Kinetic Resolution of Amines

Kinetic Resolution of Amines
B4 Rumiko Shimabara
optical resolution
crystallization
preferential crystallization (優先晶出法)
inclusion complex (包接錯体法)
preferential enrichment (優先富化)
diastereomeric methods
diastereomereric salt formation
covalent diastereomeric derivatives
enzymatic methods
chiral column chromatography
kinetic resolution
preferential enrichment
Kinetic Resolution
Me
NH2
acylating reagent
Me
N
H
Me
O
R
+
NH2
１組のエナンチオマーのそれぞれで反応速度が異なる不斉反応を利用
し，反応性の低い方のエナンチオマーを未反応のままで残す手法
速度論的光学分割は 1899年に Marckwald と McKenzie によって最初に観察された
carboxylic acid
O
HO
OH
OH
O
O
HO
HO
+
O
OH
(S)-mandelic
acid
amine
Me
NH2
chiral reagent (0.25 eq)
Me
!
rt
O
O
N
H
CH3
N
O
H
N
48%ee
s=3
25% conv.
O
chiral reagent
K. Kondo, T. Kurosaki, Y. Murakami, Synlett 1998, 725
Dynamic Kinetic Resolution
NH2
R
R'
O
enzyme, ROAc
fast
HN
R
CH3
R'
cat.
NH2
R
R'
O
enzyme, ROAc
slow
HN
R
CH3
R'
in situで反応速度の遅い方のエナンチオマーを金属触媒
などでラセミ化させることで, 50％を超える収率が可能
になる
Kinetic Resolution methods
enzymes
stoichiometric chiral reagents
small-molecule catalysts
achiral nucleophiles + chiral hydrogen-bond donors
enantiomeric exess (ee)
ee = ¦R ‒ S¦/(R + S)
100
conversion (C)
C = ees/(ees + eep)
ees : ee of the recoverd substrate fraction
eep : ee of the product fraction
enantioselectivity factor (E)
E = ln[(1 ‒ C)(1 ‒ ees)]/ln[(1 ‒ C)(1 + ees)]
selectivity factor (s)
s = kfast/kslow
kfast : rate of fast-reaction enantiomer
kslow : rate of slow-reacton enantiomer
Charles J. Sih* et al. J. Am. Chem. Soc. 1982, 104, 7294-7299
3t
3u
COCH3
COn-C11H23
CH3
CH3
Figure 2. Structures of docked potential acyl donor candidates.
mately 55% conversion decreased while E increased (see entries 10 ,
40 , 60 ).25
5
6
7
8
9
10
11
GLY47/MET50
SER249
THR164/LYS170
THR244/
ARG247
ASP60/LYS94
ARG186
GLN10
4
3
2
2
À3.85
À3.39
À3.58
À3.61
À1.71
À1.64
À1.42
À2.12
1
1
1
À2.60
À3.03
À3.53
À1.28
À1.52
À1.47
3. Computational data
site triad, that is, SER221/HIS64/ASP32 accounts for 43% of the conformations. The second most populated site (21%) is close to
As aforementioned, automated docking was used to explore the
LEU235. The third one (14%) is located around the LYS43/VAL44/
binding affinities of different acyl donors for Subtilisin Novo that
2828
A.-L.
Bottalla
et
al.
/
Tetrahedron:
Asymmetry
20
(2009)
2823–2834
PRO86 triad. The other eight sites account for 1–7% of the docked
was selected as prototype for serine proteases. Blind docking sim26
conformations. For each site a mean binding free energy and the
ulations were performed with the AUTODOCK 4.0 program with five
minimum binding free energy are given.
different structures of Subtilisin Novo issued from the Protein Data
The three most populated sites correspond to the lowest miniBank (PDB). It has been shown recently that the docking approach
mum free energy, and thus to the strongest interactions.
