An efficient synthetic method of N-protected dipeptide acids using amino acid calcium carboxylates in an organic solvent

Article abstract of DOI:10.1055/s-0030-1260569

The syntheses of N-protected dipeptide acids using alkaline earth metal (Mg, Ca, and Ba) carboxylates of an amino acid in organic solvents were investigated. It was found that amino acid calcium carboxylates are the most effective among the carboxylates of the amino acids tested for coupling with active esters of Boc-Ala-OH in organic solvents. The coupling of Boc-Ala-ONp or Boc-Ala-ONSu with amino acid calcium carboxylates in DMF gave the desired N-protected dipeptide acids in high yields (92-100%). Georg Thieme Verlag Stuttgart - New York.

Full text of DOI:10.1055/s-0030-1260569

LETTER
1427
An Efficient Synthetic Method of N-Protected Dipeptide Acids Using Amino
Acid Calcium Carboxylates in an Organic Solvent
S
ynthetic Method
h
of N-Protec
i
ted Dip
k
eptide
A
cid
a
o Hashimoto,*a Kazunobu Takeguchi,b Mitsuo Kodomarib
a
Department of Chemistry, The Jikei University School of Medicine, 8-3-1 Kokuryo-cho, Choufu-shi, Tokyo 182-8570, Japan
Fax +81(3)34804591; E-mail: hashimoto@jikei.ac.jp
b
Department of Bioscience and Engineering, Shibaura Institute of Technology, 307 Fukasaku, Minuma-ku, Saitama 337-8570, Japan
Received 17 February 2011
Scheme 1. Consequently, the amino acid or the peptide
carboxylates protected by the alkaline earth metal ions
could be expected to possess a higher hydrophobicity
compared to the corresponding carboxylates protected by
Abstract: The syntheses of N-protected dipeptide acids using alka-
line earth metal (Mg, Ca, and Ba) carboxylates of an amino acid in
organic solvents were investigated. It was found that amino acid
calcium carboxylates are the most effective among the carboxylates
of the amino acids tested for coupling with active esters of Boc-Ala- alkaline metal ions. Therefore, we anticipated that the use
OH in organic solvents. The coupling of Boc-Ala-ONp or Boc-Ala-
of alkaline earth metal carboxylates of amino acid will en-
ONSu with amino acid calcium carboxylates in DMF gave the de-
sired N-protected dipeptide acids in high yields (92–100%).
able the condensation with the carboxyl components in or-
ganic solvents and that also dipeptide salts, (Boc-Ala-
AA)2M, generated by the coupling would be liable to dis-
solve in organic solvents. In this paper, we report on the
Key words: N-protected peptide acid, amino acid calcium carbox-
ylate, organic solvent, active ester method, synthesis
effectiveness of amino acid calcium carboxylates for the
synthesis of N-protected dipeptide acids.
N-Protected peptide acids are intermediates available as a
The general synthetic procedure of N-protected dipeptide
carboxyl component for fragment condensation in peptide
acids using amino acid alkaline earth metal salts as an
synthesis. In the synthesis of N-protected peptide acids,
amino component was carried out according to the meth-
the carboxyl group of an amino component is usually pro-
od shown in Scheme 1. An N-protected amino acid active
tected by an ester which is removed after the coupling
ester, Boc-Ala-X,4 was dissolved in an organic solvent.
with the carboxyl component.1 In general, when peptide
To this solution was added an amino acid alkaline earth
esters are treated with deblocking reagents, incomplete re-
metal salt, (H-AA-O)2M.5 After the coupling at 25 °C for
moval of the esters or unexpected side reactions can oc-
a certain period of time, the yield of the obtained N-pro-
cur. In contrast, protection of the carboxyl group by a
tected dipeptide acid was determined by isolation and
metal ion can easily be achieved by dissolving amino ac-
HPLC using an internal standard method.6
ids in an aqueous solution of metal hydroxides; the metal
carboxylates are converted into the free carboxyl groups
by acidification of the reaction mixture after the coupling.
