A general scheme for synthesis of substrate-based polyketide labels for acyl carrier proteins

Article abstract of DOI:10.1016/j.bmcl.2010.05.089

A general strategy to enzymatically label acyl carrier proteins (ACPs) of polyketide synthases has been developed. Incorporation of a chloromethyl ketone or vinyl ketone moiety into polyketide chain elongation intermediate mimics allows for the synthesis of CoA adducts. These CoA adducts undergo enzymatic reaction with Sfp, a phosphopantetheinyl transferase, to afford labeled CurB carrier proteins.

Full text of DOI:10.1016/j.bmcl.2010.05.089

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5939–5942
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A general scheme for synthesis of substrate-based polyketide labels for
acyl carrier proteins
Erick K. Leggans a, David L. Akey b, Janet L. Smith b, Robert A. Fecik a,
*
a Department of Medicinal Chemistry, University of Minnesota, 717 Delaware Street S.E., Room 456, Minneapolis, MN 55414-2959, USA
b Life Sciences Institute and Department of Biological Chemistry, University of Michigan, 210 Washtenaw Avenue, Ann Arbor, MI 48109-2216, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A general strategy to enzymatically label acyl carrier proteins (ACPs) of polyketide synthases has been
developed. Incorporation of a chloromethyl ketone or vinyl ketone moiety into polyketide chain elonga-
tion intermediate mimics allows for the synthesis of CoA adducts. These CoA adducts undergo enzymatic
reaction with Sfp, a phosphopantetheinyl transferase, to afford labeled CurB carrier proteins.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 27 April 2010
Revised 19 May 2010
Accepted 21 May 2010
Available online 25 May 2010
Keywords:
Acyl carrier proteins
Labels
Biosynthesis
Polyketides
The engineering of natural product biosynthetic pathways
through combinatorial biosynthesis holds great promise for the
discovery of new biologically active lead compounds. Polyketide
natural products continue to be important for drug discovery,
and >10% of the top 200 drugs by prescriptions dispensed in the
United States were of polyketide origin.1,2 Modular polyketide syn-
thases (PKSs) are well suited for combinatorial biosynthesis be-
cause of the rich chemical and biological diversity of polyketides.
However, little is known about the structural basis for substrate
specificity and stereochemical outcome for the individual enzy-
matic reactions of PKSs. We previously demonstrated the power
of affinity labels that mimic the substrates of pikromycin thioester-
ase (Pik TE) to determine its mechanism of macrolactonization.3,4
Labels for acyl carrier protein (ACP) domains could be effective
tools for study of substrate specificity and stereochemistry of other
PKS catalytic domains because ACP domains are the natural deliv-
ery systems for PKS substrates.
Whereas FAS operates through an iterative process, type I PKSs
are modular and have individual enzymatic domains for each reac-
tion of the pathway.5 Each module catalyzes the condensation and
modification of one ketide building ‘block’ and contains at mini-
mum a ketosynthase (KS), acyltransferase (AT), and ACP domain.
Additional b-processing domains may be present, including ketore-
ductase (KR), dehydratase (DH), and enoyl reductase (ER). Each
ACP of a type I PKS system passes the chain elongation intermedi-
ate to the KS domain of the next module. A thioesterase (TE)
domain releases the final intermediate from the PKS.
Lack of information about the structural determinants of sub-
strate specificity for nearly all catalytic domains of PKSs is a crucial
barrier for combinatorial biosynthesis. Three-dimensional struc-
tures of domains from modular PKSs have only recently been
reported. These include two KS–AT didomains,6,7 two KR domains,8,9
five DH domains,10,11 two TE domains,3,4,12,13 an ACP domain,14
a
pair of fused docking domains,15 and two non-classical domains.16,17
Among these, only Pik TE has an analogs of a chain elongation inter-
PKSs make polyketides in a manner similar to fatty acid con-
struction by human fatty acid synthase (FAS). The polyketide chain
elongation intermediate is linked to the phosphopantetheine group
of an ACP. Each intermediate is extended by a two- or three-carbon
building block, and then modified by other enzymatic domains.
mediate bound in the active site3,4
: incorporation of diph-
enylphosphonate groups led to covalent modification of the active
siteSer residue. These affinitylabeled structuresmimicked thecova-
lent reaction intermediates, providing unprecedented insights to
the mechanism and substrate specificity of Pik TE.
