Lipase-catalyzed enantiomer separation of 3-hydroxy-4-(tosyloxy) butanenitrile: Synthesis of (R)-GABOB (=(3R)-4-amino-3-hydroxybutanoic acid) and (R)-carnitine hydrochloride (= (2R)-3-carboxy-2-hydroxy-N,N,N-trimethylpropan- 1-aminium chloride)

Article abstract of DOI:10.1002/hlca.200790180

Enzymatic resolution of racemic 3-hydroxy-4-(tosyloxy)butanenitrile ((±)-5) by using various lipases in different solvents were studied. The obtained optically pure (3R)-3-(acetyloxy)-4-(tosyloxy)-butanenitrile ((R)-6), upon treatment with aqueous ammonia followed by cone. HCl solution, provided (R)-GABOB (=(3R)-4-amino-3-hydroxybutanoic acid; (R)-1). Similarly, reaction of (R)-6 with aqueous trimethylamine solution followed by cone. HCl solution provided (R)-carnitine hydrochloride (=(2R)-3-carboxy-2-hydroxy-N,N,N- trimethylpropan-1-aminium chloride; (R)-2·HCl) in an expeditious manner.

Full text of DOI:10.1002/hlca.200790180

Helvetica Chimica Acta – Vol. 90 (2007)
1723
Lipase-Catalyzed Enantiomer Separation of 3-Hydroxy-4-
(tosyloxy)butanenitrile: Synthesis of (R)-GABOB (¼(3R)-4-Amino-3-
hydroxybutanoic Acid) and (R)-Carnitine Hydrochloride (¼(2R)-3-Carboxy-
2-hydroxy-N,N,N-trimethylpropan-1-aminium Chloride)
by Ahmed Kamal*, G. B. Ramesh Khanna, and Tadiparthi Krishnaji
Biotransformation Laboratory, Division of Organic Chemistry, Indian Institute of Chemical Technology,
Hyderabad 500007, India
(phone: þ 914027193157; fax: þ 914027193189; e-mail: ahmedkamal@iict.res.in)
Enzymatic resolution of racemic 3-hydroxy-4-(tosyloxy)butanenitrile ((ꢀ)-5) by using various
lipases in different solvents were studied. The obtained optically pure (3R)-3-(acetyloxy)-4-(tosyloxy)-
butanenitrile ((R)-6), upon treatment with aqueous ammonia followed by conc. HCl solution, provided
(R)-GABOB (¼(3R)-4-amino-3-hydroxybutanoic acid; (R)-1). Similarly, reaction of (R)-6 with
aqueous trimethylamine solution followed by conc. HCl solution provided (R)-carnitine hydrochloride
(¼(2R)-3-carboxy-2-hydroxy-N,N,N-trimethylpropan-1-aminium chloride; (R)-2 · HCl) in an expedi-
tious manner.
Introduction. – g-Amino-b-hydroxybutanoic acid (GABOB; 1) is a compound of
great pharmacological importance because of its biological function as a neuro-
modulator in the mammalian central nervous system [1 – 6]. The (R)-form of GABOB
is shown to have greater biological activity than its (S)-enantiomer [1b][7]. Moreover,
the (R)-isomer of GABOB serves as a precursor for (R)-carnitine ((R)-2), a compound
having a high level of medical significance. Carnitine is a vitamin-like substance and
plays an important role in converting stored body fat into energy. Its primary
physiological function is to transport long-chain fatty acids through the mitochondrial
membrane [5a] [8] into the cellular compartments for oxidation where these fats can be
converted into energy. Moreover, there are several medical indications for which
carnitine has been prescribed [9][10]. These include its usefulness as a pharmaceutical
for hemodialysis [11] (hypolipidemic agent), heart diseases [9], and myopathic
deficiencies [5a] [8e,f], etc.
The discovery of the adverse effects of (S)-carnitine [12] ((S)-2) and the
questionable effectiveness of the racemic mixture in treating angina pectoris [13],
coupled with the recent growing interest in therapeutic application of carnitine, has
prompted its use in only the (R)-form. The high biological importance associated with
(R)-GABOB ((R)-1) and (R)-carnitine ((R)-2) continue to stimulate the interest of
ꢀ 2007 Verlag Helvetica Chimica Acta AG, Zürich

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Helvetica Chimica Acta – Vol. 90 (2007)
many research groups [14a] for the development of a facile, efficient, and economic
approach to their preparation.
