Improved enzymatic syntheses of valuable β-arylalkyl-β-amino acid enantiomers

Article abstract of DOI:10.1039/b920731g

The enantioselective (E～ 200) Burkholderia cepacia-catalysed hydrolyses of β-amino esters with H₂O (0.5 equiv.) in t-BuOMe or in i-Pr₂O at 45 °C are described. The enantiomers of biologically relevant β-arylalkyl-substituted β-amino acids, and especially (R)-3-amino-3-(2,4,5-trifluorophenyl)butanoic acid, the intermediate of the new antidiabetic drug sitagliptine, were prepared with high enantiomeric excesses (ee≥96%) and in good yields (≥42%). The Royal Society of Chemistry 2010.

Full text of DOI:10.1039/b920731g

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Improved enzymatic syntheses of valuable b-arylalkyl-b-amino acid
enantiomers†
Ga´bor Tasna´di,a Eniko˝ Forro´a and Ferenc Fu¨lo¨p*a,b
Received 5th October 2009, Accepted 13th November 2009
First published as an Advance Article on the web 17th December 2009
DOI: 10.1039/b920731g
The enantioselective (E ~ 200) Burkholderia cepacia-catalysed hydrolyses of b-amino esters with H2O
(0.5 equiv.) in t-BuOMe or in i-Pr2O at 45 ◦C are described. The enantiomers of biologically relevant
b-arylalkyl-substituted b-amino acids, and especially (R)-3-amino-3-(2,4,5-trifluorophenyl)butanoic
acid, the intermediate of the new antidiabetic drug sitagliptine, were prepared with high enantiomeric
excesses (ee ≥96%) and in good yields (≥42%).
Introduction
In recent years, extensive investigations have been carried out on
the chemistry of b-amino acids, in particular because of their im-
portance in pharmaceutical research.1 Optically pure b-arylalkyl-
substituted b-amino acids have wide-ranging applications, e.g.
b-peptides containing an (S)-homo-b-phenylalanine unit (5c),
Fig. 1 JanuviaTM (sitagliptin phosphate).
such as a matrix metalloproteinase-2 inhibitor b-tetrapeptide,
utilized for the diagnosis of cancer and atherosclerosis,2 or
b-dipeptides, used for studying the conformational behaviour of
foldamers.3 Moreover, various heterocyclic compounds have been
tested as modulators of protein kinase B, a potential therapeutic
target for diseases associated with abnormal cell growth, can-
cer, inflammation or metabolic disorders.4 (S)-b-Phenylethyl-b-
alanine (6c) has been built into a heat shock protein 70 (Hsp70)
modulator.5 Hsp70 probably contributes to a number of diseases,
including cancer and neurodegeneration. 6c has also been applied
in an a-helical peptide mimetic compound,6 while a derivative of
the (R) enantiomer (6d) has been tested as a hepatitis C virus
inhibitor.7 Type 2 diabetes is a major health problem in the
21st century. Unfortunately, the current modes of therapy are
associated with undesirable side-effects, such as hypoglycaemia
or cardiovascular abnormalities. Dipeptidyl peptidase IV is a
new therapeutic target for the treatment of this form of diabetes.
Inhibition of this peptidase results in increased levels of incretins
(glucagon-like peptide 1 and gastric inhibitory polypeptide), which
control the blood glucose concentration.8 JanuviaTM (sitagliptin
phosphate) (Fig. 1), the first approved drug for the inhibition
of dipeptidyl peptidase IV, contains a b-amino acid subunit,
(R)-3-amino-4-(2,4,5-trifluorophenyl)butanoic acid (11b).9 Be-
sides JanuviaTM, numerous derivatives of 11b have been synthesized
and tested as potential antidiabetic drugs.10
their preparation, e.g. (i) enantioselective hydrogenation of an
enamine,10e,11 (ii) cross-coupling of an enantiomeric organozinc
reagent with aryl iodides,12 (iii) homologation of an optically pure
a-amino acid by the Arndt–Eistert method,10a,13 (iv) homologation
of an optically pure a-amino acid by the formation of a b-
amino alcohol following substitution of the OH group with a
CN group,2,14 (v) conversion of L- or D-aspartic acid to a lactone,
followed by reduction and substitution,15 (vi) conjugate addition
of an amine to an a,b-unsaturated carbonyl compound,16 (vii) SN2
ring opening of an enantiopure b-lactone with a nitrogen-based
nucleophile,17 or (viii) ring opening of an enantiopure b-lactam.18
Enzymatic methods have also been used for the preparation
of enantiopure b-amino acids. Indirect methods are based on
acylation of the corresponding N-hydroxymethyl-b-lactam or
hydrolysis of the N-acyloxymethyl-b-lactam.19 However, indirect
methods have some disadvantages, such as the addition and elimi-
nation of the hydroxymethyl group or the separation of the product
enantiomers by column chromatography.20 These additional steps
can cause relatively low yields. We recently developed an efficient
direct enzymatic method for the synthesis of carbocyclic and
aryl-substituted b-amino acid enantiomers through the selective
(E > 200) ring opening of racemic b-lactams.21 On extension
of this method to 4-arylalkyl-substituted b-lactams, surprisingly,
low E values (≤12) were observed.22 Preparative-scale resolutions
were carried out in two steps, which led to relatively low yields
(≤36%). Later, we devised a highly selective direct enzymatic
method (E usually >100) through the lipase-catalysed hydrolysis
of carbocyclic-,23a aryl-23b and heteroaryl23c-substituted b-amino
esters.
