Chemoenzymatic synthesis of enantiomerically pure (2S,3R,4S)-4- hydroxyisoleucine, an insulinotropic amino acid isolated from fenugreek seeds

Article abstract of DOI:10.1002/ejoc.200300434

A short six-step synthesis of (2S,3R,4S)-4-hydroxyisoleucine (1a) with total control of stereochemistry is reported, the last step being the enzymatic resolution by hydrolysis of a N-phenylacetyl lactone derivative using the commercially available penicillin acylase G immobilized on Eupergit C (E-PAC). Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004.

Full text of DOI:10.1002/ejoc.200300434

FULL PAPER
Chemoenzymatic Synthesis of Enantiomerically Pure (2S,3R,4S)-4-Hydroxy-
isoleucine, an Insulinotropic Amino Acid Isolated from Fenugreek Seeds
[a]
[a]
[a]
Val e´ rie Rolland-Fulcrand,* Marc Rolland, Marie-Louise Roumestant, and
Jean Martinez[
a]
Keywords: Amino acids / Asymmetric synthesis / Bioconversions / Insulinotropic agent / Aminooxydases / Penicillin acylase
A short six-step synthesis of (2S,3R,4S)-4-hydroxyisoleucine
1a) with total control of stereochemistry is reported, the last
available penicillin acylase G immobilized on Eupergit C
(E-PAC).
(
step being the enzymatic resolution by hydrolysis of a N- ( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
phenylacetyl lactone derivative using the commercially
Germany, 2004)
Introduction
ous that the search for new improved antidiabetic drugs is
of real interest. A few stereoselective syntheses of 4-
hydroxyisoleucine have been reported in the literature, in-
cluding the enantioselective synthesis of the (3R,4R,5R)-
4-Hydroxyisoleucine was first isolated as a new amino
[
1]
acid lactone from γ-aminitin hydrolysate and later from
[
2]
ε-amanitin, one of the cyclic octapeptides found in the
[10]
and the (3R,4S,5S)-lactone diastereomers by Schöllkopf
[
4]
highly poisonous mushroom, Amanita phalloides. In 1973
using a reaction between a chiral glycine anion equivalent
and epoxybutane. However the stereochemical assignment
of the (3R,4R,5R)-lactone was erroneous as demonstrated
[
3]
Fowden et al. reported the isolation and identification of
free 4-hydroxyisoleucine from fenugreek seeds, Trigonella
foenum graecum L. (Leguminosae).
[11]
by Fredj et al. who achieved the enantiospecific synthesis
Their results suggested that 4-hydroxyisoleucine in fenu-
greek and in γ-amanitin have the same absolute configura-
tions at the three chiral carbon atoms. Gieren et al. had
correctly determined the configurations which were
of the 3R,4R,5R and 3S,4R,5R diastereomers starting from
benzyl 2,3-anhydro-4-O-(tert-butyldimethylsilyl)-β--ribo-
pyranoside.
[
4]
We described in 2001[12] the synthesis of (2R,3R,4R)-4-
hydroxyisoleucine lactone with good yields and total con-
trol of the stereochemistry. The key intermediate was the
didehydroamino acid derivative arising from an aldol dehy-
dration reaction between a glycine anion equivalent and bu-
tan-2,3-dione using an optically pure oxazinone as the chi-
ral auxiliary. X-ray structures of each intermediate have
been obtained and the stereochemistry of each asymmetric
[
5]
2S,3R,4S; this result was confirmed by Alcock who car-
ried out an X-ray crystal structure determination of 4-
hydroxyisoleucine. In fenugreek, (2R,3R,4S)-4-hydroxyiso-
leucine is also present as a minor component. Dardenne et
al.[ reported the presence of the same isomer in the fruit
6]
[7]
bodies of Lactarius camphoratus. Ribes et al. showed that
2S,3R,4S)-4-hydroxyisoleucine extracted from fenugreek
(
seeds, known for their antidiabetic properties in traditional
medicine, was an insulinostimulant compound which could
be used in diabetes mellitus treatment. More recently they
tested synthetic analogues on insulin secretion in compari-
son with the natural one: (2S,3R,4S)-4-hydroxyisoleucine
and its minor isomer (2R,3R,4S)-4-hydroxyisoleucine:
[
13]
center clearly identified.
Very recently, Wang et al.[14] described a practical eight-
step synthesis of (2S,3R,4S)-4-hydroxyisoleucine. The key
steps involved the biotransformation of ethyl 2-methyl-
acetoacetate to (2S,3S)-3-hydroxy-2-methylbutanoate with
Geotrichum candidum and an asymmetric Strecker synthesis.
