06 m
1.31 d (7.3)
—
60.7
68.4
20.3
a
b
g
4.22 d (7.0)
4.02 m
1.26 d ovl
—
60.4
68.0
20.1
171.6
8.18a br s
172.8 CONH
171.9 CONH
Arg
a
b
g
d
Arg
a
b
Arg
4.32 ovl
54.0
29.5
25.5
41.7
4.35 ovl
1.98 m
1.68, 1.92 m
3.19 m
—
53.9
29.5
25.9
41.7
a
b
g
d
4.35 ovl
1.93, 1.68 m
1.67 m
3.18 m
—
53.9
29.5
26.2
42.2
173.8
na
1.97, 1.67 m
1.68, 1.92 m
3.19 m
g
d
CONH
CONH
Guan
7.71b br s
7.40b br s
na
157.8 Guan
174.7 CONH
157.8 Guan
—
—
Leu
a
b
Leu
Leu
4.73 dd (10.2, 4.6)
1.67, 1.45 m
1.67 m
0.92 d (6.1)
0.95 d (6.5)
7.99b br s
50.1
40.7
25.8
a
b
g
4.83 m
1.67, 1.45 m
1.74 m
0.99 d (7.2)
0.99 d (7.0)
—
49.8
40.6
26.0
a
b
g
4.76 m
1.70, 1.40 m
1.70 m
0.94 d (7.4)
0.94 d (7.4)
—
48.9
40.6
25.7
21.6
23.9
na
g
Me-g
Me-c
CONH
21.4 Me-g
23.5 Me-c
172.9 CONH
22.2 Me-g
22.2 Me-c
174.2 CONH
NMeGln
a
b
NMeGln
NMeGln
4.93 dd (10.4, 4.5)
1.70 m
1.98, 1.92 m
3.09 s
57.3
26.4
32.4
a
b
g
5.26 dd (9.7, 3.1)
2.20 m
2.19, 2.22 m
2.99 s
56.4
24.2
31.8
a
b
g
4.90 dd (10.1, 4.7)
1.94, 1.70 m
1.94 m
56.9
24.6
32.4
31.3
169.7
na
g
NMe
CO
CONH2
32.7 NMe
na CO
176.9 CONH2
31.7 NMe
169.9 CO
176.9 CONH2
2.80 s
na
—
—
b-OMeTyr
a
b
C4
C5
C6
C7
OMe
CONH
b-OTyr
a
b
b-OMeTyr
a
b
5.23 d (8.4)
4.33 d (8.4)
54.4
85.2
4.35 br d (2.6)
6.17 br d (2.6)
60.3
77.7
5.23 d (8.1)
4.32 d (8.1)
54.1
85.0
128.7
129.6
116.1
158.5
129.7 C4
130.6 C5
116.0 C6
157.9 C7
56.7
128.7 C4
126.0 C5
116.0 C6
158.7 C7
7.21 d (7.9)
6.82 d (7.9)
7.16 d (8.4)
6.84 d (8.4)
7.21 d (8.1)
6.79 d (8.1)
3.14 s
7.71b br s
174.9 CONH
—
164.8 CONH
—
172.1
MeAla
a
b
NMe
MeAla
a
b
MeAla
a
b
5.14 q (7.1)
1.41 d (7.1)
2.83 s
53.3
14.7
3.92 q (6.8)
1.53 d (6.8)
2.98 s
56.6
18.6
5.14 q (6.5)
1.42 d (6.5)
3.10 s
53.9
14.7
32.6
31.4 NMe
32.3 NMe
COOH
174.2 COOH
169.4 COOH
173.3
ovl: overlapped.
Coupling constants are in parentheses and given in hertz. 1H and 13C assignments aided by COSY, TOCSY, HMQC and HMBC experiments.
Data recorded in CD3OH.
a
b

134
M. V. D’Auria et al. / Tetrahedron 63 (2007) 131–140
NH2
To determine the stereochemistry of the 3-glutamine residue
O
in callipeltins J and K, both diastereoisomers of 3-methyl-
glutamic acid were prepared through diastereoselective
synthesis (Scheme 1).
NH
OH
O
O
H
N
H2N
N
N
H
OH
H
O
NH OH
458
O
The introduction of the b-methyl group in the above amino
acid was achieved by diastereoselective methylation of
a suitable L-aspartic acid derivative. Thus, the perbenzylated
L-aspartate 6 was enolised with KHMDS and then alkylated
with CH3I affording an unseparable 3:2 mixture of syn/anti
adducts. To assure the separation of the two diastereomers
and the regioselective deprotection of the b-carboxyl group,
the fully deprotected aspartate derivatives 8 were trans-
formed into the cyclic anhydrides 9. The alcoholysis of the
latter with 2-propanol gave exclusively the diastereomeric
mixture of a-ester b-acids 10a and 10b that were easily sep-
arated by HPLC. Each b-carboxylic acid was converted into
the corresponding diazo ketone by way of the b-acid
chloride (oxalyl chloride) and the obtained diazo ketones
11a and 11b were subjected to Wolff rearrangement fol-
lowed by methanolysis to afford the protected 3-methylglu-
tamic derivatives 12a and 12b. Concomitant removal of
the protecting groups through acidic hydrolysis gave the re-
quired (2S,3S)- and (2S,3R)-3-methyl glutamic acids, 13a
and 13b.25 A small sample (100 mg) of the two synthetic
3-methyl glutamates was derivatised with both enantiomers
of Marfey’s reagent.26
HO
600
HN
242
O
OH
Figure 1. Callipeltin J (2) with ESI MS/MS fragmentations.
