Synthesis of (ε-13C-,ε-15N)-enriched L-lysine - Establishing schemes for the preparation of all possible 13C and 15N isotopomers of L-lysine, L-ornithine, and L-proline

Article abstract of DOI:10.1002/ejoc.200400370

In this paper we describe a simple synthetic strategy that, with the right rational adaptation, gives direct access to any ¹³C/¹⁵N isotopomer of L-glutamate, L-ornithine, L-proline, L-lysine, and L-α, amino adipic acid. This strategy also allows access to nonproteinogenic amino acids like L-citrulline in high yields and optical purity. Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004.

Full text of DOI:10.1002/ejoc.200400370

FULL PAPER
Synthesis of (ε-13C-,ε-15N)-Enriched
L
-Lysine ؊ Establishing Schemes for the
13
15
Preparation of All Possible C and N Isotopomers of
and -Proline
L
-Lysine, -Ornithine,
L
L
Arjan H. G. Siebum,[a] Robert K. F. Tsang,[a] Rob van der Steen,[b] Jan Raap,[a] and
Johan Lugtenburg*[a]
Keywords: Amino acids / Carbon labels / Asymmetric synthesis / Rhodopsin
In this paper we describe a simple synthetic strategy that,
with the right rational adaptation, gives direct access to any
access to nonproteinogenic amino acids like
high yields and optical purity.
( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
L-citrulline in
13C/15N isotopomer of
lysine, and
L-glutamate,
L-ornithine,
L-proline,
L-
L-α amino adipic acid. This strategy also allows
Germany, 2004)
Introduction
In this paper we describe a modular strategy that is able
to give access to any site-directed isotopically labeled -ly-
sine, -ornithine and -proline, with the use of commercially
available stable isotopically enriched synthons or synthons
that are available in any site-directed enriched form by stra-
Rhodopsin is the photosensitive protein of the rod
photoreceptor in the vertebrate retina that mediates dim
light vision. Rhodopsin represents a paradigm for the large
and diverse family of G-protein coupled membrane recep-
tors (GPCR’s).[1] Lysine-296 in the active site of rhodopsin
connects the 11-Z protonated Schiff base of retinal to the
protein.[2] In order to study the role of this residue in the
rhodopsin photoreceptor process with 1H and 13C solid-
state NMR techniques we needed access to (ε-13C,ε-15N)lys-
ine (Figure 1, compound 1a). Lysine is also an essential am-
ino acid in human (and animal) nutrition, and access to
the set of site-directed isotopomers of lysine is essential to
elucidate the metabolic role of this amino acid by mass-
spectral techniques.
1
tegies described before by us.[3,4] For the H, 13C, and 15N
MAS NMR study we need, next to (13C20)-11-(Z)-retinal,
also (ε-15N,ε-13C)lysine.[5] The one-step synthesis of the
protected -glutamate ester by the OЈ Donnell method[6]
has been published for the natural abundance materials.[7]
In our group, we have worked out schemes to prepare the
required synthons in any site-directed isotopomeric
form.[3,4] Glutamic acid has the complete carbon skeleton
of other proteinogenic amino acids such as proline and glu-
tamine. For the preparation of lysine, only one additional
carbon and nitrogen atoms are needed. The basis of using
glutamic acid as a building block for the preparation of the
aforementioned amino acids in each site-directed iso-
topically enriched form is the possibility of the selective
conversion of the 5-carboxylic acid ester group into other
functional groups. The protected glutamate obtained by the
OЈDonnell method has proved to be the perfect intermedi-
ate in this synthesis.
Results and Discussion
Synthetic Strategy
Figure 1. -lysine (1), -ornithine 2) and -proline (3)
In Scheme 1, the strategy to prepare any site-directed en-
riched isotopomer of -lysine 1 is indicated. The first step
is the OЈ Donnell coupling of the protected glycine deriva-
tive[3] 4 with methyl acrylate 5, which can be prepared in
any isotopomeric form, as has been previously published by
our group.[4] The product of the coupling after treatment
[a]
Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden
University,
P. O. Box 9502, 2300 RA Leiden, The Netherlands
[b]
ARC Laboratories B.V.,
Minden 62, 7327 AW Apeldoorn, The Netherlands
Fax: (internat.) ϩ31-71-5274488
E-mail: Lugtenbu@chem.leidenuniv.nl
Eur. J. Org. Chem. 2004, 4391Ϫ4396
DOI: 10.1002/ejoc.200400370
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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A. H. G. Siebum, R. K. F. Tsang, R. van der Steen, J. Raap, J. Lugtenburg
FULL PAPER
Scheme 1. The synthesis of -lysine (1) starting from the achiral scaffold N-diphenylmethylene glycine tert-butyl ester (4)
with aqueous citric acid gives the tert-butyl, methyl ester of microorganisms,[11] and access to the full set of isotopomers
glutamic acid 6. The free amino group in 6 is subsequently is beneficial to metabolic research.
converted into the Boc derivative in 85% yield. This fully
For the synthesis of proline 3, the internal alkylation re-
protected glutamate is then treated with NaBH4/LiCl in action of the α-amino group with CδϪBr leads directly to
ethanol at 0 °C.[8] This reagent is a mild and selective reduc- -proline (3) (Scheme 2). The reduction of 6 with NaBH4/
ing agent, capable of selectively reducing the methyl ester LiCl to the amino alcohol is directly followed by treatment
to the alcohol 7, without concomitant reduction of the tert- with the adduct of triphenylphosphane and bromine in
butyl ester. Conversion of the alcohol function in 7 with dichloromethane in the presence of imidazole as proton
triphenylphosphane and bromine in dichloromethane scavenger. The resulting bromide 10 reacts intramolecularly
(DCM) gives the corresponding bromide in 81% yield. Sub- with the amino group to form the proline tert-butyl ester.
