The first asymmetric syntheses of L-homocysteine and L-homocystine

Article abstract of DOI:10.1016/S0957-4166(99)00453-X

Asymmetric syntheses of L-homocysteine 1 and L-homocystine 2 are described. Alkylation of the carbanion derived from Schollkopf reagent 3 and ensuing hydrolyses gave S-triphenylmethyl-L-homocysteine 6. Removal of the triphenylmethyl group gave L-homocysteine 1 and subsequent oxidation provided L-homocystine 2.

Full text of DOI:10.1016/S0957-4166(99)00453-X

Pergamon
Tetrahedron: Asymmetry 10 (1999) 4151–4156
The first asymmetric syntheses of L-homocysteine and
L-homocystine
Maciej Adamczyk,∗ Jeffrey R. Fishpaugh and Mohan Thiruvazhi
Department of Chemistry (9NM, AP20), Diagnostics Division, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL
60064-6016, USA
Received 13 September 1999; accepted 3 October 1999
Abstract
Asymmetric syntheses of L-homocysteine 1 and L-homocystine 2 are described. Alkylation of the carbanion
derived from Schöllkopf reagent 3 and ensuing hydrolyses gave S-triphenylmethyl-L-homocysteine 6. Removal of
the triphenylmethyl group gave L-homocysteine 1 and subsequent oxidation provided L-homocystine 2. © 1999
Elsevier Science Ltd. All rights reserved.
1. Introduction
L-Homocysteine 1 is an established clinical marker.1–3 A moderate increase in plasma or serum
concentrations of 1 (hyperhomocysteinemia) constitutes an independent and reversible risk factor for
cardiovascular,4 cerebrovascular,5 and peripheral vascular diseases.6 Homocysteinuria, a rare inborn error
marked by high levels of 1, is a risk factor for premature cardiovascular disease in children and young
adults.7
Vigneaud identified homocysteine in 1935 as a reduced form of homocystine,8 which had been isolated
in 1932 by treating methionine with concentrated sulfuric acid.9 L-Homocysteine 1 and L-homocystine
2 were later found in the body (Fig. 1).10,11 Most of the known preparations for these two compounds
rely on chiral resolutions to afford L-homocysteine 1,8,12,13 which is then oxidized to give L-homocystine
2. A recent preparation14 employs HBr and H2SO4 to convert L-methionine to 2 as an improvement
of a previously published method.9 However, there are no published asymmetric syntheses of either L-
homocysteine 1 or L-homocystine 2.
∗
Corresponding author. E-mail: maciej.adamczyk@abbott.com
0957-4166/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(99)00453-X

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Figure 1.
2. Results and discussion
We have developed an asymmetric synthetic strategy that affords 1 or 2 in four steps. Our strategy
utilized Schöllkopf reagent 3,15,16 a chiral glycine anion equivalent. Successful alkylation of the lithio
derivative of Schöllkopf reagent 3 with 2-bromoethyl triphenylmethyl sulfide required the presence of
two equivalents of N,N-dimethylethyleneurea (DMEU).17 Alkylation at −78°C for 20 h gave two dia-
stereomers, 4a and 4b, in a 1:4.3 ratio after chromatographic isolation as pure diastereomers (Scheme 1).
Acid catalyzed hydrolysis of the pyrazine nucleus of the desired diastereomer 4b gave S-triphenylmethyl-
L-homocysteine methyl ester 5 in 89% yield after separation from valine methyl ester by flash chro-
matography. The Mosher amide derivative of 5 revealed only a single 19F resonance, δ=−6.15 ppm
vs α,α,α-trifluorotoluene.18 Hydrolysis of 5 with lithium hydroxide in 1:1 water:dioxane afforded S-
triphenylmethyl-L-homocysteine 6 in 92% yield. Removal of the triphenylmethyl group was achieved
by reduction using excess sodium in liquid ammonia at −33°C to form an intermediate disodium salt
7. L-Homocysteine 1 was isolated in 52% yield from 6 after adding acid to the disodium salt 7.19 Air
oxidation of the intermediate 7 and ensuing acidic workup furnished L-homocystine 2 as a powder in
40% yield from 6.
Scheme 1.

