Efficient asymmetric hydrogenation of the C-C double bond of 2-methyl- and 2,3-dimethyl-N-phenylalkylmaleimides by Aspergillus fumigatus

Article abstract of DOI:10.1016/j.tetasy.2010.02.017

Eight N-phenylalkylmaleimides (four 2-methyl-N-phenylalkylmaleimides and four 2,3-dimethyl-N-phenylalkylmaleimides with an alkyl chain (CH₂)_n (n = 1-4) between the imide N and the benzene ring) were subjected to biotransformation usi

Full text of DOI:10.1016/j.tetasy.2010.02.017

Tetrahedron: Asymmetry 21 (2010) 535–539
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Efficient asymmetric hydrogenation of the C–C double bond of 2-methyl-
and 2,3-dimethyl-N-phenylalkylmaleimides by Aspergillus fumigatus
*
Maximiliano Sortino, Susana Alicia Zacchino
Pharmacognosy, Faculty of Biochemical and Pharmaceutical Sciences, National University of Rosario, Suipacha 531, Rosario 2000, Argentina
a r t i c l e i n f o
a b s t r a c t
Article history:
Eight N-phenylalkylmaleimides (four 2-methyl-N-phenylalkylmaleimides and four 2,3-dimethyl-N-
phenylalkylmaleimides with an alkyl chain (CH2)n (n = 1–4) between the imide N and the benzene ring)
were subjected to biotransformation using the fungal strain Aspergillus fumigatus ATCC 26934. All com-
pounds were reduced enantioselectively to their respective succinimides: (R)-2-methyl-N-phenylalkyl-
succinimides and (2R,3R)-2,3-dimethyl-N-phenylalkylsuccinimides, with satisfactory conversion rates
and high stereoisomeric excesses. NMR analysis using the chiral shift reagent Eu(hfc)3 showed that enan-
tiomeric excesses were >99%.
Received 25 January 2010
Accepted 19 February 2010
Available online 10 April 2010
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
stereogenic centers into an achiral structure with high enantiose-
lectivity. Culture plant cells of N. tabacum, Cinechococcus sp., and
Biocatalysis has become an increasingly valuable tool for the
easy preparation of chiral compounds, which are greatly needed
in organic synthesis as well as in the pharmaceutical and agro-
chemical fields, due to the great differences observed in the biolog-
ical activity of stereoisomers (enantiomers and diastereoisomers).1
In these conversions, a particular compound is modified by trans-
forming functional groups that are difficult or impossible to
achieve through conventional chemical procedures.1 In addition,
biotransformations have great potential because of their sustain-
able methodology for the production of chemicals, that is, green
chemistry.
To perform bioconversions, whole microorganisms or isolated
enzymes can be utilized, but the use of microbial cells is very
attractive since they simultaneously provide a number of stereose-
lective enzymes. Among them, whole fungal cells are highly advan-
tageous biocatalysts because of their rapid growth in natural and
synthetic media, their ease of handling, and simple scale-up. They
play a leading role in ‘chemo-enzymatic syntheses’ because of their
great diversity which produces a range of useful enzymes with cat-
alytic abilities.2
Marchantia polymorpha have been shown to possess the ability of
hydrogenating the double bond of 1 to afford (R)-N-phenyl-3-
methylsuccinimide 3 with a 99% ee.3 In turn, M. polymorpha has
been shown to enantioselectively hydrogenate 2 to yield trans-
(2R,3R)-N-phenyl-2,3-dimethylsuccinimide 4 with 99% ee.4 In a
more recent paper, we have found that Aspergillus flavus, Aspergillus
fumigatus, and Aspergillus niger are also efficient biocatalysts for the
enantioselective hydrogenation of 1 and 2 into (R)-(+)-2-methyl-N-
phenyl- and (2R,3R)-(+)-2,3-dimethyl-N-phenylsuccinimides 3 and
4 (Schemes 1 and 2) with 99% ee and conversions higher than 96%,
thus demonstrating that Aspergillus spp. are efficient tools for the
production of chiral succinimides, which could be used as asym-
metric synthons for organic synthesis or useful biologically active
compounds.5 In the same paper, we reported that Fusarium grami-
nearum and Penicillium sp. converted 2 to 4 with a lower reduction
capacity and the same enantioselectivity.
