Broad substrate specificity of the amide synthase in S. hygroscopicus -new 20-membered macrolactones derived from geldanamycin

Article abstract of DOI:10.1021/ja2087147

The amide synthase of the geldanamycin producer, Streptomyces hygroscopicus, shows a broader chemoselectivity than the corresponding amide synthase present in Actinosynnema pretiosum, the producer of the highly cytotoxic ansamycin antibiotics, the ansamit

Full text of DOI:10.1021/ja2087147

Article
pubs.acs.org/JACS
Broad Substrate Specificity of the Amide Synthase in S.
hygroscopicusNew 20-Membered Macrolactones Derived from
Geldanamycin
Simone Eichner,† Timo Eichner,‡ Heinz G. Floss,§ Jorg Fohrer,† Edgar Hofer,† Florenz Sasse,∥
̈
Carsten Zeilinger,⊥ and Andreas Kirschning*,†
†Institute of Organic Chemistry and Center of Biomolecular Research (BMWZ), Schneiderberg 1B, Leibniz University Hannover,
D-30167 Hannover, Germany
‡The Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, U.K.
§Department of Chemistry, University of Washington, Seattle, Washington 98195-1700, United States
∥Department of Chemical Biology, Helmholtz Center for Infection Research (HZI), Inhoffenstraße 7, D-38124 Braunschweig,
Germany
⊥Institute of Biophysics, Herrenhauser Straße 2, Leibniz University Hannover, 30419 Hannover, Germany
̈
S
* Supporting Information
ABSTRACT: The amide synthase of the geldanamycin
producer, Streptomyces hygroscopicus, shows a broader chemo-
selectivity than the corresponding amide synthase present in
Actinosynnema pretiosum, the producer of the highly cytotoxic
ansamycin antibiotics, the ansamitocins. This was demonstrated
when blocked mutants of both strains incapable of biosynthesiz-
ing 3-amino-5-hydroxybenzoic acid (AHBA), the polyketide
synthase starter unit of both natural products, were supple-
mented with 3-amino-5-hydroxymethylbenzoic acid instead.
Unlike the ansamitocin producer A. pretiosum, S. hygroscopicus
processed this modified starter unit not only to the expected 19-membered macrolactams but also to ring enlarged 20-membered
macrolactones. The former mutaproducts revealed the sequence of transformations catalyzed by the post-PKS tailoring enzymes
in geldanamycin biosynthesis. The unprecedented formation of the macrolactones together with molecular modeling studies shed
light on the mode of action of the amide synthase responsible for macrocyclization. Obviously, the 3-hydroxymethyl substituent
shows similar reactivity and accessibility toward C-1 of the seco-acid as the arylamino group, while phenolic hydroxyl groups lack
this propensity to act as nucleophiles in the macrocyclization. The promiscuity of the amide synthase of S. hygroscopicus was
further demonstrated by successful feeding of four other m-hydroxymethylbenzoic acids, leading to formation of the expected 20-
membered macrocycles. Good to moderate antiproliferative activities were encountered for three of the five new geldanamycin
derivatives, which matched well with a competition assay for Hsp90α.
quinone moiety contains a phenolic group at C-18.
Importantly, it shows lower cytotoxicity than geldanamycin
while having a higher affinity for Hsp90.5
INTRODUCTION
■
Geldanamycin 4 is a highly potent antitumor drug1 which binds
to the N-terminal ATP-binding domain of heat shock protein
90 (Hsp90) and inhibits its ATP-dependent chaperone
activities.2 It was demonstrated that geldanamycin could
prevent restructuring of mutated cancer-relevant proteins
such as p53.3 Most geldanamycin derivatives reported to date
are 17-aminated compounds such as 5a and 5b and were
obtained by semisynthesis while total synthesis has not
provided essential information for structure−activity relation-
ship studies (SAR).4 17-Dimethylaminoethylamino demethox-
ygeldanamycin hydrochloride (17-DMAG, 5a), also known as
alvespimicin, shows improved water solubility, undergoes only
limited metabolism compared to 4, and has reached clinical
trials as an anticancer agent against several tumors. Related to
geldanamycin (4) is reblastatin (6), which instead of the
The producing microorganism Streptomyces hygroscopicus var.
