An improved synthesis of β-cyano- l -alanine esters and amides

Article abstract of DOI:10.1055/s-0035-1560489

We have developed a novel, mild, efficient, and scalable protocol for the synthesis of N-protected β-cyano-l-alanine esters or -amides from N-protected l-asparagin. This protocol avoided the use of toxic or unpleasant reagents and was easy to operate in laboratory.

Full text of DOI:10.1055/s-0035-1560489

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© Georg Thieme Verlag Stuttgart · New York
2016, 27, 309–312
letter
309
G. Wang et al.
Letter
Synlett
An Improved Synthesis of β-Cyano-L-Alanine Esters and Amides
EDC.HCl
cat. DMAP
NHPGO
NHPG
Guoxin Wang*a
Longjian Chena
Xiaodan Caia
Zigang Li*a
HO
RX
CN
+
XH
R
NH2
DCM, r.t.
O
O
PG = Boc, Fmoc,
Cbz, Ac
X = O, NH
mild
Ming Luoa,b
R = aliphatic, aromatic,
amino acid
simple operation
scalable
a Key Laboratory of Structural Biology and Laboratory of Chemical Genomics,
School of Chemical Biology and Biotechnology, Peking University Shenzhen
Graduate School, Shenzhen 518055, P. R. of China
wanggx@pkusz.edu.cn
lizg@pkusz.edu.cn
b Department of Chemistry, Georgia State University, Atlanta, GA 30302, USA
mluo@gsu.edu
Received: 31.07.2015
Accepted after revision: 13.09.2015
Published online: 30.09.2015
amide.7a In addition, β-cyano-L-alanine ester or amide can
be also prepared from L-asparagin ester or amide using
some dehydrate agents in good yield, such as phenylphos-
phonic dichloride/pyridine,7b trichloroacetyl chloride/pyri-
dine,7c ethylpolyphosphate/pyridine,7d trimethylsilyl poly-
phosphate/pyridine,7e cycanuric chloride/pyridine,7f Vils-
DOI: 10.1055/s-0035-1560489; Art ID: st-2015-w0598-l
Abstract We have developed a novel, mild, efficient, and scalable pro-
tocol for the synthesis of N-protected β-cyano-L-alanine esters or
amides from N-protected L-asparagin. This protocol avoided the use of
toxic or unpleasant reagents and was easy to operate in laboratory.
meier
reagent,7g
phosphoryl
chloride/pyridine,7h
chlorosulfonyl isocyanate/pyridine,7i trifluoromethanesul-
fonic anhydride/pyridine,7j toluenesulfonyl chloride/pyri-
dine,7k trifluroacetic anhydride/pyridine,7l Burgess re-
agent/pyridine (Scheme 1).7m However, all existing method-
ologies require the use of toxic chemical reagents or heating
and/or acidic conditions which may not be applicable to
complex molecules. Thus, a mild method for the transfor-
mation of L-asparagin derivatives into β-cyano-L-alanine
derivatives is still desirable. Here, we report a facile synthe-
sis of β-cyano-L-alanine derivatives using commercially
available N-protecting-L-asparagin with various alcohols or
amines by using common coupling reagent 1-ethyl-3-(3-di-
menthylaminopropyl)-carbodiimide hydrochloride (EDC·
HCl)8 and 4-dimethylaminopyridine (DMAP) as the catalyst.
This method is facile, eco-friendly, and highly efficient.
