New geldanamycin derivatives with anti Hsp properties by mutasynthesis

Article abstract of DOI:10.1039/c9ob00892f

Mutasynthetic supplementation of the AHBA blocked mutant strain of S. hygroscopicus, the geldanamycin producer, with 21 aromatic and heteroaromatic amino acids provided new nonquinoid geldanamycin derivatives. Large scale (5 L) fermentation provided four new derivatives in sufficient quantity for full structural characterisation. Among these, the first thiophene derivative of reblastatin showed strong antiproliferative activity towards several human cancer cell lines. Additionally, inhibitory effects on human heat shock protein Hsp90α and bacterial heat shock protein from H. pylori HpHtpG were observed, revealing strong displacement properties for labelled ATP and demonstrating that the ATP-binding site of Hsps is the target site for the new geldanamycin derivatives.

Full text of DOI:10.1039/c9ob00892f

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New geldanamycin derivatives with anti Hsp
properties by mutasynthesis†
Cite this: DOI: 10.1039/c9ob00892f
Jekaterina Hermane,‡a Simone Eichner,‡a Lena Mancuso,a Benjamin Schröder,‡a
a
Florenz Sasse, b Carsten Zeilingerc and Andreas Kirschning
*
Mutasynthetic supplementation of the AHBA blocked mutant strain of S. hygroscopicus, the geldanamycin
producer, with 21 aromatic and heteroaromatic amino acids provided new nonquinoid geldanamycin
derivatives. Large scale (5 L) fermentation provided four new derivatives in sufficient quantity for full struc-
tural characterisation. Among these, the first thiophene derivative of reblastatin showed strong anti-
proliferative activity towards several human cancer cell lines. Additionally, inhibitory effects on human
heat shock protein Hsp90α and bacterial heat shock protein from H. pylori HpHtpG were observed,
revealing strong displacement properties for labelled ATP and demonstrating that the ATP-binding site of
Hsps is the target site for the new geldanamycin derivatives.
Received 17th April 2019,
Accepted 2nd May 2019
DOI: 10.1039/c9ob00892f
rsc.li/obc
lysis with high levels of efficiency, selectivity and a broad sub-
strate tolerance when presented with non-natural precursors.
Introduction
Besides classical concepts of natural product synthesis,
Ansamycin antibiotics, specifically the ansamitocins,
namely total- and semisynthesis, strategies that rely on the rifamycin and geldanamycin, have been a prime target to create
manipulation of microbial secondary metabolites not after, natural product libraries through mutasynthesis.5 Geldanamycin
but in the course of their biosynthesis, have broadened the (3; hydroquinone form in Scheme 1), produced by Streptomyces
synthetic portfolio of chemists and biotechnologists. One strat-
egy employs mutant organisms blocked in the biosynthesis of
endogenous key precursors on a genetic level. According to
Rinehart,1 mutational biosynthesis or mutasynthesis2 aims to
produce genetic mutants of an organism in which the for-
mation of key biosynthetic building blocks required for the
assembly of natural metabolites are blocked.3 In turn, admin-
istration of structurally modified building blocks, so-called
mutasynthons, to the blocked mutant may result in their
incorporation and lead to the synthesis of novel metabolites,
which can be isolated and evaluated for their therapeutic
potential.4 In principle, mutasynthesis allows access to com-
pound libraries of pharmacologically relevant and complex
novel products. While it is simple to produce gene knock-outs
that are useful in the creation of blocked deletion mutants, it
is critical that the modified organism performs enzymatic cata-
aInstitute of Organic Chemistry, Leibniz Universität Hannover, Schneiderberg 1B,
30167 Hannover, Germany. E-mail: andreas.kirschning@oci.uni-hannover.de
bHelmholtz-Center for Infection Research, Department of Microbial Communication,
Inhoffenstr. 7, 38124 Braunschweig, Germany
cLeibniz Universität Hannover, Institute of Biophysics and Center of Biomolecular
Drug Research (BMWZ), Schneiderberg 38, D-30167 Hannover, Germany
†Electronic supplementary information (ESI) available: Syntheses of new amino
acid precursors and analytical as well as spectroscopic data including copies of
NMR spectra of new reblastatin derivatives. See DOI: 10.1039/c9ob00892f
‡These authors contributed equally to the work.
Scheme 1 Key features of geldanamycin biosynthesis.8,9
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hygroscopicus, is a potential antitumor drug6 that binds to
the N-terminal ATP-binding domain of heat shock protein 90
(Hsp90) and inhibits its ATP-dependent chaperone activity.7
Heat shock proteins are essential for survival because they
regulate a diverse set of cellular processes and consequently,
geldanamycin has been a lead structure for at least two clinical
candidates against cancer.
Fig. 1 Aromatic mutasynthons 5–9 successfully transformed into new
reblastatin derivatives in preparative amounts.
