Thalassospiramides A and B, immunosuppressive peptides from the marine bacterium thalassospira sp.

Article abstract of DOI:10.1021/ol070294u

Equation presented Two new cyclic peptides, thalassospiramides A and B (1 and 2), were isolated from a new member of the marine α-proteobacterium Thalassospira. The thalassospiramides, the structures of which were assigned by combined spectral and chemica

Full text of DOI:10.1021/ol070294u

ORGANIC
LETTERS
2007
Vol. 9, No. 8
1525-1528
Thalassospiramides A and B,
Immunosuppressive Peptides from the
Marine Bacterium Thalassospira sp.
Dong-Chan Oh, Wendy K. Strangman, Christopher A. Kauffman,
Paul R. Jensen, and William Fenical*
Center for Marine Biotechnology and Biomedicine, Scripps Institution of
Oceanography, UniVersity of CaliforniasSan Diego, La Jolla, California 92093
wfenical@ucsd.edu
Received February 5, 2007
ABSTRACT
Two new cyclic peptides, thalassospiramides A and B (1 and 2), were isolated from a new member of the marine
r-proteobacterium Thalassospira.
The thalassospiramides, the structures of which were assigned by combined spectral and chemical methods, bear unusual
γ-amino acids and
show immunosuppressive activity in an interleukin-5 production inhibition assay (IC50 M for thalassospiramide B).
)
5 µ
Marine bacteria are now being exploited as a new source
for novel secondary metabolites.1 The presence of new
actinomycete marine bacteria had not been appreciated until
the application of precise phylogenetic analysis.2 Chemical
investigations of these new actinomycetes are now resulting
in the discovery of novel secondary metabolites with
pharmaceutical potential.3 As part of a program to discover
new marine bacterial taxa, we identified a new marine
eubacterium, which is closely phylogenetically related to the
R-proteobacterium Thalassospira lucentensis.4 In culture, this
R-proteobacterium (strain CNJ-328) produced two interesting
peptides, thalassospiramides A and B (1 and 2). Here, we
report the isolation, structural determination, and biological
activity of these new compounds, the first secondary
metabolites to be reported from Thalassospira spp.
Thalassospiramide A (1)5 was isolated as a gum, which
analyzed for the molecular formula C48H75N7O13 by high-
resolution MALDI-TOF mass spectrometry coupled with 1H
1
and 13C NMR spectral data (Table 1). The H and gCOSY
NMR spectra showed the typical features for peptides with
six amide proton signals [δH: 9.24, 9.03, 8.86, 8.56, 8.48,
7.73], one N-methyl group [δH: 3.10], and seven R-amino
protons [δH: 5.33, 5.19, 4.89, 4.73, 4.62, 4.53, 4.22]. The
1H homonuclear J-resolved NMR spectrum revealed six doub-
let [δH: 0.9∼1.1] and one triplet methyl groups [δH: 0.79].
In addition to the general peptide features, the proton NMR
spectrum showed four olefinic protons at δ 6.98, 6.91, 5.89,
and 5.55, which are uncommon in ordinary peptides. Carbon-
(1) (a) Jensen, P. R.; Fenical, W. J. Indust. Microbiol. Biotechnol. 1996,
17, 346. (b) Jensen, P. R.; Mincer, T. J.; Williams, P. G.; Fenical, W. Antonie
Van Leeuwenhoek 2005, 87, 43.
(2) (a) Fenical, W.; Jensen, P. R. Nat. Chem. Biol. 2006, 2, 666. (b)
Jensen, P. R.; Williams, P. G.; Oh, D.-C.; Zeigler, L.; Fenical, W. Appl.
EnViron. Microbiol. 2007, 73, 1146. (c) Fiedler, H.-P.; Bruntner, C.; Bull,
A. T.; Ward, A. C.; Goodfellow, M.; Potterat, O.; Puder, C.; Mihm, G.
Antonie Van Leeuwenhoek 2005, 87, 37.
(3) (a) Feling, R. H.; Buchanan, G. O.; Mincer, T. J.; Kauffman, C. A.;
Jensen, P. R.; Fenical, W. Angew. Chem., Int. Ed. 2003, 42, 355. (b)
Buchanan, G. O.; Williams, P. G.; Feling, R. H.; Kauffman, C. A.; Jensen,
P. R.; Fenical, W. Org. Lett. 2005, 7, 2731. (c) Oh, D.-C.; Williams, P. G.;
Kauffman, C. A.; Jensen, P. R.; Fenical, W. Org. Lett. 2006, 8, 1021.
