Chemo-enzymatic approach to D-allo-isoleucine

Article abstract of DOI:10.1016/j.tetasy.2003.08.009

A mixture of D-alloisoleucine and L-isoleucine derivatives are selectively hydrolysed by enzymatic catalysis (alcalase) allowing the recovery of the D-allo stereoisomer in excellent d.e. and yields. Thus, N-formyl-D-allo- isoleucine benzylester is obtained after enzymatic hydrolysis of the diastereoisomeric mixture or by a crystallisation procedure.

Full text of DOI:10.1016/j.tetasy.2003.08.009

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 14 (2003) 3189–3196
Chemo-enzymatic approach to
D-allo-isoleucine
Mara Cambie`,a,* Paola D’Arrigo,a Ezio Fasoli,a Stefano Servi,a Davide Tessaro,a
Francesco Canevottib and Lucio Del Coronab
aDipartimento di Chimica, Materiali e Ingegneria Chimica G. Natta, Politecnico di Milano, Via Mancinelli 7, 20131,
Milano, Italy
bFLAMMA SpA, Chignolo d’Isola, (BG), Italy
Received 17 June 2003; accepted 6 August 2003
Abstract—A mixture of
D
-alloisoleucine and
L
-isoleucine derivatives are selectively hydrolysed by enzymatic catalysis (alcalase)
-allo-isoleucine benzylester is
allowing the recovery of the
D
-allo stereoisomer in excellent d.e. and yields. Thus, N-formyl-
D
obtained after enzymatic hydrolysis of the diastereoisomeric mixture or by a crystallisation procedure.
© 2003 Elsevier Ltd. All rights reserved.
1. Introduction
Indeed L-specific hydrolytic enzymes hydrolyse L-
isoleucine derivatives, leaving behind the
stereoisomer. Epimerization of the recovered
allows the repetition of the separation process.
D
-allo
-ileu
D
-Allo-isoleucine (
D
-allo) is a non-proteinogenic amino
L
acid, found in nature which is probably formed by time
dependent epimerisation of -isoleucine (
L
L
-ileu).1 It has
recently been found as a component unit in a number
of biologically active depsipeptides2–13 and it has been
employed in the synthesis of oxytocin analogues14 and
as a chiral synthon for the preparation of isostatins and
of natural cytotoxic depsipeptides such tamandarins.15
In view of the growing interest in the synthesis of
peptide mimetics involving non-natural amino acids,
We report here a practical preparation of
D-allo as
such, and in synthetically useful protected forms, start-
ing from 1 with an approach that is both chemical and
enzymatic.
2. Results and discussion
several synthetic schemes leading to
D-allo have been
proposed. total synthesis from (S)-2-butanol,
A
Biocatalysis has reached a stage of mature methodology
for application on the industrial scale.28,29 It is widely
recognised that the bottleneck in applications depends
on the enzymes availability.28,29 Solution for the obten-
tion of affordable biocatalysts is the in house develop-
ment of suitable enzyme sources30 or simply the
application of commercially available enzyme prepara-
tions of low cost. Alcalase is a commercial preparation
of a partly purified protease from B. licheniformis pro-
duced by NOVOzyme for detergent applications, it is
commercially available at low cost, and it has an
extremely broad substrate specificity.24–27 As most enzy-
matic systems acting on amino acid derivatives31–34 it
requires among other steps, a TEMPO catalysed oxida-
tion and condensation with (R)-(+)-menthyl p-toluene-
sulphinate (Andersen’s reagent).16 Since a roughly 1:1
mixture of
by epimerisation of the natural amino acid, separation
of -allo from that mixture is a logical approach to
D-allo and L-ileu (mix-ile 1) is easily formed
D
compound 4. Thus, several reports along this line have
been published, exploiting specific properties of com-
pounds obtained reacting the mixture of diastereoiso-
mers
with
enantiomerically
pure
compounds
(a-phenylethylamine,17
L
-phenylalanine,18 tartaric acid
derivatives19) and separating them by fractional crys-
tallisation or chromatography. Moreover the enzymatic
hydrolysis of appropriate derivatives of the
diastereoisomeric mixture has been applied using
papaine,20 hog kidney acylase21,22 or hydantoinase.23
shows L-selectivity. Most of those enzymes have been
applied in the kinetic resolution of amino acid deriva-
tives often coupled with racemisation techniques to
improve the efficiency.35 We prepared a series of deriva-
tives of the epimeric forms of isoleucine and submitted
them to alcalase hydrolysis with the aim of obtaining as
* Corresponding author. E-mail: stefano.servi@polimi.it
0957-4166/$ - see front matter © 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2003.08.009

3190
M. Cambie` et al. / Tetrahedron: Asymmetry 14 (2003) 3189–3196
a
residue from the enzymatic hydrolysis
D
-
The specificity for L-ile was quite high allowing com-
pounds of type 3 to be obtained as residues in >98%
d.e. Table 1 summarises the results observed with vari-
alloisoleucine or a synthetically useful protected form
thereof (Scheme 1).
ous L-isoleucine derivatives. It appears from these data
that compounds 1e–m allow the D-allo derivatives to be
obtained with excellent d.e.