sequent treatment of the bromide with KCN in refluxing Removal of the ester group with 10% TFA in DCM gives
acetone gives the protected adiponitrile 8 in 80% yield. -proline (3). This scheme thus gives access to all iso-
Catalytic reduction with Adam’s catalyst (PtO2) in acidified topomers of -proline (3) in a simpler and more convergent
ethanol and subsequent deprotection with 10% trifluoro- fashion than existing methods.[12]
acetic acid leads to -lysine 1, with all the characteristics of
The simple catalytic reduction of adiponitrile 8 to form
the authentic material. With the use of K13C15N, 8a can be the protected lysine motivated us to prepare -ornithine in
prepared in 76% yield from the bromide. Reduction of the a similar way through reduction of glutaronitrile 12. The
labeled nitrile and subsequent deprotection gives (ε-13C,ε- protected glycinate 4 was treated under OЈ Donnell con-
15N)-lysine in 91% yield. Introducing K13C15N in the final ditions with acrylonitrile, which can be prepared in all iso-
steps gives (ε-13C,ε-15N)lysine in 69% yield based on topomeric forms,[13] to form the protected glutaronitrile de-
K13C15N. It is clear from Scheme 1 that -lysine can be pre- rivative, which upon treatment with aqueous citric acid
pared in any isotopomeric form.
gives 12. Catalytic reduction and acid-mediated deprotec-
Before we developed the present synthetic scheme we tion leads to -ornithine 2 with the analytical properties
have prepared 1a by the OЈ Donnell method using of the authentic material. Access to all stable 13C and 15N
(13C,15N)4-iodobutyronitrile. Labeled iodobutyronitrile was isotopomers of ornithine allows preparation of the isotopo-
prepared by SN2-substitution of the iodo-substituent of 3- mers of other amino acids that share the same carbon skel-
chloro-1-iodopropane with K13C15N, followed by treatment eton. In this way, starting from ornithine, arginine and ci-
of the resulting chlorobutyronitrile with KI in refluxing ace- trulline can easily be prepared in a few simple steps. Treat-
tone. Based on this method, the overall yield of 1a from ment of -ornithine with the cyanate anion gives citrul-
K13C15N is 37%. This method does allow access to the full line.[14] Earlier, our group prepared all isotopomers of
set of isotopomers,[9] but gives a lower yield based on the cyanate and prepared site-directed 15N- and 13C-enriched
incorporated isotopes, hence the development of the syn- urea derivatives from primary amines.[15] The conversion of
thesis presented in Scheme 1. In Scheme 1, the α-aminoadi- -ornithine into -arginine with 13C and 15N isotopes has
ponitrile derivative 8 is the intermediate that can be easily also been published.[16,17] The ability to prepare all 15N and
hydrolyzed to α-aminoadipic acid.[10] α-Aminoadipic acid is 13C isotopomers of the three amino acids ornithine, argi-
an essential intermediate in the biosynthesis of -lysine in nine, and citrulline will be a boon to the metabonomics
4392
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Eur. J. Org. Chem. 2004, 4391Ϫ4396

Synthesis of (ε-13C-,ε-15N)-Enriched L-Lysine
FULL PAPER
Scheme 2. A Ϫ The synthesis of -proline (3) starting from chiral tert-butyl methyl glutamate (6); B Ϫ the synthesis of -ornithine (2)
starting from achiral glycinate 4
ing (0.2% in ethanol) or staining with a mixture of 4,4Ј-methyl-
enebis(N,N-dimethylaniline) and ninhydrin (TDM staining). Chiral
HPLC was performed with a Chiralcel ODH column (25 cm) using
hexane and 2-propanol (iPrOH) as solvent system. Dry diethyl
ether (ether, Et2O) was obtained by distilling from P2O5. Dry petro-
leum ether (PE, boiling range 40Ϫ60 °C) and dry dichloromethane
(DCM) were obtained by distilling from CaH2. All commercially
available chemicals were purchased from SigmaϪAldrich, Across
or Fluka. All chemicals were used without further purification, un-
studies of the vertebrate urea-cycle since all intermediates
of this cycle can now be prepared easily in any desired iso-
topomeric form.
Conclusion
In this paper we describe the modular and convergent
strategy to prepare -lysine (1), -ornithine (2), and -pro- less stated otherwise. (13C,15N)potassium cyanide (Ͼ 99% isotope-
enriched) was purchased from Cambridge Isotope Laboratories.
line (3) in an optically pure form such that all site-directed
13C and 15N isotopomers can easily be prepared by the al-
kylation of a protected glycine derivative using Michael-
type reactions with methyl acrylate and acrylonitrile in the
presence of a chiral phase-transfer catalyst. After the reac-
tion, the optically active catalyst can easily be isolated,
ready to be reused. With this method, no difficult separ-
N-(Benzophenoneimine) of 1-tert-Butyl 5-Methyl Glutamate: Methyl
acrylate (5) (1.98 mL, 22 mmol) dissolved in toluene (3 mL) was
added dropwise to a solution of 4 (5 g, 17 mmol) in toluene
(15 mL), and a 50% KOH solution (1/3 volume of toluene, 6 mL)
with O-allyl-N-(9-anthracenyl)methylcinchonidium bromide (0.05
equiv.) was added as chiral phase-transfer catalyst. The solution
ation of the product from an optically active scaffold is was stirred very vigorously for 14 h. The mixture was then diluted
needed.[18] We feel that this scheme can easily be adapted
to the synthesis of a whole class of new nonproteinogenic
α-amino acids in high optical purity.
