Efficient syntheses of 13C- and 14C-labelled 5-benzyl and 5-indolylmethyl L-hydantoins

Article abstract of DOI:10.1002/jlcr.1827

Robust and straightforward methods are described for the first syntheses of highly pure ¹³C- and ¹⁴C-labelled L-5-benzylhydantoin (L-BH) and L-5-indolylmethylhydantoin (L-IMH) by cyclizing the amino acids L-phenylalanine and L-trypto

Full text of DOI:10.1002/jlcr.1827

Research Article
Received 25 June 2010,
Accepted 2 August 2010
Published online 2 November 2010 in Wiley Online Library
(wileyonlinelibrary.com). DOI: 10.1002/jlcr.1827
13 14
Efficient syntheses of C- and C-labelled
5-benzyl and 5-indolylmethyl L-hydantoins
Ã
Simon G. Patching
Robust and straightforward methods are described for the first syntheses of highly pure 13C- and 14C-labelled
L-5-benzylhydantoin (L-BH) and L-5-indolylmethylhydantoin (L-IMH) by cyclizing the amino acids L-phenylalanine and
L-tryptophan, respectively, with potassium cyanate. [3-13C]-L-phenylalanine was used to prepare [6-13C]-L-BH and
[indole-2-13C]-L-tryptophan was used to prepare [indole-2-13C]-L-IMH, which we required for solid-state NMR experiments
with a hydantoin transport protein. The successful incorporation and integrity of the 13C labels was confirmed by solution-
state NMR spectroscopy. [14C]Potassium cyanate was used to prepare [2-14C]-L-BH and [2-14C]-L-IMH, which we required for
transport assays with the protein.
Keywords: hydantoins; 5-substituted; benzylhydantoin; indolylmethylhydantoin; 13C-labelled; 14C-labelled; transport proteins; solid-
state NMR
1
of 500.23 MHz for H and 125.80 MHz for 13C; chemical shifts (d)
are given in ppm relative to the internal standard TMS. High-
Introduction
5-Substituted hydantoins are important precursors to optically
pure a-amino acids for use in the food and pharmaceutical
industries, principally via their enzyme-catalysed hydrolyses.1–6
The hydantoin moiety is also found in natural products7–9 and
forms the basis of some pharmacologically active compounds
including 5-benzylhydantoin anticonvulsants such as pheny-
toin,10–12 inhibitors of the NMDA receptor,13 and modulators of
the androgen receptor.14 As part of our studies on
the structure–function relationships of hydantoin transporters
in pathogenic bacteria,15–19 we required a number of 13C- and
14C-labelled 5-monosubstituted hydantoins for solid-state NMR
experiments and for transport assays, respectively, which are not
commercially available. We have already reported the synthesis
of 14C-labelled DL-allantoin,20 but we then required optically
pure L-5-benzylhydantoin (L-BH) 1 and L-5-indolylmethylhydan-
toin (L-IMH) 2 (Figure 1), which are thought to be substrates for
the Mhp1 transporter,17–19 labelled separately with both 13C and
14C. Here, we describe our approach to the syntheses of 13C/14C-
labelled L-BH and L-IMH by cyclizing the amino acids
L-phenylalanine and L-tryptophan, respectively, with potassium
cyanate.
resolution mass spectra were obtained using a Waters GCT
Premier instrument. Liquid scintillation analysis was performed
on a Packard Tri-Carb 2100TR instrument and using an
emulsifier-safe scintillation cocktail from Perkin-Elmer.
