One-pot preparation of coenzyme a analogues via an improved chemo-enzymatic synthesis of pre-CoA thioester synthons

Article abstract of DOI:10.1039/b613527g

Coenzyme A analogues are synthesized in a one-pot preparation by biotransformation of pantothenate thioesters through the simultaneous use of three CoA biosynthetic enzymes, followed by aminolysis. The Royal Society of Chemistry.

Full text of DOI:10.1039/b613527g

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One-pot preparation of coenzyme A analogues via an improved chemo-
enzymatic synthesis of pre-CoA thioester synthons{
Marianne van Wyk and Erick Strauss*
Received (in Cambridge, UK) 18th September 2006, Accepted 24th October 2006
First published as an Advance Article on the web 14th November 2006
DOI: 10.1039/b613527g
acetyl-CoA analogues through aminolysis reactions with various
amines.7 Although this methodology offered significant advan-
tages, it also had some shortcomings: first, the chemo-enzymatic
preparation of the CoA thioester analogue was a low yielding
process, mostly due to the chemical synthesis of the phospho-
pantothenate thioester precursor (3, R9 = Pr); and second, the
propyl thioester of the analogue was relatively stable towards
aminolysis, requiring a large excess of (often costly) amine to drive
the reaction to completion.
Coenzyme A analogues are synthesized in a one-pot prepara-
tion by biotransformation of pantothenate thioesters through
the simultaneous use of three CoA biosynthetic enzymes,
followed by aminolysis.
Analogues of the acyl group-carrying cofactor coenzyme A (CoA)
have been widely used as mechanistic probes or as inhibitors of
enzymatic activity.1 Recently their utility has expanded even
further through the demonstration that analogues of CoA, which
normally serve as the source of the 49-phosphopantetheine
prosthetic group of acyl carrier proteins (ACPs), are also accepted
as alternate substrates by promiscuous phosphopantetheinyl
transferase (PPTase) enzymes.2 This allows the preparation of
so-called crypto-ACPs in which the normal pantetheine prosthetic
group is replaced by a variety of pantothenate-based analogues;
these may subsequently serve as alternate substrates for mechan-
istic studies, as fusion protein tags, or as reporter labels.3
The biggest obstacle to the widespread use of CoA analogues
remains the tedious synthetic preparation of the analogue itself.1 A
major advancement in this regard was made when it was shown
that the adenylylation and final phosphorylation steps of the
synthesis can be performed by the last two enzymes of the CoA
biosynthetic pathway (CoaD and CoaE); this allowed the
preparation of CoA analogues 6 using almost any phosphopan-
tetheine analogue 8 as substrate (Scheme 1).1,4 Recent develop-
ments have also seen the introduction of the first kinase in the
pathway (pantothenate kinase, CoaA) to the method to perform
the problematic initial phosphorylation reaction.5 However, since
CoaA acts as the rate-limiting enzyme in CoA biosynthesis
through its feedback inhibition by CoA itself,1 chemo-enzymatic
preparations that make use of this enzyme require the sequential
application of the three biosynthetic enzymes to limit the negative
effect this inhibition may have on yield.
The greatest drawback of the methods outlined above lies in
their bottom-up approach: each CoA analogue must be prepared
by the chemo-enzymatic transformation of the corresponding
pantetheine analogue, which is usually prepared synthetically.6 In
contrast, a preferred method would allow the preparation of CoA
analogues in a top-down manner from a single common precursor,
ideally in a single step (Scheme 1). Such an approach was
first developed by Drueckhammer and co-workers, who used a
single CoA thioester analogue (5, R9 = Pr) to prepare CoA and
Scheme 1 Chemo-enzymatic preparation of CoA analogues via bio-
transformation of pantothenamides 7 (bottom-up approach) or via
Department of Chemistry, Stellenbosch University, Matieland, 7602,
South Africa E-mail: estrauss@sun.ac.za; Fax: +27-21-808-3360
{ Electronic supplementary information (ESI) available: Detailed experi-
mental methods, and analytical data (NMR spectra and LC-MS traces).
See DOI: 10.1039/b613527g
aminolysis of pre-CoA thioester synthons
5 (top-down approach).
CoaA: pantothenate kinase; CoaD: phosphopantetheine adenylyltransfer-
ase; CoaE: dephospho-CoA kinase.
