Isolation and synthesis of chivotriene, a chivosazole shunt product from Sorangium cellulosum

Article abstract of DOI:10.1002/ejoc.201000781

A shunt product of chivosazole biosynthesis was isolated from the fermentation broth of Sorangium cellulosum, strain So ce 12. Its structure was elucidated by NMR spectroscopy and exhibits six chiral centers, a triene, and a labile β-hydroxy ketone. The synthesis of chivotriene confirms the structure and in particular the proposed configuration at C22, which was originally deduced for chivosazole from the combination of its genetic and spectroscopic analyses. Remarkably, chivotriene exhibits biological activity usually not observed for segments of complex natural products. However, this activity is not connected to interference with the actin cytoskeleton, the intrinsic target of the chivosazoles.

Full text of DOI:10.1002/ejoc.201000781

SHORT COMMUNICATION
DOI: 10.1002/ejoc.201000781
Isolation and Synthesis of Chivotriene, a Chivosazole Shunt Product from
Sorangium cellulosum
Tobias Brodmann,[a,b] Dominic Janssen,[a,b] Florenz Sasse,[b] Herbert Irschik,[b]
Rolf Jansen,[b] Rolf Müller,[b] and Markus Kalesse*[a,b]
Keywords: Natural products / Biosynthesis / Structure elucidation / Chirality
A shunt product of chivosazole biosynthesis was isolated
from the fermentation broth of Sorangium cellulosum, strain
So ce 12. Its structure was elucidated by NMR spectroscopy
and exhibits six chiral centers, a triene, and a labile β-hy-
droxy ketone. The synthesis of chivotriene confirms the
structure and in particular the proposed configuration at C22,
which was originally deduced for chivosazole from the com-
bination of its genetic and spectroscopic analyses. Remark-
ably, chivotriene exhibits biological activity usually not ob-
served for segments of complex natural products. However,
this activity is not connected to interference with the actin
cytoskeleton, the intrinsic target of the chivosazoles.
Introduction
Chivosazoles are in the focus of chemical research due to exhibit antifungal and cytotoxicity activity against mouse
their complex chemical structure and remarkable biological fibroblasts (L-929) and HeLa cells.[1a] Recently, Sasse et al.
profile.[l] Their structures contain a 31-membered macrolide reported the reduction of actin polymerization by chivosa-
constructed from three polyene, one oxazole, and a hydrox- zoles.[2] These studies illustrate that chivosazoles act on F-
ylated ketide segment. Additionally, chivosazoles A–E in- actin differently than that proposed for cytochalasin D,
clude a 6-deoxyglucose derivative at C11. Chivosazoles chondramide, or rhizopodin.[3]
Figure 1. Proposed chivotriene biosynthesis.
[a] Centre for Biomolecular Drug Research (BMWZ),
Leibniz Universität Hannover,
Biosynthetic studies revealed that chivosazole is biosyn-
thesized by a trans-AT polyketide synthase (PKS)/nonribo-
somal peptide synthetase (NRPS). The shunt product re-
ported here is consistent with the biosynthetic hypothesis
put forward by Perlova et al. and can be rationalized by
hydrolysis and decarboxylation of the intermediate poly-
ketide bound to ACP8 (Figure 1).[4]
Schneiderberg 1B, 30167 Hannover, Germany
E-mail: markus.kalesse@oci.uni-hannover.de
[b] Helmholtz Centre for Infection Research GmbH (HZI), Abt.
Chemische Biologie, Arbeitsgruppe Mikrobielle Wirkstoffe and
Abt. Medizinische Chemie
Inhoffenstrasse 7, 38124 Braunschweig, Germany
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201000781.
View this journal online at
wileyonlinelibrary.com
Eur. J. Org. Chem. 2010, 5155–5159
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
5155

M. Kalesse et al.
SHORT COMMUNICATION
Herein we report the isolation, structure elucidation, and signal of the oxymethine at C13 no longer appeared at δ =
synthesis of the chivosazole shunt product chivotriene (1) 5.30 ppm but at δ = 3.61 ppm due to the missing acyl shift
together with its biological data.
of the former lactone. The remaining ketone part (C1–C4)
1
was assigned by H–1H COSY correlations and chemical
shifts.