On the basis of our earlier findings, we herein expand upon the
preliminary report, by using analogues of 1 and 2 as substrates, all
possessing an alkyl chain (CH2)n between the N of maleimide and
the benzene ring, whose length (n) varies from 1 to 4 (compounds
Although the potential of fungi as biocatalysts has been demon-
strated in some previous work,1 new studies exploring the potenti-
ality of fungal species as new routes to obtain enantiopure
compounds are highly welcome.
2-Methyl-N-phenyl- and 2,3-dimethyl-N-phenylmaleimides 1
and 2 have proven to be good substrates for biocatalytic C–C
double bond reduction leading to the introduction of one or two
A. fumigatus
A. flavus
A. niger
O
O
H3C
H3C
N
N
O
O
1
(R)-(+)-3
* Corresponding author. Tel./fax: +54 341 4375315.
E-mail address: szaabgil@citynet.net.ar (S.A. Zacchino).
Scheme 1. Biotransformation of 1 by fungal species.
0957-4166/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2010.02.017

536
M. Sortino, S. A. Zacchino / Tetrahedron: Asymmetry 21 (2010) 535–539
A. fumigatus
A. flavus
commercial (R)-methyl succinic acid 21 and the respective phen-
ylalkylamines (Ph–(CH2)n–NH2, n = 1–4) 22–25, following the re-
ported procedures.7 All these synthetic enantiopure compounds
were dextrorotatory indicating that the biotransformation prod-
ucts (+)-13–16 had the (R)-configuration.
O
O
A. niger
F. graminearum
Penicillium sp
H3C
H3C
H3C
H3C
N
N
Enantiomeric excesses were determined by 1H NMR spectros-
copy using the chiral shift reagent, Eu(hfc)3. For this purpose,
rac-2-methyl-N-phenylalkylsuccinimides 13–16 were prepared
by catalytic hydrogenation from the respective monomethylated
maleimides 5–8. The methyl protons of both enantiomers were
clearly separated in the 1H NMR spectra of 13–16 upon addition
of Eu(hfc)3, allowing the calculation of the ee of the biotransforma-
tion products with satisfactory precision. The spectrum of the 1H
NMR added to the shift reagent showed that the enantiomeric
purities of (+)-13–16 were higher than 99% for all bioconversions
(Table 1).
O
O
(2R,3R)-(+)-4
2
Scheme 2. Biotransformation of 2 by fungal species.
5–12). Of these, substrates 2-methyl and 2,3-dimethyl-N-phen-
ylalkylmaleimides 6–8 and 10–12, with n = 2, 3, and 4 have not
been reported previously.
Considering that A. fumigatus ATCC 26934 showed the best effi-
ciency (99% conversion rate and >99% ee) in the previous paper, we
chose this fungus for the bioconversion of the new series of
maleimides.
2.2. Absolute configuration and enantiomeric excesses of 2,3-
dimethyl-N-phenylalkylsuccinimides 17–20
2. Results and discussion
The 2,3-dimethylsuccinimides 17–20 obtained by biotransfor-
mation of N-substituted 2,3-dimethylmaleimides 9–12 with A.
fumigatus have optical activity (all were dextrorotatory, Table 3)
clearly indicating that the (+)-trans-isomers were produced enan-
tio- and diastereoselectively.
To investigate the diastereoisomeric excess (de) of the reaction,
rac-trans- and cis-2,3-dimethyl-N-phenylalkylsuccinimides 17–20
were prepared from commercial dimethyl succinic acid 26 (mix-
ture of and meso) and the respective phenylalkylamines (Ph–
(CH2)n–NH2, n = 1–4) 22–25, according to the reported proce-
dures.7 The peaks of CH3 and 2,3-H in the 1H NMR spectra of each
diastereoisomer were determined, showing that those of the trans-
isomers appear at lower and higher fields, respectively, than those
of the cis-isomer (Table 2).
