Stereochemical determination of thuggacins A-C, highly active antibiotics from the myxobacterium Sorangium cellulosum

Article abstract of DOI:10.1002/anie.200704897

Chemistry and biology meet in the structure elucidation of the antibiotics thuggacins A-C. The absolute configuration of all stereogenic centers in the thuggacins was confirmed through a combination of chemical derivatization, NMR analysis, molecular modeling, and bioinformatic analysis of the thuggacin biosynthetic genes. (Chemical Equation Presented).

Full text of DOI:10.1002/anie.200704897

Communications
DOI: 10.1002/anie.200704897
Structure Elucidation
Stereochemical Determination of Thuggacins A–C, Highly Active
Antibiotics from the Myxobacterium Sorangium cellulosum**
Martin Bock, Kathrin Buntin, Rolf Müller, and Andreas Kirschning*
Since 1993, when the World Health Organization declared
tuberculosis “a global health emergency”, there has been new
impetus to research on tuberculosis because it can no longer
be sufficiently treated by currently available antibiotic
therapy. The increasing multidrug resistance of Mycobacte-
rium tuberculosis and its ability to persist as a latent infection
makes the development of alternative antibiotics—preferably
with novel modes of action—necessary.[1] The polyketide
natural products thuggacin A (1), B (2), and C (3) recently
isolated from the myxobacterium Sorangium cellulosum show
strong antibiotic activity against Mycobacterium tuberculosis
which targets the bacterial respiratory chain.[2] Recently, it
was found that the myxobacterium Chondromyces crocatus
Cmc5 also produces thuggacin derivatives, namely thuggacin
cmc-A (4) and thuggacin cmc-C (5).
Thuggacin A (1; Scheme 1) features a 17-membered
macrolactone with a thiazole ring, a diene (11E, 13Z), and
an a,b-unsaturated lactone with a n-hexyl side chain at C2
plus a complex side chain at C16 bearing three hydroxy
groups and a diene unit. Thuggacin B (2) shares the same
structural features except for the ring size: the cyclic lactone is
closed at O17 instead of O16 in thuggacin A. Finally,
thuggacin C (3) is macrocyclized at O18. With two additional
hydroxy groups and an aliphatic methyl group in the macro-
lactone ring, the thuggacins include eight stereogenic centers.
The structure of the thuggacins, their constitution, and
Scheme 1. Thuggacin A (1) (relative configuration at C7, C8, C10, and
C16 as published by Jansen et al.[2]) and thuggacins B (2) C (3), cmc-A
(4), and cmc-C( 5).
preliminary assignments of the relative stereochemistry
were determined by Jansen et al. on the basis of NMR
spectroscopic data.[2] However, as the analytical methods
employed strongly relied on the rigid conformation of the ring
system, they could not be extended to the stereocenters of the
freely rotating side chain.
For this purpose, synthetic derivatization combined with
NMR analysis and modeling was conducted. We also report
that bioinformatic analysis of the thuggacin biosynthetic
genes is a powerful tool for confirming the absolute config-
uration of the stereocenters first assigned by chemical
methods. Thus, the relative configurations of both stereo-
domains (C7 to C10 and C16 to C20) were first determined
separately, which was followed by establishing the correct
sterochemical relationship between these stereoclusters.
Since six out of eight stereogenic centers bear a hydroxy
group, a straightforward derivatization strategy could be
developed based on rigidifying selected areas by protection of
1,3- and 1,2-diols as acetonides (Scheme 2). The constitutions
of derivatives 6–9 were assigned by 1H and 13C NMR
spectroscopy complemented with COSY, HMQC, and
HMBC experiments and by comparison with the natural
product 1. Two chemical aspects are noteworthy, namely
1) the preferred formation of the 1,3-acetonide (C18/C20)
over the 1,2-acetonide (C17/C18) and 2) formation of the
acyclic methyl ester 9, which was important to secure the
NMR data of macrocyclic derivatives 6–8. In fact, the d (13C)
values found for the acetonide carbon atoms at C8/C10 and
[*] M. Bock, Prof. Dr. A. Kirschning
Institut für Organische Chemie
und Biomolekulares Wirkstoffzentrum (BMWZ)
Leibniz Universität Hannover
Schneiderberg 1B, 30167 Hannover (Germany)
Fax: (+49)511-762-3011
E-mail: andreas.kirschning@oci.uni-hannover.de
K. Buntin, Prof. Dr. R. Müller
Institut für Pharmazeutische Biotechnologie,
Universität des Saarlandes
Postfach 151150, 66041 Saarbrücken (Germany)
Prof. Dr. R. Müller
Helmholtz-Zentrum für Infektionsforschung (HZI)
Inhoffenstrasse 7, 38124 Braunschweig (Germany)
[**] This work was supported by the Fonds der Chemischen Industrie.
We are indebted to H. Irschik (HZI, Braunschweig) for the
fermentation of Sorangium cellulosium and R. Jansen (HZI,
Braunschweig) for helpful discussions.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
2308
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2008, 47, 2308 –2311