the presence of the signals of the spin system of 3-methyl-
glutamine in the place of 3,4-dimethylglutamine in callipel-
tin C. The sequence of amino acid units in callipeltin K was
deduced from inter-residue NOE interactions NH/CHa,
acquired in CD3OH, and HMBC correlations as shown in
Figure 2. The absolute configurations of D-Ala, D-Arg,
L-MeAla, L-Leu, L-MeGln, two D-alloThr and (2R,3R,4S)-
4-amino-7-guanidino-2,3-dihydroxyheptanoic acid residues
were determined by LC–MS analysis of the acid hydrolysate
derivatised with Marfey’s reagent and comparison with
appropriate amino acid standards. To define the chirality of
b-OMeTyr residue, a sample of 3 was ozonised and hydro-
lysed and then subjected to Marfey’s analysis. Ion selective
monitoring of L-FDAA-OMeAsp (m/z 416) showed a peak at
tR 17.3 min corresponding to (2R,3S)-b-OMeAsp, indicating
a (2R,3R)-stereochemistry of b-OMeTyr residue.20
The L- and D-FDAA derivatives 14a–14d were analysed us-
ing ESI LC–MS in the positive ion mode. By monitoring for
FDAA-b-MeGlu at m/z 414, they were detected as separate
D-erythro- OMeTyr
D-threo-MeGln
NH2
L-Leu
OH
D-alloThr-2
OH
O
AGDHE
O
MeO
O
NH
OH
O
O
O
H
N
H
Me
N
Me
N
N
COOH
H2N
N
N
H
N
H
N
H
N
H
H
NH OH
O
O
O
O
HO
L-NMeAla
D-alloThr-1
HN
O
NH
NH
O
NH2
L-NMeGln
H2N
D-Arg
NOESY in CD3OH
HMBC in CD3OD
OH
Figure 2. Callipeltin K (3) with NOESY and HMBC correlations.
O
O
O
HOOC
c
a
BnOOC
i-PrOOC
BnOOC
b
d
COOH
COOBn
COOBn
NH2
NBn2
NBn2
7, mix
R
CF3CONH
9, mix
6
8, mix
R
R
R
i-PrOOC
HOOC
COOH
i-PrOOC
COOMe
g
e, f
h
CHN2
NHCOCF3
COOH
NH2
NHCOCF3
CH3
CH3
NHCOCF3
CH3
CH3
CH3
CH3
CH3
11a, R=
11b, R=
10a, R=
10b, R=
12a, R=
12b, R=
13a, R=
13b, R=
CH3
Scheme 1. (a) CH3I, KHDMS, THF, ꢀ78 ꢁC, 89%; (b) H2, Pd(OH)2, EtOH, Parr apparatus, 80%; (c) TFAA, THF, 0 ꢁC/rt; (d) iPrOH, rt, 82% over two steps;
(e) (ClCO)2, DMF, CH2Cl2, rt; (f) CH2N2, ether, 0 ꢁC, 62% over two steps; (g) PhCO2Ag, Et3N, MeOH/THF, ꢀ15 ꢁC/rt, 57%; (h) 6 N HCl, 160 ꢁC.

M. V. D’Auria et al. / Tetrahedron 63 (2007) 131–140
135
CH3
CH3
L-FDAA
HOOC
COOH
HOOC
COOH
CH3
NH(L)-FDAA
NH(L)-FDAA
(2S, 3S)-14a
tR=21.3
HOOC
COOH
(2S, 3R)-14b
tR=16.2
NH2
CH3
CH3
(2S, 3S)-13a
(2S, 3R)-13b
D-FDAA
HOOC
COOH
HOOC
COOH
NH(D)-FDAA
NH(D)-FDAA
(2S, 3S)-14c
tR=26.0
(2S, 3R)-14d
tR=18.2
CH3
(i) HCl 6N 16 h HOOC
COOH
Callipeltin J (2)
Callipeltin K (3)
(ii) L-FDAA
(iii) LC MS
NH(L)-FDAA
(2R, 3S)-14ent-d
tR=18.2
Scheme 2. HPLC retention times (tR) of FDAA derivatives of synthetic (2S,3S)- and (2S,3R)-3-methyl-glutamic acid and configurational assignment of 3-Me-
Glu in callipeltins J and K.
peaks at 21.3, 16.2, 26.0, 18.2 min, respectively (Scheme 2).