Results and Discussion. – On the basis of a retro-synthetic pathway, 3-hydroxy-4-
(tosyloxy)butanenitrile (5), a b-hydroxy nitrile, was conceived as an interesting
precursor for the synthesis of GABOB and carnitine. History reveals that b-
hydroxynitriles are important both as technical products and as reagents in organic
chemistry [15]. They have been extensively investigated and employed in the
preparation of various intermediates for the naturally occurring bioactive compounds
[16]. In continuation of our earlier efforts towards the preparation of biologically
important compounds or their intermediates by employing biocatalysts [17 – 20], the
present investigation describes an efficient chemoenzymatic preparation of optically
pure 3-hydroxy-4-(tosyloxy)butanenitrile and its application towards the synthesis of
optically pure (R)-GABOB and (R)-carnitine. (R)-GABOB has been prepared
previously from 3-hydroxy-4-(tosyloxy)butanenitrile obtained from d-glucitol [21a]
and also by opening of the epoxide ring of glycidyl tosylate by Et2AlCN [21b]. These
reported methods are tedious, employ expensive reagents, and offer low yields.
Therefore, the newly developed method is not only versatile but provides higher yields
with excellent enantioselectivity.
The preparation of racemic 3-hydroxy-4-(tosyloxy)butanenitrile ((ꢀ)-5) required
for its enzymatic resolution has not been that trivial. Neither the nucleophilic ring
opening of glycidyl tosylate with NaCN nor the nucleophilic displacement of the Cl-
atom in 1-chloro-3-(tosyloxy)propan-2-ol with NaCN under mild or drastic conditions
afforded the desired product. Instead, an indirect approach was adopted to obtain the
required b-hydroxynitrile. Racemic glycidol (3) was subjected to ring opening by
NaCN in aqueous alcohol to afford 3,4-dihydroxybutanenitrile (4). Compound 4,
without further purification, was selectively monoprotected at the primary OH group
by p-toluenesulfonyl chloride (TsCl) to give the desired 3-hydroxy-4-(tosyloxy)buta-
nenitrile ((ꢀ)-5) (Scheme 1). However, the reaction of 3,4-dihydroxynitrile with TsCl
in the presence of Et3N and other bases in different solvents afforded only low yields of
the desired product. In an interesting development, the yield of the desired
monoprotected (ꢀ)-5 was increased to 65% by employing dibutyltin oxide [22] in
catalytic amounts. The use of dibutyltin oxide not only improved the rate of tosylation
but was also instrumental in selective monoprotection of the primary alcohol (via a
cyclic intermediate).
The resolution of racemic 3-hydroxy-4-(tosyloxy)butanenitrile ((ꢀ)-5) was carried
out by employing lipases. The preparation of optically pure b-hydroxynitriles has
generally been carried out by enzymatic approaches like the reduction of the carbonyl
Scheme 1
i) NaCN, EtOH/H2O. ii) Bu2SnO, TsCl, Et3N, CH2Cl2.

Helvetica Chimica Acta – Vol. 90 (2007)
1725
group of a ketonitrile by microbes [23] or the lipase-mediated enantioselective
hydrolysis of their corresponding acetates [24]. Lipase-mediated resolution of a-
hydroxy tosylates [25] has been reported, while this is the first report for the enzymatic
resolution of 3-hydroxy-4-(tosyloxy)butanenitrile ((ꢀ)-5) employing lipases. There-
fore, in the present investigation, the lipase-mediated esterification process was applied
as well as examined in detail to obtain the best results (Scheme 2).
Scheme 2
i) Lipase, vinyl acetate (6 equiv.), diisopropyl ether, r.t.
In the first series of experiments, the efficiency of different commercially available
lipases to catalyze the esterification of 3-hydroxy-4-(tosyloxy)butanenitrile ((ꢀ)-5)
was investigated. A variety of lipases including the immobilized ones were screened for
the esterification in the presence of vinyl acetate in diisopropyl ether. Among the
lipases studied, the immobilized lipase on diatomite from Pseudomonas cepacia (PS-
D) afforded good yields of both (S)-alcohol (S)-5 and (R)-acetate (R)-6 in high
enantiomer excess (Table 1).
