Improved specific synthesis of [1′-15N]- and [3′-15N]L-histidine

Article abstract of DOI:10.1002/jlcr.507

Specifically, ¹⁵N-enriched L-histidines have been prepared. The labelling methodology involves introduction of labels in its precursor 1-benzyl-5-hydroxy methyl imidazole, which is converted into L-histidine via the Schoellkopf method. The procedure allows the preparation of the intermediates and finally histidine with high ¹⁵N enrichment (99%) at each position, in 29% overall yield starting with ¹⁵NH₄Cl and 56% with KSC¹⁵N, respectively. Copyright

Full text of DOI:10.1002/jlcr.507

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Labelled Cpd Radiopharm 2001; 44: 831–841.
DOI: 10.1002/jlcr.507
Research Article
Improved specific synthesis of [10-15N]- and
[30-15N]l-histidine
C. Soede-Huijbregts, M. van Laren, F. B. Hulsbergen, J. Raap
and J. Lugtenburg*
Leiden Institute of Chemistry, Gorlaeus Laboratorium, Leiden University,
P.O. Box 9502, 2300 RA Leiden, The Netherlands
Summary
Specifically, 15N-enriched l-histidines have been prepared. The labelling
methodology involves introduction of labels in its precursor 1-benzyl-5-
hydroxy methyl imidazole, which is converted into l-histidine via the
.
Schollkopf method. The procedure allows the preparation of the intermediates
and finally histidine with high 15N enrichment (99%) at each position, in 29%
overall yield starting with 15NH4Cl and 56% with KSC15N, respectively.
Copyright # 2001 John Wiley & Sons, Ltd.
Key Words: 15N-labelling; 13C-labelling; 1-benzyl-5-hydroxymethylimidazole;
l-histidine
Introduction
l-histidine residues play an important role in life processes. In many
enzymes, the t- and p-nitrogen atoms of the heterocyclic imidazole side
chain play a key-role in both protonation and tautomerization
reactions. In photosynthesis,1–11 the energy and material generating
process in the biosphere, the Mg2+ ions of the (bacterio)chlorophylls
are axially co-ordinated to the 10-N atom of l-histidine residues both in
*Correspondence to: J. Lugtenburg, Leiden Institute of Chemistry, Gorlaeus Laboratorium,
Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
E-mail: lugtenbu@chem.leidenuniv.nl.
Contract/grant sponsor: Netherlands Foundation for Chemical Research (SON).
Copyright # 2001 John Wiley & Sons, Ltd.
Received 27 April 2001
Revised 25June 2001
Accepted 10 July 2001

832
C. SOEDE-HUIJBREGTS ET AL.
the antenna complexes and photosynthetic reaction centres and special
properties of the imidazole ring system in the conversion of the light
energy into chemical energy are expected.1–10 NMR spectroscopy is the
method of choice to determine the univocal protonation, tautomeric
and co-ordination states. The incorporation of highly enriched 15N-
labelled l-histidines into the protein is achieved by growing micro-
organisms on a well-defined substrate that is supplemented with the
specifically labelled amino acid.
Earlier, we reported the synthesis of 15N- and 13C-labelled l-histidines
in which 1-benzyl-5-hydroxymethyl-imidazole has been coupled via its
chloride derivative to the chiral bislactim ether of cyclo-(d-valylglycine)
to produce the enantiomerically pure labelled l-histidines.11 However,
attempted growth of photosyntheticmirco-organisms on media that
contained this [10-15N]l-histidine lead to the death of the living cells,
which meant that it contained analytically undetectable traces of toxic
substances.
In the present paper, we describe an improved synthesis of 1-benzyl-5-
hydroxymethyl-imidazole and the 15N-histidines prepared via this new
approach which do not exhibit harmful effects on the growth of
photosyntheticmicroorganisms.