could be used to predict protease specificities.18
Focusing our attention on the active site enabled a more accuFor sake of comparison, the docking of ligands like trifluoroethyl
rate comparison of the various acyl donors. For each ligand in the
N-dodecanoyl glycinate 3s, a peptide mimetic with a long hydroseries, the percentage of conformations docked in the active site,
phobic chain, and trifluoroethyl butanoate 3j, trifluoroethyl penttheir average binding free energy, and the binding energy of the
anoate 3k, or trifluoroethyl dodecanoate 3l, which are not
Figure 8. Compared enantioselectivity of ligands 3g, and 3q for the selected
most stable docked conformation are plotted in Figures 4–6,
enzymes andwas
subtilisin.
peptide mimetics,
also examined. In addition, potential peprespectively. Three groups can be distinguished.
tide mimetics candidates bearing alkanoyl chains in C8, C9, C10,
catalyzed reaction requires the ligands to dock in the active site
For ligands with a short chain length (3b, 3c, 3e, 3h, 3j, 3k, 3t),
and C11 with
a terminal phenyl group, that is, 3i, 3n, 3o, 3g, 3q,
with a preorganized structure27 close to that of the tetrahedral
competition
between
the
intermediate. In
order to
study
more accurately
the structures of
3r, were investigated
with
the
purpose
of evaluating
the Figure
interde10. Percentage of the
preorganized
docked conformations
in the active site
for active site and other binding sites is
each ligand according to its structural features.
the ligands docked near the catalytic triad, we performed new simnot in favor of the former [nconf (%) does not exceed 41%, Fig. 4].
pendence of binding
subsites.
structures
of all
ulations, where
the ligands The
were allowed
to dock only
near these
the ac- additional
tive
site
and
not
all
over
the
enzyme
as
previously.
within
thisbyfirst
group
ligands are given in Figure 2.
As the tetrahedralThus,
intermediate
is stabilized
hydrogen
bonds,of ligands, N-acetyl phenylalanine 3e
The structures for which the distance between the carbon of the
it was interesting
to seems
check if these
interactions
octoligand-receptor
be the best
ligand.
The percentage of conformations
Figure 3 shows
views
of the
enzyme
structure
by
shorter thanrepresented
3.5 Å
ester group
and SER221
oxygen
atom (r1) was
curred in the preorganized conformations (Fig. 9). Therefore, three
were
reckoned
as
preorganized
conformations
(Fig.
9).
The
number
docked
at
the
active
site
that peptide mimetics have more
its Connelly surface. The active site can easily be locateddistances
by the
between the atoms susceptible to form hydrogen shows
bonds
of these preorganized conformations for each ligand is given in Figwere monitored: first, the distance between the ester carbonyl
28
affinity
for
the
active
site
than
simple ester derivatives. Ligands
location of SER221
and
the
secondary
binding
sites
are
colored
in
ure 10. Compared to other similar studies, far less preorganized
oxygen atom and the proton of the NH group of the ASN155 resiconformations were observed. Moreover, these conformations
and
which
are the
poorest
ligands, are not peptide mimetics.
second,
the distance
between
the
due in the oxyanion3j
hole
(r2),3k,
pink.
were not necessarily the ones with the strongest binding free enoxygen atom of SER125 carbonyl group and the glycine NH proton
ergy (except
ligand 4,
3g).on
Nevertheless,
wewhen
have attempted
to
However,
if one looks at the second group of ligands 3f, 3a, 3d,
As summarized
in for
Table
average,
all ligands
all (r3), and third,
the distance between the oxygen atom
in and
the ligand
analyze these results.
the proton
of SER221
hydroxyl 3m,
proton the preference for peptide mimetics
ofconforthe CF3CH2O group
3l,and3u,
3p, 3s,
excluding
enzyme structures
were
taken together,
99%
of the
docked
The number
of preorganized
conformations
increases
with
the
(r4). In the docked structures for which r1 is less than or equal to
chain
length forin
peptide
mimetics ligands.
