Chemical probes for the functionalization of polyketide intermediates

Article abstract of DOI:10.1002/anie.201407448

A library of functionalized chemical probes capable of reacting with ketosynthase-bound biosynthetic intermediates was prepared and utilized to explore in vivo polyketide diversification. Fermentation of ACP mutants of S. lasaliensis in the presence of the probes generated a range of unnatural polyketide derivatives, including novel putative lasalocid A derivatives characterized by variable aryl ketone moieties and linear polyketide chains (bearing alkyne/azide handles and fluorine) flanking the polyether scaffold. By providing direct information on microorganism tolerance and enzyme processing of unnatural malonyl-ACP analogues, as well as on the amenability of unnatural polyketides to further structural modifications, the chemical probes constitute invaluable tools for the development of novel mutasynthesis and synthetic biology.

Full text of DOI:10.1002/anie.201407448

Angewandte
Chemie
DOI: 10.1002/anie.201407448
Biosynthesis Very Important Paper
Chemical Probes for the Functionalization of Polyketide
Intermediates**
Elena Riva, Ina Wilkening, Silvia Gazzola, W. M. Ariel Li, Luke Smith, Peter F. Leadlay, and
Manuela Tosin*
Abstract: A library of functionalized chemical probes capable
of reacting with ketosynthase-bound biosynthetic intermediates
was prepared and utilized to explore in vivo polyketide
diversification. Fermentation of ACP mutants of S. lasaliensis
in the presence of the probes generated a range of unnatural
polyketide derivatives, including novel putative lasalocid A
derivatives characterized by variable aryl ketone moieties and
linear polyketide chains (bearing alkyne/azide handles and
fluorine) flanking the polyether scaffold. By providing direct
information on microorganism tolerance and enzyme process-
ing of unnatural malonyl-ACP analogues, as well as on the
amenability of unnatural polyketides to further structural
modifications, the chemical probes constitute invaluable tools
for the development of novel mutasynthesis and synthetic
biology.
enzymes. PKSs utilize a wide range of bioavailable acyl
building blocks (e.g. acetate, propionate, butyrate, etc.) and
iterative decarboxylative Claisen condensation to generate
stereoenriched enzyme-bound polyketide chains, which are
eventually released from PKSs and can be further enzymati-
cally tailored to yield the final bioactive products.[2] Natural
product structural diversification is highly desirable to obtain
novel molecules of increased efficiency and/or novel bioac-
tivity.[3] Synthetic biology is currently seen as a highly
promising avenue to generate novel polyketide derivatives,
especially in the context of modular PKSs.[4] This potential
arises because of the relative amenability of these assembly
lines to genetic manipulation by domain swap and other
redesign.[5–7] Current approaches towards the generation of
unnatural polyketides (compounds which are still made by
enzymatic assembly but are structurally different from the
original products) include semisynthesis, precursor-directed
biosynthesis, mutasynthesis, combinatorial biosynthesis, and
chemogenetics.[8] Chemical synthesis as well as enzyme and
metabolic engineering can be utilized to provide polyketide
machineries with unnatural building blocks. Recent efforts in
these directions have highlighted both the innate and
engineered chemical flexibility of PKSs in accepting a variety
of non-native substrates to generate novel polyketide deriv-
atives in vitro and in vivo, thus opening up the exploitation of
novel chemical spaces and bioactivities for this class of natural
products.[9] We have recently developed synthetic chain
terminators capable of capturing polyketide biosynthetic
intermediates in vitro[10] and in vivo.[11] These chemical
probes (e.g., the b-ketoacids 1a,b; Scheme 1; generated
in situ from the hydrolysis of the corresponding methyl
esters)[11] compete with ACP-bound malonate units for
polyketide chain extension. By reacting with enzyme-bound
biosynthetic intermediates, intermediate-like species (2a,b)
are off-loaded from PKSs and become available for LC-MS
characterization. Further, the use of these molecules in vivo
has allowed us to reveal relatively inaccessible features of
polyketide assembly, such as the timing of ring formation in
the biosynthesis of the polyether antibiotic lasalocid A.[11b]
In characterizing the captured biosynthetic intermediate
species, it occurred to us that it might be possible, by utilizing
further derivatized chemical probes together with suitably
engineered bacterial strains, to rapidly generate a variety of
novel polyketide intermediates and products bearing bioor-
