Facile synthesis of 1,1-[2H2]-2-methylaminoethane-1-sulfonic acid as a substrate for taurine α ketoglutarate dioxygenase (TauD)

Article abstract of DOI:10.1016/j.tetlet.2008.11.063

Taurine α-ketoglutarate dioxygenase (TauD) is an archetypal α-ketoglutarate-dependent non-heme iron oxygenase. Here, we report a practical five-step synthetic route to 1,1-dideutero-2-methylaminoethanesulfonic acid hydrochloride, a useful alternative substrate for the characterization of deuterium kinetic isotope effects on TauD.

Full text of DOI:10.1016/j.tetlet.2008.11.063

Tetrahedron Letters 50 (2009) 611–613
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Facile synthesis of 1,1-[2H2]-2-methylaminoethane-1-sulfonic acid
as a substrate for taurine
a
ketoglutarate dioxygenase (TauD)
*
Kevin P. McCusker, Judith P. Klinman
Department of Chemistry, University of California, Berkeley, CA 94720, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Taurine a-ketoglutarate dioxygenase (TauD) is an archetypal a-ketoglutarate-dependent non-heme iron
Received 12 November 2008
Revised 18 November 2008
Accepted 19 November 2008
Available online 24 November 2008
oxygenase. Here, we report a practical five-step synthetic route to 1,1-dideutero-2-methylaminoethane-
sulfonic acid hydrochloride, a useful alternative substrate for the characterization of deuterium kinetic
isotope effects on TauD.
Ó 2008 Published by Elsevier Ltd.
Keywords:
Enzymes
Isotope effects
Mechanism
Stable isotopes
TauD catalyzes the hydroxylation of taurine (aminoethanesulf-
onic acid) and other alkylsulfonates, alpha to the sulfonate, with
One common feature of this reaction mechanism is the use of a
high-valent Fe(IV)-oxo species (species III in Fig. 2) to abstract a
hydrogen atom from the prime substrate—taurine in the case of
TauD. Deuterium kinetic isotope effects (KIEs) have proven to be
an invaluable tool for probing the mechanism of enzymatic hydro-
gen atom transfer.14 The ability to obtain reliable deuterium KIE
measurements is contingent on the production of specifically deu-
terium-labeled substrate molecules with high isotopic purity. Pre-
viously, Bollinger and co-workers have produced 1,1-[2H2]-taurine
from chlorosulfanylacetyl chloride in five steps with ꢀ15% overall
yield and 99% isotopic purity.15 The synthesis of the N-methylated
derivative indicated here was undertaken to allow the possible
installation of a remote label (3H, 14C or 13C) on a taurine analog
for product analysis studies. N-methyltaurine was chosen as the
simplest analog to fulfill this possibility. Prior to undertaking the
synthesis, it was confirmed that the kinetic parameters of TauD
with commercially available protio N-methyltaurine did not differ
the concomitant consumption of one equivalent each of
a
ketoglu-
tarate (
a
KG) and molecular oxygen to yield the products CO2, suc-
cinate, sulfite and aminoacetaldehyde (Fig. 1).1 The enzyme is only
expressed in microorganisms under conditions of sulfate starva-
tion, and the product sulfite is subsequently used as a sulfur
source.2 The ease of overexpression and purification of the E. coli
protein has led to its position as a dominant model system for
the study of the family of aKG-dependent non-heme iron oxygen-
ases, which have been implicated in processes as diverse as antibi-
otic biosynthesis,3 oxygen sensing,4 DNA repair5–7 and the
biodegradation of anthropogenic compounds, particularly herbi-
cides.8,9 Despite this broad array of chemical transformations,
mounting evidence indicates that this class of enzymes utilizes a
common mechanism (Fig. 2) to generate the reactive oxygen inter-
mediate necessary to carry out these reactions.10–13
Succinate
CO2
αKG
O2
O
O
O
OH
OH
H2N
S
S
Spont.
2-
TauD
H2N
SO3
H2N
O
O
OH
Figure 1. Reaction catalyzed by TauD.
* Corresponding author. Tel.: +1 510 642 2668; fax: +1 510 643 6232.
E-mail address: klinman@berkeley.edu (J.P. Klinman).
0040-4039/$ - see front matter Ó 2008 Published by Elsevier Ltd.
doi:10.1016/j.tetlet.2008.11.063

