Preparation of 2'-13c-l-histidine starting from 13c-thiocyanate: Synthetic access to any site-directed stable isotope enriched l-histidine

Article abstract of DOI:10.3390/molecules19011023

1-Benzyl-2-(methylthio)-imidazole-5-ketone is obtained in a few simple steps starting from thiocyanate and glycine amide (glycin). Subsequent treatment with diethyl phosphorocyanidate and functional group manipulations gives 1-benzyl-5-chloromethylimidazolium chloride. This compound is converted under mild O'Donnell conditions into the corresponding L-histidine derivative. After deprotection L-histidine is obtained in good yield and 99% enantiomeric excess. 2'-13C-L-Histidine has been obtained via this new scheme with high (99%) 13C incorporation starting with commercially available 13C- thiocyanate. This synthetic scheme allows access to any isotopomer of L-histidine and many other biologically important imidazole derivatives.

Full text of DOI:10.3390/molecules19011023

Molecules 2014, 19, 1023-1033; doi:10.3390/molecules19011023
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Preparation of 2'-13C-L-Histidine Starting from
13C-Thiocyanate: Synthetic Access to Any Site-Directed
Stable Isotope Enriched L-Histidine
Sarra Talab 1,*, Kamal Khalifa Taha 2 and Johan Lugtenburg 3
1
Department of Chemistry, College of Applied and Industrial Science, University of Bahri,
P.O. Box 11111, Khartoum 1660, Sudan
2
Department of Chemistry, College of Science, AI Imam Mohammad Ibn Saud Islamic
University(IMSIU), P.O. Box 5701, Riyadh 11432, Kingdom of Saudi Arabia;
E-Mail: kamaltha99@rediffmail.com
3
Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden,
The Netherlands; E-Mail: lugtenbu@chem.leidenuniv.nl
* Author to whom correspondence should be addressed; E-Mail: Stalab9@gmail.com;
Tel.: +2-499-1229-2439.
Received: 14 November 2013; in revised form: 19 December 2013 / Accepted: 20 December 2013 /
Published: 15 January 2014
Abstract: 1-Benzyl-2-(methylthio)-imidazole-5-ketone is obtained in a few simple steps
starting from thiocyanate and glycine amide (glycin). Subsequent treatment with diethyl
phosphorocyanidate and functional group manipulations gives 1-benzyl-5-chloromethyl-
imidazolium chloride. This compound is converted under mild O’Donnell conditions into
the corresponding L-histidine derivative. After deprotection L-histidine is obtained in
good yield and 99% enantiomeric excess. 2'-13C-L-Histidine has been obtained via this
new scheme with high (99%) 13C incorporation starting with commercially available
13C- thiocyanate. This synthetic scheme allows access to any isotopomer of L-histidine and
many other biologically important imidazole derivatives.
Keywords: 1-benzyl-2-(methylthio)-5-imidazole carbonitrile; ethyl-1-benzyl-5-imidazol-
carboxylate; N-diphenylmethylene glycine tert-butyl ester; O’Donnell method

Molecules 2014, 19
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1. Introduction
L-Histidine constitutes a unique amino acid residue in peptides and proteins. Its pKa value is in the
range of 6.4–7.0, so histidine residues account to a large extent for the buffer capacity of the protein
system [1]. The histidine links with the central metal in haemoglobin, myoglobin, cytochrome and
(bacterio) chlorophyll and binds with other metal ions [2]. Via its role in the above mentioned systems
it is intimately linked to vital life processes such as respiration and photosynthesis. The access to a
whole library of side directed stable isotope-enriched histidine is essential to study the role of histidine
in the proteins which are responsible for respiration and photosynthesis at the atomic level with isotope
sensitive techniques without perturbation [3]. This deeper level of knowledge obtained in this way is
essential to get a fundamental understanding of these important life processes. In this paper we
describe the synthesis of 2'-13C-L-histidine (1a) via a new scheme that also allows access to any
isotopologue and isotopomer of L-histidine. In the present scheme the O’Donnell asymmetric amino
acid synthesis is used, allowing mild reaction conditions and simple workups [4]. In the previous
schemes preparative HPLC techniques had to be used [5,6]. Benzyl isothiocyanate is formed in a
simple reaction between potassium thiocyanate and benzyl chloride.