thogonal chemical handles for site-specific modifications,
such as alkyne and azide moieties,[12] as well as pharmaceuti-
cally relevant motifs, such as fluorine.[13] To test our hypoth-
esis, we prepared a small library of second-generation methyl
ester probes characterized by the variation of the N-acyl
A
s direct outcomes of millions of years of structural and
functional evolution, natural products remain at the forefront
of drug discovery and development.[1] Polyketides in partic-
ular constitute a highly diverse family including important
pharmaceuticals and agrochemicals such as the antibiotic
erythromycin A, the cholesterol-lowering agent lovastatin,
the antiparasitic ivermectin, and the immunosuppressant
rapamycin.[2] Polyketides are biosynthesized in microorgan-
isms and plants by the polyketide synthase (PKS) multi-
[*] Dr. E. Riva,[+] Dr. I. Wilkening,[+] S. Gazzola,[$] Dr. M. Tosin
Department of Chemistry, University of Warwick
Library Road, Coventry CV4 7AL (UK)
E-mail: m.tosin@warwick.ac.uk
W. M. A. Li
Department of Chemistry, University of Cambridge
Lensfield Road, Cambridge CB2 1EW (UK)
Dr. L. Smith, Prof. P. F. Leadlay
Department of Biochemistry, University of Cambridge
80 Tennis Court Road, Cambridge CB2 1GA (UK)
[$] Current address: Dipartimento di Scienza e Alta Tecnologia,
Universitꢀ dell’Insubria, 22100 Como (Italy)
[+] These authors contributed equally to this work.
[**] We gratefully acknowledge BBSRC (project grant BB/J007250/1 to
M.T.), the Institute of Advanced Studies at Warwick (Postdoctoral
Research Fellowship to E. R.), the European Commission (Marie
Curie Intra-European Fellowship to I. W.), Dr. Lijiang Song, Philip
Aston, Dr. Rebecca Wills, Dr. Yuki Inahashi, and Prof. Greg Challis
(Warwick Chemistry) for UPLC-MS analyses (Bruker Maxis Impact).
Supporting information for this article (synthesis of probes 3–13
and their use in the fermentation of S. lasaliensis strains, as well as
detailed LC-HR-MSn analysis of all the newly generated polyketide
intermediates and products (24–43, 45, 47–48)) is available on the
WWW under http://dx.doi.org/10.1002/anie.201407448.
Angew. Chem. Int. Ed. 2014, 53, 1 – 7
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1
These are not the final page numbers!

.
Angewandte
Communications
extent of the hydrolysis being substrate-dependent (see the
Supporting Information).
The overall outcome of the feeding experiments is
summarized in Scheme 2 and Table 1 (see the Supporting
Information for details). In the ethyl acetate extracts of the
ACP12 (S970A) mutant, grown in the presence of the methyl
ester substrates 3–8 (supplemented daily to reach a 2.5–
4.0 mm final concentration), the putative lasalocid A deriva-
tives 2 and 24–28 were identified as the major polyketide
products off-loaded from the lasA PKS by micro-LC-HR-MS
analyses (Table 1, entries 1–7). The compounds 2 and 24–28
were characterized by HR-MS2 and displayed a conserved
fragmentation pattern involving McLafferty rearrangement
and subsequent formation of m/z 377, a diagnostic polyether
fragment.[11b] In addition, for the compounds 27 and 28 the
loss of the C10 acyl chain occurring during fragmentation
afforded an m/z 260 fragment, which would correspond to
a protonated cyclic imine (Figure 1c and see the Supporting
Information) and strongly supports the aromatic ketone
nature of these derivatives. The formation of 24–28 indicates
that variations of the N-acyl moiety of the pseudomalonate
substrates are well tolerated by both S. lasaliensis and the lasA
PKS in the range of concentrations utilized in these experi-
ments. Interestingly, additional formation of short-chain
products (e.g., the pentaketides 38–40) was observed for
long-chain substrates (7–8, and, later on, 13; Table 1,
entries 6, 7, and 19). The nature of these products was
independently verified by feeding of S. lasaliensis ACP5
(S3799A) with 7 and 8 (entries 9 and 10). Putative iso-
lasalocid derivatives (29–30) were also selectively obtained
when the compounds 6 and 8 were fed to S. lasaliensis DlasB-
ACP12 (S970A)[11b] (entries 11 and 12). These derivatives
displayed a similar HR-MS2 fragmentation pattern to their
lasalocid putative counterparts but eluted at significantly
Scheme 1. a) Late stages in the biosynthesis of lasalocid A by the lasA
PKS: malonyl-ACP decarboxylative condensation with the last KS-
bound intermediate, followed by aromatization and thioester hydroly-
sis, leads to natural product formation. b) The competitive decarbox-
ylative condensation of the carba(dethia) N-acetyl cysteamine probes
1a,b[11b] with the same advanced intermediate leads to the formation
of the lasalocid A unnatural derivatives 2a,b. ACP=acyl carrier protein,
AT =acyl transferase, KS=ketosynthase, TE=thioesterase.
