Chemoenzymatic Synthesis of Acyl Coenzyme A Substrates Enables in Situ Labeling of Small Molecules and Proteins

Article abstract of DOI:10.1021/acs.orglett.5b02113

A chemoenzymatic approach to generate fully functional acyl coenzyme A molecules that are then used as substrates to drive in situ acyl transfer reactions is described. Mass spectrometry based assays to verify the identity of acyl coenzyme A enzymatic products are also illustrated. The approach is responsive to a diverse array of carboxylic acids that can be elaborated to their corresponding coenzyme A thioesters, with potential applications in wide-ranging chemical biology studies that utilize acyl coenzyme A substrates.

Full text of DOI:10.1021/acs.orglett.5b02113

Letter
pubs.acs.org/OrgLett
Chemoenzymatic Synthesis of Acyl Coenzyme A Substrates Enables
in Situ Labeling of Small Molecules and Proteins
Vinayak Agarwal,†,⊥ Stefan Diethelm,‡,⊥ Lauren Ray,‡,⊥ Neha Garg,§ Takayoshi Awakawa,‡,∥
Pieter C. Dorrestein,§ and Bradley S. Moore*,†,‡,§
‡
†Center for Oceans and Human Health and Center for Marine Biotechnology and Biomedicine, Scripps Institution of
§
Oceanography, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California
92093, United States
∥Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan
S
* Supporting Information
ABSTRACT: A chemoenzymatic approach to generate fully
functional acyl coenzyme A molecules that are then used as
substrates to drive in situ acyl transfer reactions is described. Mass
spectrometry based assays to verify the identity of acyl coenzyme A
enzymatic products are also illustrated. The approach is responsive
to a diverse array of carboxylic acids that can be elaborated to their
corresponding coenzyme A thioesters, with potential applications in
wide-ranging chemical biology studies that utilize acyl coenzyme A
substrates.
Acyl-CoAs are also preeminent in their participation in
secondary metabolism, such as in the biosynthesis of polyketide
and nonribosomal peptide antibiotics.1 In each of the above-
mentioned applications, the reactive thioester moiety of acyl-
CoA is of primary importance, activating the attached
carboxylic acid for participation in downstream enzymatic
processes.
n biological chemistry, coenzyme A (CoA, 1, Scheme 1) acts
Ias a molecular shuttle for carboxylic acids linked to its
terminal thiol. S-Acylated derivatives of 1 (acyl-CoAs, Scheme
1) participate in numerous enzymatic reactions, including
primary energy metabolism, synthesis of biomolecules, post-
translational modification of proteins, and other processes.
a
As shown in Scheme 1, 1 is biosynthesized in bacteria via the
action of the five enzymes CoaA−E.2 Pantothenic acid (2,
vitamin B5) is converted into phosphopantetheine (3) by the
action of three enzymes (CoaA−C). Further adenylation,
followed by phosphorylation of the ribose 3′-hydroxyl, affords
1. Carboxylic acids are thioesterified to 1 by the action of ATP-
dependent acyl-CoA ligases to afford acyl-CoAs. While a wide
diversity of acyl-CoA ligases have been discovered, their limited
substrate promiscuity has restricted their development as
synthetically useful catalysts. As a result, the traditional
synthesis of acyl-CoAs has relied on the chemical ligation of
carboxylic acids to 1 with the aid of peptide coupling reagents
to generate the thioester linkage. However, this approach
suffers from several drawbacks, including the high cost of 1 as
the starting material, poor yields for the coupling step, and
labor-intensive HPLC-based purification. Acyl-CoAs are also
known for their inherent instability by virtue of the chemically
labile thioester and phosphoester linkages in the molecule. In
the course of our previous studies3 requiring the preparation of
acyl-CoA substrates, we encountered these problems, often
representing significant challenges. These difficulties prompted
Scheme 1. Natural Route for Biosynthesis of Acyl-CoAs
a
Reactions catalyzed by CoaB and CoaC can be bypassed by
Received: July 22, 2015
synthetically appending the β-mercaptoethylamine moiety to 2.
