N-trifluoroacetyl-β-alanine in the synthesis of carnosine

Article abstract of DOI:10.1134/S1070363207090125

Conditions have been developed for the synthesis of N-trifluoroacetyl- β-alanine, N-tifluoroacetyl-β-alanyl chloride, and N-trifluoroacetyl-β-alanine 4-nitrophenyl ester. These compounds reacted with histidine methyl ester or sodium salt to give N-trifluoroacetyl-β- alanyl-l-histidine methyl ester CF₃CONHCH₂CH ₂?CONHCH(CH₂C₃H₃N ₂)COOCH₃ and N-trifluoroacetyl-β-alanyl-l-histidine CF₃CONHCH₂CH₂CONHCH?(CH₂C ₃H₃N₂)COOH. Their hydrolysis with a solution of sodium hydroxide in aqueous ethanol, followed by acidification with trifluoroacetic acid, led to the formation of β-alanyl-l-histidine (l-carnosine).

Full text of DOI:10.1134/S1070363207090125

ISSN 1070-3632, Russian Journal of General Chemistry, 2007, Vol. 77, No. 9, pp. 1576 1579.
Pleiades Publishing, Ltd., 2007.
Original Russian Text M.S. Cherevin, Z.P. Zubreichuk, L.A. Popova, T.G. Gulevich, V.A. Knizhnikov, 2007, published in Zhurnal Obshchei Khimii,
2007, Vol. 77, No. 9, pp. 1503 1507.
N-Trifluoroacetyl- -alanine in the Synthesis of Carnosine
M. S. Cherevin, Z. P. Zubreichuk, L. A. Popova, T. G. Gulevich, 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 February 20, 2007
Abstract Conditions have been developed for the synthesis of N-trifluoroacetyl- -alanine, N-tifluoroacetyl-
-alanyl chloride, and N-trifluoroacetyl- -alanine 4-nitrophenyl ester. These compounds reacted with histidine
methyl ester or sodium salt to give N-trifluoroacetyl- -alanyl-L-histidine methyl ester CF3CONHCH2CH2
CONHCH(CH2C3H3N2)COOCH3 and N-trifluoroacetyl- -alanyl-L-histidine CF3CONHCH2CH2CONHCH
(CH2C3H3N2)COOH. Their hydrolysis with a solution of sodium hydroxide in aqueous ethanol, followed by
acidification with trifluoroacetic acid, led to the formation of -alanyl-L-histidine (L-carnosine).
DOI: 10.1134/S1070363207090125
L-Carnosine ( -alanyl-L-histidine) exhibits a broad
spectrum of biological activity and is used in the
synthesis of medicines [1]. Numerous methods for the
preparation of carnosine have been reported [2 11];
the amino group in -alanine is mainly protected
using phthalyl [2, 3], triphenylmethyl [4], or carba-
mate-like group [5, 6]. Although trifluoroacetyl group
has long been used for the protection of amino groups,
they have not found wide application in peptide
syntheses, in particular due to racemization processes.
On the other hand, the ease of introduction and re-
moval of trifluoroacetyl protection makes such pro-
cedures quite promising in those cases when no ra-
cemization is possible. In the present work we made
an attempt to synthesize carnosine on the basis of N-
trifluoroacetyl- -alanine and its derivatives.
By reaction of -alanine sodium salt (I) with ethyl
trifluoroacetate (reactant molar ratio 1:1.3) in an-
hydrous ethanol, followed by acidification of the re-
action mixture with trifluoroacetic acid, we isolated
N-trifluoroacetyl- -alanine (II) which was then con-
verted into N-trifluoroacetyl- -alanyl chloride (III) by
treatment with boiling thionyl chloride. Compound
III was brought into reaction with 4-nitrophenol in
the presence of triethylamine in tetrahydrofuran to
obtain N-trifluoroacetyl- -alanine 4-nitrophenyl ester
(IV) (Scheme 1).
Scheme 1.