TheDocking
nature of thenear
pep- the active
can
be offset
by thewhile
influence of chain length. Octanoyl glymations were
located
11 binding
sites.
long nearly
(4.4 Å in average
over all ligands),
3.5 Å, hr3i is relatively
Bacillus subtilis
tide shows a higher frequency of preorganized conformations in
the order PHE < ALA < GLY which points to the influence of hydrophobic interactions in the specificity pocket. Among the ligands
with a phenyl group at the end of the acyl chain, the ones with a
chain of 9 carbons (3n and 3q) are favored. The evolution of the
free energy for these preorganized conformations according to
the structural features of the ligands shows the same tendency
as observed in the blind docking (see Fig. 5).
hr2i and hr4i are shorter (3.2 Å and 3.5 Å, respectively). Moreover, in
about half of these structures r2 and r4 are shorter than 3.5 Å at the
same time, suggesting that water molecules are not absolutely necessary to mediate proton transfers.
The relatively small number of preorganized conformations for
each ligand makes the statistical approach difficult and therefore, it
was not possible to evaluate the effect of the chain length or of the
nature of the peptide mimic on parameters r2, r3, and r4. It was possible, however, to observe that within the ligands bearing a terminal phenyl group in the GLY series (3g, 3q, 3r), ligand 3q showed
the shortest distances hr2i and hr4i, thus prefiguring its greater
affinity. However, docking simulations in the active site did not
help us to discriminate the ligands more accurately.
4. Conclusion
The screening of a large panel of commercially available proteases for the resolution of 3-amino-1-phenylbutane resulted in the
selection of four serine proteases which were used to determine
the best acyl donor from a series of trifluoroethyl esters. The most
efficient donors happened to be trifluoroethyl esters derived from
N-acyl a-amino acids bearing an aliphatic acyl chain 8 or 9 carbon
long. Blind automated docking enabled a rapid selection of the
most promising ligands, by looking at their binding affinity for
Subtilisin Novo.
A noteworthy increase in the enantiomeric ratio was observed
with alkaline protease by replacing the glycine moiety by alanine
(3a/3f; E factor up to 99).
5. Experimental
Figure 9. Preorganized docked conformation of 3q in the active site. Stabilizing
hydrogen bonding interactions occurs between the ligand amide NH and SER125,
the ester carbonyl oxygen atom and ASN155 in the oxyanion hole.
The 1H and 13C NMR spectra were recorded at 200 or 300 MHz and
50 or 75 MHz, respectively. Chemical shifts are reported in ppm, and