with 10 mL water and extracted with dichloromethane (3 ϫ
20 mL). The collected organic layers were dried with MgSO4, and
the solvents evaporated. The product was further purified over a
silica column (PE/Et2O, 90:10) to yield the product in 92% (5.97 g,
1
15.6 mmol). H NMR (300 MHz, CDCl3): δ ϭ 1.48 (s, 9 H, tBu),
2.23 (m, 2 H, 4-H), 2.41 (m, 2 H, 3-H), 3.56 (s, 3 H, OMe), 4.01
Experimental Section
3
3
(dd, JH,H ϭ 7.28, JH,H ϭ 5.50 Hz, 1 H, 2-H), 7.14Ϫ7.66 (m, 10
H, arom.) ppm. 13C NMR (75.5 MHz, CDCl3) δ ϭ 27.77 (tBu),
28.4 (C-4), 30.21 (C-3), 51.18 (OMe), 64.54 (C-2), 80.84 (tBu),
127.52Ϫ139.19 (aromatic), 170.44 (C-1 ϩ CN), 173.20 (C-5) ppm.
General Remarks: 1H NMR spectra were recorded with a Jeol FX-
200, a Bruker DPX-300 or a Bruker DPX 400 spectrometer using
tetramethylsilane (TMS: δ ϭ 0 ppm) or water (H2O: δ ϭ 4.8 ppm)
as an internal standard. 13C noise-decoupled NMR spectra were
recorded with a Jeol FX-200 at 50.1 MHz, a Bruker DPX-300 spec-
trometer at 75.5 MHz, and a Bruker DPX-400 at 100.7 MHz, using
CDCl3 (δ ϭ 77 ppm), (CD3)2CO (δ ϭ 206 ppm), or TSP [3-(tri-
1-tert-Butyl 5-Methyl Glutamate (6): The alkylated protected gly-
cinate (5.97 g, 15.6 mmol) was dissolved in THF (10 mL) and
stirred with a 10% citric acid solution (20 mL) for one night. TLC
showed the complete disappearance of the starting material, and
methylsilyl)tetradeuteriopropionic acid sodium salt, δ ϭ 0 ppm] as the mixture was extracted twice with ether (25 mL). The water layer
internal standard. A saturated solution of ammonium nitrate was
used as an external standard (15NH4NO3 δ ϭ 22.3 ppm relative to
NH3 (l) δ ϭ 0.0 ppm) for the 15N NMR spectra. All spectra were
recorded in CDCl3, except where noted otherwise. Column chroma-
tography was performed on Merck silica gel 60 (0.040Ϫ0.063 mm
230Ϫ400 mesh), and spots on the thin-layer chromatograph were
detected with UV light, KMnO4 solution spraying, ninhydrin stain-
was then brought to pH 12 and extracted with ethyl acetate (2 ϫ
30 mL) to give, after evaporation, the product 6 in 96% yield
(3.26 g). H NMR (300 MHz): δ ϭ 1.47 (s, 9 H, tBu), 1.81 (m, 1
1
H, 3-H), 2.03 (m, 1 H, 3-H), 2.36 (dd, 2 H, 4-H), 3.34 (dd 3JH,H ϭ
5.3, 3JH,H ϭ 8.2 Hz, 1 H, 2-H), 3.68 (s, 3 H, OMe) ppm. 13C NMR
(75.5 MHz, CDCl3) δ ϭ 27.35 (tBu), 29.60 (C-4) 29.80 (C-3), 50.90
(C-2), 53.65 (OMe), 80.44 (tBu), 172.95 (C-1), 174.11 (C-5) ppm.
Eur. J. Org. Chem. 2004, 4391Ϫ4396
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A. H. G. Siebum, R. K. F. Tsang, R. van der Steen, J. Raap, J. Lugtenburg
FULL PAPER
1-tert-Butyl 5-Methyl N-(Boc)glutamate: The amino ester was sub-
0.8 mmol). 1H NMR (300 MHz, CDCl3): δ ϭ 1.45 (s, 9 H, tBu),
1.47 (s, 9 H, tBu), 1.63 (m, 2 H, 4-H), 1.72 (m, 2 H, 3-H), 2.40 (m,
sequently dissolved in DMF (10 mL) and triethylamine (1 equiv.,
2.1 mL) was added, followed by di-tert-butyl dicarbonate (1.1 2 H, 5-H), 4.20 (m, 1 H, 2-H), 5.05 (m, 1 H, NH) ppm. 13C NMR
equiv., 3.53 mL), and the mixture was stirred until TLC showed
the completion of the reaction. The DMF was evaporated, and
ethyl acetate (25 mL) was added. The organic layer was washed
with KHSO4 solution (pH 2, 3 ϫ 15 mL ), water, brine, and dried
with MgSO4. The raw product was purified over a short silica col-
(75.5 MHz, CDCl3) δ ϭ 16.44 (C-5), 21.26 (C-4), 28.0 (tBu), 28.3
(tBu), 31.12 (C-3), 52.8 (C-2), 79.5 (tBu), 82.0 (tBu), 118.70 (C-6),
155.2 (CO), 171.5 (C-1) ppm.
tert-Butyl (5-13C,15N)-N-Boc-2-amino-5-cyanopentanoate (8a): Pre-
pared as described for 8. Yield: 260 mg, 0.87 mmol 8a. Starting
from bromide (0.36 g, 1.04 mmol) and K13C15N (77 mg, 1.1 equiv.).
