Synthesis of novel N-protected β3-amino nitriles: study of their hydrolysis involving a nitrilase-catalyzed step

Article abstract of DOI:10.1016/j.tetasy.2009.07.045

Several commercially available nitrilases were investigated with regard to their potential to hydrolyze N-protected β³-amino nitriles into their corresponding N-protected β³-amino acids. The biotransformations were obtained in different proportions depending on the nitrilase involved. The best hydrolysis results were achieved for the N-Cbz-β³-amino nitrile from l-alanine using the NIT-107, in a phosphate buffer at 0.05 M. However, no biotransformation into the corresponding acids was observed for the N-sulfonylamide β³-amino nitriles. Two simple and efficient procedures to prepare the β³-amino nitriles from their analogous α-amino acids are described. Thirty four new substances were synthesized and characterized over the course of this work.

Full text of DOI:10.1016/j.tetasy.2009.07.045

Tetrahedron: Asymmetry 20 (2009) 2077–2089
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Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Synthesis of novel N-protected b3-amino nitriles: study of their hydrolysis
involving a nitrilase-catalyzed step
*
*
Maité Sylla-Iyarreta Veitía , Pierre Louis Brun , Pierre Jorda, Annie Falguières, Clotilde Ferroud
Laboratoire de Transformations Chimiques et Pharmaceutiques, UMR 7084, Case 303, Conservatoire National des Arts et Métiers, 2 rue Conté 75003 Paris, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Several commercially available nitrilases were investigated with regard to their potential to hydrolyze N-
protected b3-amino nitriles into their corresponding N-protected b3-amino acids.
Received 6 May 2009
Accepted 6 July 2009
Available online 1 October 2009
The biotransformations were obtained in different proportions depending on the nitrilase involved.
The best hydrolysis results were achieved for the N-Cbz-b3-amino nitrile from
L-alanine using the NIT-
107, in a phosphate buffer at 0.05 M. However, no biotransformation into the corresponding acids was
observed for the N-sulfonylamide b3-amino nitriles. Two simple and efficient procedures to prepare
the b3-amino nitriles from their analogous
a
-amino acids are described. Thirty four new substances were
synthesized and characterized over the course of this work.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
or basic conditions, high temperatures, the formation of undesir-
able by-products, racemization, low yields, and environmental
problems due to the generation of waste salts.12
In recent years, there has been an increasing interest in the
synthesis of b-amino acids due to their significant effects, such as
antibiotic,1 antifungal,2 antitumor,3 antihelminthic,4 cytotoxic,5 and
other important pharmacological properties.6 Many natural products
withab-aminoacidmoietyarepotentialleadstructuresforthedevel-
As a result, in recent years, considerable attention has been paid
to the enzymatic hydrolysis of nitriles as an alternative route to the
chemical synthesis of amides and carboxylic acids.13 High conver-
sion yields and selective hydrolysis of the –CN functionality of com-
pounds containing labile groups as well as high chemo-, regio- and
stereoselectivities can be obtained. On the other hand, biocatalysis
frequently use a biodegradable catalyst and can be performed under
mild reaction conditions (neutral pH, low temperature, and water as
solvent).14 Furthermore, the use of enzymes usually generates less
waste and, hence, is both environmentally and economically more
attractive than traditional organic syntheses.14c
opment of new drugs.7 The replacement of
a-amino acids in biologi-
cally active peptides by certain b-counterparts can have pronounced
effects on their folding properties. For instance, cyclic and linear olig-
omersofb-aminoacids haverevealedhighbiologicalactivityasmim-
ics for the peptide hormone somatostatin showing antiproliferative
activities against human cancer cell lines.6a
Several routes to prepare b3-amino acids have been devel-
oped,6b,c,8 some from the corresponding
a
-amino acids.9 The best
The recent availability of ‘ready to use’ nitrilase preparations15
which avoids the laborious handling of whole cell biotransforma-
tion systems has prompted us to search for an efficient and short
synthesis of b3-amino acids from the readily available correspond-
ing nitriles.
known is the Arndt–Eistert methodology.6b,10 Unfortunately, the
Arndt–Eistert conditions for direct homologation are not suitable
for large scale preparations and, besides, are not recommended
for preserving the N-protected group necessary in peptidomimetic
synthesis.9b
In the drive toward Green, sustainable methodologies for chemi-
Several methods using b3-amino nitriles as precursors of b3-
amino acids have been described.11 Nitriles have been extensively
used in the industry as precursors for the production of a wide
variety of amides and carboxylic acids by chemical synthesis. How-
ever, the conventional chemical hydrolysis of nitriles suffers from
several disadvantages, including the requirement for harsh acidic
cals manufacture, we herein report the synthesis of b3-amino nitriles
from their natural a-amino acids counterparts and their bioconver-
sion into N-protected b3-amino acids by purified nitrilases. These
b3-amino acids will be ready to use in peptidomimetic syntheses.
2. Results and discussion
2.1. Synthesis of b3-amino nitriles
* Corresponding authors. Tel.: + 33 1 58 80 84 82; fax +33 1 42 71 05 34 (M.S.-I.V.).
E-mail addresses: maite.sylla@cnam.fr (M.S.-I. Veitía), clotilde.ferroud@cnam.fr
(C. Ferroud).
Initially, we were interested in the preparation of N-protected
b3-amino nitriles 7a–f from their corresponding
a-amino acids
Present address: ISOCHEM, 32 rue Lavoisier 91710, Vert-Le-Petit, France.
0957-4166/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2009.07.045

2078
M.S.-I. Veitía et al. / Tetrahedron: Asymmetry 20 (2009) 2077–2089
and then in their hydrolysis by a bioconversion step using
nitrilases.
this type of Mitsunobu reaction (35–60%) (Table 1) could be attrib-
uted to the low acidity of the amine moiety resulting in the slow
formation of the oxyphosphonium intermediate and hence product
formation.19 Additionally, the instability due to the risk of poly-
merization, and in some cases, the volatile nature of N–H aziridines
lead to a loss of more than 50% after work-up and during the puri-
fication step.
Aliphatic
and aromatic
L
-amino acids: alanine 1a, valine 1b, and isoleucine 1c,
-amino acids: phenylalanine 1d, O-tert-butyl-tyro-
L
sine 1e, and O-benzyl-tyrosine 1f were employed. Either classical
N-protecting groups such as Cbz and Boc were used in the peptide
synthesis or uncommon protecting groups such as tosyl, nosyl, and
diphenylphosphinyl (Dpp) were employed. We focused our atten-
tion on the study of the influence of these protecting groups in the
biocatalysis step by nitrilases.
Next, our first attempt to prepare the N-Cbz-b3-amino nitriles
involved a one-pot procedure whereby the aziridines 3a–f were di-
rectly transformed into the corresponding opening compounds 7a–
f-Cbz by the reaction with benzyl cyanoformate under phase trans-
fer catalysis conditions. Recently Moss et al. have developed an
efficient enantioselective alkylation of N-sulfonyl-protected aziri-
dines using sophisticated phase transfer catalysts and a variety of
base combinations.20
Taking into account that homologation of the parent chiral
a-
amino acids is one of the most powerful strategies to prepare the
b-homologues, we designed two straightforward procedures, in
which the key step of the whole conversion is represented by the
synthesis of enantiomerically pure N-protected b3-aminonitriles
7a–f (Scheme 1).
In our case, a most commonly used phase transfer agent, tetra-
butylammonium bromide (TBABr), was employed in the presence
of NaOH in stoichiometric quantities. As a result, the ring-opening
reaction of intermediate aziridines 5a–f-Cbz was carried out with
NaCN in the presence of a catalytic amount of TBABr (10%, relative
to the nucleophile) in a mixture of toluene/water (5:1) to obtain
the corresponding N-Cbz-b3-amino nitriles 7a–f-Cbz in yields of
45–75%. It should be noted that significant amounts of polymeric
materials were generated when this procedure was scaled up.
Subsequently, in order to improve the yields and make the
work-up more easy, we decided to carry out the ring-opening reac-
tion of aziridines 5a–f-Cbz under mild conditions.11b,21 First, aziri-
dines 3a–f were reacted with benzyl cyanoformate in acetonitrile
to afford compounds 5a–f-Cbz. The reaction was monitored by
TLC and GC–MS and after removal of the solvent the activated azir-
idines were used in the next step without any purification. The
crude N-Cbz aziridines 5a–f-Cbz were reacted with additional
NaCN (3 equiv) in a mixture of acetonitrile/water (9:1) at 80 °C
and the reaction was completed in 16–21 h. The desired N-pro-
tected b-amino nitriles 7a–f-Cbz were obtained with yields of
55–89% (Table 1). According to the NMR spectra, the aforemen-
tioned conditions produced a regioselective ring-opening of the
heterocycle.
Firstly the amino alcohols required are obtained in enantiome-
rically pure form via the reduction of the corresponding commer-
cially available enantiopure
achieved by sodium borohydride in tetrahydrofuran using ZnCl2
as a Lewis acid catalyst.16 The tendency of
-amino alcohols to
a-amino acids. The reduction was
a
form stable borate esters, and chelates with metal cations makes
their isolation difficult.17 Therefore, the key step of this protocol
is the hydrolysis of the complex amino alcohol/borate by treatment
of the crude reaction with 2 equiv of a solution of NaOH 50% (w/w)
at 70 °C for 4 h. The amino alcohols 2a–f were obtained in good
yields (86–95%) and used in the next step without further
purification.
The extent of racemization at the stereogenic center of the
starting
a-amino acids and/or b-amino alcohols was checked at
various stages of the whole process by chiral HPLC analyses. Under
our conditions, no trace amounts of racemized products could be
detected.
The first strategy developed (method A) was employed in the
preparation of the N-Cbz-b3-amino nitriles 7a–f-Cbz. This ap-
proach involved the formation of N–H aziridines, their activation
by a benzyloxycarbonyl group, and their regioselective ring-open-
ing using a cyanide ion as a nucleophile (Scheme 1).11a,b
Chiral amino alcohols 2a–f were converted into aziridines 3a–f
via a Mitsunobu reaction18 using diethyl azodicarboxylate (DEAD)
(1.05 equiv) and triphenylphosphine (Ph3P) (1.05 equiv) in diethyl
ether, toluene, or tetrahydrofuran. The modest yields obtained in
In order to improve the yields, the reaction time, and to avoid
any intermediate work-up, a one-pot reaction was attempted.
Unfortunately, even if the overall reaction time in the one-pot reac-
tion decreased by half compared to the multi-step protocol (20 h vs
d
method A
R
R
R
R
a
c
b
HO
NH2
HO
NH
N
G
NH2
86-95%
35-60%
O
2
3
1
5
method B
40-90%
G= Cbz, Dpp
R
f
e
44-81%
1a Ala
CH3
1b Val
1c Ile
1d Phe
iPr
sec-Bu
Bn
R
g
R
R
h
HO
G
MsO
G
N
N
H
NC
G
1e O-tBu-Tyr O-tBu-Ph-CH2
1f O-Bn-Tyr
O-Bn-Ph-CH2
N
H
70-100%
H
71-87%
4
7
6
G= Cbz, Boc, Ns,Ts
G= Cbz, Boc, Dpp, Ns,Ts
Scheme 1. Synthesis of b-amino nitriles. Reagents and conditions: (a) NaBH4, ZnCl2, THF; (b): PPh3/DEAD, THF or toluene (c) CbzCN, CH3CN; (d): DppCl, TEA, THF, 0 °C; (e)
NaCN, CH3CN/H2O 9:1, reflux; (f) CbzCl, TEA CH2Cl2 (or THF) or Boc2O, NaOH, dioxane or TsCl (or 4NsCl), NaHCO3, THF (g) MsCl, TEA, THF; (h) NaCN, DMF.