2. Results and Discussion
For the access to stable isotope labeled 2-13C 1-benzyl-5-chloromethylimidazolium chloride (9a)
the reactions depicted in Scheme 1 were optimized with natural abundance reagents. Potassium
thiocyanate (2) is reacted with benzyl chloride in o-dichlorobenzene in the presence of the phase
transfer catalyst bis(triphenyl)phosphoranylidene ammonium chloride. According to a known procedure
a mixture containing mainly benzyl isothiocyanate and some benzyl thiocyanate is obtained [7]. This
isomeric mixture is treated with glycine amide HCl 3 at pH 8.5 [8]. The product is recrystallized from
acetone and benzyl thiourea 4 is thus obtained.
Scheme 1. The preparation of 1-benzyl-5-chloromethylimidazolium chloride 9 starting
from potassium thiocyanate 2 giving stable isotope incorporation in 9 at position 1 and/or 2.
CH3
S
S
NH2
conc. HCl
CH3I
a
Cl
a
N
N
H
N
H
KSCN
a
N
O
Catalyst
O
2
2a
NH2CH2CONH2HCl
3
4
/ K2CO3
5
4a= 13C
5a = 13
C
S
CH3
S
a
N
(C H O) P(O)CN
EtOH/ H
C2H5S Na
LiCN/
2
5
2
a
N
NH
N
BF3.(C2H5)2O
O
6
7
OC2H5
6a= 13
C
N
7a= 13
C
1
N
3
1'
a
2a
N
NaNO2
N
LiAlH4
SOCl2
HNO
/
NH Cl
3
5
4
Na2CO3
O
8
9
OC2H5
8a= 13C
9a= 13C
Cl

Molecules 2014, 19
1025
For the cyclisation of 4 it is dissolved in acetone and aqueous HCl [9]. When the reaction is finished
the 1-benzyl-2-thioxoimidazolium-4-one is obtained by extraction (not shown in Scheme 1). The
conversion of benzyl isothiocyanate and 1-benzyl-2-thioxoimidazolium-4-one is analogous to the
reaction that takes place in the Edman sequencing [10] of proteins and is a high yielding, efficient
process. The conversion can also be effected with glycine instead of 3 [8].
1-Benzyl-2-thioxoimidazolium-4-one dissolved in acetonitrile is treated with methyl iodide in the
presence of potassium carbonate under known conditions to form 1-benzyl-2-(methylthio)-imidazol-5-
ketone (5) [10]. A conversion of aromatic ketones into unsaturated nitriles has been described [11].
We hoped that the amide function of 5 would undergo a similar conversion with diethyl
phosphoro-cyanidate. Compound 5 is treated with diethyl phosphorocyanidate in the presence of LiCN
in THF. After evaporation of THF the product extracted with ethyl acetate is the cyanohydrin
phosphate corresponding to 5. Treatment of this product in benzene with boron trifluoride etherate
results in the elimination of diethyl phosphate giving the aromatic nitrile 6, which is then dissolved in
ethanol and after the addition of concentrated sulphuric acid, the solution is refluxed. After the workup
crystalline ethyl-1-benzyl-2-ethyl thio-5-imidazole carboxylate is obtained (not shown in Scheme 1).
During the reaction of 6 with acidified ethanol not only the expected conversion of the nitrile
function into the ethyl carboxyl function, but also acid catalyzed conversion of the methylthio group
into an ethylthio function had taken place. This simple conversion of 6 into ethyl-1-benzyl-2-ethyl
thio-5-imidazole carboxylate was found after failed attempts of DIBAL-H reduction and Raney nickel
desulphurization of 6.
Ethyl-1-benzyl-2-ethyl thio-5-imidazole carboxylate is treated with sodium ethylthiolate in DMF [12].
This gives ethyl-1-benzyl-2,3-dihydro-2-thioxo-5-imidazole-carboxylate 7 after crystallization. This
compound was an intermediate in our earlier preparation of [1'-5' N] and [3'-5' N] L-histidine [6] and
can be converted into 1-benzyl-5-chloromethylimidazolium chloride (9).
The imidazolium chloride salt 9 is treated according to Scheme 2 with tert-butyl(N-diphenyl
methylene) glycinate (10) under O’Donnell conditions [4]. The protected histidine derivative 11 is
obtained in high yield. Acid catalysis removes the tert- butyl ester and the amino protection. A final
hydrogenlysis with palladium on activated charcoal gives L-histidine (1) with 99% chemical purity and
99% optical purity (via HPLC as described in the Experimental).
Scheme 2. The conversion of 1-benzyl-5-chloromethylimidazolium chloride (9) under
O’Donnell conditions into the protected L-histidine compound 11. Final deprotection leads
to stable isotope enriched L-histidine (1a).
