Cyclodysidins A-D, cyclic lipopeptides from the marine sponge-derived Streptomyces strain RV15

Article abstract of DOI:10.1016/j.tetlet.2011.10.051

Four new cyclic lipopeptides, cyclo-(AFA-Ser-Gln-Asn-Tyr-Asn-Ser-Thr), named cyclodysidins A-D, were isolated from the broth culture of Streptomyces strain RV15 associated with the marine sponge Dysidea tupha. The sequences of the amino acid building blocks in the compounds and their structures were determined by 1D- and 2D-NMR techniques and CID-MS/MS experiments. The absolute configurations of all α-amino acids were determined by HPLC analysis after derivatization with Marfey's reagent and comparison with commercially available reference samples, while those two of the β-amino fatty acids were determined by using racemic and enantiopure reference samples synthetically prepared.

Full text of DOI:10.1016/j.tetlet.2011.10.051

Tetrahedron Letters 53 (2012) 23–29
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Cyclodysidins A–D, cyclic lipopeptides from the marine sponge-derived
Streptomyces strain RV15
Usama Ramadan Abdelmohsen a, , Guoliang Zhang b, , Allan Philippe b, Werner Schmitz c,
b,
Sheila Marie Pimentel-Elardo a, Barbara Hertlein-Amslinger b, Ute Hentschel a, , Gerhard Bringmann
⇑
⇑
a Julius-von-Sachs Institute for Biological Sciences, University of Würzburg, Julius-von-Sachs-Platz 3, D-97082 Würzburg, Germany
b Institute of Organic Chemistry, University of Würzburg, Am Hubland, D-97074 Würzburg, Germany
c Department of Physiological Chemistry II, Theodor-Boveri Institute, University of Würzburg, Am Hubland, D-97074 Würzburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Four new cyclic lipopeptides, cyclo-(AFA-Ser-Gln-Asn-Tyr-Asn-Ser-Thr), named cyclodysidins A–D, were
isolated from the broth culture of Streptomyces strain RV15 associated with the marine sponge Dysidea
tupha. The sequences of the amino acid building blocks in the compounds and their structures were
determined by 1D- and 2D-NMR techniques and CID-MS/MS experiments. The absolute configurations
Received 1 September 2011
Revised 7 October 2011
Accepted 10 October 2011
Available online 17 October 2011
of all
a-amino acids were determined by HPLC analysis after derivatization with Marfey’s reagent and
comparison with commercially available reference samples, while those two of the b-amino fatty acids
were determined by using racemic and enantiopure reference samples synthetically prepared.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Cyclic lipopeptides
Cyclodysidins
Dysidea tupha
Streptomyces
Marfey’s analysis
b-amino fatty acid
Rodionov reaction
Reformatsky reaction
Sponges are reservoirs of dense and phylogenetically diverse
microbial communities.1 These marine microorganisms have been
a rich source for a multitude of biomedically relevant secondary
metabolites.2 Bacteria of the clade actinomycetes account for
approximately half of the bioactive secondary metabolites that
have so far been discovered from the marine invertebrate-associ-
ated bacteria.3,4 These metabolites exhibit various activities
including anti-infective, anti-inflammatory, and anticancer activi-
ties and represent a plethora of structurally diverse natural prod-
ucts, among them polyketides, alkaloids, fatty acids, terpenes,
and peptides.5–8 The cyclic lipopeptides attract significant atten-
tion due to their antimicrobial and anticancer activities9–11 and
they have been reported from different bacterial genera including
Mycobacterium, Streptomyces, Pseudomonas, and Bacillus.12–15 We
have previously reported the isolation and phylogenetic identifica-
tion based on the 16S-rRNA gene sequence information of Strepto-
myces strain RV15 associated with the sponge Dysidea tupha,
collected from Rovinj, Croatia.16 Here, we describe the isolation
and full structural elucidation of four new cyclic lipopeptides from
this marine strain (Fig. 1).
Streptomyces sp. RV15 strain was fermented in ISP 2 medium and
the secondary metabolites were collectedwith XAD-16resin.17 After
elution of the resin with acetone, the crude extract was fractionated
by normal-phase silica gel chromatography followed by gel filtra-
tion on Sephadex LH-20 to give a fraction enriched with peptides. Fi-
nal purification was performed on reversed-phase HPLC to afford
four pure new compounds, named cyclodysidins A–D. Cyclodysidin
A18 showed a molecular formulaof C45H71N11O15 by HR-ESIMS (TOF)
analysis (m/z 1006.5207 for [M+H]+), with 16 degrees of unsatura-
tion. The presence of several doublets and doublets of doublets at
4-5 ppm, attributed to a-protons of amino acids, and the character-
istic chemical shifts for the amide carbonyls at 170–180 ppm sug-
gest the peptidic nature of the molecule. In agreement with the
possible occurrence of amide functionalities, the IR spectrum
showed bands at 3345 and 1638 cmꢀ1. The compounds turned pur-
ple with ninhydrin reagent only upon hydrolysis with 6 M HCl,
which gave a first hint at a cyclic structure.
The 13C NMR spectrum displayed 11 ester/amide-type carbon-
yls (d 172.4, 172.9, 173.0, 173.7, 174.1, 174.3, 174.4, 174.5, 174.9,
⇑
Corresponding authors. Tel.: +49 931 31 85323; fax: +49 931 31 84755 (G.B.);
tel.: +49 931 31 82581; fax: +49 931 31 86235 (U.H.).
E-mail addresses: bringman@chemie.uni-wuerzburg.de (G. Bringmann),
ute.hentschel@uni-wuerzburg.de (U. Hentschel).
175.1, and 176.6), seven
56.9, 57.2, 57.4, and 60.0), two primary carbinols (d 62.3 and
62.4), secondary carbinol (d 67.3), aromatic signals for
a-methine carbons (d 52.3, 52.4, 55.6,
a
a
These authors contributed equally to this work.