In the course of our studies on the synthesis of L-aspar-
agine moiety containing peptides, we observed that the re-
action of N-Boc-L-asparagine (1a, 1.0 equiv) and benzyl al-
cohol (2a, 1.2 equiv) in CH2Cl2 leads to N-Boc-L-asparagine
benzyl ester (3a) in 40% yield by using DCC (2.0 equiv) as
the coupling reagent and DMAP (0.20 equiv) as the catalyst,
accompanying with minor N-Boc-β-cyano-L-alanine benzyl
ester (4a) in ca. 10% yield (Scheme 2). This result is as ex-
pected. In order to avoid interference by the byproduct di-
cyclohexylurea (DCU) during purification, DCC was re-
placed with a water-soluble dehydrate agent EDC·HCl (2.0
equiv) to conduct this coupling reaction in the presence of
DMAP (0.20 equiv). Surprisingly, this same coupling reac-
Key words L-asparagin, β-cyano-L-alanine, EDC·HCl, DCC
β-Cyano-L-alanine derivatives are found in the seeds of
common vetch and function as lathyrism causing neuro-
toxins.1 β-Cyano-L-alanine is a potent inhibitor of bacterial
L-glutamate decarboxylase, an enzyme thought to modulate
neuronal activities in high species.2 Previous studies re-
ported that the proteins containing the β-cyano-L-alanine
moiety exhibit moderate inhibitory activities to Schistoso-
ma japonica glutathione S-transferase.3 Understanding
β-cyano-L-alanine’s functions could help on elucidating the
structural and biogenetic relationship between lathrogenic
factors, β-aminopropionitrile (in Lathyrus odoratus) and
α,γ-diaminobutyric acid (in L. latifolius and L. sylvestris
Wagneri),4 additionally, it could also help on elucidating the
possible metabolic pathway of β-cyano-L-alanine from the
abundant amino acid L-asparagine.5 The amide group of
L-asparagine could be converted into a cyano group which
generates β-cyano-L-alanine in plants.6 However, the de-
tailed mechanism on how β-cyano-L-alanine is biosynthe-
sized in other species remains unknown.
Several methods exist for the conversion of asparagine
and glutamine derivatives into their corresponding cyano
compounds.7 Usually, using N,N′-dicyclohexylcarbodiimide
(DCC) as a dehydrate agent in the presence of pyridine as
base in a proper solvent, L-asparagin can be converted into
β-cyano-L-alanine, with good yield, that can be further
transformed into the respective β-cyano-L-alanine ester or
© Georg Thieme Verlag Stuttgart · New York — Synlett 2016, 27, 309–312

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G. Wang et al.
Letter
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effect on this reaction (Table 1, entries 4–7). This reaction
can be conducted under mild reaction conditions and in ex-
cellent yield.
NH2
O
NH2
pyridine, r.t.
HO
O
HO
RO
CN
CN
+
DCC
NH2
NH2
78%
O
O
NHPGO
NHPG
Table 1 Screening Reaction Conditions in the Synthesis of N-Boc-β-cy-
ano-L-alanine Benzyl Ester 4a from N-Boc-L-asparagin (1a) and Benzyl
Alcohol (2a)a
dehydrating agent
RO
O
dehydrating agent = PhP(=O)Cl2/py,
POCl3/py,
Entry
Solvent
EDC·HCl
(equiv)
DMAP
(equiv)
Conv.
(%)
3ab
4ab
PG = Boc, Fmoc,
Cbz, Ac
PhSO2Cl/py,
(CF3(C=O))2O/py,
cyanuric trichloride/py,
Burgess reagent/py
1
2
3
4
5
6
7
CH2Cl2
CH2Cl2
CH2Cl2
CH2Cl2
THF
2.0
3.0
2.0
3.0
3.0
3.0
3.0
0
50
60
10
10
10
0
90
0
90
90
0.2
0.2
0.2
0.2
0.2
65
Scheme 1 The reported transformation of L-asparagin into β-cyano-L-
alanine
>95
>95
>95
>95
100
100
100
100
0
DMF
0
tion generated the converse result: N-Boc-β-cyano-L-ala-
nine benzyl ester (4a) as the major product in 45% yield,
and N-Boc-L-asparagine benzyl ester (3a) as the minor
product in ca. 5% yield (Scheme 2).
EGc
0
a Reaction conditions: 1a (0.10 mmol), 2a (0.12 mmol), EDC·HCl, DMAP,
and solvent (10 mL) for 12 h at r.t.
b Ratio 3a/4a determined by GC.
c EG = ethyl glycol.
