COMMUNICATION
One-pot chemo-enzymatic synthesis of reporter-modified proteins
Andrew S. Worthington and Michael D. Burkart*
Received 9th September 2005, Accepted 10th November 2005
First published as an Advance Article on the web 24th November 2005
DOI: 10.1039/b512735a
To meet recent advancements in the covalent reporter labeling
of proteins, we propose a flexible synthesis for reporter
analogs. Here we demonstrate a one-pot chemo-enzymatic
synthesis of reporter-labeled proteins that allows the covalent
tethering of any amine-terminal fluorescent or affinity label
to a carrier protein or fusion construct. This two-reaction se-
quence consists of activated panthothenate coupling, biosyn-
thetic conversion to the coenzyme A (CoA) analog, and enzy-
matic carrier protein modification via phosphopantetheinyl-
transferase (PPTase). We also probe substrate specificity for
CoAA, the first enzyme in the pathway. With this approach
CoA analogs may be rapidly prepared, thus permitting the
regiospecific attachment of reporter moieties from a variety
of molecular species.
eukaryotic pathways been fully elucidated.6 CoA is biosynthesized
from pantothenate (vitamin B5) through a series of five enzymes,
CoAA–E. CoA synthesis has also been shown to be possible
via an abbreviated approach beginning with pantetheine.7,8 We
found that CoAA, which is understood to accept pantothenate as
a metabolic substrate, will also catalyze the phosphorylation of
pantetheine and pantetheine derivatives at the 4ꢀ-hydroxyl group.9
Subsequently, CoAD (Phosphopantetheine adenylyltransferase,
PPAT) and CoAE (dephosphocoenzyme A kinase, DPCK) can be
used to complete the CoA molecule from 4ꢀ-phosphopantetheine.
The human version of these enzymes exists as a bifunctional fusion
protein (PPAT–DPCK)6b, 10 and is utilized in our studies. Here
we introduce a synthetic scheme that allows any amine-terminal
reporter for covalent protein attachment. A one-pot, two-reaction
sequence, this scheme uses only two recombinant proteins to make
reporter-modified CoA analogs (Scheme 1), and a third enzyme
carries out carrier protein attachment (Scheme 2).
Introduction
The post-translational modification of carrier protein domains
from polyketide, non-ribosomal peptide, and fatty acid biosynthe-
ses has recently attracted the attention of the biological chemistry
community for the selective covalent modification of proteins with
reporter molecules. Since introduction of this technology,1 it has
been used as a multiplex functional assay,2 as a fusion protein in
vitro and in vivo,3 and as a phage library reporter.3b Each of these
studies utilized reporters attached to coenzyme A through selective
reactivity of the CoA thiol to a maleimide functionality incorpo-
rated into each reporter molecule.4 While convenient, this coupling
method shows limited flexibility for diverse reporter identity, as
many desirable reporters require multi-step syntheses to introduce
the maleimide functionality. Additionally, excess maleimide can
lead to non-specific protein labeling if not rigorously purified
from unreacted starting materials after coupling to CoA. The
resulting sulfanylsuccinimide linkage may also not be compatible
with selected downstream applications, where a less-bulky or more
natural linkage is preferred. We have therefore developed the
following one-pot sequence for carrier protein labeling that may be
used with any amine-terminal reporter and allows great flexibility
in reporter and linkage choice.
Scheme 1 Pantothenate 1 activation and coupling to amine-terminal re-
porters 4a–c, followed by deprotection of 3 and in vitro enzymatic synthesis
of CoA analogs 7a–c by E. coli CoAA, H. sapiens PPAT–DPCK in one
pot. Reagents: (i) acetone, p-toluenesulfonic acid; (ii) EDC, N-hydroxy-
succinimide, iPr2NEt, DMF; (iii) Et3N, THF; (iv) 1 M HCl, THF.
In order to produce modified CoA analogs, we sought a linear
sequence via synthetic modification of CoA precursors. Along
with the introduction of a modular approach to panthetheine
and phosphopantetheine synthesis,5 CoA biosynthetic enzymes
came to our attention with the potential of demonstrating
promiscuous substrate specificity akin to phosphopantetheinyl-
transferases (PPTases).1 CoA biosynthesis has been a topic of
research for decades, yet only recently have prokaryotic and
Results and discussion
The chemo-enzymatic synthesis begins with the chemical synthesis
of pantetheine analogs via activated panthothenate coupling with
an N-terminal reporter. Subsequent enzymatic incubation with
two enzymes, recombinant E. coli CoAA and human PPAT–
DPCK, completes the synthesis of labeled CoA analogs. PPAT–
DPCK was chosen as a convenient natural fusion protein, thus
abbreviating recombinant enzyme production. Human DPCK
Department of Chemistry and Biochemistry, University of California,
San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0358, USA.
E-mail: mburkart@ucsd.edu; Fax: +1 858 822 2182; Tel: +1 858 534 5673
4 4 | Org. Biomol. Chem., 2006, 4, 44–46
This journal is
The Royal Society of Chemistry 2006
©