One-pot preparation of N-carbamate protected amino acids via the azide

Article abstract of DOI:10.1021/op049917z

A convenient and efficient method for the preparation of fluorenylmethyloxycarbonyl (Fmoc) and allyloxycarbonyl (Alloc) amino acids is proposed. This method is particularly attractive due to the fact that the reaction sequence Fmoc/Alloc-chloride to Fmoc/Alloc-azide to Fmoc/Alloc-amino acid can readily be carried out in one pot. A further advantage is the minimization of byproducts, which are easily removed during the workup. Most important, this strategy minimizes the formation of dipeptides that are difficult to remove by crystallization. Thus, Fmoc and Alloc amino acids are obtained in high yield (60-90%) and purity as evidenced by thin-layer chromatography, reversed-phase high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance.

Full text of DOI:10.1021/op049917z

Organic Process Research & Development 2004, 8, 920−924
“One-Pot” Preparation of N-Carbamate Protected Amino Acids via the Azide
Luis J. Cruz,† Natalia G. Beteta,† Ariel Ewenson,‡ and Fernando Albericio*,†,§
Barcelona Biomedical Research Institute, Barcelona Science Park, UniVersity of Barcelona, 08028-Barcelona, Spain,
Luxembourg Industries Ltd., 27 Hamered Street, Tel AViV 61000, Israel, and Department of Organic Chemistry,
UniVersity of Barcelona, 08028-Barcelona, Spain
Abstract:
to Boc chemistry for the synthesis of peptides. However,
their attachment through powerful chloroformate reagents
under Schotten-Baumann conditions can lead to the forma-
tion of protected dipeptides as side products.9 In most cases
the identified protected dipeptide impurities for Fmoc
represent 1-5% of the total. As an example, even when the
relatively hindered Alloc-Val-OH was prepared, 14% of the
corresponding dipeptide was obtained.8 This high incidence
of protected dipeptide can lead to the insertion of an extra
amino acid in the final peptide synthesis, which cannot be
tolerated for use in therapeutic applications for some kinds
of drug. Several approaches based on the use of different
activated formates have been proposed to minimize this
problematic side reaction. For instance, azides,9-11 mixed
carbonates such as the succinimidyl,12-15 the 1,2,2,2-tetra-
chloroethyl,16,17 and the 5-norbornene-2,3-dicarboximido,18
and the symmetrical pyrocarbonates.19 Although, the suc-
cinimidyl carbonate is recognized as the method of choice,
it is not practical when protected amino acids are prepared,
because the N-hydroxysuccinimide formed can contaminate
the final product. Alternatively, the Bolin method, which
involves the in situ preparation of the trimethylsilyl amino
acid derivatives and their subsequent reaction with the
chloroformate, is tedious and impractical for large scale.20
This paper describes a new protocol for preparing Fmoc
and Alloc amino acids using sodium azide as reagent and
free amino acids. The syntheses of N-protected amino acids
are carried out in one pot removing the byproducts during
the workup, so it is not necessary to use a crystallization
step. Moreover, it was demonstrated that the amounts of the
undesired protected dipeptide produced by the present
A convenient and efficient method for the preparation of
fluorenylmethyloxycarbonyl (Fmoc) and allyloxycarbonyl (Al-
loc) amino acids is proposed. This method is particularly
attractive due to the fact that the reaction sequence Fmoc/Alloc-
chloride to Fmoc/Alloc-azide to Fmoc/Alloc-amino acid can
readily be carried out in one pot. A further advantage is the
minimization of byproducts, which are easily removed during
the workup. Most important, this strategy minimizes the
formation of dipeptides that are difficult to remove by crystal-
lization. Thus, Fmoc and Alloc amino acids are obtained in high
yield (60-90%) and purity as evidenced by thin-layer chro-
matography, reversed-phase high-performance liquid chroma-
tography, mass spectrometry, and nuclear magnetic resonance.1
Introduction
Nowadays synthetic peptides such as T20,2 R15K (V3
peptide from HIV-1 IIIB gp120),3 Somatostatin,4 and Oxy-
tocin5 are widely recognized as potential pharmaceutical
agents.6 The ultimate purity of any peptide, whether prepared
by solution or solid-phase methodology, depends heavily on
the purity of the initial starting materials. The most com-
monly utilized amino acid derivatives for both solution and
solid-phase procedures are the N-urethane blocked amino
acids, in particular Z (benzyloxycarbonyl), Boc (tert-bu-
toxycarbonyl), Fmoc (fluorenylmethyloxycarbonyl), and Al-
loc (allyloxycarbonyl).7,8 Therefore, there is a growing
demand for the preparation of those with a high level of
purity.