However, activity of the benzoquinone form of geldan-
amycin first depends on reductive activation to hydroquinone 3
by the enzyme NAD(P)H/quinone oxidoreductase 1 (NQO1).7,10–12
Thus, it is the hydroquinone itself that is regarded to be the
motif, that binds to the ATP binding domain. The benzo-
quinone-based geldanamycins are far from being ideal antitu-
mor drugs, because they show hepatotoxic side effects, in part,
because of their Michael-acceptor properties toward natural
thiols such as glutathione.12,13
Herein, we provide a full evaluation of substrate tolerance
for an AHBA blocked mutant strain of S. hygroscopicus using a
wide variety of aromatic and heteroaromatic amino acids as
mutasynthons, and subsequently report the preparation of
new reblastatin derivatives by mutasynthesis. Antiproliferative
properties of the new metabolites, which were available in
sufficient amounts, are compared with their inhibitory effects
on the ATP binding domain in human and bacterial heat
shock proteins using a microarray competition assay.20
Results and discussion
Mutasynthetic experiments (preliminary screening)
5-Substituted 3-aminobenzoic acids 6 and 7 were found to be
Reblastatin 4, a phenolic analogue of geldanamycin, shows the most promising candidates for accessing new reblastatin
similar Hsp90 inhibitory effects and antiproliferative activity. derivatives using mutasynthesis.18a We extended the scope
Beneficially, it lacks the redox-sensitive quinone or hydro- of substrates to 5-substituted-3-aminobenzoic acids 10–15
quinone moiety14 and such macrolactam derivatives have (Table 1)21 that included halo-substitution (10 and 11) as well
emerged as promising alternatives in the development of as C-substitution (12–15) Typically, the free acids can be
cancer chemotherapies.15 The biosynthesis of geldanamycin administered as they are activated to the corresponding
(3)8 begins with 3-amino-5-hydroxybenzoic acid (AHBA 1), adenylates in Streptomyces hygroscopicus. Next, these are
which is derived from D-glucose through a modified shikimate attacked by phosphopantheteine-thiol to form the PKS-bound
pathway. Loading onto the starter module and processing thioesters. Preliminary experiments were performed on a small
through seven PKS modules yields seco-progeldanamycin, scale (0.038 mmol mutasynthon, 30 mL fermentation broth, 7
which is subsequently cyclised and released from the PKS by d). This set of mutasynthetic experiments revealed that carba-
an amide synthase. The resulting progeldanamycin (2) is then moylation always takes place, and in selected cases hydroxy-
transformed to geldanamycin by a set of tailoring enzymes lation at C17 as tailoring modifications can occur. In our
that induce amidination, several oxidation reactions of the aro- hands, desaturation at C4,C5 is a rarely observed transform-
matic moiety, O-methylation and desaturation at C4,C5.8,9
ation when feeding unnatural aminobenzoic acids and for the
As mentioned previously, mutasynthesis has been success- new examples reported here, it was not observed at all. Amide
fully employed to access new geldanamycin analogues, particu- 12 was not processed as judged by UPLC-HRMS analysis but
larly by the groups of Lee, Hong,16,17 by Menzella13 and the functionally related nitrile 13 provided one macrolactam
through our own efforts.9,18 All published mutasyntheses of metabolite after feeding to the AHBA(−) mutant strain of
new phenolic derivatives of geldanamycin, which we call S. hygroscopicus. Other C-substituents such as the vinyl group
reblastatin derivatives in this work as they commonly lack the present in benzoic acid 14 or the cyclopropyl group as in 15
double bond at C4–C5 or a quinone/hydroquinone moiety, rely were also converted into the amidinated macrolactams. In
on the disruption of genes coding for AHBA formation and several cases, we detected the presence of seco acids (for muta-
feeding of unnatural 3-aminobenzoic acid derivatives. In our synthons 10, 11 and 15) and in one case also the “seco amide”
preliminary studies, we demonstrated that aromatic mutasyn- (for mutasynthon 15). The “seco amide” is the amide deriva-
thons 5–9 are successfully processed to new reblastatin deriva- tive of the corresponding seco acid, which cannot serve as sub-
tives (Fig. 1). These results showed that modifications at posi- strate for macrolactamisation anymore.
tions 4–6 (AHBA numbering),19 as well as heteroaromatic
mutasynthons, are in principle tolerated by the biosynthetic tuted aminobenzoic acids 16–20 in which the third and in
The second series of experiments utilised diversely substi-
assembly line, although the scope is still unknown.
some cases a fourth substituent are located at positions 4 and
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Table 1 Test mutasyntheses with (−)-AHBA blocked mutant of
S. hygroscopicus using 5-substituted-3-aminobenzoic acids 10–15
Table 2 Test mutasyntheses with (−)-AHBA blocked mutant of
S. hygroscopicus using diversely substituted 3-aminobenzoic acids
16–20
Proposed
Mutasynthon21 Formula
C29H44N2O7Cl[M + H]+: calc.:
structurea,b
Mutasynthon21 Formula
C31H50N3O6[M + H]+: calc.:
Proposed structurea,b
555.2837, found: 555.2834
C28H42N2O7Cl[M + H]+, calc.:
537.2731, found: 537.2748
C28H41N2O6ClNa[M + Na]+: calc.:
575.2500, found: 575.2505
560.3700, found: 560.3701
C28H40N2O6FBrNa[M + Na]+:
calc.: 621.1951, found:
621.2000
C28H44N2O7I[M + H]+: calc.:
647.2193, found: 647.2184
C28H42N2O6I[M + H]+: calc.:
629.2088, found: 629.2072
C29H46N3O8[M + H]+: calc.:
564.3285, found: 564.3288
C30H42N2O7Na[M + Na]+: calc.:
565.2890, found: 565.2884
—
Not processed
—
Not processed
C29H42N3O6[M + H]+: calc.:
528.3074, found: 528.3070
a Analysis by UPLC-HRMS. b s.b. = single bond.
5 or 5 and 6, respectively (Table 2). With the successful bio-
transformation of 4-fluoro-3-aminobenzoic acid 8, precedence
for the acceptance of 4-substituted mutasynthons was already
at hand.18b 3-Amino-4-cyclopropylbenzoic acid 16 was pro-
cessed to the carbamoylated seco acid (amide form) so that
macrolactamisation did not occur, which is likely due to steric
hindrance in the ortho-position of the aniline group.
4,5-Disubstituted aminobenzoic acid 18 behaved in the same
manner, whereas the 4-fluoro-5-bromo derivative 17 yielded
the expected amidinated macrolactam. Obviously, a small
fluorine substituent at the 4 position as is also the case for
benzoic acid 8 does not hamper macrolactamisation by the
amide synthase (GdmF; Scheme 1).