geldanus NRRL 3602 generates geldanamycin through a
biosynthetic machinery based on a polyketide synthase (PKS)
and additional post-PKS enzymes (Scheme 1), encoded by the
gdm biosynthetic gene cluster.6 The biosynthesis of 4 is primed
by the starter unit, 3-amino-5-hydroxybenzoic acid (AHBA, 1).6
Chain extension by the PKS using one malonate, four
methylmalonate, and two methoxymalonate extender units
gives seco-progeldanamycin (2), which is cyclized and released
from the PKS by an amide synthase, a key enzyme. The
Received: September 15, 2011
Published: December 5, 2011
© 2011 American Chemical Society
1673
dx.doi.org/10.1021/ja2087147 | J. Am. Chem.Soc. 2012, 134, 1673−1679

Journal of the American Chemical Society
Article
formed in sufficient amounts for full structural analysis and
preliminary biological evaluation. One aspect of the present
report covers the unprecedented observation that the
mutasynthon (hydroxymethyl)aminobenzoic acid (7) yields a
large variety of new geldanamycin derivatives when fed to
fermentations (1.6 L) of Streptomyces hygroscopicus (strain
K390-61-1). We first collected five new geldanamycin
derivatives 8−12 in amounts that allowed full characterization
(Scheme 2).
Scheme 1. Biosynthesis Starting from 3-Amino-5-
hydroxybenzoic Acid (AHBA, 1) of Geldanamycin (4) via
seco-(2) and Progeldanamycin (3)
Scheme 2. Mutasynthesis with S. hygroscopicus K390-61-1
Using 3-Aminobenzoic Acid (7) and Our Proposed
Sequence of Postketide Transformations
resulting progeldanamycin 3 is then further modified by a set of
tailoring enzymes.
It was proposed that this sequence starts with the oxidation
of C-21 and C-17, followed by O-methylation at C-17 and
introduction of the carbamoyl moiety, and finalized by
dehydrogenation across C-4,5.7a,b The oxidation of the
hydroquinone moiety to the quinone only takes place after
oxidation at C-21.7c Surprisingly, it still remains unclear at
which stage during the biosynthesis bioactive geldanamycin
analogues are formed or what the simplest biosynthetic
intermediate is that inhibits Hsp90 activity.
The formation of this set of modified advanced biosynthetic
intermediates is particularly interesting from two aspects: (a)
The new products can be arranged in a linear biosynthetic
sequence of postketide transformations which slightly differs
from the one proposed in the literature7 and (b) SAR studies
can be conducted on structurally closely related derivatives
along a postketide biosynthetic sequence.
With respect to point (a), we propose that the post-PKS
modifications are initiated by carbamoylation at C-7 to yield
mutaproduct 8,14 which is then oxidized at C-17 to yield phenol
9, followed by dehydrogenation across C-4,5, which furnishes
geldanamycin derivative 10. The next post-PKS transformation
is the O-methylation at C-17 to yield benzyl alcohol 11. Finally,
we also isolated the fully substituted mutaproduct 12, which is
formed upon oxidation at C-21 of 11. This sequence of events
differs from the proposed one,7 which postulates that
carbamoylation takes place at a later stage.
Remarkably, oxidation at C-21 takes place despite the fact
that the para position is hydroxymethylated instead of being
functionalized with a phenolic moiety. It is noteworthy that
benzoic acid 7 is the first mutasynthon that is fully processed by
the geldanamycin producer S. hygroscopicus, except for the
oxidation of the aromatic ring to the quinone form. Up to the
present study, our mutasynthetic experiments had only afforded
new geldanamycin derivatives that are processed to the level of
functionalization found in derivative 10. Lee, Hong, and co-
workers9 as well as Menzella et al.10 disclosed related studies
and also only described new geldanamycin mutaproducts that
were incompletely processed.
We and two other groups8−10 have utilized mutants of the
geldanamycin producer blocked in AHBA formation [AHBA-
(−) mutants] to prepare several new geldanamycin derivatives
in a mutasynthetic approach,11,12 a synthetic method that is
inspired by the detailed biosynthetic knowledge of geldanamy-
cin formation.7,13
In this report, we disclose the first mutasynthetic preparation
of a metabolic series of geldanamycin derivatives using an
AHBA-(−) mutant of S. hygroscopicus (strain K390-61-1),
which reveals the order of the post-PKS modifications in
geldanamycin biosynthesis and sheds light on structure−activity
relationships among the post-PKS modification products. We
also disclose the unprecedented finding that ring-enlarged
macrolactones instead of macrolactams can be generated by the
amide synthase present in S. hygroscopicus. We finally discuss
the possible molecular basis of this unusual behavior of the
amide synthase involved.
