Enzymatic resolution of α-alkyl β-amino acids using immobilized penicillin G acylase

Full text of DOI:10.1021/jo9613500

J . Org. Chem. 1996, 61, 8651-8654
8651
Notes
Sch em e 1a
En zym a tic Resolu tion of r-Alk yl â-Am in o
Acid s Usin g Im m obilized P en icillin G
Acyla se
Giuliana Cardillo,* Alessandra Tolomelli, and
Claudia Tomasini*
Dipartimento di Chimica “G. Ciamician” and CSFM,
Universita` di Bologna, Via Selmi 2, 40126 Bologna, Italy
Received J uly 16, 1996
In recent years, increasing attention has been paid to
the synthesis of enantiomerically pure R-substituted
â-amino acids,1 owing to their role as precursors of
3-substituted â-lactams and in the preparation of natural
and unnatural biologically active polypeptides.
The synthesis of enantiomerically pure R-alkyl â-amino
acids has been recently accomplished by the addition of
chiral secondary amines to R,â-unsaturated esters,2 by
the condensation of disubstituted imines,3 by homologa-
tion of the corresponding R-analogues,4 or by the func-
tionalization at C-5 of chiral 6-alkylperhydropyrimidin-
4-ones.5 This method allowed us to obtain (S,S)- or (R,R)-
R-alkyl â-amino acids with high stereocontrol and in high
yields.
A very simple alternative procedure for the preparation
of these compounds is reported here, as the enzymatic
hydrolysis of racemic N-phenylacetyl R-alkyl â-amino
acids with penicillin G acylase (PGA). This enzyme is
widely distributed among microorganisms and is used on
an industrial scale for the production of 6-aminopenicil-
lanic acid.6 Since it exihibits a high affinity for the
phenylacetyl moiety,7 the hydrolysis of different phen-
ylacetic acid derivatives has been reported. Among them,
the resolution of racemic â-amino acids is catalyzed by
PGA,8 preferentially hydrolyzing the S isomer.
a
Reagents and conditions: (i) PhCH2COCl (1.2 equiv), NaOH
(2 equiv), acetone/H2O, 0 °C, 1 h; (ii) SOCl2 (2 equiv), MeOH, -15
°C, 2 h; (iii) LiHMDS (1 M solution in THF) (2 equiv), 0 °C, 1 h;
(iv) RX (1.5 equiv), -78 °C to rt, 16 h; (v) K2CO3 (2 equiv), H2O,
reflux, 2 h, then 1 M HCl.
Ta ble 1. Dia ster eom er ic P r od u ct Ra tios a n d Ch em ica l
Yield s for th e Alk yla tion Rea ction of Meth yl
(()-3-[(P h en yla cetyl)a m in o]bu ta n oa te 1
entry
RX
yield (%)
anti/syn ratioa (%)
1
2
3
4
MeI
EtI
AllylBr
BnBr
68
75
80
84
93:7
>99:1
>99:1
>99:1
a
Determined by 1H NMR and GC-MS analysis.
In order to test the versatility of PGA toward R-sub-
stituted â-amino acids and to depict a simple method for
the preparation of these substrates, the N-(phenylacetyl)-
amides 2a -d were synthesized from the commercially
available (()-3-aminobutanoic acid, following the proce-
dure reported by Seebach9 (Scheme 1). This alkylation
reaction occurs with high anti selectivity. Indeed, by
treatment of the lithium salt of 1 with alkyl halides at
-78 °C in dry THF, the corresponding alkyl derivatives
were obtained with high yield and selectivity (Table 1).
Analysis by 1H NMR and GC-MS of the reaction
mixtures for entries b-d show, in each case, the presence
of a single product with the anti configuration, which was
assigned by comparisons of the 1H NMR spectra with the
data reported in the literature.9 Only for the smaller
methyl group was a small amount of syn adduct obtained.
The methyl ester of 2a -d was then simply hydrolyzed
in quantitative yield by reaction with potassium carbon-
ate in refluxing water to afford amide acids 3a -d .
This procedure is particularly convenient because the
phenylacetyl moiety acts both as a protecting group of
the amino functionality during the alkylation reaction
and as a fragment that can be hydrolyzed during the
subsequent enzymatic resolution.
