An efficient method for the synthesis of phenacyl ester-protected dipeptides using neutral alumina-supported sodium carbonate 'Na2CO3/n-Al2O3'

Article abstract of DOI:10.1002/psc.2544

In the synthesis of dipeptides (Boc-AA¹-AA²-OPac: AA¹ and AA² represent amino acids) protected by phenacyl (Pac) ester, amines and solid bases as the base for the conversion of the trifluoroacetic acid (TFA) sal

Full text of DOI:10.1002/psc.2544

Research Article
Received: 14 February 2013
Revised: 7 July 2013
Accepted: 10 July 2013
Published online in Wiley Online Library: 28 August 2013
(wileyonlinelibrary.com) DOI 10.1002/psc.2544
An efficient method for the synthesis of
phenacyl ester-protected dipeptides using
neutral alumina-supported sodium carbonate
‘Na2CO3/n-Al2O3’
Chikao Hashimoto,a* Kazuhiro Sugimoto,b Youhei Takahashib
and Mitsuo Kodomarib
In the synthesis of dipeptides (Boc-AA1-AA2-OPac: AA1 and AA2 represent amino acids) protected by phenacyl (Pac) ester,
amines and solid bases as the base for the conversion of the trifluoroacetic acid (TFA) salt of the amino component (TFA·H-
AA2-OPac) into the corresponding free amino component (H-AA2-OPac) were examined. The synthesis of a dipeptide (Boc-
Ala-Gly-OPac) using amines for the conversion afforded an unsatisfactory yield with by-products. On the other hand, the use
of neutral alumina-supported Na2CO3 (Na2CO3/n-Al2O3) as a solid base for the conversion provided the dipeptide in a quanti-
tative yield without by-products. The application of Na2CO3/n-Al2O3 to the synthesis of some dipeptides protected by Pac ester
gave the desired peptides in excellent yields. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.
Keywords: neutral alumina-supported sodium carbonate; solid base; phenacyl ester; peptide synthesis; amine
of time that the H-AA2-OPac produced by the treatment with
an amine in an organic solvent exists in those solution.
Introduction
Phenacyl (Pac) esters offer a useful means by which to protect
the carboxy groups of amino acids and other organic
compounds. They are easily and selectively removed by special
conditions, such as zinc powder in acetic acid [1], sodium
thiophenoxide [2], and photolysis [3], without affecting other
protective groups such as t-butoxycarbonyl (Boc) and ethyl
ester (OEt), which are eliminated by treatment with acid or
base, respectively. Additionally, a magnesium reduction with
acetic acid has been reported for the cleavage of Pac esters
[4]. Because of these unique means of deprotection, Pac esters
have been successfully applied to the protection of carboxy
groups during peptide and other organic syntheses [5,6]. Also,
it is an advantage that N-protected amino acid Pac esters are
easy to handle because they crystallize easily. When a Pac
ester-protected dipeptide (Boc-AA1-AA2-OPac: AA1 and AA2
represent the carboxy and amino component amino acids,
respectively) is synthesized by a solution-phase method, the
trifluoroacetic acid (TFA) salt of the amino component
(TFA·H-AA2-OPac) derived from Boc-AA2-OPac is usually
converted in advance into the corresponding free amino
component (H-AA2-OPac) by treatment with amines such as
triethylamine (Et3N), N-methylmorpholine (NMM), and N,N-
diisopropylethylamine (DIEA) in an organic solvent. Next, the
desired peptide (Boc-AA1-AA2-OPac) is synthesized by adding
a solution containing the produced H-AA2-OPac to a solution
including the carboxy component (Boc-AA1-OH). In our previ-
ous synthesis of Pac ester-protected peptides, the use of
amines in the conversion occasionally brought about unsatis-
factory yields of the desired peptides [6]. It was suggested to
the authors that such lower yields are affected by the length
Hence, in order to improve the yields, the use of solid bases
in the synthesis of dipeptides using Pac ester-protected
amino acids was examined. If a solid base exists in the
reaction solution, it is assumed that TFA·H-AA2-OPac is
converted into H-AA2-OPac on the solid base, and the
resulting H-AA2-OPac will couple in situ with a carboxy com-
ponent which dissolves in the same solution. The use of solid
bases will therefore shorten the time in which H-AA2-OPac
exists in the solution and should improve the coupling yields
using Pac esters. In this paper, we report on the effectiveness
of neutral alumina-supported Na2CO3 (Na2CO3/n-Al2O3) as a
solid base for the synthesis of dipeptides that are protected
by Pac esters (Scheme 1).