3
N
1'
2a
N
O
1
'
O
5
N
4
3
1'
HN
2
3'
2'a
O
1
OH
H
9
4'
5'
O
N
N
Pd/ C, H2
NH2
O
11
11a= 13C
10
Compound 7 is treated briefly with NaNO2/HNO3 giving desulphurization to 1-benzyl-5-carbo-
ethoxyimidazole (8). Subsequently reduction of 8 with LiAlH4 converts the ethyl function into the

Molecules 2014, 19
1026
methylene alcohol function. Treatment of the latter product with SOCl2 gives the imidazolium chloride
salt 9. The analytical data of 9 are within experimental error in agreement with those reported by us
before [6].
3. Experimental
3.1. General
All chemicals were purchased from Sigma-Aldrich (Zwijndrecht, The Netherlands) or Acros
Chimica (Geel, Belgium). In all experiments anhydrous solvents were used. Ether refers to diethyl ether
and all solvent mixtures are given in volume ratio (v/v). All solvents were removed by evaporation
in vacuo.
Reactions were monitored by thin-layer chromatography [TLC, on Merck F 254 silica gel
60 aluminum sheets, 0.2 mm: spots were visualized by treatment with an oxidizing spray (2 g of
KMnO4 and 4 g of NaHCO3 in 100 mL of water)]. Column chromatography was performed on Merck
1
silica gel 60. H- and 13C spectra were recorded with a Bruker WM-300 or WM-400 with
tetramethylsilane (TMS: δ 0.00 ppm) as internal standard for spectra recorded in CDCl3. A Perkin-Elmer
Paragon 1000 FTIR spectrometer and Varian Cary 50 series UV spectrometer were used for IR and
UV measurement, respectively.
The optical purities of the histidines were determined by HPLC and are given as molar fractions (%).
The apparatus consisted of a Spherisorb ODS-2 (5 μm) column (250 × 4 mm), a Pharmacia LKB
gradient delivery system, a Reheodyne injection valve fitted with a 5-μL loop, and a Spectro Vision FD-300
fluorescence detector. Prior to chromatography the histidines were derivatized using o-phthalaldehyde
(OPA, Janssen, vosselaar Beerse, Belgium) and N-acetyl-L-cysteine (NAC, Aldrich) [13]. The
excitation and emission wavelengths were 360 and 405 nm, respectively. Derivatization was carried
out using a solution of 2 mg of histidine in H2O (I), a 0.2 M borax buffer (pH 10.4) (II), a solution of
10 mg of OPA in 5 mL of methanol (III) and a solution of 10 mg of NAC in 5 mL of methanol (IV).
10 μL of I, 20 μL of II, 10 μL of III and 10 μL of IV were added together. The mixture was shaken for
2 min and injected. The diastereomers were separated employing a linear elution gradient of 100%
buffer A2 (30 mM sodium acetate pH 4) to 80% buffer A2 and 20% buffer B2 (50% 30 mM sodium
acetate pH 7.6/50% acetonitrile) in 60 min (0.4 mL/min). The retention time was 50 min.
Mass spectra were recorded on a Finnigan MAT-90 mass spectrometer (CI-MS, CH4) or on a
Finnigan MAT ITD 700 mass spectrometer (EI, 70 eV) coupled to a Hewlett Packard 438 A gas
chromatograph equipped with a Hewlett Packard 25-m SE 30 capillary column (GC-MS). Prior to
EI-MS and CI-MS the amino acids were derivatized to their corresponding n-butyl esters. The sample
was treated with 3 M HCl in n-BuOH for 15 min at 100 °C under an anhydrous nitrogen atmosphere in
a sealed Kimax tube. The solvent was evaporated and the sample was injected as a solution in
dichloromethane. The procedures used for the preparation for the preparation of the isotopically
enriched compound were the same as those described for the corresponding unlabelled compounds.
Only spectroscopic characteristics of the enriched compounds that differ from those of the unlabelled
compound are given.

Molecules 2014, 19
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3.2. Preparation of Compounds
N-Benzyl-N'-(2'-acetamido) thiourea (4). First benzyl isothiocyanate is prepared. Benzyl chloride
(3.0 g, 23.7 mmol, 2.27 mL) is dissolved in o-dichlorobenzene (10 mL) to which the phase transfer
catalyst bis(triphenyl) phosphoranylidene ammonium chloride (0.57 g, 0.5 mmol) and solid potassium
thiocyanate (2, 2.5 g, 25.7 mmol) is added. The mixture is heated under reflux for three hours at 180 °C.