RESULTS AND DISCUSSION
■
Feeding Experiments with Mutasynthon 7 and
Biosynthetic Consequences. In our previous mutasynthetic
studies, we found that when feeding aminobenzoic acid
analogues to a growing culture of an AHBA-(−) mutant of
Streptomyces hygroscopicus, only one or two mutaproducts were
1674
dx.doi.org/10.1021/ja2087147 | J. Am. Chem.Soc. 2012, 134, 1673−1679

Journal of the American Chemical Society
Article
However, the most striking result of these feeding studies is
the formation of the additional 20-membered macrolactone 13
and its metabolites 14 and 15 (Figure 1). Structural proof for
Scheme 3. Macrolactamization of seco-Proansamitocin 16a
and Derivative 16b to Proansamitocin 17a and Derivative
17b, Respectively, by the Amide Synthase (Asm9) from
Actinosynnema pretiosum (for X Refer to the Legend of
Figure 1)15
Asm9 revealed 42% identity and 58% similarity when
accounting for conservative mutations.
Therefore, any of a large number of residues (105) could
potentially be responsible for the different specificity of GdmF
compared to Asm9 when handling the aromatic hydroxymethyl
group in 7 (Figure 2A).18
Figure 1. New 20-membered macrolactones 13−15 and precursors A/
B responsible for macrolactamization and macrolactonization,
respectively (X; The nature of the leaving group at C-1 is unknown,
so that X principally could be −OH, −SCoA, or −SPKS, with the
latter two being the more likely cases).15
the formation of these macrolactones which are expanded in
ring size compared to 4 was gained from HRMS spectrometric
1
data and H NMR studies. The benzyl ether protons at C-22
undergo a downfield shift (δ = 5.07 and 4.95 ppm for 13
compared to δ = 4.57 ppm for 8) which is diagnostic for
acylated benzyl alcohols. The macrolactone formation was also
confirmed by long-range proton carbon couplings in HMBC
spectra. The additionally isolated compounds 14 and 15 result
from further metabolism of 13 by, respectively, nonspecific N-
acetylation, a common biotransformation in Actinomycetes,
and carbamoylation, presumably catalyzed by the carbamoyl-
transferase GdmN of the gdm gene cluster.7a
Obviously, the amide synthase, that is responsible for the
macrolactamization, is able to recognize and utilize alcohols for
cyclization as long as they are sufficiently nucleophilic. This is
not the case for the phenolic moiety in seco-progeldanamycin 2,
but in marked contrast, it is for its hydroxymethyl derivative A/
B.
Figure 2. (A) Sequence alignment of GdmF and Asm9 using web-
based resources (http://blast.ncbi.nlm.nih.gov). Amino acids in bold
indicate a perfect match. Yellow ground displays the catalytic triad:
Cys72, His110, and Asp125. Green ground, Pro/Ala129 (for GdmF
and Asm9, respectively). (B) Surface representations of the homology
model of GdmF using SWISS-MODEL.18−20 Note the tight channel
leading to the catalytic triad (Cys72, His110, and Asp125 are colored;
compare to C and D). (C, D) Zoom-in of Figure 2B showing the
catalytic triad and Pro129.
Molecular Modeling of Amide Synthases. Studies on
the formation of ring-enlarged macrolactones reveal that the
amide synthase GdmF carries a non-native functional group
specificity when dealing with seco acids as substrate. In the
non-natural seco-progeldanamycin A/B, the benzylic alcohol
serves as a potential nucleophile for cyclization to about the
same extent as the naturally occurring aryl amine. So far, our
mutasynthetic experiments with the AHBA-(−) mutants of S.
hygroscopicus and A. pretiosum, the ansamitocin producer,16,17
unequivocally demonstrated that both amide synthases
involved in macrolactamization accept a wide range of seco
acids modified in the aromatic moiety.5 But when feeding
aminobenzoic acid 7 to a AHBA-(−) blocked mutant of A.
