Synthesis of L-leucyl-L-isoleucine and L-isoleucyl-L-leucine

Article abstract of DOI:10.1134/S1070428015050048

New preparative procedures were developed applying tert-butoxycarbonyl or trifluoroacetyl protection of amino groups in the synthesis of L-leucyl-L-isoleucine and L-isoleucyl-L-leucine.

Full text of DOI:10.1134/S1070428015050048

ISSN 1070-4280, Russian Journal of Organic Chemistry, 2015, Vol. 51, No. 5, pp. 615–618. © Pleiades Publishing, Ltd., 2015.
Original Russian Text © V.A. Gaidukevich, L.A. Popova, Z.P. Zubreichuk, V.A. Knizhnikov, 2015, published in Zhurnal Organicheskoi Khimii, 2015,
Vol. 51, No. 5, pp. 637–640.
Synthesis of L-Leucyl-L-isoleucine and L-Isoleucyl-L-leucine
V. A. Gaidukevich, L. A. Popova, Z. P. Zubreichuk, and V. A. Knizhnikov
Institute of Physical Organic Chemistry, National Academy of Sciences of Belarus,
ul. Surganova 13, Minsk, 220072 Belarus
e-mail: knizh@ifoch.bas-net.by
Received December 17, 2014
Abstract—New preparative procedures were developed applying tert-butoxycarbonyl or trifluoroacetyl
protection of amino groups in the synthesis of L-leucyl-L-isoleucine and L-isoleucyl-L-leucine.
DOI: 10.1134/S1070428015050048
Peptides and their derivatives are biologically
active compounds. They underlie the preparation of
drugs successfully used in the treatment of
cardiovascular diseases, of antiulcer pharmaceuticals,
analgesics, immune modulators, cardioprotectants. The
more extensive application of peptides in drugs
designing is impeded by the difficulties in the
production of a considerable amount of peptides of a
high purity. Therefore the optimization of the known
procedures of peptides preparation and the
development of new preparative syntheses of this class
compounds is an urgent task. The published methods
of synthesis of L-leucyl-L-isoleucine [1, 2] and L-
isoleucyl-L-leucine [2] utilized for the protection of
amino groups the benzyloxycarbonyl group, and its
removal was performed by treatment with hydrogen in
the presence of palladium catalyst. In this study we
aimed to develop the method of preparation of these
dipeptides using for amino groups’ protection the tert-
butoxycarbonyl and the cheaper trifluoroacetyl groups
which were readily removable at the treatment with
available reagents.
butoxycarbonyl-L-isoleucyl-L-leucine 4. The removal
of the tert-butoxycarbonyl protection in compounds 3
and 4 with dioxane solution of hydrogen chloride and
the treatment of the obtained hydrochlorides of
L-leucyl-L-isoleucine and L-isoleucyl-L-leucine with
an equimolar amount of triethylamine led to the
formation of L-leucyl-L-isoleucine 5 and L-isoleucyl-
L-leucine 6.
The use of excess methyl esters of amino acids in
the condensation stage and the selected sequence of
protective groups removal made it possible to avoid
the formation of notable amounts of free amino acid
impurities in the target products.
At the application of the trifluoroacetyl protective
group in reactions of N-trifluoroacetyl-L-leucine or N-
trifluoroacetyl-L-isoleucine with methyl esters of L-
isoleucine or L-leucine respectively in the presence of
dicyclohexylcarbodiimide as the condensation agent
we obtained methyl esters of N-trifluoroacetyl-L-
leucyl-L-isoleucine
7 and N-trifluoroacetyl-L-iso-
leucyl-L-leucine 8. One-stage removal of the
protective groups in compounds 7 and 8 by the
treatment with sodium hydroxide followed by
acidifying of the reaction mixtures with trifluoroacetic
acid led to the formation of the target dipeptides 5
and 6.
The condensation of tert-butoxycarbonyl-L-leucine
or tert-butoxycarbonyl-L-isoleucine with the methyl
ester of L-isoleucine or L-leucine respectively under
the action of dicyclohexylcarbodiimide afforded
methyl ester of tert-butoxycarbonyl-L-leucyl-L-
isoleucine 1 and methyl ester of tert-butoxycarbonyl-
L-isoleucyl-L-leucine 2. The treatment of ethanol
solutions of compounds 1 and 2 with a water solution
of sodium hydroxide followed by acidification of the
reaction mixtures with citric acid provided tert-
Physicochemical characteristics of dipeptide
samples 5 and 6 obtained both at the use of tert-
butoxycarbonyl and trifluoroacetyl protection virtually
coincide with the characteristics of compounds
described in the literature thus showing the absence of
racemization processes in all the stages of the
butoxycarbonyl-L-leucyl-L-isoleucine
3
and tert-
6
15

6
16
GAIDUKEVICH et al.