© XXXX American Chemical Society
A
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Scheme 2. Synthetic Strategy for the Preparation of S-Acyl Pantetheine Derivatives
us to seek an alternative and more convenient access to acyl-
CoAs.
We herein report a straightforward and efficient method-
ology for the preparation of a wide range of acyl-CoAs. Our
strategy relies on the combination of an initial chemical
preparation of S-acylated pantetheine derivatives, followed by a
one-pot enzymatic transformation into the desired acyl-CoA
end product. We also establish a mass spectrometric assay to
characterize acyl-CoA products. Furthermore, we demonstrate
the applicability of chemoenzymatically generated acyl-CoAs as
acyl donors in acyl-transfer reactions.
Begley and co-workers have shown that Escherichia coli
CoaA, CoaD, and CoaE enzymes are promiscuous, in that they
can accept substrates modified either at the carboxy terminus of
2 or at the cysteamine moiety of 3.4 Burkart and co-workers
reported that this observation allows for an access route to
amide and ester analogues of acyl-CoAs that relies on bypassing
the first enzymatic steps (Scheme 1) by employing
appropriately designed derivatives of 2 that can be extended
by CoaA, CoaD, and CoaE enzymes.5 Despite these advances,
it should be noted that functional acyl-CoAs with a thioester
linkage that are capable of acting as physiological acyl-donors
have not been previously elaborated. We have chosen a similar
strategy to efficiently prepare thioester linked acyl-CoA
derivatives by a chemoenzymatic route.
We first sought to identify an efficient synthetic strategy for
the preparation of functionally diverse S-acyl pantetheines. As
shown in Scheme 2, we chose acetonide 4 as a starting point.6
Peptide coupling of cysteamine to 4 delivered thiol 5 in 96%
yield.7 Intermediate 5 can now be used to attach a variety of
carboxylic acids (Figure 1) to the thiol headgroup elaborating
the reactive thioester linkage. For alkyl, aryl, heteroaryl, and
alkenyl carboxylates, we identified an EDC-based protocol as
the most efficient and general.8 In particular, some challenging
substrates were successfully coupled to 5 in synthetically useful
yields. These include crotonic acid (12),9 pyrrole carboxylic
acid (11), and α- and β-hydroxyacids (8, 9) (Figure 1).
Subsequent removal of the acetonide protecting group
proceeded smoothly using AcOH/H2O (route A, Scheme
2).10 This mild deprotection protocol did not affect sensitive
functional groups, such as β-hydroxy carbonyls (9) or alkenes
(12−14).11 Furthermore, no HPLC purification step was
needed for most products. For the synthesis of an aminoacyl-S-
pantetheine derivative, we found that a trityl-protected
precursor was the most convenient among several tested.12
Despite the lower coupling yield, mild conditions were
sufficient for the additional deprotection step required to
Figure 1. Substrate scope for the synthesis of S-acyl pantetheine
derivatives (R groups shown). Yields given correspond to the coupling
reaction (first yield) followed by the deprotection (second yield) as
outlined in Scheme 2. a Yields following HPLC purifica-
tion.b Combined yield for both deprotection steps.
access this compound class (15, 1% TFA in CH2Cl2, route B,
Scheme 2). Initial attempts to couple β-ketoacids to 5 remained
unsuccessful. Gratifyingly, we were able to prepare derivatives
16−18 by reaction of 5 with masked ketoacids (route C,
Scheme 2) in refluxing toluene.13 Using this protocol, the
corresponding β-keto thioesters were obtained in acceptable
yields (Figure 1).
In order to elaborate pantetheine thioesters 6−18 into the
corresponding acyl-CoAs, we next cloned, expressed, and
purified recombinant E. coli CoaA, CoaD, and CoaE enzymes.