C30H44N2O6Na[M + Na]+: calc.:
551.3097, found: 551.3105
C31H49N2O7[M + H]+: calc.:
561.3540, found: 561.3527
C31H50N3O6[M + H]+: calc.:
560.3700, found: 560.3710
C31H46N2O6Na[M + Na]+: calc.:
565.3254, found: 565.3254
C31H46N2O7Na[M + Na]+: calc.:
581.3203, found: 581.3208
Subtleties concerning the type of substitution at C4 become
evident when examining benzoic acid 19, which also gave
the corresponding macrolactam ring. Compared to the 2,2-
dimethyl-1,3-dioxole ring in 18, the furan moiety is planar and
sterically less congested. Finally, substituents located at posi-
tion 6 as in benzoic acid 20 suppress PKS processing probably
due to inefficient loading onto the PKS starter module.
a Analysis by UPLC-HRMS. b s.b. = single bond.
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One important result of our previous studies was that, not fermentation conditions, we found the following general
only are 3-aminobenzoic acid derivatives processed by the trends. One sulfur atom must be part of the five-membered
PKS of S. hygroscopicus, 5-aminonicotinic acid 9 is also ring in order to yield mutaproducts, either as macrolactam
accepted as a PKS substrate. This unprecedented finding derivatives or as seco acids. Consequently, thiophene 22, thia-
initiated a study in our laboratories on the scope and limit- zole 28 and thiazine 33 were accepted and processed.
ations of this new result using a series of six- and five- However, in the case of the thiazole core the positions of the
membered hetarenes 21–34 as mutasynthons (Table 3). The amino- and carboxyl substituents are crucial, because muta-
pyrimidine derivative 21, bearing the same substitution synthon 27 did not provide new products as judged by HRMS.
pattern as AHBA, failed to serve as a mutasynthon. When Therefore, also imidazole 25, oxazole 26 and pyrazine 32 did
exposing a series of five membered hetarenes to the typical not serve as mutasynthons. Additional substitution as
present in thiophenes 23 and 24 as well as in thiazoles 29
and 30 is also not tolerated by S. hygroscopicus. In view of
these findings triazole 31 represents an unusual case, as the
Table 3 Test mutasyntheses with (−)-AHBA blocked mutant of
S. hygroscopicus heteroaromatic aminobenzoic acids 21–34
sulfur atom is missing in the hetarene ring but UPLC-HRMS
revealed formation of a macrocyclic mutaproduct. It has to be
noted that the thiophene moiety is a bioisostere for the
benzene ring22 and this is also reflected in the present work
as 5-aminothiophene-3-carboxylic acid 22 was processed to
the corresponding macrolactam by the polyketide synthase.
Finally, the benzotriazole derivative 34, which bears a meta
orientation between the carboxylate and in this case a hetarene
NH group, was also processed to the amidinated seco acid. We
did not encounter macrolactamisation, likely due to the
presence of a secondary sp2-hybridised amine instead of an
aniline.
Mutasynthon21 Formula
Proposed structurea,b
Not processed
—
For R = H C26H40N2NaO6S
[M + Na]+: calc.: 551.2505,
found: 551.2510
At this point, one can conclude that the PKS machinery
involved in the biosynthesis of geldanamycin shows a rather
broad substrate flexibility with respect to structural altera-
tions in the aromatic starter group. Additional substituents at
the 4, 5 and 6 positions as well as disubstitution of selected
3-aminobenzoic acids are tolerated as long as these substitu-
ents are not too large. In several cases, we encountered seco
acid formation which included formation of carboxamides, a
process that stops macrolactamisation. The open chain
metabolites indicate that the amide synthase can act as a
bottle neck for further processing, while substrate tolerance
of the PKS up to that point must have been rather broad.
Advanced intermediates, whether they are acyclic or cyclic, all
serve as substrates for the tailoring amidination. Other post
PKS transformations, such as oxidations of the aromatic core
as well as 4,5-desaturation, are much more sensitive to struc-
tural variation in progeldanamycin precursors and in most
cases, they do not occur.
—
Not processed
For R = H C25H37N3NaO6S
[M + Na]+: calc.: 530.2301,
found: 530.2298
C26H41N5NaO7 [M + Na]+:
calc.: 558.2904, found:
558.2911
Mutasynthetic experiments (preparative scale)
For X = S C25H39N3NaO6S
[M + H]+: calc.: 532.2457,
found: 532.2451
Principally, all new seco acids and ansa macrolactams that are
listed in Tables 1–3 should be accessible as pure compounds,
provided that the fermentation is appropriately upscaled.
However, substantial HPLC purification is required to separate
all metabolites and mg quantities are needed for full structural
characterisation as well as biological evaluation. These require-
ments may therefore necessitate fermentation at a 10–50 L
scale.
C28H43N4O7 [M + H]+:
calc.: 547.6730,
found:547.6725
After close inspection of all chromatograms of the crude
fermentation extracts, we decided that mutasynthons 15, 22
and 34 provided sufficient amounts of new reblastatin deriva-
a Analysis by UPLC-HRMS. b s.b. = single bond.
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tives for complete structure elucidation and biological evalu-
ation. For this purpose, the fermentations were repeated in up
to 5 L scale.