L-5-benzylhydantoin (unlabelled)
A mixture of L-phenylalanine (1.0 g, 6.05 mmol) and potassium
cyanate (1.0 g, 12.33 mmol) in water (50 ml) was heated (501C)
and rotated on a rotary evaporator until all the phenylalanine
had dissolved. The solution was then reduced under vacuum to
ꢀ10 ml. Dilute HCl (1 M) was added dropwise with stirring until
the precipitate remained in solution (final pHꢀ5). The
precipitate was collected by filtration to give N-carbamoyl-
L-phenylalanine (634 mg, 3.05 mmol, 50%) as colourless crystals
[Mp 1921C (lit. 188–1901C21, 1931C22). 1H NMR (300 MHz, DMSO-
d6): d = 11.45 (s, 1 H, OH), 7.31–7.17 (m, 5 H, Ar–H), 6.16 (d, 1 H,
J = 8.3 Hz, NH), 5.63 (s, 2 H, NH2), 4.32 (m, 1 H, J = 5.2 Hz and
10.8 Hz, CHCO), 2.92 (m, 2 H, J = 5.2 Hz, 7.8 Hz, and 13.9 Hz, CH2).
13C NMR (75 MHz, DMSO-d6) d = 174.3 (COOH), 158.5 (CONH2),
137.9 (Ar–C), 129.6 (Ar–C), 128.5 (Ar–C), 126.7 (Ar–C), 54.1
(CH2CH), 37.9 (CH2). MS (ES1/TOF) m/z 209.33 (MH1, 100%),
231.34 (MNa1, 35%), 247.30 (MK1, 71%), 120.34 (63%), 166.36
(58%). Anal. calcd for C10H12N2O3: C, 57.69; H, 5.81; N, 13.45.
Found: C, 56.45; H, 5.55; N, 13.45]. 500 mg (2.40 mmol) of the
crystals were suspended in 2 M HCl (10 ml) and heated under
Experimental
General
Chemicals and NMR solvents were from Sigma-Aldrich (unless
stated otherwise), reaction solvents were of analytical grade and
all used without further purification. Centrifugation was
performed using an Eppendorf 5804 R bench-top centrifuge.
Melting points were measured using a STUART Melting Point
Apparatus. NMR spectra were recorded on a Bruker Avance 300
spectrometer at frequencies of 300.13 MHz for 1H and 75.48 MHz
for 13C and on a Bruker Avance 500 spectrometer at frequencies
Astbury Centre for Structural Molecular Biology and Institute of Membrane and
Systems Biology, University of Leeds, Leeds LS2 9JT, UK
*Correspondence to: Simon G. Patching, Astbury Centre for Structural Molecular
Biology and Institute of Membrane and Systems Biology, University of Leeds,
Leeds LS2 9JT, UK.
E-mail: s.g.patching@leeds.ac.uk
J. Label Compd. Radiopharm 2011, 54 110–114
Copyright r 2010 John Wiley & Sons, Ltd.

S. G. Patching
2.7193 mCi; therefore, 78 mg of the crystals has a total of
106.1 mCi and a 14C specific activity of 258.6 mCi/mmol. The
[14C]potassium cyanate used in the reaction had 500 mCi; hence,
the radiochemical yield was 106.1 mCi/500 mCi  100 = 21%.
L-5-indolylmethylhydantoin (unlabelled)
Figure 1. Structures of L-5-benzylhydantoin (L-BH) 1 and L-5-indolylmethylhydan-
toin (L-IMH) 2.
A mixture of L-tryptophan (204.2 mg, 1.0 mmol) and potassium
cyanate (97.3 mg, 1.2 mmol) in water (5 ml) was stirred at 701C
for 30 min (in a 25-ml round-bottomed flask). The solution was
allowed to cool to room temperature and concentrated HCl
(12 M, 1.04 ml) (final concentration = 2 M) was added followed by
stirring at room temperature for 30 min; the resultant white
precipitate is N-carbamoyl-L-tryptophan. A concentration of 2 M
HCl (5 ml) was added, then the suspension was transferred to a
50-ml Falcon tube; the precipitate was collected by centrifuga-
tion (5000 rpm, 10 min, 41C) and then washed twice with 2 M
HCl (20 ml) with centrifugation after each wash. The precipitate
was resuspended in 2 M HCl (20 ml), transferred to a 50-ml
round-bottomed flask and stirred at 1001C for 30 min (formation
of L-IMH). The mixture was transferred to a 50-ml Falcon tube
and incubated on ice overnight to completely crystallize L-IMH.