In the last two decades, the use of the Fmoc and Alloc
groups has evidenced an important increase as an alternative
(9) Tessier, M.; Albericio, F.; Pedroso, E.; Grandas, A.; Eritja, R.; Giralt, E.;
Granier, C.; Van- Rietschoten, J. Int. J. Pept. Protein Res. 1983, 22, 125.
(10) Garcia, J.; Mart´ınez-Teipel, B.; Nicola´s, E.; Michelotti, E. L.; Albericio, F.
Lett. Peptide Sci., in press.
(11) Although the existence of this side reaction was not mentioned, Fmoc-N3
was already proposed as an alternative to the chloroformate in the seminal
papers of Carpino and Han (Carpino, L. A.; Han, G. Y. J. Am. Chem. Soc.
1970, 92, 5748. Carpino, L. A.; Han, G. Y. J. Org. Chem. 1972, 37, 3404).
(12) Sigler, G. F.; Fuller, W. D.; Chaturvedi, N. C.; Goodman, M.; Verlander,
M. Biopolymers 1983, 22, 2157.
* Corresponding author. Present address: Fernando Albericio. Barcelona
Biomedical Research Institute, Barcelona Science Park, University of Barcelona,
Josep Samitier 1-5, 08028 Barcelona, Spain. E-mail: albericio@pcb.ub.es.
† Barcelona Biomedical Research Institute, University of Barcelona.
‡ Luxembourg Industries Ltd.
§ Department of Organic Chemistry, University of Barcelona.
(1) Wade, J. D. In Solid-Phase Synthesis. A Practical Guide; Kates, S. A.,
Albericio, F., Eds.; Marcel Dekker: New York, 2000; pp 103-128.
(2) Kannan, N. P.; Hengguang, L.; Alonso, H.; Haijing, A.; Lai-Xi, W. Org.
Biomol. Chem. 2004, 2, 660.
(13) Lapatsanis, L., Milias, G., Froussios, K.; Kolovos, M. Synthesis 1983, 671.
(14) Ten K. P.; Van Dijk, B. G.; Peeters, J. M.; Raaben, B. J.; Adams, P. J.;
Tesser, G. I. Int. J. Pept. Protein Res. 1986, 27, 398.
(3) Nehete, P. N.; Vela, E. M.; Hossain, M. M.; Sarkar, A. K.; Yahi, N.; Fantini,
J.; Sastry, K. J. AntiViral Res. 2002, 56, 233.
(4) Licha, K.; Hessenius, C.; Becker, A.; Henklein, P.; Bauer, M.; Wisniewski,
S.; Wiedenmann, B.; Semmler, W. Biooconj. Chem. 2001, 12, 44.
(5) Du V. V.; Ressler, C.; Swan, J. M.; Roberts, C. W.; Katsoyannis, P. G.;
Gordon, S. J. Am. Chem. Soc. 1953, 75, 4879.
(6) Bruckdorfer, T.; Marder, O.; Albericio, F. Curr. Pharm. Biotechnol. 2004,
5, 29.
(7) Lloyd-Williams, P.; Albericio, F.; Giralt, E. CRC, Boca Raton (FL), 1997.
(8) Albericio, F. Biopolymers (Peptide Science) 2000, 55, 123.