Biological studies
Antiproliferative activities and Hsp binding affinities
After harvesting, the fermentation broths were extracted For the evaluation of its antiproliferating potential the two
with ethyl acetate. Depending on the complexity of the meta- new derivative 35 and 37 that were available in sufficient
bolic spectrum, several purification steps using silica gel amounts in pure form were administered to cultured human
chromatography, Sephadex size exclusion chromatography and tumour cell lines (Table 4). The data were compared to
RP-HPLC were performed (see ESI†). In all cases, the fermenta- geldanamycin 3 and the 19-fluoro derivative 8 one of the
tion yields of new mutaproducts were significantly lower than most potent nonhydroquinone derivatives known so far. The
that obtained when the natural starter unit, AHBA 1, was fed branched cyclopropyl substituent at position 17 led to
to yield geldanamycin 3. Supplementation of cyclopropyl- reduced activity. Remarkably, the thiophene analogue 37 of
substituted aminobenzoic acid 15 to a growing culture of reblastatin shows good antiproliferative activity against all
the (−)AHBA mutant of Streptomyces hygroscopicus provided human cancer cell lines tested. Still it is not as active as
reblastatin derivatives 35 and 36 in sufficient amounts for iso- geldanamycin 3. This can be ascribed to the fact that thio-
lation and characterisation (Scheme 2). Besides amidination of phene and benzene are bioisosteres and the sulfur atom is
the alcohol at position 7, oxidation at C17 also occurred as a located at position 18 of geldanamycin 3 and reblastatin 4,
tailoring modification. Likewise, thiophene 22 provided reblas- respectively.
tatin derivative 37 in excellent yield in comparison to those
Recently, we reported a new displacement assay for the ATP
commonly observed for such mutasynthetic experiments.18 domain of heat shock proteins such as for Hsp90α.20 The
Finally, we carried out the supplementation of AHBA(−) inhibition is monitored using fluorescence-labelled Cy3- or
S. hygroscopicus with benzotriazole 34 and were able to collect Cy5-ATP in the presence of a potential inhibitor that binds to
the chemically labile seco acid derivative 38. This is the first the ATP binding domain. This approach allows to distinguish
example of a bicyclic mutasynthon undergoing successful between the bound and unbound inhibitor. The assay is per-
loading onto the starter module and being fully processed by formed by incubating different Hsp-loaded microarrays for
the PKS; although this proceeded without macrolactamisation 16 h at 4 °C with a solution containing a mixture consisting of
by the amide synthase.
100 nM of the fluorescent-labelled ATP derivatives Cy3-ATP or
Cy5-ATP, the binding buffer and the potential Hsp90 inhibitor
(500 nM), including 17-aminoallyl geldanamycin (17-AAG) 39
(line 2), reblastatin derivatives 40–43 (lines 3–6) and thiophene
reblastatin 37 (line 7). To compare different heat shock
proteins from different sources, we chose human Hsp90α
(Fig. 2a), Hsp90β (b), Xanthomonas campestris XcHtpG (c) and
Helicobacter pylori HpHtpG (d). The results are presented as a
heat map (Fig. 2). The simultaneous screening of different pur-
ified Hsps reveals that most of all synthesised reblastatin
derivatives, except for cyclopropyl derivative 35, are capable of
inhibiting human and HpHtpG heat shock proteins.
Remarkably, the clinical candidate 17-AAG 39 only shows a
minor activity for HpHtpG. The EC50 of thiophene 37 against
HpHtpG and HsHtpG were 59 nM and 128 nM, respectively.
This comparison reveals that the thiophene group shows
varying affinities to the ATP binding sites of different Hsps,
Table 4 IC50 [nM] of the antiproliferative activity of 35, 37, 3 and 8 with
human cancer cell lines (values are means of two determinations in
parallel)a
IC50 [nM]
KB-3-1
PC-3
SK-OV-3
A-431
35
37
3
960
128
53
1500
472
18
1300
159
125
54
980
85
18
8
73
42
62
a KB-3-1 = Cervix carcinoma; PC-3 = Prostate carcinoma; SK-OV-3 =
Ovarian carcinoma; A-431 = Epidermoid carcinoma.
Scheme 2 Successful upscale mutasyntheses with (−) AHBA mutant
strain of S. hygroscopicus.
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Except for the cyclopropyl reblastatin 35, all derivatives show
pronounced displacement properties for the heat shock pro-
teins tested. Reblastatin derivative 39 bearing the simplest
possible substitution pattern at the aromatic ring demonstrates
that non-quinone and even non phenolic geldanamycin deriva-
tives are very effective in replacing ATP in human Hsp90α (39,
IC50Hsp90α = 30 nM). However, in no case have we encountered
a relevant selectivity difference between human Hsp90α and
Hsp90β (Fig. 2). Interestingly, thiophene reblastatin 37 exhibits
a stronger activity for the bacterial Hsp homologue HpHtpG
than for human Hsp90α. Therefore, it can serve as a starting
point for developing antibiotics that target bacterial heat shock
proteins, especially because it was recently shown that HtpG is
required for adaptation through environmental stress.24
Conclusions
Fig. 2 Top: Structures of geldanamycin and reblastatin derivatives
screened. Bottom left: Heat map presentations of competitive Hsp
activities tested for compounds 37 and 39–43 dedicated to lines 2–7
using a microarray-based assay with purified heat shock proteins in the
presence of ATP. The normalised signals are ranked from 0 to 1 (line 1,
blank) displaying different colours. Low signal values (red) indicate
stronger competition against fluorescence dye labelled ATP and hence
higher affinity for Hsps. No competition shown in position 1 provides a
green colour. The concentrations of potential binders are 500 nM
against (a) human Hsp90a, (b) Hsp90b, (c) Xanthomonas campestris
XcHtpG and (d) Helicobacter pylori HpHtpG. These were spotted onto a
microarray as described before.20a Bottom left: Corresponding normal-
ised values derived from the optical measurements.