Since b-arylalkyl-b-amino acids are of considerable importance,
many asymmetric synthetic routes have been developed for
aInstitute of Pharmaceutical Chemistry, University of Szeged, H-6720
Szeged, Eo¨tvo¨s u. 6, Hungary. E-mail: Forro.Eniko@pharm.u-szeged.hu;
Fax: +36-62-545705; Tel: +36-62-545564
bResearch Group for Stereochemistry, Hungarian Academy of Sciences,
University of Szeged, H-6720 Szeged, Eo¨tvo¨s u. 6, Hungary. E-mail:
fulop@pharm.u-szeged.hu; Fax: +36-62-545705; Tel: +36-62-545564
Our aim was to develop a direct enzymatic method for
the resolution of racemic b-arylalkyl-substituted b-amino esters
under non-aqueous conditions, resulting in valuable b-amino acid
† This paper is part of an Organic & Biomolecular Chemistry web theme
issue on biocatalysis.
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enantiomers, e.g. (R)-3-amino-4-(2,4,5-trifluorophenyl)butanoic
acid (11b), an intermediate of sitagliptin.
First, a number of lipases were tested in t-BuOMe at 25 ◦C with
0.5 equiv. of H2O (the quantity of the racemic compound is
considered as 1 molar equivalent). The use of organic solvents
in enzymatic reactions has numerous advantages, and lipases
are able to hydrolyse ester and amide bonds in organic solvents
with high enantioselectivity.25 CAL-A (Candida antarctica lipase
A) and Lipolase (Candida antarctica lipase B) catalysed the
reaction with low E values (Table 1, entries 1 and 2), while PPL
(porcine pancreas lipase) and lipase AK (Pseudomonas fluorescens)
displayed moderate E values (entries 3 and 4). Lipase PS IM
(Burkholderia cepacia) proved to be the best enzyme for the
hydrolysis of ( )-5 (entry 5), therefore it was chosen for further
experiments and for the preparative-scale resolutions.
Results and discussion
Syntheses of ( )-5, ( )-6 and ( )-11
b-Lactams ( )-3 and ( )-4 were prepared from alkenes 1 and
2 by the addition of chlorosulfonyl isocyanate (CSI), according
to known literature methods.19b,24 b-Amino esters ( )-5 and ( )-
6 were synthesized by the ring opening of ( )-3 and ( )-4 with
22% HCl/EtOH, followed by treatment with aqueous K2CO3
(Scheme 1).
When the temperature was increased from 25 to 45 ◦C, the
conversion increased considerably (Table 1, entries 5 and 6), but
a further increase of the reaction temperature did not exert any
additional beneficial effect on the reaction rate (entry 10), though
E remained high (≥56). The enantioselective (E > 200) hydrolysis
of ( )-5 was complete in 72 h, even at 45 ◦C (entry 7); accordingly,
subsequent experiments were planned at 45 ◦C.
Starting from acid 7, enamine 10 was prepared by a slightly
modified literature method.10e b-Amino ester ( )-11 was obtained
by the reduction of enamine 10 with NaCNBH3 in the presence of
AcOH (Scheme 2).
Enzymatic hydrolysis of ( )-5, ( )-6 and ( )-11
Preliminary experiments. Preliminary experiments were per-
formed on the hydrolysis of model compound ( )-5 (Scheme 3).
When the amount of enzyme was increased from 30 to 50 and
then 75 mg mL-1, the reaction rates clearly increased, while the E
Scheme 1 Syntheses of ( )-5 and ( )-6.
Scheme 2 Synthesis of ( )-11.
Scheme 3 Enzyme-catalysed hydrolyses of ( )-5, ( )-6 and ( )-11.
794 | Org. Biomol. Chem., 2010, 8, 793–799
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Table 1 Conversion, enantiomeric excesses (ee) and enantioselectivities (E) of the hydrolysis of ( )-5a
Entry
Enzyme
Enzyme/mg mL-1
t/h
T/◦C
ees (%)b
eep (%)b
Conv. (%)
E
1
2
3
4
5
6
7
8
CAL-Ac
50
50
50
50
50
50
50
30
75
50
87
87
87
87
26
26
72
26
26
26
25
25
25
25
25
45
45
45
45
60
7
88
2
19
14
37
99
24
48
39
37
15
88
88
96
96
96
96
96
96
16
85
2
18
13
28
51
20
33
29
2
3
Lipolase
PPLc
16
19
56
70
>200
62
79
72
Lipase AKc
Lipase PS IM
Lipase PS IM
Lipase PS IM
Lipase PS IM
Lipase PS IM
Lipase PS IM
9
10
a 0.05 M substrate, 1 mL of t-BuOMe, 0.5 equiv. of H2O. b According to HPLC (Experimental section). c Contains 20% (w/w) lipase adsorbed on Celite
in the presence of sucrose.
Table 4 Effects of added H2O on the hydrolysis of ( )-5a
values were apparently not affected (Table 1, entries 6, 8 and 9).