(1) (a) Cole, D. C. Tetrahedron 1994, 50, 9517. (b) J uaristi, E.;
Quintana, D.; Escalante, J . Aldrichim. Acta 1994, 27, 3. (c) Cardillo,
G.; Tomasini, C. Chem. Soc. Rev. 1996, 25, 117.
(2) (a) Bunnage, M. E.; Davies, S. G.; Goodwin, C. J . J . Chem. Soc.,
Perkin Trans. 1 1993, 1375. (b) Bunnage, M. E.; Burke, A. J .; Davies,
S. G.; Goodwin, C. J . Tetrahedron: Asymmetry 1994, 5, 203. (c)
Bunnage, M. E.; Davies, S. G.; Goodwin, C. J . Synlett 1993, 731. (d)
Davies, S. G.; Garrido, N. M.; Ichihara, O.; Walters, I. A. S. J . Chem.
Soc., Chem. Commun. 1993, 1153. (e) Hawkins, J . M.; Lewis, T. A. J .
Org. Chem. 1994, 59, 649. (f) Asao, N.; Tsukada, N.; Yamamoto, Y. J .
Chem. Soc., Chem. Commun. 1993, 1660. (g) Enders, D.; Bettray, W.;
Raabe, G.; Runsink, J . Synthesis 1994, 1322. (h) Enders, D.; Wahl,
H.; Bettray, W. Angew. Chem., Int. Ed. Engl. 1995, 34, 455.
(3) (a) Yamasaki, N.; Murkami, M.; Mukaiyama, T. Chem. Lett.
1986, 1013. (b) Yamada, T.; Suzuki, H.; Mukaiyama, T. Chem. Lett.
1987, 293. (c) Kunz, H.; Schanzenbach, O. Angew. Chem., Int. Ed. Engl.
1989, 28, 1068. (d) Gennari, C.; Venturini, I.; Gilson, G.; Schimperna,
G. Tetrahedron Lett. 1987, 28, 227.
(4) Burgess, K.; Liu, L. T.; Pal, B. J . Org. Chem. 1993, 58, 4758.
(5) (a) Amoroso, R.; Cardillo, G.; Tomasini, C. Tetrahedron Lett.
1992, 33, 2725. (b) Braschi, I.; Cardillo, G.; Tomasini, C.; Venezia, R.
J . Org. Chem. 1994, 59, 7292. (c) J uaristi, E.; Escalante, J . J . Org.
Chem. 1993, 58, 2282.
Although the optimum rate of hydrolysis of penicillin
G acylase is reported to occur at pH ) 8.2, we found that
under these conditions complete hydrolysis of both (S)-
and (R)-3-aminobutanoic acid was accomplished by reac-
tion at 40 °C for 5 h with E. coli PGA immobilized on
(6) Baldaro, E.; Fuganti, C.; Servi, S.; Tagliani, A.; Terreni, M.
(Servi, S., Ed.) NATO ASI Ser., Ser. C 1992, 381, 175.
(7) For an exhaustive study on the structure and reactivity of
penicillin G acylase see: Duggleby, H. J .; Tolley, S. P.; Hill, C. P.;
Dodson, E. J .; Dodson, G.; Moody, P. C. E. Nature 1995, 373, 264.
(8) (a) Rossi, D.; Lucente, G.; Romeo, A. Experientia 1977, 1557. (b)
Soloshonok, V. A.; Svedas, V. K.; Kukhar, V. P.; Kirilenko, A. G.;
Rybakova, A. V.; Solodenko, V. A.; Fokina, N. A.; Kogut, O. V.; Galaev,
I. Y.; Kozlova, E.V.; Shishkina, I. P.; Galushko, S. V. Synlett 1993,
339.
(9) (a) Seebach, D.; Estermann, H. Tetrahedron Lett. 1987, 28, 3103.
(b) Seebach, D.; Estermann, H. Helv. Chim. Acta 1988, 71, 1824.