F
O
O
F
F
(1) CF3COOEt
O
N
H
H2N
O
OH
(2) CF3COOH
Na
I
II
SOCl2
F
O
O
O
F
F
HOC6H4NO2
Et3N
O
+
N
O
N
H
OH
F
F
O
NH
F
IV
II
O
1576

N-TRIFLUOROACETYL- -ALANINE IN THE SYNTHESIS OF CARNOSINE
1577
Compounds II IV were used as starting materials
in the synthesis of L-carnosine. Addition of acid chlo-
ride III to a solution of L-histidine methyl ester (V)
and triethylamine in THF led to the formation of N-
trifluoroacetyl- -alanyl-L-histidine methyl ester (VI)
in a yield of about 20%. In the reaction of compound
V with nitrophenyl ester IV, the yield of product VI
attained 65%. Finally, ester V reacted with N-trifluoro-
acetyl- alanine (II) in the presence of dicyclohexyl-
carbodiimide (DCC) as condensing agent to give
about 80% of VI (Scheme 2).
subsequent acidification of the reaction mixtures with
trifluoroacetic acid gave -alanyl-L-histidine (IX); the
optical rotation of samples of IX thus obtained coin-
cided with that reported for the best samples of L-
carnosine.
O
N
VI
(1) NaOH
OH
NH2
HN
(2) CF3COOH
VIII
N
H
O
IX
Scheme 2.
The structure of the isolated products was con-
firmed by the IR and NMR spectra and elemental
analyses.
IV
N
HN
O
NH
O
EXPERIMENTAL
N
O
O
NH2
The IR spectra were recorded in KBr on a Protege-
460 spectrometer with Fourier transform. The NMR
spectra were measured on a Bruker Avance-400 in-
strument at 20 C. L-Histidine methyl ester was syn-
thesized by the procedure reported in [3]. Ethanol was
purified by distillation over calcium hydride. Tetra-
hydrofuran, diethyl ether, and hexane were distilled
over metallic sodium. The other solvents and reagents
were used without additional purification.
O
CH3
N
H
CH3
II, DCC
V
F
F
NH
F
O
VI
We also tried to use L-histidine sodium salt (VII)
which was prepared by treatment of L-histidine with
sodium ethoxide. Sodium salt VII was subjected to
subsequent transformations without isolation as in-
dividual substance. Addition of acid chloride III to a
solution of sodium salt VII in ethanol at 0 C afforded
55% of N-trifluoroacetyl- -alanyl-L-histidine (VIII).
When a solution of VII in ethanol was treated with
p-nitrophenyl ester IV, followed by acidification of
the reaction mixture with trifluoroacetic acid, we
isolated about 65% of amino acid derivative VIII
(Scheme 3).
N-Trifluoroacetyl- -alanine
(II).
-Alanine,
8.91 g, was added to a solution of sodium ethoxide
prepared from 2.3 g of sodium and 150 ml of ethanol.
The mixture was stirred for 1 h, 18.46 g of ethyl tri-
fluoroacetate was added dropwise, and the mixture
was stirred for 10 h, treated with 14.82 g of trifluoro-
acetic acid, stirred for 1 h, and evaporated under re-
duced pressure. The residue was extracted with diethyl
ether (3 150 ml), the extracts were combined,
washed with 150 ml of water, dried over sodium
sulfate, and filtered, 50 ml of hexane was added, and
the solvent was distilled off under reduced pressure to
a volume of 70 ml. The precipitate was filtered off,
washed with hexane, and dried under reduced pressure.
Yield 14.81 g (80%), mp 114 116 C. IR spectrum, ,
Scheme 3.
N
HN
III
1
O
cm : 1716 (C=O, acid), 1692 (C=O, amide), 1561
(C N, amide). H NMR spectrum (D2O), , ppm (J,
Hz): 2.73 t (2H, J = 6.5), 3.57 t (2H, J = 6.3). Found,
%: C 32.56; H 3.48; N 7.41. C5H6F3NO3. Calculated,
%: C 32.44; H 3.27; N 7.57.