For economic reasons, preparative-scale resolutions were carried
out with 50 mg mL-1 enzyme.
Entry
H2O (equiv.)
ees (%)b
eep (%)b
Conv. (%)
E
We next analysed the effects of solvents on the reaction rate and
E (Table 2). The highest E values and conversions were observed
in i-Pr2O and in t-BuOMe (entries 1 and 2). None of the other
solvents tested were suitable for the hydrolysis of ( )-5 (entries
3–5). In view of our earlier results on the vapour-assisted ring
opening of carbocyclic cis-b-lactams,26 the hydrolysis of ( )-5 was
attempted under solvent-free conditions (entry 6): the reaction rate
increased considerably, while E decreased.
1
2
3
4
5
0
0.5
1
5
10
38
35
31
22
8
96
96
96
96
96
28
27
24
19
8
71
69
66
61
53
a 0.05 M substrate, 1 mL of t-BuOMe, 50 mg mL-1 lipase PS IM at 45 ◦
C
after 25 h. b According to HPLC (Experimental section).
Certain additives can influence the enantioselectivity or the
reaction rate of lipase-catalysed reactions (Table 3).27 However,
no enhancement relative to H2O was achieved by the addition of
i-Pr2EtN, Et3N or 2-octanol.
We then attempted to increase the reaction rate by increasing
the amount of H2O (1–10 equiv.) (Table 4). In contrast with our
previous experience,23b,c the reaction rate decreased on increasing
the amount of H2O; moreover, the degree of hydrolysis was
complete and the reaction was fastest without the addition of any
H2O (entry 1). We presume that the poorer solubility of ( )-5 in the
presence of more H2O can cause a decrease in the reaction rate. In
good correlation with our earlier observation,21 the H2O present
in the reaction medium (<0.1%) or at the surface of the enzyme
preparation (<5% w/w H2O) was responsible for the hydrolysis of
( )-5.
Racemic 6 and 11 were hydrolysed under the optimized condi-
tions for ( )-5: with 0.5 equiv. of H2O in the presence of 50 mg mL-1
lipase PS IM in t-BuOMe or in i-Pr2O at 45 ◦C. When the reaction
was performed in i-Pr2O instead of t-BuOMe, higher reaction rates
and better E values were observed (Table 5).
With regard to the results of the preliminary experiments,
preparative-scale resolutions were performed with lipase PS IM
in t-BuOMe or in i-Pr2O with 0.5 equiv. of H2O at 45 ◦C. The
reactions were stopped at close to 50% conversion, and the
products were obtained in good yields (≥42%) and with good ee
values (≥96%).
Enantiomeric 5a, 6a and 11a were hydrolysed with aqueous
HCl, affording 5c, 6c and 11c (ee ≥96%) (Scheme 4). Treatment of
5b, 6b and 11b with 22% HCl/EtOH resulted in the corresponding
enantiopure 5d, 6d and 11d (ee ≥96%) (Scheme 5).
Table 2 Effects of solvents on the hydrolysis of ( )-5a
Entry
Solvent (1 mL)
ees (%)b
eep (%)b
Conv. (%)
E
1
2
3
4
5
6
i-Pr2O
60
50
8
50
5
96
96
74
89
82
66
38
34
10
36
6
90
81
7
28
11
11
t-BuOMe
Et2O
n-Hexane
Toluene
Solvent-free
74
53
a 0.05 M substrate, 50 mg mL-1 lipase PS IM, 0.5 equiv. of H2O at 45 ◦
after 35 h. b According to HPLC (Experimental section).
C
Table 3 Effects of additives on the hydrolysis of ( )-5a
Entry Additive (1 equiv.)
ees (%)b
eep (%)b
Conv. (%)
E
1
2
3
4
H2O
i-Pr2EtN
Et3N
44
41
43
39
96
96
96
96
31
30
31
29
76
73
75
72
2-Octanol
a 0.05 M substrate, 1 mL of t-BuOMe, 50 mg mL-1 lipase PS IM at 45 ◦
C
after 31 h. b According to HPLC (Experimental section).
Scheme 4 Syntheses of 5c, 6c and 11c.
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Table 5 Effects of solvents on the hydrolyses of ( )-6 and ( )-11a
Entry
Substrate
t/h
Solvent (1 mL)
ees (%)b
eep (%)b
Conv. (%)
E
1
2
3
4
( )-6
43
43
65
65
i-Pr2O
79
54
85
61
96
91
97
97
45
37
47
39
119
36
179
123
( )-6
t-BuOMe
i-Pr2O
( )-11
( )-11
t-BuOMe
a 0.05 M substrate, 50 mg mL-1 lipase PS IM, 0.5 equiv. of H2O at 45 ◦C. b According to HPLC (Experimental section).
(5 g) were dissolved in Tris-HCl buffer (0.02 M; pH 7.8) in the
presence of sucrose (3 g), followed by adsorption on Celite (17 g)
(Sigma). Allylbenzene, 4-phenylbut-1-ene and Meldrum’s acid
were from Aldrich. (2,4,5-Trifluorophenyl)acetic acid was from
Matrix Scientific. The solvents were of the highest analytical grade.