Results and Discussions
The effect of various solid bases and amines on the conversion of
TFA·H-Gly-OPac [7] into H-Gly-OPac was examined by the
coupling between Boc-Ala-OH and TFA·H-Gly-OPac in the
presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hy
*
Correspondence to: Chikao Hashimoto, Department of Chemistry, The
Jikei University School of Medicine, 8-3-1 Kokuryo-cho, Choufu-shi, Tokyo
182-8570, Japan. E-mail: hashimoto@jikei.ac.jp
a Department of Chemistry, The Jikei University School of Medicine, 8-3-1
Kokuryo-cho, Choufu-shi, Tokyo, 182-8570, Japan
b Department of Bioscience and Engineering, Shibaura Institute of Technology,
307 Fukasaku, Minuma-ku, Saitama, 337-8570, Japan
J. Pept. Sci. 2013; 19: 659–662
Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.

HASHIMOTO ET AL.
Scheme 1. Synthetic method of dipeptides protected by Pac ester using Na2CO3/n-Al2O3 as a solid base. AA1 and AA2 represent the carboxy and amino
component amino acids, respectively. R1 and R2 represent the side chains of amino acids. Reagents and conditions: TFA·H-AA2-OPac (1.00 equiv),
Na2CO3/n-Al2O3 0.530 g (Na2CO3 0.500 equiv for TFA·H-AA2-OPac), Boc-AA1-OH (1.10 equiv for TFA·H-AA2-OPac), EDC·HCl (1.20 equiv for Boc-AA1-
OH), HOBt (1.50 equiv for Boc-AA1-OH), DMF (4.0 ml).
drochloride (EDC·HCl)-1-hydroxybenzotoriazole (HOBt) in N,N-
dimethylformamide (DMF) at 0 °C for 6.0 h followed by room tem-
perature (r. t.) for 18 h. These results are shown in Table 1.
The coupling using powdered Na2CO3 (0.500 mmol) as a solid
base gave Boc-Ala-Gly-OPac (1) in an 83% yield. The use of
neutral alumina (n-Al2O3, 0.480 g) afforded 1 in a 78% yield.
Also, the coupling using a mixture of powdered Na2CO3
(0.500 mmol) and n-Al2O3 (0.480 g) (Na2CO3: n-Al2O3 = 1 : 10
weight ratio) gave 1 in a much higher yield of 96%. These re-
sults suggest that the coexistence of Na2CO3 and n-Al2O3 in
the reaction system is necessary to produce 1 in a high yield.
On this basis, the coupling using neutral alumina-supported
10% Na2CO3 (Na2CO3/n-Al2O3) [8] as a solid base yielded 1
in a quantitative yield of 99%. On the other hand, the cou-
plings employing the amines such as Et3N, NMM, and DIEA
gave 1 in an 87%, 85%, and a 71% yield, respectively. This
shows that Na2CO3/n-Al2O3 is a very effective base compared
with the amines for the coupling using the Pac ester-
protected amino acids. Although the results of Table 1 also
suggested that the n-Al2O3 itself acts as base, the insufficient
conversion to H-AA2-OPac of TFA·H-AA2-OPac will result in a
lowering of coupling yields. Therefore, subsequent experi-
ments were performed with an equivalent amount of sodium
carbonate to TFA·H-AA2-OPac. The specific rotation data of 1
obtained by the couplings employing Na2CO3/Al2O3 and
general Et3N were nearly identical values (Table 1).