BocHN
BnO
O
NH2
With the optimized conditions in hand, we investigated
the scope of the transformation of N-Boc-L-asparagin into
N-Boc-β-cyano-L-alanine ester or amide with various alco-
hols and amines (Scheme 3). First, different primary and
secondary alcohols were examined. It was observed that al-
cohols would not disturb the transformation of N-Boc-L-as-
paragin into N-Boc-β-cyano-L-alanine ester, and the reac-
tions afforded the corresponding products 4a–f in excellent
yield (87–95%).10,11 Next, N-Boc-L-asparagin (1a) was al-
lowed to react with different amines, aiming to produce the
corresponding N-Boc-β-cyano-L-alanine amide 4g–m in ex-
cellent yields (80–95%). Furthermore, it was observed that
the reaction of 1a with glycine ethyl ester 2n also furnished
the product 4n in a 76% yield. It is valuable that this reac-
tion could be successfully employed in a gram-scale synthe-
sis for β-cyano-L-alanine esters and amides. Under the opti-
mized reaction conditions, the reaction of 1a (11.6 g, 0.10
mol) with 2a (13.0 g, 0.12 mol) proceeded to isolate the
product 4a in 90% yield (27.4 g, 0.09 mol).
Subsequently, we investigated on how an N-protecting
group of L-asparagin could affect the transformation of L-as-
paragin into β-cyano-L-alanine under the EDC·HCl/DMAP
reaction conditions. It was found that the N-protecting
group of L-asparagin showed no effect on this transforma-
tion, and the reaction of N-Cbz-L-asparagin (1b) or N-Fmoc-
L-asparagin (1c) with 2a both offered the respective N-pro-
tected β-cyano-L-alanine ester 5a and 5b in excellent yields
(Scheme 4).
BocHN
HO
O
O
dehydrating agent
CH2Cl2, r.t., 12 h
3a
+
BnOH
NH2
NHBoc
O
BnO
1a
2a
N
O
4a
Scheme 2 The reaction of N-Boc-L-asparagine (1a) with benzyl alcohol
(2a)
As we already know, DCC and EDC·HCl as common de-
hydrate reagents are universally used to form the amide or
ester bond in laboratory.9 Our question was whether the re-
action of N-Boc-L-asparagine (1a) with benzyl alcohol (2a)
can deliver N-Boc-β-cyano-L-alanine benzyl ester (4a) as
the sole product using EDC·HCl as the dehydrate agent. If
the reaction result was as expected after screening the reac-
tion conditions, this protocol would be useful for the syn-
thesis of β-cyano-L-alanine esters or amides. Therefore, we
screened reaction conditions, and the results are summa-
rized in Table 1. It was observed that the reaction between
1a (1.0 equiv) and 2a (1.2 equiv) with EDC·HCl as the dehy-
drate reagent resulted in 4a as the major product (Table 1,
entries 1–6). Increasing the amount of EDC·HCl could im-
prove the reaction conversion (Table 1, entries 1–2, rising
from 50% to 60%; entries 3–4, rising from 65% to >95%). The
presence of DMAP dramatically increased the yield of 4a
(Table 1, entries 4–6). Very promising results were obtained
using 3.0 equivalents of EDC·HCl as the dehydrate agent and
DMAP (0.20 equiv) as the catalyst, and the yield of 4a
reached 98% (Table 1, entry 6). Various solvents were also
screened, and the results revealed that the solvent has little
In order to investigate the scope of this transformation
of the carboxamide group into the cyano group under the
EDC·HCl/DMAP reaction conditions, different carbox-
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G. Wang et al.
Letter
Synlett
cyano compounds either, and just the starting materials
were recovered (Scheme 5). These results indicated that
this methodology was specific to some particular carbox-
amides with β-acid group.
BocHN
HO
O
NHBoc
EDC.HCl, DMAP
CH2Cl2, r.t., 12 h
RX
+
RXH
EtO
NH2
N
N
O
O
4a–n
1a
2a–n
76–95%
O
NHBoc
NHBoc
NHBoc
EDC.HCl, DMAP
O
BnO
no reaction
no reaction
NH2
NH2
CH2Cl2, r.t. 12 h
N
N
O
O
O
4a, 95% (90%)a
4b, 90%
4c, 87%
6a
6b
O
EDC.HCl, DMAP
CH2Cl2, r.t. 12 h
NHBoc
O
NHBoc
NHBoc
O
O
O
N
N
N
O
O
4d, 93%
4e, 88%
4f, 95%
O
EDC.HCl, DMAP
CH2Cl2, r.t. 12 h
NHBoc
NHBoc
NHBoc
H
N
H
N
N
no reaction
no reaction
BnHN
NH2
N
N
N
N
O
Ot-Bu
6c
O
O
O
4g, 92% (90%)c
4h, 88%
4i, 90%
EDC.HCl, DMAP
CH2Cl2, r.t. 12 h
NHBoc
N
NHBoc
NHBoc
O
H
H
N
N
Et2N
O
N
N
NH2
O
O
O
O
Ot-Bu
4j, 93%
4k, 85%
4l, 82%
6d
NHBoc
N
O
NHBoc
H
N
N
Scheme 5 The scope of the transformation of carboxamide group into
cyano group using EDC·HCl/DMAP reaction conditions
EtO
N
O
O
4m, 80%
4n, 76%
Scheme 3 Synthesis of N-Boc-β-cyano-L-alanine esters and amides
from N-Boc-L-asparagin Isolated yields are given. Reagents and condi-
tions: 1a (1.0 mmol, 1.0 equiv), 2a–n (1.2 mmol, 1.2 equiv), EDC·HCl
(3.0 mmol, 3.0 equiv), DMAP (0.20 mmol, 0.20 equiv), and solvent (10
mL) for 12 h at r.t. a Large-scale (0.10 mol) reaction.