Starting with
L
-ile, an approximately equimolar mixture
(mix-ile) of the two epimers was generated as described
in the experimental. The corresponding esters 1a–d
were prepared as substrates for the enzyme. Preliminary
assays at various pH and temperatures ranging from 20
to 40°C in the absence of the enzyme, showed those
substrates which were prone to spontaneous hydrolysis
above pH 7.5 and 25°C. Accordingly, alcalase catalysed
hydrolysis of a 0.2 M solution of substrates 1a–d
allowed the recovery of the corresponding derivatives 3
with unsatisfactory d.e.s. Thus under the conditions
described in the experimental part, compound 1c was
hydrolysed at a rate of 4 mmol/h living at approxi-
mately 50% conversion 3c of 70% d.e., as determined
by HPLC. Compound 1d, which was transformed at a
rate approximately 25 times slower, gave the corre-
sponding compound 3d with about the same d.e. as
before. This result confirms that the lack of selectivity is
due to concomitant chemical hydrolysis.
Table 1. Results of the alcalase catalysed hydrolysis of
-isoleucine derivatives
L
Substrate
Product
Yield (%)
D.e. (%)
Time (h)
1c
1d
1e
1f
1g
1h
1j
3c
3d
3e
3f
3g
3h
3j
50
40
30
30
40
41
50
20
70
60
98
98
96
98
99
98
2
40
20
21
2.5
22
3.3
28
1m
3m
In Figure 1, the enzymatic hydrolyses of the N-formyl
derivatives 1f–h are compared. The graph suggests that
recovery of
D-allo of high d.e. is possible for high
We turned our attention to the N-acyl derivative of
mixtures of 1 and substrates 1e–m were prepared
through conventional methods. Although the substrates
are poorly soluble in water, the enzymatic hydrolysis
requires no added cosolvent and the reactions are run
in a suspension with a formal concentration of 0.2 M.
Higher concentrations were occasionally equally
efficient. Preliminary experiments in the absence of the
enzyme showed that no detectable chemical hydrolysis
occurred on those substrates at pHs up to 9 and
temperatures up to 40°C. Among esters 1e–h,
chloroethyl esters were hydrolysed about 25 times faster
than benzyl esters, methyl and ethyl lying in between.
conversion in the f and g series, while the hydrolysis
profile of the benzyl ester indicates a sharp selectivity.
Indeed at 50% conversion (as determined from NaOH
uptake at pH 7.8), compound 3h was obtained in
almost quantitative yield and a diastereomeric excess
higher than 98%. Comparison of benzyl esters of differ-
ently N-protected compounds are reported in Figure 2.
Mixture 1j proved to be the best substrate for the
hydrolysis. Reaction was fast and extremely selective
allowing the obtainment of 3j with a d.e. higher than
98%. Moreover the remaining 2i is easily recycled in the
preparation of the epimeric mixture 1j. However, the
results with 1h should also be considered for the easier
Scheme 1.

M. Cambie` et al. / Tetrahedron: Asymmetry 14 (2003) 3189–3196
3191
Figure 1. Enzymatic hydrolysis (alcalase) of N-formyl-
1g, ꢁ O-benzyl 1h).
D-allo- and N-formyl-L-isoleucine esters (" O-methyl 1f, ꢀ O-chloroethyl
Figure 2. Enzymatic hydrolysis (alcalase) of
N-Cbz 1m).
D-allo- and L-isoleucine benzylester derivatives (ꢀ N-acetyl 1j, ꢁ N-formyl 1h, ꢂ
solid difficult to crystallise. An epimeric mixture of 1d
dissolved in ethyl ether at 0°C, was saturated with
gaseous HCl. 3d·HCl separated in white needles, while
the epimer remained in solution. The solid was thus
recovered in two successive experiments, accounting for
>40% of the expected compound. The d.e. of the solid
compound was >98% from HPLC. Thus, this simple
crystallisation method complemented well the above
separations based on selective enzymatic hydrolysis.
removal of the formyl group compared with the N-ace-
tyl derivative. The hydrolysis of mixture 1m is exceed-
ingly slow. Thus the procedure from 1j/1h to D-allo has
been applied on a multigram scale. The effect of sub-
strate and enzyme concentration was evaluated. Best
selectivity was obtained with low enzyme/substrate
ratio (see Section 4). L-ile obtained from the hydrolysis
could be easily epimerised under several conditions
both at the acid and at the ester level. Thus N-formyl-
O-benzylester was epimerised to give the thermody-
namic mixture in the presence of catalytic KtBuO in
MTBE as solvent. Thus in successive steps
principle be converted to the diastereoisomer
quantitative yield.
L
-ile can in
-allo in
3. Conclusions
D
N-Acyl-esters of mixtures of the epimeric isoleucines
are easily enzymatically hydrolysed in water to the
N-acyl-L-forms allowing the recovery of the D-allo
compounds in high diastereoisomeric excess. Although
similar methods have been applied before,20–23 the
present approach seems convenient for large scale
Complementary to this method, we found that the two
diastereomers often exhibit quite different physical
properties. Compound 3d·HCl for instance, is a nice
crystalline material, while the L-liso epimer is a glassy

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M. Cambie` et al. / Tetrahedron: Asymmetry 14 (2003) 3189–3196
4.2.