(2S,4R)- and (2S,4S)-4-hydroxynorvalines as well as
In 2001, Sauvaire et al. published a patent concerning
the use of amino acids for making medicines for treating
(
2S,3S)- and (2S,3R)-3-hydroxynorvalines prepared in our
[
8a,8b]
laboratory.
They proved that insulinotropic properties
[
15]
insulin resistance.
In 1989, Englisch-Peters[16] described the synthesis of 4-
were shown by only the major isomer (2S,3R,4S)-4-
hydroxyisoleucine in the micromolar range. By 2010, it
[
9]
has been estimated that more than 200 million of the hydroxy analogues of leucine and isoleucine via 1,4-Michael
world’s population will have diabetes mellitus and it is obvi- addition and 4-hydroxy valine by a modified Erlenmeyer
strategy. The diastereomers of 4-hydroxy valine were sepa-
[
a]
Laboratoire dЈAminoacides, Peptides et Prot e´ ines, UMR Ϫ
CNRS 5810 Ϫ Universit e´ Montpellier I et II,
Place E Bataillon, 34095 Montpellier Cedex 5, France
E-mail: rolland@univ-montp2.fr
rated by column chromatography and the 4-hydroxy-Leu
and 4-hydroxy-Ile by reversed-phase HPLC. The enanti-
omers of allo- and iso-4-hydroxyvaline were enzymatically
Eur. J. Org. Chem. 2004, 873Ϫ877
DOI: 10.1002/ejoc.200300434
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
873

V. Rolland-Fulcrand, M. Rolland, M.-L. Roumestant, J. Martinez
FULL PAPER
separated using - or -amino acid oxidase which selec- and 4(a,b) were separated using silica-gel chromatography
tively oxidized the α-amino group of the - or - enanti- and separately identified (dr ϭ 60:40) as racemates. Apply-
omer to the corresponding α-keto acid.
ing the same strategy described in Scheme 1, the final prod-
We describe in this paper a new strategy in six steps com- uct prepared from the major racemate 3(a,b) was inactive in
1
bining a glycine enolate approach with the cis-2,3-dimeth- the biological antidiabetic tests. H NMR studies and op-
yloxirane ring opening and a final enantioselective enzy- tical rotation values confirmed the 2R,3S,4R configuration
matic hydrolysis using immobilized penicillin acylase (E- of other isomer.
PAC).
We describe below the strategy which led to the biologi-
cally active compound 1a.
Starting from the minor racemate 4(a,b), catalytic hydro-
genolysis and tert-butyl ester hydrolysis gave racemate
Results
6
(a,b), (3S,4R,5S)- and (3R,4S,5R)-3-amino-4,5-dimethyldi-
In our case racemic 4-hydroxyisoleucine lactone was ob-
tained in four steps as described in Scheme 1.
hydrofuran-2-one in 90% yield.
First commercially available α-aminooxidases were
[
16]
tested.
As shown in Scheme 2, -AAO (EC, 1.4.3.3) from hog
kidney, a very cheap and easy to use enzyme, could catalyze
the enantioselective aminooxidation of the (D) α-amino ac-
ids leading to the corresponding five-membered ring keto
acid (4R,5R)-4,5-dimethyldihydrofuran-2,3-dione (7b) and
the natural compound 1a (2S,3R,4S)-2-amino-4-hydroxy-3-
methylpentanoic acid. One 7b derivative is a very strong
[
17]
smelling furanone called sotolone
and in this step the
absence of an odour after stirring for three days indicated
that aminooxidation had been unsuccessful.
-AAO (EC, 1.4.3.2) is an expensive and toxic enzyme,
isolated from Crotalus adamanteus venom and it catalyzed
enantioselective aminooxidation of the -α-amino acids in
the corresponding five-membered ring keto acid (4S,5S)-
4,5-dimethyldihydrofuran-2,3-dione (7a), also very favour-
Scheme 1. Synthesis of (3S,4R,5S)-3-amino-4,5-dimethyldihydro- ous and (2R,3S,4R)-2-amino-4-hydroxy-3-methylpentanoic
furan-2-one (6a) and (3R,4S,5R)-3-amino-4,5-dimethyldihydro-
acid (1b), but in very low yield (6%). (2R)-4-hydroxyiso-
furan-2-one (6b): a) 2.2 mmol of dibenzylamine; b) 1) LiHMDS/
leucine (1b) was also a minor product from the fenugreek
THF (1 , 1.5 mmol), 30 min, Ϫ30 °C; 2) cis-2,3-dimethyloxirane
(
3 2 4
0.3 mmol/BF ·Et O/TMSCl); 3) NH Cl; 4) diastereomers separ- seeds extraction; after purification on ion-exchange resin
ation by silica gel column chromatography; c) Pd(OH)
2
/C/MeOH;
DOWEX 50WX8, 1b was tested for its antidiabetic activity
with no effect.
