Efficient enzymatic amine resolution at high substrate input using diethyl malonate as an acyl donor of low hazard potential

Article abstract of DOI:10.5560/ZNB.2012-0127

Diethyl malonate turned out to be both a green and highly efficient acyl donor in the lipase-catalyzed resolution of amines, thus representing an attractive alternative to currently applied acyl donors. By means of this acyl donor a highly efficient enzymatic process for the resolution of amines, running at high substrate input of up to 200 g/L in an organic solvent classified as usable according to the Pfizer Solvent Selection Guide, is presented.

Full text of DOI:10.5560/ZNB.2012-0127

Note
1123
Efficient Enzymatic Amine Resolution
at High Substrate Input Using Diethyl
Malonate as an Acyl Donor of Low
Hazard Potential
quires the use of enantiomerically pure amines as
building blocks [1]. In order to provide enantiomer-
ically pure amines as drug intermediates in an eco-
nomically attractive fashion, the development of effi-
cient methodologies for their enantioselective synthe-
sis is a key task for organic chemists. Among exist-
ing synthetic approaches, comprising classic chemical
resolution via diastereomeric salts as well as asym-
metric chemo- and biocatalysis, lipase-catalyzed res-
olution of amines developed at BASF certainly ranks
among the top processes for chiral amine synthesis
in terms of both synthetic efficiency and technical at-
tractiveness [2 – 4]. This is underlined by the impres-
sive production volume of this technology at BASF
being in the multi-thousand tons range [4]. Key fea-
ture of this outstanding BASF process technology is
the use of an α-heteroatom-substituted acetate such as
a methoxyacetate (1) as an acyl donor for the enan-
tioselective acylation of (racemic) amines as substrates
(Scheme 1) [2 – 7].
What makes this process additionally attractive
from the perspective of sustainability is the opportunity
to recycle the unwanted enantiomer through racemiza-
tion. To further improve the sustainability of the res-
olution process, the search for alternative acyl donors
showing a low hazard potential (which ideally means
being listed as “no hazardous product”) and at the
same time excellent reactivity is desirable. For compar-
ison, ethyl methoxyacetate is classified as flammable,
and the corresponding methoxyacetic acid is listed as
a toxic compound [8, 9]. On the other hand, ethyl ac-
etate [10] (and related non-activated aliphatic esters)
show a tremendous decrease of reactivity compared to
methoxyacetate [2 – 6]. Thus, identifying acyl donors,
which are highly reactive acylating agents and attrac-
tive from both a hazard potential and economical per-
Sabine Simona, Steffen Oßwaldb, Ju¨rgen Roosb, and
Harald Gro¨gera,c
a
Department of Chemistry and Pharmacy, University
of Erlangen-Nu¨rnberg, Henkestr. 42, 91054 Erlangen,
Germany
Evonik Industries AG, Health & Nutrition,
b
Rodenbacher Chaussee 4, 63457 Hanau-Wolfgang,
Germany
Current address: Faculty of Chemistry, Bielefeld
c
University, Universita¨tsstr. 25, 33615 Bielefeld,
Germany
Reprint requests to Prof. Dr. Harald Gro¨ger. Fax: +49 521
1066146. E-mail: harald.groeger@uni-bielefeld.de
Z. Naturforsch. 2012, 67b, 1123 – 1126
DOI: 10.5560/ZNB.2012-0127
Received May 17, 2012
Dedicated to Professor Dr. Dr. Heribert Offermanns on the
occasion of his 75th birthday
Diethyl malonate turned out to be both a “green” and
highly efficient acyl donor in the lipase-catalyzed resolu-
tion of amines, thus representing an attractive alternative to
currently applied acyl donors. By means of this acyl donor
a highly efficient enzymatic process for the resolution of
amines, running at high substrate input of up to 200 g/L in an
organic solvent classified as “usable” according to the Pfizer
Solvent Selection Guide, is presented.
Key words: Amines, Amides, Enantioselective Synthesis,
Biocatalysis, Enzymatic Resolution
Introduction
A large percentage of today’s drugs contain chi- spective, still represents a challenging task. In our at-
ral amine frameworks, and their synthesis often re- tempts addressing this issue we became interested in
O
OMe
NH2
rac
NH2
Me
HN
O
lipase
+
Me O
+
R
Me
R
R
Me
OR´
1
Scheme 1. Industrial process technology at BASF for amine resolution.
c
ꢀ 2012 Verlag der Zeitschrift fu¨r Naturforschung, Tu¨bingen · http://znaturforsch.com