M.S.-I. Veitía et al. / Tetrahedron: Asymmetry 20 (2009) 2077–2089
2079
Table 1
Mitsunobu reaction, ring-opening of aziridines in CH3CN/H2O, and overall yield of method A
R
R
R
PPh3/DEAD
R
CbzCN
CH3CN
NaCN
NC
Cbz
HO
NH
N Cbz
N
H
NH2
THF or toluene
CH3CN/H2O 9:1
7
2
3
5
2
R
3-Cbz
7-Cbz
Overall yield % (from 2)
Time (h)
Yield %
Time (h)
Yield %
a
b
c
d
e
f
CH3
iPr
sec-Bu
Bn
O-tBu-Ph-CH2
O-Bn-Ph-CH2
4.5
40
18
21
19
25a
35b
60b
53c
43c
30c
21
21
16
21
18
18
55
60
66
81
89
73
14
21
40
43
38
22
22
a
b
c
Prepared in diethyl ether.
Prepared in tetrahydrofurane.
Prepared in toluene.
40 h), the overall yields obtained (17–35%) were not significantly
better.
N-protected-b3-amino nitriles. Nevertheless, to circumvent the dif-
ficulties found during the synthesis of compounds 7a–f-Cbz using
method A, an alternative route (method B) was designed starting
from amino alcohols 2a–f to prepare the N-protected b-amino
nitriles 7a–f-Cbz. To extend the application of this method we
wished to prepare other N-protected-b-amino nitriles 7a–c-Boc,
7a–c-4Ns, and 7a–c-Ts.
The new approach in method B involved amine group protec-
tion, activation of the alcohol function by mesylation, and SN2
displacement by cyanide ion13m,24 (Scheme 1). The synthesis of
N-protected amino alcohols was carried out according to the liter-
ature procedures25 and the results are reported in Table 3. To pre-
pare the N-Cbz amino alcohols 4a–f-Cbz, compounds 2a–f were
reacted with benzylchloroformate and triethylamine in THF or
CH2Cl2 to afford the desired compounds 4a–f-Cbz with yields rang-
ing from 45% to 94%.
To obtain the N-Boc amino alcohols 4a–c-Boc, the correspond-
ing amino alcohols 2a–c were reacted with (Boc)2O and NaOH
1 M in dioxane.25 In the case of compounds 4a–c-4Ns and 4a–c-
Ts, b-amino alcohols 2a–c were reacted with 4NsCl or TsCl
(1.1 equiv) in the presence of NaHCO3 (4 equiv) at room tempera-
ture in THF. The reaction times and the yields of the N-protection
steps are summarized in Table 3.
In the next step, the N-protected amino alcohols 4a–f-Cbz, 4a–
c-Boc, 4a–c-Ts, and 4a–c-4Ns were treated with methanesulfonyl
chloride in THF to afford the solid intermediates 6a–f-Cbz, 6a–c-
Boc, 6a–c-Ts, and 6a–c-4Ns with yields ranging from 70% to
100%. These compounds were unstable at room temperature and
were used in the following reactions without further purification.
Finally, N-protected b-amino nitriles 7a–f-Cbz, 7a–c-Boc, 7a–
c-4Ns, and 7a–c-Ts were obtained by nucleophilic substitution of
The N-Dpp b-amino nitriles 7a-Dpp, 7c-Dpp, 7d-Dpp, and 7e-
Dpp were prepared following a modification of the procedure de-
scribed above for method A. The use of a diphenylphosphinyl
group as an alternative of protecting group in amino acid and pep-
tide chemistry is well-known,22 although, a few reports regarding
N-Dpp aziridines synthesis are described.22a,23 Firstly, the activated
aziridines 5a-Dpp, 5c-Dpp, 5d-Dpp, and 5e-Dpp were synthesized
from amino alcohols in a one-pot transformation by ring-closure of
the corresponding N,O-diphenylphosphinylated intermediates.
Amino alcohols 2a, 2c, 2d, and 2e were reacted with diphenyl-
phosphinyl chloride (DppCl) (2 equiv) and Et3N (3 equiv) in THF at
25 °C for 20 h. Next, sodium hydride (5 equiv) was required to al-
low the cyclization to afford N-Dpp aziridines 5a-Dpp, 5c-Dpp,
5d-Dpp, and 5e-Dpp with yields of 55%, 76%, 67%, and 74%,
respectively.
The ring-opening reaction of N-Dpp aziridines 5a-Dpp, 5c-Dpp,
5d-Dpp, and 5e-Dpp occurred smoothly using phase transfer con-
ditions (TBABr 10 mol % in toluene/water 5:1 as described above
for 5(a–f)-Cbz) for 18 h and afforded the product as a single stereo-
isomer. The reaction gives the expected ring-opened product
resulting from nucleophilic attack on the less substituted carbon
atom. The N-Dpp amino nitriles were obtained regioselectively in
good yields (74–86%, HPLC purity of 85–98%) (Table 2). To our
knowledge, no ring-opening reaction of N-Dpp aziridines using
such phase transfer conditions to afford N-Dpp b3-aminonitriles
is described. This method represents an interesting way to synthe-
size such N-Dpp b3-aminonitriles.
As described above, the regioselective ring-opening reaction of
aziridines (method A) is a synthetically powerful route to prepare
Table 2
Reaction times and yields of the N-Dpp aziridines 5a,c,d,e-Dpp and 7a,c,d,e-Dpp
R
R
R
NaCN
DppCl/TEA
THF, 0°C
NC
Dpp
HO
N
H
N
Dpp
NH2
CH3CN/H2O 9:1
7
5
2
2
R
5-Dpp
7-Dpp
Overall yield % (from 2)
Time (h)
Yield %
Time (h)
Yield %
a
c
d
e
CH3
sec-Bu
Bn
20
20
20
20
55
76
67
74
18
18
18
18
86
74
75
74
47
56
50
74
O-tBu-Ph-CH2

2080
M.S.-I. Veitía et al. / Tetrahedron: Asymmetry 20 (2009) 2077–2089
Table 3
Yields of N-protected b-amino alcohols
R
R
Cbz, TEA, CH2Cl2( or THF)
HO
G
HO
N
H
NH2
or Boc2O, NaOH, dioxane
4
or TsCl or 4NsCl, NaHCO3, THF
2
G= Cbz, Boc, Ns,Ts
2
R
4-Cbz
4-Boc
4-4Ns
4-Ts
Time (h)
Yield %
Time (h)
Yield %
Time (h)
Yield %
Time (h)
Yield %
a
b
c
d
e
f
CH3
iPr
sec-Bu
Bn
O-tBu-Ph-CH2
O-Bn-Ph-CH2
12
12
3
10
1.5
3.5
60
82
55
3
2.4
2.6
85
99
65
5
7
5
95
70
78
28
24
24
71
97
83
92
50a
94a
a
Prepared in CH2Cl2.
the corresponding b-amino mesylates by cyanide ion (NaCN,
1.5 equiv) in DMF at 70 °C without any deprotection. The reaction
times and the yields obtained in the preparation of N-protected
b-amino nitriles 7 are reported in Table 4. In the case of 7e-Cbz,
the low yield of 30% could be explained by the slow rate of the
reaction (28 h) that induces the formation of polymers.
On the other hand, it was noticed that the required condi-
tions for the preparation of N-Ts-b-amino nitriles 7b-Ts and
7c-Ts from their corresponding b-amino mesylates 6b-Ts and
6c-Ts afforded the corresponding N-tosyl aziridines in yields of
66% and 100%, respectively. Consequently, an additional step
was necessary. The N-tosyl aziridines were opened using a cya-
nide ion as a nucleophile in a mixture of acetonitrile/water
(9:1) at 80 °C. The desired N-Ts b-amino nitriles 7b-Ts and 7c-
Ts were afforded in excellent yields of 92% and 94%, respectively.
The reaction times and the yields of the N-protected-b-amino ni-
triles are summarized in Table 4.
In summary, the strategies described in this study (method A
and method B) constitute as efficient approaches to prepare b-ami-
no nitriles 7a–f-Cbz, 7a–c-Boc, 7a–c-4Ns, and 7a–c-Ts from the
corresponding amino alcohols. Moreover, a large number of com-
pounds synthesized are reported here for the first time. The spec-
troscopic and physical data of these new compounds are given in
Section 4.
Method A has shown that the regioselective ring-opening of
aziridines is a potential tool to obtain b-amino nitriles in an easy
way. The ring opening of aziridines by a cyanide ion requires an
activating group and in our particular case, this reaction was
particularly efficient with the diphenylphosphinyl group. However,
our study shows that method B (applied to other protecting
groups) is the best strategy for preparing N-protected-b-amino
nitriles. This method used inexpensive reagents, and was cleaner
and faster. On the other hand, the work-up was easier than that
required for the method A and these operative conditions can be
applicable on a large-scale. The reaction times and overall yields
of the three steps for method B are reported in Table 5.
2.2. Biotransformation screening
The aim of the present study was to develop the hydrolysis of
N-protected b-amino nitriles by a bioconversion step using nitri-
lases in order to avoid the harsh conditions required in the chem-
ical hydrolysis of nitriles. We were also interested in studying the
influence of the nature of N-protecting groups during the biotrans-
formation process by nitrilases and in consequently evaluating
their use for biocatalytic screening.
The recent availability of nitrilases ‘ready for use’ simplifies the
reaction protocol considerably. To the best of our knowledge, very
few publications showing the utilization of commercial nitrilases
in biotransformation screening are available.13m,o,r
Most of the biotransformation reactions were monitored by re-
versed phase HPLC and UV-detection. For N-Boc-b-amino nitriles,
the biotransformation is severely hindered by the problems arising
in terms of reaction monitoring and product separation of strongly
polar compounds. The detection of the aliphatic nitriles/amides is
limited because of their poor UV-sensibility, especially on a screen-
Table 4
Yields of N-protected b-amino nitriles
R
R
NaCN
DMF
NC
MsO
G
G
N
H
N
H
7
6
G= Cbz, Boc, Ns,Ts
6
R
7-Cbz
7-Boc
7-Ns
7-Ts
Time (h)
Yield %
Time (h)
Yield %
Time (h)
Yield %
Time (h)
Yield %
a
b
c
d
e
f
CH3
iPr
sec-Bu
Bn
O-tBu-Ph-CH2
O-Bn-Ph-CH2
1
4
4
1
28
1.5
81
85
87
88
30
89
4
1
5.5
66
43
47
4
4
48
22
81
40
0.5
2.5
4
73
92a
94a
a
Yields obtained from the N-tosyl aziridine intermediates.

M.S.-I. Veitía et al. / Tetrahedron: Asymmetry 20 (2009) 2077–2089
2081
Table 5
Table 6
Reaction times and overall yields of method B in three steps
Yields calculated by HPLC data during biotransformation screening of compounds 7a-
Cbz and 7b-Cbz
R
R
R
O
R
O
R
NIT 101 - 112
NC
Cbz
NC
G
Cbz
Cbz
HO
N
H
H2N
N
H
N
H
HO
N
H
NH2
7a-f-Cbz
9a-f-Cbz
8a-f-Cbz
7
2
Entry
Enzyme
Concn (M)
Nitrile %
Amide %
Acid %
G= Cbz, Boc, Ns,Ts
7a
7b
8a
8b
9a
9b
Compound 7
Time (h)
Overall yield % (from 2)
1
2
3
4
5
6
7
8
NIT-104
NIT-107
NIT-107
NIT-107
NIT-108
NIT-109
NIT-110
NIT-112
0.2
0.2
0.1
0.05
0.2
0.2
0.2
0.2
54
48
28
0
80
54
55
55
96
59
52
14
96
96
95
95
—
—
—
—
—
—
—
—
2
46
52
72
100
20
46
45
45
2
7a-Cbz
7b-Cbz
7c-Cbz
7d-Cbz
7e-Cbz
7f-Cbz
7a-Boc
7b-Boc
7c-Boc
7a-4Ns
7b-4Ns
7c-4Ns
7a-Ts
15
7
8
11.5
29
7.5
11
5.4
14
19.5
19
58.5
29.5
29
46
54
47
57
14
74
42
40
30
19
53
30
51
88a
74a
26
31
43
2
3
5
15
17
43
2
1
0
3
2
Taking into account that the nitrilase NIT-107 showed the best
results, we decided to limit the screening tests using only this
enzyme at different concentrations of substrate (0.1 and 0.05 M).
The results are depicted in Table 6 (entries 3 and 4). The best con-
ditions for the biotransformation of 7a-Cbz into the corresponding
amino acid 9a-Cbz were obtained with the nitrilase NIT-107, at a
substrate concentration of 0.05 M, without any cosolvent
(yield = 100%).
On the basis of these screening results, the optimized transfor-
mation protocols were established to synthesize 7a-Cbz on a
preparative scale with the appropriate nitrilase in high enantio-
meric purity. The reactions were monitored by HPLC and stopped
at the time established by the catalytic activity of enzyme. As
expected, the biotransformation by the NIT-107 into the corre-
sponding N-Cbz-b-amino acid 7a-Cbz was complete without any
formation of the amide. The yield of 98% obtained was determined
after isolation by extraction and chromatographic purification (see
Section 4).
7b-Ts
7c-Ts
26
a
Overall yield from 2 in four steps.
ing scale. When UV-detection was not possible, Micromass ZQ
detection was used.
Initially, all the amino nitriles 7a–f-Cbz (Scheme 2) were sub-
jected to biotransformations on a screening level employing 12
nitrilases. The reactions were monitored by HPLC and for better
comparability, all screening experiments were stopped at the time
of the expected maximum conversion of the Cbz-b-amino nitriles
7a–f-Cbz into the corresponding Cbz-b-amino acids 9a–f-Cbz
(after 18 h).
The initial biotransformation screening tests were achieved
according to a modification of a protocol from the literature.13m,o
In our study, the b-amino nitriles 7a–f-Cbz (0.2 M) were subjected
to a biotransformation on a screening level with twelve nitrilases
(NIT-101–NIT-112)15 in a phosphate buffer (pH 7) and DMSO or
methanol as cosolvent when necessary at 30 °C.
On the basis of screening results, the reaction protocols were
established for each b-amino nitrile. Therefore, new conditions
were tested with each nitrilase (NIT-101–NIT-112) and different
parameters were studied. The concentration of the substrate rang-
ing of 0.2, 0.1, and 0.05 M was evaluated. On the other hand, the
nature and concentration of the cosolvent (DMSO or methanol)
at 3%, 4%, 5%, 10%, and 60% were also screened.
The first biocatalysis results dealing with the formation of
N-Cbz-b-amino acids 9a–b-Cbz from the corresponding N-Cbz-b-
amino nitriles 7a–b-Cbz with the different purified nitrilases are
shown in Table 6.
An unexpected result was obtained for the N-Cbz-b-amino ni-
trile 7c-Cbz derived from the
L-isoleucine which showed a lower
affinity for the purified nitrilases. The best rate of biotransforma-
tion of 7c-Cbz into the corresponding acid 9c-Cbz was observed
using the NIT-107, methanol as cosolvent (5%) at a 0.1 M concen-
tration of substrate (yield = 5.2%).
The biotransformation reactions of 7b-Cbz were accompanied
by the formation of the corresponding amide 8b-Cbz (up to 43%
for NIT-107). Such a nitrile hydratase activity has been noticed in
the past and principally described for
a
-amino nitriles.13j,m,26 Re-
cently, Sheldon et al. have suggested a rational mechanism for
amide formation.27
The specific rotations of 9a-Cbz and 9b-Cbz were measured and
were in agreement with the data reported in the literature.28
Hence, the absolute configuration of these compounds could be
assigned as the (S)-isomer by comparison with the reported spe-
cific rotation value (see Section 4). These results clearly show that
there is no racemization during the reaction sequences.
In the case of the N-Cbz-b-amino nitriles 7d–e-Cbz, the results
were very dissimilar. The first problem we had to face was the low
solubility of the substrates. The insolubility of nitrile substrates in
The best results of the biotransformation screenings for the
substrates 7a-Cbz and 7b-Cbz (0.2 M) were obtained for NIT-104,
NIT-107, NIT-108, NIT-109, NIT-110, and NIT-112 without any
cosolvent (Table 6, entries 1, 2, 5–8). It was noticed that the homo-
geneity of the media was of primary importance, rendering the use
of a cosolvent unnecessary.
R
O
R
O
R
NC
Cbz
Cbz
NIT 101 - 112
Cbz
HO
N
H
N
H
H2N
N
H
8a-f-Cbz
9a-f-Cbz
7a-f-Cbz
Scheme 2. Biotransformation of b-amino nitriles 7a–f-Cbz into amides and into carboxylic acids.