d) TFA (50% DCM)
The last chemoenzymatic strategy applied afforded
tert-Butyl bromoacetate was substituted by dibenzyla- (2S,3R,4S)-2-amino-4-hydroxy-3-methylpentanoic acid (1a)
mine in basic conditions to obtain tert-butyl di-N-benzyl- as (2S,3R,4S)-4-hydroxyisoleucine (Scheme 3).
glycinate (2) in 90% yield. Direct ring opening of the com-
The NH group of racemic lactone 6a؊b was protected
2
mercially available cis-butylene oxide with the lithium enol- by a phenylacetic group, the best hydrolytic group for the
ate of tert-butyl dibenzylglycinate afforded a diastereomeric commercially available metalloenzyme, penicillin acylase G
mixture in 60% yield. The couples of diastereomers 3(a,b) which is immobilized on Eupergit C. E-PAC enantioselec-
Scheme 2. Enzymatic enantioselective aminooxidation of 6a and 6b by -AAO and -AAO: e) phosphate buffer pH 8.3, O
% H , catalase and -AAO (or -AAO), 1 day, RT; f) purification of mother solution liquor by DOWEX 50WX8 ion-exchange resin
form)
2
bubbling,
1
2 2
O
ϩ
(
H
874
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2004, 873Ϫ877

An Insulinotropic Amino Acid Isolated from Fenugreek Seeds
FULL PAPER
3
Scheme 3. Synthesis of (2S,3R,4S)-2-amino-4-hydroxy-3-methyl-pentanoic acid 1a; g) phenylacetic chloride/Et N; h) phosphate buffer
pH 9, penicillin acylase G immobilized on Eupergit C, 1 day, room temp.; i) purification of mother solution liquor by DOWEX 50WX8
ϩ
ion-exchange resin (H form); j) 1  NaOH in dioxan
tively hydrolyzed the phenylacetic amide bond of an -am-
Conclusion
ino acid in the pH range 5Ϫ7 when its carboxyl group com-
2
ϩ
plexed the metal (Co ) present inside the active site of this
An efficient synthesis of optically pure (2S,3R,4S)-4-
enzyme. At pH 5Ϫ7 lactone 8a؊b was formed spon- hydroxyisoleucine has been performed in only six steps. The
taneously. This resolution was performed in unusual con- optically pure (2S,3R,4S)-4-hydroxyisoleucine has been
ditions (pH 8.5Ϫ9).
tested for its antidiabetic activity by Prof. Sauvaire et al.
The extent of bioconversion was stopped at 40%, before and presented exactly the same activity as the compound
the 2R lactone 8b became substrate by default in these basic extracted from fenugreek seeds.
conditions. The reaction was followed by HPLC on a C-18
column and by Chiral HPLC on a Chiralcel OJ column (2S,3S,4R)- and (2R,3R,4S)-4-hydroxyisoleucines, have
ee ϭ 99Ϯ1%). Both amino acids 1a and 8b were separated been also characterized and tested for their biological ac-
on DOWEX 50WX8 and the pure product 1a was obtained tivity with no positive responses.
The other isomers obtained from the compound, 3a؊b
(
in 38% yield. After recrystallization from 95% ethanol, op-
Using E-PAC to immobilize the enzyme presents the pos-
tically pure (2S,3R,4S)-4-hydroxyisoleucine 1a was charac- sibility for it to be reused by simple filtration or in continu-
1
terized by its H NMR spectrum, m.p., optical activity, and ous flow in a batch reactor. This process will be optimized
chiral HPLC, which were in complete agreement with the to decrease the cost of this efficient synthesis.
data reported in the literature.[
3,5]
As shown in Scheme 3, the ring opening of 8a؊b in 1 
Experimental Section
NaOH afforded in quantitative yield N-phenylacetyl amino
acids 9aϪb as racemate sodium salts easily separated on
chiral HPLC [Table 1 column (b)]. The identical absorbance
and the different retention time of 9a and 9b were deter-
mined by chiral-HPLC analysis without any purification or
General Remarks: Melting points were obtained using a Büchi 510
capillary apparatus and are uncorrected. Infrared spectra were re-
corded using a PerkinϪElmer Fourier transform spectrometer. H
NMR spectra were recorded at 250 MHz or at 400 MHz using a
1
1
13
other analysis. This experiment was performed to identify Bruker AC400 instrument. For H NMR or C spectra recorded
each isomer.
