An efficient enzymatic synthesis of thiamin pyrophosphate

Article abstract of DOI:10.1016/j.bmcl.2003.07.026

Thiamin pyrophosphate was synthesized in 71% yield, on a multi-milligram scale, using overexpressed thiazole kinase, pyrimidine kinase, thiamin phosphate synthase, and thiamin phosphate kinase. This provides a facile route to isotopically labeled thiamin pyrophosphate from its readily available pyrimidine and thiazole precursors.

Full text of DOI:10.1016/j.bmcl.2003.07.026

Bioorganic & Medicinal Chemistry Letters 13 (2003) 4139–4141
An Efficient Enzymatic Synthesis of Thiamin Pyrophosphate
Jonathan S. Melnick, K. Ingrid Sprinz, Jason J. Reddick,
Cynthia Kinsland and Tadhg P. Begley*
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14850, USA
Received 21 May 2003; revised 24 July 2003
Abstract—Thiamin pyrophosphate was synthesized in 71% yield, on a multi-milligram scale, using overexpressed thiazole kinase,
pyrimidine kinase, thiamin phosphate synthase, and thiamin phosphate kinase. This provides a facile route to isotopically labeled
thiamin pyrophosphate from its readily available pyrimidine and thiazole precursors.
# 2003 Elsevier Ltd. All rights reserved.
Thiamin pyrophosphate utilizing enzymes are found in
all living systems. For example, in Escherichia coli,
seven such enzymes have been identified; these play key
roles in the citric acid cycle, in carbohydrate metabo-
lism, and in the biosynthesis of isopentenyl pyrophos-
phate, menaquinone, pyridoxal phosphate, thiamin
pyrophosphate and branched chain amino acids.1 While
the mechanistic enzymology of thiamin-utilizing
enzymes has been extensively studied,2 NMR has not
been widely used as a mechanistic tool for this family of
Our synthesis strategy overcomes the chemically diffi-
cult thiazole/pyrimidine coupling and the low yielding
phosphorylations of thiamin to thiamin pyrophos-
phate.11 The overall yield compares favorably with the
less versatile synthesis of thiamin pyrophosphate from
thiamin using thiamin pyrophosphokinase. This synth-
esis will be of use for the preparation of isotopically-
labeled thiamin pyrophosphate from the readily synthe-
sized pyrimidine and thiazole moieties for NMR studies
on thiamin-utilizing enzymes.
enzymes
because
isotopically-labeled
thiamin
pyrophosphate is difficult to synthesize.3,4
Standard methods were used for DNA restriction
endonuclease digestion, ligation and transformation of
DNA.12,13 Automated DNA sequencing was performed
at the Cornell BioResource Center. Plasmid DNA was
purified with the WizardTM Plus SV DNA miniprep kit
(Promega, Madison, WI, USA). DNA fragments were
separated by agarose gel electrophoresis, excised and
purified with the QIAEX II Gel Extraction Kit
(Qiagen, Valencia, CA, USA). E. coli strain DH5a
was used as a recipient for transformations during
plasmid construction, propagation and storage. PCR
was catalyzed with Platinum Pfx DNA polymerase and
performed in a Perkin-Elmer GeneAmp PCR System
2400 (Gibco BRL, Rockville, MD, USA). All restriction
endonucleases and T4 DNA ligase were purchased
from New England Biolabs (Beverly, MA, USA). E.
coli strain BL21(DE3) and the pET overexpression sys-
tem were purchased from Novagen (Madison, WI,
USA).
The later steps in the thiamin biosynthesis and salvage
pathways are outlined in Figure 1.5 2-Methyl-4-amino-
5-hydroxymethyl pyrimidine pyrophosphate (3) is
formed by the iterative phosphorylation of (2) and 4-
methyl-5-(2-hydroxyethyl)thiazole phosphate (5) is
formed by the phosphorylation of (4). The pyrimidine
pyrophosphate (3) and the thiazole phosphate (5) are
then coupled, in a reaction catalyzed by thiamin phos-
phate synthase, to give (6). A final phosphorylation
gives thiamin pyrophosphate (1). Alternatively, thiamin
pyrophosphate can be formed by the direct pyrophos-
phorylation of thiamin (7).6 Since all of the enzymes
required for the catalysis of these reactions have now
been overexpressed and characterized, and the pyr-
imidine7,8 and thiazole moieties9,10 are readily synthe-
sized, we are able to report here the development of a
facile enzymatic synthesis of thiamin pyrophosphate.
Thiazole (4) and thiamin alcohol (7) were purchased
from Sigma, pyrimidine (2) was synthesized as pre-
viously described.7,8
*Corresponding author. Tel.: +1-607-255-7133; e-mail: tpb2@cornell.
edu
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.07.026