Optical rotations were measured with a Perkin-Elmer 341
1
polarimeter. H NMR and 13C NMR spectra were recorded on
a Bruker Avance DRX 400 spectrometer. Melting points were
determined on a Kofler apparatus. Elemental analyses (CHNS)
corresponded closely (within 0.3%) with the calculated ones in
all cases.
In a typical small-scale enzyme test, ( )-5, ( )-6 or ( )-11 (0.05 M
solution) in an organic solvent (1 mL) was added to the enzyme
tested (30, 50 or 75 mg mL-1), followed by H2O, i-Pr2EtN, Et3N
or 2-octanol (0, 0.5, 1, 5 or 10 equiv.). The mixture was shaken at
25, 45 or 60 ◦C.
Scheme 5 Syntheses of 5d, 6d and 11d.
The absolute configurations and E values were proved by
comparing the [a] values with literature data (Experimental
section).
Conclusions
The ee values for the unreacted b-amino ester and the b-amino
acid enantiomers produced were determined by HPLC as follows:
5a, 6a, 11a, 11b: [11b was pre-column derivatized with CH2N2
A simple and efficient direct enzymatic method has been developed
for the synthesis of pharmacologically valuable optically active
b-arylalkyl-substituted b-amino acids via enantioselective hydrol-
ysis of the corresponding racemic b-amino esters in an organic
medium. The R-selective hydrolyses of ( )-5, ( )-6 and ( )-11 were
performed with H2O (0.5 equiv.) as a nucleophile, using Burkholde-
ria cepacia lipase (lipase PS IM) as an enzyme in t-BuOMe or in
i-Pr2O at 45 ◦C (E ~ 200). The enantiomers of 5a, 6a and 11a
(ee ≥96%), and 5b, 6b and 11b (ee ≥96%) were isolated in good
yields (≥42%) and could be easily separated. Ester enantiomers
5a, 6a and 11a were readily hydrolysed with 18% aqueous HCl,
resulting in acids 5c, 6c and 11c (ee ≥96%).
This method offers a better choice for the preparation of
b-arylalkyl-substituted b-amino acid enantiomers as compared
with the ring opening of the corresponding lactam.22 It should be
mentioned that, although derivatives of 11b have been prepared
by asymmetric routes (see ref. 10a, 11 and 18), to the best of
our knowledge they have not yet been obtained by enzymatic
procedures.
28
(Caution! derivatization with CH2N2 should be performed under
a well-working hood)]: a Chiralpak IA column (4.6 mm ¥
250 mm); eluent: n-hexane (0.1% DEA)–i-PrOH (95 5); flow rate:
0.3 mL min-1; detection at 276 nm; retention times (min) for 5a:
35.2 (antipode: 33.9); for 6a: 37.5 (antipode: 36.6); for 11a: 48.5
(antipode: 39.6); for 11b: 44.4 (antipode: 50.0).
5b: a Chirobiotic TAG column (4.6 mm ¥ 250 mm); eluent:
MeOH–AcOH–TEA (100 : 0.1 : 0.1); flow rate: 0.8 mL min-1;
detection at 205 nm; retention times (min): 17.2 (antipode: 19.2).
6b: an APEX Octadecyl 5 m column (0.04 cm ¥ 25 cm); pre-
column derivatization with (S)-NIFE according to the literature;29
the mobile phases were H2O (A) and MeCN (B), both of which
contained 0.1% TFA; the gradient slopes were: 95% A + 5% B
at 0 min, increased to 25% A + 75% B within 60 min; flow
rate: 0.8 mL min-1; room temperature; detection at 205 nm;
retention times (min): 42.7 (antipode: 41.9).
Syntheses of b-amino esters ( )-5 and ( )-6
Experimental section
Racemic b-lactams ( )-3 and ( )-4 were prepared by the addition
of chlorosulfonyl isocyanate to allylbenzene (1) or 4-phenylbut-1-
ene (2), respectively, according to known literature methods.19b,24
( )-3 and ( )-4 (6.0 mmol) were then refluxed with 22% HCl/EtOH
(15 mL) for 8 h, after which the solvent was evaporated off,
resulting in the corresponding b-amino ester hydrochlorides
( )-5·HCl and ( )-6·HCl, which were immediately treated with
aqueous K2CO3 to afford ( )-5 and ( )-6, as oils.
Materials and methods
Lipase PS IM (Burkholderia cepacia, immobilized on diatoma-
ceous earth) was a gift of Amano Enzyme Europe Ltd. Lipase
AK (Pseudomonas fluorescens) was from Amano Pharmaceuti-
cals, Lipolase (lipase B from Candida antarctica, produced by
submerged fermentation of a genetically modified Aspergillus
oryzae microorganism and adsorbed on a macroporous resin)
and PPL (porcine pancreas lipase type II) were from Sigma,
and Chyrazyme L-5 (lipase A from Candida antarctica) was
from Novo Nordisk. Before use, lipase AK, CAL-A and PPL
Ethyl ( )-3-amino-4-phenylbutanoate [( )-5]. Yield: 1.07 g
(86%), a pale-yellow oil; dH(400 MHz; CDCl3; Me4Si) 1.27-1.31
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(3 H, t, J 7.1, CH2CH3), 2.32-2.38 (1 H, dd, J 8.7 and 15.9,
CH2COOH), 2.49-2.54 (1 H, dd, J 4.2 and 15.9, CH2COOH),
2.62-2.68 (1 H, dd, J 8.1 and 13.4, CH2Ar), 2.78-2.82 (1 H, dd, J
5.6 and 13.4, CH2Ar), 3.49-3.52 (1 H, m, CH), 4.15-4.20 (2 H, q,
J 7.1 and 14.3, CH2CH3), 7.22-7.36 (5 H, m, Ar); dC(100.62 MHz;
CDCl3; Me4Si) 14.6, 42.4, 44.4, 50.1, 60.8, 126.9, 129.0, 129.7,
139.0, 172.8.