3 2
in CDCl , DCl, D O, or MeOD; chemical shifts are quoted in parts
per million, referenced to the residual solvent peak. The following
abbreviations are used: s, singlet; d, doublet; t, triplet; q, quad-
ruplet; m, multiplet; br, broad. Coupling constants are reported in
Table 1. HPLC conditions: (a) isocratic conditions on C-18 column
inverse phase, λ ϭ 214 nm, 60% H O TFA 1‰ and 40% CH CN
2 3
1
Hertz (Hz). Diastereoisomeric ratios (dr) were determined by H
TFA 1‰; flow rate: 1 mL/min; (b) isocratic conditions on Chiralcel
OJ column (DAICEL) normal phase, λ ϭ 214 nm, 85% hexane
NMR or HPLC analysis on the crude product. Low-resolution
mass spectra were recorded on a micromass electrospray instru-
ment with only molecular ion and other major peaks being re-
ported. Optical rotations were determined on a Perkin-Elmer 241
Retention polarimeter at room temperature. Flash chromatography was car-
15% 2-propanol flow rate: 0.8 mL/min
Products
α
D
Retention
time (a)
time (b)
ried out using E-Merck Silica Gel (Kieselgel 60, 230Ϫ400 mesh) as
stationary phase. Thin-layer chromatography was carried out on
aluminum plates pre-coated with Merck Silica-gel 60F254 and were
visualized by quenching of UV fluorescence, by iodine vapor or
by ninhydrin spray. THF was distilled from sodium/benzophenone
ketyl. Reagents were supplied from commercial sources (Aldrich,
Fluka). The immobilized acylase E-PAC (EC, 3.5.1.11), -AAO
(EC, 1.4.3.3) from hog kidney and -AAO (EC, 1.4.3.2) from Crot-
alus adamanteus venom were from Fluka.
1
a (2S,3R,4S)
b (2R, 3S,4R)
ϩ31 (c ϭ 1, D
Ϫ30 (c ϭ 1, D
2
O)
O)
8 min
8 min
6.5 min
/
10 min
12 min
12 min
7.3 min
5.5 min
/
22 min
14 min
15 min
17 min
1
2
phenylacetic acid
6
8
9
9
a
b
a
b
/
/
/
/
Eur. J. Org. Chem. 2004, 873Ϫ877
www.eurjoc.org
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
875

V. Rolland-Fulcrand, M. Rolland, M.-L. Roumestant, J. Martinez
FULL PAPER
tert-Butyl N,N-Dibenzyl Glycinate (2): Dibenzylamine (2.12 mL,
CDCl
3 H, CH
J ϭ 8.2 Hz, 1 H, CH), 3.01Ϫ3.26 (br, 2 H, OH), 3.55Ϫ3.64 (m, 1
3
): δ ϭ 0.74 (d, J ϭ 6.8 Hz, 3 H, CH
3
), 1.08 (d, J ϭ 6.0 Hz,
11 mmol) was added dropwise over 5 min to a stirred solution of
3
), 1.38 (s, 9 H, tBu), 1.47Ϫ1.55 (m, 1 H, CH), 3.17 (d,
tert-butyl bromoacetate in ethanol (0.74 mL, 5 mmol) and the mix-
ture was then stirred at room temperature for 4 h. The resulting H, CH).
mixture was filtered, the salt washed with a mixture of diethyl
4-Hydroxyisoleucine Lactone: (3S,4R,5S)-3-Amino-4,5-dimethyldi-
ether/hexane (1:1). The expected compound 2 (1.4 g, 90% yield)
hydrofuran-2-one (6a) and (3R,4S,5R)-3-Amino-4,5-dimethyldi-
hydrofuran-2-one (6b): The racemic compound 5a-b (406 mg,
2 mmol) was dissolved in TFA/DCM (50:50 v/v) (10 mL) and mag-
netically stirred overnight at room temperature. The solvents were
evaporated under reduced pressure and the residue was dissolved
in ethyl acetate (15 mL); the organic layer was washed with 10%
was obtained as a colorless solid after purification by chromato-
graphy on silica gel (eluent Et
2
O/hexane, 10:90). R
f
ϭ 0.50 (Et
2
O/
ϩ
ϩ
hexane, 10:90), m.p. 68 °C. ES MS: m/z ϭ 312 [M ϩ H ], 334
ϩ
1
[
M ϩ Na ]. H NMR (250 MHz, CDCl
3
): δ ϭ 1.53 (s, 9 H, tBu),
3
.24 (s, 2 H, CH ), 3.87 (s, 4 H, CH ), 7.26Ϫ7.49 (10 H, m, Ph).