After cooling to room temperature the solvent is removed under reduced pressure, the residue is
extracted with chloroform (3 × 10 mL). The chloroform solution is washed with water and then dried
over MgSO4. After drying the solvent is removed under reduced pressure to yield benzyl
1
isothiocyanate as a yellow oil 2.9 g, 19.46 mmol (82.1%). H-NMR spectroscopy shows that the
product consists of a major compound (benzyl isothiocyanate , 83.0%) and a minor compound (benzyl
thiocyanate, 17.0%). Benzyl isothiocyanate: 1H-NMR (300 MHz, DMSO, δ in ppm): 4.91 (2H, benzyl
13
CH2), 7.31–7.42 (5H, aromatic CH); and (2) C-NMR (75.5 MHz, DMSO, δ in ppm): 47.9 (benzyl
CH2), 127.0 (CHPh), 128.0 (CHPh), 128.5 (CHPh), 129.2 (CS), 134.6 (Cq). Benzyl thiocyanate: 1H-NMR
(300 MHz, DMSO, δ in ppm): 4.34 (2H, benzyl CH2), 7.31–7.42 (5H, aromatic CH) and (2) 13C-NMR
(75.5 MHz, DMSO, δ in ppm): 37.0 (benzyl CH2), 127.0 (CHPh), 128.0 (CHPh), 128.5 (CHPh), 129.2
(CS), 133.5 (Cq).
Glycinamide hydrochloride (3, 4.0 g, 36.1 mmol) is dissolved in water (24 mL). To this mixture a
50% solution of KOH (35.7 mmol, 2 g in 3.0 mL of water) is added to adjust the pH to 8.5, and then
ethanol (72.0 mL) is added. The solution is cooled in ice bath to 0 °C, and then treated dropwise with
the previously prepared mixture containing 83.0% benzyl isothiocyanate and 17.0% benzyl thiocyanate
(5.39 g, 4.7 mL, 36.1 mmol). The reaction mixture is stirred for 3 h, and frozen at −30 °C overnight.
The mixture is allowed to reach room temperature, the solvent is decanted from the solid compound,
and the solid compound is dried in vacuo to remove the remaining ethanol and water. The product is
recrystallized from acetone to afford 7.0 g (31.4 mmol, 86.98%) of N-benzyl-N'-(2'-acetylamido)
thiourea as pale yellow crystals. 1H-NMR (300 MHz, DMSO, δ in ppm): 4.04 (2H, C-5), 4.66 (2H, C-1),
7.32 (5H, aromatic CH), 8.30 (1 H, N-2), 7.54 (1H, N-4), 7.11 (2H, N-7); 13C-NMR (75.5 MHz,
DMSO, δ in ppm): 42.3 (C-5), 45.1 (C-1), 126.8, 127.2, 128.2, 134.19 (Cq), 170.5 (C6, Cq), 175.0
(C3, Cq).
13
[13C] N-Benzyl-N'-(2'-acetamido) thiourea (4a). First C benzyl isothiocyanate (1.2 g, 8.0 mmol) is
1
prepared (80.0% yield) as described above from KS13CN (1.0 g, 10.3 mmol). H-NMR: the signal at
4.91 ppm was split into a doublet (3JC-H1 = 2.6 Hz).13C-NMR: enhanced signal at 129.2 ppm. Then
[13C]-N-benzyl-N'-(2'-acetamido)thiourea (4a, 1.5 g, 6.5 mmol) is prepared in 89.04% yield from
1
13C-benzyl isothiocyanate (1.1 g, 7.3 mmol) as described for 4. H-NMR: the signals at 4.11 and 4.65
were split into doublets (3JC3H5 = 4.8 Hz, 3JC3H1 = 5.6 Hz); 13C-NMR: enhanced signal at 175.0 ppm.
1-Benzyl-2-(methylthio)-imidazol-5-ketone (5). Benzyl thiourea (4, 7.0 g, 31.3 mmol) is dissolved in
acetone (25 mL) and treated carefully with concentrated HCl (3 mL for each gram of compound) and
the mixture is then heated overnight in an oil bath at 40 °C (the reaction is followed with TLC until the
starting compound has disappeared). The acetone is removed in vacuo and the residue is treated with
sat. aq. NaHCO3 to neutralize the excess acid and the mixture is extracted three times with CH2Cl2.

Molecules 2014, 19
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Then the combined organic layers are washed with water and brine (100 mL), the dichloromethane
layer is dried with MgSO4, filtered and concentrated in vacuo. The product is purified on a column of
silica gel (dichloromethane/methanol = 95/5) to give a yellow crystalline compound (5.05 g, 78.1%).