moiety and 1,3-dicarbonyl functionalization (3–13;
Scheme 2). We then employed these molecules as substrates
in small-scale fermentations (10 mL) of ACP mutant strains
of Streptomyces lasaliensis. The strains, constructed from S.
lasaliensis NRRL 3382 by point mutation of the active serine
(for 4’-phosphopantetheine attachment) of ACP12 and ACP5
to alanine residues as previously reported,[11b] do not produce
the natural polyketide product lasalocid A. However they still
harbour enzyme-bound biosynthetic intermediates.[11b] The
esters 3–13 were hydrolyzed in situ to the corresponding b-
ketoacids 1 and 14–23 by endogenous esterases, with the
Scheme 2. Generation of putative functionalized polyketide intermediates and the products 2 and 24–43 by fermentation of S. lasaliensis in the
presence of 1 and 14–23 (derived from in vivo hydrolysis of the corresponding methyl esters 3–13; see boxed inset). The stereochemistry of the
newly formed compounds is yet to be established.
2
www.angewandte.org
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2014, 53, 1 – 7
These are not the final page numbers!

Angewandte
Chemie
Table 1: Generation of putative unnatural polyketides from the lasA PKS and the substrates 1 and 14–23 (generated from in vivo hydrolysis of 3–13;
Scheme 2).
Entry
S. lasaliensis strain
Substrate[a]
Major product(s)[b]
Ester
acid
R1
R2
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
ACP12 (S970A)
ACP12 (S970A)
ACP12 (S970A)
ACP12 (S970A)
ACP12 (S970A)
ACP12 (S970A)
ACP12 (S970A)
ACP5 (S3799A)
ACP5 (S3799A)
ACP5 (S3799A)
DlasB-ACP12 (S970A)
DlasB-ACP12 (S970A)
ACP12 (S970A)
WT (NRRL 3382)
WT (NRRL 3382)
ACP12 (S970A)
ACP12 (S970A)
ACP12 (S970A)
ACP12 (S970A)
3a
3b
4
5
6
7
8
5
7
8
6
8
9
1a
1b
14
15
16
17
18
15
17
18
16
18
19
19
20
21a
21b
22
CH3
CD3
CF3
(CH2)2C CH
(CH2)3N3
(CH2)8CH3
(CH2)9N3
(CH2)2C CH
(CH2)8CH3
(CH2)9N3
(CH2)3N3
(CH2)9N3
(CH2)2C CH
(CH2)2C CH
H
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
CH3
F
F
F
2a[c]
2b[c]
24[c]
25[c]
ꢀ
26[c]
27,[c] 38[c,e]
28,[c] 39[c]
not detected
38[c]
ꢀ
39[c]
29[c]
30[c]
31[c]
ꢀ
35, 31[d]
36, 32,[d]
37a, 33a[d]
37b, 33b[d]
not detected
40–43, 34[d]
ꢀ
9
10
11a
11b
12
13
CH3
CH3
CD3
(CH2)2C CH
(CH2)8CH3
ꢀ
23
F
[a] Methyl ester hydrolyzed in vivo to the corresponding acid (see boxed inset in Scheme 2). [b] Major product(s) as deduced by LC-HRMS analysis.
[c] Double peaks (for both sodium and ammonium adducts) detected, possibly arising from isomers/conformers.[16] [d] Detected in minor amounts.
[e] Products of different chain lengths and oxidations states detected in minor amounts.
different retention times, as previously observed[11b] (see the
Supporting Information).