2
2
tert-Butyl (2S,3S,4R)-2-Dibenzylamino-4-hydroxy-3-methylpentano-
2
sodium hydrogencarbonate (10 mL) then with H O until neutral
ate (3a), (2R,3R,4S)-... (3b), (2S,3R,4S)-... (4a), (2R,3S,4R)-... (4b): pH (2 ϫ 10 mL). The expected compound 6a-b (220 mg, 80Ϫ90%
A solution of 1  LiHMDS in anhydrous THF (9.6 mL, yield) was obtained as a colorless solid after purification by chro-
9
6
.60 mmol) was added dropwise to a stirred solution of 2 (2 g,
.42 mmol) in dry THF (12 mL) at 0 °C. After 1 h stirring at Ϫ30
matography on silica gel (eluent: diethyl ether/hexane, 20:80 v/v).
ϭ 0.58 (diethyl ether/hexane, 1:2 v/v); m.p. 240Ϫ245 °C. MS
R
f
ϩ
ϩ
1
°
(
C
cis-2,3-dimethyloxirane (0.33 mL, 3.8 mmol), BF
0.8 mL), and TMSCl (0.87 mL) were added. After 15 h stirring at
Ϫ30 °C the reaction mixture was neutralized with saturated aque-
ous NH Cl. (20 mL). The aqueous layer was extracted with ethyl
acetate (3 ϫ 50 mL). The combined organic layers were dried with
MgSO and the solvents were evaporated under reduced pressure.
3
·OEt
2
(FAB ): m/z ϭ 130 [M ϩ H ]. H NMR (400 MHz, DCl): δ ϭ
1.22 (d, J ϭ 7.3 Hz, 3 H, CH ), 1.51 (d, J ϭ 6.5 Hz, 3 H, CH ),
2.81 (dqd, J ϭ 8.3, 7.3, 2.0 Hz, 1 H, CH), 4.66 (qd, J ϭ 6.5, 2.0 Hz,
3
3
1
3
4
1 H, CH), 4.67 (d, J ϭ 8.3 Hz, 1 H, CH) ppm. C NMR
(300 MHz, DCl): δ ϭ 12.5, 19.0, 37.6, 51.8, 84.2, 173.8 ppm. IR
(KBr): ν˜ ϭ 3500Ϫ2500 (3340, 2900), 1769, 1494, 1384, 1259, 1207,
1133, 1023, 1003, 944.
4
The oxirane opening was afforded in 60% yield (1.4 g). Both racem-
ates (3a-b and 4a-b) were separately identified after chromatogra-
Resolution by Aminooxidases (d-AAO and l-AAO): Standard Pro-
cedure: Phosphate buffer (0.2 , 25 mL) pH 8.3 was saturated with
oxygen by bubbling the gas through the solution for 20 min. After-
phy on silica gel, eluting with hexane/diethyl ether/CH
2
Cl
2
(50 mL
9
7
6
0:5:5, then 500 mL 85:7.5:7.5, then 500 mL 80:10:10, and 500 mL
5:12.5:12.5). HPLC analysis proved a diastereomeric ratio of
0:40.
2 2
wards, 1% H O (2 mL), Catalase (10 µL) (260000 U/mL; 30% gly-
cerol; 10% ethanol), 4-hydroxyisoleucine lactone 6a-b (140 mg,
3a؊b (Major isomers): (2S,3S,4R)-tert-Butyl 2-Dibenzylamino-4- 1.09 mmol) and aminooxidase (2.5 mg) were simultaneously added
hydroxy-3-methylpentanoate (3a); (2R,3R,4S)-... (3b): 885 mg, yield to the buffer. This mixture was incubated for one day at room tem-
1
6
0%; m.p. 99Ϫ100 °C; R
NMR (250 MHz, CDCl ): δ ϭ 1.03 (d, CH
.05 (d, J ϭ 6.3 Hz, 3 H, CH ), 1.6 (s, 9 H, tBu), 1.80Ϫ1.90 (br, 1
H, OH), 3.13 (d, J ϭ 9.6 Hz, 1 H, CH), 2.13Ϫ2.32 (m, 1 H, CH), The amino acid was eluted by 2  NH
f
ϭ 0.22 (hexane/DCM/Et
, J ϭ 6.9 Hz, 3 H), 2% acetic acid and applied to the ion-exchange resin DOWEX 50
WX8 previously washed with water and methanol (2 ϫ 20 mL).
OH solution (2 ϫ 10 mL),
then the solvent was evaporated under reduced pressure and the
): amino acid was crystallized from water/ethanol.