Therefore, not only does the protection of a carboxyl
group by a metal ion shorten the time for introduction and
removal of the protecting group, but also side reactions
caused by acid- or base-catalyzed deprotection, and so on,
can be prevented. Coupling using amino acid–alkaline
metal salts as an amino component has been carried out in
an aqueous organic solvent, as these salts are insoluble in
organic solvents.1 However, the desired peptides are not
obtained in satisfactory yield. As means to increase the
solubility of amino acid carboxylates in an organic sol-
vent, methods using tertiary amines1 and phase-transfer
reagents2,3 have been reported. However, the disadvan-
tage of these methods is that the amino acid carboxylates
cannot be prepared in advance, and also phase-transfer
reagents are relatively expensive.
Figure 1 Effect of alkaline earth metal ions, M2+, in the synthesis of
Boc-Ala-Leu-OH by the coupling of Boc-Ala-ONp with (H-Leu-
O)2M. Yields were determined by HPLC. Reagents and conditions:
Boc-Ala-ONp (0.30 mmol), (H-Leu-O)2M (0.165 mmol), DMF (4.0
mL), 25 °C, and 1.0 h.
The reactivity of the amino acid alkaline earth metal salts,
(H-AA-O)2M (M: Mg, Ca, or Ba), which were prepared
from the corresponding metal hydroxides, Mg(OH)2,
Ca(OH)2, or Ba(OH)2, was examined for the coupling be-
tween (H-Leu-O)2M and the Boc-Ala-p-nitrophenyl ester,
Boc-Ala-ONp, in dimethylformamide (DMF) at 25 °C for
one hour. These results are shown in Figure 1. The cou-
Since alkaline earth metals form a bivalent ion, one of the
metal ions combines with two molecules of an amino acid
or a peptide having a free carboxyl group as shown in
SYNLETT 2011, No. 10, pp 1427–1430
x
x
.x
x
.2
0
1
1
Advanced online publication: 26.05.2011
DOI: 10.1055/s-0030-1260569; Art ID: U01811ST
© Georg Thieme Verlag Stuttgart · New York

1428
C. Hashimoto et al.
LETTER
NH2
R
O
O
O
O
25 °C
X
+
+
M2
2 Boc-HN
organic solvent
H2N
R
O
Boc-Ala-X
X = ONp or ONSu
(H-AA-O)2M
M = Mg2+, Ca2+, or Ba2
+
R = side chain
AA = amino acid residue
Boc-HN
NH-Boc
O
H
NH
R
O
O
O
O
HN
R
H+
N
+
M2
2 Boc-HN
OH
O
O
O
R
(Boc-Ala-AA-O)2M
2 Boc-Ala-AA-OH
Scheme 1 Synthetic scheme of Boc-Ala-AA-OH by the coupling of Boc-amino acid active esters with amino acid alkaline earth metal salts
in an organic solvent. Reagents and conditions: Boc-Ala-X (1.0 equiv), (H-AA-O)2M (0.55 equiv), organic solvent (4.0–20 mL), 25 °C, and a
certain period of time.
pling of Boc-Ala-ONp with (H-Leu-O)2Ca gave the de-
sired N-protected dipeptide acid, Boc-Ala-Leu-OH, in
high yield (88%), but the couplings of Boc-Ala-ONp with
(H-Leu-O)2Mg or (H-Leu-O)2Ba resulted in the N-pro-
tected dipeptide acid in low yields (24% and 25%, respec-
tively). These results indicated that the amino acid
calcium carboxylate is the most effective among the salts
tested.
Figure 3 Effect of the reaction time in the synthesis of Boc-Ala-
Leu-OH by the coupling of Boc-Ala-ONp with (H-Leu-O)2Ca. Yields
were determined by HPLC. Reagents and conditions: Boc-Ala-ONp
(0.30 mmol), (H-Leu-O)2Ca (0.165 mmol), DMF (4.0 mL), and 25
°C.
The effect of the reaction time for the formation of the N-
protected dipeptide acids was examined by the coupling
between Boc-Ala-ONp and (H-Leu-O)2Ca in DMF at 25
°C. Figure 3 shows the yields for each reaction time. Al-
though a yield of over 90% was obtained by the reaction
for five hours, subsequent experiments were performed
Figure 2 Effect of the organic solvents in the synthesis of Boc-Ala-
with a reaction time of 24 hours because having as little
Leu-OH by the coupling of Boc-Ala-ONp with (H-Leu-O)2Ca. Yields
starting material as possible remaining in the reaction
were determined by HPLC. Reagents and conditions: Boc-Ala-ONp
mixture can be expected to facilitate the isolation of the
(0.30 mmol), (H-Leu-O)2Ca (0.165 mmol), solvent (4.0 mL), 25 °C,
and 1.0 h.
desired N-protected dipeptide acids.