(15) Milton, R. C. de L.; Becker, E.; Milton, S. C. F.; Baxter, J. E. J.; Elsworth,
J. F. Int. J. Pept. Protein Res. 1987, 30, 431.
(16) Barcelo, G.; Sente´, J.-P.; Sennyey, G. J. Org. Chem. 1985, 50, 3953.
(17) Barcelo, G.; Sente´ J.-P.; Sennyey, G.; Bensoam, J.; Loffet, A. Synthesis
1986, 627.
(18) Henklein, P.; Heyne, H,-V.; Halatsch, W. R.; Niedrich, H. Synthesis 1987,
166.
(19) Sennyey, G.; Barcelo, G.; Sente´, J. P. Tetrahedron Lett. 1986, 44, 5375.
(20) Bolin, D. R.; Sytwu, I. I.; Humiec, F.; Meienhofer, J. Int. J. Pept. Protein
Res. 1989, 33, 353.
920
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Vol. 8, No. 6, 2004 / Organic Process Research & Development
10.1021/op049917z CCC: $27.50 © 2004 American Chemical Society
Published on Web 09/18/2004

Table 1. N-Protected amino acids prepared by the azide procedure
commercial
Fmoc-
dipeptide
(%)
reaction
conditions
(1:1, v/v)
Fmoc-
dipeptide
(%)
time
(day)
yield
(%)
purity
(%)
no.
compound
Fmoc-Gln
0.39
1
2
3
4
5
6
7
8
9
10
10% Na2CO3-dioxane
10% Na2CO3-DMF
5
5
5
5
5
5
5
3
3
3
59
68
65
47
71
75
56
50
82
90
97.6
95.7
98.2
97.8
96.6
98.8
94.2
94.4
89.7
92.2
0.19
0.35
0.86
0.15
not detectable
not detectable
0.38
2.41
2.31
10% Na2CO3-acetone
10% Na2CO3-ACN
5% Na2CO3-dioxane
1% Na2CO3-dioxane
1% Na2CO3-acetone
acetone-H2O, DIEA (1 equiv)
acetone-H2O, DIEA (2 equiv)
acetone-H2O, DIEA (3 equiv)
2.59
Fmoc-Asn
Fmoc-Ala
Fmoc-Gly
0.65
0.59
0.31
11
12
13
1% Na2CO3-dioxane
1% Na2CO3-dioxane
1% Na2CO3-dioxane
5
3
2
79
90
93
98.1
96.2
98.6
0.11
0.45
0.25
method were insignificant. In addition, a series of detailed
improvements over the previous synthesis of Fmoc/Alloc
amino acid were developed, which resulted in an optimized
protocol for the preparation of these derivatives. Furthermore,
the azide method is less expensive than other acyl derivatives
and therefore shows a better suitability for the preparation
of N-carbamate protected amino acids in large scale.
solution is added. The mixture is left stirring at room
temperature, the reaction progress being followed by reversed-
phase HPLC. Reaction completion is typically achieved after
1 h. Contrary to previous reports,9,11 in which acetone was
used instead of dioxane, the Fmoc-N3 does not precipitate,
favouring the reaction with the free amino acids. This second
reaction was investigated in depth by varying temperatures,
solvents, reaction times, and pH.
Results and Discussion
Using glutamine as a model, an amino acid that can
present solubility problems, initial experiments were per-
formed in solvents such as acetonitrile (ACN), N,N-dimethyl-
formamide (DMF), dioxane, and acetone. Also, different
amounts of inorganic base (Na2CO3) were added (entries
1-7) (Table 1). According to these results, the use of dioxane
and variable amounts of aqueous Na2CO3 produced the most
favourable results (entries 1, 5, and 6).