The L-FDAA derivative of b-MeGlu unit in callipeltins J and
K was co-eluted with the D-FDAA derivative of (2S,3R)-
3-methyl glutamic acid. Thus, the (2R,3S) configuration
for the b-MeGln residue in 2 and 3 was unambiguously
established (Scheme 2).
and 2D NMR data and comparison with those of callipeltin
C disclosed the presence of the same residues of callipeltin C
with a modification of the NMeGln-Tyr-NMeAla C-termi-
nus moiety in callipeltin L, as evidenced by the strong per-
turbation of all the resonances of these residues (see Table
1). In particular, in the TOCSY spectrum an a-aminoacyl
proton at d 4.35 (dC 60.3) was found to be coupled with
a broad doublet (J¼2.6 Hz) carbinol proton at dH 6.17 (dC
77.7). The chemical shift of this proton and HMBC cross
peak correlations with signals at d 126.0 and 128.7 were con-
sistent with the linkage of a p-hydroxyphenyl ring to the
b-carbon of the spin system. In addition, the proton at dH
6.17 was found to correlate with the C1 carbon (dC 169.4)
of the residue of NMeAla indicating that callipeltin L fea-
tures an unusual 3-lactone system, likely arising from the nu-
cleophilic displacement of the methoxy group at Cb of the
b-OMeTyr in callipeltin C by the carboxyl group of NMeAla
C-terminus. As a consequence, the stereochemistry of the
Cb of tyrosine unit in callipeltin L is inverted with respect
to 1 as suggested from the coupling constants’ analysis
(Table 1). Callipeltin L showed a high instability even in neu-
tral conditions and at low temperature. In the ESIMS spec-
trum no peaks relative to the molecular ion were observed,
whereas a peak at m/z 1245.7 corresponding to the loss of
dipeptide azepan-2-one subunit was present. ESIMS/MS
analysis of the peak at m/z 1245.7 revealed a fragmentation
pattern compatible with the structure in Figure 3 and super-
imposable to those reported for callipeltin G.4 The
As concerning the absolute configuration of some non-pro-
teinogenic units in callipeltins there are some apparent
discrepancies that should be mentioned. The b-OMeTyr resi-
due has the (2R,3R) configuration in callipeltins and in papu-
amide B27 and the (2S,3R) configuration in neamphamide
A,27 despite the high degree of structural homology with
callipeltin A. The 3,4-dimethylglutamine has a (2S,3S,4R)
configuration in callipeltins and in papuamide, whereas a
(2R,3S) configuration was found for the 3-methylglutamine
residue in callipeltins J and K. The anti-dehydration of
D-allo-Thr would afford the E-dAbu residue whereas in cal-
lipeltins H and I a Z-dAbu residue was found.4 These find-
ings confirm the important role of the epimerase domains
in non-ribosomal peptide synthetases (NRPSs) that catalyse
the conversion of a PCP bound, L/D-amino acid (in initiation
modules) or L/D-peptidyl (in elongation modules) moiety by
de- and re-protonating the Ca atom of the substrate.28
1
In the H NMR spectrum of callipeltin L (4), the signal
assigned to the b-methoxy group of the tyrosine residue
(dH 3.15–3.20, 3H, s) was absent. The analysis of the 1D
HO
NH2
O
O
O
OH
NH
OH
O
O
O
O
H
N
H
Me
N
N
N
H2N
N
N
H
N
H
N
H
N
H
Me
H
O
NH OH
O
O
O
O
HO
HN
NH
O
NH2
O
H2N
NH
OH
Figure 3. Callipeltin L (4).

136
M. V. D’Auria et al. / Tetrahedron 63 (2007) 131–140
degradation of the natural metabolite hampered any stereo-
chemical investigation. We, therefore, assumed that the
configuration of all residues was the same as in callipeltin C.
products were purified by column chromatography on silica
gel 70–230 mesh. All reactions were carried out under argon
atmosphere using flame-dried glassware.
Callipeltin M (5) is related to callipeltin B. In the ESIMS
spectrum, the molecular ion peak at m/z 1049 is 18 mass
units higher than in callipeltin B. The analysis of the 1D
and 2D NMR spectra suggested callipeltin M to be the acy-
clic callipeltin B. In particular, the absence of a macrocyclic
lactone closure was clearly indicated by the NMR reso-
nances of the b centres in Thr residues (Table 1), whereas
the cyclisation of 3,4-dimethylglutamine N-terminus as
3,4-dimethylpyroglutamic was evidenced mainly by the
downfield shift of the Ca to 63.4 compared with the value
of 57.0–57.7 of the same carbon in 3,4-dimethylglutamine
residue in other callipeltin derivatives.
3.2. Sponge material and separation of individual
peptides
Latrunculia sp. (Class Demospongiae, Order Poeciloscler-
ida, Family Latrunculidae) was collected at a depth of 15–
20 m at Emae, Vanuatu South Pacific, in June 1996. The
samples were frozen immediately after collection and
lyophilised to yield 800 g of dry mass. Taxonomic identifica-
tion was performed by Prof. John Hooper of Queensland
Museum, Brisbane, Australia and reference specimens are
on file (R1642) at the ORSTOM Centre of Noumea. Prelim-
inary tests of bioactivity on polar extracts showed antifungal
activity against C. albicans and cytotoxic activity against
L16 cells (10 mg/mL, 100% inhibition).