O
O
O
OH
NH
H
H
N
O
N
O
1
2
. NaOH
. citric acid
O
OCH3
NH2
OCH3
OH
DCC
R1
R2
O
R2
O
+
R1
NH
R1
NH
R2
O
O
O
O
3
, 4
1
, 2
1. HCl
2
. Et N
O
3
H
N
O
OH
O
O
OCH3
1. NaOH
H
O
OCH3
NH2
2
O
N
R2
O
2. CF COOH
DCC
3
OH
R1
F3C
NH
R1
NH
+
R2
R2
R1
NH2
O
F3C
7, 8
5, 6
1
1
2
1
, 3, 5, 7, R = (CH
3
)
2
CHCH
2
, R = (CH
3
CH
2
)(CH
3
)CH; 2, 4, 6, 8, R = (CH
3
CH
2
)(CH
3
)CH, R = (CH
3
)
2
2
CHCH .
dipeptide preparation utilizing the trifluoroacetyl
protective group.
solvent was removed at a reduced pressure, the
reaction product was extracted from the residue with
ethyl ether, the solution obtained was filtered, the
solvent was removed at a reduced pressure, residue
was washed with cold hexane and dried in a vacuum.
EXPERIMENTAL
2
0
In all experiments anhydrous organic solvents were
used. Methyl esters of L-leucine and L-isoleucine were
obtained by treating with trimethylamine amino acids
methyl esters hydrochlorides prepared by procedure
Yield 29.04 g (81%), mp 118–120°С, [α] –32° (c 3,
D
–
1
MeOH). IR spectrum, ν, cm : 1752, 1688, 1655
(С=О), 1555, 1527 (N–Hamide). Н NMR spectrum
1
(CDCl ), δ, ppm: 0.77–0.86 m (12Н, 4СН ), 1.05–1.12
3
3
[1]. Trifluoroacetyl and tert-butoxycarbonyl deri-
m (1Н, СН), 1.34 s (9Н, 3СН ), 1.55–1.62 m (4Н,
3
vatives of L-leucine and L-isoleucine were obtained by
standard procedures [3]. The intermediate compounds
isolated from the reaction mixtures were used without
additional purification. IR spectra were recorded on an
IR Fourier spectrophotometer Protégé-460 from pellets
with KBr. Н and С NMR spectra were registered on
a spectrometer Bruker Avance-400, the residual proton
signals of solvent were used as internal reference. The
optical activity was measured on a polarimeter
Polamat A.
2СН ), 1.77–1.82 m (1Н, СН), 3.63 s (3Н, СН ), 4.05–
4.11 m (1Н, СН), 4.42–4.46 m (1Н, СН). С NMR
2
3
3
1
spectrum (CDCl ), δ, ppm: 11.29, 15.19, 21.93, 22.63,
3
24.47, 24.79, 28.09, 37.56, 40.61, 51.77, 52.83, 56.23,
79.63, 155.59, 171.95, 172.34. Found, %: С 60.53; Н
9.81; N 7.62. C H N O . Calculated, %: С 60.31; Н
1
13
1
8
34
2
5
9.56; N 7.81.