(4) (a) Lo´pez-Lo´pez, A.; Pujalte, M. J.; Benlloch, S.; Mata-Roig, M.;
Rossello´-Mora, R.; Garay, E.; Rodr´ıguez-Valera, F. Int. J. System EVol.
Microbiol. 2002, 52, 1277. (b) The most closely related bacterium that has
been identified is Thalassospira lucentensis (98% identity, AF358664), and
thus CNJ-328 may represent a new Thalassospira species.
(5) Thalassospiramide A (1): oil; [R]D -29 (c 0.07, CH3CN); IR (neat)
νmax 3354, 2966, 1740, 1643, 1519 cm-1; UV (CH3CN) λmax (log ꢀ) 221
(4.3), 278 (3.3) nm; NMR spectral data, see Table 1 and Table S1 in the
Supporting Information; HR-MALDI-TOFMS [M + H]+ m/z 958.5528
(C48H76N7O13, calcd [M + H]+ 958.5496).
10.1021/ol070294u CCC: $37.00
© 2007 American Chemical Society
Published on Web 03/21/2007

Table 1. NMR Spectral Data for 1 in Pyridine-d5
a
b
C/H
δH
mult (J in Hz)
δC
HMBC
1, 10
1
2
170.2
67.6 CH
34.7 CH2 2, 5, 9
2, 5, 9
129.3
131.0 CH
116.6 CH
157.6
C
4.22 dd (11.0, 4.5)
3.58 dd (14.0, 4.5)
3.77 dd (14.0, 11.0)
3a
3b
4
C
5
7.31 d (8.0)
7.08 d (8.0)
3
3
6
7
C
7-OH
8
11.44 br s
7.08 d (8.0)
7.31 d (8.0)
116.4 CH
130.7 CH
3
3
9
10
3.10
s
40.0 CH3 1, 11
11
171.9
C
12
4.53 dd (6.5, 4.0)
7.73 d (6.5)
59.0 CH
11, 13
12, 13, 16
12-NH
13
2.07
m
29.8 CH
14
0.94 d (7.0)
1.14 d (7.0)
20.2 CH3 12, 13, 15
17.1 CH3 12, 13, 14
15
16
170.9
C
17
6.91
6.98
m
m
126.8 CH
139.6 CH
49.7 CH
16, 19
18
16, 19
19
5.19 br m
17, 18
19-NH
20a
20b
21
8.56 d (7.5)
17, 18, 19, 21
Interpretation of gCOSY, TOCSY, and gHMBC NMR
data allowed eight distinct fragments to be constructed. These
included unusual amino acids such as 4-amino-5-hydroxy-
penta-2-enoic acid (Ahpea), 4-amino-3,5-dihydroxy-pen-
tanoic acid (Adpa), as well as N-methyltyrosine, three valines,
serine, and dec-3-enoic acid. The Adpa unit showed no three-
bond COSY correlations between H-28 and H-29, and 1D
and 2D TOCSY also indicated discontinuity of this spin
system. HMBC correlations from H-27 to C-29, however,
allowed the assignment of this unit. ROESY correlations
between H-28 and H-29 also supported this partial structure.
The geometry of the double bond (C-17, C-18) in Ahpea
4.79 dd (11.0, 2.5)
4.30 dd (11.0, 2.5)
64.0 CH2 1, 18
18
171.8
C
22
4.73 dd (8.0, 8.0)
9.03 d (8.0)
60.0 CH
21, 22
22, 26
21, 22, 25
22-NH
23
2.42
m
30.3 CH
24
0.94 d (7.0)
1.02 d (7.0)
20.2 CH3 22, 23, 24
19.0 CH3 22, 23, 24
25
26
172.7
C
27a
27b
28
28-OH
29
29-NH
30
30-OH
31
32
3.10 dd (14.0, 2.5)
2.99 dd (14.0, 5.5)
5.09 br m
42.2 CH2 26, 28, 29
26, 28, 29
68.0 CH
6.95
4.62
m
m
56.2 CH
31
62.2 CH2
8.48 d (9.0)
4.13
m
1
was assigned as E by the H coupling constant (15.5 Hz)
6.61 br t (5.0)
between H-17 and H-18 measured in DMSO-d6 (these proton
signals are second order in pyridine-d5). The double bond
geometry at C-41 and C-42 was established as Z also by the
11.0 Hz coupling constant observed between H-41 and H-42.
This geometry was confirmed by a strong ROESY cross-
peak between these protons. The sequence of the amino acid
residues was readily determined by analysis of HMBC and
ROESY spectral data.