pretiosum in our previous studies, we had only encountered
formation of lactams and never lactones, when screening the
fermentation broth by UPLC HR-MS.16 Thus, the amide
synthase Asm9 catalyzing the cyclization of seco-ansamitocin
16a to proansamitocin 17a (Scheme 3) shows clearly a
narrower reaction specificity compared to that of GdmF. This
specificity difference led us to analyze the specific amino acid
composition at and around the catalytic site of the two amide
synthases. An initial sequence alignment between GdmF and
For the amide synthase to perform the cyclization of seco-
progeldanamycin 2 or the corresponding hydroxymethyl
analogue, both ends of the PKS-bound polyketide have to
access the catalytic pocket containing the catalytic triad, Cys72,
His110, and Asp125 (Figure 2B−D). Hydrogen-bonding
between those three residues enhances the nucleophilic
power (partial charge) of the Cys72 sulfur atom, enabling it,
as the first reaction step, to covalently link to C-1 of an
incoming precursor molecule via nucleophilic attack, displacing
it from the PKS. In the second reaction step, the aromatic ring
enters the catalytic pocket through a tight channel. Once the
aromatic ring is in close proximity to C-1 (covalently bound to
Cys72), one of the two functional groups present (−NH2 vs
−OH or −NH2 vs −CH2OH) at the aromatic ring can
1675
dx.doi.org/10.1021/ja2087147 | J. Am. Chem.Soc. 2012, 134, 1673−1679

Journal of the American Chemical Society
Article
nucleophilically attack C-1. Cys72 then serves as a leaving
group while the macrocycle is formed.
Restrained molecular dynamics (MD) simulations on a
nanosecond time scale using explicit solvent allowed us to
probe qualitatively the conformational space available to the
aromatic ring while penetrating the catalytic pocket and thus
the distance of the nucleophilic groups −NH2 vs −OH/−
CH2OH, respectively, relative to C-1 (Figure 318). The
Figure 4. Restrained MD simulations of the aromatic ring while
penetrating the catalytic site of GdmF (and its variant P129A) using a
distance restraint (5 Å) with linearly increasing energy penalty
between C-1 and C-19, with the latter being located halfway between
the nucleophilic groups (−NH2, −OH, or −CH2OH) decorating the
aromatic ring (Figure 3). Using this approach, we were able to
qualitatively probe the conformational space available to the aromatic
ring and thus the distance of the nucleophilic groups to C-1. The
dashed red line indicates close proximity of the NH2 nucleophile to C-
1 after penetration, as observed for the natural ligand seco-
progeldanmycin (in black). In contrast, the approach of the NH2
nucleophile of the hydroxymethyl derivative of seco-geldanamycin
toward C-1 appears hindered (in blue), which might explain the
observed slower lactam cyclization rates for this non-natural substrate.
In fact, detailed analyses of the dynamic trajectories imply that the
naturally occurring compound is still mobile within the catalytic
pocket of GdmF, while the hydroxymethyl derivative shows static
properties (compare Figure 4A and B and data not shown). The static
ligand properties can be overcome once proline 129 is mutated to an
alanine (P129A, in green), rationalizing the observed turnover
specificity for Asm9 (compare Figure 4B and 4C). Note that P129A
mimics the catalytic pocket of Asm9 (see Figures 2 and 3).
Figure 3. Starting and end trajectory after 25 ns of the restrained MD
simulations of GdmF covalently linked to seco-progeldanamycin (top
panel) and its hydroxymethyl derivative (bottom panel). The distance
restraint (5 Å) between C-1 and C-19 contained a linearly increasing
energy penalty during the simulation. Note that, for the
hydroxymethyl derivative, the aromatic ring moves out of plane in
order to avoid clashes with P129.
dynamic trajectories obtained imply that the natural substrate
2 has some degree of mobility within the catalytic pocket, while
the hydroxymethyl derivative is more statically trapped. Further
detailed analysis of the trajectories pointed toward Pro129
being a key residue for the specific macrocyclization of seco-
progeldanamycins via the amino group by introducing stiffness
inside the catalytic pocket of GdmF (Figure 3). Intriguingly, the
sequence alignment of GdmF and Asm9 shows that the latter
amide synthase contains Ala129 instead of proline, which could
explain the different substrate specificity of this enzyme.