In conclusion, we reported on the mutasynthetic generation
and biological evaluation as heat shock protein inhibitors of
several new reblastatin derivatives. The cyclopropane deriva-
tives 35 and 36 are remarkable in that this is the first example
of introducing such structural elements into complex natural
products by a mutasynthetic approach. More striking is the
high yielding uploading and PKS-processing of 5-aminothio-
phene-3-carboxylic acid 22 to the corresponding thiophene
reblastatine derivative 37, the first mutaproduct of this kind
reported so far.25
Importantly, this broad study also demonstrates the scope
and limitations of mutasynthesis as a synthetic tool in natural
product chemistry. The structural diversity of aromatic building
blocks including hetarenes that are accepted by the starting
module of Gdm-PKS and fully processed up to that stage of the
macrolactam is astounding. A promising new candidate is the
thiophene derivative of reblastatin. It shows inhibitory activity
against HtpG from Helicobacter pylori and this derivative clearly
can serve as a starting point to develop new selective inhibitors
against bacterial heat shock proteins. Finally, the limitation of
producing sufficient quantities of new derivatives by mutasynth-
esis has to be kept in mind26–29 and is also in issue in the
present study. This becomes particularly evident when calculat-
ing the isolated yields of new mutaproducts based on the
amount of mutasynthons fed: 35 (0.15% from 15), 36 (0.04%
from 15), 37 (1.1% from 22) and 38 (0.09% from 34).
which can serve as a starting point for developing Hsp specific
inhibitors.
Additionally, the measurements were conducted at different
concentrations (50 µM, 5 µM, 0.5 µM, 50 nM, 5 nM, 0.5 nM, 50
pM) to estimate the IC50 value. Unbound fluorescently-labelled
ATP was removed by several washing steps and then the
amount of bound Cy3-labelled ATP was determined. The
signal intensity was analysed and normalised for the unbound
value and hence visualised. These studies18,20 reveal that the
inhibitory properties found for new reblastatin derivatives 35
and 37 match those of the antiproliferative activity (Tables 4
and 5).
Table 5 Summary of the calculated IC50 values [nM] of Hsp90α from H.
sapiens and of HpHtpG from H. pylori and the specific Z-factors23 of
each protein microarray (in brackets position on the microarray in Fig. 2)
Experimental procedures
IC50(nM)
Mutasynthetic experiments
General parameters. Streptomyces hygroscopicus mutant
K390-61-1 was stored as stock cultures at −80 °C as a cryocul-
ture in the refrigerator. 100 µL of the cryoculture of K390-61-1
mutant was used to inoculate the GYP precultures which were
incubated at 28 °C for 2 days. Precultures were prepared in
GYP-medium (40 mL per flask, 2.5 g L−1 yeast extract, 10 g L−1
peptone, 10 g L−1 glucose, 3 g L−1 xanthane gum, distilled
Hsp90α
Z23
HpHtpG
Z23
35
39
40
41
42
43
37
n.b.
0.72
0.81
0.54
0.78
0.77
0.68
0.45
n.b.
0.53
0.53
0.79
0.69
0.55
0.78
0.77
30.03 6.56
59.41 30.55
57 15.51
18.22 4.11
10 2.7
80.6 7.34
20.99 5.91
163.26 31.77
38.48 7.48
64 2.34
127.5 42.2
56.63 25.0
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water). Main cultures were prepared in GP-medium (40 g L−1 (s, 1H, Ar–H), 5.88–5.80 (m, 1H, 3-H), 5.69 (br. s, 2H, CONH2),
glucose, 2.5 g L−1 peptone, 2.5 g L−1 tryptone, 5 g L−1 oatmeal, 5.30 (d, J = 9.9 Hz, 1H, 9-H), 5.06 (d, J = 5.8 Hz, 1H, 7-H), 4.54
2.5 g L−1 yeast extract, 3 g L−1 xanthane gum, distilled water). (br. s., 1H, 11-OH), 3.54–3.48 (m, 1H, 11-H), 3.35 (s, 3H,
Liquid culture fermentations were incubated at 28 °C with 6-OMe), 3.31 (s, 3H, 12-OMe), 3.30–3.24 (m, 1H, 6-H),
vigorous shaking (200 rpm) in 500 mL Erlenmeyer flasks 3.15–3.08 (m, 1H, 12-H), 2.65 (dd, J = 13.2, 5.0 Hz, 1H, 15-Ha),
equipped with a baffle. Main cultures were inoculated with 2.47–2.40 (m, 1H, 10-H), 2.37–2.25 (m, 1H, 15-Hb), 2.37–2.25
1 mL preculture per 30 mL culture broth. The production cul- (m, 1H, 4-Ha), 2.17–2.05 (m, 1H, 4-Hb), 1.91–1.82 (m, 1H,
tures were harvested after 5 d of fermentation and extracted 14-H), 1.91–1.82 (m, 1H, CH-cyclopropyl) 1.79 (s, 3H, 2-Me),
twice with EtOAc. The EtOAc extracts were concentrated, and 1.60–1.52 (m, 1H, 13-Ha), 1.48 (s, 3H, 8-Me), 1.40–1.32 (m, 2H,
the residue was dissolved in MeOH (1 ml) and used directly 5-H), 1.27–1.20 (m, 1H, 13-Hb), 1.00 (d, J = 6.6 Hz, 3H, 10-Me),
for ESI-MS analysis.