The absolute configurations of the amino acids in 1 were
assigned by application of the advanced Marfey method.6
Prior to acid hydrolysis, the double bonds in Ahpea and Dea
were hydrogenated to enhance the acid stability of the Ahpea
unit by converting it to 4-amino-5-hydroxy-pentanoic acid
(Ahpa). Acid hydrolysis of the hydrogenated product of 1
yielded the expected free amino acids, which were deriva-
tized using L- and D-FDLA (advanced Marfey’s reagent,
1-fluoro-2,4-dinitrophenyl-5-leucine amide) and analyzed by
LC/MS.
172.5
C
4.89 dd (8.0, 7.0)
9.24 d (8.0)
60.4 CH
31
32-NH
33
32, 36
31, 32, 34, 35
2.42
m
31.0 CH
34
1.02 d (7.0)
1.02 d (7.0)
20.1 CH3 32, 33, 35
18.7 CH3 32, 33, 34
35
36
172.6
C
37
5.33 td (7.5, 7.0)
8.86 d (7.5)
56.4 CH
36
37-NH
38
37, 39
4.26
7.20
m
m
63.0 CH2
38-OH
39
171.9
C
40
3.32 d (7.0)
5.89 dtt (11.0, 7.0, 1.5) 123.1 CH
5.55 dtt (11.0, 7.5, 2.0) 133.2 CH
35.5 CH2 39, 41, 42
41
40, 43
40, 43, 44
42
43
1.98
1.22
1.12
1.10
1.13
m
m
m
m
m
28.0 CH2 44
29.9 CH2 45
29.4 CH2
32.2 CH2
23.1 CH2
14.6 CH3 46, 47
44
45
46
47
48
0.79 t (5.0)
a
b
300 MHz. 100 MHz.
The absolute configurations of R-amino acid residues,
assigned as L, were established in a straightforward fashion
by LC/MS analysis. The determination of the absolute
configuration at C-19 in Ahpa required an additional experi-
13 NMR and gHSQC spectra displayed eight amide or ester
carbonyl carbons, ten olefinic carbons, eight methines, and
three methylenes bearing oxygen or nitrogen between 49 and
68 ppm, three aliphatic methines, eight aliphatic methylenes,
one downfield methyl at 40.0 ppm, and seven upfield methyl
signals. Analysis of gHSQC spectral data allowed all one-bond
carbon and proton correlations to be assigned (Table 1).
(6) (a) Fujii, K.; Ikai, Y.; Oka, H.; Suzuki, M.; Harada, K. Anal. Chem.
1997, 69, 3346. (b) Fujii, K.; Ikai, Y.; Oka, H.; Suzuki, M.; Harada, K.
Anal. Chem. 1997, 69, 5146. (c) Marfey, P. Carlsberg Res. Commun. 1984,
49, 591.
1526
Org. Lett., Vol. 9, No. 8, 2007

Scheme 1. Reaction Sequence for Determination of the Absolute Configuration of 4-Amino-3,5-dihydroxy-pentanoic Acid
ment (see the Supporting Information). Initially, the L- and
D-FDLA derivatives of Ahpa were detected by LC/MS
analysis at 23.2 and 22.3 min, respectively. Because Ahpa
is a γ-amino acid, which bears two additional methylenes,
the elution sequence for the L- and D-FDLA derivatives is
difficult to predict. This problem was solved by comparing
the retention times of the FDLA derivatives of 4-amino-
butyric acid and ethanolamine, which are the fragments of
Ahpa. The FDLA derivative of 4-amino-butyric acid eluted
later than that of ethanolamine, indicating that the acid chain
of Ahpa is more hydrophobic than the serine side chain. This
result allowed the prediction of the elution sequence of the
L- and D-FDLA derivatives of Ahpa, the D-derivative eluting
earlier than the L-derivative. On the basis of this result, the
absolute configuration at the C-19 chiral center in Ahpa was
established as R. Subsequently, the original amino acid
Ahpea was assigned the 19R configuration.