Results and Discussion
We started a synthetic program on the shunt product to
confirm the proposed structure and in particular the config-
uration at C5. In our retrosynthetic disconnection we took
advantage of our established route of the northern fragment
of chivosazole A.[6] To achieve a convergent route we de-
cided to perform a Wittig olefination between C8 and C9
(Scheme 1). As a result of our synthetic endeavors we real-
ized that the correct choice of the α,β-unsaturated aldehyde
and corresponding phosphonium salt on either the eastern
or western fragment was pivotal for obtaining good (E)
selectivities. Additionally, different reaction parameters
were screened to avoid elimination of the TBS-protected β-
hydroxy ketone.
Isolation
Chivotriene (1) was isolated by silica gel chromatography
as a byproduct (1 mg) from the purification of disorazole B2
(fraction 1.1).[5] Comparison of the NMR spectroscopic
data of 1 with those of chivosazole A identified the struc-
ture from C5 to C20 including the double bond configura-
1
tions of the E,E,Z-triene (Table 1). However, the H NMR
Table 1. NMR spectroscopic data of chivotriene (1) in CD3OD [1H/
13C: 400/100 MHz].
Position
∆
C
[ppm]
δH [ppm]
1
2
3
4
7.7
1.01 (t, J = 7.3 Hz)
2.52 (q, J = 7.3 Hz)
37.6
212.1
50.6
2.67 (dd, J = 15.6, 8.2 Hz)
2.58 (dd, J = 15.6, 4.9 Hz)
4.60 (ddd, J = 8.2, 6.4, 4.9 Hz)
5.71 (dd, J = 15.2, 6.4 Hz)
6.33 (dd, J = 10.5, 15.2 Hz)
6.19 (dd, J = 10.5, 14.7 Hz)
6.58 (dd, J = 11.4, 14.7 Hz)
6.07 (dd, J = 11.4, 11.4 Hz)
5.45 (m)
2.92–2.82 (m)
3.61 (dd, J = 7.0, 4.1 Hz)
1.72–1.62 (m, 1 H)
3.87 (ddd, J = 9.0, 5.6, 4.5 Hz)
1.54–1.47 (m, 2 H)
4.01 (m)
5
6
7
8
69.5
136.1
131.8
133.4
130.1
129.9
136.7
37.1
76.5
42.2
71.3
43.8
Scheme 1. Retrosynthesis of 1.
9
The β-hydroxy ketone group in fragments 2 and 3 was
synthesized by a diastereoselective aldol reaction by using
the Nagao protocol.[7] Subsequent TBS protection and
treatment with N,O-dimethylhydroxylamine in combination
with trimethylaluminum lead to Weinreb amide 8.[8] Ad-
dition of ethylmagnesium bromide and PMB deprotection
installed the ethyl ketone moiety and afforded allyl alcohol
9. Conversion to the corresponding α,β-unsaturated alde-
hyde 2 or phosphonium bromide 3 provided both possible
segments for the Wittig olefination (Scheme 2).
10
11
12
13
14
15
16
17
18
19
20
65.5
24.6
10.3
18.5
1.21 (d, J = 6.3 Hz)
0.96 (d, J = 7.0 Hz)
0.95 (d, J = 6.8 Hz)
Scheme 2. Synthesis of β-hydroxy ketones 2 and 3.
5156
www.eurjoc.org
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2010, 5155–5159

Isolation and Synthesis of Chivotriene
Ethyl ester 14 previously synthesized[1d] was obtained af- Table 2. Wittig olefination.
ter reductive opening of PMB acetal 11, Swern oxidation,
and a (Z)-selective olefination by using the Ando proto-
col.[9] However, PMB deprotection at the end of this route
turned out to be problematic. Various reaction conditions
led to decomposition of the starting material. Therefore, we
changed the PMB protecting group to a TMS ether at an
earlier stage of the synthesis. Complementary to the func-
tionalities of ethyl ketones 2 and 3 we generated both phos-
phonium bromide 4 and α,β-unsaturated aldehyde 5 from
alcohol 15 (Scheme 3).
Entry
Base
R1
R2
E/Z
T
[°C]
Yield[a]
[%]
1
2
3
4
5
KOtBu
KOtBu
LiOtBu
LiHMDS CHO
KOtBu
CHO
CHO
CHO
CH2PBu3Br 1:1
0
57, 36[b]
78
71
80
73
CH2PBu3Br 2:1 –30
CH2PBu3Br 2:1 –30
CH2PBu3Br 2:1 –30
CH2PBu3BrCHO
7:1 –30
[a] Calculated over two steps from the corresponding allyl bromide.
[b] Elimination product.
Scheme 3. Synthesis of segments 4 and 5.