The covalent coupling of polyketide chain elongation intermedi-
ates to tethered ACP domains ensures a high effective concentra-
tion of these substrates with respect to the catalytic domains
and, conversely, decreases the pressure to ensure a low ‘natural’
Km. In addition, some substrate specificity of catalytic domains
may be due to interactions with ACP domains although origins of
Abbreviations: ACP, acyl carrier protein; AT, acyltransferase; CoA/CoASH,
coenzyme A; DEBS, 6-deoxyerythronolid B synthase; DH, dehydratase; ER, enoyl
reductase; FAS, fatty acid synthase; KR, ketoreductase; KS, ketosynthase; NRPS,
nonribosomal peptide synthetase; Pik TE, pikromycin thioesterase; PKS, polyketide
synthase; PPTase, phosphopantetheinyl transferase; TE, thioesterase.
* Corresponding author. Tel.: +1 612 625 9335; fax: +1 612 626 6318.
E-mail address: fecik001@umn.edu (R.A. Fecik).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.089

5940
E. K. Leggans et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5939–5942
specificity are not clear.18,19 Thus, solution techniques may be inef-
fective for trapping substrates or analogs within catalytic domains.
PKS labels can help to overcome these obstacles. The capture of
substrate mimics delivered to virtually all PKS domains is possible
using ACP-bound labels. Electrophilic CoASH derivatives have been
reported by others to be useful in labeling carrier protein domains
for crosslinking to TE20 and KS21–23 domains. Liu and Bruner cou-
pled amino-CoA with chloroacetic acid to give a CoA derivative
that inhibited the catalytic activity of the TE domain of EntF.20
Burkart and coworkers have used the chemoenzymatic synthesis
of amino-CoA analogs with electrophilic groups (primarily vinyl
chlorides) to crosslink ACP and KS domains of the 6-deoxyerython-
olide B synthase (DEBS) PKS, type II FAS, and type II PKS systems.21–
23
This approach is useful for trapping and studying protein–pro-
tein interactions between ACP and catalytic domains. The cross-
linking systems, however, lack incorporation of PKS-like
substrates targeted to the active sites of the domain they modify,
and the degree of crosslinking varies widely.23 A method for the
acylation of ACPs with simple b-lactones was recently reported,
however, at the large excess concentration of b-lactone required
for >90% ACP loading (50 equiv) the selectivity for ACP domains
versus a KS–AT didomain is low (ꢀ1.5:1).24 Additionally, the load-
ing of a b-lactone with a C2-methyl group present in many polyke-
tide chain elongation intermediates onto ACPs was low (ꢀ30%).24
Here we describe the design and synthesis of two new classes of
polyketide-based ACP labels, halomethyl ketones and vinyl ke-
tones, that can be readily incorporated into mimics of polyketide
chain elongation intermediates. A similar chloromethyl ketone-de-
rived CoA analog was used to trap a biosynthetic intermediate in a
type III stilbene synthase pathway.25 These compounds were de-
signed as labels for ACP domains with a substrate recognition ele-
ment for delivery to the catalytic sites of other PKS domains. We
show that both classes of labels react with CoASH, and we demon-
strate enzymatic labeling of a model ACP, CurB.
