En route to (S)-selective chemoenzymatic dynamic kinetic resolution of aliphatic amines. One-pot KR/racemization/KR sequence leading to (S)-amides

Full text of DOI:10.1021/jo900074w

matic DKR of amines. In most cases, racemization was based
on metal-catalyzed reversible dehydrogenation of the amine into
the corresponding imine. Our approach was different since the
racemization process relied on reversible hydrogen atom
abstraction at the stereocenter (directly adjacent to the reactive
amine moiety) by sulfanyl radical.5,6 The known examples of
(S)-selective chemoenzymatic DKRs involving hydrolases refer
exclusively to alcohols.7 We report herein chemoenzymatic
conversion of aliphatic amines into the corresponding (S)-amides
through a one-pot three-step sequence (Scheme 1).
En Route to (S)-Selective Chemoenzymatic
Dynamic Kinetic Resolution of Aliphatic Amines.
One-Pot KR/Racemization/KR Sequence Leading
to (S)-Amides
Lahssen El Blidi,† Malek Nechab,†,‡ Nicolas Vanthuyne,‡
Ste´phane Gastaldi,*,† Miche`le P. Bertrand,*,† and
Ge´rard Gil*,‡
Equipe Chimie Mole´culaire Organique, LCP UMR 6264,
Boite 562, UniVersite´ Paul Ce´zanne, Faculte´ des Sciences St
Je´roˆme, AVenue Escadrille Normandie-Niemen, 13397
Marseille Cedex 20, France, and Chirosciences, ISM2, UMR
6263, UniVersite´ Paul Ce´zanne, Faculte´ des Sciences St
Je´roˆme, AVenue Escadrille Normandie-Niemen, 13397
Marseille Cedex 20, France
SCHEME 1. One-Pot Sequential Procedure
michele.bertrand@uniV-cezanne.fr
ReceiVed January 13, 2009
Our aim was to devise a DKR process. To reach such an
objective, several conditions had to be fulfilled. First of all, the
racemization procedure had to be compatible with the kinetic
resolution conditions. Besides the requirement of the highest
possible enantioselectivity for the KR, it was also essential for
the rate of racemization of the slow reacting amine enantiomer
to be fast compared to its enzyme-mediated conversion into the
corresponding amide.8
The synthesis of (S)-amides via kinetic resolution of a racemic
mixture can be achieved with commercially available proteases.9
The catalytic triad of the latter is the mirror image of that
encountered in lipases.10 We have shown recently that the use
of carbamoyl glycine trifluoroacetates derived from octanoic
acid, as acyl donors, enabled alkaline protease-catalyzed resolu-
tion of most aliphatic amines to proceed within 15 min to 6 h
depending on the substrate, with good to high enantioselectiv-
ity.11 This enzyme has less thermal stability than CAL-B. It
was necessary to find out how to perform the radical racem-
ization at a temperature that did not exceed 40 °C. This condition
was fulfilled by generating sulfanyl radical via photolysis of
A one-pot sequential process, involving a radical racemiza-
tion and an enzymatic resolution, provides access to (S)-
amides, from racemic amines, with ee and yields ranging
from 78 to 94% and 58 to 80%, respectively.
Dynamic kinetic resolution (DKR) of racemic amines enables
their conversion into enantiomerically pure amides.1 Our group2
and others3,4 have recently reported (R)-selective chemoenzy-
(5) (a) Escoubet, S.; Gastaldi, S.; Vanthuyne, N.; Gil, G.; Siri, D.; Bertrand,
M. P. J. Org. Chem. 2006, 71, 7288. (b) Escoubet, S.; Gastaldi, S.; Timokhin,
V. I.; Bertrand, M. P.; Siri, D. J. Am. Chem. Soc. 2004, 126, 12343. (c) Escoubet,
S.; Gastaldi, S.; Vanthuyne, N.; Gil, G.; Siri, D.; Bertrand, M. P. Eur. J. Org.
Chem. 2006, 3242. (d) Bertrand, M. P.; Escoubet, S.; Gastaldi, S.; Timokhin,
V. I. Chem. Commun. 2002, 216.
(6) Routaboul, L.; Vanthuyne, N.; Gastaldi, S.; Gil, G.; Bertrand, M. P. J.
Org. Chem. 2008, 73, 364.
(7) (a) Kim, M.-J.; Chung, Y. I.; Lee, H. K.; Kim, D.; Park, J. J. Am. Chem.
Soc. 2003, 125, 11494. (b) Kim, M.-J.; Kim, H. M.; Kim, D.; Ahn, Y.; Park, J.