Various amino acid calcium carboxylates and amino acid
derivative calcium carboxylates as an amino component
were coupled with Boc-Ala-ONp in DMF at 25 °C for 24
hours. The yields by HPLC are shown in Table 1. The
yields from the couplings of the calcium salts of Gly and
Ala having a small side chain were 100% and 92%, re-
spectively, and the yields from the couplings of the calci-
um salts of Leu and Ile having a bulky side chain were
99% and 100%, respectively. The use of the calcium salt
of Phe, which has a benzene ring at the side chain, also
provided the desired peptide in a 98% yield. The yield
from the coupling of the calcium salt of Pro, which is an
a-imino acid with a cyclic structure, was 98%. In the case
The effect of organic solvents in the syntheses of the N-
protected dipeptide acids was evaluated by the coupling of
Boc-Ala-ONp with (H-Leu-O)2Ca at 25 °C for one hour.
Figure 2 shows the yields from the coupling in various or-
ganic solvents. In highly polar aprotic solvents such as
DMF and dimethylsulfoxide (DMSO), Boc-Ala-Leu-OH
was formed in high yields (88% and 80%, respectively).
The organic solvents such as acetonitrile (MeCN), ethyl
acetate (EtOAc), 1,2-dichloroethane (DCE), and tetra-
hydrofuran (THF) provided the desired N-protected
dipeptide acid in very low yields (<1%, 2%, 1%, and 3%,
respectively).
Synlett 2011, No. 10, 1427–1430 © Thieme Stuttgart · New York

LETTER
Synthetic Method of N-Protected Dipeptide Acids
1429
Table 2 Synthesis of Boc-Ala-AA-OH by the Coupling of Boc-Ala-
Table 1 Synthesis of Boc-Ala-AA-OH by the Coupling of Boc-Ala-
ONp with (H-AA-O)2Ca
ONSu with (H-AA-O)2Caa
AA
Gly
Ala
Leu
Ile
Yield (%)
100
AA
Yield (%)
HPLCa
[a]D22 (°)c
Isolationb
Gly
100
92
94
92
97
99
96
98
93
98
–23.9d
–36.9
–41.3e
–25.1
–1.07
–92.5f
+8.50
–10.1
96
Ala
97
Leu
99
100
Ile
100
98
Phe
94
a Yields were determined by HPLC. Reagents and conditions: Boc-
Ala-ONSu (0.30 mmol), (H-AA-O)2Ca (0.165 mmol), DMF (4.0
mL), 25 °C, and 24 h.
Phe
Pro
98
Ser(Bzl)
Lys(Z)
96
In conclusion, it was found that a variety of amino acid
calcium carboxylates and/or amino acid derivative calci-
um carboxylates are extremely efficient as amino compo-
nents in the synthesis of the N-protected dipeptide acids
using the active esters, Boc-Ala-ONp and Boc-Ala-ON-
Su, in DMF, and that the present method gives the desired
N-protected dipeptide acids in almost quantitative yield
by the use of a small excess of those calcium salts. Also,
it is advantageous that those amino acid calcium salts are
easy to prepare, and preserve very well. Therefore, this
synthetic method may prove a very useful tool for the syn-
thesis of a small amount of stable isotope-labeled N-pro-
tected dipeptide acids and dipeptides, and also for the
synthesis of those dipeptides on an industrial scale and at
low cost.