Table 1. Transesterification of ( ꢀ )-3-Hydroxy-4-(tosyloxy)butanenitrile ((ꢀ)-5) with Various Lipases in
Diisopropyl Ether
Lipase
Time
[h]
Alcohol (S)-5
yielda) [%]
Acetate (R)-6
yielda) [%]
Conversion
C [%]
Enantiomer
ratio E
eeb) [%]
eeb) [%]
PS-D
PS
PS-C
AK
13
24
7
25
67
67
67
67
67
44
55
52
44
80
88
69
68
90
> 99
66
50
90
15
06
13
14
03
46
37
40
47
12
08
23
25
02
> 99
97
93
90
98
89
54
51
23
0.5
0.4
1046
130
58.4
106
102
18.28
3.78
4
0.35
0.5
0.13
0.06
0.2
P
Lipozyme
AYS
CRL
CAL-B
0.22
0.12
1.4
a) Isolated yield. b) Determined by chiral HPLC (chiral column ADH; Daicel) employing hexane/iPrOH
93 : 07 as a mobile phase at 0.7 ml/min and monitored by UV (235 nm).
The stereochemical preference of PS-D during kinetic resolution was in line with
Kazlauskasꢁ rule (see Figure) and further, this can also be predicted based on the
existing 3D models of the lipase from Pseudomonas cepacia reported by Theil and co-
workers [26]. According to this model, the substrate binding and orientation is due to
two hydrophobic pockets, which are varying by shape – one with a very limited
diameter and the other with a spherical shape near the active serine.

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Helvetica Chimica Acta – Vol. 90 (2007)
Figure. Kazlauskasꢁ rule for kinetic resolution
The enantiomer excess was calculated from the enantiomer ratios obtained by
employing a Chiralpak-ADH column. The absolute configuration of the alcohol and
the acetate was determined by comparison of their chiroptical and chromatographic
properties with those of the corresponding known compounds. In this process,
comparison of the specific rotation of the unreacted alcohol (S)-5 with that of the
reported [16] hydroxy nitrile (S)-5 established the (S)-configuration of the alcohol and
(R)-configuration of the acetate.
As the solvent variation in many cases of lipase-catalyzed kinetic resolution
influences the enantiomer or enantiotopic-face selectivity as well as the reaction rates,
the effect of the solvents on the substrate (ꢀ)-5, was also studied (Table 2). The solvent
effect can be rationalized by the polarity of the solvent. Among the solvents tested for
the esterification of (ꢀ)-5, the hydrophobic solvents gave better results compared to
the hydrophilic solvents in terms of enantioselectivity and conversion rates. As the
polarity of the solvent increases, the catalytic activity of enzyme diminishes. Hydro-
philic solvents may denature enzymes by penetrating into the hydrophobic core of
proteins. The use of diisopropyl ether as the solvent provided the best results (Table 2).
Empirical rules have been developed for the optimization of biocatalytic activity in
different organic solvents. The quantitative measurement of solvent can be determined
by the parameter P, the partition coefficient of the solvent between octanol and H2O,
and the log P values for the solvents examined are also given in Table 2. The catalytic
activity is low in polar solvents having a log P < 2, moderate in solvents having a log P
between 2 and 4, and high in apolar solvents having a log P > 4.
Optically pure (3R)-3-(acetyloxy)-4-(tosyloxy)butanenitrile ((R)-6) is a highly
functionalized chiral compound. It was utilized in the present investigation for the
preparation of (R)-GABOB ((R)-1) and (R)-carnitine hydrochloride ((R)-2 · HCl). In
this process, (R)-1 and (R)-2 were prepared in a single pot starting from optically pure
(R)-6 by treating with aqueous ammonia in refluxing EtOH and then treating with
conc. HCl solution at 808 to afford (R)-1, which upon methylation gave (R)-2. In a
similar manner, the treatment of (R)-6 with aqueous trimethylamine and then with
conc. HCl solution at 808 yielded optically pure (R)-carnitine hydrochloride ((R)-2 ·
HCl) (Scheme 3).