2082
M.S.-I. Veitía et al. / Tetrahedron: Asymmetry 20 (2009) 2077–2089
aqueous reaction media decreased the enzymatic reaction time. A
lot of remarks dealing with the substrate solubility and the cosol-
vent compatibility in the biotransformation of nitriles is noticed in
the literature.29 However, this knowledge is hardly applicable in an
unexplored area, such as the microbial transformation of b-amino
nitriles.13h In our study, the nature and amount of the cosolvent
had to be developed.
New conditions were tested for each nitrilase (NIT-101–NIT-
112) requiring us to reestablish the concentration of the substrate
and the cosolvent. Unfortunately, in spite of the high number of
biotransformation reactions performed, no results were obtained
for the Cbz-b-amino nitriles 7e-Cbz and 7f-Cbz. On the other hand,
among more than forty screening tests carried out for 7d-Cbz, only
a poor biotransformation into the expected acid 9d-Cbz was ob-
served (2–5.5%) using the NIT-106.
At this stage in order to control the enantiospecificity of each
nitrilases, we studied the same reaction using two racemic nitriles
7b-Cbz and 7d-Cbz. No biotransformation results were obtained
with ( )-7d-Cbz. These first results of biotransformation suggested
to us that Cbz-b-amino nitriles in the aromatic series were not suit-
able substrates for the purified nitrilases tested. The hydrolysis of
( )-7b-Cbz by the NIT-107 gave 23% of 9b-Cbz which corresponds
Cbz have been successfully biotransformed. In spite of the b-amino
nitriles with sulfonamide-like as well as diphenylphosphinyl-like
N-protecting groups not being transformed by these nitrilases,
their study contributes to a better understanding of nitrilase sub-
strate specificities.
4. Experimental section
4.1. General
4.1.1. Chemicals
All reagents were obtained from commercial sources unless
otherwise noted, and used as received. All reactions were moni-
tored by thin layer chromatography (TLC) performed on precoated
silica gel plates (60 F254, Merck). TLC plates were viewed under UV
(254 nm) and developed with ninhydrine or in an iodine chamber;
frontal retention values Rf have been mentioned when necessary.
Flash chromatography was performed on Silica Gel 60 (particle size
0.063–0.200 mm, Merck). The enzymes NIT-101–NIT-112 were
purchased from a commercial supplier.15 The enzymes used were
delivered with the following specifications: NIT-101 (9 U/mg
solid), NIT-102 (17 U/mg solid); NIT-103 (2.4 U/mg solid); NIT-
104 (3.1 U/mg solid); NIT-105 (5.8 U/mg solid); NIT-106 (65 U/
mg solid); NIT-107 (2.8 U/mg solid); NIT-108 (12 U/mg solid);
NIT-109 (19 U/mg solid); NIT-110 (19 U/mg solid); NIT-111
(2.2 U/mg solid); and NIT-112 (11 U/mg solid).
to the expected L-isomer.
Next, we studied the biotransformation of other N-protected-b-
amino nitriles previously synthesized. As a result of the protocols
previously established for N-Cbz-b-amino nitriles 7a-Cbz and 7b-
Cbz (NIT-107, 0.05 M without cosolvent), the N-protected-b-amino
nitriles 7a–c-Boc, 7a–c-Ts, 7a–c-4Ns, and 7a,c-Dpp were also sub-
mitted to the biotransformation screenings.
4.1.2. Physical measurements
1H and 13C-NMR spectra were acquired on a Bruker BioSpin
GmbH spectrometer 400 MHz, at room temperature. Chemical
shifts d are given in ppm and coupling constants J are measured
in hertz. Coupling patterns are described by abbreviations: s (sin-
glet), d (doublet), t (triplet), dd (doublet of a doublet), m (multi-
plet). GC analyses and EI-mass spectra were performed with an
Agilent 6890N instrument equipped with a 15 m ꢀ 0.25 mm HP-
5MS column and an Agilent 5973 N MS detector-column tempera-
ture gradient 50–300 °C for compounds 5c-Cbz, 5a-Dpp, 5e-Dpp,
and 7(a–c)-Cbz; gradient 60–300 °C for compound 6a-Boc; gradi-
ent 80–300 °C for compounds 3e, 3f, 4e-Cbz, 4a-Ns, 5(d-f)-Cbz,
5d-Dpp, 6c-Cbz, 6f-Cbz, 6b-Boc, 6b-Ts, 7(e–f)-Cbz, 7c-Boc, and
7c-Ts, gradient 130–300 °C for compounds 4(b–c)-Ns, and gradient
160–300 °C for compounds 6a-Ts and 6c-Ts. Infrared spectra were
measured as KBr discs with a Nicolet FT-IR Avatar 320 spectrome-
ter. High resolution mass spectra were performed with a Thermo
Scientific LTQ Orbitrap mass spectrometer. The mass spectra were
taken using electrospray (ESI) in the positive-ion mode. The m/z
resulting from fragmentation processes were indicated, and some-
times assigned; the corresponding ionic abundances were reported
in percentage relative to the more abundance. Specific rotations
were determined on a Perkin Elmer 241. Melting points were
determined on a Leica VMHB system Kofler apparatus. The HPLC
analyses were performed using different columns. The chemical
purity was determined using either a normal phase column
Hypersil Si60 or Lichrosorb Si 60 or Krom Si 250, AME-OC
Once more, the biocatalytic conditions had to be newly estab-
lished. We were again confronted with the low solubility of the
substrates; as a result DMSO and methanol were tested as cosol-
vents in 5%, 10%, and 20%. Under these new screening conditions,
the nitrilases were used as cocktails: cocktail 1 (NIT-101–NIT-
104), cocktail 2 (NIT-105–NIT-108), and cocktail 3 (NIT-109–NIT-
112). Unfortunately, no biotransformation into the corresponding
acids was observed under the newly explored conditions.
The nature of the N-protecting groups of b-amino nitriles on
nitrilase activity has been investigated by a few authors.13h The
protecting functional group could be a tool to modulate the sub-
strate acceptance and selectivity of an enzyme. In terms of the
requirements mentioned above, an alkoxycarbonyl N-protecting
group, in particular the benzyloxycarbonyl group would be prefer-
able. The b-amino nitriles N-protected by sulfonyl amide group did
not undergo enzymatic hydrolysis with the nitrilases investigated.
These results could be attributed to the inherent structural features
of the substrates, especially the high substrate specificity of nitri-
lases regarding the nature of the protecting groups such as sulfon-
amides and aromatic groups.
Since there has not been an X-ray analysis of the nitrilases avail-
able, so that the actual structure of their active site remains
unknown, such a difference in substrate reactivity cannot be easily
rationalized. The same is true for the substrate specificity of NIT-
107 toward b-amino nitriles 7a–c-Cbz resulting in remarkably dif-
ferent biotransformation results.
110205 (250 mm ꢀ 4.6 mm, 5
lm stationary phase) or with a
reverse phase column (Hypersil ODS-C18, 5
lm stationary phase,
3. Conclusion
150 mm ꢀ 4.6 mm). The HPLC analyses for enantiomeric purity
were performed with a chiral normal phase (Column AS chiralpak,
The efficient preparation of b-amino nitriles was described by
two procedures featuring a maximum of four steps from the corre-
250 mm ꢀ 4.6 mm, 10
for the study were carried out with a reverse phase Column
(YMC ODS AQ 2185, 250 mm, 5 m) in an isocratic system of elu-
lm stationary phase). The HPLC analyses
sponding commercially available
a
-amino acids. Both the effi-
l
ciency and enantioselectivity of the commercial nitrilases used
for the b-amino nitriles hydrolysis were strongly dependent upon
the nature of the N-protecting group. The nitriles from the aliphatic
tion using a Photodiode Array Detector (PDA) Waters 996 (220–
350 nm) or using a Waters Micromass ZQ detector (ESI/APCI
source). The retention times tR are expressed in minutes in the dec-
imal system.
a-amino acids with the protecting benzyloxycarbonyl group 7a–b-