This compound (3-benzyl-2-thioimidazolidin-4-one) is dissolved in acetonitrile (30 mL). To this
mixture iodomethane (5.24 g, 36.9 mmol) and potassium carbonate (1.7 g, 12.3 mmol) are added. The
mixture is stirred at 40 °C for 14 h. The reaction mixture is cooled to room temperature and then
filtered. The solvents are removed in vacuo. The residue is dissolved in a mixture of dichloromethane
and hexane (1:1, 50 mL), filtered and the solvents are removed in vacuo. The product is purified on a
column of silica gel (dichloromethane/methanol = 97/3) yielding 1-benzyl-2-(methylthio)-imidazol-5-
1
ketone as a green crystalline compound (5.15 g, 23.4 mmol, 75%). H-NMR (300 MHz, DMSO, δ in
ppm): 2.22 (3H, methyl thiogroup CH3), 2.99 (2H, 5-CH2), 4.65 (2H, benzyl CH2), 7.21–7.33 (5H,
13
aromatic CH); C-NMR (75.5 MHz, DMSO, δ in ppm): 12.0 (CH3), 43.0 (C-5), 58.3 (benzyl CH2),
127.0, 127.6, 128.5, 136.1 (Cq), 162.1 (C2), 179.8 (C4, Cq).
[13C] 1-Benzyl-2-(methylthio)-imidazol-5-ketone (5a). 13C-1-benzyl-2-(methylthio)-imidazol-5-ketone
(5a, 1.25 g, 5.6 mmol, 96.5% yield) is prepared as described for 5 from 13C-benzylthiourea (1.3 g,
1
5.8 mmol). H-NMR: the signals at 2.22 and 4.65 ppm were split into doublets (3JC2H5 = 4.8 Hz,
3JC2H3' = 5.6 Hz); 13C-NMR: enhanced signal at 162.1 ppm.
1-Benzyl-2-(methylthio)-5-imidazolcarbonitrile (6). 1-Benzyl-2-(methylthio)-imidazol-5- ketone (5, 3 g,
13.6 mmol) is dissolved in THF (130 mL), and to this mixture diethyl phosphorocyanidate (DEPC,
6.67 g, 40.9 mmol) and lithium cyanide (1.3 g, 39.3 mmol) are added gradually [the lithium cyanide is
prepared by using a 250 mL round bottomed flask equipped with a magnetic stirrer, nitrogen inlet and
a 60 mL addition funnel; lithium hydride (1.0 g, 126 mmol) is dissolved in anhydrous tetrahydrofuran
(100 mL). The stirred suspension is cooled in an ice bath and acetone cyanohydrin (9.14 g, 9.14 mL,
100 mmol) is added dropwise over 15 min. After the addition is completed, the ice bath is removed
and the mixture stirred for 2 h at room temperature. The magnetic stirring bar is removed and the
solvent evaporated in vacuo to yield lithium cyanide as a white powder (4.07 g, 123.4 mmol, 99.2%)].
After the addition of LiCN is completed the reaction mixture is stirred at room temperature for
10 min. After removal of THF by evaporation the residue is dissolved in water (130 mL) and a mixture
of benzene/ethyl acetate (1/1, 400 mL). The organic layer is separated and washed with water
(2 × 100 mL) and saturated aqueous sodium chloride (1 × 100 mL). The organic layer is separated and
then dried over MgSO4, followed by concentration to give green oil, which is dissolved directly in
benzene (20 mL) followed by addition of boron trifluoride etherate (5.8 g, 40.9 mmol). The reaction
mixture is stirred at room temperature for 2 h under nitrogen. After 2 h benzene (200 mL) and water
(40 mL) are added to the reaction mixture, the organic layer is separated, and washed with water
(2 × 100 mL) and saturated aqueous sodium chloride (150 mL). The organic layer is separated, dried
over MgSO4 followed by concentration giving a brown oil of the nitrile, which is purified on a silica
gel column (CH2Cl2/MeOH = 95/5). The result is 2.3 g (10.04 mmol, 73.8%) of the desired compound 6
1
as a brown oil. H-NMR (300 MHz, DMSO, δ in ppm): 2.49 (3H, CH3), 5.15 (2H, benzyl CH2), 6.92
13
(H, C4), 7.16 (2H, aromatic CH), 7.41 (3H, aromatic CH); C-NMR (75.5 MHz, DMSO, δ in ppm):

Molecules 2014, 19
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15.9 (methyl thio group CH3), 46.1 (benzyl CH2), 104.1 (C5), 111.9 (CN), 126.5, 127.1, 128.6, 136.1
(Cq), 136.4 (C4, Cq), 138.0 (C2).