Figure 3. Connelly surface views of the two faces of Subtilisin Novo structure showing the active site (SER221), the S1–S4 channel (blue), and secondary binding sites (pink).
Subtilisin Novo
S. Queyroy*, G. Gil* et al. Tetrahedron Asymmetry 2009, 20, 2823-2834
Subtilisin Novo (6 mg)
acyl donor (1.0 eq)
NH2
O
HN
3-methyl-3-pentanol (0.25 M)
rt
Ph
R
R1
NH2
+
Ph
R2
Ph
1
H
1N
H
N
(R)-1
(S)-2
O
(S)
O
CF3
R2
acyl donor
acyl
donor
a
b
c
d
e
f
g
h
i
R1
COn-C7H15
COCH3
COPh
COn-C7H15
COCH3
COn-C7H15
COn-C7H14Ph
Ph
COn-C8H16Ph
R2
H
H
H
CH2Ph
CH2Ph
CH3
H
H
H
entry
acyl
donor
time
(min)
1 ee
(%)
2 ee or dr
(%)
C (%)
E
1
2
3
4
5
6
7
8
9
a
b
c
d
e
f
g
h
i
60
50
150
20
30
25
90
50
240
52.2
62.9
46.7
77.3
89.6
86.8
73.2
64.5
62.0
83.3
77.8
76.3
88.5
72.1
92.5
59.7
62.2
84.7
38.5
44.7
38.0
46.6
55.4
48.4
55.0
50.9
42.3
19
15
11
39
18
73
8
8
22
Me
Me
O
N
H
tBuO
Peptide
NHBoc
N
H
N
O
O
O
Base
N
N
H
OtBu
Acid
OtBu
OtBu
Me
Me
N
H
O
Peptide
O
4
3
O
Me
O
O
tBuO
O
(±)-2
O
N
H
Me
H
Boc
N
H
5
2
NHBoc
N
H
Me
1
N
N
Scott J. Miller*, et al. J.Am.Chem.Soc. 2010, 132, 2870-2871
Scott J. Miller
O
H
Me
Boc2O (0.6 eq)
catalyst (10 mol%)
O
N
H
H
Me
N
H
Boc
CDCl3 (0.3 M), 4 Å MS
25 ºC, 36 h
O Me Me
N
H
O
N
O
HN
Ph
O
NH
NH
Ac
Me N
+
6d
β-turn
H
(S)-2
entry
catalyst
i–1
i+1
i+2
i+3
i+4
s-factor
1
2
3
4
5
6a
6b
6c
6d
6e
Boc
Boc
Boc
Ac
Boc
D-Pro
D-Pro
D-Pip
D-Pro
D-Pro
Sp6
Aib
Aib
Aib
Aib
L-Val
L-Val
L-Val
L-Phg
L-Phg
D-Phe-OMe
D-Phe-OMe
D-Phe-OMe
D-Phe-OMe
NMe2
4.6
7.8
5.3
9.6
3.2
OMe
Ph
N
O
N
H
4
(±)-2
H
Me
O
O
CO2H
NH
D-Pip
NH2
Ph
CO2H
L-Phg
H2N
CO2H
Aib
NH2
CO2H
Sp6
Kinetic Resolution methods
enzymes
stoichiometric chiral reagents
small-molecule catalysts
achiral nucleophiles + chiral hydrogen-bond donors
NH2
Ph
Me
1 (0.5 eq)
solvent, rt
NHAc
!
Ph
Me
Tf
N
NH2
+
!
Ph
Me
N
H
Ac
Tf
(1S,2S)-1
chiral reagent
solvent
ee (%) s-factor
toluene
CHCl3
DMF
DMPU
58 (R)
56 (R)
72 (S)
84 (S)
6.6
6.1
13.1
30.3
Wagner, A.*; Mioskowski, C.* et al. J Am. Chem. Soc. 2005, 127, 6138-6139
Wagner, A.*; Mioskowski, C.* et al. Angew. Chem. Int. Ed. 2004, 43, 3314 –3317
N
N
DMPU
O
Tf
N
N
H
Tf
N
Ac
Tf
Ac
Tf
N
Me
(1S,2S)-2
(1S,2S)-1
(1S,2S)-1
O
Tf
NHAc
NN
H Me
Tf
NH2
Ph
!
Ph
NH2
+
Me
!
Ph
Me
Me
(1S,2S)-2
O
Tf
NN
Me Me
Tf
Tf
Tf
N NMe
Me
O
Me
NN
O
Tf
Tf
Me
Me
Tf
N N Tf
Me
O
1 (0.5 eq)
NH2
Ph
salt (1 M)
THF, rt
Me
NHAc
!
Ph
Me
NH2
+
!
Ph
entry
time (min)
salt (1 M)
ee (%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
60
20
10
10
10
20
10
20
20
20
10
20
20
10
LiBr
LiClO4
TFSI-Li
n-BuPyBr
n-Et4NBr
n-Et4NCl
n-Bu4NCl
n-Bu4NBr
n-Bu4NI
n-Bu4NBF4
n-Bu4PCl
n-Bu4PBr
n-Oct3NMeCl
42 (R)
42 (S)
68 (S)
54 (S)
68 (S)
22 (S)
74 (S)
82 (S)
88 (S)
54 (S)
76 (S)
44 (S)
70 (S)
90 (S)
Me
Li
Tf
N
Tf
TFSI-Li
1 (0.5 eq)
NH2
Ph
NHAc
n-Oct3NMeCl (1 M)
solvent, temp.
