Enzyme-catalyzed cascade synthesis of hydroxyiminoacetamides

Article abstract of DOI:10.1016/j.tetlet.2014.06.027

In order to synthesize N-(3-azido-1-phenylpropyl)-2-hydroxyiminoacetamide, a key compound for the preparation of acetylcholinesterase (AChE) reactivators of the N-substituted 2-hydroxyiminoacetamide type, it was necessary to develop a method for forming a

Full text of DOI:10.1016/j.tetlet.2014.06.027

Tetrahedron Letters 55 (2014) 4338–4341
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Tetrahedron Letters
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Enzyme-catalyzed cascade synthesis of hydroxyiminoacetamides
a,
a
b
b
⇑
ˇ
´
´
´
Anamarija Knezevic , Vladimir Vinkovic , Nikola Marakovic , Goran Šinko
a
-
ˇ
Division of Organic Chemistry and Biochemistry, Ruder Boškovi´c Institute, Bijenicka 54, HR-10000 Zagreb, Croatia
b Biochemistry and Organic Analytical Chemistry Unit, Institute for Medical Research and Occupational Health, P.O. Box 291, HR-10000 Zagreb, Croatia
a r t i c l e i n f o
a b s t r a c t
Article history:
In order to synthesize N-(3-azido-1-phenylpropyl)-2-hydroxyiminoacetamide, a key compound for the
preparation of acetylcholinesterase (AChE) reactivators of the N-substituted 2-hydroxyiminoacetamide
type, it was necessary to develop a method for forming an amide bond between an ethyl glyoxylate oxime
and an amine. Using Candida antarctica lipase B (CAL-B) in a cascade enzyme-BOP catalyzed reaction, the
efficient synthesis of the target hydroxyiminoacetamide was achieved.
Received 10 April 2014
Revised 20 May 2014
Accepted 5 June 2014
Available online 14 June 2014
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Amide formation
Chemoenzymatic synthesis
Coupling reagents
Enzyme catalysis
Oxime protecting groups
Organophosphorus (OP) nerve agents are extremely toxic
compounds used as chemical warfare agents in armed conflicts
and terrorist attacks1 and as pest control agents.2 The acute toxic-
ity of these compounds is due to their irreversible inhibition of
acetylcholinesterase (AChE; EC 3.1.1.7).3 Current therapy in cases
of OP nerve agent poisoning includes an antimuscarinic drug
(e.g., atropine), an anticonvulsant drug (e.g., diazepam), and an
AChE reactivator from the quaternary pyridinium oxime family
(2-PAM, trimedoxime, obidoxime, HI-6, Hlö-7).4 However, current
therapy directed at the reactivation of inhibited AChE is limited to
peripheral circulation because commonly used quaternary pyridi-
nium oximes do not cross the blood–brain barrier due to their
permanent positive charge.5 In order to achieve an efficient reacti-
vation of central nervous system AChE, attempts have been made
to develop efficient uncharged reactivators.6 Recently, a new series
of AChE reactivators, including N-substituted 2-hydroxyiminoace-
tamides, were reported.7 A few N-substituted 2-hydroxyiminoace-
tamides have shown high reactivation potential toward sarin,
cyclosarin, and VX inhibited AChE.8 Because of the significant role
of non-bonding interactions between the quaternary pyridinium
ring of 2-PAM or HI-6 and the surrounding aromatic amino acids
in the AChE active site in the overall stabilization of these com-
pounds, it is reasonable to assume that the introduction of a phenyl
ring into the structure of N-substituted 2-hydroxyiminoacetamides
would help their stabilization and possibly improve the geometry
of the oxime group access to phosphylated serine. Thus, the aim
of our work was to synthesize several structurally diverse aromatic
N-substituted 2-hydroxyiminoacetamides 2a–c (Fig. 1).
The synthesis of key compound 1, [N-(3-azido-1-phenylpropyl)-
2-hydroxyiminoacetamide], from which the target molecules 2a–c
could be prepared by the well-known copper catalyzed azide–
alkyne cycloaddition,9 started from readily available cinnamyl
alcohol (3) (Scheme 1).
1-Phenyl-allylamine (4) was prepared from alcohol 3 according
to a procedure described in the literature10 and an azide group was
then introduced via a three-step process. The final reaction was the
formation of an amide bond between 3-azido-1-phenylpropyl-
amine (5) and ethyl glyoxylate oxime, which was previously
⇑
Corresponding author. Tel.: +385 1 456 1029; fax: +385 14571300.
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E-mail address: Anamarija.Knezevic@irb.hr (A. Knezevic).
Figure 1. Targeted aromatic N-substituted 2-hydroxyiminoacetamides.
http://dx.doi.org/10.1016/j.tetlet.2014.06.027
0040-4039/Ó 2014 Elsevier Ltd. All rights reserved.