Results and discussion
For the synthesis of [1-15N]- and [3-15N]1-phenyl-5-hydroxymethylimi-
dazole, 8a and 8b respectively, we optimized the new route depicted in
the scheme, which is partially based on a literature method.12 The
[1-15N]Im- and [3-15N]Im-atoms are introduced via [15N]-labelled
benzylamine (which is prepared from 15NH4Cl and benzoylchloride)
and KSC15N, respectively. However, first the scheme was optimized
using synthons with natural isotopicabundance.
In the first step, methyl bromoacetate (2) is treated with benzylamine
%
to give N-benzylglycine methyl ester (3). Via treatment with formicacid
%
and acetic acid anhydride, 3 is N-formylated to N-benzyl-N-formylgly-
%
cine methyl ester (4).
%
Compound 4 is subsequently carbon formylated with methyl formate
%
in the presence of sodium methoxide, which gives the sodium salt 5.
%
Treatment of 5 with potassium thiocyanate in the presence of aqueous
%
HCl gives the imidazole derivative 6. The compound is converted by
%
treatment with nitric acid in the presence of sodium nitrite, followed by
Copyright # 2001 John Wiley & Sons, Ltd.
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IMPROVED SPECIFIC SYNTHESIS
833
addition of sodium carbonate to give methyl 1-benzyl-5-imidazole-
carboxylate (7).
%
LiAlH4 reduction the ester function gives the required 1-benzyl-5-
hydroxymethylimidazole (8). The latter compound has been converted
%
into the corresponding chloromethyl derivative and finally to the
isotopomers of l-histidine by the method described before.11 The
isotopic enrichments at the chosen positions, determined by mass
spectrometry, were the same (99%) as that of the starting materials. The
conversions could be executed without scrambling and isotope dilution.
The syntheticscheme has also been optimized to label the 13C-atoms of
histidine by using reagents (that can be commercially obtained in 13C-
enriched form) in stoichiometric quantities. This means that 8 and l-
%
histidine can now be obtained in isotopically enriched form (13C, 15N)
on each position and combinations of positions.
Via the present scheme, 6.7 g of [30-15N]l-histidine has been prepared
15
in a 29% overall yield based on NH4Cl. Similarly 6.2 g of [10-15N]l-
histidine has been obtained in a 56% overall yield based on KSC15N.
All experiments to incorporate [15N]-l-histidine, made via the present
scheme, in photosynthetic organisms did not show any harmful effect on
the organisms, meaning that the biological quality of isotopically
enriched histidine made via the present scheme is excellent.
Experimental
General details
All chemicals were purchased from Acros Organics or Aldrich, except
for [15N]-ammonium chloride (99% 15N) and the [15N]-potassium
thiocyanate (99% 15N), which were obtained from Cambridge Isotope
Laboratories. [15N]benzylamine (99% 15N) was prepared from [15N]am-
monium chloride as has been described previously.11 In all experiments
distilled anhydrous solvents were used. Tetrahydrofuran (THF) was
freshly distilled from sodium, prior to use. Ether refers to diethylether
and all solvent mixtures are given in volume ratios (v/v). All solvents
were removed by evaporation in vacuo.
All reactions were optimized using non-isotopically enriched starting
materials and the procedures are all described for unlabelled
compounds. The procedures that were used for the preparation of the
isotopically enriched compounds were the same as those described for
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C. SOEDE-HUIJBREGTS ET AL.
the corresponding unlabelled compounds. Only those spectroscopic
characteristics or the enriched compounds are given that are different
from the unlabelled compounds.
The specificity of the isotope labelling has been determined by NMR
1
spectroscopy. All H- and 13C-NMR spectra of 15N compounds were
1
recorded using a Jeol NM FX-200 or a Bruker WM-300. H, 13C and
15N spectra of the labelled 15N products were recorded on a Bruker
WM-300 or on a Bruker WM-600 with tetramethylsilane (TMS:
d 0.00 ppm) as internal standard for spectra recorded in CDCl3, and
3-(trimethylsilyl)tetradeuteropropionicidac sodium salt (TPS:
d 0.00 ppm) for spectra recorded in D2O. For the 15N-NMR spectra
we used a saturated solution of ammonium nitrate as an external
standard (15NH4NO3 d 22.3 ppm relative to NH3 (l): 0.00 ppm).
