larged coenzyme A structure. Hence, complementary, chemi-
cally synthesised, enlarged coenzymes and site-specifically mu-
tated enzymes that accept the enlarged cofactor could be
combined and used to determine the natural substrate portfo-
lio and hence function of PCAF.
to generate novel CoA analogues in high yields from pante-
theine derivatives by using PPAT and DPCK in conjunction with
pantothenate kinase.[37]
The majority of CoA analogues reported to date are modi-
fied at the thiol position, which is likely to decrease their cata-
lytic activity. We chose the 3’’-hydroxyl, as this position is suffi-
ciently far away from the reactive thiol to minimise any reduc-
tion in the ability of the enlarged CoA to act as an acetyl
donor and is close to several residues in the active site with
moderate to large side chains (Figure 1B.). In this paper, we
describe the chemoenzymatic preparation of 3’’-O-benzyl-
acetyl-coenzyme A, from readily available d-pantethine
through a seven-step reaction including a coupled enzymatic
reaction by PPAT and DPCK (Scheme 2). This is the first report
of the synthesis of this analogue and the evaluation of its
activity with the transcriptional acetylase KAT2B/PCAF.
Extra groups need to be appended to CoA in order to
render it specific to PCAF mutants and to prevent it from bind-
ing to the natural form of the enzyme or other acetyltransfer-
ases. We initially designed and evaluated an enlarged CoA
with a modified benzyl group: 3’’-O-benzyl-coenzyme A (com-
pound 1), which was identified on the basis of an examination
of the first sphere of residues that contact CoA in the KAT2B/
PCAF active site and taking into account the catalytic activity
of mutants that had been previously reported.[17,18] The PCAF
mutant proposed to be best suited to accommodating this an-
alogue was created by mutating Gln581 to alanine or glycine,
thus creating extra space close to the 3’’-hydroxyl that could
accommodate the additional benzyl group (Figure 1). The
benzyl group was chosen in this “proof-of-principle study” as it
has a rigid bulky structure that would not significantly change
the lipophilicity of coenzyme A.
2-O-Benzyl-4-O-phospho-d-pantethine (5) was prepared
chemically from the commercially available starting material d-
pantethine. Compound 2 was prepared by selectively protect-
ing the primary and secondary hydroxyl groups of d-pante-
thine with tert-butyldimethylsilyl (TBDMS) and benzyl groups,
respectively (90% overall yield). Removal of TBDMS to release
the primary hydroxyl group led to 2-O-benzyl-d-pantethine (3)
in a high yield (88%). Phosphorylation of 3 with di-tert-butyl
N,N’-diethyl phosphoramidite, activated by 1H-tetrazole,
Numerous coenzyme A analogues have been synthesised by
chemical or chemoenzymatic methods including seleno-CoA,[19]
oxy-CoA,[20] decysteamine-CoA,[21] desulfo-CoA,[22] succinyl-
CoA,[23] crotonyl-CoA,[24] bromoacetyl-CoA,[25] S-acetonyl-CoA,[26]
S-(3-oxobutyl)-CoA,[27] acetonyldethio-CoA,[28] methylmalonyl-
carba(dethia)-CoA,[29] keto-CoA[21] and guano-CoA.[30] The enzy-
matic synthesis of CoA analogues was first achieved in 1964
when Michelson utilised ribonuclease T2.[31] Drueckhammer and
Strauss have since, independently used the CoA biosynthetic
enzymes phosphopantetheine adenylyltransferase (PPAT) and
dephospho-coenzyme A kinase (DPCK) to synthesise a range of
analogues that are modified at the thiol position.[32–36] More re-
cently, a one-pot procedure has been developed by Nazi et al.
formed
2-O-benzyl-d-pantethine-1-O-di-tert-butylphosphite,
which was oxidised in situ with meta-chloroperbenzoic acid
(m-CPBA) to produce the di-tert-butyl phosphate 4 in a 50%
yield. The corresponding phosphate 5 was obtained quantita-
tively from 4 after the removal of the tert-butyl groups in acid
(>98%).
Due to a lack of success in synthesising 3’’-O-benzyl-coenzy-
me A from 5 by the procedure of Moffatt and Khorana[38]
through chemical coupling of the modified pantethine and ad-
Scheme 2. Synthesis of acetyl-3’’-O-benzyl-coenzyme A. Reagents and conditions: a) TBDMSCl, 4-dimethylaminopyridine (DMAP), dry DMF, RT, overnight, 95%;
b) i: potassium tert-butoxide (1m in THF), THF, À788C, 0.5 h, ii: benzyl bromide, RT, overnight, 90%; c) tetrabutyl ammonium fluoride, THF, RT, 4 h, 88%; d) i:
1H-tetrazole, di-tert-butyl N,N’-diethylphosphoramidite, THF, RT, 4 h; ii: m-CPBA (70%), À408C, 10 min, warm to RT, 2 h, 52%; e) HCl/dioxane (0.1m), 4 h,
100%; f) dithiothreitol (DTT), ATP, MgCl2·6H2O, PPAT (0.02 U), DPCK (0.40 U), Tris buffer (50 mm, pH 7.6), 378C, 2 d, 10%; g) i: R’=14CH3, 14CH3COONa, 2,6-di-
chlorobenzoic acid, THF, reflux, 30 min; R’=CH3, CH3COOH, THF, reflux, 30 min; ii: 1,1’-carbonyldiimidazole, THF, 658C, 1.5 h; iii: 6, buffer (1 mm DTT, 0.5m imi-
dazole, pH 7.0), RT, 30 min.
ChemBioChem 2010, 11, 2100 – 2103
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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