To explore the mechanism of this transformation, some
control experiments were performed (Scheme 6). Based on
the results obtained above, N-protected L-asparagin reacted
with benzyl alcohol to offer the benzyl β-cyano-L-alanine
ester when the standard reaction conditions (EDC·HCl/
DMAP) was used, no matter what the protecting group was.
According to these results, we speculated that the amino
group in α-position of carboxylic acid is unnecessary for the
transformation of carboxamides into the corresponding cy-
ano group. Based on this speculation, the reaction of succi-
namic acid (7) and 2a using the standard reaction condi-
tions (EDC·HCl/DMAP) was performed (Scheme 5, eq. 1),
and this reaction afforded the benzyl 3-cyanopanoate (8a)
as the only product in a 95% yield. Conversely, using
DCC/DMAP as the reaction conditions, this reaction could
deliver benzyl succinamate (8b) as the only product in a
92% yield (Scheme 5, eq. 1). In addition, for the reaction of
N-Boc-L-glutamine (9) with 2a under the standard reaction
conditions, a 55% yield of benzyl 4-cyano-2-[(1,1-dimethy-
lethoxy)carbonyl]aminobutyrate (10a) and a 40% yield of
benzyl N-Boc-L-glutamine ester (10b) were generated
(Scheme 5, eq. 2). Notably, benzyl N-Boc-L-asparagin ester
(3a) could not be converted into its corresponding benzyl
β-cyano-L-alanine ester (4a) with the treatment of EDC·HCl/
DMAP (Scheme 5, eq. 3). It is expected that the acid group
in β-position of carboxamide should play an important role
in the transformation of carboxamides into the correspond-
ing cyano group. However, we cannot draw a plausible
NHPGO
NHPG
EDC.HCl, DMAP
CH2Cl2, r.t., 12 h
HO
BnO
2a
+
NH2
N
O
O
1b–c
5a–b
NHFmoc
NHCbz
BnO
BnO
N
N
O
O
5b, 90%
5a, 95%
Scheme 4 One-pot synthesis of N-Cbz-β-cyano-L-alanine benzyl ester
5a and N-Fmoc-β-cyano-L-alanine benzyl ester 5b from N-Cbz-L-aspar-
agin 1b and N-Fmoc-L-asparagin 1c, respectively. Isolated yields are giv-
en. Reagents and conditions: 1b or 1c (1.0 mmol, 1.0 equiv), 2a (1.2
mmol, 1.2 equiv), EDC·HCl (3.0 mmol, 3.0 equiv), DMAP (0.20 mmol,
0.20 equiv), and solvent (10 mL) for 12 h at r.t.
amides, such as aromatic carboxamides 6a and aliphatic
carboxamides 6b, were treated using the standard reaction
conditions. Unfortunately, these carboxamide groups in 6a
and 6b could not be converted into the corresponding cya-
no group, and the starting materials were recovered
(Scheme 5). Similar to the aromatic carboxamides 6a and
6b, N-Boc-L-prolinamide (6c) and N-Boc-L-pyroglutamyl-
amide (6d) could not be converted into the corresponding
© Georg Thieme Verlag Stuttgart · New York — Synlett 2016, 27, 309–312

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G. Wang et al.