Additionally, the substitution of an inorganic base (Na2-
CO3) by an organic base such as N,N-diisopropylethylamine
(DIEA) was studied. The DIEA base was added in increasing
amounts, and when 2 or 3 equiv were used, an improvement
in the yield and reaction rate were detected (entries 9, 10 vs
3; Table 1). However, as a concomitant result, the Fmoc
amino acid became contaminated with a substantial amount
of protected dipeptide (2-3%). Besides, the use of organic
base leads to the premature cleavage of the Fmoc group,
and its use was discontinued.
It has been reported that the Fmoc group can be
introduced into amino acids using allyloxycarbonyl chloride
(Fmoc-Cl) as acylating agent under Schotten-Baumann
conditions in the presence of NaHCO3 or Na2CO3. However,
the formation of protected dipeptide contaminants in high
amount represents a real problem in peptide chemistry. This
side product is difficult to detect by TLC but appears clearly
on C18 chromatography.9 It was demonstrated that a simple
crystallization is not effective, making the removal of the
contaminant a tedious and expensive process. Most of the
reported methods are based on modifications of the acylating
agent used for introducing the urethane-type protecting
groups. However, the new methods developed were more
expensive than the chloroformate method.
To develop an efficient, competitive, and inexpensive
procedure, the azide method was studied and optimized. In
earlier reports,9,10 the intermediate 9-fluorenylmethylazido-
formate (Fmoc-N3) was isolated and the formation of the
Fmoc-amino acid was accomplished in a separate step. The
main drawback of this method is that Fmoc-N3 needs to be
isolated and stored, with the corresponding handling prob-
lems that this can cause especially at large scale. Furthermore,
the elimination of a reaction step reduces the cost and the
time of the total process.
Several Fmoc amino acids were synthesized using the
reaction conditions described above (free amino acids were
dissolved in 1% aqueous Na2CO3), and good yields were
obtained (entries 11-13, Table 1). Moreover, in all cases
studied, the amounts of Fmoc dipeptide contaminant found
in these compounds was less than those in commercial
samples.21
Herein, the protocol is based on the synthesis of these
compounds in one pot. Thus, Fmoc-Cl reacts with NaN3
without isolating the Fmoc-N3, the reaction with the free
amino acids leading to the target compounds. In detail, the
Fmoc-Cl is dissolved in dioxane, and an aqueous NaN3
(21) To quantify the formation of protected dipeptide as contaminants, the
corresponding protected dipeptides (Fmoc-Gln-Gln-OH, Fmoc-Asn-Asn-
OH, Fmoc-Ala-Ala-OH, Fmoc-Gly-Gly-OH, Alloc-Gly-Gly-OH, and Alloc-
Val-Val-OH) were synthesized on solid phase using Fmoc/Alloc chemistry
and ClTrt-Cl-resin. The protected dipeptides were cleaved from the resin
and characterized by analytical HPLC.
Vol. 8, No. 6, 2004 / Organic Process Research & Development
•
921

Table 2. Synthesis of Fmoc/Alloc amino acids via the azide
All the Fmoc amino acids prepared by this procedure were
found to be exceptionally pure (Table 3). Analysis by TLC,
using conditions that separate monomer from dimer deriva-
tives, indicated the presence of only one spot. Figure 1 shows
the HPLC profiles of some of these compounds.
methoda
reaction
condition
(1:1, v/v)
Fmoc-
dipeptide
(%)
yield purity
(%) (%)
no. compound
To extend this method to other N-protecting amino groups,
Alloc amino acids were prepared. Alloc-Gly-OH and Alloc-
Val-OH were obtained using the azide method in one-pot.
Thus, starting from allyloxycarbonyl chloride (Alloc-Cl),
after 1 h of reaction with NaN3, allyloxycarbonyl azide
(Alloc-N3) was obtained. Then, the free amino acid, dissolved
in 1% aqueous Na2CO3-dioxane, was added and stirred for
48 h. The Alloc-Gly-OH and Alloc-Val-OH were obtained
with good yields and high purities (entries 19 and 20, Table
2). These were characterized satisfactorily by reversed-phase
HPLC, TLC, and other physical methods (Table 3). The
Alloc-Gly-OH and Alloc-Val-OH prepared by the azide
method did not contain significant amounts of protected
dipeptide as determined by HPLC.