D-Allo- and L-isoleucine methyl ester hydrochloride
application due to the nature of the biocatalyst
(alcalase, an industrial grade proteolytic enzyme), the
possibility of running the reaction in water at concen-
1a·HCl
To a stirred suspension of the mixture of
and -alloisoleucine (15 g,114 mmol) in methanol
L-isoleucine
trations of 30–50 g/l, and for the useful
tives obtained.17,41,42 In fact both N- or C-protected
forms of -allo can be easily prepared in one step
D-allo deriva-
D
(100 ml) 5 ml of thionyl chloride were dropped at
0°C within 15 min. The solution was refluxed for 7 h.
After removal of the solvent, the residue was dis-
solved in water (100 ml) and adjusted to pH 10 with
50% aqueous potassium carbonate. After extraction
with diethyl ether (4×200 ml), the organic layers were
dried over sodium sulphate and evaporated. The liq-
uid was taken up in diethyl ether (150 ml) and con-
verted to the hydrochloride with gaseous hydrochloric
acid at 0°C. The precipitate was filtered and washed
with diethyl ether, giving 16.6 g (91.8 mmol, 84%) of
a white solid. NMR (D2O) l: 0.9–1.1 ppm, 6H, m;
1.2–1.6 ppm, 2H, m; 2.0–2.2 ppm, 1H, m; 3.9 ppm,
3H, s; 4.2 ppm, 1H, m HPLC (Method 1a) tR: 4.79
min 2a and 4.96 min 3a.
D
from the material surviving to enzymatic hydrolysis.
A simple crystallisation technique allows to obtain 3d
directly from the epimeric mixture.
4. Experimental
Alcalase 2.5L DX from NOVOZYMES was used in
all experiments. 1H NMR analyses were carried out
on a 250 MHz Varian EMX instrument. EI-MS were
recorded on a TSQ mass spectrometer. Optical rota-
tion data were measured with a Propol automatic dig-
ital polarimeter at 20°C. Gradient HPLC was
performed on
a Agilent 1100 Series instrument
4.3.
D-Allo- + L-isoleucine ethyl ester hydrochloride
equipped with a SEDERE Sedex 75 evaporative light
scattering detector (T=40°C, Gain=4, P=3 bar)
using a reversed phase column (Agilent ZORBAX
XDB-C8) at 25°C and with a 1 ml/min flow. Method
1a: eluent TFA 0.1%/acetonitrile; from 0 to 35% of
acetonitrile in 7 min, followed by acetonitrile 35% for
2 min. Method 1b: eluent TFA 0.1%/acetonitrile 75/
25. HPLC evaluation of the diastereoisomeric excess
of the N-protected esters was carried out on a
Merck-Hitachi L6000 Intelligent Pump equipped with
a L600 UV-detector using a chiral column (Daicel
Chiralcel OD) with a 0.6 ml/min flow. Method 2a:
u=220 nm, eluent hexane/isopropanol 95/5. Method
2b: u=240 nm, eluent hexane/isopropanol 90/10.
Method 2c: u=254 nm, eluent hexane/isopropanol 90/
10. In enzymatic reactions automatic pH control was
1b·HCl
A suspension of the epimeric mixture of
and -alloisoleucine (15 g,114 mmol) in ethanol (250
L-isoleucine
D
ml) was treated with gaseous hydrochloric acid and
refluxed for 8 h. After evaporation of the solvent, the
oily residue was taken up in water (150 ml), adjusted
to pH 9.5 with NH3 and extracted with ethyl ether
(3×100 ml). The organic layers were washed with
brine (2×100 ml), dried over sodium sulphate, filtered
and concentrated (200 ml). The solution was treated
with gaseous hydrochloric acid at 0°C and evaporated
to give 18.52 g (95 mmol, 83%) of a white solid.
NMR (D2O) l: 0.85–1.05 ppm, 6H, m; 1.2–1.3 ppm,
3H, t; 1.2–1.6 ppm, 2H, m; 2.0–2.2 ppm, 1H, m; 4.10
ppm, 1H, m; 4.30 ppm, 2H, m. HPLC (Method 1a)
tR: 6.26 min 2b and 6.46 min 3b.
performed by
a
Metrohm 718 STAT Titrino
Isoleucine derivatives were prepared according to gen-
eral procedures found in the literature.36–40
4.4.
D-Allo- and L-isoleucine chloroethyl ester hydro-
chloride 1c·HCl36
4.1.
D
-allo- and
L
-isoleucine mix-ile 1
To a chilled suspension of the epimeric mixture of
-isoleucine and -alloisoleucine (15 g,114 mmol) in
L
D
50 g of
L
-isoleucine were dissolved in 250 g of glacial
2-chloroethanol (80 ml) thionyl chloride (96 g, 1.19
mol) was added dropwise within 30 min. The suspen-
sion was kept at rt for 3 days and then heated at
50°C for 7 h. After evaporation of the solvent, the
residue was treated according to the procedure
described for the ethyl ester 2 to give 18.6 g (81
mmol, 71%) of a white solid. NMR (D2O) l: 0.80–
1.15 ppm, 6H, m; 1.15–1.6 ppm, 2H, m; 2.0–2.3 ppm,
1H, m; 3.8–4.0 ppm, 2H, t; 4.20 ppm, 1H, m; 4.55
ppm, 2H, m. HPLC (Method 1a) tR: 7.36 min 2c and
7.54 min 3c.
acetic acid in the presence of 2 g of benzaldehyde,
and the mixture heated at 100°C in a nitrogen atmo-
sphere for 2 h. The mixture was cooled to 50°C and
filtered on a Celite pad. The solution was concen-
trated under vacuum to a volume of 50 ml. 160 ml of
2-propanol were then added and the mixture stirred
for 1 h. The mixture was filtered and washed with
2×40 ml of 2-propanol. The solid was dried under
vacuum to give 45 g (90%) of the epimeric mixture.