Conclusion. – In summary, an efficient method for the preparation of racemic 3-
hydroxy-4-(tosyloxy)butanenitrile ((ꢀ)-5) and its successful enzymatic resolution
employing lipase-catalyzed esterification was reported. This esterification process
employing lipase PS-D in diisopropyl ether provided a good conversion with high

Helvetica Chimica Acta – Vol. 90 (2007)
1727
Table 2. Effect of Solvents on the Transesterification of ( ꢀ )-3-Hydroxy-4-(tosyloxy)butanenitrile ((ꢀ)-5)
by Lipase PS-D
Solvent
Log P Time Alcohol (S)-5
Acetate (R)-6
Conversion Enantio-
[h]
C [%]
meric
ratio E
yielda) [%] eeb) [%] yielda) [%] eeb) [%]
iPr2O
Hexane
Et2O
Toluene
CHCl3
Acetone
Tetrahydrofuran
Dioxane
1.9 13
3.5 15
0.85 19
2.5 19
2.0 72
ꢁ 0.23 76
0.49 69
ꢁ 1.1 78
44
44
48
40
54
86
90
91
> 99
86
83
99
69
11
04
04
46
46
44
53
38
07
03
03
> 99
86
86
84
95
> 99
> 99
> 99
0.5
0.5
1046
37
1.5
59
0.49
0.54
0.42
0.1
0.04
0.04
86
223
397
397
a) Isolated yield. b) Determined by chiral HPLC (chiral column ADH; Daicel) employing hexane/iPrOH
93 : 07 as a mobile phase at 0.7 ml/min and monitored by UV (235 nm).
Scheme 3
i) Aq. NH3 soln., EtOH, reflux. ii) Conc. HCl soln. iii) Aq. Me3N soln., EtOH, reflux. iv) MeI, KOH,
MeOH/H2O.
enantioselectivities. Moreover, this investigation also established that immobilized
lipases are better than nonimmobilized lipases for this process. Further, optically pure
(3R)-3-(acetyloxy)-4-(tosyloxy)butanenitrile ((R)-6) was employed in a short and
efficient synthesis of the biologically important (R)-GABOB ((R)-1) and (R)-
carnitine hydrochloride ((R)-2 · HCl). This chemoenzymatic protocol involves very few
steps compared to earlier methods, hence it is efficient and practical.
The authors G. B. R. K. and T. K. thank CSIR, New Delhi, for the award of a research fellowship.
Experimental Part
1. General. Unless specified, all solvents and reagents were reagent grade and used without
purification. Reactions involving moisture-sensitive reagents were performed under N2 in glassware that

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Helvetica Chimica Acta – Vol. 90 (2007)
had been oven-dried. M.p.: electrothermal melting-point apparatus; uncorrected. Anal. TLC: Merck
prepared plates (silica gel 60 F-254 on glass). Column chromatography (CC): Acme silica gel (100 – 200
mesh). Chiral HPLC: Shimadzu LC-10AT system controller, SPD-10A fixed-wavelength UV monitor as
detector; chiral column ADH (Daicel), hexane/iPrOH 93 :07 as mobile phase, flow 0.7 ml/min, UV
monitoring at 235 nm. Optical rotations: SEPA-300 (Horiba) digital polarimeter. IR Spectra: Perkin-
Elmer 683 or 1310 spectrometer; cmꢁ1. 1H-NMR Spectra: Gemini 200 (200 MHz) or AV 300 (300 MHz)
instrument; CDCl3 or (D6)DMSO solns.; chemical shifts d in ppm with Me4Si as an internal standard,
coupling constants J in Hz. Low-resolution MS: CEC-21-100B Finnigan Mat-1210 or VG-7070 H
micromass mass spectrometer; in m/z.
Lipases. Pseudomonas cepacia lipase immobilized on diatomite (PS-D), Pseudomonas cepacia (PS),
Pseudomonas cepacia lipase immobilized on modified ceramic particles (PS-C), and Pseudomonas
fluorescens lipase (AK) were obtained from Amano Pharmaceutical Company, Japan, Pseudomonas
fluorescens lipase immobilized in Sol-Gel-AK on sintered glass (P), lipase immobilized from Mucor
meihei (MML), and Candida antartica lipase immobilized in Sol-Gel-AK on sintered glass (CAL B) were
from Fluka, and Candida rugosa lipase (CRL) was from Sigma.