Me
Ph
entry
temp. (ºC)
solvent
eec/eed (%)
1
2
3
4
5
6
25
25
25
25
–20a
–20b
THF
1,4-dioxane
CH2Cl2
toluene
THF
THF
90e (S)/42 (R)
72 (S)/50 (R)
76 (S)/40 (R)
78 (S)/58 (R)
94 (S)
95 (S)
NH2
Ph
Me
1 (0.5 eq)
n-Oct3NMeCl
THF, rt
entry
conc. (M)
salt:1
eea (%)
1
2
3
4
5
6
1
0.1
0.01
0.001
0.0001
-
25:1
2.5:1
1:4
1:40
1:400
0
90 (S)b
80 (S)
30 (S)
0
26 (R)
42 (R)
NH2
+
!
Me
!
Ph
a) 0.5 eq of 1 (s = 115, 50% conversion)
b) 0.33 eq of 1 (s = 62, 33% conversion)
c) Enantiomeric exess of the acetamide determined
by HPLC analysis using a chiral phase column.
d) Enantiomeric exess observed without using a salt.
e) Absolute configuration of the major enantiomer.
NHAc
!
Ph
Me
Me
NH2
+
!
Ph
Me
a) Enantiomeric exess of the acetamide determined
by HPLC analysis using a chiral phase column.
b) Enantiomeric exess observed without using a salt.
Kinetic Resolution methods
enzymes
stoichiometric chiral reagents
small-molecule catalysts
achiral nucleophiles + chiral hydrogen-bond donors
OAc
tBu
O
(0.65 eq)
N
Ph
5% (–)-1
R1
R1
N
H
+
R1
N
Ac
N
H
LiBr (1.5 eq)
18-crown-6 (0.75 eq)
toluene
N
N
R
R
R
O
(–)-1
(–)-2
(–)-3
(–)-4
R
R
OMe
O
tBu
Fe
R = 3,5-dimethylphenyl
Me
Ph
3,5-diethylphenyl
O
(0.65 eq)
N
2-naphthyl
NH2
Ar
R
O
NH2
10% (–)-2
CHCl3, –50 ºC
Ar
+
HN
OMe
R
Ar
R
s = 11-27
Gregory C. Fu*, Yutaka I. Chem. Commun. 2000, 119-120
Gregory C. Fu*, Forrest O. Arp J. Am. Chem. Soc. 2006, 128, 14264-14265
Gregory C. Fu
N
N
O
tBu
O
N
O
N
R
OMe
O
N
2-naphthyl
Fe
R
MeO
R
R
R
NH2
R
Ar
R
tBu
O
Fe
O
HN
N
R
R
O
2-naphthyl
R
R
ion pair
Ar
OMe
R
OAc
tBu
O
(0.65 eq)
N
Ph
5% (+)-1
Me
N
H
Me
+
N
H
LiBr (1.5 eq)
18-crown-6 (0.75 eq)
toluene, 0 ºC, 5 days
Me
N
Ac
optimized conditions
entry
N
N
R
Fe
R
R = 3,5-dimethylphenyl
Me
Ph
3,5-diethylphenyl
R
R
R
(–)-1
(–)-2
(–)-3
(–)-4
1
2
3
4
5
6
7
8
9
10
11
12
change from optimized conditions
none
(+)-2 instead of (+)-1
(+)-3 instead of (+)-1
(+)-4 instead of (+)-1
15-crown-5 instead of 18-crown-6
12-crown-4 instead of 18-crown-6
no 18-crown-6
no LiBr
Bu4NBr instead of LiBr/18-crown-6
LiCl instead of LiBr
LiI instead of LiBr
room temp. instead of 0 ºC (2 days)
conv. (%)
s-factor
54
4
48
58
54
43
16
55
49
43
12
49
23
<2
10
19
20
3
6
<2
2
14
12
11
OAc
tBu
O
(0.65 eq)
N
Ph
R1
R1
5% (–)-1
R1
R
R
N
H
s-factor
ee of resolved
indoline (%)
conv. (%)
R = Me
Ph
CH2CH2Ph
CH2OTBS
25
26
18
14
94
90
98
90
55
53
60
56
n
n=1
2
9.8
31
91
91
64
51