2
O, 3:1:1). H
perature. After evaporation the residue was dissolved in 10 mL of
3
3
1
3
4
3
7
.56Ϫ3.68 (m, 1 H, CH), 3.80 (dd, J
1
ϭ J
2
ϭ 13.9 Hz, 4 H, 2 CH
2
),
.28Ϫ7.46 (m, 10 H, 2 Ph) ppm. 13C NMR (300 MHz, CDCl
3
δ ϭ 8.5, 20.2, 29.0, 36.7, 56.8, 62.0, 68.0, 73.5, 127.0, 128.2, 128.8,
ϩ
ϩ
(2R,3S,4R)-2-Amino-4-hydroxy-3-methylpentanoic
Acid
(1b):
1
36.2, 173.7. MS ESI Ͼ 0: 384 [M ϩ H ], 767 [2M ϩ H ].
(383): calcd. C 75.19, H 8.61, N 3.65. found C 75.14,
H 8.56, N 3.63.
9
.7 mg, yield 6%, m.p. 223Ϫ226 °C. MS (FABϩ): m/z ϭ 148 [M ϩ
24 3
C H33NO
ϩ
1
H ], [α
.0 (d, J ϭ 7.1 Hz, 3 H, CH
a؊b (Minor isomers): tert-Butyl (2S,3R,4S)-2-Dibenzylamino-4- 2.00 (qdd, CH, J ϭ 7, 4, 8 Hz, 1 H), 3.85 (dq, J ϭ 6.3, 8.1 Hz, 1
D
] ϭ Ϫ30 (c ϭ 1, H
2
O). H NMR (400 MHz, D
2
O): δ ϭ
1
3 3
), 1.30 (d, J ϭ 6.4 Hz, 3 H, CH ),
4
1
3
hydroxy-3-methylpentanoate (4a); (2R,3S,4R)-... (4b): 515 mg, yield H, CH), 3.93 (d, J ϭ 4 Hz, 1 H, CH) ppm. C NMR (300 MHz,
1
4
0%; m.p. 96Ϫ97 °C; R
f
ϭ 0.36 (hexane/DCM/Et
): δ ϭ 0.72 (d, J ϭ 6.6 Hz, 3 H, CH
.11 (d, J ϭ 6.6 Hz, 3 H, CH ), 1.63 (s, 9 H, tBu), 2.04Ϫ2.17 (m,
H, CH), 3.22 (d, J ϭ 11 Hz, 1 H), 3.31Ϫ3.44 (m, 1 H), 3.73 (dd,
ϭ J ϭ 13.8 Hz, 4 H, 2 CH ), 6.45 (s, 1 H, OH), 7.29Ϫ7.4 (m,
0 H, 2 Ph) ppm. C NMR (300 MHz, CDCl ): δ ϭ 9.0, 20.4,
9.0, 36.5, 56.8, 62.2, 68.3, 73.5, 127.0, 128.2, 128.8, 136.2, 173.5.
2
O, 3:1:1).
H
D
2
6 3
O): δ ϭ 12.6, 21.1, 41.6, 57.2, 70.1, 174.0. C H13NO (147.17):
NMR (400 MHz, CDCl
3
3
), calcd. C 49.9, H 8.9, N9.5; found C 49.5, H 9.0, N 9.4.
1
1
J
1
2
3
Phenylacetic Protection: N-[(3S,4R,5S)-4,5-Dimethyltetrahydro-2-
oxofuran-3-yl]-2-phenylacetamide (8a) and N-[(3R,4S,5R)-4,5-Di-
methyltetrahydro-2-oxofuran-3-yl]-2-phenylacetamide (8b): Racemic
lactone 6a-b (323 mg, 2.5 mmol) was dissolved in anhydrous di-
chloromethane (15 mL) and the solution was cooled to 0 °C in an
ice bath. Then triethylamine (0.452 mL, 3.25 mmol) was added and
the mixture was magnetically stirred at 0 °C for 15 min. Phenylace-
tyl chloride (0.43 mL, 3.25 mmol) diluted in anhydrous DCM
1
2
2
13
3
ϩ
ϩ
3
MS ESI Ͼ 0: 384 [M ϩ H ], 767 [2M ϩ H ]. C24H33 NO (383):
calcd. C 75.19, H 8.61, N 3.65. found C 75.46, H 8.87, N 3.58.
tert-Butyl (2S,3R,4S)-2-Amino-4-hydroxy-3-methylpentanoate (5a)
and tert-Butyl (2R,3S,4R)-2-Amino-4-hydroxy-3-methylpentanoate (3 mL) was added dropwise. The mixture was stirred for 1 h at 0
(
5b): A solution of racemate 4a-b (524 mg, 1.17 mmol) in methanol
°C and 16 h at room temperature. The mixture was washed with
saturated solution
(20 mL) was hydrogenated with 10% palladium hydroxide on char- 15% citric acid solution (10 mL) and NaHCO
3
coal (150 mg) and the reaction followed by TLC. When the reaction
was finished (4Ϫ6 h) (disappearance of TLC spot), the catalyst was
(10 mL). The organic layer was dried with magnesium sulfate, con-
centrated under reduced pressure, and the residue was purified by
removed by filtration through celite and the filtrate was evaporated silica-gel column chromatography (eluent: Et
2
O/hexane, 1:1 v/v;
then 2:1, 3:1, and 4:1 gradients). The compound 8a-b was obtained
as white crystals in 78% yield (482 mg). R ϭ 0.37 (Et O). m.p.