398 | Chem. Commun., 2007, 398–400
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We set out to address these shortcomings by establishing an
improved method for the top-down synthesis of CoA analogues
using the method of Drueckhammer as our starting point. To
address the low overall yield of the preparation we decided to
investigate the feasibility of the enzymatic phosphorylation of the
thioester precursors of the CoA thioester analogues 5 (which we
will refer to as pre-CoA synthons). To increase the reactivity of the
original propyl thioester analogue toward aminolysis we prepared
the equivalent but sterically less hindered methyl and ethyl
thioesters, as well as the activated phenyl thioester. However,
since the aminolysis reactions are performed in aqueous solutions,
we were keenly aware that these changes might also increase the
rate of hydrolysis of the thioester, a competitive side reaction that
takes place under these conditions.8 Our aim was thus to find a
balance between low reactivity and a high rate of hydrolysis.
The S-methyl (2a), S-ethyl (2b) and S-phenyl (2c) thiopantothe-
nate precursors were subsequently all successfully synthesized in a
simple one-step thioesterification procedure using unprotected
pantothenic acid as starting material.{
Fig. 1 Three-step biosynthesis of pre-CoA synthon 5a from S-methyl
thiopantothenate 2a as monitored by HPLC. A: Reaction mixture of 2a
and SaCoaA in the presence of ATP forms 3a. B: Reaction mixture of
2a, SaCoaA, EcCoaD and ATP forms 4a. C: Reaction mixture of 2a,
SaCoaA, EcCoaD, EcCoaE and ATP forms the pre-CoA synthon 5a. The
peaks between 2 and 3 min represent excess ATP and the ADP formed
during the course of the reaction.
Our initial investigation of the enzymatic phosphorylation of the
pantothenate thioesters 2a–c was focused on making use of the
CoaA enzyme from Escherichia coli (EcCoaA). However, when
this enzyme was assayed for its ability to catalyze the ATP-
dependant phosphorylation of its native substrate pantothenate 1
compared to the thioesters 2a–c, no evidence of kinase activity
with the unnatural substrates was found (results not shown). This
was surprising, since EcCoaA is known to accept a variety of
pantothenamides as alternate substrates.9,10 However, when the
same analysis was performed using the recently characterized
CoaA protein from Staphylococcus aureus (SaCoaA) the enzyme
exhibited activity towards all the substrates with specificity
constants within an order of magnitude of that of the natural
substrate (Table 1). This result was particularly encouraging, not
only because it confirmed SaCoaA’s lack of substrate specificity,
but also because this particular pantothenate kinase enzyme has
been shown to be refractory towards feedback inhibition by
CoA.11 This specific trait suggested that SaCoaA could be used
simultaneously with the CoaD and CoaE enzymes in the
biotransformation reactions, without any concern about the
impact it may have on yield. We set out to demonstrate that this
was indeed the case, using the chemo-enzymatic preparation of the
pre-CoA synthons 5a–c as a model.
to confirm the formation of the 39-dephospho-CoA analogues
4a–c, and finally by analysis of reaction mixtures in which the
thioester substrate, the three biosynthetic enzymes (SaCoaA,
EcCoaD and EcCoaE) and ATP are all incubated together to
form the expected thioester synthons 5a–c. Fig. 1 shows HPLC
traces of these analyses for the methyl thioester as representative of
the obtained results: panel A shows the formation of 3a as a single
product, panel B the formation of 4a, and panel C the formation
of the methyl pre-CoA synthon 5a. Similar results were obtained
for the ethyl and phenyl pre-CoA synthons, although longer
incubation times were required in the case of the latter.§ LC-MS
analyses were also used to determine if the thioesters are stable
under these conditions; however, no hydrolysis products could be
detected in any of the reaction mixtures.