In order to further clarify the effectiveness, each solution
extracted by ethyl acetate (EtOAc) after the couplings be-
tween Boc-Ala-OH and TFA·H-Gly-OPac using both Na2CO3/
n-Al2O3 and Et3N bases were analyzed by reversed phase
HPLC (RP-HPLC), as shown in Figure 1. The RP-HPLC profile af-
ter the coupling using Na2CO3/n-Al2O3 showed only one peak
(a) corresponding to 1 without peaks of by-products [Figure 1
(A)]. In contrast, the RP-HPLC profile after the coupling using
Et3N showed peaks corresponding to by-products, b and c,
along with a [Figure 1(B)]. These RP-HPLC profiles strongly
indicate that Na2CO3/n-Al2O3 is preferable to Et3N for the
coupling employing the Pac ester-protected amino acids. In
addition, the RP-HPLC results show that Na2CO3/n-Al2O3 is a
very favorable base for inhibiting the formation of by-
products. The by-products, b and c, in Figure 1(B) were isolated,
and their respective structures were characterized by mass
spectrometry (MS). Each by-product was additionally confirmed
by direct comparison with authentic samples synthesized
independently. By these analyses, b and c were identified as
Boc-Ala-OPac and Boc-Ala-Gly-Gly-OPac, respectively. On the
basis of these results, it was suggested that the Pac ester in H-
Gly-OPac is hydrolyzed into glycine and 2-hydroxyacetophenone
by water, which will be included in the commercial grade
solvent (DMF), and then b and c are formed by the couplings
among the degradation products and a small excess carboxy
component (Boc-Ala-OH). In the synthesis of 1, an intramolecular
cyclized by-product was not observed [9,10].
Table 1. Effect of various bases in the synthesis of Boc-Ala-Gly-OPac
(1) by the coupling of Boc-Ala-OH with TFA·H-Gly-OPaca
Bases
Yield/%
Boc-Ala-Gly-OPac (1)
Na2CO3/n-Al2O3
99b
96
Powdered Na2CO3 + n-Al2O3
Powdered Na2CO3
83
n-Al2O3
Et3N
78
87c
NMM
DIEA
85
71
Table 2 shows the results using Na2CO3/n-Al2O3 in various
couplings between Boc-AA1-OH and TFA·H-AA2-OPac in the
presence of EDC·HCl-HOBt in DMF at 0 °C for 6.0 h followed
by r. t. for 18 h. For the couplings of Boc-Ala-OH and TFA·H-
AA2-OPac (AA2 = Gly, Ala, Leu, and Val), the products (Boc-Ala-
AA2-OPac) were obtained in high yields of 99%, 87%, 89%,
and 85%, respectively. The coupling between Boc-Ala-OH and
TFA·H-Ile-OPac using EDC·HCl (1.30 mmol) produced Boc-Ala-
Ile-OPac in an unsatisfactory yield of 75%. By increasing the
amount of EDC·HCl to 1.65 mmol, the yield increased to 91%.
aYield was determined by RP-HPLC using an internal standard
sample. Reagents and conditions: TFA·H-Gly-OPac (1.00 mmol),
Boc-Ala-OH (1.10 mmol), EDC·HCl (1.30 mmol), HOBt (1.70 mmol);
bases: Na2CO3/n-Al2O3 (0.533 g, Na2CO3: 0.500 mmol), powdered
Na2CO3 (0.500 mmol) + n-Al2O3 (0.480 g), powdered Na2CO3
(0.500 mmol), n-Al2O3 (0.480 g), Et3N (1.00 mmol), NMM
(1.00 mmol), DIEA (1.00 mmol); reaction temperature and time:
0 °C for 6.0 h followed by r. t. for 18 h.
27
b
½αꢀD 28.4 (MeOH, c = 1.00).