M. Adamczyk et al. / Tetrahedron: Asymmetry 10 (1999) 4151–4156
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3. Conclusion
In conclusion, we have developed a synthetic route for the asymmetric syntheses of L-homocysteine
1 and L-homocystine 2. This flexible strategy is amenable for the synthesis of unnatural amino acids,
homologs of 1 and 2, needed for biochemical studies, building blocks for combinatorial chemistry and
assay development.
4. Experimental
All chemicals were purchased from Aldrich except (R)-2,5-dihydro-3,6-dimethoxy-2-
isopropylpyrazine (3, Schöllkopf reagent) which was purchased from Fluka. THF was freshly distilled
1
from sodium–benzophenone ketyl. H, 13C and 19F NMR spectra were recorded on a Varian Gemini
2300 spectrometer using tetramethylsilane as internal standard (1H, 13C) or α,α,α-trifluorotoluene
as external standard (19F). Electrospray mass spectra (ESMS) were obtained on a PE Sciex API 100
system. Optical rotations were obtained on an AUTOPOL III automatic polarimeter. Melting points are
uncorrected and obtained on an Electrothermal® melting point apparatus. Thin layer chromatography
was carried out on silica gel (Whatman MK6F) plates purchased from VWR and spots located either
by UV, PMA stain or I2 visualization. Analytical HPLC runs were performed using a Waters µ-porasil
8×100 column, λ=225 nm, flow rate=1.8 mL/min.
4.1. Preparation of (2R,5R)-2-isopropyl-3,6-dimethoxy-5-[2-(tritylsulfanyl)ethyl]-2,5-dihydropyrazine
4a and (2R,5S)-2-isopropyl-3,6-dimethoxy-5-[2-(tritylsulfanyl)ethyl]-2,5-dihydropyrazine 4b
n-Butyl lithium (n-BuLi, 2.5 M in hexanes, 22 mL, 55 mmol) was added to a −78°C solution
of triphenylmethyl mercaptan (15.0 g, 54.3 mmol) in dry THF (300 mL) and stirred for 2 h. 1,2-
Dibromoethane (22.0 mL, 255 mmol, 4.7 equiv.) was added all at once to the −78°C reaction solution.
After 30 min, the reaction mixture was warmed to room temperature, stirred for 3 h and concentrated in
vacuo. Residue was partitioned between ether (500 mL) and water (500 mL), the layers were separated
and the aqueous layer was extracted with CH2Cl2 (500 mL). Organic extracts were combined, dried over
Na2SO4, decanted and concentrated in vacuo to afford the desired 2-bromoethyl triphenylmethyl sulfide
as a white solid (20.1 g, 97%). 1H NMR (CDCl3) δ 7.64–7.60 (m, 5H), 7.52–7.38 (m, 10H), 3.09–3.03
(m, 2H), 2.95–2.86 (m, 2H); 13C NMR (CDCl3) δ 144.4, 129.5, 128.0, 126.9, 67.4, 34.1, 29.9.
n-BuLi (1.6 M in hexanes, 9.6 mL, 15.4 mmol) was added to a −78°C solution of (R)-2,5-dihydro-
3,6-dimethoxy-2-isopropylpyrazine (3, 2.5 mL, 14 mmol) and 1,3-dimethyl-2-imidazolidinone (DMEU,
3.1 mL, 28 mmol) in dry THF (140 mL). After 45 min, a solution of 2-bromoethyl triphenylmethyl
sulfide (6.32 g, 16.5 mmol, 1.2 equiv.) in dry THF (30 mL) was added over 25 min. After 20 h at −78°C,
the cherry colored reaction mixture was quenched with 5 mL of 100 mmol, pH 7.2 phosphate buffer
and warmed to ambient temperature. The yellow solution was concentrated in vacuo and the resulting
residue was partitioned between ethyl acetate (EtOAc, 100 mL) and water (100 mL). The aqueous
layer was separated and extracted with EtOAc (50 mL). Combined organic extracts were dried over
Na2SO4, decanted and concentrated in vacuo to give an oil. Purification by flash chromatography (5%
EtOAc/hexanes) gave 0.37 g (11% based on recovered starting material) of (2R,5R)-2-isopropyl-3,6-
dimethoxy-5-[2-(tritylsulfanyl)ethyl]-2,5-dihydropyrazine 4a, 1.60 g (47% based on recovered starting
material) of (2R,5S)-2-isopropyl-3,6-dimethoxy-5-[2-(tritylsulfanyl)ethyl]-2,5-dihydropyrazine 4b and
3.64 g recovered 2-bromoethyl triphenylmethyl sulfide.