The yield was 76% based on the labeled KCN, and 84% based on
1
umn (PE/Et2O, 80:20). The yield was 94% (4.48 g, 14.1 mmol). H
NMR (300 MHz): δ ϭ 1.44 (s, 9 H, tBu), 1.47 (s, 9 H, tBu), 1.91
(m, 1 H, 3-H), 2.16 (m, 1 H, 3-H), 2.34 (m, 2 H, 4-H), 3.68 (s, 3
1
the bromide. H NMR (300 MHz, CDCl3): δ ϭ 1.45 (s, 9 H, tBu),
3
H, OMe), 4.19 (m, 1 H, 2-H), 5.10 (d, JH,H ϭ 8.1 Hz, 1 H, N-H)
1.47 (s, 9 H, tBu), 1.63 (m, 2 H, 4-H), 1.72 (m, 2 H, 3-H), 2.40 (m,
ppm. 13C NMR (75.5 MHz, CDCl3): δ ϭ 27.87 (tBu), 27.96 (C-4)
28.20 (tBu), 30.00 (C-3), 51.61 (OMe), 53.31 (C-2), 79.63 (tBu),
82.04 (tBu), 155.27 (NCO), 171.20 (C-1), 173.20 (C-5) ppm.
2 H, 5-H), 4.20 (m, 1 H, 2-H), 5.05 (m, 1 H, NH) ppm. 13C NMR
1
(75.5 MHz, CDCl3) δ ϭ 16.44 (d, JC,C ϭ 56.6 Hz, C-5), 21.26 (d,
2JC,C ϭ 2.6 Hz, C-4), 28.0 (tBu), 28.3 (tBu), 31.12 (C-3), 52.8 (C-
1
2), 79.5 (tBu), 82.0 (tBu), 118.70 (d, 1.6, JC,N ϭ 16.8 Hz, C-6),
tert-Butyl N-Boc-2-amino-5-hydroxypentanoate (7): NaBH4 (4
equiv., 278 mg) was added to a suspension of LiCl (4 equiv.,
311 mg) in THF/EtOH (10 mL, 1:1), and the mixture was stirred
for 10 min. After cooling to 0 °C, a solution of the glutamate
(1.8 mmol, 580 mg) dissolved in THF/EtOH (5 mL) was added
dropwise. The reaction mixture was stirred overnight, and the tem-
perature was allowed to rise to room temperature. Water was added
(30 mL), and the reaction mixture was extracted with EtOAc (3 ϫ
30 mL). The collected organic layers were extracted with brine and
dried with MgSO4. Subsequently, the solvents were evaporated, and
the raw product was purified using column chromatography (15%
to 25% EtOAc in PE) to give 7 in 70% yield (361 mg, 1.26 mmol).
1H NMR (300 MHz): δ ϭ 1.47 (2*s, 18 H, 2*tBu), 1.54Ϫ1.95 (m,
4 H, 3-H, 4-H), 3.66 (m, 2 H, 5-H), 4.20 (m, 1 H, 2-H), 5.15 (d,
3JH,H ϭ 8.3 Hz, 1 H, NH) ppm. 13C NMR (75.5 MHz, CDCl3) δ ϭ
27.88 (tBu), 27.90 (C-4) 28.23 (tBu), 29.53 (C-3), 53.55 (C-2), 61.95
(C-5), 79.63 (tBu), 81.80 (tBu), 155.88 (NCO), 171.88 (C-1) ppm.
155.2 (CO), 171.5 (C-1) ppm.
L-Lysine·TFA (1): The purified protected nitrile (237 mg, 0.8 mmol)
was dissolved in 2-propanol (30 mL), together with concentrated
HCl (2 mL) and a catalytic amount of platinum oxide. Using a
Parr-apparatus, the mixture was then shaken vigorously under 50
psi H2 for 16 h. Thereafter, the mixture was filtered through celite
to remove the platinum catalyst and concentrated in vacuo to give
the free amine in 95% yield (226 mg, 0.76 mmol). The raw product
was redissolved in a mixture of 10% trifluoroacetic acid in dry
DCM (20 mL) and stirred overnight. The mixture was subsequently
extracted with water (3 ϫ 15 mL), and the combined water layers
were evaporated, and the sample was lyophilized to give the
lysine·TFA salt 1 in 97% yield (190 mg, 0.73 mmol). 1H NMR
(400 MHz, D2O): δ ϭ 1.55 (m, 2 H, 4-H), 1.75 (m, 2 H, 3-H), 1.99
(m, 2 H, 5-H), 3.2 (dt, 3JH,H ϭ 7.6 Hz, 2 H, 6-H), 3.7 (dd, 3JH,H ϭ
6.4 Hz, 1 H, 2-H) ppm. 13C NMR (100 MHz, D2O): δ ϭ 22.0 (C-
4), 26.83 (C-5), 29.8 (C-3), 39.7 (C-6), 53.7 (C-2), 117.1 (q, CF3),
163.6 (q, CO), 175.3 (C-1) ppm.