to dryness to afford the corresponding α-amino ester 5a-b as a
racemic mixture in 95% yield (226 mg). MS ESI Ͼ 0: 204 [M ϩ
f
2
ϩ
ϩ
ϩ
1
ϩ
ϩ
H ], 407 [2M ϩ H ], 429 [2M ϩ Na] . H NMR (250 MHz, 131Ϫ132 °C. MS ESI Ͼ 0: 248 [M ϩ H ], 270 [M ϩ Na] , 517
876  2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.eurjoc.org
Eur. J. Org. Chem. 2004, 873Ϫ877

An Insulinotropic Amino Acid Isolated from Fenugreek Seeds
FULL PAPER
[
(2M ϩ Na) ]. 1H NMR (250 MHz, CDCl
ϩ
3
): δ ϭ 0.81 (d, J ϭ 70.2, 174.1 ppm. IR (KBr): ν˜ ϭ 3500Ϫ2500, 1634, 1490, 1415,
), 1.40 (d, J ϭ 6.7 Hz, 3 H, CH ), 2.53Ϫ2.65 (m, 1259, 1356, 1312, 1270, 1180, 1104, 1053, 1017, 962, 931, 899, 858,
H, CH), 3.62 (2 H, s, CH ), 4.3Ϫ4.4 (m, 1 H, CH), 4.7 (dd, J ϭ 817, 794. C (147.17): calcd. C 49.9, H 8.9, N 9.5; found
7
1
6
.2 Hz, 3 H, CH
3
3
2
6 3
H13 NO
.0, J ϭ 8 Hz, 1 H, CH), 5.8 (br, 1 H, OH), 7.25Ϫ7.4 (m, 5 H, Ph). C 49.3, H 9.1, N 9.4.
9a-b: Sodium (2S,3R,4S)- and (2R,3S,4R)-2-[(N-Phenylacetyl)-
amino]-4-hydroxy-3-methylpentanoate: Racemate 8a-b (350 mg,
.42 mmol) was stirred in a dioxan/H O mixture (9:1 v/v, 15 mL).
Acknowledgments
We thank Prof. Yves Sauvaire and Prof. G e´ rard Ribes for perform-
1
2
After solubilization 1  NaOH was added (3.25 mmol). The ring
opening was followed by TLC. The solvent was concentrated under
reduced pressure and the racemate 9a-b was obtained as an oil in
quantitative yield (375 mg). Both isomers were eluted on HPLC
on a Chiralcell OJ column (eluent: 85% hexane, 15% 2-propanol,
flow rate: 0.8 mL/min). The elution results are given in Table 1. MS
ing biological tests.
[1]
Th. Wieland, H. Wehrt, Justus Liebigs Ann. Chem. 1966, 700,
120.
[
[
[
2]
3]
4]
Th. Wieland, A. Bukin, Justus Liebigs Ann. Chem. 1968, 717,
215.
L. Fowden, H. M. Pratt, A. Smith, Phytochemistry 1973, 12,
ϩ
ϩ
1
ESI Ͼ 0: 265 [M ϩ H ], 287 [M ϩ Na ]. H NMR (400 MHz,
O): δ ϭ 1.05 (d, J ϭ 7.0 Hz, 3 H, CH ), 1.35 (d, J ϭ 6.3 Hz, 3
H, CH ), 2.10 (qdd, CH, J ϭ 7, 4, 8 Hz, 1 H), 3.58 (s, 2 H, CH ),
D
2
3
1
707.
3
2
A. Gieren, Th. Wieland, Justus Liebigs Ann. Chem. 1974,
3
7
.86 (dq, J ϭ 6.3, 8.0 Hz, 1 H, CH), 3.94 (d, J ϭ 4.0 Hz, 1 H, CH),
.20Ϫ7.4 (m, 5 H, Ph).
1561Ϫ1569.
[5]
N. W. Alcock, D. H. G. Crout, M. V. M. Gregorio, E. Lee, G.
Pike, C. J. Samuel, Phytochemistry 1989, 28, 1835.
G. Dardenne, J. Casimir, S. I. Hatanaka, T. Aoki, Bull. Rech.
Agron. Gembloux 1986, 21, 125.