Scheme 1 depicts our general strategy for the labeling of ACPs. A
phosphopantetheine linker derived from CoASH serves as the point
of attachment for polyketide chain elongation intermediates. The
sulfhydryl group of CoASH (1) is modified by SN2 reaction with a
chloromethyl ketone affinity label (2) or by Michael addition to a
vinyl ketone affinity label (3) to give stable, non-hydrolyzable
CoA derivatives 4 or 5. The labels are mimics of the substrates
and products of a particular PKS catalytic domain. Similar Michael
reactions of CoASH are known,26 and modifications of the sulfhy-
dryl group of CoASH have also been reported.27,28 Each CoA deriv-
Scheme 1. General strategy for covalent modification of ACP domains by polyke-
tide-based labels.
ative (4 or 5) is then attached enzymatically to
a non-
phosphopantetheinylated ACP (apo-form) by the promiscuous
phosphopantetheinyl transferase (PPTase) Sfp.29
We sought to establish this approach through the synthesis of
two model labels and CoA adducts (Scheme 2). The model chloro-
methyl ketone label we chose was chloroacetone (8). Chloroace-
tone smoothly reacted with the sulfhydryl group of CoASH to
give CoA adduct 9. A more complex model system was employed
for the vinyl ketone labels. Synthesis of the second model label
began with aldol product 10, an intermediate used in our synthesis
of Pik TE affinity labels.4 The secondary alcohol was protected as
silyl ether 11, followed by displacement of the thiazolidinethione
chiral auxiliary to afford Weinreb amide 12. Ketone formation with
vinylmagnesium bromide and deprotection gave vinyl ketone 14.
Attempts to form vinyl ketone 13 directly from silyl ether 11 were
unsuccessful. Michael addition with CoASH completed the synthe-
sis of CoA adduct 15. Compound 15 mimics the C1–C7 segment of
the hexaketide chain elongation intermediate of pikromycin.
We next examined the ability of Sfp, a non-specific PPTase from
Bacillus subtilis, to modify the apo-form of a representative ACP. We
validated this strategy with Lyngbya majuscula CurB, a stand-alone
ACP from the curacin biosynthetic pathway.30 CurB was quantita-
Scheme 2. Synthesis of CoA adducts 9 and 15.
tively modified by both CoA adducts as determined by mass spec-
trometry to give CurB adducts 16 and 17 (Scheme 3 and Figs. 1, S1,
S2). No unlabeled CurB was detected. The mass spectra of both

E. K. Leggans et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5939–5942
5941
CurB derivatives contain a phosphopantetheine group labeled with
a non-hydrolyzable polyketide chain elongation intermediate mi-
mic. Key features are the incorporation of stable chain elongation
intermediate mimics into ACP and the relative ease of synthesis
of the required vinyl ketone precursors.
We expect this labeling method to be broadly useful for the
modification of more complex PKS systems, such as full PKS mod-
ules. Labeled constructs could be a powerful means to trap ACP-
bound chain elongation intermediate mimics in the active sites
of catalytic domains. The use of these and similar ACP labels in
the study of ACP-containing PKS systems will be the subject of
future investigations. This labeling technique also can be adapted
readily to the study of mechanistically similar FAS and nonriboso-
mal peptide synthetases (NRPSs) to expand their utility for a large
number of primary and secondary metabolic pathways important
for diseases and for combinatorial biosynthesis.
Scheme 3. Enzymatic labeling of the ACP CurB with CoA adducts 9 and 15.
Acknowledgments
The authors thank Liangcai Gu and David Sherman for plasmid
pET28b::CurB encoding CurB, K. R. Noon (Biomedical Mass
Spectrometry Facility, University of Michigan) for assistance with
LC–MS, and Jamie Razelun for preparation of CurB. Financial
support from NIH DK-42303 (to J.L.S.) and NIH GM081544-01 (to
J.L.S. and R.A.F.) is gratefully acknowledged.
Supplementary data
Experimental procedures and spectral data for all new com-
pounds and CurB production and labeling, and copies of 1H and
13C NMR spectra for compounds 11–14. Supplementary data asso-
ciated with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2010.05.089.