Mass spectrometry is used to determine the exact molecular mass and
the isotope enrichment of the synthesized histidines. The exact
molecular mass is determined with double focus mass spectroscopy
(Finnigan MAT 900 mass spectrometer, equipped with a direct insertion
probe (EI-MS, 70 eV), using a soft ionization (ESI) technique, which
results in a protonated molecule. For [10-15N]- and [30-15N]l-histidines
the experimental m=z values are 157.07421 and 157.07426, respectively.
Both values correspond to the calculated one (m=z 157.07434) of the
1
protonated histidine molecule 12C6 H9 14N2 15N16O2).
Prior to the determination of the isotope enrichment the labelled
histidines are derivatized to form the N,O-ethoxycarbonyl ethyl ester
(N,O-ECEE) derivatives and the enrichment is calculated from the
intensities of the peaks measured by GC/MS. The measurements were
performed on a Finnigan MAT ITD 700 (EI, 70 eV) coupled to a
Chrompack 438A gas chromatograph equipped with a 25 m fused silica
column (CP-Sil-5CB; 0.25 mm i.d.). The amino acids were derivatized
by reaction with ethyl chloroformate (ECF).13 The sample was dissolved
in 200 ml of a mixture of water/ethanol/pyridine (60/32/8) and 10 ml of
ECF was added. The tube was vortexed for 5 s.; gas evolution occurred
(CO2 release). A solution of 1% ECF in chloroform (200 ml) was added
and the derivatives were extracted into the organic phase by vortexing
for 15 s.; 1 ml of this organic layer was injected (Scheme 1).
Preparation of the compounds
N-Benzylglycine methylester (3). Methyl bromoacetate (2) (7.66 g,
%
%
50 mmol, 4.74 ml) in 50 ml THF was treated with 13.9 ml (10.1 g,
Copyright # 2001 John Wiley & Sons, Ltd.
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IMPROVED SPECIFIC SYNTHESIS
835
Scheme 1. Synthesis of 1-benzyl-5-hydroxymethylimidazole (8) and the [1-15N]-
and [3-15N]-isotopomers (8a and 8b), the precursor for the synthesis of [10-15N]-
and [30-15N]histidine (1a and 1b). Labelled positions are indicated by italics.It
should be noticed that in the literature two different systems of numbering the
atoms of the imidazole ring of histidine had been used for a long time (biochemists
generally numbering as 1, the nitrogen atom adjacent to the side chain, and
organic chemists designed it as 3). For this reason the by IUPAC recommended
notation pros (‘near’, abbreviated by p ) and tele (‘far’ abbreviated t) has also
been included14
100 mmol) Et3N. To this solution 5.58 g (52 mmol, 5.69 ml) of
benzylamine was added. After stirring (1 night, at room temperature)
the precipitated Et3NHBr was removed by filtration and the filtrate was
concentrated under vacuum to yield pure N-benzylglycine methylester
(3) (8.26 g, 46 mmol, 92% yield).
%
1H NMR (300 MHz, CDCl3, d in ppm): 2.03 (bs, NH), 3.37 (s, 2H,
CH2), 3.67 (s, 3H, CH3), 3.76 (s, 2 H, PhCH2), 7.29 (m, 5 H, CHPh).
13C NMR (75.5 MHz, CDCl3, d in ppm): 49.9 (CH2), 51.4 (CH3), 53.1
(CH2), 126.8 (CHPh), 127.8 (CHPh), 127.9 (CHPh), 139.2 (CPh,q), 172.5
(CO).
[15N]Benzylglycine methylester (3a). 20.4 g (114 mmol) of [15N]benzyl-
glycine methylester (3a) was prepared as described for 3 from 18.7 g
(122 mmol) [15N]benzylamine (94% yield).