OH
MeO
O
OH
The lyophilised material (800 g) was extracted with metha-
nol (4ꢂ2.5 L) at room temperature and the crude methanolic
extract (80 g) was subjected to a modified Kupchan’s parti-
tioning procedure as follows. The methanol extract was dis-
solved in a mixture of MeOH/H2O containing 10% H2O and
partitioned against n-hexane. The water content (%, v/v) of
the MeOH extract was adjusted to 20 and 40%, and parti-
tioned against CCl4 and CHCl3, respectively. The aqueous
phase was concentrated to remove MeOH and then extracted
with n-BuOH. The butanol-soluble material (ca. 4 g) was
chromatographed by DCCC in five runs (CHCl3/MeOH/
H2O, 7:13:8, ascending mode) and fractions of 4 mL were
collected.
O
O
H
N
H
Me
N
Me
N
N
COOH
N
H
N
H
N
H
N
H
O
O
O
O
O
HO
NH
O
NH2
H2N
NH
Callipeltin M (5)
The stereochemistry of the amino residues was determined to
be D-allo-Thr, D-Arg, L-Leu, L-MeGln, L-MeAla, (2S,3S,4R)
diMeGln, (2R,3R)-b-OMeTyr by Marfey’s analysis.
Fraction 10 was purified by HPLC on a Vydac C18 column
(10m, 250ꢂ10 mm, 4 mL/min) with 28% CH3CN/
H2O (0.1% TFA) as eluent to give 16.8 mg of pure callipeltin
K 3 (tR¼5.4 min). The additional peaks at tR¼2.4 and
3.1 min were further individually purified by HPLC on a
Thermo-Hypurity column (5m, 250ꢂ4.6 mm) eluting with
28% CH3CN/H2O containing 0.05% TFA (flow rate
1.2 mL/min) to give 6.9 mg of pure callipeltin M 5
(tR¼3.4 min) and 1.2 mg of pure callipeltin J 2 (tR¼5.4 min),
respectively.
Callipeltins K (2) and J (3) inhibit the growth of Candida
albicans (ATCC 24433) in the standard disk assay with
MIC values of ca. 10ꢀ4 M.
3. Experimental
3.1. General experimental procedures
Specific rotations were measured on a Perkin–Elmer 243 B
polarimeter. High-resolution ESIMS spectra were per-
formed with a Micromass QTOF Micromass spectrometer.
ESIMS experiments were performed on an Applied Biosys-
tem API 2000 triple-quadrupole mass spectrometer. NMR
spectra were obtained on a Varian Mercury-400 and Inova-
500 NMR spectrometers (1H at 400 and 500 MHz, 13C at
100 and 125 MHz, respectively) equipped with a Bruker
X-32 hardware, d (ppm), J in hertz, spectra referred to
CD2HOD as internal standards (dH¼3.30). HPLC was
performed using a Waters Model 6000 A pump equipped
with U6K injector and a differential refractometer, model
401.
Fractions 11 and 12 were purified by HPLC on a Vydac C18
column (10m, 250ꢂ10 mm, 4 mL/min) with 28% CH3CN/
H2O (0.1% TFA) as eluent to give a peak at tR¼10.6 min
containing mainly callipeltin L (4) that was further purified
by HPLC on a Thermo-Hypurity column (5m, 250ꢂ4.6 mm)
eluting with 27% CH3CN/H2O containing 0.05% TFA (flow
rate 1.2 mL/min) to give 5.0 mg of pure 4 (tR¼4.2 min).
3.3. Characteristic data for each compound
3.3.1. Callipeltin J (2). White amorphous solid; [a]2D5 ꢀ1.2
(c 0.09, methanol); IR (KBr) 3300, 1760, 1650 cmꢀ1; 1H and
13C NMR data in CD3OD given in Table 1. ESIMS: m/z
(%) 719.6 (25) [M+H]+, 360.2 (100) [M+2H]++; HRMS
(ESI) calcd for C31H59N8O11: 719.4303; found: 719.4297
[M+H]+.
All reagents were commercially obtained (Aldrich, Fluka) at
highest commercial quality and used without further purifi-
cation except where noted. Dichloromethane, ether, tetra-
hydrofuran and triethylamine were distilled from calcium
hydride immediately prior to use. All reactions were moni-
tored by TLC on silica gel plates (Macherey, Nagel). Crude
3.3.2. Callipeltin K (3). White amorphous solid; [a]2D5 ꢀ7.0
(c 1.12, methanol); IR (KBr) 3340, 3080, 1760, 1625 cmꢀ1
;

M. V. D’Auria et al. / Tetrahedron 63 (2007) 131–140
137
1H and 13C NMR data in CD3OD given in Table 1. ESIMS:
m/z (%) 1509.7 (25) [M+H]+, 754.5 (100) [M+2H]++; HRMS
(ESI) calcd for C67H117N18O21: 1509.8641; found:
1509.8635 [M+H]+.
D-Ala (20.5), D-aThr (14.5), (2R,3S)-3-methyl-glutamic
acid (18.2).
The hydrolysate of callipeltin K (3) contained: (2R,3R,4S)-
4-amino-7-guanidino-2,3-dihydroxyheptanoic acid (19.5),
D-Ala (20.5), D-aThr (14.5), (2R,3S)-3-methyl-glutamic
acid (18.2), D-Arg (13.0), L-Leu (29.3), L-NMeAla (18.5).