tert-Butoxycarbonyl-L-isoleucyl-L-leucine
methyl ester (2) was obtained similarly from 23.13 g
(
100 mmol) of tert-butoxycarbonyl-L-isoleucine,
tert-Butoxycarbonyl-L-leucyl-L-isoleucine
methyl ester (1). To a solution of 23.13 g (100 mmol)
tert-butoxycarbonyl-L-leucine in 150 mL of THF
cooled to 0°С at vigorous stirring were added in
succession solutions of 21.63 g (105 mmol) of
dicyclohexylcarbodiimide in 150 mL of THF and
21.63 g (105 mmol) of dicyclohexylcarbodiimide and
15.97 g (105 mmol) of L-leucine methyl ester. Yield
2
0
27.60 g (77%), mp 140–141°С, [α] –47.5° (с 2.5,
D
–
1
MeОН). IR spectrum, ν, cm : 1760, 1685, 1650
1
(С=О), 1555, 1525 (N–Hamide). Н NMR spectrum
(CDCl ), δ, ppm: 0.78–0.86 m (12Н, 4СН ), 1.04–1.09 m
3
3
1
1
5.97 g (110 mmol) of L-isoleucine methyl ester in
00 mL of THF. The reaction mixture was warmed to
(1Н, СН), 1.34 s (9Н, 3СН ), 1.48 t (2Н, СН , J 6.1 Hz),
3 2
1.56–1.65 m (2Н, СН ), 1.71–1.74 m (1Н, СН), 3.63 s
2
room temperature, and the stirring was continued for
0 h. The separated precipitate was filtered off, the
(3Н, СН ), 3.92 t (1Н, СН, J 6.4 Hz), 4.48–4.52 m
3
1
3
2
(1Н, СН). С NMR spectrum (CDCl ), δ, ppm: 11.09,
3
RUSSIAN JOURNAL OF ORGANIC CHEMISTRY Vol. 51 No. 5 2015

SYNTHESIS OF L-LEUCYL-L-ISOLEUCINE AND L-ISOLEUCYL-L-LEUCINE
617
1
5
5.13, 21.68, 22.57, 24.56, 24.65, 28.12, 37.0, 40.94,
0.55, 51.92, 58.91, 79.40, 155.64, 171.67, 172.94.
0°С at vigorous stirring were added in succession
solutions of 10.3 g (50 mmol) of dicyclohexylcarbodi-
imide in 70 mL of THF and 7.99 g (55 mmol) of L-
isoleucine methyl ester in 50 mL of THF. The reaction
mixture was stirred for 36 h, filtered, and the solvent
was removed at a reduced pressure. The residue was
washed with hexane, dissolved in ethyl ether, the
solution obtained was filtered, the solvent was
removed at a reduced pressure, residue was washed
Found, %: С 60.49; Н 9.71; N 7.52. C H N O .
Calculated, %: С 60.31; Н 9.56; N 7.81.
1
8
34
2
5
tert-Butoxycarbonyl-L-leucyl-L-isoleucine (3).
To a solution of 25.09 g (70 mmol) of tert-butoxy-
carbonyl-L-leucyl-L-isoleucine methyl ester in 70 mL
of ethanol a solution was added of 3 g (75 mmol) of
sodium hydroxide in 100 mL of water. The reaction
mixture was stirred for 10 h, ethanol was distilled off
at a reduced pressure. On adding 100 mL of water the
mixture was filtered, the solution obtained was
acidified with citric acid till рН 4.5, the separated
precipitate was filtered off, washed with water, and
dissolved in ethyl ether. The ether solution was dried
over sodium sulfate, filtered, and the solvent was re-
moved at a reduced pressure. The residue was washed
with hexane and dried in a vacuum. Yield 24.1 g
with hexane and dried in a vacuum. Yield 13.46 g
20
(
76%), mp 134–135°С, [α] –48.5° (c 2.5, MeOH).
D
–1
IR spectrum, ν, cm : 1754, 1718, 1663 (С=О), 1563,
547 (N–Hamide). Н NMR spectrum (CDCl ), δ, ppm:
.77–0.86 m (12Н, 4СН ), 1.05–1.15 m (1Н, СН),
.55–1.65 m (4Н, 2СН ), 1.76–1.82 m (1Н, СН), 3.66
1
1
0
1
3
3
2
s (3Н, СН ), 4.44–4.48 m (1Н, СН), 4.67–4.72 m (1Н,
3
13
СН). С NMR spectrum (CDCl ), δ, ppm: 11.27,
3
1
5
3
5.13, 21.84, 22.52, 24.56, 25.01, 37.46, 40.92, 51.95,
2.14, 56.76, 115.71 q (JС–F 228.5 Hz), 157.24 q (J
1.7 Hz), 171.29, 171.86. Found, %: С 50.63; Н 7.38;
2
D
0
(
81%), mp 90–93°С, [α] –23.7° (c 3, MeOH). IR
–
1
spectrum, ν, cm : 1722, 1689, 1659 (С=О), 1526 br
N 7.81. C H F N O . Calculated, %: С 50.84; Н
1
15 25
3
2
4
(
N–Hamide). Н NMR spectrum (CDCl ), δ, ppm: 0.83–
3
7
.11; N 7.91.