Indeed, our modeling indicates that a proline-to-alanine
mutation at position 129 in GdmF (which mimics the catalytic
pocket of Asm9) changes the dynamic penetration trajectory
for the hydroxymethyl derivative of seco-geldanamycin
substantially (see Figures 3 and 4).18
It needs to be stressed that proline residues are the most rigid
amino acids due to ϕ and ψ torsion angle restrictions in the
protein backbone. Thus, our results suggest that catalysis of
seco-progeldanamycin 2 cyclization via the amino group by
GdmF is fast due to smooth penetration into the catalytic
channel as well as the ideal distance and flexibility of the amino
group toward C-1. In contrast, several factors, such as
penetration hindrance, less mobility, and a nonideal distance
of the amino group with respect to C-1 for the hydroxymethyl
derivative of seco-progeldanamycin, all reduce the efficiency of
the nucleophilic attack dramatically. In addition to these
bioinformatic considerations, the differences in nucleophilicity
of the three different relevant functional groups can also
account for the observed reactivity pattern, as it is well
established that anilines and benzyl alcohols are more reactive
toward electrophiles than phenols.21 However, further
computational and biochemical experimental data are needed
to fully understand the molecular mechanisms underlying
catalysis of macrocyclization of these enzymes.
Formation of Other Macrolactones. These unprece-
dented results along with our preliminary computational
analyses paved the way to the design of alternative benzoic
acids 18, 20, 22, and 24 that upon feeding to S. hygroscopicus
(strain K390-61-1) under the typical fermentation conditions
were expected to be transformed into lactones. In fact, the
expected lactones 19, 21, 23, and 25 were isolated and
characterized (Scheme 4). Again, post-PKS carbamoylation
occurred in all cases. Notably, benzyl amine 26 was also
processed to the corresponding 20-membered macrolactam 27,
as judged by HRMS-MS analysis.22 In contrast, the ortho- and
para-substituted benzoic acids 29 and 30, regioisomers of the
meta-substituted mutasynthon 24, did not serve as substrates
1676
dx.doi.org/10.1021/ja2087147 | J. Am. Chem.Soc. 2012, 134, 1673−1679

Journal of the American Chemical Society
Article
Scheme 4. New 20-Membered Geldanamycin-Derived
Macrolactones 19, 21, 23, and 25 (Isolated Yields) and 20-
Membered Macrolactam 30
Table 1. Antiproliferative Activity (IC50 [ng/mL]),
Compared to Geldanamycin 4, of New Geldanamycin
Derivatives 8−12 against Different Cell Lines: A Permanent
Mouse Line, Four Human Cancer-Derived Lines, and a
Primary HUVEC Line (Values Shown Are Means of Two
Parallel Determinations)
cell line
L-929
mouse
cell line
KB-3-1
cervix
carc
PC-3
prostate
carc
SK-OV-3
ovarian
carc
A-431
epider-
moid carc
HUVEC
primary
cells
4
5.0
470
2.7
22
21
130
4.1
60
2.0
30
2.1
38
8
9
3200
>4000
880
210
640
210
75
600
380
270
210
840
190
95
10
11
12
2800
440
1900
280
1300
240
980
400
170
240
Figure 5. Dose−response curve for the binding of 10 nM
geldanamycin−FITC conjugate to human Hsp90α. Different amounts
of purified protein were incubated with geldanamycin−FITC at 4 °C,
and fluorescence polarization was measured after 16 h. Data were
imported into Origin 6.0 to determine the dissociation constant from a
dose−response curve as a function of the Hsp90α concentration.22
The tracer had a Kd of 2.3 nM for HSP90α. The assay was performed
under conditions at or above 130 mP (inset: purified full length
Hsp90α).
for the biosynthetic assembly line of the geldanamycin
producer S. hygroscopicus (Figure 3). Obviously, only the
meta architecture provides a geometric environment for
successful PKS-loading and PKS-processing of unnatural starter
units. One cannot exclude the possibility that charging onto the
loading module does happen but that further processing is
inefficient or is blocked. However, we were never able to detect
any oligoketide intermediates in cases of unsuccessful
mutasyntheses.
The modeling analyses and the feeding experiments are also
in line with the following observation. Benzoic acid 28
containing two meta-oriented phenolic hydroxyl groups does
not act as a substrate for mutasynthetic formation of
macrocyclized geldanamycin derivatives. This would require
that one of the two phenolic groups has to act as nucleophile
during amide synthase-catalyzed macrocyclization, but ring
closure via a phenolic moiety has never been observed.
Biological Evaluation. The new geldanamycin derivatives
8−12 were tested for their inhibitory effects on the
proliferation of different mammalian cell lines. The cell lines
tested are cancer derived, permanentbut not cancer derived,
and primary cells. In general, all derivatives show a lower
activity than geldanamycin 4, with the least active derivative
being 10 and the most active one 8, but still less active than 4
by a factor of 10−100.
