G. Han M. Tamaki V.J. Hruby
Fast, ef®cient and selective deprotection of the tert-butoxycarbonyl (Boc) group using HCl/dioxane (4 m)
Authors' af®liations:
Key words: Boc; dioxane; hydrogen chloride; selective
G. Han, M. Tamaki and V.J. Hruby,
deprotection; tert-butyl ester
Department of Chemistry, University of Arizona, Tucson, Arizona, USA.
Abstract: Fast, ef®cient and selective deprotection of the tert-butoxycarbonyl (Boc) group of various amino acids and
Correspondence to:
V. J. Hruby
peptides was achieved by using hydrogen chloride (4 M) in anhydrous dioxane solution for 30 min at room temperature. In
Department of Chemistry
the cases studied in our laboratory, this protocol provided
University of Arizona
superior selectivity to deprotect Na-Boc groups in the presence
Tucson AZ 85721 USA
of tert-butyl esters and tert-butyl ethers, including thio-tertbutyl ethers, but not phenolic tert-butyl ethers.
Tel.: 1-520-621-6332 Fax: 1-520-621-8407 E-mail:
[email protected]
The tert-butyl group has been widely applied for the protection of a variety of functional groups in natural product synthesis, including amino acid, peptide and protein chemistry. A variety of methods for protection with tertbutyl-related groups has been studied extensively in the past (1). However, evaluation of methods for selective deprotection of different functionalized tert-butyl-containing protecting groups has not been widely examined, except by a few groups. For example, using (CH3)3SiClO4 in a mixture of benzene and acetonitrile (1:1), Na-Boc-protecting groups were selectively removed in the presence of benzyloxycarbonyl (Cbz, Z) groups (2). However, selective deprotection of Na-Boc groups vs. tert-butyl ester and other groups has not
Dates:
Received 18 April 2001 Revised 11 May 2001
been well studied. In 1969, it was reported that selective deprotection of Boc could be accomplished by using acidic
Accepted 9 July 2001
ionic-exchange resins (3). The procedure required long reac-
To cite this article:
tion times (6 h) using complicated procedures, and the yields
Han, G., Tamaki, M. & Hruby, V. J. Fast, ef®cient and selective deprotection of the tert-butoxycarbonyl (Boc) group using HCl/dioxane (4 m).
(47±87%) were not suf®cient for most applications. p-Toluenesulfonic acid (p-TsOH, PTSA) in ethanol has
J. Peptide Res., 2001, 58, 338±341.
also been used to selectively deprotect Na-Boc groups in
Copyright Munksgaard International Publishers Ltd, 2001
the presence of tert-butyl esters (4). Although the yields
ISSN 1397±002X
(81±93%) were improved compared with those obtained
338
Han et al . Deprotection of the Boc group using HCl/dioxane
using acidic resins, this method required dropwise addition
reaction was almost complete in 15 min). After evaporating
of PTSA solution at cold temperatures (±10 to 08C), followed
the solvent at room temperature under high vacuum, the
by 3 h at room temperature. In 1972, it was reported that a
residue was examined directly, without puri®cation, and
TFA-CHCl3 (1:1) mixture could selectively remove Na-Boc
was checked for purity by NMR, which demonstrated that
groups in the presence of tert-butyl ethers with high yields
the product was pure. Further treatment of the product by
around 90% for the very limited examples studied (5).
trituration with dry ethyl ether provided pure H-Ala-OtBu
However, this procedure required a much lower tempera-
HCl salt in a yield of .95%. Similar results were obtained
ture, ±208C, and a much longer reaction time, 10 h. In addi-
for other naturally occurring amino acids with primary
tion, selectivity against tert-butyl esters was not explored
amino groups, such as Na-Boc-Phe-OtBu and Na-Boc-Asp
a
using this method. Recently, selective deprotection of N -
(OtBu)-OtBu (Table 1). Thus, tert-butyl-protected esters
Boc groups in the presence of tert-butyl esters was reported
were safe under the conditions used in this study. How-
using dry HCl in ethyl acetate (1 m) (6). This procedure
ever, for Boc-Glu(OtBu)-OtBu, the result was complicated
required 5 h for the reaction to be complete, and was applied
because the free a-amino group nucleophilically attacked
only to amino acids. In addition, ethyl acetate is not
the side-chain ester group, and consequently, tert-butyl
applicable in many situations, including peptide and protein
pyroglutamate was formed as the main product.