The crystals were collected by centrifugation, the supernatant
was removed, and ice-cold water (20 ml) was added. The pH was
adjusted to 7.0 by the addition of NaOH (2 M at first then 0.5 M)
to dissolve any residual N-carbamoyl-L-tryptophan (Be very
careful! L-IMH will be damaged at pH 9 and above). The crystals
were collected by centrifugation, the supernatant was removed,
and the crystals were washed with ice-cold water (20 ml).
The crystals were collected by centrifugation, the supernatant
was removed, and water (50 ml) was added. The suspension
was transferred to a 100-ml round-bottomed flask and heated
at 1001C until the crystals had dissolved, followed by hot
filtration. The filtrate was incubated on ice overnight and
the resultant crystals were collected by filtration (on a glass-frit
filter with no vacuum), washed with ice-cold water (2 Â 10 ml),
air-dried under vacuum on the filter and then dried under
vacuum in a dessicator over P2O5 to give L-IMH (112 mg,
0.49 mmol, 49% from L-tryptophan) as colourless fine needles.
Mp ꢀ2501C (lit. 244, 248–2511C24). 1H NMR (500 MHz, DMSO-d6):
d = 10.91 (s, 1 H, N3-H), 10.36 (s, 1 H, N1-H), 7.91 (s, 1 H, indole-
NH), 7.56 (d, 1 H, J = 8.0 Hz, indole-H), 7.33 (d, 1 H, J = 8.0 Hz,
indole-H), 7.14 (d, 1 H, J = 3.0 Hz, indole-H2), 7.07À6.96 (m, 2 H,
2 Â indole-H), 4.32 (t, 1 H, J = 4.7 Hz, H-5), 3.08 (d, 2 H, J = 4.7 Hz,
CH2). 13C NMR (75 MHz, DMSO-d6) d = 176.1 (C-2), 157.8 (C-4),
136.2 (indole-C), 127.8 (indole-C), 124.5 (indole-C2), 121.2
(indole-C), 118.9 (indole-C), 118.7 (indole-C), 111.6 (indole-C),
108.3 (indole-C1), 58.7 (C-5), 26.9 (CH2). HRMS (ES-/TOF): m/z
calcd for C12H11N3O2-H = 228.0779; found: 228.0787. Anal. calcd
for C12H11N3O2: C, 63.15; H, 4.42; N, 18.41. Found: C, 62.15; H,
4.85; N, 18.50.
reflux for 2 h. The reaction mixture was allowed to cool to room
temperature and the resultant precipitate was collected by
filtration, washed with water, air-dried under vacuum on the
filter and then dried under vacuum in a dessicator over P2O5 to
give L-BH (357 mg, 1.88 mmol, 31% from L-phenylalanine, 78%
from N-carbamoyl-L-phenylalanine) as colourless plates. Mp
1811C (lit. 181–1831C25,24). 1H NMR (500 MHz, DMSO-d6):
d = 10.43 (s, 1 H, H-3), 7.92 (s, 1 H, H-1), 7.31–7.19 (m, 5 H,
Ar-H), 4.34 (t, 1 H, JH5,H6 = 5.1 Hz, H-5), 2.94 (d, 2 H, JH6,H5 = 5.1 Hz,
CH2). 13C NMR (75 MHz, DMSO-d6) d = 175.5 (C-2), 157.5 (C-4),
136.0 (C-7), 130.1 (C-9), 128.4 (C-8), 127.0 (C-10), 58.7 (C-5), 36.8
(C-6). HRMS (ESI1/TOF): m/z calcd for C10H10N2O21H1
191.0815; found: 191.0823 and calcd for C10H10N2O21Na1
=
=
213.0634; found: 213.0638. Anal. calcd for C10H10N2O2: C, 63.15;
H, 5.30; N, 14.73. Found: C, 63.00; H, 5.20; N, 14.45.