S0022-3263(96)01350-3 CCC: $12.00 © 1996 American Chemical Society

8652 J . Org. Chem., Vol. 61, No. 24, 1996
Notes
Sch em e 2
Sch em e 3
Ta ble 3. Sp ecific Rota tion s a n d En a n tiom er ic Excesses
for P r od u cts (2S,3S)-4 a n d (2S,3S)-5
entry product
[R]D
product
[R]D
eea
a
b
c
d
(2S,3S)-4a +10.0
(2S,3S)-5a -8.1
100
Ta ble 2. Ch em ica l Yield s a n d Rea ction Con d ition s for
th e Hyd r olysis of a n ti-(()-2-Alk yl-3-[(p h en yla cetyl)-
a m in o]bu ta n oic Acid s 3a -d
(c 1.2, H2O)
(c 0.1, CHCl3)
(2S,3S)-4b +2.4
(c 0.5, H2O)
(2S,3S)-4c +12.5
(c 0.8, H2O)
(2S,3S)-4d -14.1
(c 0.5, H2O)
(2S,3S)-5b -16.0
(c 0.2, CHCl3)
(2S,3S)-5c -37.5
(c 0.2, CHCl3)
(2S,3S)-5d -46.7
(c 0.7, CHCl3)
100
time
(h) (°C)
T
phosphate buffer/ conversn
94.5
100
entry
R
EtOH ratio
(%)a
a
b
c
d
CH3
CH2CH3
CH2CHdCH2
CH2Ph
5
5
6
6
30
30
35
35
10:1
10:1
5:1
50
47
53
49
a
The enantiomeric excesses have been determined with chiral
5:1
GC utilizing a MEGADEX 5 column with 2,3-dimethyl-5-pentyl-
â-cyclodextrin.
a
The conversion refers to the amount of phenylacetic acid
1
produced and was determined by comparison of the H NMR peak
Sch em e 4
of methylene group of starting material (δ 3.59 ppm) and of
phenylacetic acid (δ 3.67 ppm).
Eupergit. To overcome this reactivity problem, several
reaction conditions were tested.
The amide acids 3a -d (100 mg) were treated with
immobilized PGA (10 mg) in a solution of 0.1 M phos-
phate buffer (10 mL) with a variable amount of ethanol
as a cosolvent (Scheme 2). The phosphate buffer was
adjusted to pH ) 7 before addition of the cosolvent and
the enzyme, and thereafter the pH was not regulated.
The reaction proceeded for 5-8 h at 30 or 35 °C,
depending on the substrate. With an increase in the
steric hindrance of the R group, the reaction temperature,
the reaction time, and the amount of ethanol used were
increased, owing to the reduced water solubility of the
substrate (Table 2).
For each reaction, after the scheduled reaction times,
the mixture was concentrated under reduced pressure
to eliminate the ethanol, the pH was adjusted to 3, and
the water layer was extracted three times with ethyl
acetate. The unhydrolyzed amide acids 2a -d and the
phenylacetic acid were recovered from the organic layers,
while the water layers were concentrated under reduced
pressure to afford the free amino acids (2S,3S)-4a -d .
These acids were dissolved in a small amount of water
and purified on a cation-exchange resin, using 1.5 M
NH4OH as eluant.
The specific rotations of (2S,3S)-4a -d were measured
and compared with the data reported in the literature.5c,10
As expected, the absolute configuration of the hydrolyzed
amino acids was confirmed to be 2S,3S; the determina-
tion of the enantiomeric purity was troublesome because
the specific rotations of these molecules are sensitive to
conditions of purification and of measurement. Since the
gas chromatographic separation of racemic phenylacet-
amido derivatives 2a -d and 3a -d on a chiral support
failed, (2S,3S)-4a -d were transformed into the corre-
sponding acetamide methyl esters (2S,3S)-5a -d , as
reported in Scheme 3. The products were analyzed by
chiral GC chromatography on a MEGADEX 5 column
Ta ble 4. Sp ecific Rota tion s a n d En a n tiom er ic Excesses
for P r od u cts (2R,3R)-4 a n d (2R,3R)-5
entry product
[R]D
product
[R]D
eea
a
b
c
d
(2R,3R)-4a -8.7
(2R,3R)-5a +7.5
100
(c 1.2, H2O)
(c 0.1, CHCl3)
(2R,3R)-4b -3.5
(c 0.3, H2O)
(2R,3R)-4c -6.6
(c 1.2, H2O)
(2R,3R)-4d +21.0
(c 0.1, H2O)
(2R,3R)-5b +14.3
(c 0.3, CHCl3)
(2R,3R)-5c +39.4
(c 0.1, CHCl3)
(2R,3R)-5d +47.5
(c 0.1, CHCl3)
94.3
100
100
a
The enantiomeric excesses have been determined with chiral
GC utilizing a MEGADEX 5 column with 2,3-dimethyl-5-pentyl-
â-cyclodextrin.
filled with 2,3-dimethyl-5-pentyl-â-cyclodextrin (Table 3).