Green Chem. 2004, 6, 471. (c) Bore´n, L.; Mart´ın-Matute, B.; Xu, Y.; Cordova,
A.; Ba¨ckvall, J.-E. Chem. Eur. J. 2006, 12, 225.
(8) Mart´ın-Matute, A. B.; Ba¨ckvall, J.-E. Curr. Opin. Chem. Biol. 2007, 11,
226.
(9) (a) Bornscheuer, U. T.; Kazlauskas, R. J. Hydrolases in Organic Synthesis,
Regio-and StereoselectiVe Biotransformations, 2nd ed.; Wiley-VCH: Weinheim,
Germany, 2006; Chapter 6. (b) Bordera, F. Chem. ReV. 2002, 102, 4817, and
refs cited therein.
(10) (a) Fitzpatrick, P. A.; Klibanov, A. M. J. Am. Chem. Soc. 1991, 113,
3166. (b) Kazlauskas, R. J.; Weissfloch, A. N. E. J. Mol. Catal. B 1997, 3, 65.
(c) Savile, C. K.; Kazlauskas, R. J. J. Am. Chem. Soc. 2005, 127, 2104. (d)
Mugford, P. F.; Wagner, U. G.; Jiang, Y.; Faber, K.; Kazlauskas, R. J. Angew.
Chem., Int. Ed. 2008, 47, 8782.
(11) Nechab, M.; El Blidi, L.; Vanthuyne, N.; Gastaldi, S.; Bertrand, M. P.;
Gil, G. Org. Biomol. Chem. 2008, 3917.
† Equipe Chimie Mole´culaire Organique.
‡ Chirosciences.
(1) For general reviews, see: (a) Faber, K. Chem. Eur. J. 2001, 7, 5004. (b)
Pellissier, H. Tetrahedron 2003, 59, 8291. (c) Pellissier, H. Tetrahedron 2008,
64, 1563.
(2) Gastaldi, S.; Escoubet, S.; Vanthuyne, N.; Gil, G.; Bertrand, M. P. Org.
Lett. 2007, 9, 837.
(3) (a) Parvulescu, A. N.; Jacobs, P. A.; De Vos, D. E. AdV. Synth. Catal.
2008, 350, 113. (b) Roengpithya, C.; Patterson, D. A.; Livingston, A. G.; Taylor,
P. C.; Irwin, J. L.; Parrett, M. R. Chem. Commun. 2007, 3462. (c) Parvulescu,
A. N.; Jacobs, P. A.; De Vos, D. E. Chem. Eur. J. 2007, 13, 2034. (d) Crawford,
J. B.; Skerlj, R. T.; Bridger, G. J. J. Org. Chem. 2007, 72, 669. (e) Blacker,
A. J.; Stirling, M. J.; Page, M. I. Org. Process Res. DeV. 2007, 11, 642. (f)
Veld, M. A. J.; Hult, K.; Palmans, A. R. A.; Meijer, E. W. Eur. J. Org. Chem.
2007, 5416. (g) Kim, M.-J.; Kim, W.-H.; Han, K.; Choi, Y. K.; Park, J. Org.
Lett. 2007, 9, 1157. (h) Stirling, M.; Blackerb, J.; Page, M. I. Tetrahedron Lett.
2007, 48, 1247. (i) Hoben, C. E.; Kanupp, L.; Ba¨ckvall, J.-E. Tetrahedron Lett.
2007, 49, 977. (j) Paetzold, J.; Ba¨ckvall, J.-E. J. Am. Chem. Soc. 2005, 127,
17620. (k) Reetz, M. T.; Schimossek, K. Chimia 1996, 50, 668.
(4) For the preparation of (R)-amines from chemoenzymatic deracemization,
see also: (a) Bailey, K. R.; Ellis, A. J.; Reiss, R.; Snape, T. J.; Turner, N. J.