3.3.3. Callipeltin L (4). White amorphous solid; 1H and 13
NMR data in CD3OD given in Table 1. ESIMS: m/z (%)
C
1245.7 (100) [M+H]+.
The hydrolysate of callipeltin M (5) contained: D-aThr
(14.5), (3S,4R)-3,4-diMe-L-Glu (17.7), D-Arg (13.0), L-Leu
(29.3), L-NMeAla (18.5).
3.3.4. Callipeltin M (5). White amorphous solid; [a]D ꢀ1.9
(c 0.38, methanol); IR (KBr) 3340, 3080, 1760, 1625 cmꢀ1
;
1H and 13C NMR data in CD3OD given in Table 1. ESIMS:
m/z (%) 1049.5 (30) [M+H]+, 525 (100) [M+2H]++; HRMS
(ESI) calcd for C47H77N12O15: 1049.5631; found:
1049.5623 [M+H]+.
3.4.3. Determination of the absolute stereochemistry of
b-OMeTyr residue in callipeltins K (3) and M (5). A
stream of ozone in O2 was bubbled through a cooled solution
of callipeltin K (0.5 mg), callipeltin M (0.5 mg) or of all four
diastereomers of b-OMeTyr (1 mg)20 in MeOH (0.5 mL) at
ꢀ78 ꢁC for 1 h. Hydrogen peroxide (35%, 10 drops) was
added to the reaction mixture and then allowed to stand at
room temperature overnight. The solvent was removed un-
der a stream of N2 and the resulting b-methoxyaspartates
were immediately subjected to Marfey’s derivatisation. The
ozonolysis products of callipeltins K and M were then dis-
solved in degassed 6 M HCl (0.5 mL) in an evacuated glass
tube and heated at 160 ꢁC for 16 h. The solvent was removed
in vacuo and the resulting material was subjected to
Marfey’s derivatisation and LC–MS analysis.
3.4. Determination of absolute stereochemistry
3.4.1. Peptide hydrolysis. Peptide samples (200 mg) were
dissolved in degassed 6 M HCl (0.5 mL) in an evacuated
glass tube and heated at 160 ꢁC for 16 h. The solvent was
removed in vacuo and the resulting material was subjected
to further derivatisation.
3.4.2. LC–MS analysis of Marfey’s (FDAA) derivatives.
A portion of the hydrolysate mixture (800 mg) or the amino
acid standard (500 mg) was dissolved in 80 mL of a 2:3 solu-
tion of TEA/MeCN and treated with 75 mL of 1% 1-fluoro-
2,4-dinitrophenyl-5-L-alaninamide (FDAA) in 1:2 MeCN/
acetone. The vials were heated at 70 ꢁC for 1 h, and the
contents were neutralised with 0.2 M HCl (50 mL) after
cooling to room temperature. An aliquot of the L-FDAA
derivative was dried under vacuum, diluted with MeCN/
5% HCOOH in H2O (1:1) and separated on a Vydac C18
(25ꢂ1.8 mm i.d.) column by means of a linear gradient
from 10 to 50% aqueous acetonitrile containing 5% formic
acid and 0.05% trifluoroacetic acid, over 45 min at 1 mL/
min. The RP-HPLC system was connected to the electro-
spray ion source by inserting a splitter valve and the flow
going into the mass spectrometer source was set at a value
of 100 mL/min. Mass spectra were acquired in positive ion
detection mode (m/z interval of 320–900) and the data were
analysed using the suite of programs Xcalibur (Thermo-
Retention times of authentic L-FDAA-b-OMeAsp (min):
(2S,3S)-b-OMeAsp (10.2), (2S,3R)-b-OMeAsp (16.1),
(2R,3R)-b-OMeAsp (9.9), (2R,3S)-b-OMeAsp (17.3). The
hydrolysate of ozonolysis products of 3 and 5 contained:
(2R,3S)-b-OMeAsp (17.3).
3.5. Synthetic procedures for L-b-methyl-glutamic acids
3.5.1. L-N,N-Dibenzyl-b-methylaspartic acid dibenzyl
ester 7. A solution of L-N,N-dibenzyl-aspartic acid dibenzyl
ester 6 (2.0 g, 4.06 mmol) in dry THF (15 mL) was cooled
to ꢀ78 ꢁC under argon and stirred. Iodomethane (1.72 g,
12.1 mmol) was added followed by a slow addition of a solu-
tion of KHDMS (17.8 mL of a 0.5 M solution in toluene,
8.9 mmol). After the addition, the mixture was stirred at
ꢀ78 ꢁC for 30 min. A saturated solution of NH4Cl (5 mL)
was added and the mixture was warmed slowly to room tem-
perature. The organic layer was separated and the aqueous
layer was extracted several times with AcOEt. The collected
organic layers were washed with saturated NaHCO3 and
brine. After evaporation of the solvent, silica gel chromato-
graphy (hexane/AcOEt, 95:5) gave 7 as an approximately
3:2 mixture of diastereoisomers (1.83 g, 89%). White amor-
phous solid; IR (KBr) 1790, 1310 cmꢀ1; HRMS (ESI) calcd
for C33H34NO4: 508.2488; found: 508.2466 [M+H]+.