27
c
½αꢀD 28.3 (MeOH, c = 1.00).
wileyonlinelibrary.com/journal/jpepsci Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2013; 19: 659–662

SYNTHESIS OF PHENACYL ESTER-PROTECTED DIPEPTIDES USING NA2CO3/AL2O3
Table 2. Yield of the couplings of carboxy components, Boc-AA1-OH,
(A)
a (1)
with amino components, TFA·H-AA2-OPac, using Na2CO3/n-Al2O3
a
Products
Boc-AA1- TFA·H-AA2-
Yield/%
OH
OPac
AA1
AA2
Na2CO3/n- Et3Nc
Al2O3
b
Boc-Ala-Gly-OPac (1)
Boc-Ala-Ala-OPac
Boc-Ala-Leu-OPac
Boc-Ala-Val-OPac
Boc-Ala-Ile-OPac
Boc-Ala-Ile-OPac
Boc-Ala-Phe-OPac
Boc-Ala-Phe-OPac
Boc-Gly-Gly-OPac
Boc-Pro-Gly-OPac
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Gly
Pro
Gly
Ala
Leu
Val
Ile
99
87
89
85
75
91d
70
70d
99
99
87
80
73
83
62
73d
65
—
87
84
0
20
40
60
80
90
Ile
Retention time/min
Phe
Phe
Gly
Gly
(B)
a (1)
aYield was determined by RP-HPLC using an internal standard
sample. Reagents and conditions: Boc-Ala-OH (1.10 mmol),
TFA·H-Gly-OPac (1.00 mmol), EDC·HCl (1.30 mmol), HOBt
(1.70 mmol), DMF (2.0 ml), reaction temperature and time: 0 °C
for 6.0 h followed by r. t. for 18 h.
bNa2CO3/n-Al2O3 of 0.533 g was used.
cEt3N of 1.00 mmol was employed.
dEDC·HCl of 1.65 mmol was used.
c
b
0
20
40
60
80
90
Retention time/min
Figure 1. HPLC profiles of each solution extracted by EtOAc after
couplings used (A) Na2CO3/Al2O3 and (B) Et3N in the synthesis of
Boc-Ala-Gly-OPac (1). Peak a: 1, peak b: Boc-Ala-OPac, peak c: Boc-
Ala-Gly-Gly-OPac. Elution conditions: column, Waters μ-Bondasphere
5 μm C18-300 Å (3.9 × 150 mm); eluent, 14–68% CH3CN/H2O-0.1% TFA
(v/v/v); running conditions, 90 min linear gradient; flow rate, 1.0 ml/
min; detection, 210 nm.
acid salts of amino acid esters (X·H-AA2-OY: X represents acids
such as TFA, hydrogen chloride, and p-toluenesulfonic acid,
etc.; Y represents Pac, Et, and benzyl, etc.). However, as a
limitation of this method, we think that the system may not
contribute toward controlling the racemization of the proline
Pac ester during the synthesis of Boc-AA1-Pro-OPac.
Conclusions
The improvement in the yield will be attributed to couple
with Boc-Ala-OH before the decomposition of H-Ile-OPac through
the addition of excess EDC·HCl. The coupling of Boc-Ala-OH and
TFA·H-Phe-OPac with EDC·HCl (1.30 mmol) also gave Boc-Ala-Phe-
OPac in an unsatisfactory yield of 70%. However, the yield
was 70% despite using a higher excess level of EDC·HCl
(1.65 mmol). This result suggests that H-Phe-OPac is liable
to sustain the degradation. The coupling between Boc-AA1-
OH (AA1 = Gly and Pro) and TFA·H-Gly-OPac afforded Boc-
Gly-Gly-OPac and Boc-Pro-Gly-OPac in a quantitative yield
of 99%. On the other hand, as can be seen from Table 2,
the synthetic yields of the dipeptides (Boc-AA1-AA2-OPac)
using Et3N were 62–87%. These couplings using Et3N gave
low yields of 2–18% compared with those employing
Na2CO3/n-Al2O3. These results showed that Na2CO3/n-Al2O3
is an excellent base compared with Et3N for the synthesis
of the Pac ester-protected dipeptides. Although Pac esters
are utilized in the synthesis of peptide fragments, the lower-
ing of the synthetic yields employing Et3N is disadvanta-
geous in terms of cost. Accordingly, the present method
using Na2CO3/n-Al2O3 should extend the possibility for the
use of Pac esters in the synthesis of peptide fragments.