M.S.-I. Veitía et al. / Tetrahedron: Asymmetry 20 (2009) 2077–2089
2083
4.2. General procedure for the synthesis of chiral 2-substituted
aziridines 3 (Mitsunobu reaction)
4.3.2. (S)-Benzyl-2-(4-tert-butoxybenzyl)aziridine-1-carboxylate
5e-Cbz
The aziridine 3e (0.29 g, 1.41 mmol) led to the expected N-Cbz-
aziridine 5e according to the general procedure previously
described. Rf = 0.3 (CH2Cl2/MeOH 96:4); GC: tR = 10.75; m/z (EI):
339 (M), 192 (48), 148 (14), 131 (45), 107 (22), 91 (100), 77 (8),
65 (7).
The amino alcohol was dissolved in toluene (1 mL per mmol)
and added dropwise to a solution of triphenylphosphine (1 equiv)
and DEAD (1 equiv) in toluene at 0 °C. After completion, the
reaction mixture was poured into water and diluted with diethyl
ether. The layers were separated, and the organic layer was dried
over anhydrous MgSO4, filtered, and concentrated. The crude
residue was diluted with diethyl ether and kept in a freezer
overnight. Precipitated triphenylphosphine oxide was filtered off.
The precipitation step was repeated and the combined filtrates
were concentrated in vacuo and purified by flash column chroma-
tography on silica gel to afford the corresponding aziridines 3.
Yields: (S)-3a, 25%; (S)-3b, 35%; (S)-3c, 60%; (S)-3d, 53%; (S)-3e,
43%; (S)-3f, 30%.
4.3.3. (S)-Benzyl 2-(4-(benzyloxy)benzyl)aziridine-1-
carboxylate 5f-Cbz
Aziridine 3f (0.27 g, 1.1 mmol) led to the expected N-Cbz-aziri-
dine 5f according to the general procedure previously described.
Rf = 0.52 (CH2Cl2/MeOH 96:4); GC: tR = 11.69; m/z (EI): 283 (5);
207 (3), 197 (16), 107(4), 91 (100), 77 (2), 65 (6).
4.4. General procedure for the synthesis of chiral 2-substituted
N-Dpp aziridines 5-Dpp
4.2.1. (S)-2-(4-tert-Butoxybenzyl) aziridine 3e
Amino alcohol 2e (1.13 g, 5.07 mmol) led to the expected azi-
ridine 3e after heating at reflux for 18 h according to the general
procedure previously described. The crude product was purified
by flash column chromatography on silica gel (CH2Cl2/MeOH
96:4) to afford the aziridine 3e as a yellow powder. Yield:
440 mg (43%); Rf = 0.12 (CH2Cl2/MeOH 96:4); GC: tR = 6.52; 1H
NMR (CDCl3, 400 MHz): d = 1.26 (s, 9H), 1.37 (d, 1H, J = 3.5 Hz),
1.74 (d, 1H, J = 6 Hz), 2.10–2.17 (m, 1H), 2.56 (dd, 1H,
J = 14.6 Hz and J = 5.5 Hz), 2.68 (dd, 1H, J = 14.6 Hz and
J = 6 Hz), 6.85 (d, 2H, J = 8.5 Hz), 7.07 (d, 2H, J = 8.5 Hz); 13C
NMR (CDCl3, 100 MHz): d = 24.8, 28.8, 30.9, 39.3, 78.2, 124.5,
129.1, 133.8, 153.7; m/z (EI): 205 (2), 149 (26), 107 (100), 91
To a solution of the corresponding amino alcohols 2 with Et3N
(3 equiv) in THF (3 mL per mmol) at 0 °C was added diphenylphos-
phinyl chloride (2 equiv). The resulting mixture was stirred for
20 h at 25 °C. Next, a suspension of NaH 60% in oil (5 equiv) was
added and the solution was stirred for 20 h. Then 0.5 mL of water
was added and the salts were filtered over Na2SO4. The crude res-
idue was washed with EtOAc (3 ꢀ 25 mL) and filtered on silica gel.
The organic layers were combined and concentrated to afford the
corresponding N-Dpp aziridines 5-Dpp. Yields: (S)-5a-Dpp, 55%;
(S)-5c-Dpp, 76%; (S)-5d-Dpp, 67%; (S)-5e-Dpp, 74%.
4.4.1. (S)-2-(4-tert-Butoxybenzyl)-1-(diphenylphosphoryl)aziridine
5e-Dpp
(9), 77 (8), 65 (5); IR (KBr):
m 3030, 2975, 1505, 1233, 1159
1063 cmꢁ1; HRMS: calcd for C13H19 NO [M+H]+ (206.15394);
found (206.15384).
The O-tBu-tyrosinol 2e (1.0 g, 4.5 mmol) led to the expected N-
Dpp-aziridine 5e-Dpp according to the general procedure previ-
ously described. Yield: 1.1 g (74%); mp 77–78 °C; ½a D20
¼ ꢁ6:5 (c
ꢂ
4.2.2. (S)-2-(4-(Benzyloxy)benzyl)aziridine 3f
5, CH2Cl2); 1H NMR (CDCl3, 400 MHz): d = 1.29 (s, 9H), 1.99 (dd,
1H, J = 12.5 Hz and J = 3.5 Hz), 2.56 (dd, 1H, J = 12.5 Hz and
J = 5.5 Hz); 2.77–2.81 (m, 2H), 2.95–3.00 (m, 2H), 6.80–6.79 (d,
2H, J = 8.6 Hz), 6.93–6.98 (d, 2H, J = 8.6 Hz), 7.33–7.55 (m, 6H),
7.89–7.95 (m, 4H); 13C NMR (CDCl3, 100 MHz): d = 28.9, 29.1 (d,
J = 6.4 Hz), 36.2 (d, J = 6.39 Hz), 37.0 (d, J = 4.8 Hz), 78.3, 124.2,
129.1, 128.3, 128.4, 128.5, 131.5, 131.6, 131.7, 131.7, 131.8,
131.8, 132.1 (d, J = 22.1 Hz), 133.4 (d, J = 22.1 Hz), 132.5,
Amino alcohol 2f (1.31 g, 5 mmol) led to the expected aziridine
3f after heating at reflux for 22 h according to the general proce-
dure previously described. The crude product was purified by flash
column chromatography on silica gel (CH2Cl2/MeOH 96:4) to af-
ford the aziridine 3e as a yellow oil. Yield: 365 mg (30%);
Rf = 0.16 (CH2Cl2/MeOH 96:4); GC: tR = 9.23; 1H NMR (CDCl3,
400 MHz): d = 1.06 (s, 1H, N–H), 1.44 (d, 1H, J = 3.5 Hz), 1.80 (d,
1H, J = 6 Hz), 2.16–2.21 (m, 1H), 2.62 (dd, 1H, J = 14.6 Hz and
J = 6.0 Hz), 2.75 (dd, 1H, J = 14.6 Hz and J = 6 Hz), 5.05 (s, 2H),
6.93 (d, 2H, J = 8.6 Hz), 7.18 (d, 2H, J = 9 Hz), 7.30–7.46 (m, 5H);
13C NMR (CDCl3, 100 MHz): d = 24.7, 31.1, 39.1, 70.1, 115.1,
127.5, 128.0, 128.6, 129.8, 131.3, 137.2, 157.5 ppm; m/z (EI): 239
153.8 ppm; m/z (ESI+): 406 (M+H) (100); IR (KBr):
m 2963, 2930,
1152 (P@O) cmꢁ1 HRMS: calcd for C25H28NO2P [M+H]+
;
(406.18577); found (406. 18586).
4.5. General procedure for the synthesis of chiral N-Cbz amino
(9), 197 (18), 91 (100), 77 (2), 65 (8); IR (KBr):
m
3031, 2991,
alcohols 4
2906, 1509, 1233, 1175, 1016 cmꢁ1; HRMS: calcd for C16H17NO
[M+H]+ (240.13101); found (240.13111).
To a solution of the corresponding amino alcohols 2 in CH2Cl2
(3.3 mL per mmol) at 0 °C was added dropwise benzyl chlorofor-
mate (1.2 equiv), followed by Et3N (5 equiv). The resulting mixture
was stirred at room temperature (monitoring by GC and TLC). After
concentration, the crude residue was purified by flash column
chromatography on silica gel to afford the corresponding N-Cbz
amino alcohols 4-Cbz. Yields: (S)-4a-Cbz, 60%; (S)-4b-Cbz, 82%,
(S)-4c-Cbz, 55%; (S)-4d-Cbz, 92%; (S)-4e-Cbz, 50%, (S)-4f-Cbz, 94%.
4.3. General procedure for the synthesis of chiral 2-substituted
N-Cbz aziridines 5-Cbz
Benzyl cyanoformate (1 equiv) was added to a solution of the
appropriate 2-substituted aziridine 3 in acetonitrile (2 mL). The
reaction mixture was stirred at room temperature and controlled
by GC-MS. After 5 min of stirring, the solvent was removed in va-
cuo to give the crude 5-Cbz which was used directly in the next
step.
4.5.1. (S)-Benzyl 1-(4-tert-butoxyphenyl)-3-hydroxypropan-2-
ylcarbamate 4e-Cbz
The O-tBu-tyrosinol 2e (5.36 g, 15.03 mmol) led to the expected
N-Cbz amino alcohol 4e-Cbz after heating at reflux for 3 h accord-
ing to the general procedure previously described. The crude resi-
due was purified by flash column chromatography on silica gel
(CH2Cl2/MeOH 96:4) to afford 4e-Cbz as a pale white solid. Yield:
2.7 g (50%); mp 121–123 °C; Rf = 0.32 (CH2Cl2/EtOAc 96:4); GC:
4.3.1. (S)-Benzyl-2-benzylaziridine-1-carboxylate 5d-Cbz
Aziridine 3d (0.28 g, 2.1 mmol) led to the expected N-Cbz-aziri-
dine 5d according to the general procedure previously described.
Rf = 0.16 (CH2Cl2/MeOH 96:4); GC: tR = 9.10; m/z (EI): 267 (1);
176 (9), 132 (11), 91 (100), 77 (15); 65 (8).

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M.S.-I. Veitía et al. / Tetrahedron: Asymmetry 20 (2009) 2077–2089
tR = 13.4; 1H NMR (CDCl3, 400 MHz): d = 1.27 (s, 9H), 2.79 (d, 2H,
J = 4 Hz), 3.54 (m, 1H), 3.64 (m, 1H), 3.85–3.95 (m, 1H), 5.09 (s,
1H), 6.90 (d, 2H, J = 8 Hz), 7.05 (d, 2H, J = 8.1 Hz), 7.30–7.36 (m,
5H);13C NMR (CDCl3, 100 MHz): d = 28.9, 36.7, 54.2, 63.9, 66.8,
78.4, 124.2, 128.0, 129.7, 136.4, 140.1, 154.0; m/z (EI): 357(M),
194 (23), 150 (29), 107(90), 91(100); m/z (ESI+): 380.1 [M+23]
was purified by flash column chromatography on silica gel (cyclo-
hexane/EtOAc 1:1) to afford 4b-4Ns as a white solid. Yield: 2.27 g
(70%); mp: 120–122 °C; Rf = 0.27 (cyclohexane/EtOAc 1:1);
½
a 2D0
ꢂ
¼ þ17:6 (c 1.0, MeOH); GC: tR = 10.6; 1H NMR (CDCl3,
400 MHz): d = 0.82 (d, 3H, J = 6.8 Hz), 0.83 (d, 3H, J = 6.9 Hz),
1.77–1.90 (m, 1H), 3.11–3.19 (m, 1H), 3.58 (dd, 1H, J = 4.1 Hz and
J = 11.3 Hz); 3.63 (dd, 1H, J = 5.4 Hz and JB–A = 11.2 Hz), 8.08 (m,
2H), 8.36 (m, 2H); 13C NMR (CDCl3, 100 MHz): d = 18.7, 19.3,
29.8, 61.6, 62.9, 124.5, 128.5, 146.9, 150.1; m/z (EI): 257(100),
(100); IR (KBr):
m
3416, 3338 (N–H), 2929, 1695 (C@O), 1507,
;
1233, 1156 cmꢁ1
HRMS: calcd for C21H27NO4 [M+H]+
(358.194009); found (358.194209).
245 (19), 186 (14), 122 (25); IR (KBr):
m 3534, 3166 (N–H), 2959,
4.5.2. (S)-Benzyl 1-(4-(benzyloxy)phenyl)-3-hydroxypropan-2-
ylcarbamate 4f-Cbz
2926, 1524, 1355 (masSO2), 1166 (m
sSO2) cmꢁ1; HRMS: calcd for
C11H16 N2O5SNa [M+Na]+ (311.06721); found (311.06728).
The O-Bn-tyrosinol 2f (0.7, 2.72 mmol) led to the expected N-
Cbz amino alcohol 4e-Cbz according to the general procedure pre-
viously described. The crude residue was purified by flash column
chromatography on silica gel (CH2Cl2/MeOH 96:4) to afford 4f-Cbz
as a white powder. Yield: 1 g (94%); Rf = 0.33 (CH2Cl2/MeOH 96:4);
4.6.3. N-(2S)-1-Hydroxy-3-methylpentan-2-yl-4-nitrobenzene-
sulfonamide 4c-4Ns
The isoleucinol 3c (1.32 g, 11.22 mmol) led to the expected
N-4Ns amino alcohol 4c-4Ns after heating at reflux for 5 h accord-
ing to the general procedure previously described. The crude resi-
due was purified by flash column chromatography on silica gel
(cyclohexane/EtOAc 1:1) to afford 4c-4Ns as a white powder. Yield:
2.71 mg (78%); mp: 125–127 °C; Rf = 0.25 (cyclohexane/EtOAc
GC: tR = 11.69; ½a D24
ꢂ
¼ ꢁ13:3 (c 0.33, CHCl3); 1H NMR (CDCl3,
400 MHz): d = 2.52 (s, 1H, OH), 2.80 (d, 2H, J = 7 Hz), 3.56 (d, 1H,
J = 9.5 Hz), 3.65 (d, 1H, J = 9.5 Hz), 3.91 (m, 1H), 5.04 (s, 2H), 5.08
(s, 2H), 6.90 (d, 2H, J = 8.5 Hz), 7.11 (d, 2H, J = 8 Hz), 7.31–7.45
(m, 10H);13C NMR (CDCl3, 100 MHz): d = 37.1, 54.0, 54.9, 64.5,
67.4, 115.6, 128.1, 128.5, 128.6, 128.7, 129.1, 129.2, 130.9, 137.0,
137.7, 157.1, 158.2; m/z (EI): 281 (5); 207 (33), 107 (6), 91 (100),
1:1); ½a 2D0
ꢂ
¼ þ9:7 (c 1.0, MeOH); GC: tR = 11.1; 1H NMR (CDCl3,
400 MHz): d = 0.81 (m, 6H), 0.96–1.07 (m, 1H), 1.37–1.47 (m,
1H), 1.52–1.59 (m, 1H), 1.82 (sb, 1H, OH), 3.20–3.26 (m, 1H), 3.58
(dd, 1H, J = 3.9 Hz and J = 11.2 Hz), 3.63 (dd, 1H, J = 5.6 Hz and JB–
A = 11.2 Hz), 5.12 (d, 1H, J = 8 Hz, NH), 8.09 (m, 2H); 8.36 (m,
2H); 13C NMR (CDCl3, 100 MHz): d = 11.6, 15.3, 25.5, 36.7, 60.3,
62.4, 124.4, 128.5, 146.9, 150.1. m/z (EI): 271 (100), 245 (58), 215
(65), 186 (40), 122 (38), 76 (22); m/z (ESI+): 325.1 [M+23] (100);
77 (2); 65 (6); IR (KBr):
m 3464, 3311, 3056, 3031, 2949, 2921,
1687, 1541, 1156, 1008 cmꢁ1
; HRMS: calcd for C24H25 NO4
[M+H]+ (392.17835); found (392.17846).
4.6. General procedure for the synthesis of chiral N-4Ns
amino alcohols 4
IR (KBr):
m 3441, 3151 (N–H), 2961, 2915, 1530, 1350 (masSO2),
1159 (
m
sSO2) cmꢁ1; HRMS: calcd for C12H18 N2O5SNa [M+Na]+
A solution of p-nitrobenzenesulfonyl chloride (1.1 equiv) in THF
(1.9 mL per mmol) was added dropwise to a solution of the corre-
sponding amino alcohol and NaHCO3 (4 equiv) in dry THF (1.5 mL
per mmol) at 0 °C. The resulting mixture was stirred at room tem-
perature and monitored by GC and TLC. The solution was concen-
trated and the yellow solid obtained was taken up in EtOAc/H2O
(7:3 v/v, 100 mL). The layers were separated, and the aqueous
phase was back-extracted with EtOAc (3 ꢀ 100 mL). The combined
organic layers were dried over anhydrous Na2SO4, filtered, and
concentrated. The crude residue was purified by flash column
chromatography on silica gel to afford the corresponding N-4Ns
amino alcohols 4(a–c)-4Ns. Yields: (S)-4a-4Ns, 95%; (S)-4b-4Ns,
70%, (S)-4c-4Ns, 78%.
(325.08286); found (325.08279).
4.7. General procedure for the synthesis of N-protected amino
alcohol methanesulfonates 6
To a stirred solution of the corresponding N-protected amino
alcohols 4 and Et3N (3 equiv) in THF (4 mL per mmol) at 0 °C was
added dropwise methanesulfonyl chloride (1.1 equiv for 4a–e-
Cbz and 4a–b-Boc or 2.2 equiv for 4a–c-4Ns and 4a–c-Ts). The
reaction mixture was stirred at room temperature and monitored
by GC and TLC. The salts were filtered, the solution was concen-
trated, and the mixture was taken up in EtOAc/H2O (2:1, v/v,
15 mL per mmol). The layers were separated and the aqueous
phase was back-extracted with EtOAc. The combined organic lay-
ers were dried over anhydrous MgSO4, filtered, and concentrated
under reduced pressure to afford the corresponding N-protected
amino alcohol methanesulfonates 6, which were used in the next
step without any further purification. Yields: (S)-6a-Boc, 74%;
(S)-6b-Boc, 95%; (S)-6c-Boc, 99%; (S)-6a-Cbz 94%, (S)-6b-Cbz
77%; (S)-6c-Cbz, 98%; (S)-6d-Cbz, 70%; (S)-6e-Cbz, 94%; (S)-6f-
Cbz, 88%; (S)-6a-4Ns, 92%; (S)-6b-4Ns, 94%; (S)-6c-4Ns, 75%; (S)-
6a-Ts, 99%; (S)-6b-Ts, 99%; (S)-6c-Ts, 95%.
4.6.1. (S)-N-(1-Hydroxypropan-2-yl)-4-nitrobenzenesulfonamide
4a-4Ns
Alaninol 2a (843 mg, 11.22 mmol) led to the expected N-4Ns
amino alcohol 4a-4Ns after heating at reflux for 5 h according to
the general procedure previously described. The crude residue
was purified by flash column chromatography on silica gel (cyclo-
hexane/EtOAc 3:7) to afford 4a-4Ns as a pale white solid. Yield:
2.78 g (95%); mp: 121–123 °C; Rf = 0.31 (cyclohexane/EtOAc 3:7);
½
a 2D0
ꢂ
¼ ꢁ8:4 (c 1.0, MeOH); GC: tR = 9.32; 1H NMR (CD3OD,
400 MHz): d = 1.20 (d, 3H, J = 6.4 Hz), 3.48–3.50 (m, 3H), 8.15 (m,
2H), 8.44 (m, 2H); 13C NMR (CD3OD, 100 MHz): d = 14.5, 52.8,
66.8, 125.3, 129.4, 149.4, 151.3; m/z (EI): 229 (100), 186 (36),
4.7.1. (S)-2-(tert-Butoxycarbonylamino)propyl methanesulfonate
6a-Boc
The (S)-tert-butyl-1-hydroxypropan-2-ylcarbamate 4a-Boc
(0.86 g, 4.89 mmol) led to the expected compound 6a-Boc after
4 h of stirring according to the general procedure previously de-
scribed. Yellow powder, yield: 1.15 g (74%); Rf = 0.1 (cyclohex-
ane/EtOAc 7:3); GC: tR = 10.79; 1H NMR (CDCl3, 400 MHz):
d = 1.24 (d, 3H, J = 6.9 Hz), 1.46 (s, 9H), 3.05 (s, 1H), 3.94–4.05 (m,
1H), 4.16 (dd, 1H, J = 4.3 Hz and J = 10 Hz), 4.2 (dd, 1H, J = 3.3 Hz
and J = 10 Hz, 4.6 (sb, 1H, NH); 13C NMR (CDCl3, 100 MHz):
d = 17.6, 29.2, 28.9, 38.0, 72.7, 80.2, 155.5; m/z (EI): 180 (4), 144
122 (38); IR (KBr):
m 3441, 3386 (N–H), 2941, 2877, 1542, 1351
(masSO2), 1161 (m
sSO2) cmꢁ1; HRMS: calcd for C9H12N2O5SNa
[M+Na]+ (283.03591); found (283.03587).
4.6.2. (S)-N-(1-Hydroxy-3-methylbutan-2-yl)-4-
nitrobenzenesulfonamide 4b-4Ns
The valinol 2b (1.16 g, 11.22 mmol) led to the expected N-4Ns
amino alcohol 4b-4Ns after heating at reflux for 7 h according to
the general procedure previously described. The crude residue

