Dynamic kinetic resolution of N-benzoyl-DL-amino acids via peptide bond forming reactions

Article abstract of DOI:10.2174/092986610790963582

Dynamic kinetic resolution (DKR) was demonstrated in the carbodiimide-mediated couplings of N-benzoyl-DL-amino acids with L-amino acid esters: the yields of the D-L-peptides significantly exceeded 50% in some cases. N-benzoyl-DL-t-leucine afforded the D-L-peptide almost exclusively (up to 96% yield) in the reaction with methyl L-prolinate, which is the most efficient DKR obtained in the field of amino acids and derivatives.

Full text of DOI:10.2174/092986610790963582

Protein & Peptide Letters, 2010, 17, 419-422
419
Dynamic Kinetic Resolution of N-Benzoyl-DL-Amino Acids via Peptide
Bond Forming Reactions
Toshifumi Miyazawa* and Takashi Hamada
Department of Chemistry, Faculty of Science and Engineering, Konan University, Higashinada-ku, Kobe 658-8501,
Japan
Abstract: Dynamic kinetic resolution (DKR) was demonstrated in the carbodiimide-mediated couplings of N-benzoyl-DL-
amino acids with L-amino acid esters: the yields of the D-L-peptides significantly exceeded 50% in some cases. N-
Benzoyl-DL-t-leucine afforded the D-L-peptide almost exclusively (up to 96% yield) in the reaction with methyl L-
prolinate, which is the most efficient DKR obtained in the field of amino acids and derivatives.
Keywords: Dynamic kinetic resolution (DKR), carbodiimide-mediated couplings, N-benzoyl-DL-amino acids, 5(4H)-
oxazolones.
INTRODUCTION
MATERIALS AND METHODS
Kinetic resolution of racemic mixtures is still one of the
most common ways of obtaining enantiomerically enriched
compounds in industry in spite of the successful develop-
ment of asymmetric syntheses. With this process, however,
the maximum yield of one enantiomer is intrinsically only
50%. This limitation can be overcome by dynamic kinetic
resolution (DKR) [1], where the enantiomers undergo rapid
interconversion under the reaction conditions and the desired
enantiomer can be theoretically obtained in a quantitative
yield with enantiomeric excess approaching 100%. There-
fore, increasing attention has been drawn to the discovery of
DKR processes. N-Acyl-amino acids such as N-Benzoyl
(Bz)-amino acids are known to be extremely prone to race-
mization during the coupling process. Their chiral lability is
the basis for the well-known Young racemization test [2],
which addresses the degree of racemization during the cou-
pling of Bz-L-Leu with Gly-OEt by different coupling meth-
ods and conditions. This was followed by the Davies test
utilizing the coupling of Bz-L-Val [3]. Of all racemization
tests, those involving N-Bz-amino acids are known to be the
most sensitive [4]. We have already reported [5] that in the
EDC-mediated coupling of Bz-L-Val with L-Val-OMe in
DMF the percentage of the D-L-isomer in the diastereomeric
dipeptide products greatly exceeded 50% (D-L-isomer: 64%),
and that the use of racemization-suppressing N-hydroxy ad-
ditives such as HOBt [6] had only a limited effect (D-L-
isomer: 48%). In view of these facts, we have come to an
expectation that there will be a fair chance of discovering
DKR during the peptide bond formation of N-Bz-DL-amino
acids by the carbodiimide method to preferentially afford
one of the diastereomers of the resulting peptide. In practice,
we obtained the most efficient example of DKR of amino
acid derivatives.
N-Bz-DL-amino acids were prepared using benzoyl chlo-
ride (Bz-Cl) under the Schotten-Baumann conditions [7].
They were converted to 5(4H)-oxazolones using DCC ac-
cording to the literature [8]. The methyl esters of L-amino
acids were mainly prepared as their hydrochlorides by the
use of MeOH and thionyl chloride [9]. L-Pro-OMeꢀTosOH
was purchased from Peptide Institute, Inc. Other chemicals
were obtained from Wako Pure Chemical Industries, Tokyo
Chemical Industry or Aldrich Chemical Co. All solvents
were distilled and dried over molecular sieves prior to use.