1124
Note
an evaluation of diethyl malonate as an acyl donor due ethyl malonate is a “green” and highly efficient acyl
to the following features which appear to make this donor in the lipase-catalyzed resolution of amines, thus
compound promising: (i) cheap and readily available representing an attractive alternative to currently ap-
chemical produced on bulk scale, (ii) (relatively) low plied acyl donors. In addition, based on this sustain-
hazard potential (classification as no dangerous mate- able donor we report a highly efficient process running
rial according to the MSDS of Merck Chemicals; how- at high substrate concentration of 1 M in an organic sol-
ever, in the MSDS of Sigma-Aldrich this compound is vent classified as usable according to the Pfizer Solvent
classified as irritating to eyes) [11], and (iii) an acti- Selection Guide [15].
vating moiety as a substituent for the carbon atom in
α-position, namely a carboxylate ester moiety, which Results and Discussion
could result in an increased reactivity.
So far malonates are rarely known as acyl donors
At first we chose the lipase-catalyzed acylation of
in enzymatic amine resolution, and only dimethyl mal- racemic (1-phenylethyl)amine (rac-4) as a model reac-
onate was used in a few earlier experimental studies tion for our work. Initial reaction conditions were cho-
with bifunctionalized compounds as substrates such sen according to a recent process by Gastaldi and Gil
as diamines and amino alcohols [12 – 14]. In the fol- et al. for enzymatic resolution of various amines [6],
lowing, we report our results demonstrating that di- which proceeds efficiently with even non-activated
Table 1. Resolution of (1-phenylethyl)amine (rac-4) with ethyl acetate (2) and diethyl malonate (3) as acyl donors.
NH2
O
Ph
(S)-4
Me
lipase
Me
O
OEt
O
NH2
rac
CAL-B
2
or
+
+
Ph
Me
n-heptane,
80°C, t
O
O
O
rac-4
EtO
OEt
HN
Me
HN
OEt
3
or
Ph
Me
(R)-5
Ph
Me
(R)-6
Entrya
Donor
c (M)b CAL-B
t (h)
conv. (%)d ee 5 / 6 (%)e Ef
loadingc
1
2
3
4
5
6
7
2
2
2
3
3
3
3
0.1
0.1
0.1
0.1
0.1
0.1
1.0
200
200
50
200
200
40
4.5
2
4.5
4.5
2
50
32
33
50
47
45
46
98 (R)
98 (R)
99 (R)
97 (R)
96 (R)
96 (R)
97 (R)
>100
>100
>100
>100
>100
>100
>100
4.5
19
10
a
General biotransformation protocol: After dissolving rac-(1-phenylethyl)amine (rac-4,
0.1 – 1.0 M) in n-heptane, acyl donor 2 or 3 (1 equiv.) and lipase CAL-B (Novozym 435;
10 – 200 mg mmol−1) are added, and the mixture is stirred at 80 ◦C for a reaction time of
2 – 19 h. Then, the mixture is cooled, and the enzyme immobilisate is filtered and washed
with methylene chloride. After removing the volatile components in vacuo the conversion is
determined from the resulting crude product. After work-up (consisting of acidification with
HCl and subsequent extraction with methylene chloride for separation of the amide from the
amine) the enantiomeric excess of amide (R)-5 or (R)-6 is determined; b concentration of
substrate rac-4; c the catalyst loading is given in mg of lipase CAL-B per mmol of substrate
rac-4; d the conversion is determined via 1H NMR spectroscopy; e the enantiomeric excess
is determined via chiral HPLC (column: Daicel Chiralpak AD-H; eluent: isohexane : iso-
propanol (95:5 (v/v)); 210 nm; flow: 0.8 mL min−1); f E-value determined from conversion
and ee-value of amide (R)-5 or (R)-6.