Unfortunately, initial experiments with α,β-unsaturated
aldehyde 2 and phosphonium bromide 4 with the use of
KOtBu at 0 °C provided a 1:1 mixture of E/Z isomers and
elimination product (36%). Changing the conditions such
as performing the reaction at –30 °C prevented elimination
but provided only a modest increases in selectivity (Z/E =
2:1). Also, changing the bases in the olefination reaction
did not increase the selectivity either (Table 2).
Nevertheless, when we exchanged the functional groups
of both coupling partners by employing 3 and 5 we ob-
served an improved E/Z selectivity of 7:1 in 73% yield
(Scheme 4). With the optimized reaction conditions in
hand, we were able to proceed to the global deprotection
of the silyl ethers. The spectroscopic data (1H NMR, 13C
NMR, HRMS) were in all respect identical to the data of
the isolated compound. As the relative and absolute stereo-
chemistry of fragment 5 was already known from compari-
son of chemical degradation of chivosazole A, this also con-
firms the stereochemistry of C22.
Scheme 4. Endgame to shunt product 1.
showed at least weak activities in reducing cell proliferation
(Table 3). Interestingly, isomer 17 was more active than the
shunt product itself. By staining F-actin of treated PtK2
potoroo and L-929 mouse cells we looked for a possible
Table 3. Antiproliferative activity IC50 [µg/mL] of 1 and 17. Values
are means of two determinations in parallel.
Cell line
Origin
1
17
L-929
KB-3–1
PC-3
U-937
HUVEC
murine connective tissue
human cervix carcinoma
human prostate carcinoma
human lymphoma
human umbilical vein
endothelial cells
50
Ͼ100
100
n.d.
100
23
Ͼ100
Ͼ100
25
In a subsequent set of experiments, chivotriene (1) and
its E,Z,Z-isomer 17 were tested with an array of different
mammalian cell lines. Surprisingly, both compounds
25
Eur. J. Org. Chem. 2010, 5155–5159
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5157

M. Kalesse et al.
SHORT COMMUNICATION
15.0, 6.2 Hz, 1 H), 5.54 (dd ≈ t, J = 10.4 Hz, 1 H), 4.67–4.59 (m,
1 H), 4.05–3.95 (m, 1 H), 3.89–3.81 (m, 1 H), 3.63 (dd, J = 7.0,
3.9 Hz, 1 H), 2.86 (dt, J = 9.4, 6.8 Hz, 1 H), 2.68 (dd, J = 15.7,
8.2 Hz, 1 H), 2.59 (dd, J = 15.7, 4.8 Hz, 1 H), 2.52 (q, J = 7.3 Hz,
2 H), 1.72–1.62 (m, 1 H), 1.57–1.44 (m, 2 H), 1.20 (d, J = 6.5 Hz,
3 H), 1.01 (t, J = 7.3 Hz, 3 H), 0.95 (d, J = 6.8 Hz, 6 H) ppm. 13C
NMR (125 MHz, CDCl3): δ = 212.0, 137.4, 137.3, 129.7, 126.4,
126.2, 124.8, 76.5, 71.4, 69.5, 65.5, 50.6, 44.0, 42.1, 37.6, 36.7, 24.6,
18.4, 10.3, 7.8 ppm. HRMS (ESI): calcd. for C20H35O5 [M + H]+
355.2484; found 355.2469.
disintegration of the actin cytoskeleton of the cells, but we
could not identify any specific interference with actin fila-
ment stability.
Conclusions
We have presented the isolation and total synthesis of
chivotriene, a chivosazole shunt product from Sorangium
cellulosum. The convergent synthesis provides substantial
material, which enables detailed analysis of the antiprolifer-
ative activity. The fact that this shunt product is liberated
at the transition from polyketide to nonribosomal biosyn-
thesis is noteworthy from an evolutionary point of view.
In particular, the observation that the mode of action of
chivotriene differs fundamentally from that of chivosazole
adds additional aspects on the interpretation of the evolu-
tionary origin of natural products.
Supporting Information (see also the footnote on the first page of
this article): Analytical and spectroscopic data of all new com-
pounds.
Acknowledgments
The authors thank Wera Collisi (HZI) for performing cell prolifera-
tion assays. Generous financial support by the Deutsche For-
schungsgemeinschaft (DFG) (KA 913/14–1) and the Fonds der
Chemischen Industrie for T. B. is gratefully acknowledged.