0.90–0.86 (m, 2H, CH2-cyclopropyl), 0.82 (d, J = 6.8 Hz, 3H,
14-Me), 0.66–0.61 (m, 2H, CH2-cyclopropyl) ppm; 13C NMR
(125 MHz, T = 323 K, THF-d8, THF-d8 = 25.5 ppm): δ 171.2 (s,
Feeding experiments for small scale transformations
Benzoic acid derivatives 10–20 and hetarenes 21–34 C-1), 157.3 (s, CONH2), 145.4 (s, C-Ar), 141.8 (s, C-Ar), 141.3 (s,
(37.5 µmol, in 2 mL sterile solution in DMSO/H2O = 1 : 1) were C-Ar), 134.7 (d, C-3), 133.0 (s, C-2), 133.0 (s, C-9), 131.7 (d, C-8),
added in three 670 µL portions at 48, 72 and 96 h after 124.3 (d, C-Ar), 123.5 (d, C-Ar), 116.3 (d, C-Ar), 82.7 (d, C-12),
inoculation to 30 mL of the main culture of S. hygroscopicus 81.6 (d, C-6), 81.3 (d, C-7), 75.1 (d, C-11), 58.9 (q, 6-OMe), 57.0
mutant K390-61-1. After having fed precursors according to (q, 12-OMe), 43.9 (t, C-15), 35.5 (d, C-10), 34.6 (t, C-13), 32.8 (d,
the fermentation conditions described above, UPLC-ESI-HRMS C-14), 31.0 (t, C-5), 25.5 (t, C-4), 19.8 (q, 14-Me), 17.3 (d,
analysis was employed for detecting new products as [M + H]+ CH-cyclopropyl), 16.2 (q, 10-Me), 13.6 (q, 2-Me), 13.2 (q, 8-Me),
or [M + Na]+ peaks.
9.7 (t, CH2-cyclopropyl), 9.6 (t, CH2-cyclopropyl) ppm; HRMS
Feeding of benzoic acid 15 and formation of reblastatin (ESI) m/z for C31H46N2O6Na [M + Na]+: calculated: 565.3254
derivatives 35 and 36. For scale-up fermentation 3-amino-5- found: 565.3254.
cyclopropylbenzoic acid 15 (287.0 mg, 1.50 mmol, 20 mL
sterile solution in DMSO/H2O = 1 : 1) was fed in three equal
17-Demethyl-18-cyclopropyl-reblastatin 36
portions at 48, 72 and 96 h after inoculation to sixteen 75 mL HPLC conditions employed for the first purification step were
main cultures of S. hygroscopicus mutant K390-61-1. Extraction (a) column: Reprosil-Pur 120 C18 AQ, 250 × 25 mm, 5 µm,
with EtOAc afforded 17-demethoxy-18-cyclopropyl-reblastatin endc.; (b) guard: Reprosil-Pur 120 C18 AQ, 30 × 20 mm, 10 µm,
35 (1.3 mg, 2.3 µmol) and 17-demethyl-18-cyclopropyl-reblasta- endc.; (c) solvents: methanol/H2O and (d) program:
tin 36 (0.3 mg, 0.5 µmol) after silica gel filtration and three H2O : MeOH = 60 : 40 {5 min}, 60 : 40 → 50 : 50 {15 min},
HPLC purification steps.
H2O : MeOH = 50 : 50 → 30 : 70 {65 min}, H2O : MeOH =
HPLC conditions employed for the first purification step 30 : 70 → 0 : 100 {15 min}, H2O : MeOH = 0 : 100 {10 min},
were (a) column: Reprosil-Pur 120 C18 AQ, 250 × 25 mm, 5 µm, (15.0 ml min−1) (tR = 65.0 min). For the second purification
endc.; (b) guard: Reprosil-Pur 120 C18 AQ, 30 × 20 mm, step the conditions were (a) column: Reprosil-Pur 120 C18-ISIS
10 µm, endc.; (c) solvents: methanol/H2O and (d) program: AQ, 250 × 8.0 mm, 5 µm, endc.; (b) guard: Reprosil-Pur 120
H2O : MeOH = 60 : 40 {5 min}, 60 : 40 → 50 : 50 {15 min}, C18-ISIS AQ, 40 × 8 mm, 5 µm, endc.; (c) solvents: MeOH/H2O
H2O : MeOH = 50 : 50 → 30 : 70 {65 min}, H2O : MeOH = 30 : 70 → and (d) program: H2O : MeOH = 80 : 20 → 65 : 35 {5 min},
0 : 100 {15 min}, H2O : MeOH = 0 : 100 {10 min}, (15.0 ml min−1
)
H2O : MeOH = 65 : 35 → 40 : 60 {65 min}, H2O : MeOH = 40 : 60
(tR = 65.0 min). For the second purification step the conditions → 0 : 100 {10 min}, H2O : MeOH = 0 : 100 {10 min}, (2.5 mL
were (a) column: Reprosil-Pur 120 C18-ISIS AQ, 250 × 8.0 mm, min−1) (tR = 61.0 min). For the third purification step the con-
5
µm, endc.; (b) guard: Reprosil-Pur 120 C18-ISIS AQ, ditions were (a) column: Reprosil-Pur 120 C18-ISIS AQ,
40 × 8 mm, 5 µm, endc.; (c) solvents: MeOH/H2O and 250 × 8.0 mm, 5 µm, endc.; (b) guard: Reprosil-Pur 120 C18-
(d) program: H2O : MeOH 80 : 20 65 : 35 {5 min}, ISIS AQ, 40 × 8 mm, 5 µm, endc.; (c) solvents: MeOH/H2O
=
→
H2O : MeOH = 65 : 35 → 40 : 60 {65 min}, H2O : MeOH = and (d) program: H2O : MeOH = 80 : 20 → 70 : 30 {5 min},
40 : 60 → 0 : 100 {10 min}, H2O : MeOH = 0 : 100 {10 min}, H2O : MeOH = 70 : 30 → 40 : 60 {80 min}, H2O : MeOH = 40 : 60
(2.5 mL min−1) (tR = 63.0 min). For the third purification step → 0 : 100 {5 min}, (3.0 ml min−1) (tR = 60.2 min).