These transannular ROESY correlations are not consistent
with a possible 19S configuration.7
To establish the relative configurations at C-28 and C-29
of Adpa, the methanolysis product of 1 (3) was converted
to the acetonide 4 with 2,2-dimethoxypropane and pyridinium
p-toluenesulfate in 1:1 methanol/dichloromethane (Scheme
1). The 13C NMR shifts of the two methyl acetonide chemical
shifts [δC: 19.5; 29.8] revealed that the methyl groups were
in axial and equatorial positions in a six-membered 1,3-
dioxane chair conformation.8 Analysis of gCOSY and
TOCSY spectral data allowed the assignment of the proton
signals. In the ROESY NMR spectrum, the axial methyl
group [δC: 19.5; δH: 1.36] clearly showed correlations with
H-28 [δH: 4.22] and H-30a [δH: 4.09], indicating these
protons are axial. Proton H-29 [δH: 3.68] displayed ROESY
correlations with H-27a [δH: 2.33], H-27b [δH: 2.01], H-28
[δH: 4.22], H-30a [δH: 4.09], and H-30b [δH: 3.47], which
established its equatorial position and indicated a 28S*,29S*
assignment. A ROESY NMR cross-peak between 29-NH
[δH: 7.85] and H-27a [δH: 2.33] also supported this
assignment (Figure 2).
This configuration at C-19 is also supported by transan-
nular ROESY correlations in the 12-membered ring (Figure
1). The methyl group protons (H-10) of N-methyltyrosine
The absolute configuration of 4-amino-3,5-dihydroxy-
pentanoic acid was determined by the modified Mosher
method.9 Deprotection of the acetonide after acetylation of
4 furnished the triacetate 5. Timed acetylation of 5 allowed
esterification mainly at the primary alcohol at C-30 to yield
the tetraacetate 6. The intact secondary hydroxyl group at
C-28 in 6 was then treated with R-(-) and S-(+)-R-methoxy-
R-(trifluoromethyl)phenyl acetyl chloride (MTPA-Cl) to yield
the S- and R-MTPA esters (7a and 7b), respectively (Scheme
1). The proton chemical shifts of relevant protons were
1
1
assigned by analysis of H, H-1H gCOSY, and TOCSY
Figure 1. Transannular key ROESY correlations in the 12-
membered ring in 1.
NMR spectra. Calculation of ∆δS-R values clearly defined
(7) The MM2 energy-minimized structures by Chem3D for both 19R
and 19S configurations are shown in the Supporting Information.
(8) Rychnovsky, S. D.; Rogers, B. N.; Richardson, T. I. Acc. Chem. Res.
1998, 31, 9.
(9) Se´co, J. M.; Quin˜oa, E.; Riguera, R. Tetrahedron: Asymmetry 2001,
12, 2915.
showed clear ROESY correlations with H-17 and 19-NH,
indicating this methyl group, H-17, and 19-NH are oriented
into the ring. Another key correlation between H-12 and H-17
also defined the ring conformation as shown in Figure 1.
Org. Lett., Vol. 9, No. 8, 2007
1527

Figure 3. Key ROESY correlations in 4-amino-3-hydroxy-5-
phenylpentanoic acid (Ahppa).
Figure 2. Chemical shifts (a) and key ROESY correlations (b) in
the acetonide 4 of 4-amino-3,5-dihydroxy-pentanoic acid (Adpa).
4-amino-3,5-dihydroxy-pentanoic acid (Ahpa), and 4-amino-
3-hydroxy-5-phenylpentanoic acid (Ahppa). The rare Ahpea
and Ahpa units were reported only once interestingly in
peptides from the marine R-proteobacterium Oceanospirillum
(strain SANK 70992).12 The Ahppa unit was previously
reported in compounds from the actinomycete Streptomyces13
and the marine cyanobacterium Symploca sp.10 The structural
features of 1 and 2 are similar to those for compound B1371B
from strain SANK 70992, with respect to the alternative
combination of valines and λ-amino acids and the presence
of the hydrocarbon chains.
the absolute configuration of C-28 as S and subsequently
established the 29S configuration on the basis of their relative
stereochemistry (see the Supporting Information).
Thalassospiramide B (2)10 was obtained as an oil, which
analyzed for the molecular formula C56H83N7O13 by ESI high-
resolution mass spectral data. This molecular formula was
also supported by 1H and 13C NMR data (see the Supporting
Information), which displayed a high degree of similarity to
thalassospiramide A (1). Further analysis of 1D and 2D NMR
spectra allowed the assignment of N-methyltyrosine, three
valine residues, Ahpea, Adpa, and Dea, but not serine as in
thalassospiramide A. The difference in 2 was the replacement
of a serine unit in 1 with 4-amino-3-hydroxy-5-phenylpen-
tanoic acid (Ahppa), another γ-amino acid. Long-range
carbon-proton correlations in the HMBC NMR spectrum
of 2 defined the same connectivity of the eight discrete spin
systems as in 1. The sequence of these subunits was identical
to that of 1 except for the incorporation of Ahppa instead of
serine.