7.09-7.15 (1 H, m, Ar); dC(100.62 MHz; CDCl3; Me4Si) 14.9, 35.1,
59.3, 85.8, 105.6, 119.2, 145.7, 147.9, 154.4, 158.8, 169.8.
10 (1.56 g, 6.0 mmol)◦was dissolved in EtOAc (15 mL), the
mixture was cooled to 0 C, and NaCNBH3 (1.13 g, 18.0 mmol)
and glacial AcOH (1.0 mL, 18.0 mmol) were added. After 6 h,
the mixture was extracted with 10% Na2CO3 (3 ¥ 15 mL), and the
organic phase was dried with Na2SO4 and evaporated, resulting in
crude ( )-11. This was then dissolved in 22% HCl/EtOH (10 mL),
and the solution was evaporated, resulting in ( )-11·HCl, which
was immediately treated with aqueous K2CO3 to afford ( )-11:
0.85 g (54%), a pale-yellow oil; dH(400 MHz; CDCl3; Me4Si) 1.28-
1.32 (3 H, t, J 7.1, CH2CH3), 2.32-2.39 (1 H, dd, J 8.5 and 15.9,
CH2COOH), 2.48-2.54 (1 H, dd, J 4.2 and 15.9, CH2COOH),
2.65-2.71 (1 H, dd, J 7.7 and 13.7, CH2Ar), 2.76-2.81 (1 H, dd,
J 5.8 and 13.7, CH2Ar), 3.46-3.52 (1 H, m, CH), 4.16.4.21 (2 H,
q, J 7.1 and 14.2, CH2CH3), 6.93-6.96 (1 H, m, Ar), 7.08-7.11 (1
H, m, Ar); dC(100.62 MHz; CDCl3; Me4Si) 14.6, 36.8, 42.1, 49.1,
61.0, 105.6, 120.3, 145.3, 147.7, 155.0, 157.0, 172.0.
Ethyl ( )-3-amino-5-phenylpentanoate [( )-6]. Yield: 1.10 g
1
(83%), a pale-yellow oil. The H NMR and 13C NMR data are
in accordance with those reported in the literature.30
Syntheses of b-amino ester ( )-11
To a mixture of 2,4,5-trifluorophenylacetic acid 7 (2.28 g,
12.0 mmol), 2,2-dimethyl-1,3-dioxane-4,6-dione (Meldrum’s acid)
(1.90 g, 13.2 mmol), N,N-dimethylaminopyridine (0.12 g,
0.96 mmol) and N,N-diisopropylethylamine (4.7 mL, 27.0 mmol)
in MeCN (8 mL), trimethylacetylchloride (1.6 mL, 13.2 mmol)
was added at 40 ◦C. The reaction mixture was stirred at
45 ◦C for 3 h, cooled to 0 ◦C, and 1 M HCl (20 mL)
was then slowly added to the reaction mixture to form a
solid. The resulting solid was washed with 20% CH3CN–H2O
(50 mL) to give 5-(1-hydroxy-2-(2,4,5-trifluorophenyl)ethylidene)-
2,2-dimethyl-1,3-dioxane-4,6-dione (8): 3.37 g (85%), white crys-
tals; mp 99-101 ◦C (from MeCN) [lit.,31 117 ◦C (decomp.)]. The 1H
NMR and 13C NMR data are in accordance with those reported
in the literature.31
In the next step, 8 (3.30 g, 10.0 mmol) was refluxed in
EtOH–toluene 1 : 4 (80 mL) for 3 h. The reaction mixture was
subsequently diluted with EtOAc (35 mL), and washed with brine
(2 ¥ 50 mL). The organic layer was dried with Na2SO4 and
evaporated to give ethyl 3-oxo-4-(2,4,5-trifluorophenyl)butanoate
(9): 2.45 g (94%), white crystals; mp 29-30 ◦C (from n-hexane).
dH(400 MHz; CDCl3; Me4Si) 1.30-1.34 (3 H, t, J 6.9, CH2CH3),
3.55 (2 H, s, CH2COOCH2CH3), 3.88 (2 H, s, CH2Ar), 4.22-4.27
(2 H, q, J 7.1 and 14.3, CH2CH3), 6.94-7.01 (1 H, m, Ar), 7.04-
7.11 (1 H, m, Ar); dC(100.62 MHz; CDCl3; Me4Si) 14.6, 42.4, 49.1,
62.1, 106.1, 119.9, 145.8, 147.8, 155.1, 157.1, 167.1, 198.5.