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C. SOEDE-HUIJBREGTS ET AL.
1H NMR (300 MHz, CDCl3): the signal at 2.00 ppm is broadened and
2
the signal at 3.36 ppm is split into a doublet, JNH 1.0 Hz.
13C NMR (75.4 MHz, CDCl3): the signals at 49.9 and 53.1 ppm
1
1
appear as doublets, JNC 4.4 Hz and JNC 4.4 Hz, respectively.
15N NMR (30.4 MHz, CDCl3): 32.4 (bs) ppm.
N-Benzyl-,N-formylglycine methylester (4). To a solution of 3.43 g
%
(19.2 mmol) N-benzylglycine methylester (3) in 15 ml 98% formicacid,
%
15 ml of acetic anhydride was added in small portions. When the
vigorous reaction had subsided, the mixture was heated on an oil bath
(1008C) for 1 h and then evaporated under vacuum to remove the formic
and acetic acids. The residual material was taken up into dichlor-
omethane. This solution was washed four times with 1 M NaHCO3
solution to remove the last traces of acid, once with water and once with
a saturated solution of NaCl and subsequently dried over anhydrous
MgSO4. Concentration in vacuo yielded 3.67 g (17.71 mmol, 92% yield)
of N-benzyl-N-formyl-glycine methylester (4).
%
1H NMR (300 MHz, CD3S(O)CD3, d in ppm): rotamer A: 3.62 (s, 3H,
CH3), 4.10 (s, 2H, CH2), 4.50 (s, 2H, CH2Ph), 7.21 (m, 2H, CHPh), 7.31
(m, 3H, CHPh), 8.22 (s, 1H, OCH). Rotamer B: 3.60 (s, 3H, CH3), 3.90
(s, 2H, CH2), 4.56 (s, 2H, PhCH2), 7.21 (m, 2H, CHPh ), 7.31 (m, 3H,
CHPh), 8.42 (s, 1H, OCH).
13C NMR (50.1 MHz, CD3S(O)CD3, d in ppm): rotamer A: 42.6
(CH2), 51.1 (CH2Ph), 51.6 (CH3), 127.4 (CHPh), 127.8 (CHPh), 128.4
(CHPh), 134.7 (Cq,Ph), 162.7 (HCO), 168.3 (CO). Rotamer B: 45.8 (CH2),
47.3 (CH2Ph), 51.6 (CH3), 128.0 (CHPh), 128.2 (CHPh), 128.4 (CHPh),
135.1 (Cq,Ph), 162.4 (HCO), 169.0 (CO).
N-benzyl,N-formyl-[15N]glycine methylester (4a). Compound 4a
(21.5 g, 165 mmol) was prepared as described for 4 from 20.4 g
%
(184 mmol) of N-benzyl-[15N]glycine methylester (3a) (91% yield).
1H NMR (300 MHz, CDCl3): rotamer A: signal at 8.42 ppm was split
2
into a doublet, JNH 15.8 Hz. Rotamer B: signal at 8.22 ppm was also
2
split into a doublet, JNH 15.5 Hz.
13C NMR (75.4 MHz, CDCl3): Rotamer A: signals at 42.6 and 51.1 ppm
1
were split into doublets, JNC 13.2 Hz and JNC 11.7 Hz, respectively.
1
Rotamer B: signals at 45.8 and 47.3 ppm were also split into doublets,
1
1JNC 13.2 Hz and JNC 12.8 Hz respectively.
15N NMR (30.4 MHz, CDCl3, d in ppm): rotamer A: 121.8 (s).
Rotamer B: 122.5 (s).