M.S.-I. Veitía et al. / Tetrahedron: Asymmetry 20 (2009) 2077–2089
2085
(37), 57 (100); IR (KBr):
m
3359 (N–H), 2975, 2937, 1687 (C@O),
38.4, 46.2, 51.5, 71.6, 124.4, 128.2, 128.6, 129.7, 131.1, 136.3,
154.4, 155.7.
1335 ( asSO2), 1161 ( .
m
m
sSO2), 1003 cmꢁ1
4.7.2. (S)-2-(tert-Butoxycarbonylamino)-3-methylbutyl methane-
sulfonate 6b-Boc
4.7.7. (S)-3-(4-(Benzyloxy)phenyl)-2-(ethoxycarbonylamino)propyl
methanesulfonate 6f-Cbz
The
(S)-tert-butyl-1-hydroxy-3-methylbutan-2-ylcarbamate
(S)-Benzyl-1-(4-(benzyloxy)phenyl)-3-hydroxypropan-2-ylcar-
bamate 4f-Cbz (0.38 g, 0.97 mmol) led to the expected compound
6f-Cbz after 2.5 h of stirring according to the general procedure
previously described. White powder, yield: 0.4 g (88%); Rf = 0.75
(CH2Cl2/MeOH 96/4); GC: tR = 11.68; 1H NMR (CDCl3, 400 MHz):
d = 2.83 (dd, 1H, J = 14.6 Hz and J = 7.5 Hz), 2.88 (dd, 1H,
J = 14.6 Hz and J = 4.5 Hz), 2.95 (s, 3H), 4.12 (dd, 1H, J = 15.1 Hz
and J = 7 Hz), 4.15 (dd, 1H, J = 15.1 Hz and J = 3.5 Hz), 4.24–4.26
(m, 1H), 4.92 (sb, 1H, N–H), 5.04 (2H, s), 5.09 (s, 2H), 6.91 (d, 2H,
J = 8.5 Hz), 7.10 (d, 2H, J = 8.6 Hz), 7.31–7.44 (m, 10H); 13C NMR
(CDCl3, 100 MHz): d = 37.6, 52.1, 60.7, 67.3, 69.8, 70.4, 115.6,
127.8, 128.3, 128.5, 128.6, 128.9, 128,9, 130.6, 136.8, 137.3,
156.0, 158.3; m/z (EI): 283 (5), 197 (20), 107 (10), 91 (100), 77
4b-Boc (1.0 g, 4.92 mmol) led to the expected compound 6b-Boc
after 1 h of stirring according to the general procedure previously
described. Yellow powder, yield: 1.32 g (95%); Rf = 0.43 (cyclohex-
ane/EtOAc 7:3); GC: tR = 6.94; 1H NMR (CDCl3, 400 MHz): d = 1.05
(d, 1H, J = 6.9 Hz), 1.10 (d, 1H, J = 6.8 Hz), 1.42 (s, 9H), 2.08–2.17
(m, 1H), 3.20 (s, 1H), 4.38–4.49 (m, 3H); 13C NMR (CDCl3,
100 MHz): d = 28.1, 29.7, 32.6, 37.4, 56.1, 66.6, 79.5, 160.3; m/z
(EI): 281 (M), 172 (10), 57 (100); IR (KBr):
2876, 1687 (C@O), 1356 ( asSO2), 1174 ( .
m 3395 (N–H), 2966,
m
m
sSO2) cmꢁ1
4.7.3. (2S)-2-(tert-Butoxycarbonylamino)-3-methylpentyl
meth- anesulfonate 6c-Boc
The tert-butyl-(2S)-1-hydroxy-3-methylpentan-2-ylcarbamate
4c-Boc (0.9 g, 4.14 mmol) led to the expected compound 6c-Boc
after 5.5 h of stirring according to the general procedure previously
described. Yellow powder, yield: 1.21 g (99%); Rf = 0.67 (cyclohex-
ane/EtOAc 7:3); GC: tR = 7.44; 1H NMR (CDCl3, 400 MHz): d = 0.86–
0.98 (m, 8H), 1.09–1.25 (m, 2H), 1.43 (s, 9H), 3.02 (s, 3H), 3.67–3.71
(m, 1H), 4.15–4.32 (m, 2H); 13C NMR (CDCl3, 100 MHz): d = 11.1,
15.6, 25.2, 28.2, 35.5, 37.4, 53.8, 69.7, 155.4; m/z (EI): 238 (2),
(2); 65 (11); IR (KBr):
m
3384 (N–H), 3031, 2958, 2929, 2872,
1696 (C@O), 1344 ( asSO2), 1180 (
m
m
sSO2) cmꢁ1
.
4.7.8. (S)-2-(4-Nitrophenylsulfonamido)propyl methanesulfonate
6a-4Ns
(S)-N-(1-Hydroxy
propan-2-yl)-4-nitrobenzenesulfonamide
4a-4Ns (1.0 g, 3.84 mmol) led to the expected compound 6a-4Ns
after 4.5 h of stirring according to the general procedure previously
described. Yellow powder, yield: 1.2 g (92%); Rf = 0.7, Al2O3, (cyclo-
hexane/EtOAc 1:1); 1H NMR (DMSO-d6, 400 MHz): d = 0.97 (d, 3H,
J = 6.7 Hz), 3.11 (s, 3H), 3.52–3.62 (m, 1H), 4.00 (dd, 1H, J = 5.9 Hz
and J = 10.2 Hz), 4.04 (dd, 1H, J = 4.9 Hz and J = 10.3 Hz), 8.05–
8.08 (m, 2H), 8.41 (m, 2H); 13C NMR (DMSO-d6, 100 MHz):
d = 17.2, 36.6, 48.4, 72.2, 124.6, 127.9, 147.1, 149.5 ppm; m/z
186 (5), 138 (43), 86 (49), 57 (100); IR (KBr):
m 3339 (N–H),
2965, 2875, 1677 (C@O), 1291 ( asSO2), 1174 ( .
m
m
sSO2) cmꢁ1
4.7.4. (2S)-2-(Benzyloxycarbonylamino)-3-methylpentyl
methanesulfonate 6c-Cbz
The benzyl (2S)-1-hydroxy-3-methylpentan-2-ylcarbamate 4c-
Cbz (1 g, 3.9 mmol) led to the expected compound 6c-Cbz after
1.5 h of stirring according to the general procedure previously de-
scribed. Yellow oil, yield: 1.17 g (89%); Rf = 0.94 (CH2Cl2/MeOH
97:3); GC: tR = 7.03; 1H NMR (CDCl3, 400 MHz): d = 0.96 (d, 3H,
J = 7.0 Hz), 0.98 (d, 3H, J = 7.1 Hz, 1.84–1.93 (m, 1H), 2.95 (s, 3H),
3.68–3.74 (m, 1H), 4.28 (s, 2H), 4.84 (sb, 2H, NH), 5.08–5.15 (m,
2H), 7.28–7.40 (m, 5H); 13C NMR (CDCl3, 100 MHz): d = 11.1, 15.4,
25.2, 35.5, 37.3, 54.4, 66.9, 69.4, 128.8, 136.34, 156.1; m/z (EI): 233
(ESI+): 361.1, M+23 (100), IR (KBr):
m 3062, 2083, 2894, 1360 (mas-
SO2), 1169 (
m
sSO2) cmꢁ1
.
4.7.9. (S)-3-Methyl-2-(4-nitrophenylsulfonamido)butyl
methanesulfonate 6b-4Ns
(S)-N-(1-Hydroxy-3-methylbutan-2-yl)-4-nitrobenzenesulfona-
mide 4b-4Ns (1.0 g, 3.47 mmol) led to the expected compound
6b-4Ns after 2.5 h of stirring according to the general procedure pre-
viously described. Yellowpowder, yield: 1.2 g (94%); Rf = 0.78, Al2O3,
(cyclohexane/EtOAc 1:1); 1H NMR (CDCl3, 400 MHz): d = 0.85 (d, 3H,
J = 6.8 Hz), 0.88 (d, 3H, J = 6.8 Hz), 1.83–1.92 (m, 1H), 2.98 (s, 3H),
3.39 (m, 1H), 4.15 (dd, 1H, J = 10.7 Hz and J = 4.2 Hz); 4.18 (dd, 1H,
J = 5.1 Hz and J = 10.6 Hz), 8.08 (m, 2H), 8.35 (m, 2H); 13C NMR
(CDCl3, 100 MHz): d = 18.4, 19.1, 29.7, 37.4, 58.8, 69.2, 124.5,
128.4, 146.6, 150.2; m/z (ESI+): 361.1, M+23 (100).
(3), 176 (17), 91 (100), 77 (1); IR (KBr):
m
3327 (N–H), 3064, 3031,
2965, 2935, 2878, 1716 (C@O), 1355 ( asSO2), 1175 (
m
m
sSO2) cmꢁ1
.
4.7.5. (S)-2-(Ethoxycarbonylamino)-3-phenylpropyl
methanesulfonate 6d-Cbz
(S)-Benzyl-1-hydroxy-3-phenylpropan-2-ylcarbamate 4d-Cbz
(0.84 g, 2.95 mmol) led to the expected compound 6d-Cbz after
30 min of stirring according to the general procedure previously de-
scribed. Yellow powder, yield: 750 mg (70%); Rf = 0.67 (CH2Cl2/
MeOH 96:4); 1H NMR (CDCl3, 400 MHz): d = 2.79 (d, 2H, J = 7.0 Hz),
2.87 (s, 3H), 4.01–4.15 (m, 2H), 4.16–4.25 (m, 1H), 4.85–5.95 (sb,
1H), 7.05–7.35 (m, 10H); 13C NMR (CDCl3, 100 MHz): d = 37.1, 37.3,
51.4, 66.8, 69.5, 126.7, 128.1, 128.2, 128.6, 128.7, 129.4, 136.5,
4.7.10. (2S)-3-Methyl-2-(4-nitrophenylsulfonamido)pentyl
methanesulfonate 6c-4Ns
(N-((2S)-1-Hydroxy-3-methylpentan-2-yl)-4-nitrobenzenesulf-
onamide 4c-4Ns (570 mg, 1.89 mmol) led to the expected com-
pound 6c-4Ns after 5 h of stirring according to the general
procedure previously described. Yellow oil, yield: 541 mg (75%);
Rf = 0.4, SiO2, (CH2Cl2/MeOH 97/3); 1H NMR (CDCl3, 400 MHz):
d = 0.81 (t, 3H, J = 7.3 Hz), 0.86 (d, 3H, J = 6.9 Hz), 0.98–1.08 (m,
1H), 1.36–1.48 (m, 1H), 1.58–1.68 (m, 1H), 2.97 (s, 3H), 3.40–
3.47 (m, 1H), 4.09–4.20 (m, 2H), 8.08 (m, 2H), 8.35 (m, 2H); 13C
NMR (CDCl3, 100 MHz): d = 11.2, 15.1, 25.2, 36.5, 37.4, 57.5, 68.9,
137.8, 156.6; IR (KBr):
m
3347 (N–H), 3084, 3060, 2946, 1693
(C@O), 1536, 1349 ( asSO2), 1275 (
m
m
sSO2), 1186, 1067 cmꢁ1
.
4.7.6. (S)-3-(4-tert-Butoxyphenyl)-2-(ethoxycarbonylamino)-
propyl methanesulfonate 6e-Cbz
(S)-Ethyl-1-(4-tert-butoxyphenyl)-3-hydroxypropan-2-ylcarba-
mate 4e-Cbz (600 mg, 1.68 mmol) led to the expected compound
6e-Cbz after 10 min of stirring according to the general procedure
previously described. Yellow powder, yield: 638 mg (93%); Rf = 0.5
(cyclohexane/EtOAc 4:6); 1H NMR (CDCl3, 400 MHz): d = 1.26 (s,
9H); 2.88 (s, 2H), 3.13 (s, 3H), 4.04–4.08 (m, 2H), 4.15–4.25 (m,
1H), 5.01 (s, 1H), 6.85 (d, 2H, J = 8.5 Hz), 7.02 (d, 2H, J = 8.5 Hz),
7.24–7.34 (m, 5H); 13C NMR (CDCl3, 100 MHz): d = 28.8, 31.6,
124.5, 128.4, 146.6, 150.2; IR (KBr):
m 3293 (N–H), 2968, 2937,
1530, 1351 ( asSO2), 1169 ( .
m
m
sSO2) cmꢁ1
4.7.11. (S)-2-(4-Methylphenylsulfonamido)propyl methanesulfo-
nate 6a-Ts
(S)-N-(1-Hydroxy propan-2-yl)-4-methylbenzenesulfonamide
4a-Ts (1.5 g, 6.55 mmol) led to the expected compound 6a-Ts after