Substrates 6−18 were assayed using a premixed enzyme
cocktail of purified CoaA, CoaD, and CoaE as the catalyst. In
addition to the substrate and the enzyme cocktail, the only
other component that needed to be provided was freshly
prepared ATP (see Supporting Information for detailed assay
procedures). Using a 3-fold molar excess of ATP, the enzyme
cocktail catalyzed the stoichiometric conversion of a 1000-fold
molar excess of substrates to their corresponding acyl-CoA
products in 3 h at 30 °C, while no conversion was observed in
the absence of either ATP or the enzymes (Figure 2a). The
identity of the enzymatically synthesized benzoyl-CoA (Figure
2a), generated using 10 as the substrate, was verified by NMR
(see Supporting Information).
B
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Figure 3. Chemoenzymatically synthesized acyl-CoAs are acyl donors
for in situ labeling of small molecules. (a) Scheme for the conversion of
6 to acetyl-CoA, followed by the transfer of the acetyl group to 19. (b)
HPLC characterization at 280 nm of standards of acetyl-CoA (i) and
19 (ii) and the enzymatic reaction (iii) demonstrating the formation of
two acetyl-19 products (also see Figure S14). Absence of ATP (iv) or
CoaADE enzymes (v) abolished acetylation of 19, while absence of
CAT (vi) or 19 (vii) led to the production of acetyl-CoA but no
acetylated-19 products.
Figure 2. Enzymatic synthesis and characterization of acyl-CoAs
starting from S-acyl pantetheine substrates. (a) Illustratively, HPLC
analysis of reaction substrate 10 and the product benzoyl-CoA at 280
nm demonstrates that product formation is dependent on the presence
of ATP and CoaA/D/E enzyme catalysts. (b) Mass spectrometric
characterization of the enzymatic product benzoyl-CoA. In the MS1
spectra, dominant [M + H]1+ and [M + 2H]2+ ions (labeled with red
diamonds) are observed that correspond to the molecular formula of
benzoyl-CoA (C28H40N7O17P3S). Upon fragmentation of the [M +
H]1+ ion, characteristic benzoyl-(cyclo)pantetheine and (cyclo)-
pantetheine MS2 product ions are observed. A proposed chemical
route for the generation of the observed (cyclo)pantetheine MS2
product ions is shown in Figure S29.
facilitated a second acetylation event at the 3-hydroxyl position,
leading to production of diacetylated 19 (Figure S14).
A second physiological role of 1 is to donate its
phosphopantetheine moiety, such as in the conversion of
apo-acyl carrier proteins (-ACPs) to their holo forms. Substrate
promiscuity of the phosphopantetheinyl transferase enzyme
Sfp, which allows for the transfer of the acyl-phospho-
pantetheine moiety from acyl-CoAs to generate acyl-ACPs,
has been widely used to study assembly line biosynthesis of
natural products, among several other biochemical trans-
formations.16 We next queried whether the chemoenzymatic
acyl-CoA synthetic scheme described above can be used to
drive in situ production of acyl-ACPs using Sfp.
Illustratively, in a single-pot reaction starting from 10 and
apo-ACP as substrates, and CoaA/D/E and Sfp as catalysts
(Figure 4a), we observed the ATP-dependent stoichiometric
formation of benzoyl-S-ACP as the product. The mass
difference between the MS1 ions observed in the absence
and presence of ATP in the reaction correspond to the benzoyl-
phosphopantetheine moiety that is transferred by Sfp (Figure
4b). Furthermore, fragmentation of the peptidyl MS1 ions
demonstrated identical benzoyl-(cyclo)pantetheine MS2 prod-
uct ions that we previously observed for the benzoyl-CoA
enzymatic product (Figure 4c). Hence, without intermediary
purification of the acyl-CoAs, we could achieve in situ labeling
of protein substrates by acyl-CoAs. Additionally, each of the S-
acyl pantetheine substrates 6−18 was responsive to CoaA/D/
E-Sfp loading onto apo-ACP, thus underscoring the vast
chemical space that can be explored by the above-mentioned
methodology (Figures S15−S27). Furthermore, a preparative
scale synthesis of acyl-ACP using the scheme described above,
followed by purification by size exclusion chromatography,
Next, we developed a mass spectrometric assay to query the
identity of the acyl-CoA products in order to circumvent their
preparative isolation and NMR characterization. Guided by our
previous reports describing ‘phosphopantetheine ejection’ MS2
product ions,14 we observed characteristic acyl-(cyclo)-
pantetheine and (cyclo)pantetheine MS2 product ions upon
fragmentation of the acyl-CoA [M + H]1+ parent MS1 ion
(Figure 2b and Figures S1−S13). Note that the observation of
the (cyclo)pantetheine MS2 ion is indicative of the thioester
linkage present in the acyl-CoA enzymatic product. Modulation
of MS/MS parameters demonstrated that, with increasing
fragmentation energy, the abundance of the (cyclo)pantetheine
MS2 product ion increased relative to that of the acyl-
(cyclo)pantetheine ion (Figure S6).