tert-Butyl N-Boc-2-amino-5-cyanopentanoate (8): Triphenylphos-
phane (0.66 g, 2 equiv.) was dissolved in dry dichloromethane
(5 mL), the solution was cooled to 0 °C and stirred under a dry
nitrogen while bromine (128 µL, 1.99 equiv.) dissolved in DCM
(2 mL) was added dropwise. After 20 minutes, a mixture of 7
(361 mg, 1.26 mmol) and imidazole (0.18 g, 2 equiv.) dissolved in
DCM (5 mL) was slowly added to the pale yellow solution. After
1 min, a white solid became visible. The reaction mixture was
stirred for another 2 h while the temperature was maintained at 0
°C. Subsequently, the solids were filtered off, and the solvent was
evaporated. The resulting product was redissolved in DCM
(10 mL) and the solution filtered quickly over a glass-filter filled
with some silica. The silica was rinsed with EtOAc (50 mL), all
solutions were combined. After evaporation of the solvents the bro-
mide was obtained in 81% yield (0.35 g, 1.0 mmol). 1H NMR
(300 MHz, CDCl3): δ ϭ 1.43 (s, 9 H, tBu), 1.47 (s, 9 H, tBu),
1.78Ϫ199 (m, 4 H, 3-H,4-H), 3.41 (m, 2 H, 5-H), 4.18 (m, 1 H, 2-
H), 5.31 (d, 1 H, NH) ppm. 13C NMR (75.5 MHz, CDCl3) δ ϭ
28.0 (tBu), 28.3 (tBu), 30.3 (C-3), 31.6 (C-4), 33.0 (C-5), 53.2 (C-2),
79.8 (tBu), 82.14 (tBu), 155.3 (CO), 171.5 (C-1) ppm. The bromide
(0.35 g, 1.0 mmol) was dissolved in ethanol (30 mL), and after ad-
ding potassium cyanide (1.1 equiv., 74 mg) dissolved in ethanol/
water (5 mL, 90:10 v/v), the mixture was refluxed for 6 h. The solu-
tion was cooled to room temperature and concentrated to a quarter
of its volume, after which the precipitate was filtered off, and the
solution was extracted with dichloromethane (2 ϫ 40 mL). Sub-
sequently, the collected organic layers were washed with water
(20 mL). The solution was dried with MgSO4, and after removing
the solvent in vacuo, the raw product was purified by column chro-
matography (PE/Et2O, 85:15) to yield 8 in 80% yield (237 mg,
(ε-13C,15N)-
L-Lysine (1a): Prepared as described above. The yield
was 91% (220 mg, 0.84 mmol) starting from 8a. 1H NMR
(400 MHz, D2O): δ ϭ 1.55 (m, 2 H, 4-H), 1.75 (m, 2 H, 3-H), 1.99
(m, 2 H, 5-H), 3.2 (dt, 3JH,H ϭ 7.6, 1JC,H ϭ 143 Hz, 2 H, 6-H), 3.7
3
(dd, JH,H ϭ 6.4 Hz, 1 H, 2-H) ppm. 13C NMR (100 MHz, D2O):
1
3
δ ϭ 22.0 (s, C-4), 26.83 (d, JC,C ϭ 35.2 Hz, C-5), 29.8 (d, JC,C
ϭ
1
5.3 Hz, C-3), 39.7 (d, JC,N ϭ 4.9 Hz, C-6), 53.7 (C-2), 175.3 (C-
1) ppm.
(4-13C,15N)-1-Chlorobutyronitrile: A mixture of 1-bromo-3-chlorop-
ropane (22.0 g, 14.0 mL, 140 mmol) and (13C,15N)potassium cyan-
ide (2.39 g, 35.6 mmol) dissolved in ethanol/water (100 mL, 90:10
v/v) was refluxed for 3 h. The solution was cooled to room tem-
perature, the precipitate was filtered off, and the solution was ex-
tracted with dichloromethane and washed with water (3 ϫ). After
removing the solvent in vacuo, the solution was further purified by
vacuum distillation to remove the excess 1-bromo-3-chloropropane.
The product (a yellow oil) was obtained in 76% yield (2.92 g,
3
27 mmol). 1H NMR (400 MHz, CDCl3): δ ϭ 2.11 (ddt, JH,H
ϭ
6.0, 3JH,H ϭ 7.0, 3JC,H ϭ 6.6 Hz, 2 H, 2-H), 2.57 (ddt, 3JH,H ϭ 7.0,
2JC,H ϭ 10, JN,H ϭ 1.6 Hz, 2 H, 3-H), 3.67 (t, JH,H ϭ 6.0 Hz, 2
3
3
H, 1-H), ppm. 13C NMR (100 MHz, CDCl3): δ ϭ 14.59 (dd,
1JC,C ϭ 56.7, JC,N ϭ 3.0 Hz, C-3), 28.9 (d, JC,C ϭ 2.5 Hz, C-2),
2
2
42.5 (d, 3JC,C ϭ 3.8 Hz, C-1), 118.4 (d, 1JC,N ϭ 16.9 Hz, C-1) ppm.
15N NMR (40 MHz, CDCl3): δ ϭ 248.6 (dt, JN,H ϭ 1.6, JC,N
3
1
16.8 Hz, 15N) ppm.
(4-13C,15N)-1-Iodobutyronitrile:
27 mmol) and NaI (3 equiv.) were dissolved in warm acetone
1-Chlorobutyronitrile
(2.92 g,
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Synthesis of (ε-13C-,ε-15N)-Enriched L-Lysine
FULL PAPER
1
3
(150 mL) and refluxed for 24 h, during which the mixture gradually
became yellow. The mixture was cooled to 20 °C. After distilling
27.04 (d, JC,C ϭ 35.9 Hz, C-5), 27.80 (tBu), 30.5 (d, JC,C
ϭ
1
5.3 Hz, C-3), 39.7 (d, JC,N ϭ 4.9 Hz, C-6), 53.6 (s, C-2), 82.52
off the solvent, the residue was diluted with water and extracted (tBu), 173.0 (C-1) ppm.
with dichloromethane. The organic layer was successively washed
(ε-13C, ε-15N)-
L-Lysine·HCl (1a): -lysine tert-butyl ester was dis-
with 1  Na2SO4 and dried with MgSO4. The product was concen-
trated in vacuo and purified over a silica column (PE/Et2O, 90:10)
to give a colorless liquid (5.05 g, 19.4 mmol) in 94% yield. 1H NMR
(400 MHz, CDCl3): δ ϭ 2.09 (dt, 3JH,H ϭ 7.0, 3JH,H ϭ 6.0, 3JC,H ϭ
solved in 6  HCl (100 mL). The mixture was then refluxed for
24 h. After removing the solvent in vacuo, the yellow residue was
taken up in water (100 mL), treated with a small amount of char-
coal (Norit), and refluxed for 2 h to decolorize the solution. After
filtering off the charcoal, the solution was concentrated in vacuo.