1,3-dicarbonyl functionalization of the chemical probes
led to further unexpected results. From the fermentation of S.
lasaliensis ACP12 (S970A) in the presence of the N-pentynoyl
2-methyl hexanoate substrate 9, the novel putative polyether
derivative 31 was obtained and characterized by HR-MS2
(Table 1, entry 13). This product indicates that the last
ketosynthase (KS) domain of the lasA PKS is capable of
carrying out chain extension with a methyl malonate mimic in
place of the natural malonate, followed by ketoreduction and
dehydration. As a result, the normal chain aromatization does
not take place and a chemically derivatized linear polyether is
generated instead. Oxidized undecaketides such as 35 and 36
were initially identified as the major species generated from
the lasA PKS in wild-type S. lasaliensis grown in the presence
of the methylmalonate mimics 9 and 10 (entries 14 and 15).
Conversely, the use of an engineered strain in conjunction
with a newly optimized feeding protocol for liquid cultures
has herein favored the unprecedented formation of linear
oxidized dodecaketides such as 31. Intriguingly, when the N-
acetyl- and deuteroacetyl-2-fluoro hexanoates 11a and 11b,
respectively, were employed as substrates for S. lasaliensis
ACP12 (S970A), the corresponding fluorinated undecaketi-
des 37a and 37b were identified as the main products off-
loaded from the PKS (entries 16 and 17), with the oxidized
Figure 1. UPLC-HR-MS analyses of the ethyl acetate extracts of S.
lasaliensis ACP12 (S970A) grown in the presence of substrate 8
linear dodecaketides 33a,b barely detectable. No obvious
products were identified from the feeding of S. lasaliensis
(Table 1, entry 7). a) TIC and extracted ion chromatogram traces
ACP12 (S970A) with the N-pentynoyl 2-fluoro substrate 12
showing the presence of the compound 28 (Acquity HSS T3 column,
78-minute gradient). b) HR-MS and c) HR-MS2 of the putative lasalo-
cid derivative 28. The double LC-MS peak for 28 and all the other
putative lasalocid derivatives is under investigation.
(entry 18), whereas from the N-decanoyl 2-fluoro substrate
13, the fluorinated putative polyether 34 was afforded, albeit
in minor amount compared to the short-chain products 40–43
Angew. Chem. Int. Ed. 2014, 53, 1 – 7
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
3
These are not the final page numbers!

.
Angewandte
Communications
(entry 19; all products characterized by HR-MSn, see the
Supporting Information).
Staudinger-phosphite[15] reactions in crude and purified
organic extracts of S. lasaliensis cultures (Figure 2). In
particular, the crude putative lasalocid alkyne derivative 25
was reacted with the N-Boc-cadaverine azide 44 in the
presence of copper(I) to afford the putative lasalocid
conjugate 45. The same transformation was carried out on
HPLC-purified fractions of 25, thus leading to identical click
products (Figure 2a). The putative azide polyethers 26 and 28
were converted into glucose conjugates (e.g., 47) by click
reactions with the alkyne 46, and to phosphoramidates (e.g.,
48) by reaction with trimethyl phosphite (Figures 2b,c).
Overall the compounds 2, 24–28, 31–36 and their deriv-
atives were identified and characterized as putative lasalo-
cid A species by HR-MS2, all displaying the conserved
m/z 377 fragment previously mentioned[11b] and a consistent
LC-MS double-peak pattern. This pattern is possibly due to
isomerization or the adoption of different monomeric/dimeric
structures of the polyethers in complex with sodium.[16]
Because of their formation in limited amounts and the
complexity of the samples background, it has not always been
straightforward to detect the presence of the unnatural
polyketides in the bacterial crude extracts (Figure 1). From
comparative LC-MS analyses with samples of lasalocid A of
These results show for the first time that, by directly
utilizing fluoromalonate surrogate substrates in vivo, fluorine
can be successfully incorporated into advanced polyketide
biosynthetic intermediates and products at specific a-carbon-
yl positions in place of hydrogen atoms, and methyl and ethyl
groups with unexpected consequences on the natural product
assembly. Indeed for the lasA PKS the stage and the extent of
fluorine incorporation is dependent on the N-acyl length of
the probe (Table 1). Additionally, the absence of oxidized
undecaketide species bearing fluorine seems to suggest that 2-
fluorinated undecaketide dienes may not be suitable sub-
strates for epoxidation/epoxide hydrolysis leading to poly-
ether formation. These findings are of particular relevance in
view of engineering the production of fluorinated polyketi-
des[9d,13d] for medicinal chemistry and chemical biology
applications.