Four 2-methyl-N-phenylalkylmaleimides 5–8 and four 2,3-di-
methyl-N-phenylalkylmaleimides 9–12 with
(n = 1–4) between the imide N and the benzene ring, obtained
through reported procedures,6 were submitted to biotransforma-
tion with the growing cells of A. fumigatus ATCC 26934 in Czapek
medium under controlled conditions.
a (CH2)n chain
The results showed that A. fumigatus reduced the substrates 5–
12 into their respective dextrorotatory succinimides 13–20 with
the conversion rates ranging from 9.44% to 93.81% (Tables 1 and
3). Among them, dimethylated maleimide 12 (n = 4) was trans-
formed to the highest extent (93.81%) followed by 11 (n = 3,
61.40%), 9 (n = 1, 52.12%), and 10 (n = 2, 29.20%). Similar results
were obtained with monomethylated maleimides, although they
were less readily accepted: maleimide 8 (n = 4) was transformed
to the highest extent (42.07%) followed by 7 (n = 3, 18.91%), 5
(n = 1, 12.14%), and 6 (n = 2, 9.44%). Figure 1 shows the conversion
rates of maleimides 5–12, along with those obtained with malei-
mides 1 and 2 (reported previously) with the same strain of A.
fumigatus.5 For each series, a dependence of the conversion with
the length of the alkyl chain was observed, with the highest rate
occurring when n = 0 and the lowest one when n = 2, for each
mono- and dimethylated maleimides.
The 1H NMR spectroscopy data of the biotransformation prod-
ucts demonstrated that only trans-dimethylsuccinimides (+)-17–
20 were produced (de >99%) indicating the anti-addition of the
hydrogen atoms to the C–C double bond of maleimides, in accor-
dance with the fungal biotransformation of 2 recently reported.5
This is an important result, since it shows a clear difference from
the cis-product obtained by the catalytic hydrogenation of the
double bond and could be a very useful tool for synthetic
procedures.
Considering that the absolute configuration of trans-(+)-N-phe-
nylsuccinimide 4 has been assigned as (2R,3R) in the previous re-
ports,4,5 (+)-17–20 should be (2R,3R), since (+)-17–20 differs from
(+)-4 only in the N-alkyl chain and not in the chiral imide ring.
The enantiomeric excesses were determined by 1H NMR spec-
troscopy with Eu(hfc)3 with the aid of synthetic rac-trans-2,3-di-
methyl-N-phenylalkylsuccinimides 17–20. The peak analyses of
2.1. Absolute configuration and enantiomeric excesses of 2-
methyl-N-phenylalkylsuccinimides 13–16
To assist in the assignment of the absolute configuration of (+)-
13–16 obtained in the biotransformation process, (R)-2-methyl-N-
phenylalkylsuccinimides 13–16 were prepared as standards from
Table 1
Biotransformation of monomethylated maleimides 5–8
O
O
H3C
H3C
A. fumigatus
N
(CH2)n
N
(CH2)n
O
O
½ ꢀ
a 2D7
Substrate
Product
Conv (%)
% ee
5: n = 1
6: n = 2
7: n = 3
8: n = 4
(R)-(+)-13: n = 1
(R)-(+)-14: n = 2
(R)-(+)-15: n = 3
(R)-(+)-16: n = 4
12.14
9.44
18.91
42.07
+19.7 1.2 (c 0.73, CHCl3)
+7.0 1.5 (c 0.73, CHCl3)
+1.5 0.8 (c 1.50, CHCl3)
+1.3 0.3 (c 0.45, CHCl3)
>99
>99
>99
>99
Conversion rates (Conv) were determined by GC by using the following equation: % of conversion = product TIC/(product TIC + substrate TIC) ꢁ 100; % ees were determined by
1H NMR using Eu(hfc)3.

