Direct acylation of carrier proteins with functionalized β-lactones

Article abstract of DOI:10.1021/ol100684s

As the key component of many biosynthetic assemblies, acyl-carrier proteins offer a robust entry point for introduction of small molecule probes and pathway intermediates. Current labeling strategies primarily rely on modifications to the phosphopantethei

Full text of DOI:10.1021/ol100684s

ORGANIC
LETTERS
2
010
Vol. 12, No. 10
330-2333
Direct Acylation of Carrier Proteins with
Functionalized ꢀ-Lactones
2
Jon W. Amoroso, Lawrence S. Borketey, Gitanjeli Prasad, and
Nathan A. Schnarr*
Department of Chemistry, UniVersity of Massachusetts, 710 North Pleasant Street,
Amherst, Massachusetts 01003
schnarr@chem.umass.edu
Received March 23, 2010
ABSTRACT
As the key component of many biosynthetic assemblies, acyl-carrier proteins offer a robust entry point for introduction of small molecule
probes and pathway intermediates. Current labeling strategies primarily rely on modifications to the phosphopantetheine cofactor or its
biosynthetic precursors followed by attachment to the apo form of a given carrier protein. As a greatly simplified alternative, direct and
selective acylation of holo-acyl-carrier proteins using readily accessible ꢀ-lactones as electrophilic partners for the phosphopantetheine-thiol
has been demonstrated.
Site-specific labeling of intact proteins remains a formidable
challenge largely due to the multitude of potential nucleo-
philic sites in most biomacromolecules. ꢀ-Lactones have
received significant attention as electrophilic tagging agents
transferase (AT), ketoreductase (KR), dehydratase (DH),
enoyl reductase (ER), and in some cases thioesterase (TE)
domains during polyketide maturation. To function, each
apo-ACP is posttranslationally modified with a coenzyme
A-derived PPant group to produce holo-ACP. The specific
transferases responsible for this transformation have continu-
ally displayed broad tolerance for acyl groups on the terminal
1,2
capable of reacting with typical amino acid functionalities.
Our interest in polyketide biosynthesis has spurred questions
of whether these structures might be used to directly label
acyl carrier proteins (ACPs) via the phosphopantetheine
4
thiol. As a result, several groups have taken advantage of
(
Ppant)-thiol. ꢀ-Hydroxythioesters, formed by addition of the
this for loading ACPs with a variety of substrates. However,
we reasoned that the Ppant group may be uniquely reactive,
thiol to a ꢀ-lactone, are common components of polyketide
intermediates rendering this strategy particularly appealing.
Acyl carrier proteins, the primary point of attachment for
the growing polyketide chain, are inarguably the workhorse
of modular polyketide synthases (PKSs). They must rec-
(3) Khosla, C.; Tang, Y.; Chen, A. Y.; Schnarr, N. A.; Cane, D. E. Annu.
ReV. Biochem. 2007, 76, 195.
(
4) (a) Mercer, A. C.; Meier, J. L.; Torpey, J. W.; Burkart, M. D.
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ChemBioChem 2009, 10, 1091. (b) Kapur, S.; Worthington, A.; Tang, Y.;
Cane, D. E.; Burkart, M. D.; Khosla, C. Bioorg. Med. Chem. Lett. 2008,
ognize and properly associate with ketosynthase (KS), acyl
1
8, 3034. (c) Worthington, A. S.; Rivera, H.; Torpey, J. W.; Alexander,
M. D.; Burkart, M. D. ACS Chem. Biol. 2006, 1, 687. (d) Meier, J. L.;
Mercer, A. C.; Rivera, H.; Burkart, M. D. J. Am. Chem. Soc. 2006, 128,
12174. (e) Clarke, K. M.; Mercer, A. C.; La Clair, J. J.; Burkart, M. D.
J. Am. Chem. Soc. 2005, 127, 11234. (f) Sieber, S. A.; Walsh, C. T.;
Marahiel, M. A. J. Am. Chem. Soc. 2003, 125, 10862.
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1) (a) Drahl, C.; Cravatt, B. F.; Sorensen, E. J. Angew Chem., Int. Ed.
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N. K.; Han, J. H. Bioorg. Med. Chem. 2002, 10, 2553
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2
.
(
.
10.1021/ol100684s  2010 American Chemical Society
Published on Web 04/30/2010