Most of the polyketide products arising from these
experiments also bear an alkyne or an azide moiety. To
show how these molecules can be creatively diversified, the
putative lasalocid A derivatives 25, 26, and 28 were utilized as
substrates for azide–alkyne Huisgen cycloaddition[14] and
Figure 2. a) Conversion of the putative lasalocid alkyne derivative 25 into the cadaverine conjugate 45 by copper-catalyzed azide–alkyne
cycloaddition (CuAAC) with the azide 44 (carried out in crude and purified organic extracts, as shown in upper and lower selected LC-MS ion
traces, respectively (Acquity HSS T3 column, 68-minute gradient). b) The putative lasalocid azide derivative 26 is converted into the glucose
conjugate 47 by CuAAC with the alkyne 46 (Acquity HSS T3 column, 68-minute gradient). c) The putative lasalocid azide derivative 28 is converted
into the phosphoramidate 48 by a Staudinger-phosphite reaction (Eclipse C18 column, 35-minute gradient). The presence of a double LC-MS peak
for all the newly generated putative lasalocid derivatives is under investigation.
4
www.angewandte.org
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2014, 53, 1 – 7
These are not the final page numbers!

Angewandte
Chemie
known titre, we have preliminarily estimated the production
of the unnatural polyethers to be in the range of 100–
800 mglÀ1, depending on the probe utilized and in the
conditions here reported (data not shown). Nonetheless, we
have shown here that such diverse and functionalized
products can be straightforwardly generated from the fer-
mentation of a minimally engineered Streptomyces strain in
the presence of varied synthetic malonate surrogates (3–13),
they can be isolated by HPLC purification and they can be
further derivatized (Figure 2).
enhance their production in vivo. The application of the
probes 3–13 for the investigation and exploitation of other
PKS systems will be reported in due course.
Received: July 21, 2014
Published online: && &&, &&&&
Keywords: biosynthesis · enzymes · drug discovery ·
.
polyketides · synthetic methods
To the best of our knowledge, 2, 24–28, 31–36, 45, 47, and
48 together constitute the first example of an unnatural
polyether library characterized by the replacement of the aryl
carboxylate functionality with variable aryl ketones (2, 24–28,
45, 47, and 48) and with linear polyketide chains flanking the
polyether portion of the natural product (31–36). Polyether
ionophores are widely utilized as antibiotics in veterinary
medicine, and they are also rapidly emerging as promising
bioactive molecules for cancer therapy[17] and for the treat-
ment of tropical diseases.[18] Structural modification of
polyethers can change the ability and the selectivity of
metal cations and small-molecule binding.[19] Therefore this
approach may give access to novel molecules of therapeutic
interest, as well as helping illuminate their mechanism of
action.
On the basis of the results reported here, it is also
appealing to envisage the generation of a library of polyketide
derivatives by a novel mutasynthetic approach involving
chain re-initiation of stalled biosynthetic intermediates on
any ketosynthase with a variety of malonyl-ACP mimics. N-
acetyl cysteamine analogues of malonate extender units and
of advanced biosynthetic intermediates are widely utilized in
current mutasynthetic approaches in place of the natural
substrates bound to the pantetheinyl cofactor of acyl carrier
proteins.[8,9,20b] It is now clear that the pantetheinyl portion of
acyl carrier proteins can be replaced by a variety of N-acyl
groups of variable chain length and nature to generate
functionalized polyketides. This feature is of particular
interest in relation to the newly available three-dimensional
structure of a whole PKS module,[20] as molecular modeling
should be now employed to design unnatural malonyl-ACP
analogues capable of best interacting with all the catalytic
partners in the PKS reaction chamber. In light of all these
considerations, we are currently exploring whether the
mutasynthetic approach to the putative lasalocid precursor/
derivative structures reported herein is an effective and
general approach applicable to other PKSs and modular
assembly lines for the generation of novel unnatural products.
In summary, the functionalized chemical probes 3–13
constitute first-hand chemical biology tools for exploring
novel polyketide diversification by providing direct informa-
tion on PKSs tolerance and processing of unnatural malonyl-
ACP analogues, as well as on the amenability of the newly
generated unnatural polyketides to further synthetic modifi-
cations.
[1] D. Newman, G. M. Cragg, J. Nat. Prod. 2012, 75, 311 – 335.
[2] K. J. Weissman, Methods Enzymol. 2009, 459, 3 – 16.
[3] G. T. Carter, Nat. Prod. Rep. 2011, 28, 1783 – 1789.
[4] M. Cummings, R. Breiting, E. Takano, FEMS Microbiol. Lett.
2014, 351, 116 – 125.