0.92 m (12Н, 4СН ), 1.12–1.21 m (1Н, СН), 1.48 s (9Н,
3
N-Trifluoroacetyl-L-isoleucyl-L-leucine methyl
3
СН ), 1.52–1.58 m (2Н, СН ), 1.66–1.70 m (2Н,
3
2
ester (8) was similarly obtained from 11.36 g (50 mmol)
of N-trifluoroacetyl-L-isoleucine, 10.3 g (50 mmol) of
dicyclohexylcarbodiimide, and 7.99 g (55 mmol) of L-
leucine methyl ester in 50 mL of THF. Yield 13.11 g
СН ), 1.70–1.78 m (1Н, СН), 3.99 t (1Н, СН, J 6.8 Hz),
2
1
3
4
.63 t (1Н, СН, J 6.8 Hz). С NMR spectrum
(
CDCl ), δ, ppm: 11.08, 15.41, 21.89, 23.05, 24.91,
3
2
1
7
8
5.01, 28.41, 36.84, 41.40, 50.76, 59.28, 80.46,
56.40, 172.50, 175.90. Found, %: С 59.61; Н 9.58; N
.76. C H N O . Calculated, %: С 59.28; Н 9.36; N
2
0
(
74%), mp 120–122°С, [α] –55° (с 2, MeОН). IR
D
–
1
spectrum, ν, cm : 1757, 1713, 1659 (С=О), 1556 (N–
Hamide). Н NMR spectrum (CDCl ), δ, ppm: 0.78–0.89
1
7
32
2
5
1
.13.
3
m (12Н, 4СН ), 1.02–1.13 m (1Н, СН), 1.44–1.61 m
3
tert-Butoxycarbonyl-L-isoleucyl-L-leucine (4)
(
4
4Н, 2СН ), 1.80–1.88 m (1Н, СН), 3.65 s (3Н, СН ),
.42–4.50 m (2Н, 2 СН). С NMR spectrum (CDCl ),
3
2
3
1
3
was analogously prepared from 23.30 g (65 mmol) of
tert-butoxycarbonyl-L-isoleucyl-L-leucine methyl
ester and 2.72 g (68 mmol) of sodium hydroxide. Yield
δ, ppm: 10.84, 14.78, 21.57, 22.37, 24.69, 24.84, 37.18,
0.57, 51.06, 52.07, 58.02, 115.71 q (JС–F 229 Hz),
157.25 q (J 32 Hz), 170.21, 172.74. Found, %: С
4
2
0
1
7.91 (80%), mp 150–152°С, [α]
–41.6° (c 2,
D
–
1
MeOH). IR spectrum, ν, cm : 1721, 1686, 1639
С=О), 1561, 1522 (N–Hamide). Н NMR spectrum
CDCl ), δ, ppm: 0.86–0.92 m (12Н, 4СН ), 1.16–1.22
5
1.07; Н 7.35; N 7.68. C H F N O . Calculated, %:
15 25 3 2 4
1
(
(
С 50.84; Н 7.11; N 7.91.
3
3
L-Leucyl-L-isoleucine (5). a. To a solution of
7.22 g (50 mmol) of tert-butoxycarbonyl-L-leucyl-L-
m (1Н, СН), 1.39 s (9Н, 3СН ), 1.52–1.61 m (2Н,
3
1
СН ), 1.66–1.74 m (2Н, СН ), 1.76–1.81 m (1Н, СН),
2
2
1
3
isoleucine in 100 mL of dioxane was added 20 mL of
.3 N solution of hydrogen chloride in dioxane. The
3
.98 t (1Н, СН, J 6.8 Hz), 4.60–4.64 m (1Н, СН). С
5
NMR spectrum (CDCl ), δ, ppm: 10.83, 15.19, 21.68,
3
reaction mixture was vigorously stirred for 5 h. the
formed precipitate was filtered off, washed several
times with ethyl ether, and dried in a vacuum. 11.79 g
of substance obtained was dissolved in 50 mL of
methanol. To this solution was added at vigorous
2
8
9
9
2.83, 24.71, 24.83, 28.20, 36.59, 41.19, 50.54, 59.09,
0.21, 156.22, 172.37, 175.63. Found, %: С 59.53; Н
.61; N 7.81. C H N O . Calculated, %: С 59.28; Н
1
7
32
2
5
.36; N 8.13.