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.10.051

24
U. R. Abdelmohsen et al. / Tetrahedron Letters 53 (2012) 23–29
D-Ser-1
OH
L-Asn-1 H N
2
H
H
N
O
L-Thr
O
H
O
NH
Me
Me
Me
Me
( )8
( )9
( )
O
Cyclodysidin A (1): R =
Cyclodysidin B (2): R =
Cyclodysidin C (3): R =
Cyclodysidin D (4): R =
HN
H
N
H
H
O
HO
HO
-AFA
O
R
Me
3
D-Tyr
NH
10
O
H
NH
NH
( )
H
H
12
N
N
D-Ser-2
H
O
O
HO
H
O
O
H N
2
L-Gln
2
L-Asn-2
Figure 1. Structures of the new compounds 1–4.
Table 1
NMR-spectroscopic data of cyclodysidin A (1) in methanol-d4 (1H: 600 MHz; 13C: 150 MHz)
NMR-spectroscopic data of cyclodysidin A dC
(1) in methanol-d4 (1H: 600 MHz;
13C: 150 MHz)
dH, mult
COSY (J in Hz) HMBC
ROESY
Asn1
CO
a
b
173.7
52.4
37.1
174.9
4.65, dd
2.76/2.72, m
b (7.5, 6.4)
a
CO, b,
CO, a, c
c
, Tyr-CO
Tyr-b
c
Asn2
CO
a
174.3
52.3
37.4
4.51, dd
2.52/2.44, m
b (7.9, 5.7)
a
CO, b,
CO,
c
c
, Gln-CO
b
a
,
Gln-a
c
175.1
Gln
CO
a
b
c
174.1
55.6
27.1
4.19, dd
2.17/2.10, m
2.47, m
b (8.3, 5.8)
CO, b,
CO,
, b, d
c
c
, Ser2-CO
, d
Asn2-b
a,
c
a
,
32.9
b
a
d
176.6
Ser1
CO
a
172.9
57.4
62.4
4.37, t
3.84, m
b (5.6)
a
CO, b, Asn1-CO
CO,
Asn1-b
Thr-b
b
a
Ser2
CO
a
173.0
57.2
62.3
4.31, t
3.92, m
b (5.3)
a
CO, b, b-AFA-CO
CO,
b-AFA-2
Ser1-b
b
a
Thr
CO
a
b
c
172.4
60.0
67.3
20.4
4.28, d
4.40, m
1.16, d
b (2.6)
CO, b,
CO,
, b
c, Ser1-CO
c
a,
c
a,
b (6.5)
a
Tyr
CO
a
174.4
56.9
36.4
4.46, dd
3.14/2.85, dd
b (9.2, 4.8)
CO, b, Asn2-CO, 1
CO, , 1, 2, 6
b
a
(14.2,4.7)
a
Asn1-a
Bz-I
129.0
131.3
116.4
157.3
Bz-o
Bz-m
Bz-p
b-AFA
CO
2
3
4
7.06, d
6.71, d
Bz-m (8.5)
Bz-o (8.4)
Bz-I, Bz-m, Bz-p
Bz-I, Bz-o, Bz-p
174.5
42.1
48.8
35.7
2.43, m
4.14, m
1.5, m
3
2, 4
3, 5–12
CO, 3, 4
CO, 2, 4, Thr-CO
2, 3
Ser2-a
5–12
13
23.6, 30.1–30.4, 33.1 1.25–1.31, m 4, 13
14.4 0.88, t 5–12 (7.1)
4, 13
5–12
di-substituted phenyl ring (131.3, 129.0, 157.3, and 116.4), and dis-
tinct signals at d 23.6, 30.1–30.4, 35.7, and 14.4, which is consistent
with a terminal hydrocarbon side chain supporting the presence of
a lipopeptide metabolite. Analysis of the COSY, HSQC, ROESY, and

U. R. Abdelmohsen et al. / Tetrahedron Letters 53 (2012) 23–29
25
D-Ser-1
homologs of 1, differing from 1 by the presence of one or more
additional CH2 groups. In the NMR spectra the only differences be-
tween these four new metabolites were in the aliphatic part (d
1.25–1.28 ppm), that is, in the signals corresponding to the hydro-
carbon part of the b-amino fatty acid, which, despite a large signal
overlap, suggested the same composition in the b-amino acid parts.
The UV spectra of all compounds were nearly identical, indicating
the same array of chromophores. By CID-MS/MS experiments, the
connectivities of the amino acids, as qualitatively determined by
NMR (see above), were established to be cyclo-(b-AFA-Ser-Gln-
Asn-Tyr-Asn-Ser-Thr) for all these new cyclic peptides (Fig. 1).
These compounds had not previously been isolated from other
sources.
H
H
O
OH
H N
L-Asn-1
2
H
H
N
O
L-Thr
O
H
O
H
N
O
HN
H
H
H
H
H
NH
HO
HO
-AFA
R =
3
O
R
Me
D-Tyr
NH
H
Me
O
( )8
H
NH
H
H
H
N
N
H
O
HO
D-Ser-2
H
H
O
H
O
NH
H N
2
O
2
L-Gln
Given the established constitution of the new metabolites 1–4,
the absolute configurations of the constituting amino acids were
determined by acid hydrolysis followed by chiral derivatization
with Marfey’s reagent.23 HPLC analysis of the Marfey derivatives
in comparison to their analogs of authentic amino acids revealed
L-Asn-2
1
Figure 2. Selected HMBC (single arrows) and ROESY (double arrows) interactions
indicative of the constitution of cyclodysidin A (1).
that all of the new cyclic peptides consisted of
L-asparagine, D-ser-
ine, -glutamine, -threonine, and -tyrosine. Moreover, Marfey’s
L
L
D
HMBC data assigned seven partial structures (Table 1): two aspar-
agine (Asn) units, one glutamine (Gln), two serine (Ser), one tyro-
sine (Tyr), and one threonine (Thr) moiety.