4140
J. S. Melnick et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4139–4141
Figure 1. The later steps in the thiamin pyrophosphate biosynthesis and salvage pathways.
grown at 15 ꢀC. These induced cultures were then grown
Construction of the Thiamin Monophosphate Kinase
(thiL) Overexpression Plasmid
for 6 h at 37 ꢀC with shaking and the thiazole kinase,
HMP-P kinase, thiamin phosphate synthase and thia-
min phosphate kinase were partially purified as descri-
bed below.
The thiL gene was PCR amplified from E. coli genomic
DNA using the following primers: upstream primer 50-
GGC ATA ACA TATGGC ATG TGG CGA GTT
CTC CCT G-30 (inserts an NdeI site at the start codon
of the thiL open reading frame); downstream primer 50-
CAC CAC CCT CGAGTA GAG CTG CCA GGG
CAA AAA GGT-30 (inserts an XhoI site after the end of
the thiL open reading frame). The purified PCR product
was digested with NdeI and XhoI, purified and ligated
into similarly digested pET-16b(+) vector. Colonies
were screened for the presence of the insert and a
representative plasmid was designated pCLK710. The
PCR-derived DNA was sequenced and shown to con-
tain no errors.
Partial Purification of Thiazole Kinase, HMP-P
Kinase, Thiamin Phosphate Synthase and Thiamin
Phosphate Kinase
The four 500-mL cultures prepared above were com-
bined and the cells were pelleted by centrifugation at
10,000g for 30 min. The cell pellet was resuspended in
40 mL of 50 mM Tris–HCl, 2mM DTT, 2mM EDTA,
pH 8, and the cells were lysed by sonication (Heat Sys-
tems Ultrasonics model W-385 sonicator equipped with
a 0.5-inch tip on a 2-s cycle, 50% duty, 3 min). Cellular
debris was removed by centrifugation at 27,000g for 30
min, followed by passing the supernatant through a
0.45-mm filter. The cell free extract buffer was then
exchanged with a volatile buffer (100 mM NH4HCO3,
pH 8) by gel filtration on a PD-10 column (Amersham).
Protein overexpressed from pCLK710 was not active,
presumably due to the presence of the amino-terminal
His10 tag. The thiL gene was therefore excised with
BamH1/Nde1 and transferred to the pET-22b(+) vector
to give pKIS100.
Overexpression of Thiazole Kinase, HMP-P Kinase,
Thiamin Phosphate Synthase and Thiamin Phosphate
Kinase
Overexpression and Purification of Thiamin
Pyrophosphokinase
Plasmid pTPK-1, containing the S. cerevisiae gene for
thiamin pyrophosphokinase in a pET28a(+) vector,
was transformed into E. coli BL21(DE3).16 A single
colony of the recombinant cells was grown overnight,
with shaking at 37 ꢀC in 3 mL LB broth containing
kanamycin (25 mg/mL). This starter culture (0.5 mL)
was used to inoculate 500 mL LB broth containing
kanamycin (25 mg/mL). Once this culture reached an
OD600 of 0.5, IPTG was added to a final concentration
of 1 mM. This induced culture was then grown for 6 h at
37 ꢀC with shaking. The cells were pelleted by cen-
trifugation at 10,000g for 30 min and the supernatant
discarded. The pellet was then resuspended in 5 mL of
50 mM Tris–HCl, 2mM DTT, 2mM EDTA, pH 8, and
the cells lysed by sonication. Cellular debris was
removed by centrifugation at 27,000g for 30 min, fol-
lowed by passing the supernatant through a 0.45 mm
filter. The cell free extract buffer was then exchanged
with a volatile buffer (100 mM NH4HCO3, pH 8) by gel
filtration on a PD-10 column (Amersham).
Plasmids pYZK3 and pYZC6927 containing the Bacil-
lus subtilis genes for thiazole kinase and thiamin phos-
phate synthase in pQE30 and pQE32(Qiagen),
respectively, were transformed separately into E. coli
SG13009(pREP4).14 Plasmids pCLK601b containing
the E. coli gene for HMP-P kinase in pET22b(+),15 and
pKIS100, containing the E. coli gene for thiamin phos-
phate kinase in pET22b(+) were separately trans-
formed into E. coli BL21(DE3). A single colony of each
was grown overnight in 3 mL LB broth containing
either ampicillin (200 mg/mL) for E. coli BL21(DE3) or
ampicillin (200 mg/mL) and kanamycin (25 mg/mL) for
E. coli SG13009. These starter cultures (0.5 mL) were
used to inoculate 500 mL of LB broth containing the
same concentration of antibiotics as the starter culture.
Once these cultures reached an OD600 of 0.5, IPTG was
added to a final concentration of 1 mM. All cultures
were grown at 37 ꢀC with the exception of the thiamin
phosphate kinase overexpression strain which was