ꢀ
Quest, San Jose, California); all masses were reported as
average values. Capillary temperature was set at 280 ꢁC,
capillary voltage at 37 V, tube lens offset at 50 V and ion
spray voltage at 5 V.
Retention times of authentic FDAA-amino acids (min): L-Thr
(12.5), D-Thr (17.6), L-aThr (13.1), D-aThr (14.1), L-Ala
(16.6), D-Ala (20.0), L-NMeAla (18.7), D-NMeAla (19.4),
L-Arg (11.7), D-Arg (13.08), L-Leu (28.9), D-Leu (34.8).
To determine the absolute configuration of 3,4-diMeGln and
4-amino-7-guanidino-2,3-dihydroxyheptanoic acid (AGDHE)
an authentic sample of callipeltin C was used as standard.
The hydrolysate of callipeltin C contained: (2R,3R,4S)-
4-amino-7-guanidino-2,3-dihydroxyheptanoic acid (19.5),
D-Arg (13.1), D-aThr (14.0), (3S,4R)-3,4-diMe-L-Glu (17.7),
L-NMeAla (18.5), D-Ala (20.1), L-Leu (28.7).
1
NMR data for major diastereoisomer: H NMR (400 MHz,
CDCl3) d: 1.21 (3H, d, J¼7.1 Hz), 3.06 (1H, m), 3.37 (2H,
d, J¼13.5 Hz), 3.55 (2H, d, J¼9.0 Hz), 4.00 (1H, d,
J¼11.7 Hz), 4.93 (2H, m), 7.2–7.4 (20H, m); 13C NMR
(100 MHz, CDCl3) d: 15.4, 39.6, 55.1, 66.4, 66.5, 128.5,
129.4, 129.5, 138.9, 139.3, 171.4, 171.7.
1
The hydrolysate of callipeltin J (2) contained: (2R,3R,4S)-
4-amino-7-guanidino-2,3-dihydroxyheptanoic acid (19.5),
NMR data for minor diastereoisomer: H NMR (400 MHz,
CDCl3) d: 1.06 (3H, d, J¼7.2 Hz), 3.24 (1H, m), 3.28 (2H,

138
M. V. D’Auria et al. / Tetrahedron 63 (2007) 131–140
d, J¼13.5 Hz), 3.53 (2H, d, J¼9.0 Hz), 3.66 (1H, d,
J¼11.2 Hz), 5.11 (2H, m), 7.2–7.4 (20H, m); 13C NMR
(100 MHz, CDCl3) d: 14.3, 35.4, 57.7, 66.4, 66.5, 127.4,
128.8, 129.2, 136.3, 137.6, 175.3, 176.8.
(100 MHz, CDCl3) d: 13.5, 21.7, 41.9, 54.5, 71.4, 168.6,
178.3.
3.5.4. Isopropyl (2S,3R)-5-diazo-4-oxo-N-trifluoroacetyl-
isoleucinate 11a. Carboxylic acid 10a (90 mg, 0.32 mmol)
was dissolved in 5 mL of CH2Cl2 to which was added
(ClCO)2 (1.6 mL, 2 M in CH2Cl2) followed by the addition
of 4 mL of DMF (0.05 mmol). After stirring for 15 min at
room temperature the reaction mixture was concentrated in
vacuum and the resulting residue was dissolved in ether
and the resulting solution was added dropwise to an ethereal
solution of CH2N2 (ca. 20 equiv) at 0 ꢁC. The cloudy yellow
mixture was stirred for 45 min and then concentrated under
reduce pressure to give an oil, which was purified on silica
gel (8% EtOAc/hexane) affording 58.4 mg of 11a as yellow
oil (62% yield).
3.5.2. L-b-Methylaspartic acid 8. The mixture 7 (1. 0 g,
2.83 mmol) was dissolved in EtOH (20 mL) and the solution
was poured in the pressure bottle of a Parr hydrogenation ap-
paratus. Pd(OH)2 Degussa E101 (100 mg) was added and the
mixture was shaken under 6 atm of H2 for 12 h. The bottle
was degassed, the catalyst was filtered and washed several
times with MeOH. The solvent was evaporated and the crude
product was purified by RP-18 eluting with water to give 8 as
an approximately 3:2 mixture of diastereoisomers (451 mg,
80%). White amorphous solid; HRMS (ESI) calcd for
C5H10NO4: 148.1367; found: 148.1355 [M+H]+; IR (KBr)
3350, 1760 cmꢀ1
.
HRMS (ESI) calcd for C10H15F3N3O3: 282.1066; found:
1
1
NMR data for major diastereoisomer: H NMR (400 MHz,
D2O) d: 1.08 (3H, d, J¼10.8 Hz), 2.94 (1H, m), 3.76 (1H,
d, J¼4.4 Hz); 13C NMR (100 MHz, D2O) d: 14.2, 57.1,
65.1, 174.7.