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A. Knezevic et al. / Tetrahedron Letters 55 (2014) 4338–4341
Table 1
Testing different conditions for the synthesis of compound 1
Scheme 1. Retrosynthetic pathway toward 1.
Solvent
Base
T (°C)
t (h)
Conversiona (%)
described for similar compounds.7 However, as in numerous other
cases, amide bond formation turned out to represent a significant
challenge.11
The preparation of 1-phenyl-allylamine (4) from cinnamyl
alcohol (3) includes an Overman reaction, which has been
well-described in the literature.12 The hydroxyl group was intro-
duced to Boc protected 1-phenyl-allylamine (6) via hydrobora-
tion–oxidation using 9-BBN as the hydroboration reagent
(Scheme 2).
The hydroxyl group in 7 was replaced with an azide using
diphenylphosphoryl azide (DPPA) as the azide source.13 The
reaction was performed in dry toluene with 1,8-diazabicycloun-
dec-7-ene (DBU) as the base. The first step of the reaction, which
was performed at room temperature, was the formation of the
diphenylphosphate, which can easily be isolated, and the azide salt
of DBU. By raising the temperature, nucleophilic substitution of the
diphenylphosphate group with azide occurs. The resulting
Boc-protected 3-azido-1-phenyl-propylamine (8) was difficult to
purify from excess DPPA, so the crude residue was deprotected
with trifluoroacetic acid (TFA) in dichloromethane to afford pure
3-azido-1-phenylpropylamine (5).
The remaining step for the synthesis of 1 was assumed to follow
the same path as the synthesis of structurally similar reported
compounds RS41A and RS194B.7,14 The previously described con-
densation of ethyl glyoxylate with hydroxylamine provided ethyl
glyoxylate oxime (9).15 The formation of an amide between ethyl
glyoxylate oxime and an amine was reported in EtOH at 50 °C.
However, in our case, reaction at 50 °C, as well as in boiling EtOH,
did not produce the desired product. Addition of Et3N as a base
provided a somewhat poor conversion. The search for a solvent
and base that would result in a satisfactory conversion of the reac-
tants into the desired product was unsuccessful.
EtOH
EtOH
—
Et3N
Et3N
Reflux
Reflux
100
80
80
80
100
110
100
24
72
24
24
24
24
48
48
48
2
0
26
0
0
0
0
0
8
0
1,4-Dioxane
1,4-Dioxane
1,4-Dioxane
1,4-Dioxane
Diglyme
Diglyme
DMSO
THF
—
—
DIPEA
1,2,4-Triazole/DBU
DMAP
DIPEA
DIPEA
DIPEA
KOt-Bu
—
rt
0
17
18
MW-110
MW-95
0.5
0.5
DIPEA
a
Conversion of the product was determined using HPLC with UV detection at
220 nm.
our reaction. CAL-B is a very selective biocatalyst in enzymatic res-
olution of primary amines, as well as an excellent tool for amide
bond formation.18 However, the test enzymatic reaction of amine
5 and ester 9 using CAL-B did not proceed as desired. The target
product was obtained, but in very low yield. We first assumed that
these poor results were the consequence of the somewhat ambig-
uous placement of ester 9 in the active site of the enzyme due to its
oxime group. In fact, oximes are used as nucleophiles in enzyme-
catalyzed oximolysis for the preparation of oxime esters.19
Although these activated oxime esters are known to react with
amines to give amides,20 the success of amide formation depends
on the structures of the amines and oxime esters.21
Therefore, to exclude the possibility of other side reactions, the
oxime group was protected using a 2-methoxyethoxymethyl ether
(MEM) protecting group (Scheme 3), starting from ethyl glyoxylate
(12). Next, the reaction of the obtained ester 10 with amine 5 was
examined. As was the case with unprotected oxime 9, the desired
product was obtained in low yield using the classic [EtOH,
N,N-diisopropylethylamine (DIPEA), reflux for 48 h, 16% yield], as
well as the enzymatic approach (Table 2). However, some solid
was isolated which was insoluble in methyl tert-butyl ether
(MTBE) and diisopropyl ether (DIPE), but was very soluble in
dichloromethane. NMR and MS analysis of the solid showed that
this substance (13, see Scheme 4) was actually the salt of amine
5 and hydrolyzed ester 10.
Microwave-assisted reactions, which are known to be very fast
and straightforward, also proved unsuccessful.16,17 MW irradiation
of 5 and 9, with or without an additional base, for 30 min under
solvent-free conditions did not demonstrate enhanced conversion
compared to reactions in boiling EtOH. Longer reaction times did
not increase the conversion, however new peaks appeared in the
HPLC chromatogram indicating the formation of by-products. The
results of several attempts to carry out this reaction are presented
in Table 1.
Considering the results listed in Table 2, it was clear that hydro-
lysis of ester 10 catalyzed by the enzyme was faster than the
expected amide bond formation reaction. It is generally known
that during enzyme-catalyzed aminolysis, even traces of water
cause hydrolysis of the acyl donor, which is why such reactions
must be performed in dry solvents.18,23 DIPE is a solvent which
probably contains a sufficient amount of water to prevent amide
formation. Hence, salt 13 was isolated in almost quantitative yield
(96%), indicating that under these reaction conditions complete
Bearing in mind our previous experience with Candida
antarctica lipase B (CAL-B),10 we decided to test this enzyme for
Scheme 2. Synthesis of 3-azido-1-phenylpropylamine (5).
Scheme 3. Synthesis of the MEM-protected oxime 10.