Previous attempts at preparing CoA analogues by aminolysis of
a relatively stable propyl pre-CoA synthon required a large excess
of amine (up to 400 mol equivalents) and prolonged incubation
times (up to 48 hours). These reactions were also performed at pH
y10, which increased the rate of the undesired hydrolytic cleavage
of the pre-CoA synthon.7c In contrast, we decided to optimize the
aminolysis conditions of the pre-CoA synthons by using no more
than 10 equivalents of amine, an incubation time of less than 6 h
and a reaction pH of 9. To determine which of the three pre-CoA
synthons 5a–c would completely be converted under these
conditions, aminolysis reactions were initially performed using
the simple primary amine, pentylamine (Scheme 2). This amine
was chosen since we had previously prepared the expected CoA
Pantothenate thioesters 2a–c were subsequently treated with
SaCoaA in the presence of ATP and the reaction mixtures were
analyzed by LC-MS for the formation of the respective
49-phosphopantothenate thioesters 3a–c as products of the
phosphorylation reaction. This was followed by LC-MS analyses
of the reaction mixtures of each of the thioesters 2a–c in the
presence of SaCoaA, EcCoaD (i.e. CoaD from E. coli) and ATP
Table 1 The kinetic parameters of SaCoaA with pantothenic acid 1
in comparison to the pantothenate thioesters 2a–c as substrates
21
k
catKM
/
Substrate
kcat/s21 KM/mM
mM21 s21
Pantothenic acid 1
1.41
76.2 ¡ 18.5 18.5 ¡ 6.30
826.5 ¡ 108.4 3.09 ¡ 1.49
1205.5 ¡ 347.2 1.99 ¡ 1.13
936.8 ¡ 250 1.05 ¡ 0.53
S-Methyl thiopantothenate 2a 2.55
S-Ethyl thiopantothenate 2b 2.39
S-Phenyl thiopantothenate 2c 0.98
Scheme 2 Aminolysis of pre-CoA synthons 5a–c with pentylamine to
form ethyldethia-CoA 6a.
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complete disappearance of 5c, accompanied by formation of the
expected aminolysis products, a control reaction in which the poor
aminolysis substrate tert-butyl amine was used indicated that
under these conditions the pre-CoA synthon may also undergo
extensive hydrolysis. Consequently the effective conversion of 5c in
the presence of each amine was determined by integration of the
peak areas of the hydrolysis and the aminolysis products, each
identified by LC-MS analysis. The results show that while
conversion is dependent on the functional groups in the amine’s
side chain, size and complexity are not necessarily impediments to
the aminolysis reaction, as demonstrated by the successful
preparation of the biotinylated analogue 6g (Table 2). Other
analogues that were similarly prepared are the azide-containing 6b
(ideally suited for ‘‘click’’ chemistry-based reporter labeling12,13),
homocysteamine-CoA 6c,2b seleno-CoA 6d,14 the transition-state
analogue 6e,7c oxy-CoA 6f4a and the fluorescent-labeled 6h.
In summary, we have presented a general method for the one-
pot preparation of a diverse array of CoA analogues from a single
activated pre-CoA thioester synthon. Owing to its simplicity and
ease of use, this method should greatly aid the expansion of the
increasing number of applications of CoA analogues.
Fig. 2 Aminolysis of pre-CoA synthons 5a–c with 10 equivalents
pentylamine to form ethyldethia-CoA 6a. Reaction mixtures (in 50 mM
HEPES buffer, pH 9.0) were incubated for 6 h at 50 uC and analyzed by
LC-MS. A: Aminolysis of 5a shows partial conversion to 6a. B:
Aminolysis of 5b shows partial conversion to 6a. C: Aminolysis of 5c
shows its complete disappearance, and formation of 6a.
Table 2 Aminolysis of the pre-CoA synthon 5c with a variety of
amines to form the corresponding CoA analogues 6a–h
Amine
CoA analogue
Conversiona (%)
Negative control
0 (100% hydrolysis)
6a
6b
78
85
Notes and references
6c
58
{ Thioesterification reactions were performed using pantothenic acid, the
corresponding thiol (or its sodium salt) and diphenylphosphoryl azide or
diethylphosphoryl cyanide as coupling agents. See ESI{ for details.
§ The formation of the three pre-CoA synthons were subsequently
confirmed by preparation of the thioesters on a preparative scale, followed
by their purification on C18 solid phase extraction cartridges and 1H NMR
and HRMS analysis of the pure compounds. See ESI{ for details.
6d
6e
6f
94
.50b
.50b
Biotin–cadaverine
Dansylcadaverine
6g
6h
.95
.25b,c
1 P. K. Mishra and D. G. Drueckhammer, Chem. Rev., 2000, 100, 3283.
2 (a) J. J. La Clair, T. L. Foley, T. R. Schegg, C. M. Regan and
M. D. Burkart, Chem. Biol., 2004, 11, 195; (b) A. M. Gehring,
R. H. Lambalot, K. W. Vogel, D. G. Drueckhammer and C. T. Walsh,
Chem. Biol., 1997, 4, 17.