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Compound 4a: 1H NMR (CDCl3) δ 7.42–7.38 (m, 5H), 7.29–7.12 (m, 10H), 4.01–3.95 (m, 1H), 3.87
(dd, J=4.8, 3.9 Hz, 1H), 3.59 (s, 3H), 3.55 (s, 3H), 2.45–2.35 (m, 1H), 2.32–2.20 (m, 2H), 2.04–1.90
(m, 1H), 1.66–1.54 (m, 1H), 1.00 (d, J=6.6 Hz, 3H), 0.57 (d, J=6.9 Hz, 3H); ESMS (M+2H)2+ 243.5.
Analytical HPLC (2% EtOAc/hexanes): Rt=12.88 min (97%).
Compound 4b: 1H NMR (CDCl3) δ 7.42–7.38 (m, 5H), 7.29–7.12 (m, 10H), 3.96 (ddd, J=7.2, 3.6, 3.6
Hz, 1H), 3.81 (dd, J=3.6, 3.6 Hz, 1H), 3.61 (s, 3H), 3.55 (s, 3H), 2.27–2.07 (m, 2H), 1.99–1.88 (m, 1H),
1.78–1.66 (m, 1H), 1.00 (d, J=6.6 Hz, 3H), 0.65 (d, J=6.9 Hz, 3H); 13C NMR (CDCl3) δ 163.67, 163.22,
144.98, 129.60, 127.75, 126.47, 66.51, 60.73, 54.52, 52.36, 33.19, 31.78, 27.75, 18.99, 16.64; ESMS
(M+2H)2+ 243.2. [α]20=+10.88 (c=0.06, CHCl3). Analytical HPLC (2% EtOAc/hexanes): Rt=7.72 min
D
(100%). Anal. calcd for C30H34N2O2S: C, 74.04; H, 7.04; N, 5.76. Found: C, 73.88; H, 6.96; N, 5.49.
4.2. Preparation of (2S)-2-amino-4-(tritylsulfanyl)butanoic acid 6
A 0.50 M aqueous solution of hydrochloric acid (10.1 mL, 5.06 mmol, 2.1 equiv.) was added to a
solution of (2R,5S)-2-isopropyl-3,6-dimethoxy-5-[2-(tritylsulfanyl)ethyl]-2,5-dihydropyrazine (4b, 1.17
g, 2.41 mmol) in dioxane (10.1 mL) and stirred for 6 h at ambient temperature. The solution was
then adjusted to pH 10 with concentrated ammonium hydroxide and extracted with CHCl3 (3×40 mL).
Combined organic extracts were dried (Na2SO4), decanted and concentrated in vacuo. The residual oil
was purified by flash column chromatography (5% MeOH/CH2Cl2; Rf=0.37) to afford methyl (2S)-2-
amino-4-(tritylsulfanyl)butanoate 5 as a thick colorless oil (840 mg, 89%). 1H NMR (CDCl3) δ 7.43–7.39
(m, 5H), 7.31–7.18 (m, 10H), 3.64 (s, 3H), 3.43–3.38 (m, 1H), 2.30 (t, J=7.8 Hz, 2H), 1.80–1.70 (m, 1H),
1.58–1.46 (m, 1H); 13C NMR (CDCl3) δ 175.85, 144.79, 129.59, 127.86, 126.62, 66.77, 53.48, 51.96,
33.95, 28.31. [α]20=+9.03 (c=0.0113, CHCl3). Anal. calcd for C24H25NO2S: C, 73.63; H, 6.44; N, 3.58;
D
S, 8.19. Found: C, 73.20; H 6.58; N 3.70; S 7.95.
The Mosher amide of 5 was prepared by adding triethylamine (10.2 µL, 0.073 mmol) and (R)-
(−)-(α)-methoxy-(α-trifluoromethyl)phenylacetic acid chloride (13.6 µL, 0.073 mmol) in succession
to a solution of methyl (2S)-2-amino-4-(tritylsulfanyl)butanoate (5, 19 mg, 0.05 mmol) in anhydrous
dichloromethane (1 mL) and stirred for 3 h at ambient temperature then quenched by addition of water
(100 µL). After 15 min of stirring, dichloromethane (20 mL) was added and washed with water (20 mL).
The organic layer was separated, dried (Na2SO4), decanted and concentrated in vacuo to afford an oil
which was purified by flash chromatography to afford 22 mg (72%) of the Mosher amide of 5. 1H NMR
(CDCl3) δ 7.51–7.42 (m, 2H) 7.35–7.15 (m, 18H), 6.93 (d, J=8.4 Hz, 1H), 4.55–4.48 (m, 1H), 3.66 (s,
3H), 3.47 (d, J=1.5 Hz, 3H), 2.25–2.15 (m, 1H), 2.09–1.98 (m, 1H), 1.83–1.71 (m, 1H), 1.54–1.38 (m,
1H); 19F NMR (CDCl3) δ −6.15.
A 0.50 M LiOH solution (9.0 mL, 4.5 mmol, 2.02 equiv.) was added dropwise to a solution of ester 5
(772 mg, 1.98 mmol) in dioxane (9.0 mL) and stirred at room temperature for 3 h. The reaction mixture
was concentrated in vacuo and the residual solid was suspended in water (20 mL). Careful adjustment
to pH 8 with 1 M aqueous HCl afforded a precipitate. The white solid thus obtained was filtered and
dried under high vacuum at 46°C over P2O5 to give (2S)-2-amino-4-(tritylsulfanyl)butanoic acid (6, S-
1
triphenylmethyl-L-homocysteine, 687 mg, 92%). H NMR (DMSO-d6) δ 7.48–7.18 (m, 15H), 3.02 (t,
J=6.3 Hz, 1H), 2.26 (t, J=5.1 Hz, 2H), 1.90–1.73 (m, 1H), 1.69–1.53 (m, 1H); 13C NMR (DMSO-d6) δ
169.17, 144.51, 129.05, 127.93, 126.62, 65.90, 53.43, 30.40, 28.11; ESMS (M−H)− 376.