synthesis, as most peptides and proteins are insoluble in
Na-Boc derivatives of amino acids with secondary amino
ethyl acetate. Recently, it has been reported that concen-
groups were also examined under the conditions used above.
trated sulfuric acid in tBuOAc or methanesulfonic acid in a
Only one product was obtained within a 30-min reaction
tBuOAc/CH2Cl2 mixture solvent system could selectively
time at room temperature for Boc-Sar-OtBu, Boc-Pro-OtBu
remove Boc in the presence of t-butyl esters (7). However,
and Na-Boc-trans-4-Ph-Pro-OtBu, respectively.
the yields (70±100%) were highly dependent on the
Furthermore, treatment of several Na-Boc-amino acids
substrates. In addition, the reaction times varied from 2 to
with tert-butyl ether or thioether protected side-chains gave
16 h, which are relatively long. Furthermore, there is no
only a-amino selectively deprotected products. Neither
example for tert-butyl ether or tert-butyl-protected thiol
tert-butyl ethers nor tert-butylthio ethers were affected by
derivatives. Thus, all these reported protocols have very
HCl (4 m) in dioxane (Table 1) with one exception. When
limited scope. Moreover, with rapid advances in solid-phase
the substrate was Na-Boc-Tyr(OtBu)-OH, both the Na-Boc
combinatorial synthesis, which requires short reaction
and the tert-butyl phenyl ether groups were removed at
times, mild reaction conditions, easy methods of puri®ca-
room temperature within 30 min. Apparently, the tert-butyl
tion and economical reagents, the reported procedures are
phenol ether is labile under these conditions. We also tried
not useful. In fact, all failed to achieve our goal of selectively
the reaction at 08C but it was very slow and did not lead to
deprotecting tert-Boc groups during our investigations of
any selectivity.
practical peptide synthesis. Hence, there is an urgent need
We next investigated the application of the protocol
to ®nd better methods, which can have broad applications,
used above for peptide chemistry, utilizing a series of pro-
for selectively removing Na-Boc protecting groups.
tected peptides (Table 1). Only the Na-Boc groups were
Here we report the selective deprotection of Na-Boc
removed, whereas tert-butyl ethers and tert-butyl esters
groups in the presence of tert-butyl esters using HCl (4 m)
remained intact for all the peptides tested. Hence, HCl/
in dioxane at room temperature (Fig. 1), and further applica-
dioxane (4 m) also is useful for the selective deprotection
tions of this approach were also explored in our laboratory. In a typical example, Na-Boc-Ala-OtBu was dissolved in
of Na-Boc in peptide synthesis. In order to investigate whether a low concentration HCl
HCl (4 m)/dioxane at 08C and the mixture was then stirred
could selectively deprotect Na-Boc groups, we examined the
for 30 min at room temperature. According to thin-layer
use of HCl/Et2O (2 m). However, in most cases, the reac-
chromatography (TLC), only one product was present and
tions were slow, and although the reactions were initially
the starting material disappeared (in most cases the
selective, after extending the reaction time, some of the reactions became nonselective. Hence, the concentration of HCl and solvent was a signi®cant factor in discriminating the protecting groups. In conclusion, HCl (4 m) in dioxane at room temperature (initially mixed at 08C) is a superior reagent for selectively
Figure 1. Selective deprotection by HCl/dioxane (4 m).