A mixture of 9 (2.60 g, 10.0 mmol) and NH4OAc (3.85 g,
50.0 mmol) in EtOH (40 mL) was refluxed for 7 h. The reaction
mixture was evaporated, diluted with EtOAc (50 mL) and washed
with H2O (2 ¥ 50 mL). The organic layer was dried with Na2SO4
and evaporated to give ethyl 3-amino-4-(2,4,5-trifluorophenyl)but-
2-enoate (10): 2.31 g (89%), white crystals; mp 115-117 ◦C (from
n-hexane and EtOAc). dH(400 MHz; CDCl3; Me4Si) 1.27-1.31 (3
H, t, J 7.2, CH2CH3), 3.44 (2 H, s, CH2Ar), 4.12-4.17 (2 H, q, J
7.1 and 14.2, CH2CH3), 4.59 (1 H, s, CH), 6.94-7.01 (1 H, m, Ar),
General procedure for the preparative-scale resolutions of ( )-5,
( )-6 and ( )-11
Racemic ( )-5, ( )-6 and ( )-11 (3 mmol) were dissolved in t-
BuOMe [( )-5] or in i-Pr2O [( )-6 and ( )-11] (15 mL). Lipase PS
IM (0.75 g, 50 mg mL-1) and H2O (27 mL, 1.5 mmol) were added
and the mixture was shaken in an incubator shaker at 45 ◦C for 3–5
d (Table 6). The reaction was stopped by filtering off the enzyme
at close to 50% conversion. The solvent was evaporated and the
residues (S)-5a, (S)-6a and (S)-11a were immediately hydrolysed
by refluxing with 6 mL of 18% aqueous HCl solution for 7 h to
give (S)-5c, (S)-6c and (S)-11c. The filtered-off enzyme was washed
with distilled H2O (3 ¥ 15 mL), and the H2O was evaporated off,
yielding crystalline (R)-5b, (R)-6b and (R)-11b. When (R)-5b, (R)-
6b or (R)-11b (50 mg) was treated with 22% HCl/EtOH (5 mL),
then evaporated, (R)-5d, (R)-6d or (R)-11d was obtained.
(R)-3-Amino-4-phenylbutanoic acid (5b). Yield: 242 mg (45%),
white crystals; ee = 96%; [a]2D5 -4.6 (c 0.30 in H2O) [lit.,22 +7
(c 0.20 in H2O) for the (S) enant◦iomer]; mp 209-211 ◦C (from
1
H2O and Me2CO) (lit.,22 207-210 C) The H NMR data are in
accordance with those reported in the literature.22 dC(100.62 MHz;
D2O; Me4Si) 38.5, 38.6, 51.2, 128.1, 129.6, 130.0, 136.2, 178.3.
Hydrochloride salt of (S)-3-amino-4-phenylbutanoic acid (5c).
Yield: 272 mg (42%), off-white crystals; ee = 96%; [a]2D5 +4.6 (c 0.36
◦
in H2O) [lit.,22 +6 (c 0.21 in H2O)]; mp 174-176 C (from EtOH
and Et2O) (lit.,22 172-175 ◦C). The 1H NMR and 13C NMR data
are in accordance with those reported in the literature.19b
Table 6 Lipase PS IM-catalysed hydrolyses of ( )-5, ( )-6 and ( )-11a
b-Amino acid·HCl (5c, 6c, 11c)
b-Amino acid (5b, 6b, 11b)
t/d
Conv. (%)
E
Yield (%)
Isomer
ee (%)b
[a]2D5
Yield (%)
Isomer
ee (%)b
[a]2D5
( )-5
( )-6
( )-11
5
3
5
50
51
50
194
>200
>200
42
47
44
S
S
S
96
98
96
+4.6c
+11.5e
-9.8g
45
47
43
R
R
R
96
96
97
-4.6d
-12.1f
+15.5h
a 50 mg mL-1 lipase PS IM in i-Pr2O, 0.5 equiv. of H2O at 45 ◦C. b According to HPLC (Experimental section). c c 0.36. d c 0.30. e c 0.40. f c 0.34. g c 0.32.
h c 0.35.
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Hydrochloride salt of (R)-3-amino-4-phenylbutanoic acid (5d).
Quantitative yield; off-white crystals; ee = 96%; [a]2D5 -3.9 (c 0.35
2 T. Mukai, N. Suganuma, K. Soejima, J. Sasaki, F. Yamamoto and M.
Maeda, Chem. Pharm. Bull., 2008, 56, 260–265.
3 E. E. Baquero, W. H. James, S. H. Choi, S. H. Gellman and T. S. Zwier,
J. Am. Chem. Soc., 2008, 130, 4795–4807.
◦
in H2O) [lit.,22 -8 (c 0.11 in H2O)]; mp 169-172 C (lit.,22 182-
◦
1
185 C). The H NMR and 13C NMR data for 5d are similar to
4 Q. Zeng, D. Zhang, G. Yao, G. E. Wohlhieter, X. Wang, J. Rider, A.
Reichelt, H. Monenschein, F. Hong, J. R. Falsey, C. Dominguez, M. P.
Bourbeau and J. G. Allen, PCT Int. Appl., WO 011880, 2009.
5 S. Wise´n, J. Androsavich, C. G. Evans, L. Chang and J. E. Gestwicki,
Bioorg. Med. Chem. Lett., 2008, 18, 60–65.
those for 5c.