The COSY, HSQC, and HMBC experiments also correlated with a
deshielded methylene (H-2, d 2.43) and a deshielded methane sig-
nal (H-3, d 4.13), contiguous methylene resonances (H4-5–H11-12,
d 1.25–1.31, br s), and a terminal methyl signal (H-13, d 0.88),
delineating an amino fatty acid residue (b-AFA), which accounted
for the presence of a b-aminotridecanoic acid fragment. HMBC cor-
analysis evidenced that the second stereogenic center in threonine
is R, which is the usual form produced by microorganisms.24
Although several natural products containing b-amino acid units
related to the cyclodysidins have already been isolated from vari-
ous sources,13,25–27 the absolute configuration of the chiral center
in the b-amino fatty acid has been determined only rarely.13,28–30
For the stereochemical assignment of the b-amino acid compo-
nents of the new peptides 3 and 4, these amino acids were synthe-
sized as references, both in enantiomerically pure and racemic
forms. Compounds 5a and 5b were new. For their enantioselective
synthesis, the synthetic strategy presented in Scheme 1, as devel-
oped by Awasthi et al.31,32 for related amino acids, seemed prefer-
able over the synthesis described for the b-amino acid building
block of cyclodysidin B in epichlicin.13 Following the described
reaction sequence, the synthesis of the required b-amino acids,
5a–b, succeeded with good enantiomeric excess [(R)-5a: 90%;
(R)-5b: 94%; determined during Marfey’s experiments]. The
respective racemic reference compounds were prepared in one-
step syntheses using Rodionov’s reaction.33,34
With these reference compounds in hand, HPLC analyses were
performed in parallel on Marfey’s derivatives of the degradation
products of the hydrolyzed cyclodisidins C and D, on the racemic
material, and on the enantiomerically pure reference com-
pounds.35 The configuration assignment of these amino acids was
performed only with cyclodysidins C and D because of the scarcity
of cyclodysidins A and B samples. The comparison showed the
absolute configurations of these aliphatic side chain amino acids
in the natural cyclopeptides C and D to be, both, R. These results
relations from each
a-proton (b-proton in the case of b-aminotri-
decanoic acid) to the carbonyl carbon of the neighboring amino
acid and to its own carbonyl carbon were detected. Since only 15
of the calculated 16 degrees of unsaturation accounted for func-
tionalities in the eight individual fragments, it became obvious that
cyclodysidin A had a cyclic structure (Fig. 2).
By using CID (collision-induced dissociation) MS/MS, sequence
information was derived from the mass differences between the
b-series ions19 and between the y-series ions19 (Table 2).
The sequential arrangement of amino acids was accomplished
by interpretation of HMBC and ROESY data as well as fragmenta-
tion by CID-MS/MS. The structure of cyclodysidin A (1) was thus
established as cyclo-(b-AFA-Ser-Gln-Asn-Tyr-Asn-Ser-Thr).
In addition, three related compounds were isolated by reversed-
phase HPLC, showing very similar chromatographic and spectro-
scopic properties, subsequently named cyclodysidins B (2),20
C
(3),21 and D (4).22 By HR-ESIMS (TOF) analysis, compound 2 was
found to possess a molecular formula of C46H73N11O15, metabolite
3 of C47H75N11O15, and peptide 4 of C49H79N11O15, that is, each with
16 degrees of unsaturation. Thus, 2–4 were apparently higher
Table 2
Annotated MS fragments for cyclodysidin A (1)
Experimental (Da)
Predicted (Da)
Fragmentation
Sequence after
first cleavage
Fragment after
second cleavage
478.18
505.32
583.23
642.39
787.42
833.49
849.53
878.47
897.52
919.52
989.54
1006.46
478.18
505.32
583.24
642.38
787.43
833.48
849.46
878.46
897.49
919.49
989.52
1006.52
b/b4ꢀ1
b/y4
b/z5+1
b/y5ꢀH2O
b/b6ꢀH2O
b6
b/a7ꢀ1
b/y7ꢀH2O
b/c7+2
b/y7ꢀH2O
MꢀH2O+1
M
NYNSTxSQ
QNYNSTxS
xSQNYNST
YNSTxSQN
TxSQNYNS
TxSQNYNS
NYNSTxSQ
QNYNSTxS
NYNSTxSQ
STxSQNYN
NYNS
QNYN
NYNST
TxSQN
TxSQNY
TxSQNY
NYNSTxS
NYNSTxS
NYNSTxS