Chem. Commun. 2007, 3640. (b) Dunsmore, C. J.; Carr, R.; Fleming, T.; Turner,
N. J. J. Am. Chem. Soc. 2006, 128, 2224. and previous refs cited therein.
10.1021/jo900074w CCC: $40.75
Published on Web 03/10/2009
2009 American Chemical Society
J. Org. Chem. 2009, 74, 2901–2903 2901

TABLE 1. Racemization of (S)-1a with CF3CH2SHa
TABLE 2. Kinetic Resolution of (()-1a with Alkaline Proteasea
entry
solvent
time
ee % (yield %)b
entry
solvent
C% (time) 1a ee % 2a ee %
E
1
2
3
4
5
6
7
3-Me-3-pentanol
THF/3-Me-3-pentanol (1:1)
THF/3-Me-3-pentanol (1:2)
n-C6H14/3-Me-3-pentanol (1:1) 52.6 (3h)
n-C6H14/3-Me-3-pentanol (1:2) 51.6 (3h)
toluene/3-Me-3-pentanol (1:1)
toluene/3-Me-3-pentanol (1:2)
51.6 (3h)
46.7 (3h)
53.9 (3h)
96
72
96
99
96
94
96
90
82
82
88
90
88
88
75
22
40
75
75
58
61
1
2
3
4
5
6
7
8
THF
toluene
3-Me-3-pentanol
THF/3-Me-3-pentanol (1:1)
THF/3-Me-3-pentanol (1:2)
n-C6H14/3-Me-3-pentanol (1:1)
toluene/3-Me-3-pentanol (1:1)
toluene/3-Me-3-pentanol (2:1)
30 min
30 min
3-6 h
1.5 h
1.5 h
1.5 h
0 (70)c
0 (94)c
84-60 (nd)
14 (81)
14 (84)
25 (nd)
18 (95)
6 (79)
51.5 (2h)
52.2 (3h)
1 h
40 min
a All reactions were performed at 18-24 °C on 1 mL of a 0.1 M
solution (unless otherwise stated) of 1a (0.1 mmol) with 5 mg of
surfactant-coated alkaline protease (co-lyophilized in phosphate buffer
0.1 M pH 7.2 with Brij56 and R,ꢀ-D-glycopyranoside 8:1:1 w/w/w) and
n-heptylCONHCH2CO2CH2CF3 (1 equiv).
a Three milliliters of a 0.1 M solution of amine (0.3 mmol) was
irradiated at 22-24 °C in the presence of 1.2 equiv of trifluoro-
ethanethiol, in a quartz vessel, in a Rayonet apparatus.6 b Isolated after
derivatization as BOC-derivative unless otherwise stated. c Determined
by 1H NMR using pentamethylbenzene as internal standard after
irradiating for 3 h, notwithstanding that racemization was completed in
30 min. This confirmed that no degradation interfered during the
racemization process.
3-pentanol, was observed in mixtures of hexane and 3-methyl-
3-pentanol (entries 4 and 5).
Regarding the association of both processes to achieve the
DKR experiments, one of the main difficulties arose from
the rate of the photochemical transformation of the thiol into
the corresponding disulfide. Racemization efficiency is closely
related to the rate of oxidation of the thiol into the corresponding
disulfide.6 Trifluoroethane thiol is acidic, and trifluoroethyl
sulfanyl radical is easy to reduce.14 This favors the formation
of disulfide radical anion, by increasing the concentration in
thiolate anion in the reaction medium.6 This side reaction slows
the racemization. Even though the photolysis of the disulfide
generates sulfanyl radicals, after complete consumption of the
thiol, the concentration of hydrogen atom donor in the reaction
medium becomes extremely low (the thiol being only regener-
ated through hydrogen abstraction from the amine or from the
cosolvent).
The concentrations of trifluoroethane thiol and its disulfide
were monitored by 19F NMR during racemization experiments.
The thiol/disulfide ratio dropped approximately from 100:0 to
75:25 within 1 h in most solvents. Further irradiation further
decreased this ratio. As an example, in a 1:2 mixture of THF
and 3-Me-3-pentanol, the thiol/disulfide ratio that was very close
to 70:30 after irradiating for 60 min became 50:50 after 90 min.
Subtilisins contain no disulfide bridges that could interfere
with the radical process.15 This was a significant advantage
anticipated from associating resolution by a subtilisin-like
enzyme to light-induced racemization. Nevertheless, the major
problem still arose from enzyme irradiation. It has been reported
that subtilisin BPN′ was inactivated under laser flash photoly-
sis.16 The major damages were assigned to the formation of
tryptophanyl and tyrosinyl radicals. Indeed, we observed that
after irradiation the enzyme activity was reduced with respect
to the acylation of 1a.
the corresponding thiol. In the presence of thioglycolic methyl
ester, the racemization process was shown to be fast and efficient
in benzene solution under these experimental conditions;
moreover, it proceeded with little or no degradation of the amine
via oxidative side reactions.6
Having in hand two separate performant processes, we had
to test their compatibility. The kinetic resolution was not altered
in the presence of thiol. However, the best solvent for KR (i.e.,
3-methyl-3-pentanol) was a poor solvent regarding the photo-
chemically induced racemization (slowed down with respect to
toluene or THF). Moreover, thioglycolic derivatives were proven
to be inefficient regarding racemization in the presence of the
tertiary alcohol. This led us to use trifluoroethane thiol (Table
1), the S-H bond of which is slightly stronger than that of
methylthioglycolate (or the corresponding amide) that was used
in our previous studies.2,6,12
Although tertiary alcohols are not the best hydrogen bond
donors among all alcohols, they might contribute to strengthen
the R-C-H bond dissociation enthalpy of the solvated amine
(Table 1, entry 3).13
Therefore, both the racemization process (Table 1) and the
alkaline protease-mediated KR (Table 2) had to be reinvestigated
in solvent mixtures involving the tertiary alcohol and hexane,
toluene, or THF.