NH
O
1
N
O
O
NH2
HO
NH
Na
N
H
(1) IV
(2) CF3COOH
F
F
VII
F
N-Trifluoroacetyl- -alanyl chloride (III). A mix-
ture of 18.51 g of compound II and 29.75 g of thionyl
chloride was heated for 7 h under reflux. Excess thio-
nyl chloride was distilled off, and the residue was
distilled under reduced pressure. Yield 17.71 g (87%),
bp 101 102 C (6 mm), n1D7 = 1.4220. IR spectrum,
O
VIII
Compounds VI and VIII were subjected to hydro-
lysis with sodium hydroxide in aqueous ethanol. The
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 77 No. 9 2007

1578
CHEREVIN et al.
1
, cm : 1794 (C=O), 1709 (C=O, amide), 1562 (C N,
and the mixture was stirred for 12 h at room tempera-
ture, filtered, concentrated under reduced pressure to
a volume of 50 ml, and diluted with 200 ml of diethyl
ether. The precipitate was filtered off, washed with
diethyl ether and hexane, and dried under reduced
pressure. Reprecipitation from methylene chloride
with hexane gave 8.4 g (50%) of compound VI with
mp 129 132 C, [ ]2D0 = 21.0 (c = 3.0, THF). Found,
%: C 43.08; H 4.64; N 16.83.
1
amide). H NMR spectrum (CDCl3), , ppm (J, Hz):
3.22 t (2H, J = 6.0), 3.63 q (2H, J = 6.1), 7.55 br.s
(1H, NH). Found, %: C 29.39; H 2.61; N 6.73.
C5H5ClF3NO2. Calculated, %: C 29.50; H 2.48; N
6.88.
N-Trifluoroacetyl -alanine 4-nitrophenyl ester
(IV). Compound III, 16.28 g, was added dropwise to
a mixture of 11.13 g of 4-nitrophenol and 8.58 g of
triethylamine in 150 ml of THF. The mixture was stir-
red for 5 h, the solvent was removed under reduced
pressure, and the residue was extracted with diethyl
ether. The extract was filtered, diluted with 100 ml of
heptane, and concentrated under reduced pressure to a
volume of 70 ml. The precipitate was filtered off,
washed with hexane, and dried under reduced pressure.
Recrystallization from diethyl ether gave 17.15 g
(70%) of compound IV with mp 84 86 C. IR spec-
trum, , cm : 1757 (C=O, ester), 1704 (C=O, amide),
1562 (C N, amide). H NMR spectrum (CDCl3), ,
ppm (J, Hz): 2.96 t (2H, J = 6.1), 3.77 q (2H, J = 6.0),
7.31 d (2H, J = 9.1), 8.28 d (2H, J = 9.2). Found, %:
C 43.27; H 3.11; N 9.02. C11H9F3N2O5. Calculated,
%: C 43.15; H 2.96; N 9.15.
c. A solution of 18.51 g of compound II in 100 ml
of THF was cooled to 0 C, a solution of 20.6 g of
N,N -dicyclohexylcarbodiimide in 100 ml of THF and
a solution of 16.9 g of compound V in 100 ml of THF
were added, and the mixture was stirred for 2 h at 0 C
and for 15 h at room temperature. The precipitate was
filtered off, and the solvent was removed by distilla-
tion under reduced pressure. The residue was ex-
tracted with methylene chloride, the extract was fil-
tered, concentrated to a volume of 60 ml, and diluted
with 200 ml of diethyl ether, and the precipitate was
filtered off, washed with diethyl ether and hexane, and
dried under reduced pressure. Reprecipitation from
methylene chloride gave 26.9 g (80%) of compound
1
1
VI with mp 131 132 C, [ ]2D0 = 21.15 (c = 3.0,
THF). Found, %: C 42.98; H 4.54; N 16.57.