On the other hand, (2R,3R)-3a, (2R,3R)-3b, and (2R,3R)-
3d were recovered from the organic layers and dissolved
in a 1:1 6 M HCl/methanol mixture and refluxed for 30
h (Scheme 4) to obtain the free amino acids (2R,3R)-4a ,
(2R,3R)-4b, and (2R,3R)-4d . The mixture was concen-
trated under reduced pressure to eliminate the methanol
and the excess of HCl and dissolved in a small amount
of water. The free amino acids were purified on a cation-
exchange resin using 1.5 M NH4OH as eluant and fully
characterized as their acetamido methyl ester derivatives
(2R,3R)-5a , (2R,3R)-5b, and (2R,3R)-5d . These com-
pounds were analyzed by chiral GC utilizing a MEGA-
DEX 5 column with 2,3-dimethyl-5-pentyl-â-cyclodextrin
(Table 4).
Since the acid hydrolysis of the allyl derivative (2R,3R)-
(10) J auristi, E.; Escalante, J .; Lamatsch, B.; Seebach, D. J . Org.
Chem. 1992, 57, 2396.
3c afforded a cyclization product,9 the hydrolysis was

Notes
J . Org. Chem., Vol. 61, No. 24, 1996 8653
THF (10 mL) was added LiHMDS (1 M solution in THF, 4 mmol,
4 mL) in one portion under argon at 0 °C. After 1 h, the
alkylating agent (1.5 equiv) was added at -78 °C and the
solution was allowed to stir overnight while being warmed to
room temperature. The reaction was quenched with a saturated
solution of NH4Cl, and the solvent was removed under reduced
pressure and replaced with dichloromethane, which was then
washed twice with water. The organic layer was dried over Na2-
SO4 and concentrated. Compounds 2a -d were purified by flash
chromatography on silica gel (cyclohexane/ethyl acetate 9:1 as
eluant) and then added to a solution of K2CO3 (2 equiv) in water.
The mixture was refluxed for 2 h and then extracted with ethyl
acetate. To the aqueous layer was added 1 M HCl until the
solution reached pH ) 1, and the mixture was extracted twice
with ethyl acetate. After the solvent was removed under reduced
pressure, compounds 3a -d were obtained pure in 67-82%
overall yield from 1.
Sch em e 5
carried out with immobilized PGA under basic conditions.
Indeed, (2R,3R)-3c (100 mg) was hydrolyzed in water (5
mL) and ethanol (1 mL) with immobilized PGA (50 mg)
and CaCO3 (100 mg) (pH ) 11). The reaction proceeded
for 96 h at 35 °C in 95% yield (Scheme 5). The reaction
mixture was acidified to pH ) 3, and then (2R,3R)-4c
was purified on a cation-exchange resin using 1.5 M NH4-
OH as eluant and was fully characterized as its acet-
amido methyl ester derivative (2R,3R)-5c (Table 4).
In conclusion, we have demonstrated that immobilized
penicillin G acylase is an effective reagent for the kinetic
resolution of R-alkyl â-amino acids. The reaction time,
the reaction temperature, the solvent, and the pH proved
to be crucial for the yield and the enantiomeric purity of
the products and should be attentively evaluated for each
substrate.
3a : mp ) 121-124 °C; IR (Nujol) 3290, 1700, 1645 cm-1; 1H
NMR (CDCl3) δ 1.09 (d, 3H, J ) 7.2 Hz), 1.13 (d, 3H, J ) 6.8
Hz), 2.58 (dq, J ) 4.1, 7.2 Hz, 1H), 3.58 (AB, 2H, J ) 15.9 Hz),
4.15 (m, 1H), 6.49 (d,1H, J ) 9.1 Hz), 7.3 (m, 5H); 13C NMR
(CDCl3) δ 14.4, 18.6, 43.2, 43.3, 46.9, 127.0, 128.4, 129.0, 134.3,
171.8, 178.3; HRMS calcd for (M+) C13H17O3N 235.120 843 6,
found 235.120 821 3.