As exemplified in Table 1, racemization was quite efficient
in solvent mixtures. It took less than 2 h in most of them (entries
4-8). For instance, monitoring the reaction showed that 30 min
was needed to reach 14% ee in a 1:1 mixture of THF and
3-methyl-3-pentanol, whereas 60 min was needed to reach the
same ee in a 1:2 mixture of the same solvents.
The mixtures of solvents giving satisfying results for race-
mization were concomitantly tested as solvents for the KR
experiments. The results are given in Table 2.
Acceptable to good enantioselectivity factors were registered
in all solvent mixtures. However, better results were obtained
when 3-methyl-3-pentanol was associated with nonbasic, low
polarity solvents such as hexane or toluene (entries 4-7). The
highest E value (75), identical to that obtained in pure 3-methyl-
In order to circumvent the encountered obstacles to perform
a real DKR process, a one-pot sequential procedure was
devised. A first KR period in pure 3-methyl-3-pentanol was
followed by photochemically induced racemization for 2 h,
after addition of trifluoroethane thiol and THF (DKR period).
The irradiation was stopped, and KR was started again for
(14) (a) For the incidence of R-fluorine atoms on the thiolate anion
stabilization, see: De´zarnaud-Dandine, C.; Sevin, A. Chem. Phys. 1995, 195,
51. (b) For comparative evaluation of the influence of R- and ꢀ-fluorine atoms,
see also: Rusu, P. C.; Giovanetti, G.; Brocks, G. J. Phys. Chem. C 2007, 111,
14448.
(15) (a) Bode, W.; Papamokos, E.; Musil, D. Eur. J. Biochem. 1987, 166,
673. (b) Wright, C. S.; Alden, R. A.; Kraut, J. Nature 1969, 221, 235. (c) Drenth,
J.; Hol, W. G. J.; Jansonius, J. N.; Koekoek, R. Eur. J. Biochem. 1972, 26, 177.
(16) Blum, A.; Grossweiner, L. I. Photochem. Photobiol. 1982, 36, 622.
(12) BDE values calculated at 298 K according to the G3B3 methodology
are 363.2, 362.7, and 364.3 kJ mol-1 for methyl thioglycolate, N,N-dimethyl
thioglycolic amide, and trifluoroethane thiol, respectively. The variation in the
series is tiny, but the trend can be considered as significant.
(13) For examples of incidence of intramolecular hydrogen bonding on the
R-CH BDE in amines, see: (a) Laleve´e, J.; Allonas, X.; Fouassier, J.-P. J. Am.
Chem. Soc. 2002, 124, 9613. (b) See also ref 5c.
2902 J. Org. Chem. Vol. 74, No. 7, 2009

TABLE 4. Conversion of Amines 1a-f into (S)-Amides 2a-f with
Alkaline Protease (One-Pot Sequential Process)a
amine ee % amide ee % amine ee %
amide
after the
first KR
after the
first KR
after
isolated
entry amine
racemization yield % (ee %)
1
2
3
4
5
6
1a
1b
1c
1d
1e
1f
94
80
96
97
100
80
90
82
92
90
82
96
15
17
8
30
24
9
80 (86)
65 (78)
65 (85)
70 (86)
70 (80)
58 (94)
FIGURE 1. Amines structures.
TABLE 3. Kinetic Resolution of Amines 1a-f with Alkaline
Proteasea
entry amine
C% (time)
51.1 (1.5 h)
53.6 (2 h, 40 min)
53 (1.5 h)
53 (1.5 h)
53.8 (1.5 h)
45.5 (2.5 h)
amine ee % amide ee %
E
a All reactions were performed at 22-24 °C with alkaline protease
(co-lyophilized in phosphate buffer 0.1 M pH 7.2 with Brij56 and
R,ꢀ-D-glycopyranoside 8:1:1 w/w/w).
1
2
3
4
5
6
1a
1b
1c
1d
1e
1f
94
95
96
97
98
80
90
82
85
86
84
96
67
38
48
55
52
enantioselectivity and the rate of the KR. Further progress en
route to effective DKR will be reported in due course.