N-Trifluoroacetyl- -alanyl-L-histidine methyl
ester (VI). a. A solution of 8.46 g of L-histidine
methyl ester and 5.25 g of triethylamine in 100 ml of
THF was cooled to 0 C, 10.58 g of compound III was
added, and the mixture was stirred for 1 h at 0 C and
for 5 h at room temperature. The solvent was distilled
off under reduced pressure, the residue was dissolved
in water, and the solution was extracted with met-
hylene chloride (3 150 ml). The extract was dried
over sodium sulfate, filtered, and concentrated under
reduced pressure. The precipitate was filtered off,
washed in succession with diethyl ether and hexane,
and dried under reduced pressure. Reprecipitation
from methylene chloride with hexane gave 3.36 g
N-Trifluoroacetyl- -alanyl-L-histidine (VIII).
a. L-Histidine, 7.76 g, was added to a solution of
sodium ethoxide prepared from 1.15 g of metallic
sodium and 150 ml of ethanol. The mixture was stir-
red for 1 h and cooled to 0 C, 10.18 g of compound
III was added dropwise, and the mixture was stirred
for 5 h, filtered, and evaporated under reduced pres-
sure. The residue was washed with acetone and di-
ethyl ether, and dried under reduced pressure. Re-
precipitation from ethanol with diethyl ether gave
8.86 g (55%) of compound VIII with mp 190 192 C,
1
[ ]2D0 = 18.9 (c = 3.0, water). IR spectrum, , cm :
1717 (C=O, acid), 1671 (C=O, amide), 1630 (C=O,
(20%) of compound VI with mp 130 132 C, [ ]D20
=
1
amide), 1558 (C N, amide), 1538 (C N, amide). H
1
21.05 (c = 3.0, THF). IR spectrum, , cm : 1757
NMR spectrum (D2O), , ppm (J, Hz): 2.42 t (2H,
J = 4), 2.94 m (1H), 3.11 m (1H), 3.40 t (2H, J =
8.0), 4.36 q (1H, J = 4), 7.12 s (1H), 8.44 s (1H). 13C
NMR spectrum, C, ppm: 27.13, 34.19, 35.93, 53.91,
(C=O, ester), 1704 (C=O, amide), 1642 (C=O, amide),
1
1565 (C N, amide), 1536 (C N, amide). H NMR
spectrum (CD3COCD3), , ppm (J, Hz): 2.54 m (2H),
3.04 m (2H), 3.57 t (2H, J = 6.0), 3.65 s (3H), 4.70
m (1H), 6.93 s (1H), 7.62 s (1H), 7.85 br.s (1H),
1
115.69 q (CF3, JCF = 286.03 Hz), 116.50, 129.67,
133.11, 158.48, 172.54, 176.31. Found, %: C 41.24;
H 4.19; N 17.21. C11H13F3N4O4. Calculated, %: C
41.00; H 4.07; N 17.39.
9.17 br.s (1H). 13C NMR spectrum, C, ppm: 29.03,
1
34.37, 36.00, 51.32, 52.69, 113.30 q (CF3, JCF
=
287.74 Hz), 115.30, 134.76, 135.00, 156.71, 170.09,
171.64. Found, %: C 43.02; H 4.41; N 16.52.
C12H15F3N4O4. Calculated, %: C 42.86; H 4.50; N
16.66.
b. L-Histidine, 7.76 g, was added to a solution of
sodium ethoxide prepared from 1.15 g of metallic
sodium and 150 ml of ethanol. The mixture was stir-
red for 1 h, 15.31 g of compound IV was added, and
the mixture was stirred for 2 days, filtered, and treated
with 5.7 g of trifluoroacetic acid. The resulting solu-
b. Compound IV, 15.31 g, was added to a solution
of 8.46 g of L-histidine methyl ester in 150 ml of THF,
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N-TRIFLUOROACETYL- -ALANINE IN THE SYNTHESIS OF CARNOSINE
1579
tion was stirred for 2 h, filtered, concentrated under
reduced pressure to a volume of 30 ml, and diluted
with 150 ml of acetone. The precipitate was filtered
off, washed with acetone and diethyl ether, and dried
under reduced pressure. Reprecipitation from ethanol
with diethyl ether gave 10.47 g (65%) of compound
sodium hydroxide, and 0.57 g of trifluoroacetic acid
we obtained 9.27 g (82%) of compound IX with mp
256 260 C (decomp.), [ ]2D0 = 21.2 (c = 3.0, water).