3b: mp ) 91-92 °C; IR (Nujol) 3303, 1719, 1609 cm
-1
;
1H
NMR (CDCl3) δ 0.93 (t, 3H, J ) 7.4 Hz), 1.13 (d, 3H, J ) 6.7
Hz), 1.52 (m, 2H), 2.35 (ddd, J ) 6.1, 10.2, 12.3 Hz, 1H), 3.58 (s,
2H), 4.26 (m, 1H), 6.50 (d, 1H, J ) 8.9 Hz), 7.27 (m, 5H); 13C
NMR (CDCl3) δ 11.8, 19.4, 23.1, 43.4, 45.2, 50.9, 127.1, 128.7,
129.1, 134.3, 171.6, 178.1; HRMS calcd for (M+) C14H19O3N
249.136 493 7, found 249.136 658 2.
3c: mp ) 96-97 °C; IR (Nujol) 3382, 1714, 1619 cm
-1
;
1H
NMR (CDCl3) δ 1.15 (d, 3H, J ) 6.8 Hz), 2.20 (m, 2H), 2.54 (m,
1H), 3.59 (s, 2H), 4.70 (m, 1H), 5.00 (m, 2H), 5.70 (m, 1H), 6.41
(d, 1H, J ) 9.5 Hz), 7.30 (m, 5H); 13C NMR (CDCl3) δ 19.4, 34.0,
43.5, 45.2, 49.2, 117.6, 127.3, 128.5, 128.9, 134.1, 134.3, 171.7,
178.6; HRMS calcd for (M+) C15H19O3N 261.136 493 7, found
261.136 524 9.
Exp er im en ta l Section
3d : mp ) 103-106 °C; IR (Nujol) 3295, 1728, 1608 cm -1; 1H
NMR (CDCl3) δ 1.13 (d, 3H, J ) 6.7 Hz), 2.73 (m, 3H), 3.59 (AB,
2H, J ) 15.6 Hz), 4.17 (m, 1H), 6.67 (d, 1H, J ) 8.8 Hz), 7.27
(m, 10H); 13C NMR (CDCl3) δ 19.4, 35.8, 43.5, 45.2, 51.2, 126.5,
127.3, 128.3, 128.7, 128.8, 129.2, 134.3, 138.0, 172.0, 177.3;
HRMS calcd for (M+) C19H21O3N 311.152 143 8, found
311.152 192 6.
Gen er a l P r oced u r e for th e En zym a tic Hyd r olysis of (()-
N-(P h en yla cetyl)-2-a lk yl-3-a m in obu ta n oic Acid 3a -d . The
amide acid 3 (100 mg) was added to a solution of 0.1 M
phosphate buffer adjusted to pH ) 7 (10 mL) and ethanol as
cosolvent, containing penicillin G acylase from Escherichia coli
immobilized on Eupergit (10 mg). The reaction mixture was
stirred at the temperature and for the time reported in Table 2.
Then the ethanol was removed under reduced pressure, and 2
M HCl was added to the aqueous solution until pH ) 3. The
mixture was extracted twice with ethyl acetate. The organic
layer was dried over Na2SO4 and concentrated to recover
phenylacetic acid and the unhydrolyzed amide. The aqueous
layer was concentrated, and the residue was dissolved in water
(1 mL) and adsorbed on cation-exchange resin. The resin was
washed with water until the washing came out neutral and then
was eluted with 1.5 M aqueous NH4OH. The aqueous solution
was concentrated under reduced pressure to recover the (S,S)-
2-alkyl-3-amino acid in the zwitterionic form.
Gen er a l P r oced u r e for th e Hyd r olysis of (R,R)-N-(P h en -
yla cetyl)-2-a lk yl-3-a m in obu ta n oic Acid 3a ,b,d . The residue
obtained from the organic layer of the enzymatic hydrolysis was
dissolved in 6 M HCl and refluxed for 30 h. The mixture was
then extracted with ethyl acetate to remove phenylacetic acid,
and the aqueous layer was concentrated and treated with cation-
exchange resin to recover the (R,R)-2-alkyl-3-amino acid in the
zwitterionic form.