TxSQNYN

26
U. R. Abdelmohsen et al. / Tetrahedron Letters 53 (2012) 23–29
Table 3
NMR-spectroscopic data of cyclodysidin B (2) in methanol-d4 (1H: 600 MHz; 13C: 150 MHz)
Amino acid
dC
dH, mult
COSY (J in Hz)
HMBC
ROESY
Asn1
CO
a
173.9
52.5
4.66, m
b
CO, b,
c
, Tyr-CO
Tyr-b
b
37.2
2.77/2.73, m
a
CO, a, c
c
175.0
Asn2
CO
a
174.3
52.4
37.5
4.53, m
2.53/2.45, m
b
a
CO, b,
CO,
c
c
, Gln-CO
b
a
,
Gln-a
c
175.2
Gln
CO
a
b
c
174.2
55.9
27.2
4.21, m
2.16/2.10, m
2.46, m
b
a
b
CO, b,
CO,
, b, d
c
c
, Ser2-CO
, d
Asn2-b
,
c
a
,
33.2
a
d
176.7
Ser1
CO
a
173.0
57.5
62.7
4.38, t
3.86, m
b (5.6)
a
CO, b, Asn1-CO
CO,
Asn1-b
Thr-b
b
a
Ser2
CO
a
173.1
57.4
62.6
4.33, t
3.93, m
b (5.4)
a
CO, b, b-AFA-CO
CO,
b-AFA-2
Ser1-b
b
a
Thr
CO
a
b
c
172.4
60.3
67.7
20.6
4.29, d
4.42, m
1.17, d
b (2.5)
CO, b,
CO,
, b
c, Ser1-CO
c
a,
c
a
,
b (6.5)
a
Tyr
CO
a
174.4
57.1
36.7
4.48, dd
3.15/2.86, dd
b (9.3, 4.9)
CO, b, Asn2-CO, 1
CO, , 1, 2, 6
b
a
(14.3,4.8)
a
Asn1-a
Bz-I
Bz-o
Bz-m
Bz-p
b-AFA
CO
2
129.2
131.5
116.5
157.5
7.08, d
6.73, d
Bz-m (8.5)
Bz-o (8.4)
Bz-I, Bz-m, Bz-p
Bz-I, Bz-o, Bz-p
174.5
42.4
48.6
35.9
23.9, 30.3–30.8, 33.3
14.6
2.44, m
4.14, m
1.5, m
1.26–1.32, m
0.89, t
3
2, 4
3, 5–13
4, 14
5–13 (7.2)
CO, 3, 4
CO, 2, 4, Thr-CO
2, 3
4, 14
5–13
Ser2-a
3
4
5–13
14
Table 4
NMR-spectroscopic data of cyclodysidin C (3) in methanol-d4 (1H: 600 MHz; 13C: 150 MHz)
Amino acid
dC
dH, mult
COSY (J in Hz)
HMBC
ROESY
Asn1
CO
a
173.9
52.5
4.67, dd
b (7.6, 6.5)
CO, b,
c
, Tyr-CO
Tyr-b
b
37.3
2.77/2.73, m
a
CO, a, c
c
174.8
Asn2
CO
a
174.3
52.4
37.6
4.54, dd
2.53/2.44, m
b (7.9, 5.8)
a
CO, b,
CO,
c
c
, Gln-CO
b
a
,
Gln-a
c
174.9
Gln
CO
a
b
c
174.2
55.8
27.0
4.21, m
2.16/2.10, m
2.46, m
b
a
b
CO, b,
CO,
, b, d
c
c
, Ser2-CO
, d
Asn2-b
,
c
a
,
32.9
a
d
176.7
Ser1
CO
a
172.9
57.0
62.7
4.38, m
3.85, m
b
a
CO, b, Asn1-CO
CO,
Asn1-b
Thr-b
b
a
Ser2
CO
173.0

U. R. Abdelmohsen et al. / Tetrahedron Letters 53 (2012) 23–29
27
Table 4 (continued)
Amino acid
dC
dH, mult
COSY (J in Hz)
HMBC
ROESY
a
b
57.6
62.6
4.34, t
3.93, m
b (5.4)
a
CO, b, b-AFA-CO
CO, a
b-AFA-2
Thr
CO
a
b
c
172.3
60.3
67.7
20.6
4.28, d
4.40, m
1.17, d
b (2.5)
CO, b,
CO,
, b
c, Ser1-CO
c
a,
c
a
,
Ser1-b
b (6.6)
a
Tyr
CO
a
174.5
57.0
36.7
4.48, dd
3.14/2.85, dd
b (9.4, 4.9)
CO, b, Asn2-CO, 1
CO, , 1, 2, 6
b
a
(14.3,4.8)
a
Asn1-a
Bz-I
Bz-o
Bz-m
Bz-p
b-AFA
CO
2
129.2
131.5
116.5
157.5
7.06, d
6.72, d
Bz-m (8.6)
Bz-o (8.5)
Bz-I, Bz-m, Bz-p
Bz-I, Bz-o, Bz-p
174.6
42.3
48.9
35.8
23.9, 30.4–30.9, 33.2
14.6
2.43, m
4.13, m
1.5, m
1.25–1.31, m
0.88, t
3
2, 4
3, 5–11
4, 15
5–14 (6.5)
CO, 3, 4
CO, 2, 4, Thr-CO
2, 3
4, 15
5–14
Ser2-a
3
4
5–14
15
Table 5
NMR-spectroscopic data of cyclodysidin D (4) in methanol-d4 (1H: 600 MHz; 13C: 150 MHz)
Amino acid
dC
dH, mult
COSY (J in Hz)
HMBC
ROESY
Asn1
CO
a
173.7
52.4
4.65, dd
b (7.5, 6.5)
CO, b,
c
, Tyr-CO
Tyr-b
b
37.2
2.77/2.71, m
a
CO, a, c
c
175.0
Asn2
CO
a
174.3
52.3
37.3
4.53, m
2.52/2.44, m
b
a
CO, b,
CO,
c
c
, Gln-CO
b
a
,
Gln-a
c
175.2
Gln
CO
a
b
c
174.1
55.9
27.4
4.21, dd
2.18/2.10, m
2.48, m
b (8.4, 5.9)
CO, b,
CO,
, b, d
c
c
, Ser2-CO
, d
Asn2-b
a,
c
a
,
32.8
b
a
d
176.7
Ser1
CO
a
172.9
57.4
62.5
4.37, m
3.84, m
b
a
CO, b, Asn1-CO
CO,
Asn1-b
Thr-b
b
a
Ser2
CO
a
173.1
57.3
62.3
4.33, m
3.92, m
b
a
CO, b, b-AFA-CO
CO,
b-AFA-2
Ser1-b
b
a
Thr
CO
a
b
c
172.3
60.1
67.4
20.5
4.28, d
4.40, m
1.16, d
b (2.5)
a
b (6.4)
CO, b,
CO,
, b
c, Ser1-CO
c
,
c
a,
a
Tyr
CO
a
174.5
57.0
36.6
4.47, dd
3.14/2.85, dd (14.3,4.8)
b (9.3, 4.9)
a
CO, b, Asn2-CO, 1
CO, , 1, 2, 6
b
a
Asn1-a
1
129.2
131.1
116.4
157.5
2,6
3,5
4
7.06, d
6.72, d
Bz-m (8.5)
Bz-o (8.4)
Bz-I, Bz-m, Bz-p
Bz-I, Bz-o, Bz-p
b-AFA
CO
2
3
4
174.6
42.2
48.6
35.6
23.6, 30.4–30.8, 33.3
14.4
2.43, m
4.14, m
1.5, m
1.26–1.32, m
0.88, t
3
2, 4
3, 5–11
4, 17
5–16 (7.2)
CO, 3, 4
CO, 2, 4, Thr-CO
2, 3
4, 17
5–16
Ser2-a
5–16
17

28
U. R. Abdelmohsen et al. / Tetrahedron Letters 53 (2012) 23–29
7. Pimentel-Elardo, S. M.; Kozytska, S.; Bugni, T. S.; Ireland, C. M.; Moll, H.;
Ph
Hentschel, U. Mar. Drugs 2010, 8, 373–380.