The biological activities of 1 and 2 were evaluated in a
mouse mixed lymphocyte assay, which probes for noncy-
totoxic suppression of cytokine production by ovalbumin
(OVA) stimulated splenocytes. The thalassospiramides were
initially screened for inhibition of IL-5 as a representative
cytokine, which has been shown to play an important role
in TH-2 mediated inflammatory diseases such as asthma.14
In this assay, thalassospiramides A and B showed IC50 values
of 10 and 5 µM with no observable cytotoxicity at concen-
trations of 10 µM. Further studies will be needed to assess
in vivo activity and their molecular and cellular mechanisms
of action before their utility can be accessed.
The absolute configurations of the standard amino acid
units were also determined using the advanced Marfey
method, as in 1. In this experiment, the N-methyltyrosine
and three valines were assigned L configurations. The
absolute stereochemistry of the Ahpea unit was established
as 19R by transannular ROESY correlations indentical to
those shown in Figure 1. The relative configurations (C-38
and C-39) of Ahppa were proposed by analysis of 1H
coupling constants and ROESY correlations. Specifically,
no observable correlations in the gCOSY and TOCSY spectra
were indicative of zero coupling between H-38 and H-39.
This led us to establish the dihedral angle between these two
protons at near 90° and allowed ROESY spectral analysis
(Figure 3). Only the relative configurations 38S* and 39S*
were in agreement with all observed ROESY correlations.11
Thalassospiramides A and B are the first secondary
metabolites from the genus Thalassospira. These peptides
are unique in that they incorporate unusual λ-amino acids
such as 4-amino-5-hydroxy-penta-2-enoic acid (Ahpea),
Acknowledgment. This work is a result of generous
financial support provided by the NIH, National Cancer
Institute, under grant CA44848. We thank Dr. Hak Cheol
Kwon, Korea Institute of Science and Technology, for his
generous help in the determination of stereochemistry of
thalassospiramide A.
Supporting Information Available: NMR spectral data
tables and complete 2D spectra of 1 and 2, 1H NMR of 3-8,
gCOSY, gHSQC, and ROESY spectra of 4, the details of
strain CNJ-328, mouse splenocyte assay, isolation of 1 and
2, and chemical modifications of 1. This material is available
free of charge via the Internet at http://pubs.acs.org.
OL070294U
(10) Thalassospiramide B (2): oil; [R]D -39 (c 0.39, CH3CN); IR (neat)
νmax 3307, 2966, 1743, 1643, 1531 cm-1; UV (CH3CN) λmax (log ꢀ) 224
(4.2), 278 (3.4) nm; NMR spectral data, see Table S2 in the Supporting
Information; HR-ESI-TOFMS [M + Na]+ m/z 1084.5932 (C56H83N7O13-
Na, calcd [M + Na]+ 1084.5941).
(11) The consistent ROESY correlations were reported in the same unit
in tasiamide B (marine cyanobacterium), of which absolute configurations
were proposed as S and S. See: Williams, P. G.; Yoshida, W. Y.; Moore,
R. E.; Paul, V. J. J. Nat. Prod. 2003, 66, 1006.
(12) Kaneko, I.; Minekura, H.; Takeuchi, Y.; Nakamura, T.; Haruyama,
H. PCT Intl. 1994, 94-19481.
(13) (a) Sato, T.; Shibazaki, M.; Yamaguchi, H.; Abe, K.; Matsumoto,
H.; Shimizu, M. J. Antibiot. 1994, 47, 588. (b) Omura, S.; Imamura, N.;
Kawakita, K.; Mori, Y.; Yamazaki, Y.; Masuma, R.; Takahashi, Y.; Tanaka,
H.; Huang, L.-Y.; Woodruff, H. B. J. Antibiot. 1986, 39, 1079.
(14) Greenfeder, S.; Umland, S. P.; Cuss, F. M.; Chapman, R. W.; Egan,
R. W. Respir. Res. 2001, 2, 71.
1528
Org. Lett., Vol. 9, No. 8, 2007