Angewandte
Chemie
stereochemistry around C16/C17 and C17/C18
which were both determined to be 1,2-syn[5,6]
(Scheme 3).
Final proof for the relative configurations
of all stereogenic centers (C7, C8, C9, and
C16) within the ring was possible after oxida-
tive removal of the complex side chain. The
intermediate aldehyde had to be reduced
directly to the triol 10 in order to obtain
sufficiently resolved NMR spectra (Scheme 4).
Besides determination of vicinal coupling
constants, molecular modeling studies
(MMFFs) of the four possible diastereomers
generated by variation of the C7 and C16
stereogenic centers in relation to the anti 1,8-
diol unit of triol 10 were conducted.[7] To
account for the observed NOE couplings, we
constrained the distances between the atom
pairs 7-H/10-H, 3-H/12-H, and 5-H/26-H to a
range between 2 and 4.5 Š in the MonteCarlo
searches. The calculated structures for all four
diastereomers were analyzed for discrepancies
between their structures and the observed
Scheme 2. Formation of acetonides 6–9 and relevant 13CNMR signals of acetonide
carbon atoms; 2,2-DMP=2,2-dimethoxypropane, TsOH=para-toluenesulfonic acid.
C18/C20 of methyl ester 9 are similar to those of the
analogous macrocyclic derivatives 6–8.
These data unambiguously indicate that the conforma-
tions in the 1,3-dioxane rings are not distorted by the
macrocycle. Thus, Rychnovskyꢀs method[3,4] could be applied
reliably to assign the relative configuration of the 1,3-diols via
their 1,3-dioxanes. Accordingly, the 1,3-diol at C8/C10 is
configured anti. By including the information on the vicinal
coupling constants (3JHH) between 18-H/19-H and 19-H/20-H,
Scheme 4. Glycol cleavage of thuggacin A (1) and relative configura-
which both are in the range of 1.5 Hz for all derivatives, the
relative stereochemistry of the stereotriade C18 to C20 was
assigned to be all-syn. Moreover, the 1,2-diols protected in
dioxolanes 7 and 9 provide information on the relative
tion of the two stereodomains.
NMR data (i.e. vicinal 1H coupling constants and NOE
couplings).
In both C7 anti structures, the 7-H would adopt an exo
position However, the observed strong 7-H/10-H NOE
contact is not consistent with this configuration (Table 1).
This center is therefore assigned to be syn oriented with
respect to the hydroxyl group at C8. This is further supported
by a coupling constant of H-9 exo to H-10 of about 10 Hz.
Similar results were obtained for the C16 center: 15-Ha has
two vicinal coupling constants of about 10 Hz, matching
dihedral angles of 160 to 1708. The modeled dihedral angle 15-
Ha,C15,C16,16-H is about 1658 for one configuration and 608
Table 1: Selected structural features of the four modeled isomers of triol
10.
Isomer
7-H position
Dihedral angle
15-Ha,C15,C16,16-H
10
endo
exo
endo
exo
1678
1628
628
7-epi-10
16-epi-10
7,16-epi-10
648
Scheme 3. Relative configuration of 1,3-dioxolanes and 1,3-dioxanes.
Angew. Chem. Int. Ed. 2008, 47, 2308 –2311
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org 2309