OH
8. Engelhardt, K.; Degnes, K. F.; Kemmler, M. Appl. Environ. Microbiol. 2010, 76,
4969–4976.
H N
2
Ph
MgSO4
CH2Cl2,r.t.
9. Lee, H. S.; Shin, H. J.; Jang, K. H.; Kim, T. S.; Oh, K. B.; Shin, J. J. Nat. Prod. 2005, 68,
623–625.
10. Romano, A.; Vitullo, D.; Di Pietro, A.; Lima, G.; Lanzotti, V. J. Nat. Prod. 2011, 74,
145–151.
11. Kanoh, K.; Matsuo, Y.; Adachi, K.; Imagawa, H.; Nishizawa, M.; Shizuri, Y. J.
Antibiot. (Tokyo) 2005, 58, 289–292.
12. Ongena, M.; Jacques, P. Trends Microbiol. 2008, 16, 115–125.
13. Seto, Y.; Takahashi, K.; Matsuura, H.; Kogami, Y.; Yada, H.; Yoshihara, T.;
Nabeta, K. Biosci. Biotechnol. Biochem. 2007, 71, 1470–1475.
O
N
Me
Me
OH
r.t.
) n(
) n(
O
n = 10 12
THF
0°C
ZnBr
O
tBuO
Compounds = 5a 5b
O
14. Zhang, H. L.; Hua, H. M.; Pei, Y. H.; Yao, X. S. Chem. Pharm. Bull. (Tokyo) 2004, 52,
1029–1030.
tBuO
i. HCO NH , Pd/C
HO
H
2
4
Ph
H
15. Nielsen, T. H.; Sorensen, J. J. Appl. Environ. Microbiol. 2003, 69, 861–868.
16. Abdelmohsen, U. R.; Pimentel-Elardo, S. M.; Hanora, A.; Radwan, M.; Abou-El-
Ela, S. H.; Ahmed, S.; Hentschel, U. Mar. Drugs 2010, 8, 399–412.
17. Fermentation and purification: Streptomyces strain RV15 was fermented in 16
2 l-Erlenmeyer flasks, each containing 1 l of ISP 2 medium at 30 °C for 10 d
with shaking at 150 rpm. After 10 d of cultivation, sterilized XAD-16 resin
(30 g/l) was added. Resin was collected 6 h later by filtration and eluted with
acetone. The acetone was removed under reduced pressure and the aqueous
layer was extracted with ethyl acetate (3 ꢁ 500 ml). The combined extracts
were concentrated under vacuum and stored at 4 °C for chromatographic
fractionation. After fractionation of crude extract (10 g) by silica gel
chromatography, the fraction (935 mg) eluted with 40% chloroform in
methanol was further chromatographed on Sephadex LH 20. The fraction
(56 mg) eluted with 70% methanol in water, was enriched in cyclic peptides.
Further purification was carried out by preparative HPLC using an acetonitrile
(MeCN) and water solvent mixture complemented by 0.05% trifluoroacetic
acid: (10% MeCN/H2O to 100% MeCN over 20 min at a flow rate of 10 ml/min),
giving four pure compounds.
MeOH, reflux
N
H
Me
Me
OH
) n(
) n(
NH
2
ii. TFA, r.t.
(R)-5a-b
6a-b
O H
OH
O
O
O
HO
AcONH
4
O
Me
Me
) n(
) n(
R/S NH
EtOH, reflux
2
rac-5a and rac-5b
Scheme 1. Synthesis of the compounds 5a–b in enantiomerically pure and racemic
forms.
18. Cyclodysidin A (1): 2 mg of a white amorphous powder (Rt = 7.3 min); mp 290–
are consistent with the configurations found in the literature in
similar cases.13,28–30
294 °C; ½a 2D0
ꢀ19(c 0.55, MeOH); UV (MeOH): kmax (loge) = 220 (5.75), 235
ꢂ
(12.0) nm; IR: 3345, 2924, 1730, 1638, 1553, 1463, 1238, 1160, 1095, 730,
667 cmꢀ1 1H and 13C NMR data, see Table 1; HR-ESIMS: [m/z (M+H)+] found
;
In conclusion, cyclodysidins A–D, four new cyclic lipopeptides
1006.5207, calcd for C45H71N11O15 (1006.5209).
with
a- and b-amino acids, were isolated from the marine
19. Lee, J. I.; Narayan, M.; Barrett, J. S. J. Chromatogr. B 2007, 845, 95–103.
20. Cyclodysidin B (2): 1.5 mg of a white amorphous powder (Rt = 7.7 min); mp
sponge-derived Streptomyces strain RV15. Their structures were
established as cyclo-(b-AFA-Ser-Gln-Asn-Tyr-Asn-Ser-Thr) by
spectroscopic analysis using 2D NMR techniques and CID-MS/MS.