Note
1125
NH2
Me
lipase CAL-B
(10 mg mmol−1),
MTBE, Δ, 19 h
Br
NH2
rac
(S)-7
+
Me
O
O
O
O
Br
EtO
OEt
HN
OEt
rac-7
3
substrate input
200 g L−1
(1 M)
(1 equiv.)
Me
Br
(R)-8
47 % conversion
96 % ee
E > 100
Scheme 2. Enzymatic amine resolution with diethyl malonate in MTBE.
acyl donors such as ethyl acetate (2) in heptane at ele- mization with respect to an increased volumetric pro-
vated temperature (80 ◦C). When we used – in analogy ductivity at decreased catalyst demand. After a reac-
to those reaction conditions −12 g L−1 (100 mM) of tion time of 19 h even at an elevated substrate load-
racemic (1-phenylethyl)amine (rac-4) in combination ing of 121 g L−1 of rac-4 the desired amide (R)-6 was
with ethyl acetate (2) as an acyl donor in n-heptane at formed with 46% conversion and 97% ee, correspond-
80 ◦C, a conversion of 50% and 98% ee of the formed ing to a high enantioselectivity of the process with an
amide (R)-5 was obtained after 4.5 h (Table 1, entry 1). E-value [16] of > 100 (entry 7). Thus, compared to
At a decreased reaction time of 2 h, however, the bio- ethyl acetate (2), a dramatic improvement of reactiv-
transformation was incomplete with 32% (entry 2). In ity has been observed.
addition, lowering the catalyst loading by a factor of 4
Next, this type of enzymatic resolution process
led to a decrease of the conversion to 33% after 4.5 h, was further studied, varying both substrate and sol-
indicating that ethyl acetate (2) is a less efficient acyl vent component. As a substrate racemic (1-(4’-
donor (entry 3).
bromophenyl)ethyl)amine (rac-7) was used, and as
Taking these biotransformations as a “benchmark” a solvent we evaluated MTBE, which appeared to be
we next studied the acylation with diethyl malonate (3) interesting in terms of both amine substrate and amide
as an acyl donor, and we were pleased to find a sig- product solubility. When running the resolution with
nificantly increased reactivity while maintaining ex- diethyl malonate (3) as acyl donor under reflux con-
cellent enantioselectivity (Table 1, entries 4 – 6). After ditions (55 ◦C), with a lower catalyst loading of only
having demonstrated as a proof of concept the suitabil- 10 mg mmol−1 and at a high substrate input of 200 g
ity of diethyl malonate (3) in an initial “model reac- L−1 (1 M), the desired amide (R)-8 was formed with
tion” (entry 4), in further experiments at shorter reac- high conversion of 47% and 96% ee (Scheme 2). This
tion time or lower catalyst loading significantly im- corresponds to a high enantioselectivity with an E-
proved reaction rates compared to the analogous re- value of > 100, and taken together with the high re-
actions with ethyl acetate (2) were found. After a re- action rate it demonstrates that diethyl malonate is an
action time of 2 h high conversion (47%) and enan- excellent acyl donor suitable for use also in a solvent
tiomeric excess (96% ee) were obtained (entry 5), acceptable from the perspective of sustainability.
compared to only 32% when using ethyl acetate (en-
try 2). Furthermore, and in contrast to ethyl acetate, Conclusion
a lower catalyst loading led to still a high conversion
(entries 3, 6). After obtaining these encouraging re-
In summary, diethyl malonate (3) has been demon-
sults this resolution process has been subject to opti- strated to be both a “green” and highly efficient acyl