100
a Yields were determined by HPLC. Reagents and conditions: Boc-
Ala-ONp (0.30 mmol), (H-AA-O)2Ca (0.165 mmol), DMF (4.0 mL),
25 °C, 24 h.
b Yields were determined by isolation. Reagents and conditions: Boc-
Ala-ONp (1.50 mmol), (H-AA-O)2Ca (0.825 mmol), DMF (5.0–20
mL), 25 °C, and 24 h.
c Solvent: MeOH, c = 1.00.
d Ref. 9.
e Ref. 10.
f [a]D28 –93.9° (MeOH, c = 1.00). Ref. 9.
of [H-Ser(Bzl)-O]2Ca and [H-Lys(Z)-O]2Ca, which has a
hydroxy group and an amino group at the side chain, pro-
tected by a benzyl (Bzl) and benzyloxycarbonyl (Z), re-
spectively, the yields were 96% and 100%, respectively.
In all the examples tested, the application of amino acid
calcium carboxylates gave the N-protected dipeptide ac-
ids in almost quantitative yield. Also, the isolated yields in
these couplings and the values of the specific rotation for
each compound are shown in Table 1. It was possible to
isolate the desired N-protected dipeptide acids in high
yields (92–99%).7 Such excellent yields will suggest that
(H-AA-O)2Ca in the present experiment does not form a
chelate structure.8 In these couplings, the reaction solu-
tions became nearly clear with the progress of the reac-
tion, namely, this result indicated that the resulting
dipeptide calcium salts, (Boc-Ala-AA-O)2Ca, shown in
Scheme 1 are highly soluble in DMF. Such solubility of
(Boc-Ala-AA-O)2Ca in DMF may be the reason for the
excellent yields in those couplings. The results of the spe-
cific rotation confirmed that no racemization has taken
place in the couplings using (H-AA-O)2Ca.9,10
References and Notes
(1) Bodansky, M.; Klausner, S. Y.; Ondetti, A. M. Peptide
Synthesis, 2nd ed.; John Wiley & Sons: New York, 1976, 49.
(2) Lansbury, T. P. Jr.; Hendrix, C. J.; Coffman, I. A.
Tetrahedron Lett. 1989, 30, 4915.
(3) Chen, S. T.; Wang, K. T. J. Chem. Soc., Chem. Commun.
1990, 1045.
(4) The amino acids used were of L-configuration. The
abbreviations used are those recommended by the IUPAC-
IUB Joint Commission on Biochemical Nomenclature and
Symbolism for Amino Acids and Peptides. See:Eur. J.
Biochem. 1984, 138, 9.
(5) Typical Procedure for the Preparation of Amino Acid
Calcium Carboxylates; (H-Leu-O)2Ca: To a solution of
H-Leu-OH (2.623 g, 20.0 mmol) dissolved in distilled H2O
(100 mL) was added a solution of Ca(OH)2 (0.815 g, 11.0
mmol) dissolved in distilled H2O (50 mL). After the mixture
was stirred for 30 min at r.t., the H2O was removed by eva-
poration. The obtained solid was dried over SiO2 for 1 d in
vacuo. The solid was ground into a powder using a mortar
and pestle. The powder, (H-Leu-O)2Ca, was washed onto a
filter funnel with THF and Et2O, and then dried over SiO2 in
vacuo. Yield: 2.943 g (98%).
(6) Typical Procedure for the Determination of HPLC Yield
of N-Protected Dipeptide Acids; Boc-Ala-Leu-OH: To a
solution of Boc-Ala-ONp (0.0931 g, 0.300 mmol) dissolved
in DMF (4.0 mL) was added (H-Leu-O)2Ca (0.0496 g, 0.165
mmol). The mixture was stirred at 25 °C for 24 h. To the
mixture were then added MeCN (10 mL), H2O (10 mL), and
Boc-Val-OH (0.300 g, 1.38 mmol) as the internal standard
The condensation of various amino acid calcium carbox-
ylates with the Boc-Ala-N-hydroxysuccinimide ester,
Boc-Ala-ONSu, as a carboxyl component, was performed
under similar conditions to the method employing ONp.
As can be seen in Table 2, each coupling also gave the de-
sired N-protected dipeptide acids in high yields (94–
100%). These results showed that the use of the calcium
carboxylates of amino acid in the synthesis of N-protected
dipeptide acids is efficiently applicable to both the ONp
and the ONSu methods.