Hyd r olysis of (R,R)-N-(P h en yla cetyl)-2-a llyl-3-a m in obu -
ta n oic Acid (3c). The mixture of compound 3c and phenylace-
tic acid (obtained from the organic layer of the enzymatic
hydrolysis) and penicillin G acylase (50 mg) were suspended in
a solution of CaCO3 (100 mg) in water (5 mL) and ethanol (1
mL). The solution was stirred for 96 h at room temperature
and then acidified with 2 M HCl until pH ) 3. The mixture
Gen er a l P r oced u r es. 1H NMR spectra were recorded at 300
or 200 MHz. Chemical shifts are reported in ppm relative to
the solvent peak of CHCl3, defined to be δ 7.27 ppm. Infrared
spectra were recorded with an FT-IR spectrometer. Melting
points were determined in open capillaries and are uncorrected.
Flash chromatography was performed with Merck silica gel 60
(230-400 mesh). THF was distilled from sodium benzophenone
ketyl. Dichloromethane was distilled from P2O5. The enantio-
meric excesses have been determined with chiral GC utilizing a
MEGADEX 5 column with 2,3-dimethyl-5-pentyl-â-cyclodextrin
(stationary phase SE52; film thickness 0.1 µm; length 25 m;
internal diameter 0.32 mm; column material fused silica; upper
temperature limit 370 °C). Penicillin G acylase supported on
Eupergit was obtained by Recordati S.p.A. Unita` De.Bi. (Lot.
940-1606; 252.9 IU/g wet; 890 IU/g dry).
Meth yl (()-N-(P h en ylacetyl)-3-am in obu tan oate (1). Phen-
nylacetyl cloride (12 mmol, 1.59 mL) in acetone (10 mL) was
added dropwise to a stirred solution of (()-3-aminobutanoic acid
(10 mmol, 1.03 g) and NaOH (0.8 g) in distilled water (30 mL)
at 0 °C. The mixture was stirred at rt for 1 h, the acetone was
removed under reduced pressure, and the residue was extracted
with ethyl acetate. Then 2 M HCl was added to the aqueous
layer until the solution reached pH ) 1, and the mixture was
extracted with ethyl acetate. The second organic layer was dried
over Na2SO4 and concentrated. A solution of SOCl2 (20 mmol,
1.46 mL) in methanol was stirred for 2 h at -15 °C, and then
the acid previously obtained was added in one portion. The
mixture was stirred overnight and left to warm to room
temperature. The solution was concentrated under reduced
pressure, and compound 1 was obtained in 84% overall yield
after flash chromatography on silica gel (cyclohexane/ethyl
acetate 1:1 as eluant).
1: mp ) 40-41 °C; IR (nujol) 3296, 1729, 1652 cm-1; 1H NMR
(CDCl3) δ 1.18 (d, J ) 6.8 Hz, 3H), 2.51 (d, J ) 5.5 Hz, 2H), 3.57
(s, 3H), 3.67 (s, 2H), 4.33 (m, 1H), 5.98 (d, J ) 7.0 Hz, 1H), 7.30
(m, 5H); 13C NMR (CDCl3) δ 19.4, 39.6, 41.8, 42.8, 50.9, 126.3,
128.0, 128.6, 134.9, 169.9, 171.1; HRMS calcd for (M+) C13H17O3N
235.120 843 6, found 235.120 900 5.
(()-a n ti-N-(P h en yla cetyl)-2-a lk yl-3-a m in obu ta n oic Acid
3a -d . To a stirred solution of ester 1 (2 mmol, 0.47 g) in dry

8654 J . Org. Chem., Vol. 61, No. 24, 1996
Notes
was extracted twice with ethyl acetate to remove phenylacetic
acid, and then the aqueous layer was concentrated under
reduced pressure and treated with cation-exchange resin to
recover (R,R)-N-(phenylacetyl)-2-allyl-3-aminobutanoic acid (3c)
in the zwitterionic form in 95% yield.
45.1, 51.4, 51.6, 169.6, 176.2; HRMS calcd for (M+) C9H17O3N
187.120 843 6, found 187.120 936 2.