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A. Knezevic et al. / Tetrahedron Letters 55 (2014) 4338–4341
4340
Table 2
catalyzed the hydrolysis of ester 10 which produced salt 13, and
Attempts at preparing compound 1 using CAL-B
then the resulting salt was converted into amide 11 using the
BOP/Et3N catalyzed reaction. The reaction yield was similar to that
obtained when the salt was first isolated from the reaction mixture
and then subjected to BOP coupling in DMF. The desired amide 1
was now easily obtained from amide 11 by removing the MEM
protecting group under mild reaction conditions using ZnBr2.26,27
In conclusion, we have described the synthesis of hydrox-
yiminoacetamide 1 via a cascade enzyme-BOP catalyzed reaction.
After several unsuccessful attempts in forming an amide bond
between ethyl glyoxylate oxime and 3-azido-1-phenylpropyl-
amine, the problem was solved in three steps. The oxime group
was protected using the 2-methoxyethoxymethyl ether (MEM)
protecting group. The obtained MEM-protected oxime ester was
hydrolyzed using Candida antarctica lipase B (CAL-B) in an organic
solvent and then coupled with an amine using BOP as the coupling
reagent in a one-pot cascade reaction. After removing the MEM
protecting group under mild conditions, the target hydrox-
yiminoacetamide 1 was obtained.
Ester
Solvent
T (°C)
t (h)
Yield of 1 or 11 (%)
9
9
9
10
10
10
DIPE
40
30
40
40
40
45
48
24
24
72
48
48
0
9
2
31
23
0b
MTBE
MTBE
MTBE
MTBEa
DIPE
a
Et3N was added to the reaction mixure.22
Salt 13 (see Scheme 4) was isolated from the reaction mixture (96%).
b
Acknowledgments
This study was supported by the Ministry of Science, Education
and Sports, Republic of Croatia (Grant 098-0982904-2910) and
Croatian Science Foundation—Research Project ‘Design, synthesis
and evaluation of new antidotes in nerve agents and pesticides
poisoning’ (PI: Z. Kovarik).
Scheme 4. Formation of amide 11 from salt 13.
Supplementary data
hydrolysis of the MEM-protected ester 10 occurred. Furthermore,
ammoniolysis catalyzed by Novozym 435 (CAL-B immobilized on
macroporous acrylic resin) has been reported to sometimes be
accompanied by unexpected hydrolysis of the ester in spite of
the rigorous exclusion of moisture.23 It is assumed that these
results are the consequence of acrylic carrier adsorption of traces
of water flushed out by ammonia. Since we also used Novozym
435, it is reasonable to assume that this problem is also present.
The reaction yield of amide 11 could be enhanced by using a
very large excess of ester that would ensure an ample amount of
acyl donor for amide formation. However, ester 10 is not commer-
cially available and has to be synthesized, so this approach was not
suitable. Considering the above-mentioned facts and since we
needed amide 1 in racemic form and higher yield if possible, we
decided to try an alternative approach.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2014.06.
027. These data include MOL files and InChiKeys of the most
important compounds described in this article.
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Scheme 5. Synthesis of hydroxyiminoacetamide 1 from ester 10 and amine 5.

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followed by Et3N (86 lL, 0.61 mmol) and immobilized lipase B from C.
antarctica (200 mg). The mixture was stirred at 45 °C for 6 h. Next, BOP
(267 mg, 0.60 mmol) was added and the mixture was stirred at the same
temperature for another 2 h. The enzyme was filtered off and washed with
DIPE and CH2Cl2. The filtrate was extracted with saturated aqueous NH4Cl
solution, dried over Na2SO4, filtered and evaporated under reduced pressure.
The crude residue was purified by silica gel column chromatography (hexane–
EtOAc, 1:1, Rf = 0.21) to give a clear oil (125 mg, 63%).
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27. MEM-amide deprotection: MEM-protected amide 11 (68 mg, 0.20 mmol) was
dissolved in a mixture of CH2Cl2 (2 mL) and MeOH (0.4 mL). ZnBr2 (oven-dried
at 200 °C overnight) was added (400 mg, 1.8 mmol) to the solution. The
mixture was stirred at 35 °C until completion of the reaction (monitored by
HPLC). The mixture was diluted with CH2Cl2 (5 mL) and extracted with
saturated aqueous NaHCO3 solution, brine and water, dried over Na2SO4,
filtered, and evaporated under reduced pressure. Amide 1 was obtained as a
white solid (46 mg, 92%).