References and notes
1. Butler, M. S. Nat. Prod. Rep. 2008, 25, 475.
2. Lamb, E. Pharm. Times 2009, 75, 26.
3. Giraldes, J. W.; Akey, D. L.; Kittendorf, J. D.; Sherman, D. H.; Smith, J. L.; Fecik, R.
A. Nat. Chem. Biol. 2006, 2, 531.
4. Akey, D. L.; Kittendorf, J. D.; Giraldes, J. W.; Fecik, R. A.; Sherman, D. H.; Smith, J.
L. Nat. Chem. Biol. 2006, 2, 537.
5. McDaniel, R.; Welch, M.; Hutchinson, C. R. Chem. Rev. 2005, 105, 543. and
references therein.
Figure 1. Deconvoluted mass spectra of apo-CurB (A) and of CurB adduct 16 (B).
Unlabeled protein is observed with masses of 10841.9 Da for CurB (calculated mass
10842.5 Da) and 11019.6 Da for
a-N-gluconyl-CurB (calculated mass 11020.6 Da).
The PPTase reaction catalyzed by Sfp quantitatively labeled both forms of CurB,
6. Tang, Y.; Kim, C.-Y.; Mathews, I. I.; Cane, D. E.; Khosla, C. Proc. Natl. Acad. Sci.
U.S.A. 2006, 103, 11124.
with mass shifts of 393.5 Da for CurB and 393.8 Da for
(calculated shift 395.4 Da).
a-N-gluconyl-CurB
7. Tang, Y.; Chen, A. Y.; Kim, C.-Y.; Cane, D. E.; Khosla, C. Chem. Biol. 2007, 14, 931.
8. Keatinge-Clay, A. T.; Stroud, R. M. Structure 2006, 14, 737.
9. Keatinge-Clay, A. T. Chem. Biol. 2007, 14, 898.
10. Keatinge-Clay, A. J. Mol. Biol. 2008, 384, 941.
11. Akey, D. L.; Razelun, J. R.; Tehranisa, J.; Sherman, D. H.; Gerwick, W. H.; Smith, J.
L. Structure 2010, 18, 94.
12. Tsai, S.-C.; Miercke, L. J. W.; Krucinski, J.; Gokhale, R.; Chen, J. C.-H.; Foster, P.
G.; Cane, D. E.; Khosla, C.; Stroud, R. M. Proc. Natl. Acad. Sci. U.S.A. 2001, 98,
14808.
13. Tsai, S.-C.; Lu, H.; Cane, D. E.; Khosla, C.; Stroud, R. M. Biochemistry 2002, 41,
12598.
14. Alekseyev, V. Y.; Liu, C. W.; Cane, D. E.; Puglisi, J. D.; Khosla, C. Protein Sci. 2007,
16, 2093.
15. Broadhurst, R. W.; Nietlispach, D.; Wheatcroft, M. P.; Leadley, P. F.; Weissman,
K. J. Chem. Biol. 2003, 10, 723.
16. Gu, L.; Geders, T. W.; Wang, B.; Gerwick, W. H.; Hakansson, K.; Smith, J. L.;
Sherman, D. H. Science 2007, 318, 970.
17. Geders, T. W.; Gu, L.; Mowers, J. C.; Liu, H.; Gerwick, W. H.; Hakansson, K.;
Sherman, D. H.; Smith, J. L. J. Biol. Chem. 2007, 282, 35954.
18. Castonguay, R.; He, W.; Chen, A. Y.; Khosla, C.; Cane, D. E. J. Am. Chem. Soc. 2007,
129, 13758.
unmodified and modified CurB showed two peaks in the labeling
experiment with CoA adduct 9: one corresponding to the protein
and another corresponding to the protein +178 Da. This additional
mass is likely due to
a-N-gluconyl posttranslational modification
of the N-terminal hexahistidine-tag. Others have noted similar
posttranslational modification of several His-tagged proteins ex-
pressed in Escherichia coli.31–33 The CurB protein used for the label-
ing experiment with CoA adduct 15 showed no posttranslational
modification (Fig. S2). This sample was obtained from a separate
purification experiment, and the reason for differences in post-
translational modification between CurB samples is not known.