5c: IR (film) 3120, 1733, 1651 cm-1; 1H NMR (CDCl3) δ 1.14
(d, 3H, J ) 6.8 Hz), 2.00 (s, 3H), 2.33 (m, 2H), 2.58 (m, 1H),
3.70 (s, 3H), 4.28 (m, 1H), 5.07 (m, 2H), 5.73 (1H, m), 6.38 (d,
1H, J ) 8.1 Hz); 13C NMR (CDCl3) δ 19.8, 23.3, 34.4, 45.3, 49.5,
51.6, 117.4, 134.5, 169.7, 175.4; HRMS calcd for (M+) C10H17O3N
199.120 843 6, found 199.120 966 9.
Gen er a l P r oced u r e for t h e P r ep a r a t ion of Met h yl
N-Acetyl-2-a lk yl-3-a m in obu ta n oa te (S,S)-5a -d a n d (R,R)-
5a -d . The amino acid 4 was added in one portion to a stirred
solution of pyridine (2 mL), acetic anhydride (2 mL), and
(dimethylamino)pyridine (5 mg). The mixture was stirred at
room temperature for 2 h and then quenched with water and
extracted twice with ethyl acetate. The organic layer was dried
over Na2SO4 and concentrated under reduced pressure.
A solution of SOCl2 (2 equiv) in methanol (5 mL) was stirred
for 2 h at -15 °C, and then the N-acetylamino acid previously
obtained was added in one portion. The mixture was stirred
overnight while slowly being warmed to room temperature. The
solution was concentrated under reduced pressure, and com-
pound 5 was obtained in overall 80% yield after purification with
silica gel chromatography (cyclohexane/ethyl acetate 6:4 as
eluant).
5d : IR (film) 3290, 1720, 1652 cm-1; 1H NMR (CDCl3) δ 1.13
(d, 3H, J ) 6.7 Hz), 2.01 (s, 3H), 2.81 (m, 2H), 2.95 (m, 1H),
3.61 (s, 3H), 4.23 (m, 1H), 6.66 (d, 1H, J ) 8.7 Hz), 7.30 (m,
5H); 13C NMR (CDCl3) δ 19.8, 23.3, 36.1, 45.3, 51.4, 51.6, 126.6,
128.5, 128.9, 138.4, 170.5, 177.3; HRMS calcd for (M+) C14H19O3N
249.136 493 6, found 249.136 442 2.
Ack n ow led gm en t. This work was supported in part
by MURST (40%), a NATO Collaborative Research
Grant, and by University of Bologna (funds for Selected
Research Topics). We are also thankful to Recordati
S.p.A. Unita` De.Bi. for a kind gift of 50 g of penicillin G
acylase supported on Eupergit.
5a : IR (film) 3290, 1738, 1646 cm-1; 1H NMR (CDCl3) δ 1.14
(d, 3H, J ) 6.8 Hz), 1.19 (d, 3H, J ) 7.2 Hz), 1.99 (s, 3H), 2.63
(dq, 1H, J ) 4.2 Hz, 7.2 Hz), 3.70 (s, 3H), 4.20 (m, 1H), 6.25 (bs,
1H); 13C NMR (CDCl3) δ 14.9, 19.5, 23.5, 43.5, 46.8, 51.7, 169.6,
176.2; HRMS calcd for (M+) C8H15O3N 173.105 193 5, found
173.105 234 1.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR of the
crude mixture of 3c hydrolysis and gas chromatograms of
(2R,3R)-5c and (2S,3S)-5c (2 pages). This material is con-
tained in libraries on microfiche, immediately follows this
article in the microfilm version of the journal, and can be
ordered from the ACS; see any current masthead page for
ordering information.
5b: IR (film) 3350, 1742, 1657 cm-1; 1H NMR (CDCl3) δ 0.93
(t, 3H, J ) 7.4 Hz), 1.13 (d, 3H, J ) 6.8 Hz), 1.62 (m, 2H), 1.99
(s,3H), 2.40 (ddd, 1H, J ) 4.0, 6.8, 10.8 Hz), 3.72 (s, 3H), 4.25
(m, 1H), 6.4 (bs, 1H); 13C NMR (CDCl3) δ 12.0, 20.0, 23.5, 29.7,
J O9613500