Communications
for the opposite one (Table 1).[8] This clearly points to the
configuration shown in Figure 1. In fact, the 3D structure
depicted in Figure 1 looks very similar to that of thugga-
tion fragments in sufficient quantities for configurational
analysis. However, the biosynthetic genes must be identified
and analyzed. In the course of this study a method for gene
inactivation in Chondromyces crocatus Cmc5 was applied,
and the chondramide biosynthetic gene cluster was identified.
As this myxobacterium also produces thuggacin derivatives
thuggacin cmc-A (4) and thuggacin cmc-C (5) and since we
assume that the biosynthesis for thugaccin A and thuggacin
cmc-C is very similar, we set out to identify the corresponding
gene cluster in C. crocatus Cmc5. Inactivation of a candidate
polyketide synthase indeed resulted in the loss of thuggacin
cmc-C production in C. crocatus Cmc5 and enabled the
cloning and sequencing of the complete biosynthetic gene
cluster which encodes a complex hybrid of polyketide
synthases (PKSs) and a nonribosomal peptide synthetases.
By following the analysis of the ketoreductase domains (KRs)
within the PKS according to the core amino acid analysis
described to predict the absolute configuration of secondary
alcohols by Caffrey and Reid et al.[11] we could propose the
configuration at positions C18, C16, C10, and C8 (Table 2).[14]
Figure 1. Modeling of triol 10 and selected NMR data; numbering
refers to atom numbers given in Scheme 1.
Table 2: Type of ketoreductases found in the polyketide synthase
domains involved in thuggacin biosynthesis and the corresponding
stereogenic centers with their proposed absolute configuration (C20,
C18, C16, C10, C8).[a]
cin A.[2] By assembling all of data collected, we could deduce
the complete relative configuration of thuggacin A (Scheme 4
and Figure 1).
Ketoreductase
Type
Configuration
KR1 TugA
KR2 TugA
KR3 TugA
KR1 TugB
B-type
B-type
B-type
B-type
B-type
DB
DB trans
DB trans
C20 (S)
C18 (R)
C16 (S)
cis
Finally, the absolute configuration was determined by
transformation of derivative 6 containing a free hydroxy
group at C17 into esters 11a and 11b (Scheme 5) and applying
the Mosher analysis rules.[9] From the calculated dD values the
configuration at C17 was deduced to be S. Further support for
the absolute configuration was obtained after removal of the
isopropylidene protecting group yielding tetraols 12a and
12b.[10] Very similar dD values were determined, indicating
that the adjacent 1,3-dioxane ring did not invalidate the
Mosher analysis of 11a and 11b.
Recently, it has become possible to predict the stereo-
chemistry resulting from keto reduction during polyketide
biosynthesis based on a bioinformatic analysis of the respec-
tive domains in giant polyketide synthase modules.[11–13] This
method is especially useful if the availability of the natural
product is limited and it is not possible to generate degrada-
KR2 TugB
KR1 TugCA-type
KR2 TugCB-type
KR1 TugD
KR2 TugD
KR3 TugD
DB
trans
A-type
A-type
B-type
C10 (S)
C8 (S)
DB trans
[a] Action of all other KR domains is followed by elimination of water and
thus results in double bonds (DB) in the final molecule. Predicted
double-bond configurations and absolute configurations of the alcohols
are given (see the Supporting Information).
Only the assignment at C10 is somewhat ambiguous because
the conserved amino acids are not in total agreement with the
consensus. The application of these results to thuggacin A (1)
confirmed the assigned configuration.
Although the thuggacins produced by Cmc5
do not contain an OH group attached to C20,
a corresponding intermediate must be pro-
duced during PKS assembly, and therefore
the configuration at C20 can as well be
predicted.
By considering all of the data presented,
we could deduce the complete relative con-
figuration of thuggacin A (Scheme 4 and
Figure 1). As far as C7, C8, C10, and C16
are concerned, our relative assignments are in
accordance with those reported by Jansen
Scheme 5. Configurational assignment of thuggacin A by Mosher derivatization of 6 (the
R-MPTA ester was prepared from S-MPTA chloride and vice versa; see the Supporting
Information). MPTA=a-methoxy-a-(trifluoromethyl)phenylacetic acid.
et al.[2] Scheme 6 summarizes the assignments
of the complete relative and absolute config-
2310
www.angewandte.org
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2008, 47, 2308 –2311