2086
M.S.-I. Veitía et al. / Tetrahedron: Asymmetry 20 (2009) 2077–2089
30 min of stirring according to the general procedure previously
described. Yellow oil, yield: 2.01 g (100%); Rf = 0.5, SiO2, (cyclohex-
ane/EtOAc 4/6); GC: tR = 7.40; 1H NMR (CDCl3, 400 MHz): d = 1.1 (d,
3H, J = 6.9 Hz), 2.41 (s, 3H), 2.99 (s, 3H), 3.54–3.65 (m, 1H), 4.1(d,
2H, J = 4.6 Hz), 5.31 (d, 1H, NH), 7.27–7.32 (m, 2H), 7.73–7.78 (m,
2H); 13C NMR (CDCl3, 100 MHz): d = 20.4, 21.6, 26.3, 46.2, 116.8,
127.0, 129.9, 137.9, 144.0; m/z (EI): 307 [M] (1), 198 (100), 155
up in EtOAc/H2O (4:1 v/v, 35 mL per mmol). The layers were sepa-
rated, and the aqueous phase was back-extracted with EtOAc. The
combined organic layers were dried over anhydrous MgSO4, fil-
tered, and concentrated. The crude residue was purified by flash
column chromatography on silica gel. Yields: (S)-7a-Cbz, 81%;
(S)-7b-Cbz, 85%; (S)-7c-Cbz, 87%; (S)-7d-Cbz, 88%; (S)-7e-Cbz,
30%; (S)-7f-Cbz, 89%; (S)-7a-Boc, 66%, (S)-7b-Boc, 43%, (S)-7c-Boc,
47%; (S)-7a-4Ns, 22%; (S)-7b-4Ns, 81%; (S)-7c-4Ns, 40%, (S)-6a-Ts,
73%; (S)-6b-Ts, 92%; (S)-6c-Ts, 94%.
(71), 91 (69) 56(33); IR (KBr):
m 3290 (N–H), 2975, 2942, 1350 (mas-
SO2), 1168 (
m
sSO2) cmꢁ1
.
4.7.12. (S)-3-Methyl-2-(4-methylphenylsulfonamido)butyl
methanesulfonate 6b-Ts
4.8.1. (R)-Benzyl 1-cyano-3-methylbutan-2-yl carbamate 7b-
Cbz
(S)-N-(1-Hydroxy-3-methylbutan-2-yl)-4-methylbenzenesulf-
onamide 4b-Ts (1.5 g, 5.83 mmol) led to the expected compound
6b-Ts after 30 min of stirring according to the general procedure
previously described. Pale white powder, yield: 1.96 g (100%);
Rf = 0.35, SiO2, (cyclohexane/EtOAc 40/60); GC: tR = 7.44; 1H NMR
(CDCl3, 400 MHz): d = 0.80 (d, 3H, J = 5.3 Hz), 0.82 (d, 3H,
J = 5.4 Hz), 1.79–1.90 (m, 1H), 2.41 (s, 3H), 2.95 (s, 3H), 3.17–3.27
(m, 1H), 4.10 (dd, 1H, J = 4.6 Hz and J = 10.4 Hz), 4.18 (dd, 1H,
J = 4.3 Hz and J = 10.4 Hz), 5.17 (d, 1H, NH), 7.29–7.31 (m, 2H),
7.75–7.77 (m, 2H); 13C NMR (CDCl3, 100 MHz): d = 18.2, 19.1,
21.6, 29.3, 37.2, 58.0, 69.1, 137.9, 127.2, 129.8, 137.6, 143.8; m/z
(EI): 226 (1), 155 (10), 91 (32), 84 (100), 65 (12), 55 (23); IR
Method A: To a stirred solution of the (S)-benzyl 2-isopropylaz-
iridine-1-carboxylate 5b-Cbz (1.4 g, 6.4 mmol) in CH3CN/H2O 9:1
(35 mL) was added NaCN (0.43 g, 12.8 mmol). The expected com-
pound 7b-Cbz is obtained after 21 h of stirring according to the
general procedure previously described. The crude residue was
purified by flash column chromatography on silica gel (CH2Cl2/
MeOH 7:3).
Method B: (S)-2-(Benzyloxycarbonylamino)-3-methylbutyl meth-
anesulfonate 6b-Cbz (0.9 g, 2.85 mmol) led to the expected com-
pound 7b-Cbz after 4 h of stirring according to the general proce-
dure previously described. The crude residue was purified by
flash column chromatography on silica gel (CH2Cl2/MeOH 7:3).
7b-Cbz was obtained as a yellow oil. Yield: 0.93 g (60%) method
A, 0.6 g (85%) method B; Rf = 0.68 (CH2Cl2/MeOH 97:3); GC:
tR = 10.52; 1H NMR (CDCl3, 400 MHz): d = 0.98 (d, 3H, J = 3.5 Hz),
1.00 (d, 3H, J = 3.5 Hz), 1.91 (m, 1H), 2.60 (dd, 1H, J = 12 Hz and
J = 4.5 Hz), 2.70 (dd, 1H, J = 12 Hz and J = 5 Hz), 3.65–3.71(m, 1H),
4.82–4.92 (sb, 1H, NH), 5.12 (s, 2H), 7.32–7.37 (m, 5H). 13C NMR
(CDCl3, 100 MHz): d = 18.4, 19.4, 21.8, 31.0, 53.3, 60.4, 67.2,
128.4, 136.004, 155.8; m/z (EI): 246 (M), 207 (1), 162 (2), 108
(KBr):
m 3430 (N–H), 3254, 2964, 2876, 1353 (masSO2), 1178 (msSO2)
cmꢁ1
.
4.7.13. (2S)-3-Methyl-2-(4-methylphenylsulfonamido)pentyl
methanesulfonate 6c-Ts
N-((2S)-1-Hydroxy-3-methylpentan-2-yl)-4-methylbenzene-
sulfonamide 4c-Ts (1.5 g, 5.53 mmol) led to the expected com-
pound 6c-Ts after 30 min of stirring according to the general
procedure previously described. Pale white powder, yield: 1.83 g
(95%); Rf = 0.48, SiO2, (cyclohexane/EtOAc 4/6); GC: tR = 8.91; 1H
NMR (CDCl3, 400 MHz): d = 0.78 (t, 3H; J = 7.4 Hz), 0.82 (d, 3H,
J = 6.9 Hz), 0.95–1.05 (m, 1H), 1.38–1.50 (m, 1H), 1.55–1.65 (m,
1H), 2.42 (s, 3H), 2.94 (s, 3H), 3.27–3.37 (m, 1H), 4.13 (dd, 1H,
J = 7.0 Hz, J = 10.4 Hz), 4.16 (dd, 1H, J = 5.7 Hz and JB–A = 10.1 Hz),
5.07 (d, 1H, NH), 7.28–7.33 (m, 2H), 7.74–7.76 (m, 2H); 13C NMR
(CDCl3, 100 MHz): d = 11.1, 15.1, 21.7, 24.97, 36.17, 37.3, 56.8,
68.8, 127.2, 129.9, 137.6, 143.8; m/z (EI): 292 (62), 240 (64), 155
(46), 91 (100), 79 (8); IR (KBr):
m
3326 (N–H), 3034, 2964, 2929,
HRMS: calcd for
2876, 2239 (C„N) 1704, (C@O) cmꢁ1
;
C14H18N2O2Na [M+Na]+ (269.12605); found (269.12585); HPLC
purity: method A: 85%; method B: 90% on a Krom Si 250 column,
n-heptane/dioxane 3:1, 1.0 mL minꢁ1, k = 256 nm, tR = 7,78 min.
4.8.2. Benzyl (2R)-1-cyano-3-methylpentan-2-ylcarbamate 7c-
Cbz
Method A: To a stirred solution of the (S)-benzyl 2-sec-butylaz-
iridine-1-carboxylate 5c-Cbz (2.08 g, 14.5 mmol) in CH3CN/H2O
9:1 (43.5 mL) was added NaCN (0.87 g, 21.75 mmol). The expected
compound 7c-Cbz was obtained after 16 h of stirring according to
the general procedure previously described. The crude residue was
purified by flash column chromatography on silica gel (CH2Cl2/
MeOH 7:3).
(100), 91 (95) 65(20); IR (KBr):
m 3430 (N–H), 3276, 2970, 2876,
1359, 1172 ( asSO2), 1156 ( .
m
m
sSO2) cmꢁ1
4.8. General procedure for the synthesis of N-protected amino
nitriles 7
Caution: Because of the high toxicity of sodium cyanide, all of
the processes should be carried out taking into account the safety
measures.
Method B: (2S)-2-(Benzyloxycarbonylamino)-3-methylpentyl
methanesulfonate 6c-Cbz (1.17 g, 3.54 mmol) led to the expected
compound 7c-Cbz after 4 h of stirring according to the general pro-
cedure previously described. The crude residue was purified by
flash column chromatography on silica gel (CH2Cl2/MeOH 7:3).
7c-Cbz was obtained as a yellow powder. Yield: 2.5 g (66%)
method A, 0.80 g (87%) method B; Rf = 0.32 (CH2Cl2/MeOH 97:3);
Method A: To a stirred solution of the corresponding N-pro-
tected aziridines 5 in a mixture of CH3CN/H2O 9:1 (3 mL per mmol)
was added NaCN (1, 1.5, 2 or 3 equiv). The mixture was stirred at
80 °C. Upon completion of the reaction, the mixture was taken
up in EtOAc/H2O (1.5:1 v/v, 20 mL per mmol). The layers were sep-
arated, and the aqueous phase was back-extracted with EtOAc. The
combined organic layers were dried over anhydrous MgSO4, fil-
tered, and concentrated. The crude residue was purified by flash
column chromatography on silica gel. Yields: (S)-7a-Cbz, 55%;
(S)-7b-Cbz, 60%; (S)-7c-Cbz, 66%; (S)-7d-Cbz, 81%; (S)-7e-Cbz,
89%; (S)-7f-Cbz, 73%.
½
a 2D0
ꢂ
¼ ꢁ43:6 (c 1.0, MeOH); GC: tR = 11.01; 1H NMR (CDCl3,
400 MHz): d = 0.92 (t, 3H, J = 7.5 Hz), 0.97 (d, 3H, J = 7 Hz), 1.14–
1.23 (m, 1H), 1.50–1.74 (m, 2H), 2.60 (dd, 1H, J = 17 Hz and
J = 5 Hz), 2.71 (dd, 1H, J = 17 Hz and J = 5 Hz), 3.71–3.78 (m, 1H),
4.91 (d, 1H, NH), 5.11 (s, 2H), 7.30–7.39 (m, 5H). 13C NMR (CDCl3,
100 MHz): 10.1, 15.3, 21.7, 26.9, 30.9, 36.2, 44.5, 50.9, 66.1,
127.6–127.8, 135.0, 154.7; m/z (EI): 260 (M), 245 (1), 220 (1),
Method B: To a stirred solution of the corresponding N-pro-
tected amino alcohol methanesulfonates 6 in DMF (1.4 mL per
mmol) was added NaCN (1 equiv for 7a–b-Cbz and 7e–f-Cbz or
1.5 equiv for 7c-Boc, 7a-Ns, and 7c-Ns). The mixture was stirred
at 70 °C and monitored by GC and TLC. The mixture was taken
176 (2), 108 (42), 91 (100); IR (KBr): m 3350 (N–H), 2930, 2885,
2240 (C„N) 1706, (C@O) cmꢁ1; HRMS: calcd for C15H20N2O2Na
[M+Na]+ (283.14170); found (283.14141); HPLC purity: method
A = 77%; method B = 80% on a Krom Si 250 column, n-heptane/
dioxane 3:1, 1.0 mL minꢁ1, k = 256 nm, tR = 7.08 min.