[6-13C]-L-5-benzylhydantoin
A
mixture of [3-13C]-L-phenylalanine (Cambridge Isotope
Laboratories) (250 mg, 1.51 mmol) and potassium cyanate
(245 mg, 3.02 mmol) in water (12.5 ml) was heated (501C) and
rotated on a rotary evaporator until all of the phenylalanine had
dissolved. The solution was then reduced under vacuum to
ꢀ3 ml. Dilute HCl (1 M) was added dropwise with stirring until the
precipitate remained in solution (final pH ꢀ5). The precipitate was
collected by filtration to give [3-13C]-N-carbamoyl-L-phenylalanine
(151 mg, 0.73 mmol, 48%) as colourless crystals. The crystals
were suspended in 2 M HCl (2.5 ml) and heated under reflux for
2 h. The reaction mixture was allowed to cool to room
temperature and the resultant precipitate was collected by
filtration, washed with water, air-dried under vacuum on the
filter and then dried under vacuum in a dessicator over P2O5 to
give [6-13C]-L-BH (84 mg, 0.44 mmol, 29% from L-[3-13C]pheny-
lalanine, 60% from N-carbamoyl-L-phenylalanine) as colourless
1
plates. H NMR (500 MHz, DMSO-d6): d = 10.41 (s, 1 H, H-3), 7.90
(s, 1 H, H-1), 7.30–7.17 (m, 5 H, Ar–H), 4.32 (m, 1 H, JH5,H6 = 5.1 Hz,
H-5), 2.94 (m, 2 H, JH6,H5 = 5.1 Hz, and JH6,C6 = 129 Hz, CH2). 13C
NMR (75 MHz, DMSO-d6) d = 175.5 (C-2), 157.5 (C-4), 136.0 (d,
J
C7,C6 = 44.3 Hz, C-7), 130.0 (C-9), 128.4 (d, JC8,C6 = 3.8 Hz, C-8),
127.0 (C-10), 58.7 (d, JC5,C6 = 34.7 Hz, C-5), 36.8 (13C-enriched,
C-6). MS (ES1/TOF) m/z 191.8 (MH1, 100%). HRMS (ESI1/TOF):
m/z calcd for C193CH10N2O21Na1 = 214.0668; found: 214.0669.
[Indole-2-13C]-L-5-indolylmethylhydantoin
[2-14C]-L-5-benzylhydantoin
The method was as above for unlabelled L-IMH except that
205.2 mg (1.0 mmol) of [indole-2-13C]-L-tryptophan (Cambridge
Isotope Laboratories) was used in the reaction mixture. [Indole-
2-13C]-L-IMH (96 mg, 0.42 mmol, 42%) was obtained as colourless
fine needles. 1H NMR (500 MHz, DMSO-d6): d = 10.91 (s, 1 H,
N3-H), 10.36 (s, 1 H, N1-H), 7.91 (s, 1 H, indole-NH), 7.56 (d, 1 H,
J = 8.0 Hz, indole-H), 7.33 (d, 1 H, J = 8.0 Hz, indole-H), 7.14 (dd,
1 H, J = 3.0 Hz and 181 Hz, indole-H2), 7.07À6.96 (m, 2 H,
2 Â indole-H), 4.32 (t, 1 H, J = 4.7 Hz, H-5), 3.08 (dd, 2 H,
The method was as above for [6-13C]-L-BH except that 500 mCi
[14C]potassium cyanate (specific activity 40 mCi/mmol; American
Radiolabelled Chemicals) was included with the original reaction
mixture and unlabelled L-phenylalanine was used. [2-14C]-L-BH
(78 mg, 0.41 mmol) was obtained as colourless fine needles with
a chemical yield of 27% from L-phenylalanine. 2.0 mg of the
crystals gave 14C counts (Packard Tri-Carb 2100TR) with an
average of 6 036 855 dpm, which is equivalent to 100.61 kBq or
J. Label Compd. Radiopharm 2011, 54 110–114
Copyright r 2010 John Wiley & Sons, Ltd.