The absolute stereostructures were determined by Marfey’s analy-
321–325 °C; ½a 2D0
ꢀ15.3 (c 0.55, MeOH): UV (MeOH): kmax (loge) = 223 (3.82),
ꢂ
238 (13.13) nm; IR: 3351, 2935, 1740, 1633, 1549, 1455, 1243, 1165, 1089, 738,
661 cmꢀ1 1H and 13C NMR data, see Table 3; HR-ESIMS: [m/z (M+H)+] found
;
1020.5364, calcd for C46H73N11O15 (1020.5366).
sis followed by HPLC, showing that the
blocks were in all cases except for tyrosine and serine, which
found to possess -configuration. The b-amino acid blocks were as-
-configuration in the case of cyclodysidins C
a-amino acid building
21. Cyclodysidin C (3): 2 mg of a white amorphous powder (Rt = 8.2 min); mp 332–
336 °C; ½a 2D0
ꢂ
ꢀ22.5 (c 0.55, MeOH); UV (MeOH): kmax (loge) = 222 (4.3), 237
L
(14.2) nm; IR: 3340, 2920, 1735, 1629, 1560, 1450, 1235, 1158, 1090, 729,
D
665 cmꢀ1 1H and 13C NMR data, see Table 4; HR-ESIMS: [m/z (M+H)+] found
;
signed to have the
and D.
D
1034.5536, calcd for C47H75N11O15 (1034.5522).
22. Cyclodysidin D (4): 1.7 mg of a white amorphous powder (Rt = 8.5 min); mp
345–350 °C; ½a 2D0
ꢀ27.7 (c 0.55, MeOH); UV (MeOH): kmax (loge) = 224 (6.55),
ꢂ
238 (13.63) nm; IR: 3342, 2928, 1735, 1632, 1557, 1460, 1238, 1163, 1088,
Acknowledgments
733, 662 cmꢀ1 1H and 13C NMR data, see Table 5; HR-ESIMS: [m/z (M+H)+]
;
found 1062.5832, calcd for C49H79N11O15 (1062.5835).
23. Marfey, P. Carlsberg Res. Commun. 1984, 49, 591–596.
24. Bringmann, G.; Lang, G.; Steffens, S.; Schaumann, K. J. Nat. Prod. 2004, 67, 311–
315.
25. Peypoux, F.; Guinand, M.; Michel, G.; Delcambe, L.; Das, B. C.; Lederer, E.
Biochemistry 1978, 17, 3992–3996.
26. Trischman, J. A.; Jensen, P. R.; Fenical, W. Tetrahedron Lett. 1994, 35, 5571–
5574.
27. Peypoux, F.; Pommier, M. T.; Marion, D.; Ptak, M.; Das, B. C.; Michel, G. J.
Antibiot. (Tokyo) 1986, 39, 636–641.
We thank Dr. M. Büchner for the mass spectra and Dr. M. Grüne
(both University of Würzburg) for the NMR experiments. Financial
support was provided by the Egyptian Government (scholarship to
U.A.) and the Deutsche Forschungsgemeinschaft (SFB 630: ‘Recog-
nition, Preparation, and Functional Analysis of Agents against
Infectious Diseases’, projects A2 and A5). G.L.Z. was supported by
a grant of the German Excellence Initiative to the Graduate School
of Life Sciences, University of Würzburg.
28. Gerwick, W. H.; Jiang, Z. D.; Agarwal, S. K.; Farmer, B. T. Tetrahedron 1992, 48,
2313–2324.
29. Banaigs, B.; Bonnard, I.; Rolland, M.; Francisco, C. Lett. Pept. Sci. 1997, 4, 289–
292.
30. Nagai, U.; Besson, F.; Pepoux, F. Tetrahedron Lett. 1979, 25, 2359–2360.
31. Awasthi, A. K.; Boys, M. L.; Cain Janicki, K. J.; Colson, P. J.; Doubleday, W. W.;
Duran, J. E.; Farid, P. N. J. Org. Chem. 2005, 70, 5387–5397.
32. General procedure for the synthesis of (R)-5a–b:
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2011.10.051.
Imines preparation:
To a solution of 1-phenylglycinol (0.1 g, 0.54 mmol) in absolute THF (1 ml) at
ambient temperature, 1 equiv of the corresponding aldehyde34 (0.54 mmol)
was added in a dropwise manner. The mixture was stirred for 30 min at room
temperature. 0.1 g of oven-dried (110 °C) MgSO4 was added and the mixture
stirred for 2 h at room temperature. The suspension was directly transferred
into a syringe through a syringe filter and injected into the solution containing
the Reformatsky reagent.
Reformatsky reagent preparation:
A three-necked flask was charged with zinc metal (1.03 g, 15.8 mmol, 3–6
mesh) and dried at 100 °C under vacuum (0.1 mbar). Under N2 atmosphere
References and notes
1. Hentschel, U.; Usher, K. M.; Taylor, M. W. FEMS Microbiol. Ecol. 2006, 55, 167–
177.
2. Scheuermayer, M.; Pimentel-Elardo, S.; Fieseler, L.; Grozdanov, L.; Hentschel, U.
In Frontiers in Marine Biotechnology; Proksch, P., Müller, W., Eds.; Horizon
Bioscience: Norwich, 2006; pp 289–312.
3. Lam, K. S. Curr. Opin. Microbiol. 2006, 9, 245–251.
4. Thomas, T. R. A.; Kavlekar, D. P.; Loka Bharathi, P. A. Mar. Drugs 2010, 8, 1417–
1468.
5. Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2007, 70, 461–477.
6. Olano, C.; Mendez, C.; Salas, J. A. Mar. Drugs 2009, 7, 210–248.
absolute THF (1 ml) and of chlorotrimethylsilane (50 ll) was added and stirred
till the zinc metal turned dark gray. At 50 °C, tert-butyl bromoacetate (2.42 ml,
15 mmol) was added over 20 min and the solution was stirred for 3 h at 50 °C
until the zinc powder had almost disappeared. The solution was then cooled to

U. R. Abdelmohsen et al. / Tetrahedron Letters 53 (2012) 23–29
29
0 °C. After approximately 1 h, the reagent precipitated as a white solid.
