Microwave-assisted synthesis of unnatural amino acids

Article abstract of DOI:10.1016/j.bmcl.2008.09.025

Microwave irradiation has been proven to be a useful tool in the rapid assembly of racemic unnatural amino acids in only two steps. Additional benefits of this methodology are the commercial availability of the inexpensive starting materials and the high yields and high purities of the final amino acid products.

Full text of DOI:10.1016/j.bmcl.2008.09.025

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5478–5480
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Microwave-assisted synthesis of unnatural amino acids
*
Douglas D. Young, Jessica Torres-Kolbus, Alexander Deiters
Department of Chemistry, North Carolina State University, Campus Box 8204, Raleigh, NC 27695-8204, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Microwave irradiation has been proven to be a useful tool in the rapid assembly of racemic unnatural
amino acids in only two steps. Additional benefits of this methodology are the commercial availability
of the inexpensive starting materials and the high yields and high purities of the final amino acid
products.
Received 21 August 2008
Revised 4 September 2008
Accepted 5 September 2008
Available online 10 September 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Microwave
Microwave synthesis
Amino acid
Unnatural amino acid
Peptide synthesis
Protein synthesis
Unnatural amino acid
Mutagenesis
Microwave irradiation has become a very useful tool in syn-
thetic organic chemistry, and has been employed in a variety of
reactions.1 Microwave-assisted synthesis has been found to in-
crease both reaction rates and yields via more efficient heating
compared to standard thermal conduction. Moreover, in certain
cases specific microwave effects have been discussed which facili-
tate reactions that can not be conducted under purely thermal
heating.2 In order to further probe the utility of microwave irradi-
ation in organic synthesis we investigated its utility in the prepara-
tion of amino acids and unnatural amino acid analogs. The
synthesis of unnatural amino acids is especially interesting due
to technological advances in their incorporation into proteins both
in vivo and in vitro.3 Since unnatural amino acids can have a rep-
ertoire of functional groups that vastly extends beyond the com-
mon set of natural amino acids, they can be used as probes to
obtain a better understanding of biological processes. While sev-
eral routes to amino acid synthesis currently exist, microwave irra-
diation has rarely been employed.4 To the best of our knowledge,
only one synthetic route to unnatural amino acids using micro-
wave irradiation has been attempted via the Michael addition of
methyl N-(diphenylmethylene)-2,3-didehydroalaninate and nitro-
alkanes.5 Herein we explore a more general microwave-assisted
alkylation approach to afford a wide variety of unnatural amino
acid derivatives in a facile fashion.
Initial investigations commenced with the optimization of the
alkylation of diethyl acetamidomalonate (1) with benzyl bromide
(2a) in the presence of base to yield the diester 3a (Table 1). Several
bases were examined including NaH, NaOEt, K2CO3 and Cs2CO3.
Ultimately Cs2CO3 was found to be optimal as it afforded high
yields with a minimum number of side products compared to the
other bases. We next investigated the role of the solvent and the
microwave power on the alkylation reaction (Table 1). Based on
our previous experience in microwave-mediated reactions,6 tolu-
Table 1
Optimization of the microwave-assisted alkylation reaction
CO2Et
O
O
benzyl
CO2Et
bromide
EtO
OEt
NHAc
conditions,
Cs2CO3
NHAc
1
3a
Entry
Conditions
MW power (W)/temperature (°C)
Yield 3a
1
2
3
4
5
6
PhMe, 10 min
DMF, 10 min
THF, 10 min
THF, 10 min
ACN, 10 min
ACN, 10 min
300/25–120a
0–200/170b
300/25–85a
200/25–80a
0–300/130b
0/130
31%
45%
59%
47%
82%
43%
a
Fixed microwave power input and variable temperature.
Fixed temperature and variable microwave power input.
* Corresponding author.
E-mail address: alex_deiters@ncsu.edu (A. Deiters).
b
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.025