8
given an appropriate electrophilic partner, and therefore,
targetable for direct acylation. As we ideally wished to
maintain polyketide-like functionality in the product, ꢀ-lac-
tones seemed most suitable. The work described herein
provides a greatly simplified alternative to traditional means
of selectively loading ACPs.
The simplest test of selective Ppant acylation is to expose
our reagents to isolated holo- and apo-ACP domains.
Successful agents will exhibit selectivity for the holo
structures, without nonspecific acylation of the apo ones. To
begin assessing our direct acylation approach, an alkyne-
bearing ꢀ-lactone, compound 1 (Figure 1), was prepared
small molecules in vitro. Thorough trypsinolysis of apo-
and holo-ACPs produced a series of peptide fragments
including one containing the conserved DSL motif, to which
the Ppant is attached in samples derived from BAP1 as
determined by LC-MS (see the Supporting Information).
Incubation of holo-ACP2 and -3 with varied equivalents of
1 at pH 7 for 1 h followed by exhaustive trypsinolysis and
LC-MS, to determine the fraction acylated, produced satura-
tion curves which indicated that a roughly 50-fold excess of
lactone is required for complete acylation (Figure 2).
Figure 2
mass spectrometry for ACP3 (yellow), ACP2 (blue), and KS-AT6
red) with compound 1. Equivalents of lactone are per protein
molecule. Each data point is the average of three experiments.
Standard deviations are shown as black error bars. Lines are added
for clarity.
. Saturation curves as determined by tandem proteolysis-
Figure 1
acyl-carrier proteins.
. Structures of ꢀ-lactones prepared for direct acylation of
(
5
according to the method of Nelson and co-workers. Isolated
ACP domains from modules 2 (ACP2) and 3 (ACP3) of the
6
-deoxyerythronolide B synthase (DEBS) were overex-
Gratifyingly, both apo-ACPs showed no acylation with 50
equiv of lactone, providing further evidence that the reaction
is occurring on the Ppant arm as this is the only difference
between the apo and holo forms of ACP (see the Supporting
Information). The analogous saturation curve for KS-AT6
showed 25-50% less acylation than ACP for the range of
lactone equivalents tested. Most importantly, at 10 lactone
equivalents, the ACPs were approximately 35-50% loaded
while the KS was mostly unreactive after 1 h of incubation.
Standard deviations for these experiments were generally
within 1-4% suggesting that the differential reactivity
observed was genuine.
pressed and purified in both apo and holo forms, using BL21
and BAP1 cells, repectively.
Of course, to be truly useful, our probes must avoid
reaction with nucleophiles present on other common PKS
domains. Since KS active site cysteines have been previously
shown to be competent nucleophiles for ꢀ-lactone ring-
opening, a KS-AT didomain from DEBS module 6 (KS-
AT6) was overexpressed and purified as previously reported.
We were encouraged by the previous report of Sieber and
co-workers where minimal reactivity between compound 1
and the KS domain of bacterial fatty acid synthase was
6
7
6
observed. It remained to be seen, however, whether the
While tandem proteolysis/LC-MS confirms acylation of
the PPant group, not all proteolysis products readily ionize
under the conditions used. In addition, we could not rule
out the potential for acylation of the peptide fragments over
the intact protein. Therefore, an alternative, gel-based assay
was designed according to previous work by Sieber and co-
purified DEBS KS domain would behave in a similar fashion.
The stage was now set to examine both efficiency and
selectivity of ACP-acylation with ꢀ-lactones.
Tandem proteolysis-mass spectrometry has been shown
previously to be a powerful means of detecting PKS-bound
6
workers. Each ACP (apo and holo) and KS-AT6 was
(
5) Nelson, S. G.; Wan, Z.; Peelen, T. J.; Spencer, K. L. Tetrahedron
Lett. 1999, 40, 6535–6539.
(
(
6) Bottcher, T.; Sieber, S. A. Angew. Chem., Int. Ed. 2008, 47, 4600.
(8) (a) Schnarr, N. A.; Chen, A. Y.; Cane, D. E.; Khosla, C. Biochemistry
2005, 44, 11836. (b) Hicks, L. M.; O’Connor, S. E.; Mazur, M. T.; Walsh,
C. T.; Kelleher, N. L. Chem. Biol. 2004, 11, 327. (c) Hong, H.; Appleyard,
A. N.; Siskos, A. P.; Garcia-Bernardo, J.; Staunton, J.; Leadlay, P. F. FEBS
J. 2005, 272, 2373.
7) (a) Kim, C.-Y.; Alekeyev, V. Y.; Chen, A. Y.; Tang, Y.; Cane, D. E.;
Khosla, C. Biochemistry 2004, 43, 13892–13898. (b) Chen, A. Y.; Schnarr,
N. A.; Kim, C.-Y.; Cane, D. E.; Khosla, C. J. Am. Chem. Soc. 2006, 128,
3
067–3074.
Org. Lett., Vol. 12, No. 10, 2010
2331