www.jlcr.org

S. G. Patching
J = 4.7 Hz and o1 Hz, CH2). 13C NMR (75 MHz, DMSO-d6) introduction of the 13C/14C label at the final step (to maximize
d = 176.1 (C-2), 157.8 (C-4), 136.2 (d, J = 4.5 Hz, indole-C), 127.8 labelling yield and to minimize handling of radiolabelled
(d, J = 3.1 Hz, indole-C), 124.5 (13C-enriched, indole-C2), 121.2 material), and ideally a robust method that produces the final
(indole-C), 118.9 (d, J = 4.6 Hz, indole-C), 118.7 (indole-C), 111.6 hydantoins as crystalline solids with 499% purity from relatively
(d, J = 2.5 Hz, indole-C), 108.3 (d, J = 70.1 Hz, indole-C1), 58.7 small-scale (ꢀ200 mg or less) reactions and avoiding chromato-
(C-5), 26.9 (d, J = 4.5 Hz, CH2). MS (ES1/TOF) m/z 231.38 (MH1, graphy. Our approach was to first synthesize unlabelled
13
11
100%). HRMS (ESI1/TOF): m/z calcd for C CH11N3O21H1
=
=
hydantoins to develop the method and handling procedures
and to assess the identity and purity of the products before
preparing the 13C/14C-labelled versions; this is described in more
detail below and separately for L-BH and L-IMH.
13
11
231.0958; found: 231.0960 and calcd for C CH11N3O21Na1
253.0777; found: 253.0782.
[2-14C]-L-5-indolylmethylhydantoin
L-5-Benzylhydantoin
The method was as above for unlabelled L-IMH except that
500 mCi [14C]potassium cyanate (specific activity 40 mCi/mmol; In our synthesis of L-BH, we first isolated the N-carbamoyl-L-
American Radiolabelled Chemicals) was included with the amino acid using a method similar to that described by Dakin,21
original reaction mixture (added in 1 ml of water out of a total but with some modifications. Equal quantities of L-phenylala-
volume of 5 ml). [2-14C]-L-IMH (102 mg, 0.45 mmol) was obtained nine and potassium cyanate in aqueous solution were simply
as colourless fine needles with a chemical yield of 45%. 2.0 mg of heated and then the solution was acidified to ꢀpH 5 to give
the crystals gave 14C counts (Packard Tri-Carb 2100TR) with an crystals of N-carbamoyl-L-phenylalanine (a-uramido-b-phenyl-
average of 7 521 069 dpm, which is equivalent to 125.35 kBq or propionic acid) in 50% yield, which were verified by melting
3.3879 mCi; therefore, 102 mg of the crystals has a total of point, NMR, mass spectrometry, and elemental analyses. Reflux
172.8 mCi and a 14C specific activity of 386.6 mCi/mmol. The of N-carbamoyl-L-phenylalanine in hydrochloric acid followed
[14C]potassium cyanate used in the reaction had 500 mCi; hence, by cooling afforded crystals of L-BH in 31% yield from
the radiochemical yield was 172.8 mCi/500 mCi  100 = 35%.
L-phenylalanine and in 78% yield from N-carbamoyl-L-phenyla-
lanine. The straightforward method and high purity of the
unlabelled product allowed us to proceed with labelled versions
of L-BH.