D. D. Young et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5478–5480
5479
Table 3
ene was employed as a microwave transparent solvent, thus en-
Conversion of precursor 3 to amino acid 4
abling a high level of microwave power input to the substrates.
However, even at 300W of microwave irradiation overall conver-
sion was low, presumably due to the poor solubility of Cs2CO3.
When the alkylation was conducted in DMF, a polar, strongly
microwave absorbing solvent, a temperature of 170 °C was reached
very quickly; however, the yield of 3a was still relatively low at
45%. Conducting reactions in microwave absorbing solvents limits
the ability to use high microwave power inputs due to the rapid
heating of the solvents beyond their boiling point, leading to a sub-
stantial pressure formation in the sealed reaction vessel. Based on
these observations, we next performed the reaction in microwave
transparent THF, resulting in decreased temperatures but also sig-
nificant amounts of the starting material 1. However, these condi-
tions afforded slightly higher yields of 3a at high microwave
powers: 47% yield at 200W and a 59% yield at 300W. We then con-
ducted the reaction in acetonitrile, which is a common solvent em-
ployed in similar alkylations.7 Acetonitrile was found to be the
optimal solvent under standard microwave irradiation at ꢀ130 °C
for 10 min affording 3a in 82% yield. When this reaction was con-
ducted thermally at 130 °C, under otherwise identical conditions,
43% of 3a was obtained.
6N HCl (aq)
R
CO2H
NH2
4a-4g
EtO2C
R NHAc
3a-3g
CO2Et
MW, 90 oC,
10 min
Entry
Diester
Product
Yield (%)
1
2
3
4
5
6
7
3a
3b
3c
3d
3e
3f
4a
4b
4c
4d
4e
4f
98
88
90
94
86
96
92
3g
4g
be prepared in moderate to high yields within a short 10 min reac-
tion time. The methodology tolerates a wide range of functional-
ities including alkyl (2g), aryl (2a, 2d, and 2e–2f), alkene (2b and
2d), alkyne (2c), fluorine (2e), and ketone (2f) functional groups.
However, aliphatic chains (2g) lead to reduced yields despite fur-
ther optimization attempts, presumably due to their intrinsic low-
er reactivity and their propensity to undergo eliminations. In order
to increase the yield for certain bromides (2b, 2e, and 2g), sodium
iodide (20 mol%) was added to the reaction mixture to facilitate
substitution via an in situ leaving group exchange, increasing the
yields by 10–15%.
With optimized conditions in hand (Cs2CO3, CH3CN, 130 °C,
microwave irradiation), the reaction scope was investigated with
several bromides (2a–2g) to yield the alkylated products 3a–3g
(Table 2). Overall the amino acid precursors 3a–3g could typically
After generating a small array of the amino acid precursors 3a–
3g, a global deprotection of the carboxy and the amino protecting
groups followed by an instant decarboxylation was performed to
yield the amino acids 4a–4g in a single step. Typically, this reaction
is conducted in refluxing aqueous 6 N HCl for approximately 12 h.
However, we discovered that this transformation also benefits
from microwave irradiation. After just 10 min of microwave irradi-
ation of 3a–3g in 6 N HCl, the amino acids 4a–4g were obtained in
excellent yields of 85–99% and purities of >95%, as determined by
1H NMR (Table 3). The analytical data of 4a–4g was in agreement
with literature reports.8 Thermal reactions mimicking the micro-
wave reaction conditions afforded minimal product conversion
<20%, and significant amounts of the starting materials 3a–3g were
retained. Overall, the preparation of racemic unnatural amino acids
was achieved in a combined 20 min of reaction time with a single
chromatography purification between reactions.9
In conclusion, a new microwave-assisted two-step methodol-
ogy to rapidly prepare racemic unnatural amino acids in high yield
and high purity was developed. Due to the wide range of functional
groups tolerated via this approach, several important unnatural
amino acids were attained. Upon incorporation into proteins and
peptides these amino acids can potentially be employed in biocon-
jugation reactions (4c and 4f),10 as NMR labels (4e),11 and in pho-
tochemical transformations (4b).12 Due to the high convergence of
this approach and the wide range of inexpensive commercially
available bromides, the methodology will find widespread applica-
tion in the rapid assembly of unnatural amino acids and derivatives
thereof.
Table 2
Preparation of unnatural amino acid precursors 3
RBr (2 eq.)
EtO2C
CO2Et
NHAc
EtO2C
R NHAc
3a-3g
CO2Et
Cs2CO3, CH3CN,
MW, 130 oC,
10 min
1
Entry
R Group
Product
Yield
82%
1
3a
2a
2b
2
3
3b
3c
78%a
68%
2c
4
5
3d
3e
76%
2d
F
73%a
H3C
2e
This research was supported by the Department of Chemistry at
North Carolina State University. AD is a Beckman Young Investiga-
tor and a Cottrell Scholar. DDY acknowledges a graduate research
fellowship from the ACS Medicinal Chemistry Division. JTK
acknowledges AGEP for financial support.
O
6
7
q
92%
CH3
2f
References and notes
3g
33%a
H3C
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2g
a
Sodium iodide (20 mol%) was added.

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