incubated for 1 h with 10 equiv of 1 followed by 1,3-dipolar
cycloaddition (“click”) reaction with rhodamine azide. SDS-
PAGE analysis of the resulting mixtures showed bright
fluorescent bands corresponding to holo-ACP2 and -3 while
the apo-ACPs and KS-AT6 displayed only background
fluorescence (Figure 3). Not only does this provide initial
assembly. Both apo and holo forms of spinosyn module 2
were incubated for 1 h with 10, 50, and 75 equiv of 1
followed by “click” reaction with rhodamine azide and PAGE
analysis as before (Figure 4). Again, the only difference
9
Figure 3
. PAGE analysis of DEBS ACP3 (apo and holo), ACP2
(
apo and holo), KS-AT3, and KS-AT6 reaction with compound 1
and subsequent click reaction with rhodamine-azide. Lanes are
marked above with the corresponding protein component. Markers
to the left indicate expected bands for the indicated species. The
right two lanes depict transfer of the acylation product from
preloaded ACP to KSAT indicating that the intermediates are
functional. ꢀ-Lactone was applied at 10× relative to protein unless
otherwise noted. ACP2 contains a C-terminal linker region ac-
counting for its larger mass.
Figure 4. SDS-PAGE analysis of SpnB (apo and holo) reaction
with 10, 50, and 75 equiv of compound 1 and subsequent click
reaction with rhodamine-azide. Lanes are marked above with the
corresponding protein component and equivalents of lactone.
between apo and holo forms was the absence or presence of
the Ppant group, respectively. To our satisfaction, only the
holo-protein showed significant fluorescence indicating that
Ppant-thiol is the only competent partner for acylation among
a multitude of alternative nucleophiles. Interestingly, minimal
acylation of the apo form was observed even at very high
equivalents of lactone.
With increased confidence that tandem proteolysis-mass
spectrometry provides an accurate means of evaluating ACP
and KS acylation, we began examination of a diverse panel
of ꢀ-lactone structures. Compounds 2-5 were added to holo-
ACP2 and -3 as well as KS-AT6 at 10- and 50-fold excess.
Proteolysis and LC-MS anaylses were performed after 1 h
of incubation as above. Acylation efficiencies, calculated as
percent loading, were obtained from peak heights of acylated
peptides divided by the total peptide (acylated plus unreacted)
peak heights for a given LC-MS run (Table 1).
While all of the compounds tested acylate both ACP and
KS to some degree, an interesting trend emerges from this
study. The monosubstituted, alkyl-lactones, compounds 1-3,
are quite reactive toward both ACPs tested. In contrast, the
disubstituted lactone, compound 5, shows little ACP-acyla-
tion activity even at 50 equiv. KS-AT6, on the other hand,
is more reactive than ACP2 and -3 toward compound 5 but
significantly less reactive with compounds 1-3 than the
ACPs. Finally, the monosubstitued, aromatic lactone, com-
pound 4, reacts slugishly with both ACP and KS. These data
offer promising preliminary evidence that ꢀ-lactones can
indication that ꢀ-lactones can be selective for ACP over KS
but other potential competing residues such as surface
cysteines, serines, and lysines can also be avoided.
To examine the functionality of the acyl-ACP intermedi-
ates and provide evidence of thioester over thioether forma-
tion, preloaded ACP2 and ACP3 (10× compound 1) were
mixed with KS-AT6. After 1 h, the click reaction was
performed as before and the proteins separated by gel
electrophoresis. Bright bands corresponding to the KS-AT6
didomain indicate that the ꢀ-hydroxythioester is efficiently
transferred from the ACPs (Figure 3, right). It should be
noted that direct KS-acylation is not observed under the
conditions used in these experiments (Figure 3, left).
To examine ꢀ-lactone behavior with the next level of
complexity, we overexpressed and purified apo and holo
forms of the intact module 2 from spinosyn synthase (SpnB).
This particular module was chosen because it contains KS,
AT, DH, ER, KR, and ACP domains which represent the
vast majority of synthase components present in a given
(
9) (a) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int.
Ed. 2001, 40, 2004–2021. (b) Lewis, W. G.; Green, L. G.; Grynszpan, F.;
Radic, Z.; Carlier, P. R.; Taylor, P.; Finn, M. G.; Sharpless, K. B. Angew.
Chem., Int. Ed. 2002, 41, 1053–1057. (c) Rostovtsev, V. V.; Green, L. G.;
Fokin, V. V.; Sharpless, K. B. Angew. Chem., Int. Ed. 2002, 41, 2596–
2
599.
2332
Org. Lett., Vol. 12, No. 10, 2010