Angewandte
Chemie
[1] J. D. McKinney, Nat. Med. 2000, 6, 1330 – 1333.
[2] H. Steinmetz, H. Irschik, B. Kunze, H. Reichenbach, G. Höfle, R.
Jansen, Chem. Eur. J. 2007, 13, 5822 – 5832.
[3] S. Rychnovsky, D. J. Skalitzky, Tetrahedron Lett. 1990, 31, 945 –
948.
[4] Diagnostic d (13C) values for the 1,3-syn configuration: Meax
ꢀ 20 ppm, Meeq ꢀ 30 ppm; for 1,3-anti: both Me ꢀ 25 ppm.
Occasionally, also the d (13C) values for the quartenary C
atoms are analyzed: d < 100 ppm (for 1,3-syn) and d > 100 ppm
(for 1,3-anti).
[5] G. Dana, H. Denechpajouh, Bull. Soc. Chim. Fr. 1980, 395 – 399.
[6] Diagnostic d (13C) values for the 1,2-anti configuration: Meb
ꢀ 25 ppm, Meg ꢀ 28 ppm; for 1,2-syn both Me ꢀ 27 ppm.
[7] a) F. Mohamadi, N. G. J. Richards, W. C. Guida, R. Liskamp, M.
Lipton, C. Caufield, G. Chang, T. Hendrickson, W. C. Still, J.
Comput. Chem. 1990, 11, 440 – 467; b) W. C. Still, A. Tempczyk,
R. C. Hawley, T. Hendrickson, J. Am. Chem. Soc. 1990, 112,
6127 – 6129.
Scheme 6. Absolute configuration of thuggacins A–C( 1–3).
uration of thuggacin A. On the basis of a common biosyn-
thesis, and the observed rearrangement of thuggacin A (1)
into thuggacins B (2) and C (3), their configurations were
assigned accordingly.[2] This assumption is in full agreement
with the close similarities between the spectroscopic and
physical data of thuggacins A–C.[2]
In conclusion, the complete stereochemical assignment
for the antibiotic macrolides thuggacin A, B, and C has been
determined as 2E,7R,8S,10S,11E,13Z,16S,17S,18R,19S,20S,-
21E,23E on the basis of high-field NMR studies combined
with extensive chemical derivatization and molecular model-
ing. Final proof of the absolute configuration of five
stereogenic centers was collected from bioinformatic analysis
of the biosynthetic proteins for which the present work
provides one of the first examples.[12,13]
[8] Forcing a dihedral angle 15-Ha,C15,C16,16-H to be 150 – 1808 in
16-epi-10 is associated with a destabilization of about 10 kcal
molÀ1 and is not in agreement with the observed NMR data. (For
1
vicinal H coupling constants and NOE couplings, refer to the
Supporting Information.)
[9] a) J. A. Dale, H. S. Mosher, J. Am. Chem. Soc. 1968, 90, 3732 –
3738; b) J. A. Dale, D. L. Dull, H. S. Mosher, J. Org. Chem. 1969,
34, 2543 – 2449; c) J. M. Seco, E. Quinoa, R. Riguera, Chem. Rev.
2004, 104, 17 – 117.
[10] One by-product from the formation of the S ester was assigned
to be the thuggacin B derivative of diacetonide 6. The NMR data
of this acetonide were in agreement with the configuration
deduced from the initial acetalization studies (refer to the
Supporting Information).
[11] a) R. Reid, M. Piagentini, E. Rodriguez, G. Ashley, N. Viswa-
nathan, J. Carney, D. V. Santi, C. R. Hutchinson, R. McDaniel,
Biochemistry 2003, 42, 72 – 79; b) P. Caffrey, ChemBioChem
2003, 4, 654 – 657.
[12] a) D. Janssen, D. Albert, R. Jansen, R. Müller, M. Kalesse,
Angew. Chem. 2007, 119, 4985 – 4988; Angew. Chem. Int. Ed.
2007, 46, 4898 – 4901.
Received: October 22, 2007
Revised: November 26, 2007
Published online: February 14, 2008
[13] D. W. Udwary, L. Zeigler, R. N. Asolkar, V. Singan, A. Lapidus,
W. Fenical, P. R. Jensen, B. S. Moore, Proc. Natl. Acad. Sci. USA
2007, 104, 10376 – 10381.
[14] For analysis of the ketoreductase domains refer to the
Supporting Information.
Keywords: antibiotics · gene analysis · macrolactones ·
Mosher ester · structure elucidation
.
Angew. Chem. Int. Ed. 2008, 47, 2308 –2311
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
2311