Experimental Section
[1] a) H. Irschik, R. Jansen, K. Gerth, G. Höfle, H. Reichenbach,
J. Antibiotics 1995, 48, 962–966; b) R. Jansen, H. Irschik, H.
Reichenbach, G. Höfle, Liebigs Ann./Recueil 1997, 8, 1725–
1732; c) D. Janssen, D. Albert, R. Jansen, R. Müller, M. Ka-
lesse, Angew. Chem. Int. Ed. 2007, 46, 4898–4901; d) T. Brod-
mann, M. Lorenz, R. Schäckel, S. Simsek, M. Kalesse, Synlett
2009, 2, 174–192.
General Procedure for the Wittig Olefination followed by Global De-
protection: Base (35 µL, 35 mmol, 1  in THF) was added to a
solution of phosphonium bromide 3 or 4 (0.034 mmol, 1 equiv.)
and α,β-unsaturated aldehyde 2 or 5 (0.04 mmol, 1.2 equiv.) in tolu-
ene (1.5 mL) at –30 °C. The reaction mixture was stirred for 60 min
at –30 °C. Water (1 mL) was added, and the mixture was extracted
with CH2Cl2 (3ϫ2 mL). The combined organic layer was washed
with brine (5 mL) and dried with Na2SO4. The solvents were re-
moved in vacuo, and flash column chromatography provided pro-
tected chivotriene 16. The resulting mixture of E/Z isomers was
directly used for global deprotection of the silyl protecting groups.
HF·pyridine (2 mL) was added to a solution of the crude triene
(49 mg) in THF (3 mL) and pyridine (3 mL) at room temperature.
The solution was stirred for 24 h before quenching with a saturated
solution of NaHCO3. It was taken up in ethyl acetate and washed
with phosphate buffer (pH 7) solution. The organic layer was dried
with MgSO4. The solvents were removed in vacuo, and the crude
product was purified by flash column chromatography and HPLC
(Detection at 230 nm; column: Merck 50853, LiChroCART® 250–
10 LiChrospher® 100, RP-18, 10 µm; eluent A MeOH/eluent B
H2O = 55:45) to obtain chivotriene (1) and its isomer 17 (16 mg,
73%).
[2] R. Diestel, H. Irschik, R. Jansen, M. W. Khalil, H. Reichen-
bach, F. Sasse, ChemBioChem 2009, 10, 2900–2903.
[3] For cytochalasin D, see: a) D. C. Lin, S. Lin, S. Proc. Natn.
Acad. Sci. USA 1979, 76, 2345–2349; b) M. D. Flanagan, S.
Lin, J. Biol. Chem. 1980, 255, 835–838; c) S. L. Brenner, E. D.
Korn, J. Biol. Chem. 1979, 254, 9982–9985; d) S. S. Brown, J. A.
Spudich, J. Cell Biol. 1979, 83, 657–662; e) S. MacLean-
Fletcher, T. D. Pollard, Cell 1980, 20, 329–341; for chondram-
ides, see: f) B. Kunze, R. Jansen, F. Sasse, G. Höfle, H. Reichen-
bach, J. Antibiot. 1995, 48, 1262–1266; g) F. Sasse, B. Kunze,
T. M. A. Gronewold, H. Reichenbach, J. Natl. Cancer Inst.
1998, 90, 1559–1563; h) U. Eggert, R. Diestel, F. Sasse, R. Jan-
sen, B. Kunze, M. Kalesse, Angew. Chem. Int. Ed. 2008, 47,
6478–6482; for rhizopodin, see: i) F. Sasse, H. Steinmetz, G.
Höfle, H. Reichenbach, J. Antibiot. 1993, 46, 741–748; j)
T. M. A. Gronewold, F. Sasse, H. Lunsdorf, H. Reichenbach,
Cell Tissue Res. 1999, 295, 121–129; k) N. Horstmann, D. Men-
che, Chem. Commun. 2008, 41, 5173–5175.
[4] a) S. Schneiker, O. Perlova, O. Kaiser, K. Gerth, A. Alici, M. O.
Altmeyer, D. Bartels, T. Bekel, S. Beyer, E. Bode, H. B. Bode,
C. J. Bolten, J. V. Choudhuri, S. Doss, Y. A. Elnakady, B.
Frank, L. Gaigalat, A. Goesmann, C. Groeger, F. Gross, L.
Jelsbak, L. Jelsbak, J. Kalinowski, C. Kegler, T. Knauber, S.
Konietzny, M. Kopp, L. Krause, D. Krug, B. Linke, T. Mah-
mud, R. Martinez-Arias, A. C. McHardy, M. Merai, F. Meyer,
S. Mormann, J. Muñoz-Dorado, J. Perez, S. Pradella, S. Ra-
chid, G. Raddatz, F. Rosenau, C. Rückert, F. Sasse, M. Scharfe,
S. C. Schuster, G. Suen, A. Treuner-Lange, G. J. Velicer, F.-J.
Vorhölter, K. J. Weissman, R. D. Welch, S. C. Wenzel, D. E.