(R)-3-Amino-5-phenylpentanoic acid (6b). Yield: 272 mg
(47%), white crystals; ee = 96%; [a]2D5 -12.1 (c 0.34 in H2O) [lit.,22
+24 (c 0.28 in H2O) for the (S) ena◦ntiomer]; mp 214-216 ◦C (from
6 G. L. Lessene and J. Baell, US Pat., 0153802, 2008.
7 C. Bachand, M. Belema, D. H. Deon, A. C. Good, J. Goodrich,
C. A. James, R. Lavoie, O. D. Lopez, A. Martel, N. A. Meanwell,
V. N. Nguyen, J. L. Romine, E. H. Ruediger, L. B. Snyder, D. R. St.
Laurent, F. Yang, D. R. Langley and L. G. Hamann, US Pat., 0044379,
2008.
1
H2O and Me2CO) (lit.,22 215-219 C). The H NMR data are in
accordance with those reported in the literature.22 dC(100.62 MHz;
D2O; Me4Si) 31.3, 34.4, 38.7, 49.6, 127.0, 129.0, 129.4, 141.4, 178.6.
8 (a) A. E. Weber, J. Med. Chem., 2004, 47, 4135–4141; (b) S. H. Havale
Hydrochloride salt of (S)-3-amino-5-phenylpentanoic acid (6c).
Yield: 324 mg (47%), off-white crystals; ee = 98%; [a]2D5 +11.5
(c 0.40 in H2O) [lit.,22 +12 (c 0.21 ◦in H2O)]; mp 144-146 ◦C (from
accordance with those reported in the literature.22 dC(100.62 MHz;
D2O; Me4Si) 31.1, 34.1, 36.3, 48.5, 127.1, 129.0, 129.4, 141.0, 174.8.
and M. Pal, Bioorg. Med. Chem., 2009, 17, 1783–1802.
9 N. A. Thornberry and A. E. Weber, Curr. Top. Med. Chem., 2007, 7,
557–568.
1
EtOH and Et2O) (lit.,22 150-152 C). The H NMR data are in
10 (a) J. Xu, H. O. Ok, E. J. Gonzalez, L. F. Colwell, Jr., B. Habulihaz, H.
He, B. Leiting, K. A. Lyons, F. Marsilio, R. A. Patel, J. K. Wu, N. A.
Thornberry, A. E. Weber and E. R. Parmee, Bioorg. Med. Chem. Lett.,
2004, 14, 4759–4762; (b) L. L. Brockunier, J. He, L. F. Colwell, Jr., B.
Habulihaz, H. He, B. Leiting, K. A. Lyons, F. Marsilio, R. A. Patel, Y.
Teffera, J. K. Wu, N. A. Thornberry, A. E. Weber and E. R. Parmee,
Bioorg. Med. Chem. Lett., 2004, 14, 4763–4766; (c) T. Biftu, D. Feng, X.
Qian, G.-B. Liang, G. Kieczykowski, G. Eiermann, H. He, B. Leiting,
K. Lyons, A. Petrov, R. Sinha-Roy, B. Zhang, G. Scapin, S. Patel, Y.-D.
Gao, S. Singh, J. Wu, X. Zhang, N. A. Thornberry and A. E. Weber,
Bioorg. Med. Chem. Lett., 2007, 17, 49–52; (d) G.-. Liang, X. Qian, D.
Feng, T. Biftu, G. Eiermann, H. He, B. Leiting, K. Lyons, A. Petrov,
R. Sinha-Roy, B. Zhang, J. Wu, X. Zhang, N. A. Thornberry and A. E.
Weber, Bioorg. Med. Chem. Lett., 2007, 17, 1903–1907; (e) J. H. Ahn,
M. S. Shin, M. A. Jun, S. H. Jung, S. K. Kang, K. R. Kim, S. D. Rhee,
N. S. Kang, S. Y. Kim, S.-K. Sohn, S. G. Kim, M. S. Jin, J. O. Lee, H. G.
Cheon and S. S. Kim, Bioorg. Med. Chem. Lett., 2007, 17, 2622–2628;
(f) D. Kim, J. E. Kowalchick, S. D. Edmondson, A. Mastracchio, J.
Xu, G. J. Eiermann, B. Leiting, J. K. Wu, K. D. Pryor, R. A. Patel,
H. He, K. A. Lyons, N. A. Thornberry and A. E. Weber, Bioorg. Med.
Chem. Lett., 2007, 17, 3373–3377; (g) D. Kim, J. E. Kowalchick, L. L.
Brockunier, E. R. Parmee, G. J. Eiermann, M. H. Fisher, H. He, B.
Leiting, K. Lyons, G. Scapin, S. B. Patel, A. Petrov, K. D. Pryor, R.
Sinha Roy, J. K. Wu, X. Zhang, M. J. Wyvratt, B. B. Zhang, L. Zhu,
N. A. Thornberry and A. E. Weber, J. Med. Chem., 2008, 51, 589–602.
11 (a) Y. Hsiao, N. R. Rivera, T. Rosner, S. W. Krska, E. Njolito, F.