Me
cis
trans
18
9.5
91
94
55
64
19
95
58
13
11
92
90
60
60
entry
indoline
R
N
H
5
6
N
H
Me
7
8
N
H
CO2Et
MeO
9
Me
N
H
X
10
11
Me
N
H
X = OMe
Br
R
N
Ac
N
H
LiBr (1.5 eq)
18-crown-6 (0.75 eq)
toluene, –10 ºC to rt
1
2
3
4
+
Kinetic Resolution methods
enzymes
stoichiometric chiral reagents
small-molecule catalysts
achiral nucleophiles + chiral hydrogen-bond donors
current approach to nucleophilic catalyst
R'
O
R'
O
X
R'
N
N
R*
R'
O
O
N
O
R'
O
O
NMe2
NMe2
NMe2
acyl pyridinium
chiral by virtue of cation
in situ generation of chiral acyl pyridinium salts
R'
O
N
NMe2
achiral
X
S
R
N
H
N
H
R*
R'
O
N
S
R
NMe2
N
H
N
H
X
chiral by virtue of anion
D. Seidel* et al. J. Am. Chem. Soc. 2009, 131, 17060-17061
R*
Ph
+
Me
Ph
O
O
O
X
X
R
1a: X = S, R = CF3, R' = H
1b: X = O, R = CF3, R' = H
1c: X = S, R = H, R' = H
1d: X = S, R = H, R' = CF3
NH HN
R'
NH
HN
R
1
Ph
catalyst
+
NMe2
6a
R
R'
R
Ph
S
S
NH HN
Ar NH
HN Ar
S
S
NH HN
Ar NH
2
HN Ar
3
S
N
H
H
N
N
H
H
N
Ar
S
Ar
Ar
N
H
CF3
N
H
S
4
O
N
NH2
5
OH
Ar =
CF3
HN
toluene, 4 Å MS
–78 ºC, 1 h
entry
catalyst
(mol%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
1a (20)
1a (20)
1a (20)
1a (20)
1a (20)
1a (20)
1a (20)
1a (20)
1a (20)
1a (20)
1a (15)
1a (10)
1a (5)
none
none
1a (20)
1b (20)
1c (20)
1d (20)
2 (20)
3 (20)
4 (20)
5 (20)
Ph
Me
DMAP
conversion
(mol%) conc. (M)
(%)
50
50
50
50
50
40
30
20
10
5
15
10
5
none
20
none
20
20
20
20
20
20
20
0.06
0.03
0.02
0.01
0.005
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
47
47
47
45
44
47
47
45
43
44
46
46
44
<1
<2
40
40
43
21
44
42
30
38
s-factor
5.5
7.0
8.6
9.5
9.4
9.4
10
10
8.5
7.7
9.0
9.0
8.5
N/A
N/A
1.4
8.6
4.5
4.1
7.2
3.7
1.2
1.5
CF3
CF3
S
N
H
F3C
S
N
H
F3C
HN
S
Ph
HN
O
O
CF3
N
H
O
O
Ph
O
O
N
H
N
Ph
HN
S
HN
CF3
Ph
CF3
absence of DMAP
NMe2
CF3
presence of DMAP
(PhCO)2O (0.5 eq)
DMAP (20 mol%)
catalyst 1a (20 mol%)
NH2
Ar
O
HN
toluene (0.01 M), 4 Å MS
–78 ºC, 1 h
R
Ar
Ph
R
6
NH2
NH2
NH2
Me
Me
Me
Me
NH2
Me
Me
Me
6a
NH2
Me
F
6c
6b
NH2
NH2
NH2
Me
Me
6d
Cl
Cl
6e
NH2
Me
NH2
Me
Me
Cl
Br
6g
6f
NH2
NH2
6h
6j
6i
NH2
NH2
6m
6n
Me
6k
6l
entry
amine
conv. (%)
s-factor
1
2
3
4
5
6
7
8
9
10
11
12
13
14
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
45
45
46
49
43
42
47
49
48
48
46
45
45
47
10
7.2
7.1
17
16
20
20
18
13
24
13
15
7.7
15
Summary
アミンの高い求核性がエナンチオ選択性の低下を招く
立体選択性は溶媒，塩の添加，温度に依存する
様々な不斉源を利用したKRがある

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