In our experiments, CurB in both its a-N-gluconyl posttranslation-
ally modified and unmodified forms was quantitatively labeled
with CoASH analogs 9 and 15.
In summary, we have developed a general strategy for the de-
sign and synthesis of labels for ACP domains of PKS systems. Incor-
poration of a chloromethyl ketone or vinyl ketone group into a
polyketide chain elongation intermediate mimic allows for reac-
tion with CoASH. The resulting CoA adducts then undergo enzy-
matic reaction with Sfp to modify apo-CurB, a typical ACP. These
19. Chen, A. Y.; Cane, D. E.; Khosla, C. Chem. Biol. 2007, 14, 784.
20. Liu, Y.; Bruner, S. D. ChemBioChem 2007, 8, 617.
21. Worthington, A. S.; Rivera, H., Jr.; Torpey, J. W.; Alexander, M. D.; Burkart, M. D.
ACS Chem. Biol. 2006, 1, 687.
22. Kapur, S.; Worthington, A.; Tang, Y.; Cane, D. E.; Burkart, M. D.; Khosla, C.
Bioorg. Med. Chem. Lett. 2008, 18, 3034.
23. Worthington, A. S.; Hur, G. H.; Meier, J. L.; Cheng, Q.; Moore, B. S.; Burkart, M. D.
ChemBioChem 2008, 9, 2096.

5942
E. K. Leggans et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5939–5942
24. Amoroso, J. W.; Borketey, L. S.; Prasad, G.; Schnarr, N. A. Org. Lett. 2010, 12,
2330.
30. Gu, L.; Jia, J.; Liu, H.; Håkansson, K.; Gerwick, W. H.; Sherman, D. H. J. Am. Chem.
Soc. 2006, 128, 9014.
25. Spiteller, D.; Waterman, C. L.; Spencer, J. B. Angew. Chem., Int. Ed. 2005, 44,
7079.
26. La Clair, J. J.; Foley, T. L.; Schegg, T. R.; Regan, C. M.; Burkart, M. D. Chem. Biol.
2004, 11, 195.
27. Yin, J.; Straight, P. D.; Hrvatin, S.; Dorrestein, P. C.; Bumpus, S. B.; Jao, C.;
Kelleher, N. L.; Kolter, R.; Walsh, C. T. Chem. Biol. 2007, 14, 303.
28. Belshaw, P. J.; Walsh, C. T.; Stachelhaus, T. Science 1999, 284, 486.
29. Lambalot, R. H.; Gehring, A. M.; Flugel, R. S.; Zuber, P.; LaCelle, M.; Marahiel, M.
A.; Reid, R.; Khosla, C.; Walsh, C. T. Chem. Biol. 1996, 3, 923. and references
therein.
31. Geoghegan, K. F.; Dixon, H. B. F.; Rosner, P. J.; Hoth, L. R.; Lanzetti, A. J.;
Borzilleri, K. A.; Marr, E. S.; Pezzullo, L. H.; Martin, L. B.; LeMotte, P. K.; McColl,
A. S.; Kamath, A. V.; Stroh, J. G. Anal. Biochem. 1999, 267, 169.
32. Dorrestein, P. C.; Van Lanen, S. G.; Li, W.; Zhao, C.; Deng, Z.; Shen, B.; Kelleher,
N. L. J. Am. Chem. Soc. 2006, 128, 10386.
33. Sun, Y.; Hong, H.; Gillies, F.; Spencer, J. B.; Leadlay, P. F. ChemBioChem 2008, 9,
150.