Results and discussion
General strategy
We required a 13C-labelled version of L-BH for solid-state NMR
Our chosen method for synthesizing L-BH and L-IMH was to experiments to detect the binding of L-BH to the Mhp1
react the appropriate L-a-amino acid with potassium cyanate hydantoin transport protein17–19 expressed in bacterial inner
and hydrochloric acid, an approach first described by Urech in membranes, as we have successfully carried out with the
187325 and variations on the procedure have been described by substrates for other transport systems.32–36 C-6 was the target
others since then, which notably includes much work by position for a 13C label in L-BH as its 13C NMR chemical shift at
Dakin.21,23,26–28 The a-amino acid reacts with potassium cyanate 36.8 ppm has the least overlap with signals coming from lipids
to form an N-carbamoyl-a-amino acid (or b-substituted a- and proteins in 13C solid-state NMR spectra of bacterial inner
uramidopropionic acid),29,30, which is often isolated in crystalline membranes.32–36 Starting from [3-13C]-L-phenylalanine, the
form; this is cyclized under acid conditions to form the synthesis was performed on a 250-mg scale to give 84 mg of
hydantoin ring, where C-5 corresponds to the a-carbon of the [6-13C]-L-5-benzylhydantoin in a yield of 29% from the amino
starting amino acid and C-5 substituents correspond to the side acid. The 13C NMR spectrum of [6-13C]-L-BH (Figure 2(A)) shows
chain of the amino acid29 (Scheme 1). A principal reason for exclusive 13C-enrichment at C-6 along with characteristic
choosing this method is that it retains optical activity,30,31 as 13C–13C splitting of the signals for the adjacent positions C-5
demonstrated by Dakin;28 L-BH and L-IMH are therefore and C-7 with coupling constants of 34.7 Hz and 44.3 Hz,
produced from L-phenylalanine and L-tryptophan, respectively. respectively. In the 1H NMR spectrum, the H-6 methylene
1
This was an important consideration for preparing the 14C- signal at 2.94 ppm shows a H-13C coupling constant of 129 Hz
compounds as it avoids separating enantiomers of the (Figure 2(C) and (D)).
radiolabelled hydantoin products. Other important considera-
A radiolabelled version of L-BH was required for our assays to
tions in choosing the synthetic route were the availability and measure the substrate uptake into bacterial cells via hydantoin
costs of 13C/14C-labelled starting materials or reagents, avoid- transporters.17 The reagent common to the syntheses of both
ance of technically demanding procedures (due to limited L-BH and L-IMH, potassium cyanate, was used to introduce a
facilities for radiolabelled work), as few steps as possible or 14C-label at C-2 in the hydantoin ring (Scheme 1). The synthesis
Scheme 1. Synthesis of 5-substituted L-hydantoins from L-a-amino acids. For L-phenylalanine, N-carbamoyl-L-phenylalanine and L-5-benzylhydantoin R = CH2Ph; for
L-tryptophan, N-carbamoyl-L-tryptophan and L-5-indolylmethylhydantoin R = CH2-indole. The asterisk shows the position of the 14C label when [14C]potassium cyanate
was used in the reaction.
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J. Label Compd. Radiopharm 2011, 54 110–114

S. G. Patching
Unlabelled
Unlabelled
B
B
H
N
O
HN
*
O
N
H
A
A
13C chemical shift (ppm)
13C chemical shift (ppm)
H
N
*
181 Hz
O
D
HN
(D)
O
129 Hz
D
(D)
C
C
1H chemical shift (ppm)
1H chemical shift (ppm)
Figure 2. NMR analysis of [6-13C]-L-5-benzylhydantoin. (A and B) 13C NMR spectra
of [6-13C]-L-5-benzylhydantoin and unlabelled L-5-benzylhydantoin, respectively, in
DMSO-d6 obtained at 75.48 MHz. (C and D) 1H NMR spectrum of [6-13C]-L-5-
benzylhydantoin in DMSO-d6 obtained at 500.23 MHz; D shows an expansion of
the H-6 methylene signal and its coupling with the 13C label at C-6. The asterisk
shows the position of the 13C label on the structure of L-BH.
Figure 3. NMR analysis of [indole-2-13C]-L-5-indolylmethylhydantoin. (A and B) 13
C
NMR spectra of [indole-2-13C]-L-5-indolylmethylhydantoin and unlabelled L-5-
indolylmethylhydantoin, respectively, in DMSO-d6 obtained at 75.48 MHz. (C and D)
1H NMR spectrum of [indole-2-13C]-L-5-indolylmethylhydantoin in DMSO-d6
obtained at 500.23 MHz; D shows an expansion of the indole-H2 signal and its
coupling with the 13C label at indole-C2. The asterisk shows the position of the 13
C
label on the structure of L-IMH.
of L-BH was performed on a 250-mg scale, but the original
reaction mixture included 500 mCi [14C]potassium cyanate. This
produced 78 mg of [2-14C]-L-5-benzylhydantoin in a chemical
yield of 27% from L-phenylalanine and with a 14C specific
activity of 258.6 mCi/mmol; the radiochemical yield from
[14C]potassium cyanate was 21%.