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IMPROVED SPECIFIC SYNTHESIS
837
N-Benzyl,N-formyl,C-formylglycine methylester sodium salt (5). In a
%
three-necked flask provided with a stirrer and thermometer was placed
2.19 g (10.57 mmol) of N-benzyl,N-formylglycine methylester and 1.80 g
(29.98 mmol) of dry methyl formate. The reaction mixture was cooled in
an ice bath whilst the mixture was stirred. Then, 0.78 g (14.44 mmol) of
sodium methoxide and 5 ml of dry toluene were added. The temperature
of the reaction mixture was maintained below 158C, and stirring was
continued for 1 h after all the sodium methoxide had been added. The
mixture was then placed in the refrigerator and allowed to stand for
approximately 24 h. To isolate the sodium salt, extremely dry ether was
added to the reaction mixture, and the yellowish solid was collected on a
glass filter, washed with extremely dry ether and finally dried under
vacuum. The result was 2.35 g (8.66 mmol, 88% yield) of the desired
product.
1H NMR (300 MHz, CD3SOCD3, d in ppm): 3.36 (s, 3H, CH3), 4,50
(s, 2H, CH2), 7.18 (m, 3H, CHPh), 7.78 (m, 2H, CHPh), 8.48 (bs, 1H,
CHO), 8.88 (bs, 1H, HCONa). 13C NMR (75.5 MHz, CD3SOCD3, d in
ppm): 47.1 (CH2), 49.3 (CH3), 126.0 (C2), 128.7 (CHPh), 129.2 (CHPh),
129.5 (CHPh), 138.8 (CPh,q), 165.8 (NCHO), 169.3 (CO) and 173.3
(HCONa).
N-Benzyl,N-formyl,C-formyl-[15N]glycine methyl ester sodium salt (5a).
4.69 g (18.2 mmol) of (5a) was synthesized as described above from
4.30 g (20.7 mmol) of benzyl-N-formyl-[15N]glycine methylester (4a)
(yield: 88%).
1H NMR (300 MHz, CD3SOCD3): The signal at 8.50 ppm appeared
2
as a doublet: JNH 11.8 Hz.
13C NMR (75.5 MHz, CD3SOCD3: No splittings were observed.
15N NMR (30.4 MHz, CD3SOCD3: 126.9 (s) ppm.
Methyl 1-benzyl-2-mercapto-5-imidazolecarboxylate (6). 2.23g (8.67mmol)
%
of the dry sodium enolate salt of N-benzyl,N-formyl,C-formylglycine
methyl ester (5) was dissolved in 10 ml of 50% methanol and 1.66 ml
%
(19.9 mmol) of 12 M hydrochloric acid was added. After standing for
approximately 20 h at room temperature, 0.84 g (8.64 mmol) of potassium
thiocyanate was added and the mixture was heated on an oil bath at 808C
until most of the alcohol had evaporated. The methyl 1-benzyl-2-mercapto-
5-imidazolecarboxylate (6) was collected by filtration, washed with water
%
and air dried. The result was 1.79 g (7.22 mmol, 83% yield based on
KSCN).
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C. SOEDE-HUIJBREGTS ET AL.
1H NMR (200 MHz, CDCl3, d in ppm): 3.76 (s, 3H, CH3), 5.70 (s,
2H, PhCH2), 7.25 (m, 5H, CHPh), 7.44 (s, 1H, H4) 12.65 (bs, 1H, NH).
13C NMR (50.1 MHz, CDCl3, d in ppm): 48.4 (CH2Ph), 51.8 (OCH3),
120.8 (Cq, C5), 123.0 (C4), 127.5 (CHPh), 128.1 (CHPh), 128.3 (CHPh),
136.2 (CPh,q), 158.5 (Cq, CO), 164.6 (Cq, C2).
Methyl 1-benzyl-2-mercapto-[1-15N]5-imidazolecarboxylate (6a). 5.1 g
(20 mmol) of (6a) was prepared from 4.3 g (21 mmol) of benzyl-N-
formyl-[15N]glycine methylester (5a) (98% yield) as described above
for 6.
%
1H NMR (200 MHz, CDCl3): the signal at 7.44 ppm was split into a
3
doublet, JNH 2.4 Hz.