Couplings of N-Benzoyl-DL-Amino Acids (Bz-DL-Xaa)
with L-Amino Acid Methyl Esters (L-Xbb-OMe) by the
EDC Method
Bz-DL-Xaa (0.15 mmol), L-Xbb-OMeꢀHCl (or L-Pro-
OMeꢀTosOH) (0.15 mmol) and TEA (20.9 μl, 0.15 mmol)
with or without an additive (see Table 1) were suspended in
DCM (1 ml) at 0 °C. After 15 min of stirring, EDCꢀHCl
(28.8 mg, 0.15 mmol) and DCM (1 ml) were added at this
temperature. The resulting mixture was stirred at 0 °C for 2 h
and then at 25 °C for 48 h or 72 h. After evaporation of the
solvent, followed by the addition of Bz-L-Val-L-Val-OEt
(17.4 mg, 0.05 mmol) as an internal standard, the residue
was distributed between ethyl acetate and 2M HCl, and the
organic layer was further washed with 2M HCl, saturated
brine, saturated NaHCO3 and saturated brine again, and dried
over Na2SO4. The residue after evaporation of the solvent
was dissolved in acetonitrile and injected onto an ODS col-
umn for the HPLC separation of the diastereomers of the
resulting peptide. At the same time the yield of the peptide
formed was determined by the internal standard method.
Reactions of the 5(4H)-Oxazolones from N-Benzoyl-DL-
Amino Acids (Bz-DL-Xaa) with L-Pro-OMe
*Address correspondence to this author at the Department of Chemistry,
Faculty of Science and Engineering, Konan University, 8-9-1 Okamoto,
Higashinada-ku, Kobe 658-8501, Japan; Fax: +81-78-435-2539;
E-mail: miyazawa@ konan-u.ac.jp
The 5(4H)-oxazolone (0.2 mmol) from Bz-DL-Xaa, L-
Pro-OMeꢀTosOH (60.1 mg, 0.2 mmol) and Bz-L-Val-L-Val-
OEt (17.4 mg, 0.05 mmol) as an internal standard were sus-
0929-8665/10 $55.00+.00
© 2010 Bentham Science Publishers Ltd.

420 Protein & Peptide Letters, 2010, Vol. 17, No. 4
Miyazawa and Hamada
pended in DCM (2 ml). After 15 min, a solution of TEA
(27.9 μl, 0.2 mmol) in DCM (0.7 ml) was added and the re-
action mixture was stirred at 25 °C for 72 h. An aliquot of
the reaction mixture was withdrawn, diluted with acetic acid
and then injected on to the HPLC column.
compound. Table 1 shows the diastereomeric ratio (D-L : L-L)
and the yields of the D-L- and L-L-peptides during the cou-
pling of Bz-DL-Tle with L-Pro-OMe by the EDC method
with or without an additive in DCM. The coupling reaction
proceeded to completion within 48 h and the yield of the D-
L-peptide surprisingly reached 96%, clearly demonstrating
the occurrence of DKR (entry 1). The addition of some basic
compounds was next examined in expectation of facilitating
racemization. However, the use of DABCO and DBU [11]
resulted in a slight retardation of the coupling reaction (en-
tries 2 and 3). It is only when the chiral base, brucine, coex-
isted in the system that the yield of the D-L-peptide became
only a little higher (97%) (entry 4). On the contrary, when
the racemization-suppressing additive, HOBt, was employed,
the yield of the D-L-peptide dropped to 89% (entry 5). The
addition of DMAP [12] only resulted in a slight retardation
of the coupling reaction (entry 6). The solvent effect on the
same EDC-mediated coupling was next examined (Table 2).
In all the solvents used the D-L-peptide was produced in
good to excellent yields far larger than 50%, consistent with
the desired DKR process. However, the yield of the D-L-
peptide was relatively low in the most polar solvent DMF
and in the most apolar solvent o-xylene. A better solvent
than DCM was not found.
HPLC Analysis
HPLC analyses were performed on a GL Sciences PU610
instrument, equipped with a Rheodyne 8125 sample injector
and a GL Sciences UV620 variable wavelength UV monitor.