[5] J. Cortes, K. E. Wiesmann, G. A. Roberts, M. J. Brown, J.
Staunton, P. F. Leadlay, Science 1995, 268, 1487 – 1489.
[6] M. Oliynyk, M. J. B. Brown, J. Cortꢀs, J. Staunton, P. F. Leadlay,
Chem. Biol. 1996, 3, 833 – 839.
[7] a) C. J. Rowe, I. U. Bçhm, I. P. Thomas, B. Wilkinson, B. A. M.
Rudd, G. Foster, A. P. Blackaby, P. J. Sidebottom, Y. Roddis,
A. D. Buss, J. Staunton, P. F. Leadlay, Chem. Biol. 2001, 8, 475 –
485; b) A. Bhatt, C. B. W. Stark, B. M. Harvey, A. R. Gallimore,
Y. Demydchuk, J. B. Spencer, J. Staunton, P. F. Leadlay, Angew.
Chem. Int. Ed. 2005, 44, 7075 – 7078; Angew. Chem. 2005, 117,
7237 – 7240; c) S. Kushnir, U. Sundermann, S. Yahiaoui, A.
Brockmeyer, P. Janning, F. Schulz, Angew. Chem. Int. Ed. 2012,
51, 10664 – 10669; Angew. Chem. 2012, 124, 10820 – 10825; d) L.
Laureti, L. Song, S. Huang, C. Corre, P. Leblond, G. L. Challis, B.
Aigle, Proc. Natl. Acad. Sci. USA 2011, 108, 6258 – 6263.
[8] a) R. J. M. Goss, S. Shankar, A. Abou Fayad, Nat. Prod. Rep.
2012, 29, 870 – 889; b) A. Kirschning, F. Hahn, Angew. Chem. Int.
Ed. 2012, 51, 4012 – 4022; Angew. Chem. 2012, 124, 4086 – 4096.
[9] a) T. Knobloch, K. Harmrolfs, F. Taft, B. Thomaszewski, F. Sasse,
A. Kirschning, ChemBioChem 2011, 12, 540 – 547; b) J. M.
Winter, G. Chiou, I. R. Bothwell, W. Xu, N. K. Garg, M. Luo,
Y. Tang, Org. Lett. 2013, 15, 3774 – 3777; c) I. Koryakina, J. B.
McArthur, M. M. Draelos, G. J. Williams, Org. Biomol. Chem.
2013, 11, 4449 – 4458; d) M. C. Walker, B. W. Thuronyi, L. K.
Charkoudian, B. Lowry, C. Khosla, M. C. Y. Chang, Science
2013, 341, 1089 – 1094; e) U. Sundermann, K. Bravo-Rodriguez,
S. Klopries, S. Kushnir, H. Gomez, E. Sanchez-Garcia, F. Schulz,
ACS Chem. Biol. 2013, 8, 443 – 450.
[10] a) M. Tosin, D. Spiteller, J. B. Spencer, ChemBioChem 2009, 10,
1714 – 1723; b) M. Tosin, L. Betancor, E. Stephens, W. M. A. Li,
J. B. Spencer, P. F. Leadlay, ChemBioChem 2010, 11, 539 – 546.
[11] a) M. Tosin, Y. Demydchuk, J. S. Parascandolo, C. Blasco-Per,
F. J. Leeper, P. F. Leadlay, Chem. Commun. 2011, 47, 3460 –
3462; b) M. Tosin, L. Smith, P. F. Leadlay, Angew. Chem. Int.
Ed. 2011, 50, 11930 – 11933; Angew. Chem. 2011, 123, 12136 –
12139.
[12] a) E. M. Sletten, C. R. Bertozzi, Angew. Chem. Int. Ed. 2009, 48,
6974 – 6998; Angew. Chem. 2009, 121, 7108 – 7133; b) M. Gram-
mel, H. C. Hang, Nat. Chem. Biol. 2013, 9, 475 – 484.
[13] a) S. Purser, P. R. Moore, S. Swallow, V. Gouverneur, Chem. Soc.
Rev. 2008, 37, 320 – 330; b) J. Wang, M. Sꢁnchez-Rosellꢂ, J. L.
AceÇa, C. del Pozo, A. E. Sorochinsky, S. Fustero, V. A. Solo-
shonok, H. Liu, Chem. Rev. 2014, 114, 2432 – 2506; c) I. Ojima, J.
Org. Chem. 2013, 78, 6358 – 6383; d) M. C. Walker, M. C. Y.