6a–b: To the solution containing the crystallized Reformatsky reagent, the
solution of the corresponding imine in absolute THF was added slowly over
1 min at 0 °C. The mixture was then vigorously stirred for 2 d at room
temperature. A HCl solution (10 ml, 2 M) was added dropwise at 0 °C to the
mixture. MTBE (10 ml) was added and the mixture was stirred for another
30 min. The organic layer was separated and the aqueous layer was extracted
with MTBE (3 ꢁ 5 ml). The combined organic layers were washed with
saturated NH4Cl solution (5 ml), H2O (5 ml) and a saturated aqueous NaCl
solution (5 ml), and were dried over MgSO4. The solvent was evaporated and
the residue was purified by chromatography on silica gel, eluting with CH2Cl2/
MeOH/NH3 (25% in H2O) (1:0:0–95:4.8:0.2), to yield the desired products 6a
(40%) and 6b (78%).
NMR (CDCl3, 150.9 MHz) d 173.3, 49.2 (under MeOD signals), 36.3, 33.2, 32.5,
30.23, 30.21, 30.19, 30.10, 29.93, 29.84 2 C, 25.8, 23.2, 14.3 ppm; HR-ESIMS:
[m/z (M+H)+] found 258.2427, calcd for C15H32NO2 (258.2433).
(R)-3-Aminoheptadecanoic acid [(R)-5b]: Colorless crystals; mp 98–100 °C
(AcOH); ½a 2D0
ꢀ8.5 (c 0.12, AcOH); IR: 2928, 2856, 1668, 1397, 1187, 1145,
ꢂ
718 cmꢀ1 1H NMR (MeOD/CDCl3/TFA (49:49:2), 250 MHz) d 3.43 (1H, m), 2.72
;
(1H, dd (AB), J1 = 17.4 Hz, J2 = 4.6 Hz), 2.59 (1H, dd (AB), J1 = 17.4 Hz,
J2 = 7.9 Hz), 1.62 (2H, m), 1.4–1.2 (24H, m), 0.85 (3H, t, J = 6.7 Hz) ppm; 13C
NMR (CDCl3, 150.9 MHz) d 173.3, 49.2 (under MeOD signals) 36.3, 33.2, 32.5,
30.26, 30.24, 30.23 (2 C), 30.2, 30.1, 29.93, 29.92, 29.84, 25.8, 14.3 ppm; HR-
ESIMS: [m/z (M+H)+] found 286.2742, calcd for C17H36NO2 286.2746).
33. Lázár, L.; Martinek, T.; Bernáth, G.; Fülöp, F. Synth. Commun. 1998, 28, 219–224.
34. General procedure for the synthesis of rac-5a and rac-5b: 1 equiv of the
corresponding aldehyde,36 1 equiv of malonic acid, and 2 equiv of
ammonium acetate were suspended in EtOH/H2O (95:5). The solution was
vigorously stirred under reflux overnight. The solution was then cooled down
to room temperature and the precipitated solid was filtrated and recrystallized
from EtOH/H2O (95:5) to get the desired products rac-5a and rac-5b as
colorless crystals (17–21%).
(R)-5a–b:
In a dried Schlenk-tube, 1 equiv of 6a–b was dissolved in distilled MeOH
(0.1 ml for 4 mg of 6a–b) under N2. 0.1 equiv of Pd/C (10% Pd loading) and
6 equiv of NH4HCO2 were added to the solution and the mixture was stirred at
65 °C for 6 h. The mixture was filtered over Celite and the solvent was
evaporated. The crude product was then dried under high vacuum during 24 h,
dissolved in TFA (1 ml) and stirred for 1 h. The solvent was evaporated and the
residue dissolved in H2O. Several drops of concentrated aqueous NH3 were
then added to reach neutral pH. The precipitated white solid was filtered and
washed with H2O and cold EtOH to yield the desired products (R)-5a (71%) and
(R)-5b (91%). Alternatively the crude product was recrystallized from EtOH
after adding some drops of concentrated aqueous NH3. The enantiomeric
excess of the products was determined during Marfey’s experiments to be 90%
for (R)-5a and 94% for (R)-5b.
Analytical data:
( )-3-Aminopentadecanoic acid (rac-5a): Colorless crystals; mp 169–171 °C
(EtOH/H2O); IR: 2925, 2839, 1578, 1390, 697 cmꢀ1 1H NMR (MeOD/CDCl3/TFA
;
(49:49:2), 250 MHz) d 3.44 (1H, m), 2.72 (1H, dd (AB), J1 = 17.4 Hz, J2 = 4.6 Hz),
2.6 (1H, dd (AB), J1 = 17.4 Hz, J2 = 7.9 Hz), 1.62 (2H, m), 1.4–1.2 (20H, m), 0.85
(3H, t, J = 6.7 Hz) ppm; 13C NMR (CDCl3, 150.9 MHz) d 173.7, 49.2 (under MeOD
signals), 36.5, 33.2, 32.5, 30.24, 30.22, 30.19, 30.11, 30.04, 29.94, 29.85 (2 C),
23.2, 14.3 ppm; HR-ESIMS: [m/z (M+H)+] found 258.2432, calcd for C15H32NO2
(258.2433).