Recrystallization from water/ethanol yielded -lysine monohyd-
rochloride as a white hygroscopic powder in 90% yield [1.95 g,
10.8 mmol, (37% yield based on K13C15N)]. [α]2D3 ϭ ϩ24.8 (c ϭ 2,
6  HCl), ref. [α]2D0 ϭ ϩ25.2 (c ϭ 2, 6  HCl). 1H NMR (400 MHz,
D2O): δ ϭ 1.55 (m, 2 H, 4-H), 1.75 (m, 2 H, 3-H), 1.99 (m, 2 H,
2
3
3
6.6 Hz, 2 H, 2-H), 2.50 (ddt, JC,H ϭ 9.7, JH,H ϭ 6.8, JN,H
ϭ
1.8 Hz, 2 H, 3-H), 3.22 (t, JH,H ϭ 6.4 Hz, 2 H, 1-H) ppm. 13C
3
3
NMR (100 MHz, CDCl3): δ ϭ 3.3 (d, JC,C ϭ 3.7 Hz, C-1), 17.8
(dd, 1JC,C ϭ 55.7, 2JC,N 3.0 Hz, C-3), 28.1 (d, 2JC,C ϭ 2.9 Hz, C-2)
117.8 (d, 1JC,N ϭ 16.9 Hz, C-4) ppm. 15N NMR (40 MHz, CDCl3):
δ ϭ 248.7 (dt, JN,H ϭ 1.6, JC,N ϭ 16.8 Hz, 15N) ppm.
3
1
tert-Butyl (ε-13C,ε-15N)-2-(Benzhydrylideneamino)-5-cyanopentano-
ate: 1-Iodobutyronitrile (5.05 g, 25.6 mmol) was added dropwise to
a solution of tert-butyl N-(diphenylmethylene)glycinate (1.1 equiv.,
8.2 g, 28.1 mmol) in toluene (40 mL) and 50% KOH (1/3 volume
of toluene) in the presence of O-allyl-N-(9-anthracenyl)methylcin-
chonidium bromide (0.1 equiv.) as a phase-transfer catalyst. The
solution was stirred very vigorously for 12 h. The mixture was then
diluted with water, extracted with dichloromethane (3 ϫ 20 mL),
and dried with MgSO4. The product was further purified over a
silica column (PE/Et2O, 90:10) to yield a mixture of unchanged 4-
iodonitrile (which could be recovered and reused) and product in
50% yield (4.6 g, 12.8 mmol). 1H NMR (300 MHz, CDCl3): δ ϭ
1.44 (s, 9 H, tBu), 1.65 (m, 2 Η, 4-H), 1.98 (m, 2 H, 3-H), 2.31 (m,
3
2
5-H), 3.2 (dt, JH,H ϭ 7.6, JC,H ϭ 143 Hz, 2 H, 6-H), 3.7 (dd,
3JH,H ϭ 6.4 Hz, 1 H, 2-H) ppm. 13C NMR (100 MHz, D2O): δ ϭ
22.0 (s, C-4), 26.83 (d, JC,C ϭ 35.2 Hz, C-5), 29.8 (d, JC,C
5.3 Hz, C-3), 39.7 (d, JC,N ϭ 4.9 Hz, C-6), 53.7 (C-2), 175.3 (C-
1
3
ϭ
1
1) ppm.
tert-Butyl 2-[(Benzhydrylidene)amino]-4-cyanobutanoate: Acryloni-
trile 5 (116 µL, 1.77 mmol) dissolved in toluene (2 mL) was added
dropwise to a mixture of N-(diphenylmethylene)-tert-butyl glycin-
ate (400 mg, 1.36 mmol) in toluene (4 mL) and 50% KOH (2 mL)
with O-allyl N-9-anthracenyl methylcinchonidium bromide (41 mg,
0.05 equiv.) as a chiral phase-transfer catalyst. The solution was
stirred very vigorously for 14 h. The mixture was then diluted with
water (10 mL) and extracted with dichloromethane (3 ϫ 10 mL).
The collected organic layers were dried with MgSO4, and the sol-
vents evaporated. The product was further purified over a silica
column (90:10, PE/ether) to give the product in 92% yield (434 mg,
1.25 mmol). ee ϭ 91% (chiralcel ODH, 25 cm, hexane/iPrOH,
99.9:0.1 v/v, flow 0.8 mL/min, retention time: 19.05 min (R); 27.35
min (S). 1H NMR (200 MHz, CDCl3): δ ϭ 1.43 (s, 9 Η, tBu),
2.13Ϫ2.37 (m, 2 H, 3-H), 2.44Ϫ2.54 (m, 2 H, 4-H), 4.05 (dd,
3
3
2 H, 5-H), 3.94 (dd, JH,H ϭ 5.0, JH,H ϭ 7.1 Hz, 1 H, 2-H),
7.15Ϫ7.68 (m, 10 H, arom.) ppm. 13C NMR (75.5 MHz, CDCl3):
δ ϭ 16.8 (dd, 1JC,C 55.98, 2JC,N 2.8 Hz, C-5), 21.9 (d, 2JC,C 2.46 Hz,
3
C-4), 27.81 (tBu), 32.2 (d, JC,C 3.68 Hz, C-3), 64.7 (C-2), 81.09
1
(tBu), 117Ϫ139 (arom.) 119.4 (d, JC,N ϭ 16.9 Hz, C-6), 170.44
(CN), 178.10 (C-1) ppm.
tert-Butyl (ε-13C,ε-15N)-2-Amino-5-cyanopentanoate: The protected
amino acid (4.6 g, 12.8 mmol) and unchanged 4-iodobutyronitrile
were dissolved in THF (50 mL) and diluted with a 15% citric acid
solution (20 equiv.). After stirring for 3 h, the mixture was diluted
with water and extracted with diethyl ether to remove the benzo-
phenone and the unchanged iodonitrile. The water phase was then
neutralized with K2CO3 (pH 11Ϫ12) and extracted with ethyl acet-
ate (3 ϫ 20 mL). The combined organic phases were dried with
MgSO4 and concentrated in vacuo. The mixture was further puri-
fied over a silica column (PE/EtOAc, 85:15) to obtain the product
3
3JH,H ϭ 7.55, JH,H ϭ 4.80 Hz, 1 H, 2-H), 7.17Ϫ7.68 (m, 10 Η,
aromatic) ppm. 13C NMR (50.1 MHz, CDCl3): δ 13.57 (C-3), 27.96
(tBu), 29.39 (C-4), 63.69 (C-2) 81.76 (tBu), 119.32 (C-5),
127.59Ϫ135.99 (arom.), 158.10 (CN), 169.76 (C-1) ppm.