Chang, Chem. Soc. Rev. 2014, 43, 6527 – 6536.
[14] V. V. Rostovtsev, L. G. Green, V. V. Fokin, K. B. Sharpless,
Angew. Chem. Int. Ed. 2002, 41, 2596 – 2599; Angew. Chem.
2002, 114, 2708 – 2711.
[15] a) R. Serwa, I. Wilkening, G. Del Signore, M. Mꢃhlberg, I.
Claußnitzer, C. Weise, M. Gerrits, C. P. R. Hackenberger,
Our current work is focusing on the large-scale isolation
of the novel polyketide derivatives reported here, their full
characterization by NMR spectroscopy, and the evaluation of
their bioactivity, as well as on the development of tools to
Angew. Chem. Int. Ed. 2014, 53, 1 – 7
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!

.
Angewandte
Communications
Angew. Chem. Int. Ed. 2009, 48, 8234 – 8239; Angew. Chem.
2009, 121, 8382 – 8387; b) V. Bçhrsch, T. Mathew, M. Zieringer,
M. R. J. Vallꢀe, L. M. Artner, J. Dernedde, R. Haag, C. P. R.
Hackenberger, Org. Biomol. Chem. 2012, 10, 6211 – 6216; c) C. I.
Schilling, N. Jung, M. Biskup, U. Schepers, S. Brꢄse, Chem. Soc.
Rev. 2011, 40, 4840 – 4871.
[18] a) M.-H. Abdulla, D. S. Ruelas, B. Wolff, J. Snedecor, K.-C. Lim,
F. Xu, A. R. Renslo, J. Williams, J. H. McKerrow, C. R. Caffrey,
PLoS Neglected Trop. Dis. 2009, 3, e478; b) J. Adovelande, J.
Schrꢀvel, Life Sci. 1996, 59, PL309 – 315.
[19] a) W. Hꢃttel, J. B. Spencer, P. F. Leadlay, Beilstein J. Org. Chem.
2014, 10, 361 – 368; b) J. Rutkowski, B. Brzezinski, BioMed. Res.
Int. 2013, DOI: 10.1155/2013/162513.
[20] a) S. Dutta, J. R. Whicher, D. A. Hansen, W. A. Hale, J. A.
Chemler, G. R. Condgon, A. R. H. Narayan, K. Hꢅkansson,
D. H. Sherman, J. L. Smith, G. Skiniotis, Nature 2014, 510, 512 –
517; b) J. R. Whicher, S. Dutta, D. A. Hansen, W. A. Hale, J. A.
Chemler, A. M. Dosey, A. R. H. Narayan, K. Hꢅkansson, D. H.
Sherman, J. L. Smith, G. Skiniotis, Nature 2014, 510, 560 – 564.
[16] a) P. G. Schmidt, A. H.-J. Wang, I. C. Paul, J. Am. Chem. Soc.
1974, 96, 6189 – 6191; b) G. D. Schmidt, W. L. Duax, S. Fortier, J.
Am. Chem. Soc. 1978, 100, 6725 – 6727; c) R. Lyazghi, A. Cuer,
G. Dauphin, J. Juillard, J. Chem. Soc. Perkin Trans. 2 1992, 32 –
42.
[17] a) A. Huczynski, J. Rutkowski, I. Borowicz, J. Wietrzyk, E. Maj,
B. Brzezinski, Bioorg. Med. Chem. Lett. 2013, 23, 5053 – 5056;
b) P. B. Gupta, T. T. Onder, G. Jiang, K. Tao, C. Kuperwasser,
R. A. Weinberg, E. S. Lander, Cell 2009, 138, 645 – 659.
6
www.angewandte.org
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2014, 53, 1 – 7
These are not the final page numbers!

Angewandte
Chemie
Communications
Biosynthesis
E. Riva, I. Wilkening, S. Gazzola,
W. M. A. Li, L. Smith, P. F. Leadlay,
M. Tosin*
&&&& — &&&&
Chemical Probes for the
Functionalization of Polyketide
Intermediates
Towards novel “unnatural” products:
Fermentation of ACP mutants of S.
lasaliensis in the presence of functional-
ized chemical probes with subsequent
site-selective modifications generates
a library of nonnatural lasalocid A pre-
cursors and derivatives.
Angew. Chem. Int. Ed. 2014, 53, 1 – 7
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
7
These are not the final page numbers!