286.1055 [M+H]+; H NMR (400 MHz, CDCl3) d: 1.18
(6H, d, J¼6.8 Hz), 1.20 (3H, d, J¼7.4 Hz), 3.23 (1H, m),
4.61 (1H, dd, J¼3.1, 7.5 Hz), 4.97 (1H, m), 5.29 (1H, br
s), 7.71 (1H, d, J¼7.5 Hz); 13C NMR (100 MHz, CDCl3)
d: 14.7, 22.9, 23.1, 46.4, 54.8, 60.5, 69.1, 169.0, 179.0.
1
NMR data for minor diastereoisomer: H NMR (400 MHz,
D2O) d: 1.01 (3H, d, J¼7.6 Hz), 2.90–2.99 (1H, m), 3.88
(1H, d, J¼3.3 Hz); 13C NMR (100 MHz, D2O) d: 11.5,
51.8, 61.0, 172.7.
3.5.5. Isopropyl (2S,3S)-5-diazo-4-oxo-N-trifluoroacetyl-
isoleucinate 11b. The title compound 11b was prepared fol-
lowing the same procedure described for compound 11a.
HRMS (ESI) calcd for C10H15F3N3O3: 282.1066; found:
1
3.5.3. Isopropyl L-3-methyl-N-trifluoroacetylaspartates
10a and 10b. Trifluoroacetic anhydride (22.2 mg,
0.11 mmol) was added to a stirred suspension of 3-methyl-
aspartic acids 8 (420 mg, 2.11 mmol) in dry THF (10 mL)
at 0 ꢁC over 10 min under nitrogen. The reaction mixture
was then allowed to warm to room temperature and stirred
until dissolution was complete (ca. 1.5 h). The mixture
was evaporated under reduced pressure and the residue
was dried for 3–4 h under high vacuum. Dry iPrOH
(10 mL) was then added to the residue and the solution
was left at room temperature overnight. iPrOH was removed
under reduce pressure to give 389 mg (82%) of crude prod-
uct. HPLC (Macherey, Nagel; SP 250/10 Nucleosil 50-7,
10m, 250ꢂ10 mm, flow 5 mL/min) purification using hex-
ane/AcOEt (8:2, with 0.05% TFA) as eluent afforded com-
pounds 10a (106.8 mg, tR¼10 min) and 10b (188.7 mg,
tR¼13 min).
286.1072 [M+H]+; H NMR (400 MHz, CDCl3) d: 1.27
(6H, d, J¼6.0 Hz), 1.34 (3H, d, J¼7.3 Hz), 2.98 (1H, m),
4.58 (1H, dd, J¼4.4, 7.4 Hz), 5.10 (1H, m), 5.38 (1H, s),
7.38 (1H, br s); 13C NMR (100 MHz, CDCl3) d: 14.5,
21.8, 21.9, 46.2, 54.9, 55.4, 70.7, 168.6, 171.3.
3.5.6. Isopropyl (2S,3S)-3-methyl-N-trifluoroacetyl-glu-
tamate 12a. In a two-necked round-bottomed flask contain-
ing 11a (55 mg, 0.2 mmol) were added MeOH (2.8 mL) and
dry THF (2 mL). The solution was magnetically stirred
under argon with exclusion of light and cooled at ꢀ15 ꢁC.
A solution of silver benzoate (4.58 mg, 0.02 mmol) in anhy-
drous triethylamine (83.6 mL, 0.6 mmol) was added and the
reaction mixture was allowed to slowly warm up to room
temperature and stirred for 3 h. The solvents were evapo-
rated in vacuum and the residue was taken up in EtOAc.
The organic solution was successively extracted with satu-
rated aqueous solutions of NaHCO3, NH4Cl and NaCl, dried
over MgSO4, filtered and evaporated under vacuum. The
crude product was purified by HPLC (Macherey, Nagel;
ET 200/4 Nucleosil 100-5, 5m, 250ꢂ4.6 mm) with hexane/
AcOEt (75:25, with 0.05% TFA) to give pure 12a (tR¼
10 min, 35.7 mg, 57%). White amorphous solid; HRMS
(ESI) calcd for C12H19F3NO5: 314.1215; found: 314.1225
[M+H]+; 1H NMR (400 MHz, CDCl3) d: 1.04 (3H, d,
J¼7.0 Hz), 1.29 (6H, d, J¼6.3 Hz), 2.33 (1H, dd, J¼6.8,
16.0 Hz), 2.47 (1H, dd, J¼5.2, 16.0 Hz), 2.62 (1H, m),
3.70 (3H, s), 4.55 (1H, dd, J¼5.2, 8.2 Hz), 5.09 (1H, m),
7.55 (1H, d, J¼8.2 Hz); 13C NMR (100 MHz, CDCl3) d:
16.6, 21.9, 32.8, 37.5, 52.2, 57.1, 70.6, 94.6, 172.8, 175.7.
3.5.3.1. Isopropyl (2S,3R)-3-methyl-N-trifluoroacetyl-
aspartate 10a. White amorphous solid; HRMS (ESI) calcd
1
for C10H15F3NO5: 286.0902; found: 286.0911 [M+H]+; H
NMR (400 MHz, CDCl3) d: 1.26 (3H, d, J¼6.2 Hz), 1.28
(3H, d, J¼6.2 Hz), 1.33 (3H, d, J¼7.2 Hz), 3.43 (1H, dq,
J¼3.6, 7.2 Hz), 4.84 (1H, dd, J¼3.6, 9.0 Hz), 5.08 (1H,
m), 7.29 (1H, d, J¼9.0 Hz), 7.69 (1H, br s); 13C NMR
(100 MHz, CDCl3) d: 13.5, 21.7, 21.8, 41.9, 54.4, 71.4,
168.6, 178.2.