13C NMR (50.1 MHz, CDCl3): the signals at 48.5, 120.8 and
1
1
164.6 ppm are shown as doublets, JNC 10.3 Hz, JNC 13.2 Hz and
1JNC 14.7 Hz, respectively.
15N NMR (30.4 MHz, CDCl3): 173.0 (s) ppm.
Methyl 1-benzyl-2-mercapto-[3-15N]5-imidazolecarboxylate (6b). Com-
pound (6b) (8.52 g, 33.98 mmol) was prepared similarly from 9.56 g
(36.95 mmol) of the dry sodium enolate salt of N-benzyl,N-formyl,C-
formylglycine methylester (6) and 3.46 g (35.2 mmol) of KSC15N (92%
%
yield based on the labelled starting material).
1H-NMR (300 MHz, CDCl3): the signal at 7.45 and 12.65 ppm are
3
doublets, JNH 3.4 Hz and JNH 99.4 Hz, respectively.
1
13C NMR (75.5 MHz, CDCl3): the signals at 122.9 and 164.6 ppm
1
were split into doublets, JCN 10.8 Hz and JCN 14.6 Hz, respectively.
1
1
15N-NMR (30.4 MHz, CDCl3): 171.5 (d, JNH 99.3 Hz, NH) ppm.
Methyl 1-benzyl-5-imidazolecarboxylate (7). 3.53 g (14.22 mmol) of
methyl 1-benzyl-2-mercapto-5-imidazolecarboxylate (6) was dissolved
%
in 7.70 ml NaNO2 solution (2.0 g NaNO2 in 950 ml water) and 2.68 ml of
nitricacid (35% HNO 3) was added with continuous stirring. After all
the nitricacid had been added, the solution was stirred for about 10 min
or until the evolution of nitrogen oxides had completely stopped. The
reaction mixture was boiled with 40 ml of a solution of sodium
carbonate (40%) to liberate methyl 1-benzyl-5-imidazolecarboxylate.
Extraction with ether yields the maximum amount of the product. The
yield was 3.01 g (13.92 mmol, 98% yield) methyl 1-benzyl-5-imidazole
carboxylate (7).
%
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IMPROVED SPECIFIC SYNTHESIS
839
1H NMR (300 MHz, CDCl3, d in ppm): 3.79 (s, 3H, CH3), 5.50
(s, 2H, PhCH2), 7.17 (m, 2 H, CHPh), 7.30 (m, 3H, CHPh), 7.62 (d, 4JHH
4
0.8 Hz, 1H, H4), 7.77 (d, JHH 0.8 Hz,1H, H2).
13C NMR (75.5 MHz, CDCl3, d in ppm): 49.8 (CH2), 51.3 (CH3),
122.2 (C5), 127.1 (CHPh), 127.9 (CHPh), 128.7 (CHPh), 136.0 (CPh,q),
137.9 (C2), 142.1 (C4 ),160.4 (Cq (CO)).
Methyl 1-benzyl,[1-15N]5-imidazolecarboxylate (7a). Compound 7a
(6.4 g, 114 mmol) was prepared as described for 7 from 8.6 g (145 mmol)
of methyl 1-benzyl-2-mercapto-[-15N]5-imidazolecarboxylate (6a) (85%
yield).
1H NMR (200 MHz, CDCl3): the signals at 7.62 and 7.76 ppm were
2
split into double doublets, JNH 7.9 Hz en JNH 2.7 Hz.
3
13C NMR (50.1 MHz, CDCl3): the signals shown at 49.9, 122.3,
1
137.8, 142.1 and 160.4 ppm appear as doublets, JNC 8.8 Hz, JNC
1
2
1
2
18.4 Hz, JNC 5.9 Hz, JNC 10.2 Hz and JNC 2.9 Hz, respectively.
15N NMR (30.4 MHz, CDCl3): 118.1 (s) ppm.