The diastereoisomeric composition was evaluated by
polarimetry by comparison with a pure sample of
alloisoleucine {[h]D=−37.4 (c 2 in 5 M HCl)} and
-isoleucine {[h]D=+39.0 (c 2 in 5 M HCl}. The [h]D
value of 0.8 (c 2 in 5 M HCl) of the mixture showed
a slight predominance (51%) of the -allo isomer,
which is consistent with the composition found for
the N-acyl esters (generally -allo/ -iso 54:46).
D-
4.5.
D-Allo- and L-isoleucine benzyl ester p-toluene-
sulphonate 1d·p-TsOH37
L
D
To 100 ml of toluene an epimeric mixture of
L-
isoleucine and
D-alloisoleucine (10 g, 76 mmol), p-
D
L
toluenesulphonic acid monohydrate (15 g, 79 mmol)

M. Cambie` et al. / Tetrahedron: Asymmetry 14 (2003) 3189–3196
3193
and benzyl alcohol (40 ml) were added and the suspen-
sion was refluxed for 18 h collecting water with a
Dean–Stark apparatus. After cooling, toluene (150 ml)
and ethyl ether (300 ml) were added to the solution.
After 2 h at 4°C, the precipitate was filtered and
washed with diethyl ether, giving 18.6 g (47 mmol, 62%)
of a white solid. A second crop (5.6 g, 14 mmol, 19%)
was obtained after concentration of the filtered solution
and addition of ethyl acetate and hexane (24.2 g, total
yield 81%).
dd; 6.20 ppm, 1H (broad); 8.10 ppm, 0.5H, s; 8.20 ppm,
0.5H, s. MS (EI) m/z: 174 (M+1), 142 (M-OMe), 117
(Mc-Lafferty). HPLC (Method 2a) tR: 24.5 min 3f
(53%) and 26.9 min 2f (47%).
4.9. N-Formyl-
D
-allo- and
L
-isoleucine chloroethyl ester
1g
8.0 g of the mixture of L-isoleucine and D-alloisoleucine
chloroethyl ester hydrochloride 3 (35 mmol) was dis-
solved in water (100 ml). After the addition of ethyl
acetate (200 ml), the biphasic system was cooled with
an ice bath and adjusted to pH 9 by the addition of
sodium carbonate. After separation of the phases, the
product was extracted from the aqueous solution with
ethyl acetate. The combined organic layers were dried
over sodium sulphate and evaporated, yielding 6.73 g
4.6.
D
-Alloisoleucine benzyl ester hydrochloride 3d·HCl
-allo- and -isoleucine benzyl ester p-
The mixture
D
L
toluenesulphonate (24.2 g, 62 mmol) was suspended in
water (100 ml) and ethyl acetate (100 ml). The pH was
adjusted at 8.5 with K2CO3, the organic layer was
separated and the ester mixture was extracted from the
aqueous phase with other ethyl acetate (2×100 ml).
After drying and evaporation of the solvent the oily
residue was dissolved in ethyl ether and converted to
the hydrochloride with anhydrous HCl at 0°C. After
filtration and washing with ethyl ether, the solid was
used to seed the organic phase concentrated to half the
volume and the mixture was left overnight at 5°C. The
two crops were pooled and the solid crystallised from a
(35 mmol, 100%) of a mixture of
-alloisoleucine chloroethyl ester as a pale yellow oil.
L-isoleucine and
D
The mixture was dissolved in formic acid cooling with
an ice-bath between 5 and 10°C. Acetic anhydride (24
ml) was added dropwise at 5°C and the solution was
left at rt for 23 h. After evaporation of the solvent, the
residue was taken up in chilled water (100 ml) and the
product extracted with ethyl acetate (3×100 ml). The
organic layers were washed successively with brine (50
ml), aqueous 5% sodium hydrogen carbonate (50 ml)
and again with aqueous sodium chloride (50 ml). Dry-
ing over sodium sulphate and evaporation of the sol-
vent gave 7.3 g (33 mmol, 94%) of a white solid. NMR
(CDCl3) l: 0.85–1.0 ppm, 6H, m; 1.15–1.35 ppm, 1H,
m; 1.35–1.55 ppm, 1H, m; 2.0 ppm, 1H, m; 3.7 ppm,
2H, m; 4.4 ppm, 2H, m; 4.85 ppm, 0.5H, dd; 4.95 ppm,
0.5H, dd; 6.35 ppm, 1H (broad); 8.20 ppm, 0.5H, s;
8.30 ppm, 0.5H, s. HPLC (Method 2b) tR: 20.0 min 3g
and 22.5 min 2g.
mixture of ethyl ether and ethanol giving
D-
alloisoleucine benzyl ester hydrochloride 3d·HCl (8 g,
30 mmol, 41%, mp 122.6–123.8°C). NMR (D2O) l:
0.85 ppm, 3H, d; 0.85 ppm, 3H, t; 1.25 ppm, 1H, m;
1.45 ppm, 1H, m; 2.10 ppm, 1H, m; 4.20 ppm, 1H, d;
5.3 ppm, 2H, q; 7.45 ppm, 5H, m. HPLC (Method 1b)
tR: 8.20 min 3d and 10.7 min 2d.