[13C] 1-Benzyl-2-(methylthio)-5-imidazol carbonitrile (6a). [13C]-1-benzyl-2-(methylthio)-5-
imidazolcarbonitrile (6a, 1.05 g, 3.8 mmol, 70.4% yield) is prepared as described for 6 from
1
13C-1-benzyl-2-(methylthio)-imidazol-5-ketone (1.2 g, 5.4 mmol). H-NMR: the signals at 2.49 and
3
13
5.15 ppm were split into doublets (3JC2H5 = 4.6 Hz, JC2H3' = 5.6 Hz); C-NMR: enhanced signal at
138.0 ppm.
Ethyl 1-benzyl-2,3-dihydro-2-thioxo-5-imidazole-carboxylate (7). 1-Benzyl-2-(methylthio)-5-imidazol-
carbonitrile (6, 1.5 g, 6.5 mmol) is dissolved in ethanol (10 mL), the mixture is cooled to 0 °C in an ice
bath and 0.53 mL of concentrated sulfuric acid (9.825 mmol) is added dropwise and carefully. After
the addition is completed the ice bath is removed and the mixture is heated under reflux for 24 h. The
mixture is cooled to room temperature and poured into a flask containing ice water (50 mL); the pH is
adjusted to 3 with an aqueous solution of KOH. The mixture is filtered and the filtrate is concentrated
in vacuo to yield a yellow solid of the desired compound, the solid is recrystallized from dichloromethane
to give brown needles (1.7 g, 5.86 mmol, 89.9%).
The latter is added to a suspension of sodium ethanethiolate (10.6 mmol, 0.85 g) in DMF (8 mL).
The mixture is heated at reflux overnight; the DMF is removed in vacuo to afford a crude solid. The
solid is dissolved in a minimal amount of ethanol (12 mL), and then silica gel (5 g) is added to this
mixture. The mixture is filtered carefully and washed with ethanol (5 mL). The residue is treated with
a mixture of CH2Cl2/EtOH = 95/5 to yield a yellow oil (1.29 g, 4.94 mmol, 76.0%) which is used
directly in the next step.
[13C] Ethyl 1-benzyl-2,3-dihydro-2-thioxo-5-imidazolecarboxylate (7a). 13C Ethyl 1-benzyl-2,3-
dihydro-2-thioxo-5-imidazole-carboxylate (7a, 1.08 g, 4.1 mmol, 95.3% yield) is prepared as described
for 7 from 13C 1-benzyl-2-(methylthio)-5-imidazolcarbonitrile (1.0 g, 4.3 mmol).
1-Benzyl 5-carbo-ethoxy imidazole (8). The yellow compound 7 is dissolved in a NaNO2 solution (1.0 mL,
0.5 g NaNO2 in 237 mL of H2O) and then nitric acid (65%, 0.35 mL) is added with continuous stirring.
The mixture is stirred for 30 min. and then boiled with a solution of sodium carbonate (40% in water,
8 mL). The product is extracted with ether, dried over MgSO4 and concentrated. Purification is
effected by column chromatography (CH2Cl2/EtOH = 97/3) to yield a brown oil (950 mg, 4.13 mmol,
1
83.6%). H-NMR (400 MHz, DMSO, δ in ppm): 1.19 (3H, ethyl ester group CH3), 3.94 (2H, ethyl
ester group CH2), 4.45 (2H, benzyl CH2), 6.75 (1H, C5), 7.19–7.35 (5H, aromatic CH), 8.13 (1H, C2);
13C-NMR (100 MHz, DMSO, δ in ppm): 16.1 (CH3), 42.9 (CH2), 43.4 (benzyl CH2), 119.2 (C5), 126.2,
126.7, 127.9, 138.8 (Cq), 134.5 (C4, Cq), 141.2 (C2), 158.2 (CO).
[13C] 1-Benzyl 5-carboethoxy imidazole (8a). 13C 1-Benzyl-5-carboethoxy imidazole (8a, 750 mg, 3.2 mmol,
88.9% yield) is prepared as described for 8 from 13C ethyl 1-benzyl-2,3-dihydro-2-thioxo-5-
imidazolecarboxylate (950 mg, 3.6 mmol). 1H-NMR: the signals at 4.45, 6.75 and 8.13 ppm were split
into doublets (3JC2H5 = 4.8 Hz, 3JC2H3' = 5.6 Hz, 1JC2H2 = 219 Hz); 13C-NMR: enhanced signal at 141.2 ppm.