1126
Note
donor in the lipase-catalyzed resolution of amines, in an organic solvent classified as “usable” according
thus representing an attractive sustainable alternative to the Pfizer Solvent Selection Guide has been devel-
to currently applied acyl donors. By means of this oped. Future work will also address the expansion of
bulk chemical of low hazard potential as an acyl donor this protocol to the enantioselective synthesis of other
a highly efficient process for enzymatic amine resolu- amines in highly enantiomerically enriched form as
tion running at high substrate input of up to 200 g L−1 well as further process development.
[1] Overview: A. Kleemann, J. Engel, B. Kutscher, D. Rei- [10] The risk phrases and hazard symbols of ethyl ac-
chert, Pharmaceutical Substances, 5th ed., Thieme,
Stuttgart, 2009.
[2] F. Balkenhohl, K. Ditrich, B. Hauer, W. Ladner, J.
Prakt. Chem. 1997, 339, 381 – 384.
[3] K. Ditrich, Synthesis 2008, 2283 – 2287.
[4] Review: M. Breuer, K. Ditrich, T. Habicher, B. Hauer,
etate are as follows: R11 – 36-66 – 67 and hazard sym-
bols “F” and “Xi” according to the MSDS data sheet
of Sigma-Aldrich, Germany, and R11 – 36 and haz-
ard symbols “F” and “Xi” according to the MSDS
data sheet of Merck Chemicals, Germany (as stated on
March 8, 2012).
M. Keßeler, R. Stu¨rmer, T. Zelinski, Angew. Chem. Int. [11] The risk phrases and hazard symbols of diethyl mal-
Ed. 2004, 43, 788 – 824.
onate are as follows: R36 and hazard symbol “Xi” ac-
cording to the MSDS data sheet of Sigma-Aldrich, Ger-
many (as stated on March 8, 2012), and no risk phrase,
none hazard symbol and listed as “no hazardous prod-
uct” (as specified in the Directive 67/548/EEC) accord-
ing to the MSDS data sheet of Merck Chemicals, Ger-
many (as stated on March 8, 2012).
[5] Work of other authors with CALB and ethyl
methoxyacetate as an acyl donor in MTBE: I. I. Gill,
J. Das, R. N. Patel, Tetrahedron: Asymmetry 2007, 18,
1330 – 1337.
[6] Work of other authors with CALB and ethyl
methoxyacetate as well as other acyl donors at ele-
vated temperature: M. Nechab, N. Azzi, N. Vanthuyne, [12] V. Gotor-Ferna´ndez, R. Brieva, V. Gotor, J. Mol. Catal.
M. Bertrand, S. Gastaldi, G. Gil, J. Org. Chem. 2007,
72, 6918 – 6923.
[7] Overview of the different enzymatic approaches to-
B: Enzym. 2006, 40, 111 – 120.
[13] A. Luna, I. Alfonso, V. Gotor, Org. Lett. 2002, 4,
3627 – 3629.
wards chiral amines, see: S. Buchholz, H. Gro¨ger in [14] I. Alfonso, C. Astorga, F. Rebolledo, V. Gotor, Chem.
Biocatalysis in the Pharmaceutical and Biotechnology Commun. 1996, 2471 – 2472.
Industries (Ed.: R. N. Patel), CRC Press, New York, [15] Pfizer Solvent Selection Guide: K. Alfonsi, J. Col-
2006, chapter 34, pp. 829 – 847.
[8] The risk phrases and hazard symbols of ethyl methoxy-
berg, P. J. Dunn, T. Fevig, S. Jennings, T. A. John-
son, H. P. Kleine, C. Knight, M. A. Nagy, D. A. Perry,
M. Stefaniak, Green Chem. 2008, 10, 31 – 36.
acetate are as follows: R10 and none hazard symbol
according to the MSDS data sheet of Sigma-Aldrich, [16] The E-value describes the preference (selectivity)
Germany (as stated on March 8, 2012).
[9] The risk phrases and hazard symbols of methoxyacetic
acid are as follows: R60 – 61-22 – 34 and symbol “T”
according to the MSDS data sheets of Sigma-Aldrich,
Germany, and Merck Chemicals, Germany (as stated
on March 8, 2012).
of a resolution process for one enantiomer over the
other, thus indicating the enantioselectivity of this
process; for details and formula for its calculation, see:
K. Faber, Biotransformation in Organic Chemistry, 5th
ed., Springer, Heidelberg, 2004.