Methyl 1-benzyl-[3-15N]5-imidazolecarboxylate (7b). Compound 7b
(3.63 g, 16.59 mmol) was prepared as described for 7 from 4.25 g
%
(16.95 mmol) of methyl 1-benzyl-2-mercapto-[3-15N]5-imidazolecarbox-
ylate (6b) (98% yield).
1H-NMR (300 MHz, CDCl3): the signals at 7.62 and 7.77 ppm were
split into double doublets, 3JNH 11.0 Hz and 3JNH 10.0 Hz, respectively.
13C NMR (75.5 MHz, CDCl3): the signals at 122.3, 137.9 and
142.1 ppm are broadened.
15N-NMR (30.4 MHz, CDCl3): 257.6 (m) ppm.
1-Benzyl-5-hydroxymethylimidazole (8). In a 100 ml three-necked flask
%
0.13 g (3.40 mmol) lithium aluminium hydride was suspended in 15 ml of
extremely dry THF under a dry argon atmosphere. To this suspension, a
solution of 0.719 g (3.33 mmol) of methyl 1-benzyl-5-imidazolecarbox-
ylate (7) in 15 ml extremely dry THF was dropwise added with
continuous stirring, until the methyl 1-benzyl-5-imidazolecarboxylate
had disappeared (3–3.5 h, TLC: toluene/n-BuOH 80/20) The mixture
was carefully diluted with 10 ml of water and subsequently with 10 ml of
6 M HCl. The pH was adjusted to 9 using 6 M NaOH and the aqueous
layer was extracted with dichloromethane. The organic layers were
combined, washed with saturated aqueous NaCl and dried on
anhydrous MgSO4. After filtration, the solvents were evaporated in
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C. SOEDE-HUIJBREGTS ET AL.
vacuum, yielding 0.56 g (2.98 mmol, 89% yield) of 1-benzyl-5-hydro-
xymethylimidazole (8).
%
1H NMR (300 MHz, CDCl3, d in ppm): 3.55 (bs, 1H, OH), 4.48 (s,
2H, PhCH2), 5.22 (s, 2H, CH2), 6.88 (s, 1H, H4), 7.12 (m, 2H, CHPh),
7.30 (m, 3H, CHPh), 7.41 (s, 1H, H2).
13C NMR (75.5 MHz, CDCl3, d in ppm): 48.8 (CH2Ph), 54.0
(CH2OH), 127.0 (CHPh), 128.1 (CHPh), 128.4 (C4), 128.9 (CHPh),
131.4 (Cq, C5), 136.2 (CPh,q), 138.6 (C2).
[1-15N]1-Benzyl-5-hydroxymethylimidazole (8a). Compound 8a (5.7 g,
30 mmol) was prepared similarly from 6.6 g (31 mmol) of methyl
[1-15N]1-benzyl-5-imidazolecarboxylate (7a) (98% yield).
1H NMR (600 MHz, CDCl3): the signals at 4.50, 6.97 and 7.48 ppm
3
3
2
were split into doublets, JNH 1.9 Hz, JNH 2.7 Hz and JNH 7.9 Hz,
respectively.
13C NMR (150.9 MHz, CDCl3): the signals at 48.8 and 138.9 ppm
1
1
appeared as doublets, JNC 9.7 Hz and JNC 11.6 Hz, respectively.
15N NMR (60.8 MHz, CDCl3): 150.9 (s) ppm.
[3-15N]1-Benzyl-5-hydroxymethylimidazole (8b). Compound 8b (3.03 g,
16.09 mmol) was prepared from 3.63 g (16.59 mmol) of methyl [3-15N]1-
benzyl-5-imidazole-carboxylate (7b) (97% yield).
1H-NMR (300 MHz, CDCl3): the signals at 6.87 and 7.40 ppm were
3
3
split into doublets, JNH 9.4 Hz and JNH 10.8 Hz, respectively.
13C NMR (75.5 MHz, CDCl3): the signal at 128.3 ppm appeared as a
1
doublet, JNC 13.8 Hz.