A Shimadzu C-R6A data processor was used for data acqui-
sition and processing. Typical HPLC conditions were as fol-
lows: column, Inertsil ODS-3 (1.5 mm i.d. ꢀ 150 mm) (GL
Sciences); mobile phase, 40–60% (by volume) MeOH aq.;
flow rate 0.1 ml min-1; column temperature, 30–40 °C; detec-
tion, UV at 254 nm.
RESULTS AND DISCUSSION
To investigate the possibility of DKR in the coupling
process we took advantage of the good separation of the di-
astereomeric pairs of N-Bz-dipeptide esters by reversed-
phase HPLC [10], which also permitted the precise determi-
nation of the coupling yield by the use of an internal standard
Table 1. Coupling of Bz-DL-Tle with L-Pro-OMe by the EDC Method with or without an Additive in DCMa
Yield (%)
Entry
Additive (equiv.)
D-L : L-L
D-L
L-L
1
2
3
4
5
6
None
96 : 4
97 : 3
97 : 3
97 : 3
90 : 10
96 : 4
96
94
88
97
89
94
4
3
DABCO (0.67)
DBU (0.67)
Brucine (0.67)
HOBt (1)
3
3
10
4
DMAP (1)
aReaction conditions: Bz-DL-Tle/L-Pro-OMeꢁTosOH/TEA/EDCꢁHCl, 1:1:1:1 equiv.; reactant concentration, 0.075 M.; duration and temperature, 2 h at 0 °C, then 48 h at 25 °C.
Table 2. Solvent Effect on the Coupling of Bz-DL-Tle with L-Pro-OMe by the EDC Methoda
Yield (%)
Solvent
D-L : L-L
D-L
L-L
o-Xylene
Toluene
92 : 8
93 : 7
96 : 4
90 : 10
92 : 8
93 : 7
96 : 4
84 : 16
80
92
96
89
92
93
95
66
7
7
DCM
4
Chloroform
Ethyl acetate
Tetrahydrofuran
Acetonitrile
DMF
10
8
7
4
13
aReaction conditions: Bz-DL-Tle/L-Pro-OMeꢁTosOH/TEA/EDCꢁHCl, 1:1:1:1 equiv.; reactant concentration, 0.075 M.; duration and temperature, 2 h at 0 °C, then 48 h at 25 °C.

Dynamic Kinetic Resolution of N-Benzoyl-DL-Amino Acids
Protein & Peptide Letters, 2010, Vol. 17, No. 4 421
Based on the above results, the N-Bz derivatives of sev-
eral DL-amino acids (Bz-DL-Xaa) were allowed to react with
L-Pro-OMe by the EDC method in DCM (Table 3). With Val
and Leu as the carboxyl components the results clearly dem-
onstrate the DKR process, though the yields of the D-L-
peptides were lower than that obtained with Tle. The influ-
ence of the amino components (L-Xbb-OMe) was next ex-
amined on the coupling of Bz-Tle, and the results are also
included in Table 3. It is only when Xbb was Leu besides
Pro that the desired DKR process was unambiguously dem-
onstrated. The coupling reactions were retarded to a great
extent with other amino acids as the amino components.
catalyzed hydrolysis or alcoholysis [13, 14]. We previously
reported on the asymmetric induction during the aminolysis
of the oxazolones from N-Bz-DL-amino acids with a series of
L-amino acid esters [10]. In some cases the diastereomeric
ratios were found to go too far from 50:50, but it was ob-
scure whether this was ascribed to DKR or not, because the
yields of the peptide products were not quantified at that
time. Therefore, the reactions of the oxazolones from N-Bz-
DL-amino acids (Bz-DL-Xaa) with L-Pro-OMe in DCM were
investigated in connection with the corresponding EDC-
mediated couplings of Bz-DL-Xaa. The results are shown in
Table 4. The DKR processes were clearly observed in the
reactions of the oxazolones from Val, Leu and Tle. Roughly
speaking, the results are like those shown in Table 3 for the
couplings by the EDC method. However, when Xaa was Tle
or Phe, the yields of D-L-peptides were lower than those ob-
tained by the EDC-mediated couplings.