Metabolites 9, 11, and 12 show medium antiproliferative
activity. Clearly, the primary human umbilical vein endothelial
cells (HUVEC) and the cancer derived KB-3-1 cells are the
most sensitive to the geldanamycin derivatives while the
permanent mouse cell line L-929 is 5−15 times more resistant.
Surprisingly, there is no positive correlation between biological
activity and the progress of the biosynthesis. Decrease of
activity with each post-PKS transformation is particularly
striking for the desaturation step from metabolite 9 to 10,
which results in a basically inactive geldanamycin analogue. The
relative antiproliferative activities of mutaproducts 8−12
(Table 1) are unexpected because not each successive post-
PKS modification leads to metabolites with greater activity.
Starting from carbamoylated progeldanamycin 8, continuous
decrease up to derivative 10 is observed before antiproliferative
activity is partly restored in fully substituted homogeldanamy-
cin derivative 12.
1677
dx.doi.org/10.1021/ja2087147 | J. Am. Chem.Soc. 2012, 134, 1673−1679

Journal of the American Chemical Society
Article
Table 2. Competition Assay of the Geldanamycin−FITC Conjugate (10 nM) Bound to Hsp90α (50 nM) with Geldanamycin
a
Derivatives 5b and 8−12
geldanamycin derivative
GM-FICT
2.3 0.1
5b
8
9
10
11
19
12
24
fluorescence polarization (nM)
8
6
42
7
95
8
256 84
4
3
a
The IC50 values obtained from dose-response fitting curves as a function of the competitor concentration are given in the table.18.
To verify these unexpected in vivo results, we decided to
ASSOCIATED CONTENT
■
conduct an in vitro competition assay which utilizes
recombinant, purified full length human heatshock protein
Hsp90α. The assay relies on the competition of fluorescently
(fluorescein isothiocyanate (FITC)) labeled geldanamycin
bound to Hsp90α with new geldanamycin derivatives. The
geldanamycin−FITC conjugate was first incubated at different
concentrations with Hsp90α (Figure 5). Half saturation of
binding was achieved at 2.3 nM, as judged from the
fluorescence polarization signal for bound geldanamycin−
FITC. This value compares favorably to the sensitivity observed
by Kim et al. for a similar geldanamycin conjugate (6.6 nM).
This group had developed such an assay for high-throughput
screening in search of novel Hsp90 inhibitors.23
The dose−response activities of geldanamycin derivatives 5b
and 8−12 were tested in the presence of Hsp90α loaded with
the geldanamycin−FITC conjugate (Table 2).18 The data
favorably compare with the in vivo data collected and listed in
Table 1, with geldanamycin derivative 10 being the least active
new metabolite, and indicate that this binding/competition
assay well complements our in vivo results.
S
* Supporting Information
Details on the synthesis of benzoic acids and copies of 1H-, 13
C
NMR and MS spectra of mutaproducts as well as details on the
assay and modeling studies. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
Andreas.kirschning@oci.uni-hannover.de
ACKNOWLEDGMENTS
■
We thank the U.S. National Institutes of Health (Grant CA
76461) and the Deutsche Forschungsgemeinschaft (Grant Ki
397 / 13-1) and the Fonds der Chemischen Industrie. We
thank Kosan Biosciences Incorporated for providing us with
strain S. hygroscopicus (K390-61-1) and Wera Collisi (HZI)
for performing the cell proliferation assays. We are grateful to
David Agard (UCSF, USA) for a human HSP90α clone. We are
grateful to Gerald Drager for expert support in mass
̈
spectrometry analyses. We thank the reviewers for helpful
suggestions.
In contrast to the cases of the geldanamycin derivatives, the
ring enlarged 20-membered lactones 13−15 and 19, 21, and 23
did not show antiproliferative activity or Hsp90 binding affinity.
REFERENCES
■
CONCLUSIONS
(1) (a) Workman, P. Curr. Cancer Drug Targets 2003, 3, 297−300.
(b) Neckers, L.; Neckers, K. Expert Opin. Emerging Drugs 2005, 10,
137−149. (c) Whitesell, L.; Lindquist, S. L. Nat. Rev. Cancer 2005, 5,
761−772. (d) Prodromou, C.; Roe, S. M.; O’Brien, R.; Ladbury, J. E.;
Piper, P. W.; Pearl, L. H. Cell 1997, 90, 65−75.