Analytical data:
(R)-tert-butyl 3-[(S)-2-hydroxy-1-phenylethyl]aminopentadecanoate (6a): Light
( )-3-Aminoheptadecanoic acid (rac-5b): Colorless crystals; mp 177–178 °C
brown oil; ½a 2D0
ꢂ
+19.4 (c 1.16, CHCl3); IR: 3443 (br), 2921, 2857, 1721, 1464,
(EtOH/H2O); IR: 2922, 2849, 1575, 1390, 715 cmꢀ1 1H NMR (MeOD/CDCl3/TFA
;
1371, 1257, 1150, 1027, 850, 754, 700 cmꢀ1
;
1H NMR (CDCl3, 250 MHz) d 7.35–
(49:49:2), 250 MHz) d 3.42 (1H, m), 2.72 (1H, dd (AB), J1 = 17.7 Hz, J2 = 4.6 Hz),
2.6 (1H, dd (AB), J1 = 17.7 Hz, J2 = 8.2 Hz), 1.62 (2H, m), 1.4–1.2 (24H, m), 0.85
(3H, t, J = 6.7 Hz) ppm; 13C NMR (CDCl3, 150.9 MHz) d 173.3, 49.2 (under MeOD
signals), 36.4, 33.2, 32.5, 30.28, 30.27, 30.25 (2 peaks), 30.2, 30.1, 29.96, 29.94,
29.86, 25.8, 23.3, 14.3 ppm; HR-ESIMS: [m/z (M+H)+] found 286.2742, calcd for
7.25 (5H, m, aromatic H), 3.91 (1H, dd (AB), J1 = 8.5 Hz, J2 = 4.3 Hz), 3.73 (1H, dd
(AB), J1 = 11 Hz, J2 = 4.2 Hz), 3.61 (1H, dd (AB), J1 = 11 Hz, J2 = 8.5 Hz), 2.93 (1H,
quintuplet, J = 5.8 Hz), 2.46 (1H, dd (AB), J1 = 14.9 Hz, J2 = 5.5 Hz), 2.34 (1H, dd
(AB), J1 = 14.9 Hz, J2 = 6.1 Hz), 1.46 (9H, s), 1.3–1.2 (22H, m), 0.9 (3H, t, J = 7 Hz)
ppm; 13C NMR (CDCl3, 100 MHz) d 174.2, 131.2, 130.8, 130.3, 129.8, 83.5, 68.9,
64.8, 55.5, 41.8, 37.5, 34.4, 32.1, 32.1, 32.08, 32.06, 31.98, 31.91, 31.87, 31.8,
30.6, 28.2, 25.1, 16.6 ppm; HR-ESIMS: [m/z (M+H)+] found 434.3626, calcd for
C17H37NO2 (286.2746).
35. Marfey analysis of b-amino acids: The configuration analysis was performed
according to Ref. 22 but the FDLA derivatives of the b-amino acids were
dissolved in pure MeCN to ensure complete dissolution. Another HPLC method
was used for the analysis (for column and solvents see Ref. 22): linear gradient,
0 min 25% MeCN, 30 min 85% MeCN, 40 min 100% MeCN, 43 min 25% MeCN,
46 min 25% MeCN, flow rate 1 ml/min.
C
27H47NO3 (434.3629).
(R)-tert-butyl 3-[(S)-2-hydroxy-1-phenylethyl]aminopentadecanoate (6b): Light
brown oil; ½a 2D0
ꢂ
+21.1 (c 1.45, CHCl3); IR: 3423 (br), 2919, 2850, 1727, 1450,
1367, 1254, 1149, 1032, 850, 750, 700 cmꢀ1
;
1H NMR (CDCl3, 250 MHz) d 7.36-
7.28 (5H, m, aromatic H), 3.95 (1H, dd (AB), J1 = 8.2 Hz, J2 = 4.6 Hz), 3.75 (1H, dd
(AB), J1 = 11 Hz, J2 = 4.3 Hz), 3.64 (1H, bdd (AB)), 2.96 (1H, quintuplet,
J = 5.8 Hz), 2.52 (1H, dd (AB), J1 = 15 Hz, J2 = 5.5 Hz), 2.36 (1H, dd (AB),
J1 = 15 Hz, J2 = 5.8 Hz), 1.46 (9H, s), 1.35–1.1 (26H, m), 0.88 (3H, t, J = 6.7 Hz)
ppm; 13C NMR (CDCl3, 100 MHz) 171.9, 128.7, 128.3, 127.7, 127.3, 80.9, 66.6,
62.1, 52.8, 39.6, 34.8, 31.9, 29.69, 29.68, 29.65, 29.62, 29.53, 29.47, 29.35, 28.1,
25.8, 22.7, 14.1 ppm; HR-ESIMS: [m/z (M+H)+] found 462.3944, calcd for
The retention times of the b-amino acids were as follows:
(R)-5a: (S): 33:5 min, 5%; (R): 36.7 min, 95%
(R)-5b: (S): 39.7 min, 3%; (R): 43.2 min, 97%
rac-5a: (S): 33.8 min; (R): 37.2 min
rac-5b: (S): 39.7 min; (R): 43.2 min
Hydrolyzed 1c: 37.2 min (rac-5a injected in parallel: 37.0 min)
Hydrolyzed 1d: 41.6 min (rac-5b injected in parallel: 41.5 min)
36. Commercially available n-tridecanal was freshly distilled in a Kugelrohr under
20 mbar prior to use. n-pentadecanal was obtained from commercially
available 1-pentadecanol by oxidation with the chromium trioxide–pyridine
complex according to Ratcliffe et al.37
C
29H51NO3 (462.3942).
(R)-3-Aminopentadecanoic acid [(R)-5a]: Colorless crystals; mp 151–154 °C
(EtOH/H2O);
½
a 2D0
ꢂ
ꢀ9 (c 0.15, AcOH); IR: 2925, 2848, 1683, 1398, 1180,
1139,723 cmꢀ1
;
1H NMR (MeOD/CDCl3/TFA (49:49:2), 250 MHz) d 3.42 (1H,
m), 2.72 (1H, dd (AB), J1 = 17.4 Hz, J2 = 4.6 Hz), 2.59 (1H, dd (AB), J1 = 17.4 Hz,
37. Ratcliffe, T.; Rodehorst, R. J. Org. Chem. 1970, 35, 4000–4002.
J2 = 7.9 Hz), 1.62 (2H, m), 1.4–1.15 (20H, m), 0.85 (3H, t, J = 6.7 Hz) ppm; 13C