4.7. Enzymatic hydrolysis of compounds 1a–d
A solution of the substrate in distilled water (0.18 M)
was heated at 39°C and adjusted to pH 7.8 by addition
of 0.35 M sodium hydroxide. After Alcalase 2.5L was
added (0.23 g/ml) the reaction mixture was kept at pH
7.8 by the automatic pH-stat addition of 0.35 M
sodium hydroxide while the temperature was main-
tained at 39°C. The reaction course was followed by
HPLC with an evaporative light scattering detector (tR:
4.10. N-Formyl-D-allo- and L-isoleucine benzyl ester 1h
5.0 g (23 mmol) of the mixture of
-alloisoleucine benzyl ester-prepared via the p-toluen-
L-isoleucine and
D
sulphonate salt as described for product 4 was dissolved
in formic acid (100 ml) cooling with an ice-bath at
15°C. Acetic anhydride (50 ml) was added dropwise at
10°C and the solution was left at rt for 3 h. After
evaporation of the solvent, the residue is taken up in
chilled aqueous sodium chloride (100 ml) and the
product was extracted with ethyl acetate (3×100 ml).
The combined organic layers were washed successively
with aqueous sodium chloride (50 ml) solution, aqueous
5% sodium hydrogen carbonate (50 ml) and again with
aqueous sodium chloride (50 ml). Drying over sodium
sulphate and evaporation of the solvent gave 5.6 g (22
mmol, 96%) of a pale yellow oil. NMR (CDCl3) l:
0.8–1.0 ppm, 6H, m; 1.1–1.3 ppm, 1H, m; 1.3–1.5 ppm,
1H, m; 1.95 ppm, 1H, m; 4.75 ppm, 0.5H, dd; 4.85
ppm, 0.5H, dd; 5.20, 2H, m; 6.15 ppm, 1H (broad);
7.35 ppm, 5H, m; 8.20 ppm, 0.5H, s; 8.30 ppm, 0.5H, s.
MS (EI) m/z: 250 (M+1), 114 (M-PhCH2COO), 91
(PhCH2). HPLC (Method 2c) tR: 21.0 min 2h (46%)
and 22.5 min 3h (54%).
D
-allo, 3.23 min and
L
-ile 3.36 min, method 1a).
4.8. N-Formyl- -allo- and
D
L
-isoleucine methyl ester 1f
To a chilled solution of 1 (2.98 g, 23 mmol) in formic
acid (120 ml), acetic anhydride (21 ml) was added
within 15 min, keeping the temperature at 20°C. The
solution was stirred for 1 h without any further cooling
(the temperature raised to 65°C). The solvent was evap-
orated and the residue dissolved in methanol (100 ml)
and treated with a cold solution of diazomethane in
diethyl ether, at 0°C. The solvent was removed, and the
residue dissolved in ethyl acetate (100 ml). The solution
was washed with dilute hydrochloric acid (2×15 ml),
dried over sodium sulphate and evaporated, yielding
3.07 g (18 mmol, 78%) of an oil, which slowly solidified
at 4°C. NMR (CDCl3) l: 0.9–1.0 ppm, 6H, m; 1.05–
1.15 ppm, 1H, m; 1.15–1.30 ppm, 1H, m; 2.0 ppm, 1H,
m; 3.9 ppm, 3H, s; 4.85 ppm, 0.5H, dd; 4.95 ppm, 0.5H,

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M. Cambie` et al. / Tetrahedron: Asymmetry 14 (2003) 3189–3196
4.11. N-Acetyl-
D
-allo- and
L
-isoleucine benzyl ester 1j
(NHCOH)COOCH3]; 1.25 ppm [m, 1H, CH3CH2CH-
(CH3)CH(NHCOH)COOCH3]; 2.0 ppm, [m, 1H, CH3-
CH2CH(CH3)CH(NHCOH)COOCH3]; 3.9 ppm, [s, 3H,
RCOOCH3]; 4.95 ppm, [dd, J1=9.4 Hz, J2=3.5 Hz,
1H, CH3CH2CH(CH3)CH(NHCOH)COOCH3]; 6.2
ppm, [d broad, J=9.4 ppm, 1H, CH3CH2CH(CH3)-
CH(NHCOH)COOCH3]; 8.15 ppm, [s,1H, CH3-
CH2CH(CH3)CH (NHCOH) COOCH3]. Anal. calcd
for C8H15NO3: C, 55.47%; H, 8.73%; N, 8.09%. Found:
C, 55.40%; H, 8.71%; N, 8.11%
To 5.0 g (23 mmol) of 1d, dissolved in acetic anhydride
(25 ml) in an ice-bath, dimethylaminopyridine (1 mg)
and triethylamine (3.5 ml) were added and the solution
left at rt for 2.5 h. After evaporation of the solvent, the
residue was taken up in cold brine (50 ml) and the
product extracted with ethyl acetate (3×50 ml). The
combined organic layers were washed successively with
0.2 M aqueous hydrochloric acid (50 ml), aqueous 5%
sodium hydrogen carbonate (50 ml) and with sodium
chloride (50 ml). Drying over sodium sulphate and
evaporation of the solvent gave 5.74 g (22 mmol, 96%)
of a white solid. HPLC (Method 2c) tR: 13.7 min 3j
(54%) and 15.1 min 2j (46%).