3.5.3.2. Isopropyl (2S,3S)-3-methyl-N-trifluoroacetyl-
aspartate 10b. White amorphous solid; HRMS (ESI) calcd
1
for C10H15F3NO5: 286.0902; found: 286.0905 [M+H]+; H
NMR (400 MHz, CDCl3) d: 1.26 (3H, d, J¼6.3 Hz), 1.29
(3H, d, J¼6.3 Hz), 1.33 (3H, d, J¼7.4 Hz), 3.08 (1H, dq,
J¼4.2, 7.4 Hz), 4.81 (1H, dd, J¼4.2, 8.3 Hz), 5.10 (1H,
m), 7.33 (1H, d, J¼8.3 Hz), 8.63 (1H, s); 13C NMR
3.5.7. Isopropyl (2S,3R)-3-methyl-N-trifluoroacetyl-glu-
tamate 12b. The title compound 12b was prepared follow-
ing the same procedure described for compound 11b.
White amorphous solid; HRMS (ESI) calcd for

M. V. D’Auria et al. / Tetrahedron 63 (2007) 131–140
139
C12H19F3NO5: 314.1215; found: 314.1207 [M+H]+; 1H
NMR (400 MHz, CDCl3) d 1.07 (3H, d, J¼7.0 Hz), 1.29
(6H, d, J¼6.3 Hz), 2.1 (1H, dd, J¼6.7, 16.5 Hz), 2.5 (1H,
dd, J¼8.2, 16.5 Hz), 2.84 (1H, m), 3.71 (3H, s), 4.22 (1H,
d, J¼7.5 Hz), 5.11 (1H, sep, J¼6.3 Hz), 5.9 (1H, br s); 13C
NMR (100 MHz, CDCl3) d: 15.9, 22.0, 22.1, 29.9, 32.9,
37.9, 60.2, 69.5, 170.5, 177.4.
experiments were run in triplicate. Negative growth corre-
sponded to no visible growth in the well.
Acknowledgements
This work was supported by grants from Regione Campania
P.O.R. 2000–2006 Misura 3.13. NMR spectra were provided
by the CRIAS Centro Interdipartimentale di Analisi Stru-
mentale, Faculty of Pharmacy, University of Naples. The
staffs are acknowledged. We thank Dr. Anna Catania (Divi-
sione di Medicina Interna e di Trapianto del Fegato, Ospe-
dale Maggiore di Milano IRCCS, 20122 Milano, Italy) for
the antifungal tests. We thank the diving team of IRD in
3.5.8. (2S,3S)-3-Methyl-glutamic acid 13a. Protected glu-
tamic acid 12a (4 mg) was dissolved in degassed 6 M HCl
(0.5 mL) in an evacuated glass tube and heated at 160 ꢁC
for 16 h. The solvent was removed under vacuum and an al-
iquot (100 mg) was subjected to Marfey’s derivatisation26
with L-FDAA and D-FDAA and LC–MS analysis.
ꢀ
Noumea (New Caledonia) for the collection of the sponge,
White amorphous solid; HRMS (ESI) calcd for C6H12NO4:
Prof. John Hooper (Queensland Museum in Brisbane, Aus-
tralia) for the identification of the sponge and the Vanuatu
government through the Fisheries Department for allowing
the collection of biological material.
1
162.0766; found: 162.0755 [M+H]+; H NMR (400 MHz,
D2O) d: 0.97 (3H, d, J¼6.6 Hz), 2.38 (1H, dd, J¼7.7,
15.6 Hz), 2.46 (1H, m), 2.58 (1H, dd, J¼4.5, 15.6 Hz), 3.78
(1H, d, J¼3.9 Hz); 13C NMR (100 MHz, D2O) d: 14.4, 31.1,
37.6, 58.1, 163.5, 178.2.
References and notes
3.5.9. (2S,3R)-3-Methyl-glutamic acid 13b. Protected glu-
tamic acid 12b (4 mg) was dissolved in degassed 6 M HCl
(0.5 mL) in an evacuated glass tube and heated at 160 ꢁC
for 16 h. The solvent was removed under vacuum and the
resulting material was subjected to Marfey’s derivatisation26
with L-FDAA and D-FDAA and LC–MS analysis. HRMS
(ESI) calcd for C6H12NO4: 162.0766; found: 162.0772
[M+H]+; 1H NMR (400 MHz, D2O) d: 1.01 (3H, d,
J¼6.6 Hz), 2.40 (1H, dd, J¼7.0, 16.3 Hz), 2.57 (1H, dd,
J¼6.6, 16.3 Hz), 2.63 (1H, m), 4.03 (1H, d, J¼3.9 Hz); 13C
NMR (100 MHz, D2O) d: 15.0, 31.0, 37.8, 58.2, 169.3, 176.4.
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