Fmoc-Gln
14
Fmoc-Asn
15
Fmoc-Ala
16
Fmoc-Gly
17
1% Na2CO3-dioxane 71 99.8 0.09
1% Na2CO3-dioxane 60 99.37 not detectable
1% Na2CO3-dioxane 81 98.9 0.25
1% Na2CO3-dioxane 85 99.4 0.10
Fmoc-Gly
18 large scale 1% Na2CO3-dioxane 92 98.3 0.36
Alloc-Gly
19
20
1% Na2CO3-dioxane 96 98.6 0.03
1% Na2CO3-dioxane 95 98.7 0.05
Alloc-Val
a Reaction time: 48 h and pH controlled between 8 and 10.
To develop an efficient and economic methodology, the
reaction time and the basic conditions were also studied and
optimized. Long reaction times promote the formation of side
products, which are difficult to remove by workup. Another
important factor is the basic pH, which enhances the reaction
rate. Furthermore, a more basic pH improves the solubility
of the free amino acid. However, it has been observed that
highly basic conditions (pH > 11) might conceivably lead
to premature degradation of the azidoformate to dibenzo-
fulvene and other byproducts.11 Therefore, to avoid this
phenomenon, the pH reaction was kept between 8 and 10
by controlled addition of 10% aqueous Na2CO3.
With these considerations taken into account, Fmoc amino
acids were obtained in good yields (entries 14-18, Table
2). Besides, the formation of protected dipeptide contaminant
was either marginal or not detectable.
As a scaled up example of this method, the preparation
of Fmoc-Gly-OH was carried out. Due to its reduced sterical
hindrance, glycine appears to be one of the most problematic
amino acids in terms of dipeptide and tripeptide formation.
However, by the present method Fmoc-Gly-OH was obtained
in very high yield and with acceptably low formation of
Fmoc-Gly-Gly-OH (entry 18, Table 2).
Conclusions
Our studies have shown that the Fmoc/Alloc amino acids
can be obtained in one pot using Fmoc/Alloc-azide as an
acylating agent. This procedure does not require the tem-
porary protection of the carboxyl function of the amino acid.
The formation of Fmoc/Alloc-dipeptide contaminants using
the azide method in one pot is significantly reduced compared
to the higher degree of contaminant obtained employing
Fmoc/Alloc-Cl as an acylating agent.9,12,20 The contaminant
level is comparable to that obtained by the Fmoc-Cl method
in the presence of Zinc dust22 or to that of the succinimidyl
procedure.23 Handling and storage of the organic azide
reagent is circumvented, since the latter is not isolated in
this method. In addition, in the synthesis using the azide
method, the byproducts (azide) can be easily removed in the
workup. In contrast to the succinimidyl method, it is often
necessary to resort to additional crystallization steps for the
removal of byproducts (N-hydroxysuccinimide).
From these results, we conclude that the azide method is
a simple and convenient procedure to obtain Fmoc/Alloc
amino acids, with good yields, minimum side products, and
easy scalability. Other urethane-type protecting groups
Table 3. N-Protected amino acids characterization
retention
time
melting
pointc
(°C)
MWb
(m/z)
1H NMR
compound
(min)a
(δ)
Fmoc-Gln
Fmoc-Asn
Fmoc-Ala
Fmoc-Gly
Alloc-Gly
Alloc-Val
9.03
9.11
369.46 (368.14)
355.41 (354.12)
312.42 (311.12)
298.39 (297.10)
160.27 (159.05)
202.0 (201.10)
219-220
(221-223)
187-188
(186-186)
143-145
(143-144)
174-176
(173-174)
(oil)
1.74 (1H, m), 1.93 (1H, m), 2.13 (2H, t), 3.92 (1H, m),
4.19 (1H, t) 4.23 (2H, d), 6.74 (2H, broad s), 7.30-7.87 (8H, m)
2.46 (2H, m), 4.19 (1H, m), 4.24 (1H, t), 4.32 (2H, d),
6.89 (2H, broad s), 7.30-7.87 (8H, m)
10.85
10.33
4.98
1.24 (3H, d), 3.99 (1H, m), 4.19 (1H, t), 4.24 (2H, d),
7.30-7.87 (8H, m)
3.65 (2H, d), 4.19 (1H, t), 4.27 (2H, d), 7.30-7.87 (8H, m)
4.03 (2H, d), 4.60 (2H, m), 5.23 (1H, d), 5.30 (1H, m),
5.32 (1H, d), 5.92 (1H, m).