3 (a) J. Yin, F. Liu, X. Li and C. T. Walsh, J. Am. Chem. Soc., 2004, 126,
7754; (b) B. N. Cook and C. R. Bertozzi, Bioorg. Med. Chem., 2002, 10,
829; (c) K. M. Clarke, A. C. Mercer, J. J. La Clair and M. D. Burkart,
J. Am. Chem. Soc., 2005, 127, 11234; (d) A. S. Worthington and
M. D. Burkart, Org. Biomol. Chem., 2006, 4, 44; (e) N. George, H. Pick,
H. Vogel, N. Johnson and K. Johnson, J. Am. Chem. Soc., 2004, 126,
8896.
a
Conversion values relate the relative amount of aminolysis product
(compared to hydrolysis) for the reactions performed at pH 9.0.
Overlap of the chromatographic signals representing the hydrolysis
product and ATP/ADP prevented the accurate determination of the
% conversion to these analogues. Instead, conversion was
conservatively estimated by comparison to the other samples. See
b
c
ESI{ for details. Low conversion to 6h is presumed to be a result
of the low aqueous solubility of dansylcadaverine, not because of
poor aminolysis activity.13
4 (a) M. Dai, Y. Feng and P. J. Tonge, J. Am. Chem. Soc., 2001, 123, 506;
(b) E. Strauss and T. P. Begley, ChemBioChem, 2005, 6, 284.
5 I. Nazi, K. P. Koteva and G. D. Wright, Anal. Biochem., 2004, 324, 100.
6 A. L. Mandel, J. J. La Clair and M. D. Burkart, Org. Lett., 2004, 6,
4801.
7 (a) K. W. Vogel and D. G. Drueckhammer, J. Am. Chem. Soc., 1998,
120, 3275; (b) D. P. Martin, R. T. Bibart and D. G. Drueckhammer,
J. Am. Chem. Soc., 1994, 116, 4660; (c) R. T. Bibart, K. W. Vogel and
D. G. Drueckhammer, J. Org. Chem., 1999, 64, 2903.
8 (a) W. Yang and D. G. Drueckhammer, Org. Lett., 2000, 2, 4133; (b)
W. Yang and D. G. Drueckhammer, J. Am. Chem. Soc., 2001, 123,
11004.
9 K. G. Virga, Y.-M. Zhang, R. Leonardi, R. A. Ivey, K. Hevener,
H.-W. Park, S. Jackowski, C. O. Rock and R. E. Lee, Bioorg. Med.
Chem., 2006, 14, 1007.
10 E. Strauss and T. P. Begley, J. Biol. Chem., 2002, 277, 48205.
11 R. Leonardi, S. Chohnan, Y.-M. Zhang, K. G. Virga, R. E. Lee,
C. O. Rock and S. Jackowski, J. Biol. Chem., 2005, 280, 3314.
12 R. Manetsch, A. Krasinski, Z. Radic, J. Raushel, P. Taylor,
K. B. Sharpless and H. C. Kolb, J. Am. Chem. Soc., 2004, 126, 12809.
13 J. L. Meier, A. C. Mercer, H. Rivera, Jr. and M. D. Burkart, J. Am.
Chem. Soc., 2006, 128, 12174.
analogue product (i.e. ethyldethia-CoA, 6a) using the traditional
bottom-up approach;10 it was thus available for use as standard in
the subsequent analyses.
The three pre-CoA synthons 5a–c were subsequently prepared
separately by incubating the appropriate pantothenate thioester
precursor with SaCoaA, EcCoaD, EcCoaE and ATP. Upon
completion of the biotransformation the enzymes were removed,
and 10 mol equivalents of pentylamine were added to the reaction
mixture. The aminolysis reaction was allowed to proceed at 50 uC
for 6 h before the mixtures were analyzed for the disappearance of
the pre-CoA synthon and the formation of the expected product
6a. The results, as summarized in Fig. 2, show that while all three
pre-CoA synthons underwent some degree of aminolysis, only the
activated phenyl thioester 5c disappeared completely while forming
the expected aminolysis product. We thus decided to use the
phenyl pre-CoA synthon 5c for all subsequent reactions.
We finally set out to demonstrate the general utility of the
method by extending it to amines with diverse functional groups
and of increasing complexity. While initial results showed the
14 J. Seravalli, W. Gu, A. Tam, E. Strauss, T. P. Begley, S. P. Cramer and
S. W. Ragsdale, Proc. Natl. Acad. Sci. USA, 2003, 100, 3689.
400 | Chem. Commun., 2007, 398–400
This journal is ß The Royal Society of Chemistry 2007