(2S,3R,4S)-4-Hydroxyisoleucine (1a): Resolution by E-PAC (Euper-
[
[
6]
7]
git Penicillin Acylase): Racemate 8a-b (1.4 mmol, 400 mg) was dis-
solved in 0.2  NaOH solution (75 mL) at pH 9. Then the immobil-
ized amidase was added and left in suspension. The mixture was
moderately stirred at room temperature. The pH decreased during
the bioconversion and had to be maintained at pH 9 by addition
of concentrated NaOH solution. The reaction was followed by
HPLC and stopped at 40% bioconversion (after around 2 h). The
immobilized enzyme was filtered and washed with water (2 ϫ
Y. Sauvaire, P. Petit, C. Broca, M. Manteghetti, Y. Baissac, J.
Fernandez-Alvarez, R. Gross, M. Roge, A. Leconte, R. Gomes,
G. Ribes, Diabetes 1998, 47, 206.
[8] [8a]
M. Jacob, M. L. Roumestant, P. Viallefont, J. Martinez,
Synlett 1997, 691Ϫ692. [
8b]
C. Broca, M. Manteghetti, R.
Gross, Y. Baissac, M. Jacob, P. Petit, Y. Sauvaire, G. Ribes,
Eur. J. Pharmacol. 2000, 390, 339Ϫ345.
9]
[
A. F. Amos, D. J. McCarthy, P. Zimmet, Diabetic Medicine
10 mL). The filtrate was acidified to pH 3 and the phenylacetic acid
1
997, 14, 1Ϫ85.
was eliminated by extraction with dichloromethane (3 ϫ 30 mL).
The aqueous layer was concentrated under reduced pressure to
[
[
10]
11]
R. Gull, U. Schöllkopf, Synthesis 1985, 1052.
T. Inghardt, T. Fredj, G. Svensson, Tetrahedron 1991, 47, 6469.
T. Kassem, J. Wehbe, V. Rolland, M. Rolland, M. L. Roumest-
ant, J. Martinez, Tetrahedron: Asym. 2001, 12, 2657Ϫ2661.
20 mL and the solution was eluted on DOWEX 50WX8 previously
[12]
washed by three cycles of water/MeOH over 30 min. The adsorp-
tion was followed by observing the disappearance of the free amino
[13] [13a]
T. Kassem, V. Rolland, J. Martinez, M. Rolland, Acta
Crystallogr., Sect. C 2000, 56, 1037. [
sem, V. Rolland, J. Martinez, Acta Crystallogr., Sect. C 2001,
57, 1415Ϫ1417:
13b]
M. Rolland, T. Kas-
acid from the solution using TLC with EtOH/NH
eluent and ninhydrin for visualization. (2S,3R,4S)-4-Hydroxyiso-
leucine (1a) was eluted from the DOWEX resin by 2  NH OH
solution. Then after evaporation and recrystallization from 95%
ethanol, 1a was obtained in 38% yield (78 mg) (ee ϭ 99Ϯ1%). R
4
OH (4:1, v/v) as
4
[
[
14]
15]
Q. Wang, J. Ouazzani, A. Sasaki, P. Potier, Eur. J. Org. Chem.
2
002, 834Ϫ839.
f
ϭ
[5]
G Ribes, M. Taouis, P. Petit, C. Broca, Y Sauvaire, B. Pau;
patent PCT/FR00/02361 2001.
0
.68 (NH
4
OH/EtOH, 1:4, v/v). m.p. 220Ϫ225 °C (224Ϫ225 °C ).
ϩ
MS (FABϩ): m/z ϭ 148 [M ϩ H ], [α
D
] ϭ ϩ31 (c ϭ 1,H
). H NMR (400 MHz, D O): δ ϭ 1.0
d, J ϭ 7.0 Hz, 3 H), 1.3 (d, J ϭ 6.3 Hz, 3 H), 2.0 (qdd, J ϭ 7, 4,
Hz, 1 H), 3.88 (dq, J ϭ 6.3, 8.0 Hz, 1 H), 3.94 (d, J ϭ 4 Hz, 1
2
O).
[16]
S. Englisch-Peters, Tetrahedron 1989, 45, 6127Ϫ6134.
[17] [17a]
[14]
1
[
(
8
α
D
] ϭϩ31.5 (c ϭ 1, H
2
O
2
X. Fernandez, S. Kerverdos, E. Dunach, Act. Chim. 2002,
[17b]
4
Ϫ13.
H. D. Belitz, W. Grosch, Food Chemistry, 2nd ed.,
Springer-Verlag, Berlin 1999.
13
H) ppm. C NMR (300 MHz, D
2
O): δ ϭ 12.5, 21.0, 41.7, 57.3,
Received July 14, 2003
Eur. J. Org. Chem. 2004, 873Ϫ877
www.eurjoc.org
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
877