J. S. Melnick et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4139–4141
4141
Synthesis of Thiamin Pyrophosphate (1) from
Pyrimidine (2) and Thiazole (4)
thiamin pyrophosphate from (2) and (4) was 41 mg
(71%) and the yield from (7) was 26 mg (75%). The
final product for both syntheses was identical to an
authentic sample of thiamin pyrophosphate by HPLC,
MS and NMR analysis. 1H NMR (400 MHz, D2O),
7.85 (s, 1H), 5.20 (s, 1H), 3.95 (q, 2H), 3.08 (t, 2H), 2.37
(s, 3H), 2.24 (s, 3H), MS ESI (m/z, M+) 425.
An 80 mL solution containing pyrimidine (2) (19 mg,
1.7 mM), thiazole (4) (20 mg, 1.7 mM), ATP (13.3 mM),
MgCl2 (25 mM), and KCl (50 mM) was prepared in 100
mM NH4HCO3 buffer, pH 8. The pooled cell extract
(10 mL) was then added, the reaction mixture was stir-
red at room temperature for 12h and the product was
purified as described below.
Acknowledgements
We thank Frank Jordan for bringing this problem to
our attention and Robert Harris for providing us with
his thiamin pyrophosphokinase overexpression strain.
This research was supported by a grant from NIH to
TPB (DK44083), by a gift from Hoffmann-La Roche
and by a Pfizer Summer Undergraduate Research Fel-
lowship to J.M.
Synthesis of Thiamin Pyrophosphate (1) from
Thiamin (7)
A 40 mL solution containing thiamin alcohol (7) (2 1
mg, 2mM), ATP (10 mM) and MgCl (20 mM) was
2
prepared in 100 mM NH4HCO3 buffer, pH 8. Partially
purified thiamin pyrophosphokinase (5 mL) was added,
the reaction mixture was stirred at room temperature for
12h and the product was purified as described below.
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Purification of Thiamin Pyrophosphate
The reaction mixture was centrifuged at 27,000g for 30
min, the supernatant was collected and diluted to 250
mL with 25 mM NH4HCO3 buffer, pH 7.6. This was
then loaded at 0.33 mL/min onto 140 mL of DEAE-
Sephadex A25 resin that had been washed with 300 mL
of 25 mM NH4HCO3, pH 7.6. An 800 mL linear gra-
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and lyophilized to constant mass. The isolated yield of
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