Molecules 2014, 19
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1-Benzyl-5-chloromethylimidazolium chloride (9). In a 100 mL three-necked flask lithium aluminum
hydride (0.16 g, 4.4 mmol) is suspended in THF (20 mL) under a dry argon atmosphere. To this
suspension a solution of 1-benzyl 5carboethoxy imidazole (8, 930 mg, 3.5 mmol) in THF (15 mL) is
added dropwise with continuous stirring (15 min). The reaction is followed by TLC until the latter has
disappeared (2.5 h, TLC: CH2Cl2/EtOH = 95/5). The mixture is carefully diluted with water (12 mL)
and subsequently with 6 M HCl (12 mL). The pH is adjusted to 9 using a 6 M NaOH solution and the
aqueous layer is extracted with dichloromethane. The organic layers are combined, washed with
saturated aqueous NaCl and dried over anhydrous MgSO4. After filtration the solvent is removed
in vacuo, yielding a yellow oil (660 mg, 3.19 mmol, 86.8%). A portion (500 mg) of this compound is
placed in a 100 mL round bottomed flask fitted with a reflux condenser to which thionyl chloride
(1.23 g, 10.0 mmol) is added. The mixture is refluxed for three h, and then allowed to cool to room
temperature. The volatiles are evaporated in vacuo. The result is a brown oil (560 mg, 2.45 mmol,
70.0%) of the desired compound. 1H-NMR (400 MHz, CDCl3, δ in ppm): 4.36 (2H, CH2Cl), 4.69 (2H,
benzyl CH2), 6.90 (1H, C5), 7.31–7.40 (5H, aromatic CH), 7.91 (1H, C2); 13C-NMR (100 MHz,
CDCl3, δ in ppm): 44.6 (C-4'), 52.2 (benzyl CH2), 111.7 (C5), 127.5, 127.7, 129.0, 131.9 (C2), 135.6
(Cq), 144.0 (C4, Cq).
[13C] 1-Benzyl-5-chloromethylimidazolium chloride (9a). 13C 1-benzyl-5-chloromethylimidazolium
13
chloride (9a, 540 mg, 2.21 mmol, 73.7% yield) is prepared as described for 9 from C 1-benzyl-5-
1
carboethoxyimidazole (700 mg, 3.0 mmol). H-NMR: the signals at 4.69, 6.90 and 7.91 were split into
doublets (3JC2H5 = 4.6 Hz, 3JC2H3' = 5.6 Hz, 1JC2H2 = 221 Hz). 13C-NMR: enhanced signal at 131.9 ppm.
Tert-butyl-3-(1-benzyl-1H-imidazol-5-yl)-2(diphenylmethyleneamino) propanoate (11). N-(Diphenyl-
methylene) glycine tert-butyl ester (10, 570 mg, 1.92 mmol) and the chiral catalyst 2,7-bis [O-9-allyl
hydrocinchonidinium N-methyl]naphthalene dibromide (19 mg, 0.0019 mmol) are dissolved in toluene
(10 mL). To this mixture 1-benzyl-5-chloromethylimidazolium chloride (9, 500 mg, 2.05 mmol) is
added. The reaction mixture is then cooled to 0 °C, and 50% aqueous KOH (0.28 mL) is added, the
mixture is stirred at 0 °C until the starting material has been consumed (8 h). The ice bath is removed
and the mixture is diluted with ether (25 mL), washed with water (2 × 15 mL), the ether layer is
separated, dried over MgSO4, filtered and concentrated to yield a brown crystalline compound (780 mg,
1
1.67 mmol, 87.5% yield). H-NMR (300 MHz, DMSO, δ in ppm): 1.39 (9H, CH3), 4.03 (2H, benzyl
CH2), 4.48 (2H, CH2), 4.94 (1H, C3'), 6.90 (1H, C5), 7.1–7.55 (15H, aromatic CH), 7.90 (1H, C2);
13C-NMR (100 MHz, DMSO, δ in ppm): 27.6 (3C, CH3), 53.3 (C-4'), 55.7 (benzyl CH2), 67.2(C, C3'),
75.6 (C, OMe3), 125.2 (C5), 126.9–128.8 (15 C, CHPh), 135.0 (C4, Cq), 135.0 (C2), 137.3–138.6 (Cq),
168.9 (C=N), 170.0 (CO).
[13C] Tert-butyl-3-(1-benzyl-1H-imidazol-5-yl)-2(diphenylmethyleneamino) propanoate (11a). 13C
tert-Butyl-3-(1-benzyl-1H-imidazol-5-yl)-2(diphenylmethyleneamino) propanoate (11a, 1.1 g, 2.36 mmol,
13
95.9% yield) is prepared as described for 11 from C 1-benzyl-5-chloromethylimidazolium chloride
(600 mg, 2.46 mmol). 1H-NMR: the signals at 4.03, 6.90 and 7.90 were split into doublets (3JC2H5 = 4.8 Hz,
3JC2H1' = 5.6 Hz, 1JC2H2 = 220 Hz). 13C-NMR: enhanced signal at 135.0 ppm.