M.S.-I. Veitía et al. / Tetrahedron: Asymmetry 20 (2009) 2077–2089
2087
4.8.3. (S)-Benzyl 1-(4-tert-butoxyphenyl)-3-cyanopropan-2-
ylcarbamate 7e-Cbz
4.8.5. tert-Butyl (2R)-1-cyano-3-methylpentan-2-ylcarbamate
7c-Boc
Method A: To a stirred solution of the (S)-benzyl 2-(4-tert-but-
oxybenzyl)aziridine-1-carboxylate 5e-Cbz (290 mg, 1.4 mmol) in
CH3CN/H2O 9:1 (4.42 mL) was added NaCN (0.21 g, 4.23 mmol).
The expected compound 7e-Cbz was obtained after 18 h of stirring
according to the general procedure previously described. The crude
residue was purified by flash column chromatography on silica gel
(CH2Cl2/MeOH 7:3).
Method B: (S)-3-(4-tert-Butoxyphenyl)-2-(ethoxycarbonylami-
no)propyl methanesulfonate 6e-Cbz (683 mg, 1.48 mmol) led to
the expected compound 7e-Cbz after 2 h of stirring according to
the general procedure previously described. The crude residue
was purified by flash column chromatography on silica gel
(CH2Cl2/MeOH 7:3).
(2S)-2-(tert-Butoxy carbonylamino)-3-methylpentyl methane-
sulfonate 6c-Boc (1.0 g, 3.40 mmol) led to the expected compound
7c-Boc after 5.5 h of stirring according to the general procedure
previously described. The crude residue was purified by flash col-
umn chromatography on silica gel (cyclohexane/EtOAc 8:2) to give
7c-Boc as a white solid. Yield: 362 mg (47%); mp: 103–104 °C;
Rf = 0.53 (cyclohexane/EtOAc 8:2); ½a D20
¼ ꢁ4:5 (c 1.0, MeOH), GC:
ꢂ
tR = 5.52; 1H NMR (CDCl3, 400 MHz): d = 0.81 (t, 3H, J = 7.4 Hz),
0.95 (d, 3H, J = 6.7 Hz), 1.05–1.3 (m, 2H), 1.43 (s, 9H), 2.54 (dd,
1H, J = 16.9 Hz and J = 4.8 Hz); 2.68 (dd, 1H, J = 5.0 Hz and
J = 16.9 Hz), 3.62–3.69 (m, 1H), 4.69 (db, 1H, NH); 13C NMR (CDCl3,
100 MHz): d = 11.1, 15.6, 21.7, 25.3; 28.4, 37.4, 51.5, 80.2, 117.7,
155.3; m/z (EI): 211(2), 186 (5), 169 (7), 86 (14), 69 (46), 57
7e-Cbz was obtained as a white solid. Yield: 455 mg (89%)
method A, 60 mg (30%) method B; mp: 82–83 °C; Rf = 0.69
(100); IR (KBr):
(C@O) cmꢁ1
(249.15735); found (249.15720); HPLC purity: 95% on a YMC
ODS AQ 2185 column, 250, 5 ,
m, CH3CN/H2O 3:1, 0.8 mL minꢁ1
m
3367 (N–H), 2962, 2937, 2246 (C„N), 1682
;
HRMS: calcd for C12H22N2O2Na [M+Na]+
(CH2Cl2/MeOH 96/4); ½a D21
¼ ꢁ3:1 (c 1.0, EtOH); GC: tR = 11.74;
ꢂ
1H NMR (CDCl3, 400 MHz): d = 1.34 (s, 9H), 2.41 (dd, 1H,
J = 16.6 Hz and J = 4 Hz), 2.67 (dd, 1H, J = 16.6 Hz and
J = 4.5 Hz), 2.82 (dd, 1H, J = 13.6 Hz and J = 8.0 Hz), 2.93 (dd,
1H, J = 13.6 Hz and J = 6.8 Hz), 4.04–4.14 (m, 1H), 5.08 (s, 2H),
5.27 (d, 1H, J = 9.5 Hz, N–H), 6.94 (d, 2H, J = 8.5 Hz), 7.08 (d,
2H, J = 8.6 Hz), 7.28–7.38 (m, 5H); 13C NMR (CDCl3, 100 MHz):
d = 22.5, 28.9, 38.6, 49.2, 67.0, 78.6, 117.3, 124.5, 128.1, 128.3,
128.6, 129.6, 130.7, 136.1, 154.7, 155.6; m/z (EI): 366 (M), 310
(20), 219 (25), 175 (23), 159 (45), 107 (100); 91 (90); 77 (15);
l
Detector: PDA, k = 210 nm tR = 6.82 min.
4.8.6. N-((2R)-1-Cyano-3-methylpentan-2-yl)-4-nitrobenzene-
sulfonamide 7c-4Ns
(2S)-3-Methyl-2-(4-nitrophenylsulfonamido)pentyl methane-
sulfonate 6c-4Ns (571 mg, 1.5 mmol) led to the expected com-
pound 7c-4Ns after 3 days of stirring according to the general
procedure previously described. The crude residue was purified
by flash column chromatography on silica gel (cyclohexane/EtOAc
5:5) to give 7c-4Ns as a yellow oil. Yield: 190 mg (40%); Rf = 0.47
IR (KBr):
m 3351 (N–H), 3041, 2980, 2927, 2250 (C„N), 1693
(C@O), 1526, 1526, 1256, 158 cmꢁ1
;
HRMS: calcd for
C22H26N2O3Na [M+Na]+ (389.18356); found (389.18345); HPLC
purity: 96.7% on a Lichrosorb Si 60 column, 250 mm 4.6 mm,
(cyclohexane/EtOAc 1:1);
½
a 2D0
ꢂ
¼ ꢁ46:9 (c 1.0, CHCl3); GC:
tR = 10.43; 1H NMR (CDCl3, 400 MHz): d = 0.80 (t, 3H, J = 7.4 Hz),
0.86 (d, 3H, J = 6.8 Hz), 0.97 (m, 1H), 1.4 (m, 1H), 1.67 (m, 1H),
2.59 (dd, 1H, J = 17.1 Hz and J = 5.18 Hz), 2.67 (dd, 1H, J = 5.4 Hz
and J = 17.1 Hz), 3.42 (m, 1H), 8.09 (m, 2H), 8.38 (m, 2H); 13C
NMR (CDCl3, 100 MHz): d = 11.0, 15.04, 22.5, 24.9, 38.0, 55.1,
117.0, 124.7, 128.4, 146.2, 150.3; m/z (EI): 271 (16), 254 (100),
5
l
m,
Heptane/EtOAc
6:4,
1 mL minꢁ1
,
Detector
PDA,
tR = 5.79 min, k = 267 nm.
4.8.4. (S)-Benzyl 1-(4-(benzyloxy)phenyl)-3-cyanopropan-2-
ylcarbamate 7f-Cbz
Method A: To a stirred solution of the (S)-benzyl-2-(4-(benzyl-
oxy)benzyl)aziridine-1-carboxylate 5f-Cbz (265 mg, 1.11 mmol)
in CH3CN/H2O 9:1 (3.33 mL) was added NaCN (0.16 g, 3.33 mmol).
The expected compound 7f-Cbz was obtained after 18.5 h of stir-
ring according to the general procedure previously described. The
crude residue was purified by flash column chromatography on sil-
ica gel (CH2Cl2/MeOH 7:3).
186 (85), 122 (52), 76 (21); IR (KBr):
m
3284 (N–H), 2968, 2932,
asSO2), 1167 ( sSO2)
2251 (C„N), 1530 (
m
asNO2arom), 1350 (
m
m
cmꢁ1; HRMS: calcd for C13H17N3O4SNa [M+Na]+ (334.08320);
found (334.08306); HPLC purity: 99%; on a YMC ODS AQ 2185 col-
umn, 250, 5 l
m, CH3CN/H2O 6:4, 0.7 mL minꢁ1, HCOOH 0.1%,
Detector: PDA k = 220 nm, tR = 6.63 min.
Method B: (S)-3-(4-(Benzyloxy)phenyl)-2-(ethoxycarbonylami-
no)propyl methanesulfonate 6f-Cbz (0.39 g, 0.83 mmol) led to
the expected compound 7f-Cbz after 1.5 h of stirring according to
the general procedure previously described. The crude residue
was purified by flash column chromatography on silica gel
(CH2Cl2/MeOH 7:3).
4.9. General procedure for the synthesis of N-Dpp amino
nitriles 7
To a stirred solution of the corresponding N-Dpp aziridines 5a-
Dpp, 5c-Dpp, 5d-Dpp, and 5e-Dpp in toluene (5 mL per mmol)
were added TBABr (0.1 equiv), NaCN (2 equiv), and water (1 mL
per mmol). The reaction mixture was stirred at 85 °C and moni-
tored by TLC. After cooling the mixture to room temperature, it
was taken up in EtOAc/H2O (6:1 v/v, 35 mL per mmol). The layers
were separated and the organic layer was washed with water
(5 mL per mmol), and then dried over anhydrous Na2SO4, filtered,
and concentrated to afford the corresponding N-Dpp amino nitriles
7 Dpp. Yield: (S)-7a-Dpp, 86%; (S)-7c-Dpp, 74%, (S)-7d-Dpp, 75%
(S)-7e-Dpp, 74%.
7f-Cbz was obtained as a white solid. Yield: 197 mg (73%)
method A, 281 mg (89%) method B; mp = 117–118 °C; Rf = 0.82
(CH2Cl2/MeOH 96:4); ½a D20
¼ ꢁ6:4 (c 1.0, MeOH); GC: tR = 10.44;
ꢂ
1H NMR (CDCl3, 400 MHz): d = 2.46 (dd, 1H, J = 17 Hz and
J = 4 Hz), 2.71 (dd, 1H, J = 16.6 Hz and J = 4.5 Hz), 2.84 (dd, 1H,
J = 14 Hz and J = 8 Hz), 2.95 (dd, 1H, J = 14 Hz and J = 6.5 Hz),
4.04–4.15(m, 1H), 4.97 (d, 1H, J = 7 Hz, N–H), 5.05 (s, 2H), 5.10 (s,
2H), 6.94 (d, 2H, J = 8.5 Hz), 7.12 (d, 2H, J = 8 Hz), 7.30–7.45 (m,
10H); 13C NMR (CDCl3, 100 MHz): d = 22.5, 38.5, 49.2, 67.1, 70.1,
115.4, 117.1, 127.5, 128.0, 128.1, 128.3, 128.6, 128.6, 130.1,
136.0, 136.9, 155.5, 158.1; m/z (EI): 292 (10), 207 (1), 197 (2),
4.9.1. (S)-N-(1-Cyanopropan-2-yl)-P,P-diphenylphosphinic
amide 7a-Dpp
107 (3), 91 (100), 65(4); IR (KBr):
m
3368 (N–H), 3062, 2928,
(S)-1-(Diphenyl phosphoryl)-2-methylaziridine 5a-Dpp (250 mg,
0.97 mmol) led to the expected compound 7a-Dpp after 18 h of stir-
ring according to the general procedure previously described. White
solid. Yield: 238 mg (86%); mp: 178–180 °C, Rf = 0.32 (EtOAc);
2243 (C„N), 1697 (C@O), 1243, 1057 cmꢁ1; HRMS: calcd for
C25H24N2O3Na [M+Na]+ (423.16791); found (423.16752); HPLC
purity: 97.6% on a Lichrosorb Si 60 column, 250 mm 4.6 mm,
5
l
m, Heptane/AcOEt 6:4, 1 mL minꢁ1, Detector PDA, tR = 6.50 min,
½
a 2D3
ꢂ
¼ þ28:9 (c 3.6, CH2Cl2); GC: tr = 11.52; 1H NMR (CDCl3,
k = 276 nm.
400 MHz): d = 1.8 (d, 3H, J = 6.5 Hz), 2.55 (dd, 1H, J = 4.0 Hz and