4.15. N-Formyl-D-alloisoleucine chloroethyl ester 3g
[Substrate]: 0.18 M [Enzyme]: 0.043 (g/ml) The reaction
was stopped after 2.5 h (59% of sodium hydroxide
consumption). After recovery by extraction the unre-
acted substrate was purified by chromatography (hex-
ane/ethyl acetate 1:1). Yield: 40% (pale yellow oil)
d.e.(HPLC, method 2b): 96% [h]D=−13.3 (c 1 in
CHCl3); NMR (CDCl3) l: 0.9 ppm, [d, J=7.1 Hz, 3H,
CH3CH2CH(CH3)CH(NHCOH)COOR]; 0.95 ppm, [t,
J=7.5 Hz, 3H, CH3CH2CH(CH3)CH(NHCOH)-
COOR]; 1.1–1.3 ppm, [dq, J1=7.1 Hz, J2=not deter-
mined, CH3CH2CH(CH3)CH(NHCOH)COOR]; 1.35–
1.55 ppm, [dq, J1=7.1 Hz, J2=not determined,
CH3CH2CH(CH3)CH(NHCOH)COOR]; 2.0 ppm, [m,
1H, CH3CH2CH(CH3)CH (NHCOH)COOR]; 3.70
ppm, [t, J=5.6 Hz, RCOOCH2CH2Cl]; 4.40 ppm, [m,
2H, RCOOCH2 CH2Cl]; 4.85 ppm, [dd, J1=9.4 Hz,
J2=4.1 Hz, 1H, CH3CH2CH(CH3)CH(NHCOH)-
COOR]; 6.35 ppm, [d (broad), J=9.0 Hz, 1H,
CH3CH2CH(CH3)CH(NHCOH)COOBn]; 8.25 ppm, [s,
1H, CH3 CH2CH(CH3)CH(NHCOH)COOCH3]. Anal.
calcd for C9H16ClNO3: C, 48.76%; H, 7.27%, Cl
15.99%; N, 6.32%. Found: C, 48.65%; H, 7.22%, Cl
15.76%; N, 6.33%.
4.12. N-Carbobenzyloxy-D-allo- and L-isoleucine benzyl
ester 1m
To 100 ml of a 1 M NaHCO3, 5.0 g (23 mmol) of 1d,
benzyl chloroformate (4.2 ml, 30 mmol) were added
dropwise and the mixture stirred at 5–10°C. After 45
min, the reaction was quenched with 2 M aqueous
hydrochloric acid (21 ml) and, after separation of the
phases, the aqueous one was extracted with ethyl ace-
tate (2×100 ml). The combined organic layers were
washed with 0.2 M aqueous hydrochloric acid (50 ml)
and with brine (2×50 ml). Drying over sodium sulphate
and evaporation of the solvent gave a residue, which
yielded 7.65 g (22 mmol, 96%) of a pale yellow oil after
chromatography. NMR (CDCl3) l: 0.75–1.0 ppm, 6H,
m; 1.0–1.25 ppm, 1H, m; 1.25–1.5 ppm, 1H, m; 1.95
ppm, 1H, m; 4.40 ppm, 0.5H, dd; 4.95 ppm, 0.5H, dd;
5.0–5.4 ppm, 5H, m; 7.35 ppm, 10H, m. MS (EI) m/z
356 (M+1), 220 (M−PhCH2OCO), 91 (PhCH2). HPLC
(Method 2c) tR: 9.4 min 2m (47%) and 24.0 min 3m
(53%).
4.13. Enzymatic hydrolysis of compounds 1f–m
4.16. N-Formyl-D-alloisoleucine benzyl ester 3h
A suspension of the substrate in distilled water (0.18 or
0.36 M) was heated at 39°C and adjusted to pH 7.8 by
the addition of NaOH 0.35 M. Alcalase 2.5L was then
added (0.21 or 0.43 g/ml) and the reaction mixture was
kept at pH 7.8 by automatic addition of NaOH 0.35 M.
When the consumption of NaOH reached about 50%
(or more) the reaction was stopped and the unreacted
substrate was recovered by extraction with tert-butyl-
methylether. The acidic product could be recovered by
extraction or precipitation after acidification of the
aqueous phase with citric acid or dilute hydrochloric
acid.
[Substrate]: 0.18 M [Enzyme]: 0.021 (g/ml) The reaction
was stopped after 22.5 h (47% of sodium hydroxide
consumption). After recovery by extraction the unre-
acted substrate was purified by chromatography (hex-
ane/ethyl acetate 6:4). Yield: 41% (pale yellow oil) d.e.