M. Sortino, S. A. Zacchino / Tetrahedron: Asymmetry 21 (2010) 535–539
537
Table 2
1H NMR chemical shifts (d, ppm) belonging to CH3 and 2,3-H of cis and trans-2,3-dimethyl-N-phenylalkylsuccinimides 4, 17–20
H-2,3
CH3
cis
trans
cis
trans
4
17
18
19
20
3.11 (2H, m)
2.93 (2H, m)
2.84 (2H, m)
2.83 (2H, m)
2.90 (2H, m)
2.60 (2H, m)
2.42 (2H, m)
2.30 (2H, m)
2.31 (2H, m)
2.30 (2H, m)
1.33 (6H, d, J = 7.2 Hz)
1.20 (6H, d, J = 7.2 Hz)
1.12 (6H, d, J = 7.2 Hz)
1.18 (6H, d, J = 7.2 Hz)
1.20 (6H, d, J = 7.2 Hz)
1.44 (6H, d, J = 7.2 Hz)
1.32 (6H, d, J = 7.2 Hz)
1.27 (6H, d, J = 7.2 Hz)
1.28 (6H, d, J = 7.2 Hz)
1.32 (6H, d, J = 7.2 Hz)
This work provides new evidence that biocatalysis provides
great opportunities to prepare useful chiral compounds by an envi-
ronmentally viable alternative. In addition, the findings reported
herein are a valuable contribution to the challenge of discovering
new biocatalysts for the stereoselective reduction of prochiral
building blocks. In terms of diastereo- and enantioselectivities, A.
fumigatus appears as a promising fungus for deeper biochemical
and biocatalytic studies.
4. Experimental
4.1. Synthesis
4.1.1. General
Solvents and reagents were purchased from Sigma (St. Louis,
MO, USA) and were purified in the usual manner. 1H and 13C
NMR spectra were recorded on a Bruker 300 MHz (Karlsruhe, Ger-
many). Compounds were dissolved in deuterated solvents from
commercial sources (Sigma) with tetramethylsilane (TMS) as the
internal standard. Chemical shifts are reported in ppm (d) relative
to the solvent peak (CHCl3 in CDCl3 at 7.26 ppm for protons and at
77.0 ppm for carbons). Signals are designated as follows: s, singlet;
d, doublet; dd, doublets of doublets; t, triplet; m, multiplet; q,
quartet; quint, quintuplet. Gas chromatograms were obtained in
a CG–MS Turbo Mass Perkin Elmer (Waltman, USA), column PE1
Figure 1. Dependence of the conversion rates with the length of the alkyl chain
between N and the aromatic ring.
the spectra showed that the enantiomeric purities of (+)-trans-17–
20 obtained by biotransformation with A. fumigatus were higher
than 99% (Table 3).
3. Conclusions
We have shown that A. fumigatus ATCC 26934 has the ability of
enantioselectively hydrogenating the endocyclic C–C double
bond of 2- and 2,3-dimethyl-N-phenylalkylmaleimides to afford
(R)-(+)-methyl- and trans-(2R,3R)-(+)-dimethyl-N-phenylalkylsuc-
cinimides with satisfactory conversion rates and excellent stereose-
lectivities. The data obtained in the present work, added to those of
our previous paper,5 show that this fungus has the versatility to
accept not only 2-methyl or 2,3-dimethyl-N-phenylmaleimide sub-
strates but also analogues containing an alkyl chain of variable
length between the imidic N and the benzene ring.
30 m ꢁ 0.25 mm of inner diameter, film 0.1
lm, ionization energy
70 eV. Elemental analyses were performed on a Carlo Erba EA 1108
analyzer (Milano, Italy). Percentages of C, H, and N were in agree-
ment with the product formula. Melting points were obtained in an
Electrothermal apparatus (Southern on Sea-UK) and were uncor-
rected. Optical rotations were measured in a Jasco DIP-1000 polar-
imeter (Easton, USA). The reported data refer to the Na-line value
(589 nm). IR spectra were recorded using a spectrophotometer Shi-
madzu Prestige 21 (Kyoto, Japan).