the pH was neutralized to remove any residual N-carbamoyl-L-
tryptophan. Care has to be taken here as racemization of amino
acid hydantoins occurs under mild alkali conditions.26,30 Finally,
hot filtration and recrystallization from water gave L-IMH in 49%
yield from L-tryptophan. The identity and high purity of the
synthesized L-IMH was confirmed by melting point, NMR, mass
spectrometry and elemental analyses, therefore allowing us to
proceed with the labelled versions of L-IMH. The use of
centrifugation rather than filtration for the collection and
washing of crystals not only gave us higher yields but also
helped to keep materials more contained in the radiolabelled
synthesis.
L-5-Indolylmethylhydantoin
Our synthesis of L-IMH was based on that described by Suzuki
et al.24 for other amino acid hydantoins on a multigram scale,
but with major changes and additions to the procedure to tailor
it to L-IMH and to satisfy our requirements for preparing it
with 13C/14C labels and on a smaller scale. On a 200-mg scale, L-
tryptophan was heated with an excess of potassium cyanate
then the solution was acidified to give a colourless fine
precipitate of N-carbamoyl-L-tryptophan; the N-carbamoyl
amino acid had not been isolated in the reported procedure.
The fineness of the precipitate led to significant losses of
material on attempts to collect it by filtration, so the precipitate
was instead collected and washed by repeated centrifugation
and resuspension. Reflux of the precipitate in hydrochloric acid
followed by cooling produced colourless crystals of L-IMH. The
crystals were again collected and washed by centrifugation and
For solid-state NMR experiments, the target position for a 13
C
label in L-IMH was C-2 in the indole ring; this has a 13C NMR
chemical shift of 124.5 ppm, which is in a relatively clear region
of the 13C NMR solid-state NMR spectrum of bacterial inner
membranes.32–36 Starting from [indole-2-13C]-L-tryptophan, the
synthesis was performed as for unlabelled L-IMH to give 96 mg
of [indole-2-13C]-L-indolylmethylhydantoin in a yield of 42%
from the amino acid. The 13C NMR spectrum of [indole-2-13C]-
L-IMH (Figure 3A) shows exclusive 13C-enrichment at C-2 of
the indole ring along with a 13C–13C splitting of the signal for
J. Label Compd. Radiopharm 2011, 54 110–114
Copyright r 2010 John Wiley & Sons, Ltd.
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S. G. Patching
indole-C1 with a coupling constant of 70.1 Hz and long-range
1
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13C–13C splittings of other signals. In the H NMR spectrum, the
1
indole-H2 signal at 7.14 ppm shows a H–13C coupling constant
of 181 Hz (Figure 3C and D).
Radiolabelled L-IMH was also required for our transport
assays; as with L-BH, [14C]potassium cyanate was used to
introduce a 14C label at C-2 in the hydantoin ring (Scheme 1).
The synthesis was performed as for unlabelled L-IMH except that
500 mCi [14C]potassium cyanate was included in the reaction
mixture. This produced 102 mg of [2-14C]-L-5-indolylmethylhy-
dantoin in a chemical yield of 45% from L-tryptophan and with a
14C specific activity of 386.6 mCi/mmol; the radiochemical yield
from [14C]potassium cyanate was 35%.
¨
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Conclusions
This work has described the first syntheses of 13C- and 14C-
labelled 5-benzyl- and 5-indolylmethyl L-hydantoins. Robust and
straightforward procedures were developed for preparing L-BH
and L-IMH, as highly pure crystalline solids in moderate and
consistent yields from 200/250 mg scale reactions and without
using chromatography; these were then used to prepare L-BH
and L-IMH with 13C and 14C labels. The successful incorporation
and integrity of the 13C labels was confirmed by NMR spectro-
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L-IMH with other labelling patterns or of other amino acid
hydantoins with stable isotope labels or radiolabels.
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