2. 3-Hydroxy-4-{[(4-methylphenyl)sulfonyl]oxy}butanenitrile ((ꢀ)-5). To a stirred soln. of glycidol
(¼oxirane-2-methanol; 3; 14.80 g, 200.00 mmol) in EtOH (120 ml) were added H2O (240 ml) and NaCN
(9.80 g, 200.00 mmol), and stirring was continued overnight. After completion of the reaction, the solvent
was evaporated and the residue filtered through a silica gel pad. Evaporation of the solvent of the filtrate
afforded 3,4-dihydroxybutanenitrile (4). To 4 (10.10 g, 100.00 mmol) dispersed in CH2Cl2 (250 ml) was
added dibutyltin oxide (4.98 g, 20.00 mmol), Et3N (25.25 g, 250.00 mmol), and p-toluenesulfonyl
chloride (¼4-methylbenzenesulfonyl chloride; 20.95 g, 110.00 mmol) under N2. The resultant mixture
was stirred at r.t. After completion of the reaction (2 h; TLC monitoring), H2O (150 ml) was added, and
the org. layer was separated. The aq. layer was extracted with CH2Cl2 (3 ꢂ 150 ml), the combined org.
layer dried (Na2SO4) and concentrated, and the residue purified by CC (AcOEt/hexane 35 :65): pure 5
(16.5 g, 65%). IR (neat): 3474, 3059, 2933, 2902, 2220, 1584, 1349, 1169, 1098, 996. 1H-NMR (300 MHz,
CDCl3): 2.48 (s, 3 H); 2.52 – 2.67 (m, 2 H); 4.06 (d, J ¼ 5.4, 2 H); 4.15 – 4.22 (m, 1 H); 7.38 (d, J ¼ 8.3,
2 H); 7.80 (d, J ¼ 8.3, 2 H). EI-MS: 255 (Mþ), 173, 155, 139, 122, 91.
3. 3-(Acetyloxy)-4-{[(4-methylphenyl)sulfonyl]oxy}butanenitrile ((ꢀ)-6). To (ꢀ)-5 (1.27 g,
5.00 mmol) under N2 were added Ac2O (2.04 g, 20.00 mmol) and pyridine (0.43 g, 5.50 mmol), and the
resultant mixture was stirred at r.t. overnight. After completion of the reaction (TLC), the mixture was
diluted with AcOEt (25 ml) and treated with 1n HCl (20 ml). The org. layer was washed with brine and
dried (Na2SO4), the solvent evaporated, and the residue purified by CC (AcOEt/hexane 25 :75): 6 (90%,
1
1.33 g). H-NMR (200 MHz, CDCl3): 2.09 (s, 3 H); 2.49 (s, 3 H); 2.75 (d, J ¼ 5.9, 2 H); 3.67 – 4.18 (m,
2 H); 5.09 – 5.15 (m, 1 H); 7.38 (d, J ¼ 8.0, 2 H); 7.79 (d, J ¼ 8.0, 2 H). EI-MS: 297 (Mþ), 255, 185, 172,
155, 139, 91.
4. Kinetic Resolution of (ꢀ)-5. To a soln. of (ꢀ)-5 (1.5 g, 5.88 mmol) in iPr2O (15 ml) were
successively added lipase PS-D (1.20 g) and vinyl acetate (6 equiv.) and shaken at r.t. in an orbital shaker.
After ca. 50% completion of the reaction as indicated by HPLC analysis, the mixture was filtered and the
residue washed thrice with iPr2O. The combined org. layer was concentrated and the residue purified by
CC (AcOEt/hexane 30 :70): (R)-6 followed by (S)-5.
Data of (3S)-3-Hydroxy-4-{[(4-methylphenyl)sulfonyl]oxy}butanenitrile ((S)-5): [a]2D6 ¼ ꢁ14.5 (c ¼
1.45, EtOH) ([14b]: [a]2D5 ¼ ꢁ14.2 (c ¼ 1.72, EtOH)). IR, NMR, and MS: identical to those of (ꢀ)-5.
Data of (3R)-3-(Acetyloxy)-4-{[(4-methylphenyl)sulfonyl]oxy}butanenitrile ((R)-6): [a]2D8 ¼ þ22.6
(c ¼ 1.0, CHCl3). NMR and MS: identical to those of (ꢀ)-6.