cleaving Na-Boc groups, while other acid sensitive groups, J. Peptide Res. 58, 2001 / 338±341
| 339
Han et al . Deprotection of the Boc group using HCl/dioxane
Table 1. Selective deprotection of Boc in the presence of other formats of tert-butyl protection Amino acids
Protected derivative
Producta
Primary amino group
Boc-Ala-OtBu
H-Ala-OtBu.HCl
.95
Boc-Phe-OtBu
H-Phe-OtBu.HCl
.95
Boc-Asp(OtBu)-OtBu
H-Asp(OtBu)-OtBu.HCl
Boc-Glu(OtBu)-OtBu
Mixture.HCl
Boc-Sar-OtBu
H-Sar-OtBu.HCl
Boc-Pro-OtBu
H-Pro-OtBu.HCl
Boc-cis-4-Ph-Pro-OtBu
H-cis-4-Ph-Pro-OtBu.HCl
Boc-Ser(OtBu)-OtBu
H-Ser(OtBu)-OtBu.HCl
92
Boc-Cys(StBu)-OH
H-Cys(StBu)-OH.HCl
95
Boc-Tyr(OtBu)-OH
H-Tyr-OH.HCl
Boc-Ala-Ala-OtBu
H-Ala-Ala-OtBu.HCl
.95
Boc-Ala-Ser(OtBu)-OtBu
H-Ala-Ser(OtBu)-OtBu.HCl
.95
Boc-Ala-Asp(OtBu)-OtBu
H-Ala-Asp(OtBu)-OtBu.HCl
.95
Boc-Ala-Glu(OtBu)-OtBu
H-Ala-Glu(OtBu)-OtBu.HCl
.95
Secondary amino group
Side-chain tert-butyl ether
Yield (%)b
.95 ± .95 .95 < 100
< 100
Peptides
a. In the format of HCl salts, whose TLC and MS values were identical with the commercially available ones. NMR data also proved the high purity of any of the deprotected products, no additional peaks was found. b. Isolated yields, TLC for reaction mixture indicated only one product was obtained [except for the substrate of Boc-Glu(OtBu)-OtBu].
such as tert-butyl esters, tert-butyl ethers and even tert-
under argon. Na-Boc-cis-4-Ph-Pro-OtBu (0.2 mmol) was
butyl thioethers were not cleaved by this reagent. However,
added in one portion with stirring. The ice-bath was
the phenolic tert-butyl ether was completely removed by
removed and the mixture was kept stirred. After 30 min,
this reagent under the condition used to selectively remove
TLC indicated that the reaction was completed. The reac-
Na-Boc groups. In comparison with other methods reported,
tion mixture was condensed by rotary evaporation under
this reagent has many advantages in selectivity, yield, and
high vacuum at room temperature. The residue was then
also has much shorter reaction times (minutes vs. hours),
washed with dry ethyl ether and collected by ®ltration (for
ease of operation, more milder conditions and economic
oil products, a simple decantation was used instead). Yield:
materials. This provides a new, practical and ef®cient
< 100%. It was then analyzed by high-resolution mass
approach for the synthesis of peptides, alkaloids and other
spectroscopy and NMR. 1H NMR (d, p.p.m., D2O, Bruker
natural products, which frequently require orthogonal pro-
DRX-500 MHz with water suppression): 7.27±7.23 and
tection during the course of synthesis. Further applications
7.20±7.16 (m, 5H, aromatic), 4.39±4.35 (dd, 1H, a-H),
of this reagent are currently under investigation.
3.68±3.63 (dd, 1H, 5-Ha), 3.54±3.50 (m, 1H, 4-H), 3.31±3.26 (dd, 1H, 5-Hb), 2.74±2.68 (m, 1H, b-Ha), 2.11±2.04 (m, 1H, b-Hb), 1.31 (s, 9H, 3CH3). FAB-HR-MS: calcd. for C15H21NO2: 247.1572; found: 247.1573.
Experimental Procedures
Acknowledgements: This work was supported by grants from
A solution of HCl/dioxane (4 mL, 4 m, a new Aldrich
the US Public Health Service DK17420 and DA06284. The views
Sureseal2 bottle) in a 25-mL round-bottom ¯ask equipped
expressed are those of authors and not necessarily those of the
with a magnetic stir-bar was cooled by an ice-water bath
USPHS.
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