As a scope for this method, we think that the present
system using Na2CO3/n-Al2O3 will be applicable to the syn-
theses of N-protected-dipeptide-esters employing different
The application of the Na2CO3/n-Al2O3 in the synthesis of dipeptides
that are protected by a Pac ester depresses the side reactions and
produces the desired dipeptides in high yields. The superior yields
will be due to couple with a carboxy component (Boc-AA1-OH) in situ
before a free amino component (H-AA2-OPac) generated on the
Na2CO3/n-Al2O3 decompose, and these results will support the
assumption mentioned in the introduction.
Materials and Methods
All reagents and solvents were used in commercial grade. The
amino acids used, with the exception of glycine, are of the L-con-
figuration. Melting points were measured with a Type B-540
(BUCHI, Flawil, Switzerland). RP-HPLC was performed on a PU-
2080 Plus system (JASCO, Tokyo, Japan) with a Waters μ-
Bondasphere column (C18, 5 μm, and 300 Å; 3.9 I.D. × 150 mm)
using a flow rate of 1.0 ml/min and the following solvent systems:
0.1% TFA in 5% CH3CN/H2O and 0.1% TFA in 95% CH3CN/H2O.
Yields were determined by RP-HPLC results at 210 nm. Fast atom
bombardment (FAB) mass spectra were recorded using a JMS-DX
303 spectrometer (JEOL, Tokyo, Japan). Optical rotations were
determined with a DIP-360 digital type polarimeter (JASCO,
Tokyo, Japan).
J. Pept. Sci. 2013; 19: 659–662 Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

HASHIMOTO ET AL.
Preparation of Na2CO3/n-Al2O3
Boc-Ala-OPac (b)
To sodium carbonate (5.0 g) dissolved in water (80 ml),
neutral alumina (n-Al2O3; ICN Biomedical N-super 1, particle
size 63–200 μm; 45 g) was added. After the mixture was
stirred for 30 min at r. t., the water was removed by evapo-
ration. The residual solid was dried at 150 °C for 6.0 h under
vacuo to give Na2CO3/n-Al2O3.
MS (FAB) m/z [M + H]+ calculated for C16H22O5N: 308, found: 308;
[M + Na]+ calculated for C16H21O5NNa: 330, found: 330.
Boc-Ala-Gly-Gly-OPac (c)
MS (FAB) m/z [M + H]+ calculated for C20H28O7N3: 422, found: 422;
[M + Na]+ calculated for C20H27O7N3Na: 444, found: 444.
Typical Coupling Procedure Using Na2CO3/n-Al2O3;
Boc-Ala-Gly-OPac (1)
References
To a solution of Boc-Ala-OH (0.208 g, 1.10 mmol) in DMF (2.0 ml),
HOBt (0.223 g, 1.65 mmol) and EDC·HCl (0.253 g, 1.32 mmol) were
added at 0 °C. To the mixture was added Na2CO3/n-Al2O3
1 Hendrickson JB, Kendall G. Phenacyl protecting group for acids and
phenols. Tetrahedron Lett. 1970; 5: 343–348.
2 Gaudreau P, Morell JL, Gross E. Formation of aminosuccinyl derivative
from beta-phenacyl aspartyl peptides catalyzed by sodium
thiophenoxide. Int. J. Pept. Protein Res. 1982; 19: 280–283.