(HPLC, method 2c): 98% [h]D=−7.7 (c 1 in CHCl3);
NMR (CDCl3) l: 0.8 ppm, [d, J=7.2 Hz, 3H,
CH3CH2CH(CH3)CH(NHCOH)COOBn]; 0.9 ppm, [t,
J=7.2 Hz, 3H, CH3CH2CH(CH3)CH(NHCOH)-
COOBn]; 1.1–1.3 ppm, [dq, J1=7.2 Hz, J2=6.4 Hz,
CH3CH2CH(CH3)CH(NHCOH)COOBn]; 1.3–1.5 ppm,
[dq, J1=7.2 Hz, J2=6,4 Hz, CH3CH2CH (CH3)CH-
(NHCOH)COOBn]; 1.95 ppm, [m, 1H, CH3CH2CH-
(CH3)CH(NHCOH) COOBn]; 4.85 ppm, [dd, J1=8.8
Hz, J2=4.0 Hz, 1H, CH3CH2CH(CH3)CH(NHCOH)
COOBn]; 5.15 ppm, [AB system, JAB=16.0 Hz, 2H,
RCOCH2Ph]; 6.30 ppm, [d (broad), J=8,0 Hz, 1H,
CH3CH2CH (CH3)CH(NHCOH)COOBn]; 7.35 ppm,
[m, 5H, RCOCH2Ph]; 8.20 ppm, [s, 1H,
CH3CH2CH(CH3) CH(NHCOH)COOCH3]. Anal.
calcd for C14H19NO3: C, 67.45%; H, 7.68%; N, 5.62%.
Found: C, 67.35%; H, 7.72%; N, 5.60%.
4.14. N-Formyl-D-alloisoleucine methyl ester 3f
[Substrate]: 0.18 M; [Enzyme]: 0.043 (g/ml) The reac-
tion was stopped after 23 h (64% of sodium hydroxide
consumption). Yield: 30% (pale yellow oil) d.e.(HPLC,
Method 2a): 98% [h]D=−32.5 (c 1 in CHCl3) NMR
(CDCl3) l: 0.85 ppm [d, J=7.0 Hz, 3H,
CH3CH2CH(CH3)CH(NHCOH)COOCH3]; 0.95 ppm,
[t, J=7.0 Hz , 3H, CH3CH2CH(CH3)CH(NHCOH)-
COOCH3]; 1.1 ppm, [m, 1H,CH3CH2CH(CH3) CH

M. Cambie` et al. / Tetrahedron: Asymmetry 14 (2003) 3189–3196
3195
4.19.2. From N-formyl-
D
-alloisoleucine benzyl ester 3h.
4.17. N-Acetyl-D-alloisoleucine benzyl ester 3j
N-Formyl-
D
-alloisoleucine benzyl ester 3h (1.0 g, 3.8
mmol) was dissolved in dioxane (10 ml) and 5%
aqueous hydrochloric acid (10 ml) and refluxed 11 h.
After cooling, the solution was washed with ethyl acet-
ate (2×30 ml) and adjusted to pH 7–8 with sodium
carbonate. Extraction of the product with ethyl ether
(3×30 ml) gave, after drying and evaporation of the
[Substrate]: 0.18 M [Enzyme]: 0.021 (g/ml) The reaction
was stopped after 3.3 h (48% of sodium hydroxide
consumption). After recovery by extraction the unre-
acted substrate was purified by chromatography (hex-
ane/ethyl acetate 6:4). Yield: 50% (white solid) d.e.
(HPLC, Method 2c):
L-isomer was not detectable
solvent,
D
-alloisoleucine benzyl ester 3d (0.726 g, 1.2
[h]D=−13.8 (c 1 in CHCl3); NMR (CDCl3) l: 0.8 ppm,
[d, J=7.0 Hz, 3H, CH3CH2CH(CH3)CH(NHCOCH3)-
COOBn]; 0.9 ppm, [t, J=7.4 Hz, 3H, CH3CH2CH-
(CH3)CH(NHCOCH3)COOBn]; 1.1–1.3 ppm, [dq, J1=
7.4 Hz, J2=6.5 Hz, CH3CH2CH(CH3)CH(NHCO
CH3)COOBn]; 1.3–1.5 ppm, [dq, J1=7.4 Hz, J2=6.5
Hz, CH3CH2CH(CH3)CH(NHCOH)COOBn]; 1.95
ppm, [m, 1H, CH3CH2CH(CH3)CH(NHCOCH3)
COOBn]; 2.05 ppm, [s, 3H, CH3CH2CH(CH3)CH-
(NHCOCH3)COOCH3]; 4.75 ppm, [dd, J1=9.2 Hz,
J2=4.4 Hz, 1H, CH3CH2CH(CH3)CH(NHCOCH3)-
COOBn]; 5.15 ppm, [AB system, JAB=16.1 Hz, 2H,
RCOCH2Ph]; 6.05 ppm, [d (broad), J=9.2 Hz, 1H,
CH3CH2CH(CH3)CH(NHCOCH3) COOBn]; 7.35 ppm,
[m, 5H, RCOCH2Ph]. Anal. calcd for C15H21NO3: C,
68.42%; H, 8.04%; N, 5.32%. Found: C, 68.46%; H,
8.06%; N, 5.24%.
mmol, 32%) as a solid. 660 mg (6.0 mmol) of
D
-
alloisoleucine benzyl ester 3d was dissolved in water (50
ml) and ethanol (100 ml), and hydrogenated on 10%
PdꢀC (400 mg) at 70 psi for 1.5 h. The catalyst was
removed by filtration and washed with ethanol/water
1:1. Evaporation of the solvent gave
D-alloisoleucine
(360 mg, 2.7 mmol, 90%). [h]D=−36.6 (c 2 in HCl 5M)
(−37.4 on a commercial sample; lit.: −37.8).