Wang, Y. Sun, J. D. Armstrong III, E. J. J. Grabowski, R. D. Tillyer,
F. Spindler and C. Malan, J. Am. Chem. Soc., 2004, 126, 9918–9919;
(b) M. Kubryk and K. B. Hansen, Tetrahedron: Asymmetry, 2006, 17,
205–209; (c) T. M. V. D. Pinho e Melo, A. L. Cardoso, F. Palacios, J. M.
de los Santos, A. A. C. C. Pais, P. E. Abreu, J. A. Paixa, A. M. Beja and
M. R. Silva, Tetrahedron, 2008, 64, 8141–8148; (d) K. B. Hansen, Y.
Hsiao, F. Xu, N. Rivera, A. Clausen, M. Kubryk, S. Krska, T. Rosner,
B. Simmons, J. Balsells, N. Ikemoto, Y. Sun, F. Spindler, C. Malan,
E. J. J. Grabowski and J. D. Armstrong III, J. Am. Chem. Soc., 2009,
131, 8798–8804; (e) N. Perlman, M. Etinger, V. Niddam-Hildesheim
and M. Abramov, PCT Int. Appl., WO 064476, 2009.
Hydrochloride salt of (R)-3-amino-5-phenylpentanoic acid (6d).
Quantitative yield; off-white crystals; ee = 96%; [a]2D5 -10.5 (c 0.40
◦
in H2O) [lit.,22 -15 (c 0.21 in H2O)]; mp 147-149 C (lit.,22 146-
◦
1
148 C). The H NMR and 13C NMR data for 6d are similar to
those for 6c.
(R)-3-Amino-4-phenyl(2,4,5-trifluorophenyl)butanoic acid (11b).
Yield: 301 mg (43%), white crystals; ee = 97%; [a]2D5 +15.5 (c 0.35
◦
in H2O); mp 217-219 C (from H2O and Me2CO). dH(400 MHz;
D2O; Me4Si) 2.48-2.54 (1 H, dd, J 7.9 and 16.5, CH2COOH),
2.59-2.65 (1 H, dd, J 5.0 and 16.9, CH2COOH), 3.08-3.09 (2 H,
d, J 6.9, CH2Ar), 3.81-3.88 (1 H, m, CH), 7.22-7.27 (1 H, m, Ar),
7.31-7.37 (1 H, m, Ar); dC(100.62 MHz; D2O; Me4Si) 31.1, 38.0,
49.1, 105.8, 118.9, 145.6, 148.0, 155.1, 157.5, 177.3. To prove the
absolute configuration, N-Boc-11b was prepared by a literature
method11b {white crystals; [a]D25 +24.6 (c 0.46 in CHCl3) [lit.,11b
◦
+32.3 (c 1.0 in CHCl3)]; mp 112-114 C (from n-hexane) (lit.,11b
124-125 ◦C)}.
Hydrochloride salt of (S)-3-amino-4-(2,4,5-trifluorophenyl)-
butanoic acid (11c). Yield: 356 mg (44%), off-white crystals; ee =
96%; [a]2D5 -9.8 (c 0.32 in H2O); mp 177-179 ◦C (from EtOH and
Et2O). dH(400 MHz; D2O; Me4Si) 2.75-2.89 (2 H, m, CH2COOH),
3.08-3.19 (2 H, m, CH2Ar), 3.96-4.05 (1 H, m, CH), 7.21-7.28 (1
H, m, Ar), 7.32-7.38 (1 H, m, Ar); dC(100.62 MHz; D2O; Me4Si)
31.5, 36.1, 48.8, 106.5, 119.4, 146.0, 148.1, 155.4, 156.6, 174.0.
Hydrochloride salt of (R)-3-amino-4-(2,4,5-trifluorophenyl)-
butanoic acid (11d). Quantitative yield; off-white crystals; ee =
12 R. F. W. Jackson, I. Rilatt and P. J. Murray, Chem. Commun., 2003,
1242–1243.
◦
1
97%; [a]2D5 +10.6 (c 0.31 in H2O); mp 179-181 C. The H NMR
13 (a) C. Yoakim, W. W. Ogilvie, D. R. Cameron, C. Chabot, I. Guse, B.
Hach
e´, J. Naud, J. A. O’Meara, R. Plante and R. De´zil, J. Med. Chem.,
and 13C NMR data for 11d are similar to those for 11c.
1998, 41, 2882–2891; (b) C. Yoakim, W. W. Ogilvie, D. R. Cameron,
C. Chabot, I. Guse, B. Hache´, J. Naud, J. A. O’Meara, R. Plante and
R. De´zil, J. Med. Chem., 2005, 48, 141–151; (c) E. Belsito, M. L. Di
Gioia, A. Greco, A. Leggio, A. Liguori, F. Perri, C. Siciliano and M. C.
Viscomi, J. Org. Chem., 2007, 72, 4798–4802.
Acknowledgements
The authors acknowledge the receipt of grants K 71938 and T
049407 from the Hungarian Scientific Research Fund (OTKA),
and a Bolyai Fellowship for EF.
14 (a) R. Caputo, E. Cassano, L. Longobardo and G. Palumbo, Tetrahe-
dron, 1995, 51, 12337–12350; (b) M. K. Ghorai, K. Das and A. Kumar,
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1114; (b) C. W. Jefford, J. McNulty, Z.-H. Lu and J. B. Wang, Helv.
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