It transpires that the formation of 5(4H)-oxazolone in-
termediates can account for much of the racemization occur-
ring during the couplings of N-Acyl-amino acids or peptides
[4]. The oxazolones racemize easily due to the low pKa
value of the C-4 proton. They can undergo facile ring open-
ing with a variety of nucleophiles to give amino acid deriva-
tives. Thus, oxazolones make useful substrates for DKR. In
fact, the DKR reactions of oxazolones have been reported
mainly via enzymatic transformations such as lipase-
In the EDC-mediated coupling of Bz-DL-Tle with L-Pro-
OMe, the carboxyl component transforms into activated in-
termediate(s) (represented by Bz-Tle-X) by the action of
EDC (Scheme 1) [15]. The enantiomers of Bz-Tle-X un-
Table 3. Couplings of N-benzoyl-DL-Amino Acids (Bz-DL-Xaa) with L-Amino Acid Methyl Esters (L-Xbb-OMe) by the EDC
Method in DCMa
Yield (%)
Xaa
Xbbb
D-L : L-L
D-L
L-L
Ala
Val
Leu
Tle
Pro
Pro
Pro
Pro
Ala
Val
Leu
Phe
Pro
89 : 11
91 : 9
44
82
77
96
37
42
50
39
42
5
8
89 : 11
96 : 4
9
4
60 : 40
67 : 33
60 : 40
57 : 43
86 : 14
25
21
33
30
6
Phe
aReaction conditions: Bz-DL-Xaa/L-Xbb-OMeꢀHCl (or TosOH)/TEA/EDCꢀHCl, 1:1:1:1 equiv.; reactant concentration, 0.075 M; duration and temperature, 2 h at 0 °C, then 72 h at 25
°C.
bUsed in the form of hydrochlorides. L-Pro-OMe was used in the form of p-toluenesulfonate.
Table 4. Reactions of the 5(4H)-Oxazolones from N-Benzoyl-DL-Amino Acids (Bz-DL-Xaa) with L-Pro-OMe in DCMa
Yield (%)
Xaa
D-L : L-L
D-L
L-L
Ala
Val
Leu
Tle
86 : 14
87 : 13
85 : 15
90 : 10
85 : 15
48
82
74
88
33
8
12
13
10
6
Phe
aReaction conditions: 5(4H)-oxazolone from Bz-DL-Xaa/L-Pro-OMeꢀTosOH/TEA, 1:1:1 equiv.; reactant concentration, 0.074 M; duration and temperature, 72 h at 25 °C.

422 Protein & Peptide Letters, 2010, Vol. 17, No. 4
Miyazawa and Hamada
dergo isomerization mainly via the enantiomeric oxazolones
which interconvert each other quite rapidly. Then the peptide
products are produced mainly via the oxazolones and the
reaction of the D-oxazolone with L-Pro-OMe must be much
faster than that of the L-oxazolone. The same is probably true
with the reactions of the enantiomers of Bz-Tle-X. As a con-
sequence, the D-L-peptide can be formed almost exclusively.
It is worthwhile to note that this is the most efficient DKR
observed in the field of amino acids and derivatives includ-
ing oxazolones reported to date [14] and makes a unique
example of nearly exclusive formation of one epimer via the
non-enzymatic DKR of stereochemically labile enantiomers
by a chiral reagent.
DABCO = 1,4-Diazabicyclo[2.2.2]octane
DBU
=
=
1,8-Diazabicyclo[5.4.0]undec-7-ene
4-(Dimethylamino)pyridine.
DMAP
REFERENCES
[1]
Huerta, F. F.; Minidis, A. B. E.; Bäckvall, J.-E. Racemisation in
asymmetric synthesis. Dynamic kinetic resolution and related proc-
esses in enzyme and metal catalysis. Chem. Soc. Rev., 2001, 30,
321-331.
[2]
[3]
Williams, M. W.; Young, G. T. Amino-acids and peptides. Part
XVI. Further studies of racemisation during peptide synthesis. J.
Chem. Soc., 1963, 881-889.
Davies, J. S.; Thomas, R. J.; Williams, M. K. Nuclear magnetic
resonance spectra of benzoyldipeptide esters. A convenient test for
racemisation in peptide synthesis. J. Chem. Soc. Chem. Commun.,
1975, 76-77.
Bz-L-Tle
Bz-D-Tle
[4]
[5]
Benoiton, N. L. Chemistry of Peptide Synthesis; Taylor & Francis:
Boca Raton, Florida, 2006; Chap. 4.