In summary, we have successfully demonstrated direct
acylation of isolated holo-ACP domains with functionalized
Table 1. Calculated Loading for ACP2, ACP3, and KS-AT6
with Compounds 1-5 at 10 and 50 equiv of Lactone
ꢀ-lactones. The apo form of this ACP is unreactive suggest-
ing that ꢀ-hydroxythioester formation takes place on the
Ppant arm of mature carrier proteins. In addition, ACPs can
be selectively acylated in the presence of competing PKS
active sites. This method offers a synthetically straightfor-
ward means of loading key PKS components with choice
substrates.
loading, %
compd
equiv of lactone
10
ACP2
ACP3
KSAT6
1
33
93
78
97
53
96
8
32
7
28
55
97
64
97
62
94
13
36
15
35
7
64
26
65
41
62
6
14
27
58
50
2
3
4
5
10
50
10
Acknowledgment. This work was supported by start-up
funding from the University of Massachusetts, Amherst (to
N.A.S). The authors would like to thank Professor Chaitan
Khosla (Stanford University) for the generous gifts of BAP1,
pVYA05, pNW06, and pAYC11. We also thank Professors
Alan Kennan (Colorado State University) and James Chambers
50
10
50
10
50
(University of Massachusetts) for helpful discussions.
provide an efficient and selective means of acylating carrier
proteins. The differential reactivities observed for ACP versus
KS suggest that future optimization of ACP-selectivity should
focus on monosubstituted lactones. Efforts to further explore
this structure-selectivity relationship are currently underway
in our laboratory.
Supporting Information Available: Detailed descriptions
of synthetic, molecular biology, and analytical procedures.
This material is available free of charge via the Internet at
http://pubs.acs.org.
OL100684S
Org. Lett., Vol. 12, No. 10, 2010
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