Having verified the chemoenzymatic production of acyl-
CoAs, we next verified their viability to perform their
physiological roles, that is, to act as donors in acyl transfer
reactions. To illustrate, we employed chloramphenicol
acetyltransferase (CAT), an enzyme that catalyzes the
acetylation of chloramphenicol (19), using acetyl-CoA as the
acetyl donor (Figure 3a).14 Starting from 6, in a single-pot
assay, we produced acetyl-CoA that was then used as a
substrate by CAT to generate acetylated 19. Two mono-
acetylated 19 products were observed (Figure 3b, trace iii),
consistent with the slow noncatalytic transfer of the acetyl
group from 3-acetyl-19 to the 1-hydroxyl of 19.15 This then
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AUTHOR INFORMATION
Corresponding Author
■
*E-mail: bsmoore@ucsd.edu.
Author Contributions
⊥V.A., S.D., and L.R. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank our UCSD colleagues B. M. Duggan for assistance
with NMR data acquisition and analysis and P. Jordan for
helpful discussions. Funding and instrumentation support was
generously provided by the NIH (P01-ES021921, R01-
AI47818, and GMS10RR029121) and NSF (OCE-1313747),
Bruker, and postdoctoral fellowships from the Helen Hay
Whitney Foundation to V.A., the Swiss National Science
Foundation to S.D., and the Uehara Memorial Foundation to
T.A. Mass spectrometric data are made available on http://
gnps.ucsd.edu.
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■
Figure 4. In situ labeling of ACPs with enzymatically synthesized acyl-
CoAs. (a) Reaction scheme for the conversion of 10 to benzoyl-CoA
and concomitant transfer to an ACP molecule (in cartoon
representation colored red). (b) LC/MS ESI-ToF characterization of
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benzoyl-phosphopantetheine moiety that is transferred to a conserved
serine residue of the ACP (note that the difference of 1 Da between
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in Figure 2b.
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yielded several milligrams of purified and fully acylated ACP
product (Figure S28).
In conclusion, we have developed a workflow to generate a
multitude of S-acyl pantetheine molecules from a key synthetic
intermediate and demonstrated the enzymatic elaboration of
structurally diverse acyl-pantetheines to fully functional acyl-
CoA molecules that can participate in acyl transfer and acyl-
phosphopantetheine transfer reactions to small molecules and
proteins. The chemoenzymatic schemes, together with the mass
spectrometry based assays described in this study, are expected
to be broadly applicable in biochemical investigations involving
acyl-CoA substrates and intermediates. In particular, biochem-
ical and structural studies requiring large amounts of highly
pure acyl-CoAs and acyl-ACPs will benefit from the method-
ology described herein.
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ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.5b02113.
(16) Beld, J.; Sonnenschein, E. C.; Vickery, C. R.; Noel, J. P.; Burkart,
M. D. Nat. Prod. Rep. 2014, 31, 61.
Experimental details, synthetic schemes, and NMR
spectroscopy and mass spectrometry data (PDF)
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