Letter
Synlett
mechanism to this transformation. In addition, we cannot
give a reasonable explanation to illustrate the difference be-
tween EDC·HCl and DCC in this transformation process.
References and Notes
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BnO
N
O
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O
O
dehydrating agent
CH2Cl2, r.t., 12 h
HO
8a
+
(1)
NH2
+
BnOH
O
O
BnO
7
2a
NH2
8b
dehydrating agent = EDC.HCl DMAP, 8a, 95%; 8b, 0%
dehydrating agent = DCC DMAP, 8a, 0%; 8b, 95%
NHBoc
BnO
N
NHBoc
O
O
10a
+
(5) (a) Ressler, C.; Giza, Y.-H.; Nigam, S. N. J. Am. Chem. Soc. 1963,
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dehydrating agent
CH2Cl2, r.t., 12 h
HO
NH2
(2)
+
BnOH
NHBoc
O
O
BnO
NH2
(6) Warriow, A. G. S.; Hawkerford, M. J. J. Exp. Bot. 1998, 49, 1625.
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(10) General Procedures for the Synthesis of β-Cyano-L-Alanine
Esters and Amides
9
2a
O
10b
dehydrating agent = EDC.HCl DMAP, 10a, 55%; 10b, 40%
dehydrating agent = DCC DMAP, 10a, 50%; 10b, 40%
BocHN
BnO
O
NHBoc
EDC.HCl/DMAP
CH2Cl2, r.t., 12 h
BnO
(3)
N
NH2
O
O
4a, 0%
3a
Scheme 6 Control experiments
In summary, we have developed a novel, mild, efficient,
and scalable protocol for the synthesis of N-protected β-cy-
ano-L-alanine derivatives from N-protected L-asparagin.
This protocol avoided the use of toxic or unpleasant re-
agents and was easy to operate in laboratory. Hence, this
protocol may serve as an attractive alternative for the
preparation of β-cyano-L-alanine ester or amide from L-as-
paragin to the existing methods. In addition, we have
demonstrated for the first time that EDC·HCl and DCC are
different during the formation of an ester bond involving
L-asparagin, though we knew that EDC·HCl and DCC as de-
hydrate reagents can replace each other in peptide synthe-
sis. Further studies to elucidate this specific transformation
mechanism are currently under way in our laboratory.
To a stirred solution of N-protected L-asparagin 1a–c (0.10
mmol) and alcohol or amine 2a–n (0.12 mmol) in CH2Cl2 (10
mL), EDC·HCl (0.30 mmol), and DMAP (0.02 mmol) were added
at 0 °C. This reaction mixture was stirred for 12 h at r.t. Then the
reaction mixture was poured in H2O (10 mL) and extracted with
CH2Cl2 (20 mL). The organic phase was separated, dried, and
subjected to flash column chromatography (EtOAc–hexane, 1:
4) to afford the desired compounds as white solids.
Acknowledgment
(11) Selected Spectral Data for Compound Benzyl 2-[(tert-Butoxy-
carbonyl)amino]-3-cyanopropanoate (4a)
The work was supported financially by the National Natural Science
Foundation of China (Grants 81161120401) to Guoxin Wang. We also
thank the Shenzhen Science and Technology Plan Project (Grant No.
ZDSY20130331145112855) for funding.
Yield: 288 mg (95%); white crystals, mp 131–133 °C. 1H NMR
(300 MHz, CDCl3): δ = 7.45–7.35 (m, 5 H, ArH), 5.55 (d, J = 6.6
Hz, 1 H, NH), 5.26 (s, 2 H, CH2), 4.55–4.51 (m, 1 H, COCH), 3.00–
2.89 (m, 2 H, CH2CN), 1.47 [s, 9 H, OC(CH3)3]. 13C NMR (75 MHz,
CDCl3): δ = 169.13, 155.05, 134.74, 116.54, 81.06, 68.40, 50.53,
Supporting Information
28.36, 21.78. ESI-HRMS: m/z [M + Na]+ calcd for C16H20N2O4
+
Na: 327.1321; found: 327.1309.
Supporting information for this article is available online at
http://dx.doi.org/10.1055/s-0035-1560489.
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u
p
p
ortiInfogrmoaitn
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u
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ortioInfgrmoaitn
© Georg Thieme Verlag Stuttgart · New York — Synlett 2016, 27, 309–312