■
In summary, mutasynthetic feeding studies with the AHBA-(−)
mutant of the geldanamycin producer S. hygroscopicus using 3-
amino-5-hydroxymethylbenzoic acid 7 provided five new
geldanamycin derivatives differing in the degree of post-PKS
modification. This mutasynthetic experiment allowed us to
propose the sequence of post-PKS transformations by the
tailoring enzymes in geldanamcin biosynthesis. The second set
of mutaproducts, three 20-membered macrolactones, revealed
an unexpected and unprecedented chemoselectivity of the
amide synthase responsible for macrocyclization, which is not
shown by the corresponding amide synthase operating in
ansamitocin biosynthesis.15,16 This substrate flexibility with
respect to the aryl moiety of the bound nucleophile could be
extended using other m-hydroxymethylbenzoic acid starter
units. Finally, new geldanamycin derivatives 8, 11, and 12 show
good to moderate antiproliferative activities on different cell
lines while partially post-PKS processed mutaproducts 9 and 10
have lost most of this biological property. The active
mutaproducts, as the parent natural product geldanamycin 4,
act on Hsp90.
(2) Review: Biamonte, M. A.; van de Water, R.; Arndt, J. W.;
Scannevin, R. H.; Perret, D.; Lee, W.-C. J. Med. Chem. 2010, 53, 3−17.
(3) Blagosklonny, M. V.; Toretsky, J.; Neckers, L. Oncogene 1995, 11,
933−939.
(4) Janin, L. J. Med. Chem. 2005, 48, 7503−7512.
(5) (a) Stead, P.; Latif, S.; Blackaby, A. P.; Sidebottom, P. J.; Deakin,
A.; Taylor, N. L.; Life, P.; Spaull, J.; Burrell, F.; Jones, R.; Lewis, J.;
Davidson, I.; Mander, T. J. Antibiot. 2000, 53, 657−663. (b) Takatsu,
T.; Ohtsuki, M.; Muramatsu, A.; Enokita, R.; Kurakata, S. I. J. Antibiot.
2000, 53, 1310−1312.
(6) (a) Kim, C.-G.; Yu, T. W.; Fryhle, C. B.; Handa, S.; Floss, H. G. J.
Biol. Chem. 1998, 273, 6030−6040. (b) Arakawa, K.; Muller, R.;
̈
Mahmud, T.; Yu, T.-W.; Floss, H. G. J. Am. Chem. Soc. 2002, 124,
10644−10645.
(7) (a) Rascher, A.; Hu, Z.; Viswanathan, N.; Schirmer, A.; Reid, R.;
Nierman, W. C.; Lewis, M.; Hutchinson, C. R. FEMS Microbiol. Lett.
2003, 218, 223−230. (b) Hong, Y.-S.; Lee, D.; Kim, W.; Jeong, J. K.;
Kim, C. G.; Sohng, J. K.; Lee, J. H.; Paik, S. G.; Lee, J. J. J. Am. Chem.
Soc. 2004, 126, 11142−11143. (c) Lee, D.; Lee, K.; Cai, X. F.; Dat, N.
T.; Boovanahalli, S. K.; Lee, M.; Shin, J. C.; Kim, W.; Jeong, J. K.; Lee,
J. S.; Lee, C. H.; Lee, J. H.; Hong, Y.-S.; Lee, J. J. ChemBioChem 2006,
7, 246−248.
To the best of our knowledge we describe here the first
mutasynthetic experiment that yields macrocyclic metabolites
with an altered type of ring closure and ring size.24 These
results pave the way to utilize the amide synthase of S.
hygroscopicus, after overexpression and mutagenetic optimiza-
tion, as a macrocylization tool of chemically broader substrate
flexibility.
(8) Eichner, S.; Floss, H. G.; Sasse, F.; Kirschning, A. ChemBioChem
2009, 10, 1801−1805.
(9) (a) Kim, W.; Lee, J. S.; Lee, D.; Cai, X. F.; Shin, J. C.; Lee, K.;
Lee, C.-H.; Ryu, S.; Paik, S.-G.; Lee, J. J.; Hong, Y.-S. ChemBioChem
2007, 8, 1491−1494. (b) Kim, W.; Lee, D.; Hong, S. S.; Na, Z.; Shin, J.