15N-NMR (30.4 MHz, CDCl3): 251.9 (bs).
Conclusions
The synthesis of 1-benzyl-5-hydroxymethylimidazole as described is
suitable for the incorporation not only of 15N-labels but also for 13C at
all positions and combination of positions. All steps gave high yields
and did not require special purification steps. Via this new strategy, 5.7 g
of [1-15N]1-benzyl-5-hydroxymethyl imidazole (8a) with an overall yield
of 53% based on 15NH4Cl and 2.8 g of [3-15N]1-benzyl-5-hydroxy-
methyl imidazole with an overall yield of 90% based on KSC15N have
been synthesized. Both 8a and 8b could be converted into the specifically
labelled [10-15N]- and [30-15N]-l-histidines via a scheme we have
Copyright # 2001 John Wiley & Sons, Ltd.
J Labelled Cpd Radiopharm 2001; 44: 831–841

IMPROVED SPECIFIC SYNTHESIS
841
described earlier.11 The isotopically enriched l-histidine prepared via
the presented method show the required properties for optimal
biological studies.
Acknowledgements
We wish to thank C. Erkelens and F. Lefeber for assistance with the
NMR measurements and Bertil Hofte for the mass spectrometry
analyses. This research was financially supported by the Netherlands
Foundation for Chemical Research (SON).
References
1. Czarnecki G, Chynwat V, Erickson JP, Frank HA, Bocian DF. J Am
Chem Soc 1997; 119: 2594–2595.
2. Zyswilich MG, McDermott A. J Am Chem Soc 1996; 118: 5867–5873.
3. Soede-Huijbregts C, Cappon JJ, Boender GJ, Raap J, Gast P, Hoff AJ,
Lugtenburg J, de Groot HJM, In Photosynthesis: Mechanisms and Effects,
Garai G (ed.). Kluwer Academic Publishers: Dordrecht, 1998; 759.
4. Alia, Matysik J, Soede-Huijbregts C, Gast P, Raap J, Lugtenburg J, de
Groot HJM, van Gorkom HJ. In Photosynthesis: Mechanisms and Effects,
Garai G (ed.). Kluwer Academic Publishers: Dordrecht, 1998; 1009.
5. Deisenhofer J, Epp O, Miki K, Ruber R, Michel H. Nature 1985; 318:
618–624.
6. Allen JP, Feher G, Yeates JO, Kominya H, Rees DC. Proc Natl Acad Sci
USA 1987; 84: 6162–6166.
7. McDermott G, Prince SM, Freer AA, Hawthornethwaite-Lawless AM,
Papiz MZ, Cogdell RJ, Isaacs NW. Nature 1995; 374: 517–521.
8. Karrash S, Bullough PA, Gosh R, EMBO J 1995; 14: 631–638.
9. Cogdell RJ, Fyfe PK, Barrett SJ, Prince SM, Freer AA, Isaacs NW, Glynn
PM, Huentes CN. Photosynth Res 1996; 48: 55–63.
10. Alia, Matysik J, Soede-Huijbregts C, Baldus M, Raap J, Lugtenburg J,
Gast P, van Gorkom HJ, Hoff AJ, de Groot HJM. J Am Chem Soc 2001;
123: 4803–4809.
11. Cappon JJ, Witters K, Baart J, Verdegem PJE, Hoek AL, Luiten RJH,
Raap J, Lugtenburg J. Recl Trav Chim Pays-Bas 1994; 113: 318–328.
12. Jones RJ. J Am Chem Soc 1994; 71: 644–647.
$
13. Husek P. J Chromatogr 1991; 552: 289.
14. IUPAC–IUB Joint Commission on Biochemical Nomenclature (JCBN).
Biochem J 1994; 219: 345–373.
Copyright # 2001 John Wiley & Sons, Ltd.
J Labelled Cpd Radiopharm 2001; 44: 831–841