[Bz-L-Tle-X]
[Bz-D-Tle-X]
Miyazawa, T.; Otomatsu, T.; Fukui, Y.; Yamada, T.; Kuwata, S.
Simultaneous use of 1-hydroxybenzotriazole and copper(II) chlo-
ride as additives for racemization-free and efficient peptide synthe-
sis by the carbodiimide method. Int. J. Peptide Protein Res., 1992,
39, 308-314.
König, W.; Geiger, R. N-Hydroxyverbindungen als Katalysatoren
für die Aminolyse aktivierter Ester. Chem. Ber., 1973, 106, 3626-
3635.
C(CH3)3
H
C(H3C)3
H
O
O
C
C
C
C
N
O
N
O
C
C
(D)
(L)
[6]
L-Pro-OMe
L-Pro-OMe
[7]
[8]
Greenstein, J. P.; Winitz, M. Chemistry of the Amino Acids; Wiley
& Sons: New York, 1961, p. 1266.
Chen, F. M. F.; Kuroda, K.; Benoiton, N. L. A simple preparation
of 5-oxo-4,5-dihydro-1,3-oxazoles (oxazolones). Synthesis, 1979,
230-232.
Brenner, M.; Huber, W. Herstellung von ꢀ-Aminosäureestern
durch Alkoholyse der Methylester. Helv. Chim. Acta, 1953, 36,
1109-1115.
Bz-L-Tle-L-Pro-OMe
Bz-D-Tle-L-Pro-OMe
Scheme 1. Possible mechanism for the EDC-mediated coupling of
Bz-DL-Tle with L-Pro-OMe affording the D-L-peptide almost exclu-
sively. Bz-Tle-X denotes the activated species derived from Bz-Tle.
[9]
[10]
Miyazawa, T.; Otomatsu, T.; Higashi, K.; Yamada, T.; Kuwata, S.
Asymmetric induction during the aminolysis of 5(4H)-oxazolones
from N-benzoyl amino acids; almost specific formation of one
epimer in the reaction of the oxazolone from N-benzoyl-DL-t-
leucine with methyl L-prolinate. Bull. Chem. Soc. Jpn., 1988, 61,
4161-4163.
Dinh, P. M.; Williams, J. M. J.; Harris, W. Selective racemisation
of esters: relevance to enzymatic hydrolysis reactions. Tetrahedron
Lett., 1999, 40, 749-752.
Höfle, G.; Steglich, W.; Vorbrüggen, H. 4-Dialkylaminopyridines
as highly active acylation catalysts. Angew. Chem. Int. Ed. Engl.,
1978, 17, 569-583.
Crich, J.; Brieva, R.; Marquart, P.; Gu, R.-L.; Flemming, S.; Sih, C.
J. Enzymic asymmetric synthesis of ꢀ-amino acids. Enantioselec-
tive cleavage of 4-substituted oxazolin-5-ones and thiazolin-5-ones.
J. Org. Chem., 1993, 58, 3252-3258.
ABBREVIATIONS
EDC
=
1-Ethyl-3-(3-
dimethylaminopropyl)carbodiimide
DMF
HOBt
DCC
TosOH
TEA
=
=
=
=
=
=
=
=
=
N,N-Dimethylformamide
1-Hydroxybenzotriazole
N,N’- dicyclohexylcarbodiimide
p-Toluenesulfonic acid
Triethylamine
[11]
[12]
[13]
DCM
ODS
HPLC
Tle
Dichloromethane
[14]
[15]
Brown, S. A.; Parker, M.-C.; Turner, N. J. Dynamic kinetic resolu-
tion: synthesis of optically active ꢀ-amino acid derivatives. Tetra-
hedron: Asymmetry, 2000, 11, 1687-1690.
Podlech, J. In Synthesis of Peptides and Peptidomimetics, Methods
of Organic Chemistry; Goodman, M.; Felix, A.; Moroder, L.; To-
niolo, C., Eds.; Thiem: Stuttgart, 2004; Vol. E 22a, p. 517.
Octadecylsilanized
High performance liquid chromatography
t-Leucine (2-amino-3,3-dimethylbutanoic
acid)
Received: August 26, 2008
Revised: September 22, 2008
Accepted: October 28, 2008