Whitworth, S. Wilhelm, C. Wittmann, H. Blöcker, A. Pühler,
R. Müller, Nat. Biotechnol. 2007, 25, 1281–1289; b) O. Perlova,
K. Gerth, O. Kaiser, A. Hans, R. Müller, J. Biotechnol. 2006,
121, 174–191.
[5] R. Jansen, H. Irschik, H. Reichenbach, V. Wray, G. Höfle, Lie-
bigs Ann. Chem. 1994, 759–773.
[6] D. Janssen, M. Kalesse, Synlett 2007, 17, 2667–2670.
[7] a) Y. Nagao, S. Yamada, T. Kumagi, M. Ochiai, E. Fujita, J.
Chem. Soc., Chem. Commun. 1985, 1418–1419; b) Y. Nagao, Y.
Hagiwara, T. Kumagi, M. Ochiai, T. Inoue, K. Hashimoto, E.
Chivotriene (1): Rf = 0.06 (5% MeOH in CH2Cl2). [α]2D3 = +10.0 (c
= 0.10, CHCl3). 1H NMR (400 MHz, CDCl3): δ = 6.58 (dd, J =
14.7, 11.4 Hz, 1 H), 6.33 (dd, J = 15.2, 10.5 Hz, 1 H), 6.19 (dd, J
= 14.7, 10.5 Hz, 1 H), 6.07 (dd, J = 11.4, 11.4 Hz, 1 H), 5.71 (dd,
J = 15.2, 6.4 Hz, 1 H), 5.45 (m, 1 H), 4.60 (ddd, J = 8.2, 6.4,
4.9 Hz, 1 H), 4.05–3.95 (m, 1 H), 3.87 (ddd, J = 9.0, 5.6, 4.5 Hz, 1
H), 3.61 (dd, J = 7.0, 4.1 Hz, 1 H), 2.90–2.79 (m, 1 H), 2.67 (dd,
J = 15.6, 8.2 Hz, 1 H), 2.58 (dd, J = 15.6, 4.8 Hz, 1 H), 2.52 (q, J
= 7.3 Hz, 2 H), 1.72–1.63 (m, 1 H), 1.57–1.44 (m, 2 H), 1.21 (d, J
= 6.3 Hz, 3 H), 1.01 (t, J = 7.3 Hz, 3 H), 0.96 (d, J = 7.0 Hz, 3 H),
0.95 (d, J = 6.8 Hz, 3 H) ppm. 13C NMR (125 MHz, CDCl3): δ =
212.1, 136.7, 136.1, 133.4, 131.8, 130.1, 129.9, 76.5, 71.3, 69.5, 65.5,
50.6, 43.8, 42.2, 37.6, 37.1, 24.6, 18.5, 10.3, 7.7 ppm. HRMS (ESI):
calcd. for C20H34O5Na [M + Na]+ 377.2304; found 377.2299.
E,Z,Z-Isomer 17: Rf = 0.06 (5% MeOH in CH2Cl2). [α]2D3 = –104.3
(c = 0.07, CHCl3). 1H NMR (400 MHz, CDCl3): δ = 6.77 (dd, J =
14.9, 11.1 Hz, 1 H), 6.54 (dd ≈ t, J = 11.4 Hz, 1 H), 6.33 (dd ≈ t,
J = 11.3 Hz, 1 H), 6.00 (dd ≈ t, J = 11.1 Hz, 1 H), 5.74 (dd, J =
5158
www.eurjoc.org
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2010, 5155–5159

Isolation and Synthesis of Chivotriene
Fujita, J. Org. Chem. 1986, 51, 2391–2393; c) A. Gonzalez, J.
Aiguade, F. Urpi, J. Vilarrasa, Tetrahedron Lett. 1996, 37,
8949–8952.
[9] a) K. Ando, J. Org. Chem. 1997, 62, 1934–1939; b) K. Ando,
J. Org. Chem. 1999, 64, 8406–8408.
Received: May 31, 2010
[8] a) D. A. Evans, S. L. Bender, J. Morris, J. Am. Chem. Soc. 1988,
110, 2506–2526; b) A. Basha, M. Lipton, S. M. Weinreb, Tetra-
hedron Lett. 1977, 18, 4171–4174.
Published Online: ᭿
Eur. J. Org. Chem. 2010, 5155–5159
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5159