612
K. P. McCusker, J. P. Klinman / Tetrahedron Letters 50 (2009) 611–613
Figure 2. Abridged mechanism of the
a
-KG-dependent oxygenases showing the oxidation of a generic C-H bearing substrate (S) to SOX. R@CH2CH2CO2À, His and Asp denote
protein-derived histidine and aspartate ligands to the active-site iron.
Table 1
Kinetic parameters obtained with wild-type TauD and the indicated substrates
Substratea
kcat (sÀ1
)
Km(S)
l
M
kcat/Km(S)d Â10À5 MÀ1sÀ1
Km(O2)
lM
kcat/Km(O2)d Â10À5 MÀ1 sÀ1
Taurine
4.9 0.2
4.7 0.3
5.0 0.2
0.60 0.01
19.4 0.7
54 10
2.5 0.1
0.9 0.2
1.0 0.1
1.2 0.1
41
46
41
6
4
5
1.2 0.2
1.0 0.1
1.2 0.2
1.1 0.1
HMethyl taurineb
HMethyl taurinec
DMethyl taurinec
48
4
5.1 0.6
5.6 0.5
a
The superscript H indicates 1,1-[1H2] methyl taurine, and D indicates 1,1-[2H2] methyl taurine, compounds 6b and 6a, respectively.
Commercially available.
Synthetically prepared by the methods presented herein.
b
c
d
Determined in the limit of infinite concentration of the alternate substrate.
significantly from the kinetic parameters with protio taurine
(Table 1). A straightforward synthesis requiring little purification
and no strictly anhydrous conditions was sought to facilitate the
ease of synthesis in a largely biochemical lab. It was decided that
it would be advantageous to incorporate deuterium through a
reduction step with a highly isotopically enriched reducing agent,
increasing the isotopic purity of the intermediate and avoiding
possible dilution of isotopic enrichment by incorporation of pro-
tium from atmospheric water into an exchangeable position. The
overall synthesis is shown in Scheme 1.
OH
CH3I, NaH
BocN
H
1
2
THF, 0º-rT, O/N
O
O
1) NMM/ibuOCOCl
DME, 0º, 1 min
2) NaBD4
H2O/DME, 0º, 1 min
OH
BocN
CH3
OH
MsCl, Et3N
BocN
CH3
The designed synthesis begins with Boc-protected glycine (1),
which is N-methylated with methyl iodide.16 This product (2) is
converted to a mixed carbonic–carboxylic anhydride and reduced
by the action of sodium borodeuteride,17 which is commonly avail-
able at isotopic purity of >99.9%.18 The deuterio alcohol (3) is con-
verted to the mesylate (4),19 and the mesylate is displaced by
thioacetate.20 The thioacetate ester (5) is oxidized by performic
acid21 and converted to the hydrochloride salt, which is triturated
in absolute ethanol to yield the final product (6a), 1,1-[2H2]-methy-
laminoethane-1-sulfonic acid hydrochloride, with no detectable 1H
contamination as determined by 1H NMR or mass spectral analysis,
in 23% overall yield. An analogous synthesis differing only in the use
of sodium borohydride was undertaken to produce the fully protio
compound (6b) for comparative studies with similar results. The
full experimental details are given in the Supplementary data.
In order to evaluate the utility of the deuterio substrate analog,
the kinetic parameters for TauD were determined using taurine,
commercial protio N-methyltaurine, synthetic protio N-methyltau-
rine, and synthetic 1,1-dideuterio N-methyltaurine. The results are
indicated in Table 1. The indistinguishable behavior of the enzyme
with commercial and synthetic protio N-methyltaurine precludes
the possibility of inhibitory synthetic by-products, indicating the
purity and suitability of the synthetic compound for use as a sub-
strate for TauD. The moderate KIE on kcat (kcat(H)/kcat(D)) of 8.3 is
consistent with the previous result of a significant (ꢀ35) KIE in
pre-steady state experiments as well as kinetic complexity in the
form of partial rate-limitation by product release,15,22 which would
lead to the diminution of the expressed isotope effect with respect
to the ‘intrinsic’ value. The mechanism in Fig. 2 depicts an irrevers-
ible step, O–O bond scission, prior to the isotopically sensitive step
of hydrogen atom abstraction. This mechanism predicts an isotope
effect of unity on kcat/Km parameters, which include all steps from
substrate—either prime substrate or oxygen—binding through the
CH2Cl2, 0º, 30 min
3
4
D
D
D
OMs
KSAc
BocN
H3C
DMF, rT, 72 hr
D
D
1) H2O2
HCOOH, 0º-rT,O/N
SAc
BocN
H3C
5
2) HCl
D
O
O
S
HCl
HN
OH
L
L
H3C
6a L=D
6b L=H
Scheme 1. Synthetic route to 1,1-[2H2]-2-methylaminoethane-1-sulfonic acid.
first irreversible step,23 as neither kcat/Km parameter includes the
isotopically sensitive step. Also, as kcat/Km(S) is determined at sat-
urating concentrations of dioxygen, dioxygen binding effectively
makes substrate binding irreversible, again prediciting an isotope
effect on kcat/Km(S) of unity. The determined values of D(kcat
/
r
Km(S)) and D(kcat/Km(O2)) of 0.8 0.1 and 1.1 0.2 are within 2
of unity, further corroborating the concensus mechanism.
The synthetic procedure described here provides analytically
pure N-methyltaurine isotopomers for use as substrates for TauD,
allowing the determination of kinetic isotope effects. It is worth
noting that no column purification or strictly anhydrous conditions
were necessary, and that the installation of other isotopic labels
either on the N-methyl substituent or in the alpha position could

K. P. McCusker, J. P. Klinman / Tetrahedron Letters 50 (2009) 611–613
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613
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the existing methodology, make this a valuable technique for the
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Acknowledgment
This work was supported by GM025765 from the National Insti-
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Supplementary data
Experimental procedures and spectral characterization (1H
NMR) of intermediates and end products, as well as HRMS of deu-
terated end-product are available. Supplementary data associated
with this article can be found, in the online version, at
doi:10.1016/j.tetlet.2008.11.063.
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