REFERENCES
VIII with mp 188 191 C, [ ]2D0 = 18.1 (c = 3.0,
1. Boldyrev, A.A., Karnozin. Biologicheskoe znachenie
i vozmozhnosti primeneniya v meditsine (Carnosine.
Biological Role and Potential of Application in
Medicine), Moscow: Mosk. Gos. Univ., 1998.
2. Turner, R.A., J. Am. Chem. Soc., 1953, vol. 75,
no. 10, p. 2388.
3. Glemzha, A.A. and Severin, S.E., Izv. Akad. Nauk
SSSR, Ser. Khim., 1966, no. 5, p. 861.
4. Bezrukov, M.V., Panchenko, A.E., and Forma-
zyuk, V.E., Russian Patent no. 2084457, 1997; Byull.
Izobret., 1997, no. 20.
water). Found, %: C 41.21; H 4.25; N 17.48.
-Alanyl-L-histidine (IX). A solution of 4.2 g of
sodium hydroxide in 25 ml of water was added to a
solution of 16.81 g of compound VI in 75 ml of
ethanol. The mixture was stirred for 24 h, filtered,
acidified by adding 6.27 g of trifluoroacetic acid,
stirred for 2 h, and evaporated under reduced pressure.
The residue was washed with acetone and ethanol,
dried under reduced pressure, and dissolved in a
minimal amount of water, the solution was filtered
and diluted with 150 ml of acetone, and the precipitate
was filtered off and washed with acetone. Reprecipita-
tion from water with ethanol gave 8.48 g (75%) of
5. Sifferd, R.H. and du Vigneaud, V., J. Biol. Chem.,
1935, vol. 108, no. 3, p. 753.
6. Skoblik, T.I., Markin, Yu.A., Zaborina, T.G., Filip-
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vol. 35, no. 2, p. 263.
compound IX with mp 259 262 C (decomp.), [ ]D20
=
21.9 (c = 3.0, water); published data: mp 259 263 C,
[ ]2D0 = 22 [6]; 250 C (decomp.), [ ]2D0 = 20.9 [3].
1
IR spectrum, , cm : 1660 (C=O, acid), 1648 (C=O,
amide), 1584 (C N, amide). 1H NMR spectrum (D2O),
, ppm (J, Hz): 2.62 t (2H, J = 6), 3.02 m (2H), 3.18 t
(2H, J = 6.0), 4.43 m (1H), 6.92 s (1H), 7.68 s (1H).
13C NMR spectrum, C, ppm: 28.86, 32.16, 35.66,
55.09, 117.31, 133.16, 135.63, 171.48, 177.84. Found,
%: C 47.84; H 6.33; N 24.68. C9H14N4O3. Calculated,
%: C 47.78; H 6.24; N 24.76.
8. Barger, G. and Tutin, F., Biochem. J., 1918, vol. 12,
no. 4, p. 402.
9. Kroll, H. and Hoberman, H., J. Am. Chem. Soc., 1953,
vol. 75, no. 10, p. 2511.
10. Bailin, G. and Lukton, A., J. Org. Chem., 1962,
vol. 27, no. 2, p. 684.
Following an analogous procedure, from 16.11 g
of N-trifluoroacetyl- -alanyl-L-histidine, 2.2 g of
11. Vinick, F.J., US Patent no. 4359416, 1982; Chem.
Abstr., 1983, vol. 98, no. 89935c.
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