0.94 (3H, d), 1.01 (3H, d), 2.23 (1H, m), 4.33 (1H, m),
4.588 (2H, d), 5.23 (1H, dd), 5.32 (1H, d), 5.92 (1H, m).
7.57
(oil)
a Retention time was determined by RP-HPLC. b The molecular weight was measured by MS-HPLC; the values in parentheses are calculated by the ChemDraw
program. c Values in parentheses are reported by Chang. et al.24
922
•
Vol. 8, No. 6, 2004 / Organic Process Research & Development

Figure 1. HPLC chromatograms of (a) Fmoc-Gln-OH, (b) Fmoc-Ala-OH, (c) Fmoc-Gly-OH, (d) Alloc-Val-OH. All the Fmoc/Alloc
amino acids were prepared by the azide method in one pot. Samples were applied onto a Waters system C18 Symmetry column (4.6
× 150 mm, 5 µm). Conditions: λ ) 220 nm, flow ) 1 mL/min, linear gradient ) 5-100% B in 15 min (B ) ACN).
introduced through the procedure described herein are
currently being studied.
was dissolved in dioxane (5 mL), and NaN3 (0.78 g, 12
mmol) in water (4 mL) was added. The mixture was left to
stir at room temperature, and the reaction was followed by
HPLC, which showed that it was complete after 1 h (no
precipitate appeared). Next, the amino acid [H-Gln-OH,
H-Asn-OH, H-Ala-OH, and H-Gly-OH (11 mmol)] was
added to the previous mixture dissolved in 1% aqueous
Na2CO3-dioxane (1:1, v/v) (the amount of the latter depend-
ing on the solubility of the respective amino acid: 30 mL
for H-Ala-OH and H-Gly-OH; 60 mL for H-Gln-OH and
H-Asn-OH). The reaction was stirred at room temperature
for 48 h, during which the pH (controlled by pH paper) was
kept between 8 and 10 by the addition of 10% aqueous
Na2CO3. The progress of the reaction was monitored by RP-
HPLC.
Once the reaction was completed, the mixture was poured
into 100 mL of water (keeping a basic pH) and extracted 4
times with tert-butyl methyl ether to remove side products.
The aqueous solution was cooled to 4 °C and acidified to
pH 2.0 with 2 N aqueous HCl. The white precipitate was
filtered off and washed with ice water made slightly acidic
(pH 4-5) with dilute HCl. The solid is dried under vacuum
overnight, and the purity was measured by HPLC. Yields
and purities are shown in Tables 1 and 2. Additional
analytical data are shown in Table 3.
Experimental Section
General. Melting points were determined on a Buchi
capillary tube melting point B-540 apparatus and are uncor-
rected. Thin-layer chromatography (TLC) was performed on
precoated TLC plates (Merck, silica gel 60, F254), using as
eluent CH2Cl2-MeOH-AcOH (94:5:1). The plates were
visualized by using a 254 nm UV lamp. HPLC reversed-
phase Symmetry columns C18 4.6 × 150 mm, 5 µm were
from Waters (Ireland). Analytical HPLC was carried out on
a Waters 996 instrument provided with a photodiode array
detector and equipped with a Waters 2695 separation module
controlled by the Millennium software. NMR spectra were
taken on a (400 MHz) Varian spectrometer and expressed
in ppm from Me4Si as internal standard. HPLC-MS analysis
were carried out on an Alliance system (Waters 2795
Separation Module, MA, U.S.A.) coupled to a double
wavelength UV detector (Waters 2487 dual λ Absorbance
Detector, MA) and a ZQ4000 mass spectrometer (Waters,
Micromass ZQ, MA).