5. (3R)-4-Amino-3-hydroxybutanoic Acid ((¼ R)-GABOB; (R)-1). To a soln. of (R)-6 (4.07 g,
13.70 mmol) in EtOH (40 ml) was added excess aq. NH3 soln., the mixture refluxed overnight, and then
the solvent evaporated. To the resulting residue, conc. HCl soln. was added, and the mixture was heated
to 808 for 6 h. After evaporation, the residue containing crude (R)-1 was purified by an ion-exchange
chromatography (Amberlite IR-120 Hþ, H2O until neutral, then 10% NH4OH soln.). Concentration of
the basic fractions gave a thick oil, which was dissolved in a minimum amount of H2O, and abs. EtOH was
added to provide (R)-1 as a white solid (84%). Recrystallization from H2O/EtOH provided pure (R)-1
(73%). White crystals. M.p. 211 – 2138 ([14b]: 206 – 2088). [a]2D8 ¼ ꢁ20.7 (c ¼ 1.0, H2O) ([14b]: [a]2D5
¼

Helvetica Chimica Acta – Vol. 90 (2007)
1729
ꢁ18.6 (c ¼ 0.5, H2O)). 1H-NMR (200 MHz, D2O): 2.43 (d, J ¼ 5.9, 2 H); 2.95 (dd, J ¼ 13.4, 9.7, 1 H); 3.18
(dd, J ¼ 13.4, 3.7, 1 H); 4.10 – 4.30 (m, 1 H). 13C-NMR (50 MHz, D2O): 42.3; 44.0; 65.5; 178.5. EI-MS: 118
([M ꢁ H]þ), 74, 60, 43.
6. (R)-Carnitine Hydrochloride ( ¼ (2R)-3-Carboxy-2-hydroxy-N,N,N-trimethylpropan-2-aminium
Chloride (R)-2 · HCl). To a soln. of (R)-6 (4.07 g, 13.70 mmol) in EtOH (40 ml) was added Me3N (25 – 27
wt.-% in H2O; 26 ml, 109.6 mmol). The mixture was refluxed overnight and then concentrated. To the
resulting residue, conc. HCl soln. was added, and the mixture heated at 808 for 6 h. After evaporation, the
residue containing crude (R)-2 was purified by ion-exchange chromatography (Amberlite IR-120 Hþ,
H2O until neutral, then 10% NH4OH soln.). Concentration of the basic fractions gave a thick oil, which
was again acidified with conc. HCl soln. Evaporation of the solvent yielded carnitine hydrochloride.
Trituration with iPrOH afforded (R)-2 · HCl (85%). Colorless solid.
In another approach, MeI (6.82 g, 48.00 mmol) in MeOH (60 ml) was added to a soln. of (R)-1
(0.95 g, 7.98 mmol) and KOH (2.69 g, 48.00 mmol) in H2O (25 ml). After stirring overnight at r.t., the
mixture was concentrated, the residue dissolved in H2O (60 ml), and the soln. extracted with CHCl3 (3 ꢂ
60 ml). The combined org. phase was again extracted with H2O ( 2ꢂ 30 ml), and the combined aq. phase
containing crude carnitine was evaporated to ca. 20% of the total volume and then treated with 6n HCl
(20 ml) for 2 h. Then the mixture was concentrated and the residue purified by ion-exchange
chromatography (Amberlite IR-120 Hþ, H2O until neutral, then 10% NH4OH soln.). Concentration of
the basic fractions gave a thick oil, which was again acidified with conc. HCl soln. Evaporation of the
i
solvent afforded carnitine hydrochloride in 80% yield. Trituration with PrOH afforded (R)-2 · HCl.
Colorless solid. M.p. 144 – 1468 ([14b]: 144 – 1468). [a]3D0 ¼ ꢁ22.4 (c ¼ 1.5, H2O) ([14b]: [a]D20 ¼ ꢁ20.6
(c ¼ 2.1, H2O)). 1H-NMR (200 MHz, D2O): 2.60 – 2.70 (m, 2 H); 3.24 (s, 9 H); 3.48 – 3.55 (m, 2 H); 4.60 –
4.75 (m, 1 H). 13C-NMR (50 MHz, D2O): 45.8; 56.9; 66.9; 73.0; 180.9.
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Received March 22, 2007