4.19.3. From N-formyl-
D
-alloisoleucine benzyl ester 3h.
-alloisoleucine benzyl ester
A suspension of N-formyl-
D
3h (1.0 g, 4.0 mmol) in 1 M hydrochloric acid (80 ml)
was heated at 90°C for 12 h. After cooling, the solution
was washed with ethyl acetate (2×50 ml) and adjusted
to pH 9 with sodium carbonate. Extraction of the
product with ethyl acetate (3×50 ml) gave, after drying
and evaporation of the solvent,
ester 3d (0.76 g, 3.4 mmol, 85%) as an oil. 660 mg (6.0
mmol) of -alloisoleucine benzyl ester 3d was dissolved
D-alloisoleucine benzyl
D
4.18. N-Acetyl-D-alloisoleucine 3i
in water (50 ml) and ethanol (100 ml), and hydro-
genated on 10% PdꢀC (400 mg) at 70 psi for 1.5 h. The
catalyst was removed by filtration and washed with
ethanol/water 1:1. Evaporation of the solvent gave
600 mg (2.3 mmol) of N-acetyl-
D
-alloisoleucine benzyl
ester 3j were dissolved in ethanol (100 ml), and hydro-
genated on 10% PdꢀC (400 mg) at 60 psi for 2 h. The
catalyst was removed by filtration and washed with
D
-alloisoleucine (360 mg, 2.7 mmol, 90%). [h]D=−31.2
(c 2 in HCl 5 M).
ethanol. Evaporation the solvent gave N-acetyl-D-
alloisoleucine 3i, (290 mg, 1.7 mmol, 74%) as a white
solid. [h]D=−19.0 (c 2 in EtOH) (Ref.: −20.5);6 NMR
(CD3OD) l: 0.8 ppm, [d, J=6.8 Hz, 3H, CH3CH2-
CH(CH3)CH(NHCOCH3)COOH]; 0.9 ppm [t, J=7.7,
3H, CH3CH2CH(CH3)CH(NHCOCH3)COOH]; 1.1
ppm, [dq, J1=7.7, J2=7.4, 1H, CH3CH2CH-
(CH3)CH(NHCOCH3)COOH]; 1.25 ppm, [dq, J1=7.7,
J2=6.8, 1H, CH3CH2CH (CH3)CH (NHCOCH3)-
COOH]; 1.8 ppm, [m, 1H, CH3CH2CH(CH3)CH-
(NHCOCH3) COOH]; 2.1 ppm, [s, 1H, CH3CH2CH-
(CH3)CH(NHCOCH3)COOH]; 4.3 ppm, [dd, J1=8.6
Hz, J2=4.7 Hz, 1H, CH3CH2CH(CH3)CH(NH-
COCH3)COOH]; 7.9 ppm, [d broad, J=8.6 Hz, 1H,
CH3CH2CH(CH3) CH(NHCOCH3)COOH]. Anal.
calcd for C8H15NO3: C, 55.47%; H, 8.73%; N, 8.09%.
Found: C, 55.37%; H, 8.76%; N, 8.28%.
4.19.4. From N-formyl-
D
-alloisoleucine chloroethyl ester
3g.
A
suspension of N-formyl-D-alloisoleucine
chloroethyl ester 3g (1.71 g, 7.7 mmol) in HCl 1 M (70
ml) was heated at 90°C for 5 h. After cooling, the
solution was washed with ethyl acetate (2×50 ml) and
adjusted to pH 9 with sodium carbonate. Extraction of
the product with ethyl acetate (3×50 ml) gave, after
drying and evaporation of the solvent,
chloroethyl ester 3c (1.3 g, 6.7 mmol, 87%) as an oil.
1.17 g (6.0 mmol) of -alloisoleucine chloroethyl ester
D-alloisoleucine
D
3c was dissolved in dioxane (10 ml). After addition of
sodium hydroxide 1 M (10 ml) the solution was kept at
rt for 15 h, then refluxed for 1.5 h. After cooling the
solution was washed with tert-butylmethylether (2×20
ml) and then evaporated. The residue was taken up in
water (6 ml) and the pH adjusted to 6.2 with 6 M
hydrochloric acid. After filtration
D-alloisoleucine was
recovered as a white solid. [h]D=−31.2 (c 2 in HCl 5
M).
4.19.
D-Alloisoleucine 4
4.19.1. From N-acetyl-
3i. N-Acetyl- -alloisoleucine 3i (1.0 g, 3.8 mmol) was
D-alloisoleucine 3i obtained from
D
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