General Procedure for the Preparation of Fmoc Amino
Acids by the Azide Method. Fmoc-Cl (2.58 g, 10 mmol)
(22) Gopi, H. N.; Suresh B. V. V. J. Pept. Res. 2000, 55, 295.
(23) Paquet, A. Can. J. Chem. 1982, 60, 976.
Vol. 8, No. 6, 2004 / Organic Process Research & Development
•
923

Scale-up Preparation of Fmoc-Gly-OH by the Azide
Method. Fmoc-Cl (258.70 g, 1 mol) was dissolved in
dioxane (250 mL), and NaN3 (78.0 g, 1.2 mol) in water (250
mL) was added. The mixture was left to stir at room
temperature, and the reaction was complete after 1 h. Next,
the H-Gly-OH (82.6 g, 1.1 mol) dissolved in 1% aqueous
Na2CO3-dioxane (1:1, v/v) (500 mL) was added to the
previous mixture, while the solution was stirred at 25 °C.
The reaction mixture was kept at pH 8-10 by the addition
of solid Na2CO3. The progress of the reaction was monitored
by TLC and RP-HPLC.
Once the reaction was completed, the mixture was poured
into 400 mL of water (keeping a basic pH) and extracted
four times with tert-butyl methyl ether to remove side
products. The aqueous solution was cooled to 4 °C and
acidified to pH 2.0 with 6 N aqueous HCl. The white
precipitate was filtered off and washed with ice water made
slightly acidic (pH 4-5) with dilute HCl. [The cold aqueous
solution was immediately neutralized with NaOH, to avoid
subjecting the released azide anions to acidic conditions at
room temperature.] The solid was dried under vacuum
overnight, and the purity was measured by HPLC. Yields
and purities are shown in Table 2 (entries 18).
room temperature for 1 h. Next, the amino acid H-Val-OH
and H-Gly-OH (43 mmol) dissolved in 1% aqueous Na2CO3-
dioxane (1:1, v/v) (125 mL) was added to the previous
mixture. The reaction was stirred at room temperature during
48 h, and the pH of the reaction was kept between 8 and 10
(controlled by pH paper) by addition of 10% aqueous Na2-
CO3. The progress of the reaction was monitored by RP-
HPLC.
Once the reaction was completed, the mixture was poured
into water (100 mL) (keeping the pH between 9 and 10)
and extracted several times with tert-butyl methyl ether to
remove the byproducts. The aqueous solution was acidified
to pH 2.0 with 2 N aqueous HCl and was extracted several
times with ethyl acetate. The organic phase was dried with
MgSO4, filtered, and concentrated under vacuum. The purity
of the oil was measured by HPLC. Yields and purities are
shown in Table 2. Additional analytical data are shown in
Table 3.
Acknowledgment
The work carried out at the Barcelona Science Park-
University of Barcelona was partially supported by CICYT
(BQU2003-00089) and Generalitat de Catalunya. We thank
Yoav Luxembourg for his encouragement to perform the
work.
General Procedure for the Preparation of Alloc Amino
Acids by the Azide Method. Alloc-Cl (3.81 mL, 36 mmol)
was disolved in dioxane (10 mL), and NaN3 (3.5 g, 53 mmol)
in water (8 mL) was added. The mixture was left to stir at
Received for review April 22, 2004.
OP049917Z
(24) Chang, C. D.; Felix, A. M.; Jimenez, M. H.; Meienhofer, J. Int J. Pept.
Protein Res. 1980, 15, 485.
924
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Vol. 8, No. 6, 2004 / Organic Process Research & Development