M. Adamczyk et al. / Tetrahedron: Asymmetry 10 (1999) 4151–4156
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4.3. Preparation of L-homocysteine 1
Sodium metal (61 mg, 2.65 mmol) was added to a suspension of S-triphenylmethyl-L-homocysteine (6,
270 mg, 0.72 mmol) in 20 mL of liquid ammonia at −33°C. After 1 h, the reaction mixture was warmed
to ambient temperature and concentrated in vacuo. (Caution: the product is extremely air sensitive.
Exposure to air should be kept to a minimum.) The resulting solid was suspended in deaerated water
(10 mL) and extracted with deaerated ether (10 mL) to remove triphenylmethane. The aqueous layer was
adjusted to pH 6 by careful addition of deaerated 47% aqueous HI. This solution was concentrated in
vacuo and any remaining HI was removed by azeotroping with deaerated water (3×10 mL). The residue
was stirred in deaerated hot ethanol for 15 min, cooled to ambient temperature under nitrogen and filtered.
The filtered solid was isolated and dried under high vacuum for 15 h at 56°C to afford 51 mg (52%) of
1
L-homocysteine 1. H NMR (D2O) δ 3.73 (dd, J=7.4, 5.7 Hz, 1H), 2.60–2.42 (m, 2H), 2.12–1.88 (m,
2H); 13C NMR (D2O) δ 174.61, 53.97, 34.98, 20.15; ESMS (M−H)− 134. [α]24=+27.8 (c=0.0027, 1N
D
HCl) [lit.12 [α]25=+26.9 (c=1, 1N HCl)].
D
4.4. Preparation of L-homocystine 2
Sodium metal (141 mg, 6.13 mmol) was added to a suspension of 6 (200 mg, 0.53 mmol) in 40 mL
of liquid ammonia at −33°C and stirred for 3.5 h. Unreacted sodium metal was quenched by the addition
of a few crystals of NH4Cl. Ammonia was allowed to evaporate by warming to ambient temperature and
any residual ammonia was removed in vacuo. Deaerated water (50 mL) was poured into the flask and
the mixture was extracted with deaerated ether (2×25 mL). The aqueous layer was adjusted to pH 7 by
the careful addition of deaerated concentrated HCl, the mixture treated with decolorizing carbon, filtered
through Celite® and the filter pad washed with deaerated water (15 mL). The resulting filtrate was flushed
with air for 1 h and left sitting open to air for 12 h. Filtration removed trace suspended solids and the
filtrate was concentrated in vacuo. The residual solid was dissolved in water (3 mL) and filtered. Filtrate
pH was adjusted from 9.8 to 5.5 by careful addition of concentrated HCl and allowed to stand for 30
min. The solid thus obtained was washed with water (3×2 mL) and dried under high vacuum over P2O5
1
at 46°C to give 28 mg (40%) of L-homocystine 2 as an off-white solid. H NMR (D2O/NaOD) δ 3.15
(dd, J=7.4, 5.8 Hz, 2H), 2.58 (t, J=8.0 Hz, 4H), 1.90–1.64 (m, 4H); 13C NMR (D2O/NaOD) δ 183.13,
55.40, 34.78, 34.73; ESMS (M−H)− 267; mp: 266–268°C (uncorrected) [Aldrich material: 270–272°C].
[α]24=+69.3 (c=0.00284, 1N HCl) [lit.13 [α]24=+70–73 (c=1, 1N HCl)]. Anal. calcd for C8H16N2O4S2:
D
D
C, 35.81; H, 6.01; N, 10.44; S, 23.89. Found: C, 35.88; H, 6.02; N, 10.42; S, 23.56.
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