115
a All reactions were performed at 18-24 °C on 1 mL of a 0.3 M
solution of amine (0.3 mmol) in 3-Me-3-pentanol with 15 mg of
surfactant-coated alkaline protease (co-lyophilized in phosphate buffer
0.1 M pH 7.2 with Brij56 and R,ꢀ-D-glycopyranoside 8:1:1 w/w/w) and
n-heptCONHCH2CO2CH2CF3 (0.7 equiv).
Experimental Section
Immobilization of Alkaline Protease. 11 Alkaline protease (120
mg) from Valley Research was dissolved in a solution of n-octyl
R,ꢀ-D-glycopyranoside (15 mg) and Brij56 (polyethylene glycol
hexadecyl ether, 15 mg) in a phosphate buffer (pH 7.2, unless
otherwise stated, 6 mL), and the mixture was rapidly frozen in liquid
N2 and lyophilized for 24 h.
one night after adding a second portion of acyl donor (0.7
equiv) and enzyme (Scheme 1).
General Procedure for One-Pot Kinetic Resolution/Racemiza-
tion/Kinetic Resolution Sequence. Amine (0.3 mmol) was added
to a solution of N-octanoylglycine trifluoroethyl ester (60 mg, 0.21
mmol) and coated alkaline protease (15 mg) in 3-methyl-3-pentanol
(1 mL). The resulting mixture was stirred at room temperature for
1.5-3 h depending on the substrate (see Table 3). The amine ee
was determined by GC after derivatization in trifluoroacetamide
of an aliquot of the crude mixture with N-methylbistrifluoroaceta-
mide (1.5 equiv). The mixture was diluted with 3-methyl-3-pentanol
(1 mL) and THF (1 mL), and trifluoroethane thiol (12.3 mg, 0.18
mmol, 1.2 equiv with respect to the remaining amine, assuming
50% conversion for the KR) was added. The mixture was irradiated
at 22-24 °C in a quartz tube in a Rayonet apparatus (RPR-200,
16 UV lamps RPR-3000) for 2 h. The amine ee was determined
by GC. N-Octanoylglycine trifluoroethyl ester (60 mg, 0.21 mmol)
and coated alkaline protease (15 mg) were then added. The resulting
mixture was stirred at room temperature for 15 h. The enzyme was
filtered out from the solution and washed with 5 mL of dichlo-
romethane. The combined organic phases were then evaporated,
and the crude material was purified by flash chromatography on
silica gel (dichloromethane/methanol; gradient from 0 to 5%) to
isolate the amide.
Selecting 3-methyl-3-pentanol as the solvent for the very first
KR period was justified by the high enantioselectivity observed
in this solvent for most amines in the series shown in Figure 1
(Table 3). Amide’s enantiomeric excesses ranged from 82 to
96%, and enantioselectivity factor ranged from 38 to 115.
The choice of the 1:2 mixture of THF and 3-methyl-3-
pentanol offered a good compromise for the second and third
period. It was justified by the rate of racemization in this
medium; all the more because in the presence of nonbasic low
polarity solvents such as hexane or toluene racemization was
slowed down after the first KR period. The reasons for this
slowing down are not clear yet, but the amount of trifluoroet-
hanol released by the KR might be responsible for additional
hydrogen bonding interaction with the amine.
Enantiomeric excesses of the starting amine and the amide
that were monitored at each stage of the process and the overall
yield in isolated amide are reported in Table 4 for the series of
amines given in Figure 1.
Yields exceeding 70% together with ee superior to 80% were
observed in most cases except for amines 1f, for which the data
are rather close to those of a simple KR experiment.
In summary, performing successively in the same pot (i)
alkaline protease-catalyzed KR using N-octanoyl glycine trif-
luoroethyl ester as the acyl donor, (ii) light-induced radical
racemization mediated with trifluoroethane thiol in the presence
of the enzyme and the residual acyl donor, (iii) a second KR
(after stopping irradiation and adding a second portion of both
the enzyme and the acyl donor) led to (S)-amides in yields
ranging from 58 to 80%. The final enantiomeric excesses ranged
between 78 and 94%. Other enzymes and acyl donors are
currently under investigation in order to improve both the
Acknowledgment. The authors thanks Pr. Didier Siri for BDE
calculations. Gifts of alkaline protease from Valley Research
are gratefully acknowledged. This work was supported by the
Agence Nationale de la Recherche (ANR 06-Blan-0137).
Supporting Information Available: Experimental proce-
dures and full spectroscopic data for all new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
JO900074W
J. Org. Chem. Vol. 74, No. 7, 2009 2903