the conditions were (a) column: Reprosil-Pur 120 C18-ISIS AQ,
Feeding of benzoic acid 22 and formation of reblastatin
250 × 8.0 mm, 5 µm, endc.; (b) guard: Reprosil-Pur 120 derivatives 37. For scale-up fermentation 2-amino-4-carb-
C18-ISIS AQ, 40 × 8 mm, 5 µm, endc.; (c) solvents: methanol/ oxythiophene 22 (215 mg, 1.50 mmol, in 20 mL sterile solu-
H2O and (d) program: H2O : MeOH = 80 : 20 → 65 : 35 {5 min}, tion in DMSO/H2O = 1 : 1) was fed in three equal portions at
H2O : MeOH = 65 : 35 → 40 : 60 {80 min}, H2O : MeOH = 48, 72 and 96 h after inoculation to sixteen 75 mL main
40 : 60 → 0 : 100 {5 min}, (3.0 ml min−1) (tR = 64.4 min).
cultures of S. hygroscopicus mutant K390-61-1. Extraction with
EtOAc afforded thiageldanamycin-derivative 37 (8.3 mg,
16.3 µmol) after silica gel filtration and three HPLC
17-Demethoxy-18-cyclopropyl-reblastatin 35
1H NMR (500 MHz, T = 323 K, THF-d8, THF-d7 = 1.73 ppm): purification steps. HPLC conditions employed for the first
δ 8.43 (s, 1H, NH), 7.02 (s, 1H, Ar–H), 6.61 (s, 1H, Ar–H), 6.58 purification step were (a) column: Reprosil-Pur 120 C18 AQ,
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250 × 25 mm, 5 µm, endc.; (b) guard: Reprosil-Pur 120 C18 AQ, H2O : MeOH = 0 : 100 {10 min}, (15.0 ml min−1) (tR = 55.0 min).
30 × 20 mm, 10 µm, endc.; (c) solvents: methanol/H2O and For the second purification step the conditions were (a) column:
(d) program: H2O : MeOH = 60 : 40 {5 min}, 60 : 40 → 50 : 50 Reprosil-Pur 120 C18-ISIS AQ, 250 × 8.0 mm, 5 µm, endc.;
{15 min}, H2O : MeOH
= 50 : 50 → 30 : 70 {65 min}, (b) guard: Reprosil-Pur 120 C18-ISIS AQ, 40 × 8 mm, 5 µm,
H2O : MeOH = 30 : 70 → 0 : 100 {15 min}, H2O : MeOH = 0 : 100 endc.; (c) solvents: methanol/H2O and (d) program: H2O : MeOH =
{10 min}, (15.0 ml min−1) (tR = 55.0 min). For the second puri- 80 : 20 → 65 : 35 {5 min}, H2O : MeOH = 65 : 35 → 40 : 60
fication step the conditions were (a) column: Reprosil-Pur 120 {65 min}, H2O : MeOH = 40 : 60 → 0 : 100 {10 min}, H2O : MeOH
C18-ISIS AQ, 250 × 8.0 mm, 5 µm, endc.; (b) guard: Reprosil- = 0 : 100 {10 min}, (2.5 mL min−1) (tR = 52.0 min). For the third
Pur 120 C18-ISIS AQ, 40 × 8 mm, 5 µm, endc.; (c) solvents: purification step the conditions were (a) column: Reprosil-Pur
MeOH/H2O and (d) program: H2O : MeOH = 80 : 20 → 65 : 35 120 CN AQ, 250 × 8.0 mm, 5 µm, endc.; (b) guard: Reprosil-Pur
{5 min}, H2O : MeOH = 65 : 35 → 40 : 60 {65 min}, H2O : MeOH 120 CN AQ, 40 × 8 mm, 5 µm, endc.; (c) solvents: MeCN/H2O
= 40 : 60 → 0 : 100 {10 min}, H2O : MeOH = 0 : 100 {10 min}, and (d) program: H2O : MeCN = 90 : 10 → 85 : 15 {20 min},
(2.5 mL min−1) (tR = 47.0 min). For the third purification step H2O : MeCN = 85 : 15 {40 min}, H2O : MeCN = 85 : 15 → 75 : 25
the conditions were (a) column: Reprosil-Pur 120 CN AQ, {20 min}, H2O : MeCN = 75 : 25 → 55 : 45 {20 min}, H2O : MeCN =
250 × 8.0 mm, 5 µm, endc.; (b) guard: Reprosil-Pur 120 CN AQ, 55 : 45 → 0 : 100 {15 min}, (4.0 ml min−1) (tR = 53.5 min).
40 × 8 mm, 5 µm, endc.; (c) solvents: MeCN/H2O and
(d) program: H2O : MeCN 90 : 10 85 : 15 {20 min}, δ 7.72 (d, J = 7.0 Hz, 1H, Ph), 7.56 (s, 1H, Ph), 7.20 (d, J = 8.0 Hz,
1H-NMR (500 MHz, T = 323 K, THF-d8, THF-d7 = 1.73 ppm):
=
→
H2O : MeCN = 85 : 15 {40 min}, H2O : MeCN = 85 : 15 → 75 : 25 1H, Ph), 6.77–6.69 (m, 1H, 3-H), 5.53 (br. s, 2H, CONH2), 5.28
{20 min}, H2O : MeCN = 75 : 25 → 55 : 45 {20 min}, H2O : MeCN = (d, J = 9.4 Hz, 1H, 9-H), 4.90 (dd, J = 8.6, 2.9 Hz, 1H, 7-H), 3.86
55 : 45 → 0 : 100 {15 min}, (4.0 ml min−1) (tR = 40.0 min).