Proline tert-Butyl Ester (11): NaBH4 (4 equiv., 430 mg) was added
to a suspension of LiCl (4 equiv., 483 mg) in THF/EtOH (10 mL,
1:1), and the mixture was stirred for 10 min. After cooling to 0 °C,
a solution of 6 (2.7 mmol, 600 mg) dissolved in THF/EtOH (5 mL)
was added dropwise. The reaction mixture was stirred overnight,
and the temperature was allowed to rise to room temperature. A
KOH solution (pH 13, 30 mL) was added, and the reaction mixture
was extracted with EtOAc (3 ϫ 30 mL). The collected organic lay-
ers were dried with MgSO4. Subsequently, the solvents were evapo-
rated, and the raw product was purified using column chromatog-
raphy (15% to 35% EtOAc in PE) to give the product in 68% yield
(340 mg, 1.9 mmol).
1
in 96% yield (2.8 g, 12.2 mmol). H NMR (400 MHz, CDCl3): δ ϭ
1.47 (s, 9 H, tBu), 1.64 (m, 1 H, 3-H), 1.8 (m, 3 H, 3-H ϩ 4-H),
2.4 (m, 2 H, 5-H), 3.32 (m, 1 H, H-2) ppm. 13C NMR (75.5 MHz,
1
2
CDCl3) δ ϭ 16.8 (dd, JC,C ϭ 56.02, JC,N ϭ 3.0 Hz, C-5), 21.76
2
3
(d, JC,C ϭ 2.7 Hz, C-4), 27.75 (tBu), 33.4 (d, JC,C ϭ 1.6 Hz, C-
1
3), 81.14 (tBu), 119.27 (d, JC,N ϭ 16.9 Hz, C-6), 174.5 (s, CO)
ppm. 15N NMR (40 MHz, CDCl3): δ ϭ 246 (dt, JC,N ϭ 16.93,
1
3JH,N ϭ 1.61 Hz, 15N) ppm.
(ε-13C, ε-15N)-
L
-Lysine tert-Butyl Ester: The purified nitrile (2.80 g,
Triphenylphosphane (1.0 g, 2 equiv.) was dissolved in dry dichloro-
methane (5 mL), and the solution was cooled to 0 °C and stirred
under dry nitrogen while bromine (193 µL, 1.99 equiv.) dissolved
12.3 mmol) was dissolved in 2-propanol (50 mL), together with
concentrated HCl (3 mL) and a catalytic amount of platinum oxide.
The mixture was then stirred vigorously under 50 psi H2 for 16 h in DCM (2 mL) was added dropwise. After 20 minutes, a mixture
using a Parr-apparatus. Thereafter, the mixture was filtered through of the alcohol (340 mg, 1.9 mmol) and imidazole (0.27 g, 2 equiv.)
celite to remove the platinum catalyst, which was rinsed with 2- dissolved in DCM (5 mL) was slowly added to the pale yellow solu-
propanol (25 mL), and concentrated in vacuo to give the amine in tion. After 1 min, a white solid became visible. The reaction mix-
1
98% yield (2.4 g, 12 mmol). H NMR (400 MHz, D2O): δ ϭ 1.40 ture was stirred for another 3 h, while the temperature was main-
(s, 9 H, tBu), 1.45 (m, 2 H, 4-H), 1.65 (m, 2 H, 5-H), 1.85 (m, 2 tained at 0 °C. Subsequently, a 0.1  KOH solution (10 mL) and
3
1
H, 5-H), 2.9 (dt, JH,H ϭ 7.5, JC,H ϭ 143 Hz, 2 H, 6-H), 4.0 (m, DCM (10 mL) were added, the organic layer was separated, and
1 H, 2-H) ppm. 13C NMR (100 MHz, D2O): δ ϭ 22.16 (s, C-4), the solvent was evaporated. The resulting product was redissolved
Eur. J. Org. Chem. 2004, 4391Ϫ4396
www.eurjoc.org
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4395

A. H. G. Siebum, R. K. F. Tsang, R. van der Steen, J. Raap, J. Lugtenburg
FULL PAPER
in DCM (10 mL) and the solution filtered quickly over a glass-
filter with some silica. The silica was rinsed with EtOAc (50 mL),
450 mg ornithine.HCl, with all the characteristics of an authentic
sample (50%, 2.7 mmol). [α]2D0 ϭ ϩ20.5 (c ϭ 1, 6  HCl), ref.
[α]2D0 ϭ ϩ22.0 (c ϭ 4, 6  HCl). NMR (50.1 MHz, D2O): δ ϭ 23.32
all filtrates were combined and the solvents evaporated to give the
1
proline tert-butyl ester in 83% yield (0.52 g, 1.57 mmol). H NMR (C-4), 27.96 (C-3), 39.44 (C-5), 54.64 (C-2), 174.58 (C-1) ppm.
(200 MHz, D2O/DCl): δ ϭ 1.50 (s, 9 H, tBu), 2.12 (m, 3 H, 3-H,
4-H), 2.43 (m, 1 H, 4-H), 3.42 (m, 2 H, 5-H), 4.35 (m, 1 H, H-
2) ppm.