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L-Histidine dihydrochloride (1). tert-Butyl-3-(1-benzyl-1H-imidazol-5-yl)-2-(diphenylmethylene-
amino) propanoate (11, 400 mg, 0.86 mmol) is dissolved in THF (10 mL) and then stirred with 10%
citric acid solution (5 mL) overnight. TLC shows complete disappearance of the starting material. The
mixture is extracted twice with ether (20 mL). The water layer is then brought to pH 12 with a K2CO3
solution and extracted with ethyl acetate (10 mL). The combined organic layers are dried over MgSO4
and concentrated in vacuo to give brown crystals (250 mg, 0.828 mmol, 96.3%). This compound is
then treated with 1 N HCl (5 mL), and the mixture is refluxed overnight. The reaction is followed by
TLC until the starting material has disappeared (dichloromethane/ethanol = 99/1). The mixture is
allowed to reach room temperature, diluted with a small amount of dichloromethane, filtered and
concentrated in vacuo. The product is a yellow oil (230 mg, 0.709 mmol, 85.7%). The latter is
dissolved in a mixture of cyclohexene/methanol (1/1). Palladium black (100 mg) is added to the
mixture. The mixture is refluxed until the conversion is complete, which takes 5 days. On the second
day a freshly prepared Pd pellet [6] (50 mg in 10 mL of the same solvent mixture) is added to the
mixture. The suspension is cooled down to room temperature and filtered over Celite. The solvent is
evaporated in vacuo; the compound is redissolved in methanol and then concentrated to yield a yellow
1
solid (165 mg, 0.727 mmol, 84.5%). H-NMR (400 MHz, D2O/DCl, δ in ppm): 3.46 (1H, H3a), 3.62
(1H, H3b), 4.42 (1H, H2), 7.49 (1H, H5'), 8.74 (1H, H2'); 13C-NMR (100 MHz, D2O/DCl, δ in ppm):
27.9 (C3), 54.7 (C2), 118.5 (C5'), 130.0 (C4'), 135.8 (C2'), 174.0 (C1).
2'-[13C]-L-Histidine dihydrochloride (1a). 2'-13C-L-Histidine dihydrochloride (1a, 390 mg, 1.7 mmol,
80.9% yield) is prepared as described for 1 from 13C tert-butyl-3-(1-benzyl-1H-imidazol-5-yl)-2-
1
(diphenylmethyleneamino) propanoate (1.0 g, 2.1 mmol). H-NMR: the signals at 7.49 and 8.74 ppm
1
13
were split into doublets (3JC2H5' = 6.5 Hz, JC2'H2' = 221 Hz). C-NMR: enhanced signal at 135.8 ppm.
Both chemical and optical purity is 99% as found by an HPLC method, in agreement with the literature [5].
The more volatile n-butyl ester of 2'-13C-L-histidine was investigated by double focus chemical
ionization mass spectroscopy (ionization gas CH4). The signal at m/z = 213.1439 corresponds with
13
16
12C9 C1H1814N3 O2 (calculated mass: 213.1433). In the single focus EI/70 eV mass spectrum the
fragment at m/z = 82 is formed by McLafferty rearrangement involving the imidazole ring. From the
base peak and the M-1, M+1 and M+2 peaks 99% enrichment with 13C at the C2' could be established
13
13
within experimental error and in agreement with the C incorporation in C thiocyanate. No isotope
dilution has taken place during the reaction sequence.
4. Conclusions
13
A simple procedure to obtain 2'-13C-L-histidine in high optical purity and high C incorporation is
described. The analytical properties of 2'-13C-L-histidine are within experimental error in agreement
with the literature [5]. The building blocks that form the imidazole ring and L-histidine in Schemes 1
and 2 are either commercially available or easily accessible via literature procedures [3]. This means
that L-histidine is accessible in any stable isotopologic and isotopomeric form. Using the optical
antipode of the O’Donnell phase transfer catalyst will give access to all possible D-histidines in
isotopomeric form. Recently the simple incorporation of 2H and 3H in histidine has been reported [14],

Molecules 2014, 19
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which together with the work described in this paper extends the access to stable and unstable
isotopomers and isotopologues of histidine considerably.
Acknowledgments
The authors wish to thank the University of Juba for the financial support during Sarra Talab’s stay
in Leiden, H.S. Overkleeft for financing her extension time there and A.W.M. Lefeber (Leiden
Institute of Chemistry) for his help in recording the NMR spectra. R. van der Steen and R. van Nispen
are thanked for their help in preparing the text.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1 and 1a are available from the authors.
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