3 Banerjee A, Falvey DE. Protecting groups that can be removed
through photochemical electron transfer: mechanistic and product
studies on photosensitized release of carboxylates from phenacyl
esters. J. Org. Chem. 1997; 62: 6245–6251.
4 Kokinaki S, Leondiadis L, Ferderigos N. A novel and efficient method
for cleavage of phenacyl esters by magnesium reduction with acetic
acid. Org. Lett. 2005; 7: 1723–1724.
5 Kimura T, Takai M, Masui Y, Morikawa T, Sakakibara S. Strategy for the
synthesis of large peptides: an application to the total synthesis of
human parathyroid hormone [hPTH(1-84)]. Biopolymers 1981;
20: 1823–1832.
6 Hashimoto C, Muramatsu I. Synthesis and circular dichroism spectra of
sperm whale myoglobin-(57-96)-tetracontapeptide. Bull. Chem. Soc.
Jpn. 1993; 66: 181–190.
(0.533 g; Na2CO3 0.500 mmol), immediately followed by
a
solution of TFA·H-Gly-OPac (1.00 mmol) dissolved in DMF
(2.0 ml) at r. t. The mixture was stirred at 0 °C for 6.0 h and then
at r. t. for 18 h. To the mixture was then added DMF (10 ml) and
Boc-Phe-OPac as the internal standard reagent. The solution
was applied to an RP-HPLC column under the conditions
described in the succeeding text. From the RP-HPLC result, the
yield of 1 was determined using a calibration curve with a
straight line plotted against three different concentration ratios
of Boc-Ala-Gly-OPac and Boc-Phe-OPac. Yield 99%; mp 102.0–
102.4 °C; MS (FAB): m/z [M + H]+ calculated for C18H25O6N2: 365,
found: 365; [M + Na]+ calculated for C18H24O6N2Na: 387, found:
387; [α]2D7 28.4 (MeOH, c = 1.00). Retention time: Boc-Ala-Gly-OPac
9.9 min. RP-HPLC conditions: solvent, 23–46% CH3CN/H2O-0.1%
TFA (v/v/v); linear gradient, 30 min.
7 IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN).
Nomenclature and symbolism for amino acids and peptides, recom-
mendations 1983. Eur. J. Biochem. 1984; 138: 9–37.
8 Kodomari M, Nawa S, Miyoshi T. Direct allylation of aromatic
compounds with allylic chloride using the supported reagents
system ZnCl2/SiO2-K2CO3/Al2O3. J. Chem. Soc., Chem. Commun.
1995; 1895–1896.
9 Kiso Y, Kimura T, Fujiwara Y, Sakikawa H, Akaji K. Efficient solid
phase peptide synthesis on a phenacyl-resin by a methanesulfonic
acid α-amino deprotecting procedure. Chem. Pharm. Bull. 1990;
38: 270–272.
10 Kuroda H, Kubo S, Chino N, Kimura T, Sakakibara S. Unexpected
racemization of proline or hydroxy-proline phenacyl ester during
coupling reactions with Boc-amino acids. Int. J. Pept. Protein Res.
1992; 40: 114–118.
Typical Coupling Procedure Using Amines (Et3N);
Boc-Ala-Gly-OPac (1)
TFA·H-Gly-OPac (1.00 mmol) was dissolved in DMF (2.0 ml) at r. t., and
then the solution was mixed with Et3N (1.00 mmol) at 0 °C. The solu-
tion was added to another solution containing Boc-Ala-OH
(1.10 mmol), HOBt (1.65 mmol), and EDC·HCl (1.32 mmol) in DMF
(2.0 ml) at 0 °C. The mixture was stirred at 0 °C for 6.0 h and then at
r. t. for 18 h. The yield of 1 was determined by RP-HPLC by using
an internal standard sample. Yield 85%; [α]2D7 28.3 (MeOH, c =1.00).
wileyonlinelibrary.com/journal/jpepsci Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2013; 19: 659–662