(d, J = 6.3 Hz, 1H, 11-H), 3.42 (s, 3H, 6-OMe), 3.30 (s, 3H,
12-OMe), 3.35–3.25 (m, 1H, 6-H), 3.25–3.12 (m, 1H, 12-H), 2.84
(dd, J = 13.5, 5.7 Hz, 1H, 15-Ha), 2.79–2.69 (m, 1H, 10-H),
Thiophene-reblastatin 37
1H-NMR (500 MHz, T = 323 K, THF-d8, THF-d7 = 1.73 ppm): 2.61–2.50 (m, 1H, 15-Hb), 2.28–2.17 (m, 2H, 4-H), 2.14–1.98 (m,
δ 8.61 (s, 1H, NH), 6.57 (s, 1H, Ar), 6.38 (s, 1H, Ar), 5.72 (br. s., 1H, 14-H), 1.80 (s, 3H, 2-Me), 1.75–1.02 (m, 1H, 5-Ha), 1.70–1.62
1H, 3-H), 5.57 (br. s, 2H, CONH2), 5.22 (d, J = 10.0 Hz, 1H, (m, 1H, 13-Ha), 1.60 (s, 3H, 8-Me), 1.50–1.36 (m, 1H, 13-Hb),
9-H), 4.99 (d, J = 7.0 Hz, 1H, 7-H), 3.56–3.51 (m, 1H, 11-H), 1.17–1.07 (m, 1H, 5-Hb), 0.97 (d, J = 6.6 Hz, 3H, 10-Me), 0.83
3.37 (s, 3H, 6-OMe), 3.31 (s, 3H, 12-OMe), 3.28–3.19 (m, 1H, (d, J = 6.6 Hz, 3H, 14-Me) ppm; HRMS (ESI) m/z for C28H43N4O7
6-H), 3.11–3.05 (m, 1H, 12-H), 2.65 (dd, J = 13.6, 5.4 Hz, 1H, [M + H]+: calculated: 547.3132 found: 547.3137.
15-Ha), 2.45–2.35 (m, 1H, 10-H), 2.45–2.35 (m, 1H, 15-Hb),
Preparation of Hsps and competitive microarray assay
2.22–2.05 (m, 2H, 4-H), 2.05–1.91 (m, 1H, 14-H), 1.75 (s, 3H,
2-Me), 1.66–1.58 (m, 1H, 13-Ha), 1.55 (s, 3H, 8-Me), 1.24–1.14 Recombinant Hsps were produced as previously reported.18,20
(m, 2H, 5-H), 1.20–1.11 (m, 1H, 13-Hb), 1.00 (d, J = 6.6 Hz, 3H, Hsp microarrays were produced and the binding with fluo-
10-Me), 0.75 (d, J = 6.8 Hz, 3H, 14-Me) ppm; 13C-NMR rescently labelled ATP and displacement with inhibitors was
(125 MHz, T = 323 K, THF-d8, THF-d8 = 25.5 ppm): δ 172.3 (s, performed as described before.18,20
C-1), 156.9 (s, CONH2), 143.2 (s, C-Ar), 139.6 (s, C-Ar), 135.3 (d,
Experimental details on assay (Fig. 2)
C-3), 134.0 (d, C-9), 131.8 (s, C-2), 131.7 (s, C-8), 124.4 (d, C-Ar),
117.3 (d, C-Ar), 82.1 (d, C-12), 81.8 (d, C-7), 81.4 (d, C-6), 74.2 The assay was performed by incubating the Hsp-loaded micro-
(d, C-11), 59.1 (q, 6-OMe), 57.0 (q, 12-OMe), 39.0 (t, C-15), 35.7 arrays for 16 h at 4 °C with a solution containing a mixture
(d, C-10), 33.3 (t, C-13), 31.4 (t, C-5), 30.5 (d, C-14), 24.5 (t, C-4), consisting of 100 nM Cy3-ATP or Cy5-ATP, the binding buffer
18.7 (q, 14-Me), 17.9 (q, 10-Me), 14.1 (q, 2-Me), 12.7 (q, 8-Me) and the potential Hsp90 inhibitor at 500 nM for comparative
ppm; HRMS (ESI) m/z für C26H41N2O6S [M + H]+: calculated: screen on different Hsps or at different concentrations (50 µM,
509.2685 found: 509.2666.
5 µM, 0.5 µM, 50 nM, 5 nM, 0.5 nM, 50 pM) for estimation of
Feeding of benzoic acid 34 and formation of seco acid 38. the IC50 value. Unbound fluorescently-labelled ATP was
For scale-up fermentation benzotriazole 34 (245 mg, removed by several washing steps and then the amount of
1.50 mmol), in 20 mL sterile solution in DMSO/H2O = 1 : 1 was bound labelled ATP was determined. The signal intensity was
fed in three equal portions at 48, 72 and 96 h after inoculation analysed and normalised for the unbound value.
to sixteen 75 mL main cultures of S. hygroscopicus mutant K390-
61-1. Extraction with EtOAc afforded seco-progeldanamycin acid
derivative 38 (0.5 mg, 0.9 µmol) after silica gel filtration and
three HPLC purification steps. HPLC conditions employed for
the first purification step were (a) column: Reprosil-Pur 120
Conflicts of interest
There are no conflicts to declare.
C18-ISIS AQ, 250 × 25 mm, 5 µm, endc.; (b) guard: Reprosil-Pur
120 C18-ISIS AQ, 30 × 20 mm, 10 µm, endc.; (c) solvents:
MeOH/H2O and (d) program: H2O : MeOH = 60 : 40 {5 min},
Acknowledgements
60 : 40
→
50 : 50 {15 min}, H2O : MeOH
=
50 : 50
→
This
work
was
supported
by
the
Deutsche
30 : 70 {65 min}, H2O : MeOH = 30 : 70 → 0 : 100 {15 min}, Forschungsgemeinschaft. For funding (Ki397/13-1). We thank
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Wera Collisi (HZI) for performing the cell proliferation assays. 10 W. Guo, P. Reigan, D. Siegel, J. Zirrolli, D. Gustafson and
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25 Recent examples on aromatic mutasynthons employed:
(a) Y. Yan, J. Chen, L. Zhang, Q. Zheng, Y. Han, H. Zhang,
Org. Biomol. Chem.
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