1678
dx.doi.org/10.1021/ja2087147 | J. Am. Chem.Soc. 2012, 134, 1673−1679

Journal of the American Chemical Society
Article
C.; Roh, S. H.; Wu, C.-Z.; Choi, O.; Lee, K.; Shen, Y.-M.; Paik, S.-G.;
Lee, J. J.; Hong, Y.-S. ChemBioChem 2009, 10, 1243−1251.
(10) (a) PCT Int. Appl. (2008) 53 pp, Pub. No.: U.S. 2008/0188450
A1. (b) Menzella, H. G.; Tran, T.-T.; Carney, J. R.; Lau-Wee, J.;
Galazzo, J.; Reeves, C. D.; Carreras, C.; Mukadam, S.; Eng, S.; Zhong,
Z.; Timmermans, P. B. M. W. M.; Murli, S.; Ashley, G. W. J. Med.
Chem. 2009, 52, 1518−1521.
(11) (a) Shier, W. T.; Rinehart, K. L. Jr.; Gottlieb, D. Proc. Natl. Acad.
Sci. U.S.A. 1969, 63, 198−204. (b) Rinehart, L. Jr. Jpn. J. Antibiot. 1979,
32 (Suppl.), S32−46.
(12) Reviews: (a) Weist, S.; Sussmuth, R. D. Appl. Microbiol.
̈
Biotechnol. 2005, 68, 141−150. (b) Kirschning, A.; Taft, F.; Knobloch,
T. Org. Biomol. Chem. 2007, 3245−3259.
(13) Rascher, A.; Hu, Z.; Buchanan, G. O.; Reid, R.; Hutchinson, C.
R. Appl. Environ. Microbiol. 2005, 71, 4862−4871.
(14) The carbamoyltransferase shows fairly broad promiscuity. Thus,
it cannot be excluded that amidination takes place on any of the post-
PKS intermediates, and therefore, it is not necessarily the first post-
PKS modification.
(15) Harmrolfs, K.; Brunjes, M.; Drager, G.; Floss, H. G.; Sasse, F.;
̈
̈
Taft, F.; Kirschning, A. ChemBioChem 2010, 11, 2517−2520.
(16) Knobloch, T.; Harmrolfs, K.; Taft, F.; Thomaszewski, B.; Sasse,
F.; Kirschning, A. ChemBioChem 2011, 12, 540−547.
(17) (a) Taft, F.; Brunjes, M.; Knobloch, T.; Floss, H. G.; Kirschning,
̈
A. J. Am. Chem. Soc. 2009, 131, 3812−3813. (b) Taft, F.; Brunjes, M.;
̈
Floss, H. G.; Czempinski, N.; Grond, S.; Sasse, F.; Kirschning, A.
ChemBioChem 2008, 7, 1057−1060. (c) Kubota, T.; Brunjes, M.;
̈
Frenzel, T.; Xu, J.; Kirschning, A.; Floss, H. G. ChemBioChem 2006, 7,
1221−1225.
(18) For details see the Supporting Information.
(19) Arnold, K.; Bordoli, L.; Kopp, J.; Schwede, T. Bioinformatics
2006, 22, 195−201.
(20) (a) Schwede, T.; Kopp, J.; Guex, N.; Peitsch, M. C. Nucleic Acids
Res. 2003, 31, 3381−3385. (b) Guex, N.; Peitsch, M. C. Electrophoresis
1997, 18, 2714−2723.
́
(21) Perez, P.; Domingo, L. R.; Duque-Norena, M.; Chamorro, E.
̃
THEOCHEM 2009, 895, 86−91.
(22) The production was too small to isolate an amount of 27
sufficient for complete NMR assignment.
(23) Kim, J.; Felts, S.; Llauger, L.; He, H.; Huezo, H.; Rosen, N.;
Chiosis, G. J. Biomol. Screen 2004, 9, 375−381.
(24) Ring expanded 16-membered macrolactones derived from 6-
deoxyerythronolide B were reported by Cane and Koshla when
feeding a triketide to a blocked mutant of S. coelicolor. However, the
type of ring closure was notaltered: Kinoshita, K.; Williard, P. G.;
Khosla, C.; Cane, D. E. J. Am. Chem. Soc. 2001, 123, 2495−2502.
1679
dx.doi.org/10.1021/ja2087147 | J. Am. Chem.Soc. 2012, 134, 1673−1679