N-Trifluoroacetyl-L-leucyl-L-isoleucine methyl
stirring
a solution of 4.24 g (42 mmol) of
ester (7). To a solution of 11.36 g (50 mmol) of N-
trifluoroacetyl-L-leucine in 100 mL of THF cooled to
triethylamine in 200 mL of dichloromethane. The
reaction mixture was stirred for 2 h, the separated
RUSSIAN JOURNAL OF ORGANIC CHEMISTRY Vol. 51 No. 5 2015

6
18
GAIDUKEVICH et al.
precipitate was washed with dichloromethane and
dried in a vacuum. After reprecipitation from water with
butoxycarbonyl-L-isoleucyl-L-leucine, 20 mL of 5 N
solution of hydrogen chloride in dioxane, and 3.54 g
(35 mmol) of triethylamine. Yield 7.04 g (72%), mp
20
acetone yield 8.55 g (70%), mp 279–280°С, {[α]
D
24
2
2
20
18
+
+
19.4° (c 2, Н О), [α] +20.9° (с 1, Н О), [α]
289–290°С, [α]D –10.9° (c 1, 1 M. NaOH) {[α]D
2
D
2
D
2
0
1
26.1° (с 1, 1 N HCl) [1]; [α] +19° (c 1, Н О) [2];
–11° (c 1, 1 M. NaOH) [2]}. Н NMR spectrum (D O),
D
2
2
2
0
1
[
(
(
α]D +25.7° (с 1, 1 N HCl) [4]}. Н NMR spectrum
δ, ppm: 0.65–0.72 m (9Н, 3СН ), 0.82 d (3Н, СН , J
3
3
D O), δ, ppm: 0.65–0.76 m (12Н, 4СН ), 0.88–0.97 m
5.6 Hz), 0.98–1.05 m (1Н, СН), 1.30–1.49 m (4Н,
2
3
1Н, СН), 1.18–1.25 m (1Н, СН), 1.43–1.63 m (4Н,
2СН ), 1.73–1.81 m (1Н, СН), 3.66 d (1Н, СН, J
2
1
3
2
5
2
1
СН ), 3.85 t (1Н, СН, J 6 Hz), 3.88 d (1Н, СН, J
4.8 Hz), 3.96–3.99 m (1Н, СН). С NMR spectrum
2
13
.6 Hz). С NMR spectrum (D O), δ, ppm: 10.78, 15.21,
(D O), δ, ppm: 10.47, 14.15, 20.96, 22.33, 23.86,
2
2
1.19, 21.80, 23.78, 24.72, 36.85, 39.81, 51.97, 60.47,
69.64, 177.75. Found, %: С 59.17; Н 9.85; N 11.28.
24.59, 36.40, 40.50, 54.40, 57.80, 168.63, 179.21.
Found, %: С 59.22; Н 10.14; N 11.21. C H N O .
1
2
24
2
3
C H N O . Calculated, %: С 58.99; Н 9.90; N 11.47.
Calculated, %: С 58.99; Н 9.90; N 11.47.
1
2
24
2
3
b. To a solution of 10.63 g (30 mmol) of N-trifluo-
b. From 10.63 g (30 mmol) of N-trifluoroacetyl-L-
isoleucyl-L-leucine methyl ester, 2.6 g (65 mmol) of
sodium hydroxide, and 3.99 g (35 mmol) of
trifluoroacetic acid we obtained 4.91 g (67%) of
roacetyl-L-leucyl-L-isoleucine methyl ester in 70 mL
of ethanol was added a solution of 2.6 g (65 mmol) of
sodium hydroxide in 50 mL of water. After stirring for
2
0
3
0 h to the reaction mixture was added dropwise 3.99 g
compound 6, mp 288–290°С, [α]D –10.7° (c 1, 1 M
(
35 mmol) of trifluoroacetic acid, the mixture was
NaOH).
stirred for 30 min, filtered, the solvent was removed at
a reduced pressure. The obtained residue was diluted
with 150 mL of acetone, the formed mixture was
vigorously stirred for 2 h, the precipitate was filtered
off and washed in succession with acetone, ethyl ether,
and dried in a vacuum. After reprecipitation from
water with acetone yield 5.62 g (77%), mp 278–281°С,
REFERENCES
1
. Smith, E.L., Spackman, D.H., and Polglase, W.J.,
J. Biol. Chem., 1952, vol. 199, p. 801.
. Okumura, Ya. and Sakurai, A., Bull. Chem. Soc. Jpn.,
973, vol. 46, p. 2190.
3. Gershkovich, A.A. and Kibirev, V.K., Sintez peptidov.
Reagenty i metody, Kiev: Naukova Dumka, 1987.
2
1
2
0
[
α]D +19.6° (c 2, Н О).
2
L-Isoleucyl-L-leucine (6). а. Compound 6 was
4. Abderhalden, E. and Hirsch, P., Ber., 1910, vol. 43,
similarly prepared from 13.78 g (40 mmol) of tert-
p. 2435.
RUSSIAN JOURNAL OF ORGANIC CHEMISTRY Vol. 51 No. 5 2015