2088
M.S.-I. Veitía et al. / Tetrahedron: Asymmetry 20 (2009) 2077–2089
J = 16.6 Hz), 2.70 (dd, 1H, J = 5.8 Hz and J = 16.6 Hz), 3.45–3.58
(m,1H), 7.35–7.56 (m, 6H), 7.79–7.93 (m, 4H); 13C NMR (CDCl3,
100 MHz): d = 22.8 (d, J = 6,4 Hz), 28.0 (d, J = 3.9 Hz), 44.1, 17.6,
128.6, 128.7, 128.7, 128.8, 131.8, 131.9, 132.1, 132.2; m/z (EI): 284
EDTA, pH 7.5) in a round-bottomed flask. The commercial enzyme
preparation was added as a solution in phosphate buffer to sub-
strate to afford a final concentration of 0.2 M. Absolute amounts
are added for the respective compounds listed below. The reaction
mixture was stirred with a magnetic bar and the temperature was
adjusted to 30–32 °C by use of an oil bath. The conversion was
monitored by HPLC. After completion, the mixture was acidified
by the addition of HCl and the protein was removed by (NH4)2SO4
precipitation and filtration through a plug of Celite. The products
were purified by silica gel chromatography (CH2Cl2/CH3OH 95:5).
[M] (100), 269 (1), 244 (94), 201 (100), 77 (22); IR (KBr):
H), 2968, 2255 (C„N), 1125 (P@O) cmꢁ1
HRMS: calcd for
C16H17N2P [M+H]+ (285.11570); found (285.1150).
m 3146 (N–
;
4.9.2. N-((2R)-1-Cyano-3-methylpentan-2-yl)-P,P-diphenylphos-
phinic amide 7c-Dpp
(2S)-2-sec-Butyl-1-(diphenylphosphoryl)aziridine
5c-Dpp
(500 mg, 1.7 mmol) led to the expected compound 7c-Dpp after
18 h of stirring according to the general procedure previously de-
4.11.1. (S)-3-(Benzyloxycarbonylamino)butanoic acid 9a-Cbz
This compound was obtained as white solid mp: 107–110 °C,
scribed as
142.0 °C, Rf = 0.33 (EtOAc);
a
white solid. Yield: 410 mg (74%); mp: 141.5–
½
a 2D4
ꢂ
¼ ꢁ21:5 (c 0.4, CHCl3) (lit.28
½
a 2D4
ꢂ
¼ ꢁ24:1 (c 0.4, CHCl3); 1H
½
a 2D0
ꢂ
¼ ꢁ2:8 (c 2.8, CH2Cl2); GC:
NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz): conform
tr = 12.4; 1H NMR (CDCl3, 400 MHz): d = 0.85 (t, 3H, J = 7.3 Hz),
0.92 (d, 3H, J = 6.8 Hz), 1.09–1.20 (m, H), 1.56–1.66 (m, 1H),
1.73–1.79 (m, 1H), 2.60 (dd, 1H, J = 4.3 Hz and J = 15.8 Hz), 2.75
(dd, 1H, J = 5.1 and J = 16.8 Hz), 3.14–3.25 (m, 2H), 7.42–7.52 (m,
6H), 7.87–7.92 (m, 4H); 13C NMR (CDCl3, 100 MHz): d = 11.3,
15.1, 23.4 (d, J = 3.2 Hz), 25.2, 39.4 (d, J = 5.6 Hz), 52.1(d,
J = 1.6 Hz), 118.0, 128.6, 128.7, 128.7, 128.8, 131. 9, 131.9, 132.2,
132.2, 132.3, 131.3 (d, J = 30.4 Hz), 132.6 (d, J = 28.8 Hz); m/z (EI):
to data reported in the literature;30 IR (KBr):
1686 (C„O) cmꢁ1 HRMS: calcd for C12H15O4N [M+Na]+
(260.2500); found (260.01; HPLC purity: 98.6%; on a YMC ODS
AQ 2185 column, 250, 5
m, CH3CN/H2O 6:4, 1 mL minꢁ1, HCOOH
m 3432, 3311, 2952,
;
l
0.1%, Detector: PDA k = 220 nm, tR = 3.76 min.
4.11.2. (S)-3-(Benzyloxycarbonylamino)-4-methylpentanoic
acid 9b-Cbz
326 [M] (1), 286 (16), 269 (63), 201 (100), 77 (13); IR (KBr):
m
This compound was obtained as white solid mp: 83–85 °C,
3140 (N–H), 2952, 2243 (C„N), 1189 (P@O) cmꢁ1; HRMS: calcd
½
a 2D3
ꢂ
¼ ꢁ33:0 (c 0.2, CHCl3) (lit.28
½
a 2D3
ꢂ
¼ ꢁ33:6 (c 0.2, CHCl3) 1H
for C19H23N2OP [M+H]+ (327.15480); found (327.15420).
NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz): conform
to data reported in the literature.31 IR (KBr):
1695 (C„O) cmꢁ1 HRMS: calcd for C14H19O4N [M+Na]+
(288.300); found (288.3001); HPLC purity: 95%; on a YMC ODS
AQ 2185 column, 250, 5
m, CH3CN/H2O 6:4, 1 mL minꢁ1, HCOOH
m 3435, 2913, 2848,
4.9.3. (S)-N-(1-(4-tert-Butoxyphenyl)-3-cyanopropan-2-yl)-P,P-
diphenylphosphinic amide 7e-Dpp
;
(S)-2-(4-tert-Butoxybenzyl)-1-(diphenylphosphoryl)aziridine
5e-Dpp (700 mg, 1.7 mmol) led to the expected compound 7e-Dpp
after 18 h of stirring according to the general procedure previously
described as a white solid. The crude residue was purified by flash
column chromatography on silica gel. Yield: 550 mg (74%); mp:
l
0.1%, Detector: PDA k = 220 nm, tR = 4.71 min.
4.11.3. (S)-Benzyl 1-amino-4-methyl-1-oxopentan-3-ylcarbamate
8b-Cbz
155–157 °C,
½
a 2D0
ꢂ
¼ ꢁ27:8 (c 5, CH2Cl2); 1H NMR (CDCl3,
This compound was not isolated, ESI-MS: calcd for C14H20O3N2
400 MHz): d = 1.34 (s, 9H), 2.55 (dd, 1H, J = 17.1 Hz and
J = 4.0 Hz); 2.6 (dd, 1H, J = 17.1 Hz and J = 5.52 Hz); 2.99–3.00
(2H, m), 3.29–3.33 (m, 1H), 3.46–3.60 (m, 2H), 6.94–6.96 (d, 2H,
J = 8.5 Hz), 7.04–7.06 (d, 2H, J = 8.5 Hz), 7.34–7.51 (m, 6H), 7.59–
7.78 (m, 4H); 13C NMR (CDCl3, 100 MHz): d = 28.8, 25.7 (d,
J = 2.4 Hz), 41.5 (d, J = 6.4 Hz), 49.7, 78.6, 117.6, 124.4, 129.9,
128.6, 128.64, 128.7, 131.5, 131.7, 131.8, 131.7, 131.8, 132.1,
132.2, 132.2, 132.3, 132.3, 131.2, 132.8 (d, J = 88 Hz), 131.3,
[M+Na]+ (287.32); found (287.32); HPLC purity: 97%; on a YMC
ODS AQ 2185 column, 250, 5 l ,
m, CH3CN/H2O 6:4, 1 mL minꢁ1
HCOOH 0.1%, Detector: PDA k = 220 nm, tR = 3.72 min.
Acknowledgments
We cordially acknowledge Dr. Didier Buisson from the Labora-
tory of Biocatalyst of University of Paris V for his collaboration.
We are also grateful to the student Anaïs Rosaz for her contribution
to this work.
153.8; IR (KBr):
m 3140 (N–H), 2952, 2930, 2237 (C„N), 1195
(P@O) cmꢁ1; HRMS: calcd for C26H30O2N2P [M+H]+ (433.20501);
found (433.20540).
References
4.10. Screening
1. (a) Yuan, C.; Williams, R. M. J. Am. Chem. Soc. 1997, 119, 11777–11784; (b)
Rossi, C.; Tuttobello, L.; Ricci, M.; Casinovi, C. G.; Radios, L. J. Antibiot. 1987, 40,
130–133.
For screening experiments, the substrate (0.1 mmol) was dis-
solved in a phosphate buffer (125
2 mM DTT, 1 mM EDTA, pH 7.5) and in case of low solubility of the
substrate, MeOH or DMSO was used as a cosolvent. The enzyme
(0.5 mg) was dissolved in a phosphate buffer (125 lL) and added
to the substrate to afford a final concentration of 0.2 M. The reaction
l
L, 50 mM potassium phosphate,
2. (a) Carter, D. C.; Moore, R. E.; Mynderse, J. S.; Niemczura, W. P.; Todd, J. S. J. Org.
Chem. 1984, 49, 236–241; (b) Zabriskie, T. M.; Klocke, J. A.; Ireland, C. M.;
Marcus, A. H.; Molinski, T. F.; Faulkner, D. J.; Xu, C.; Clardy, J. J. Am. Chem. Soc.
1986, 108, 3123–3124.
3. Kosemura, S.; Ogawa, T.; Totsuka, K. Tetrahedron Lett. 1993, 34, 1291–1294.
4. Crews, P.; Manes, L. V.; Boehler, M. Tetrahedron Lett. 1986, 27, 2797–2800.
5. Kimura, J.; Takada, Y.; Inayoshi, T.; Nakao, Y.; Goetz, G.; Yoshida, W. Y.; Scheuer,
P. J. J. Org. Chem. 2002, 67, 1760–1767.
6. (a) Seebach, D.; Beck, A. K.; Bierbaum, D. J. Chem. Biodiv. 2004, 1, 1111–1239.
and references cited therein; (b) Juaristi, E.; Soloshonok, V. A. Enantioselective
Synthesis of b-Amino Acids, 2nd ed.; Wiley-VCH: New Jersey, US, 2005. pp 1–
634; (c) Davies, S. G.; Smith, A. D.; Price, P. D. Tetrahedron: Asymmetry 2005, 16,
2833–2891.
mixture was stirred with a magnetic bar and the temperature was
adjusted at 30 °C. After 18 h, acetone (500
tion vessels were centrifuged for 15 min at room temperature at
4000 rpm to remove the precipitated proteins. The 300 L of super-
lL) was added. The reac-
l
natant were concentrated and then analyzed by RP-18 HPLC using
7. Wani, M. C.; Taylor, H. L.; Wall, M. E.; Coggon, P.; McPhail, A. T. J. Am. Chem. Soc.
1971, 93, 2325–2327.
acetonitrile/water/formic acid (60:40: 0.1) using an isocratic mode.
8. (a) Liljeblad, A.; Kanerva, L. T. Tetrahedron 2006, 62, 5831–5854; (b) Matthews,
J. L.; McArthur, D. R.; Muir, K. W. Tetrahedron Lett. 2002, 43, 5401–5404; (c)
Cardillo, G.; Tomasini, C. Chem. Soc. Rev. 1996, 25, 117–128; (d) Lee, H.-S.; Park,
J.-S.; Kim, B. M.; Gellman, S. H. J. Org. Chem. 2003, 68, 1575–1578; (e) Caputo,
R.; Longobardo, L. Amino Acids 2007, 32, 401–404.
4.11. Preparative scale biotransformations
Approximately 100 mg of substrate was dissolved in a phos-
phate buffer (50 mM potassium phosphate, 2 mM DTT, 1 mM

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