Synlett 2011, No. 10, 1427–1430 © Thieme Stuttgart · New York

1430
C. Hashimoto et al.
LETTER
(8) Fox, S.; Büsching, I.; Barklage, W.; Strasdeit, H. Inorg.
reagent. The solution was applied to an HPLC column under
the conditions described below. From the HPLC result, the
yield of the product, Boc-Ala-Leu-OH, was determined
using a calibration curve with a straight line plotted against
three different concentration ratios of Boc-Ala-Leu-OH and
Boc-Val-OH. Yield: 99%. tR (Boc-Ala-Leu-OH) = 21.9 min,
and tR (Boc-Val-OH) = 16.4 min. HPLC conditions: column,
Waters m-Bondasphere (3.9 × 150 mm, C18, 5 mm, and 300
Å); solvent: 14–41% MeCN–H2O–0.1% TFA; linear
gradient, 60 min; flow rate: 1.0 mL/min; and detection, l =
210 nm.
Chem. 2007, 46, 818.
(9) For the values of specific rotation, see: (a) Boc-Ala-Gly-
OH·H2O: [a]D27 –23.2° (MeOH, c = 2.0): Wolman, Y.;
Schwarzberg, M.; Frankel, M. Isr. J. Chem. 1970, 8, 53.
(b) Boc-Ala-Pro-OH: [a]D28 –89.9° (EtOH, c = 1):
McDermott, J. R.; Benoiton, N. L. Can. J. Chem. 1973, 51,
2555.
(10) Alternative Synthetic Method of Boc-Ala-Leu-OH: Boc-
Ala-OH (6.24 g, 33.0 mmol) in DMF (15 mL), ice-cooled,
was coupled with TFA·H-Leu-OPac (30.0 mmol) containing
Et3N (4.17 mL, 30.0 mmol) in DMF (15 mL) ice-chilled
using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDC·HCl; 6.90 g, 36.0 mmol) and 1-
hydroxybenzotriazole (HOBt; 6.08 g, 45.0 mmol). The
solution was stirred for 6 h in an ice-bath and then for 18 h
at r.t. After the treatment of the produced Boc-Ala-Leu-
OPac (3.91 g, 9.30 mmol) with Zn powder (19.62 g, 300
mmol) in AcOH (30 mL), the desired product Boc-Ala-Leu-
OH (1.13 g, 3.74 mmol) was obtained by the recrystalliza-
tion from EtOAc–Et2O–hexane. Boc-Ala-Leu-OH: mp
137.6–137.8 °C. MS (FAB): m/z = 303 [M + H]+, 325 [M +
Na]+. tR = 13.6 min. HPLC conditions: column, Waters
m-Bondasphere (3.9 × 150 mm, C18, 5 mm, and 300 Å);
solvent: 5–95% MeCN–H2O–0.1% TFA; linear gradient, 30
min; flow rate: 1.0 mL/min; and detection, l = 210 nm;
[a]D22 –40.7° (MeOH, c = 1.00).
(7) Typical Procedure for the Determination of Isolation
Yield of N-Protected Dipeptide Acids; Boc-Ala-Ser(Bzl)-
OH: To a solution of Boc-Ala-ONp (0.465g, 1.50 mmol)
dissolved in DMF (5.0 mL) was added [H-Ser(Bzl)-O]2Ca
(0.354g, 0.825 mmol). After the mixture was stirred at 25 °C
for 24 h, the solution was condensed in vacuo. The residue
was then dissolved in EtOAc (ca. 100 mL). The organic
layer was washed with 5% aq citric acid (1 × 20 mL, 3 × 10
mL) and H2O (5 × 10 mL), and then dried over anhyd
Na2SO4. After the removal of the solid, the filtrate was
condensed in vacuo. The residue was recrystallized from
EtOAc–hexane. Yield: 0.511 g (93%); mp 155.4–156.0 °C.
MS (FAB): m/z = 337 [M + H]+ and 359 [M + Na]+. tR = 23.3
min. HPLC conditions: column, Waters m-Bondasphere (3.9
× 150 mm, C18, 5 mm, and 300 Å); solvent: 5.0–90.5%
MeCN–H2O–0.1% TFA; linear gradient, 60 min; flow rate,
1.0 mL/min; and detection, l = 210 nm.
Synlett 2011, No. 10, 1427–1430 © Thieme Stuttgart · New York