FTIR (neat, fingerprint region): ν˜ ϭ 350.2, 397.8, 458.4, 555.7,
669.9, 757.7, 780.6, 845.6, 878.6, 935.1, 981.9, 1039.7, 1098.9,
1131.0, 1146.1, 1243.3, 1287.1, 1329.4, 1345.9, 1363.4, 1419.8,
1438.3, 1474.4 cmϪ1
.
L-Proline·HCl (3): 11 was redissolved in 6  HCl (10 mL) and re-
fluxed overnight. The mixture was subsequently concentrated in
vacuo, treated with DEAE Sephadex (acetate form), concentrated,
and transferred on a Dowex WX-8 (Hϩ form) column. Hydro-
chloric acid was first eluted with water, and the amino acid sub-
sequently with 2  ammonia. The eluate was evaporated to dryness,
dissolved in water, and the solution was adjusted to pH 3. Ethanol
was then added until crystallization began. After one night at Ϫ20
°C, the crystals were collected to give 142 mg proline.HCl, with all
Acknowledgments
We gratefully acknowledge Mr. Rian van den Nieuwenberg for his
help with the determination of the optical activity of the synthe-
sized amino acids by using chiral HPLC techniques.
[1]
L. Stryer, Biochemistry, 3rd ed., W. H. Freeman and company,
New York, 1998.
R. A. Mathies, J. Lugtenburg, Molecular Mechanisms in Visual
the characteristics of an authentic sample (60%, 0.9 mmol). [α]2D3
ϭ
[2]
Ϫ83.7 (c ϭ 4, H2O), ref. (-proline) [α]2D0 ϭ Ϫ84 (c ϭ 4, H2O). H
NMR (300 MHz, D2O): δ ϭ 2.13 (m, 3 H, 3-H, 4-H), 2.44 (m, 1
H, 3-H), 3.40 (m, 2 H, 5-H), 4.42 (dd, 1 H, 2-H) ppm. 13C NMR
(75.5 MHz, D2O): δ ϭ 24.1 (C-4), 29.1 (C-3), 47.0 (C-5), 60.4 (C-
2), 172.7 (C-1) ppm.
1
Transduction (Eds.: D. G. Stavenga, W. J. DeGrip, E. N. Pugh
Jr.), Elsevier: Amsterdam, 2000.
A. H. G. Siebum, W. S. Woo, J. Lugtenburg, Eur. J. Org. Chem.
[3]
2003, 4664Ϫ4678.
J. J. Cappon, J. Baart, G. A. M. van der Walle, J. Raap, J.
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A. F. L. Creemers, S. Kiihne, P. H. M. Bovee-Geurts, W. J.
[5]
tert-Butyl 2-Amino-4-cyanobutanoate (12): The protected amino
acid (434 g, 1.25 mmol) was dissolved in THF (10 mL), and a 15%
citric acid solution (5 mL) was added. After stirring the mixture
for 3 h, it was diluted with water (10 mL) and extracted with ether
(2 ϫ 25 mL). The water phase was then neutralized with K2CO3
to pH 12Ϫ13 and extracted with ethyl acetate (3 ϫ 20 mL). The
combined organic phases were dried with MgSO4 and concentrated
in vacuo. The mixture was further purified over a silica column
(PE/ethyl acetate, 85:15) to obtain the product in 96% yield
Degrip, J. Lugtenburg, H. J. M. De Groot, P. Natl. Acad. Sci.
USA 2002, 99, 9101Ϫ9106.
M. J. O’Donnell, F. Delgado, C. Hostettler, R. Schwesinger,
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[8]
E. N. Chauvel, C. Llorens-Cortes, P. Coric, S. Wilk, B. P. Ro-
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[9]
1
Amino Acids 1995, 171Ϫ186.
J. Raap, C. M. van der Wielen, J. Lugtenburg, Recl. Trav. Chim.
(221 mg, 1.20 mmol). H NMR (200 MHz, CDCl3): δ ϭ 1.47 (s, 9
[10]
H, tBu), 1.82 (m, 1 H, 3-H), 2.09 (m, 1 H, 3-H), 2.54 (m, 2 H, 4-
H), 3.39 (m, 1 H, 2-H) ppm. 13C NMR (50.1 MHz, D2O): δ ϭ
14.19 (C-4), 28.02 (C-3), 54.03 (C-2), 84.04 (tBu), 121.43 (C5),
175.34 (C1) ppm.
Pays-Bas 1990, 109, 277Ϫ286.
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Raap, J. Lugtenburg, Recl. Trav. Chim. Pays-Bas 1992, 111,
L
-Ornithine·HCl: The purified protected nitrile (1.0 g, 5.3 mmol)
517Ϫ523.
[13]
was dissolved in acetic acid (10 mL), and platinum oxide (300 mg)
was added. The mixture was then shaken vigorously under H2 for
16 h. Thereafter, the mixture was filtered to remove the platinum
catalyst and concentrated in vacuo. The raw product was redis-
solved in 6  HCl (10 mL) and refluxed overnight. The mixture was
subsequently concentrated in vacuo, treated with DEAE Sephadex
(acetate form), concentrated, and transferred on a Dowex WX-8
(Hϩ form) column. Hydrochloric acid was first eluted with water,
and the amino acid subsequently with 2  ammonia. The eluate
was evaporated to dryness, dissolved in water, and the solution was
adjusted to pH 3. Ethanol was then added until crystallization be-
gan. After one night at Ϫ20 °C, the crystals were collected to give
E. M. M. van den Berg, E. E. Richardson, J. Lugtenburg,
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Received May 25, 2004
4396
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2004, 4391Ϫ4396