The biotransformation of 2,3-dimethyl-N-phenylalkylmalei-
mides was easy to perform and shows a clear difference with the
C–C reduction by catalytic hydrogenation, where only the cis-iso-
mer (meso) is obtained, thus being a very useful tool for synthetic
procedures.
4.1.2. Substrates
4.1.2.1. 2-Methyl-N-phenylalkylmaleimides 5–8. Monomethy-
lated maleimides were prepared from the reaction mixtures con-
taining commercial methylmaleic anhydride (5.0 mmol) in 5 mL
Table 3
Biotransformation of dimethylated maleimides 9–12
O
O
H3C
H3C
A. fumigatus
N
(CH2)n
N
(CH2)n
H3C
H3C
O
O
½ ꢀ
a 2D7
Substrate
Product
Conv (%)
% de
% ee
9: n = 1
(2R,3R)-(+)-17: n = 1
(2R,3R)-(+)-18: n = 2
(2R,3R)-(+)-19: n = 3
(2R,3R)-(+)-20: n = 4
52.12
29.20
61.40
93.81
+38.0 2.2 (c 0.31, CHCl3)
+40.5 0.6 (c 0.36, CHCl3)
+39.4 0.9 (c 0.36, CHCl3)
+38.3 0.6 (c 0.36, CHCl3)
>99
>99
>99
>99
>99
>99
>99
>99
10: n = 2
11: n = 3
12: n = 4
Conversion rates (Conv) were determined by GC by using the following equation: % of conversion = product TIC/(product TIC + substrate TIC) ꢁ 100; % ees were determined by
1H NMR using Eu(hfc)3.

538
M. Sortino, S. A. Zacchino / Tetrahedron: Asymmetry 21 (2010) 535–539
of CHCl3 and equimolar amounts of the appropriate phenylalkyl-
amines [Ph–(CH2)n–NH2; n = 1–4] 22–25 dissolved in 1 mL of
CHCl3 and stirred during 1 h. The solid, maleamic acid, which pre-
cipitated out of the reaction mixture was filtered off. The whole
amount of maleamic acid was dissolved in 5 mL of acetic anhy-
dride, and 100 mg of sodium acetate was added. The mixture
was heated for 2 h at reflux. The reaction mixture was cooled
and the reaction was quenched with water, then, the aqueous solu-
tion was extracted with Et2O, dried with Na2SO4, filtered, and the
solvent was evaporated. The product was purified by silica gel col-
umn chromatography using a mixture of hexane and ethyl acetate
(9:1) as eluent. Spectroscopic data of compound 5 were identical to
those previously reported.6,8
2,3-Dimethyl-N-butylphenylmaleimide 12: White crystals. Mp:
62–64 °C. Yield 62%. IR
max/cmꢂ1 (KBr): 1709. 1H NMR (CDCl3,
m
300 MHz): d 1.60–1.65 (4H, m, CH2(CH2)2CH2); 2.38 (s, 6H, CH3);
2.66 (2H, t, J = 7.2 Hz, ArCH2); 3.66 (2H, t, J = 7.2 Hz, NCH2); 7.10–
7.36 (5H, m, HAr) ppm. 13C NMR (CDCl3, 75 MHz): d 26.4 (CH3);
28.6 (ArCH2CH2); 28.7 (NCH2CH2); 35.4 (ArCH2); 44.8 (NCH2);
125.9 (C-40); 128.3 (C-20,60); 128.4 (C-30,50); 137.0 (C-10); 142.1
(C-2,3); 173.2 (C-1,4) ppm. MS: m/z = 257 (M+). Anal. Calcd for
C16H19NO2: C, 74.7; H, 7.4; N, 5.4. Found: C, 75.1; H, 7.6; N, 5.6.
4.1.3. Standards
4.1.3.1. (R)-(+)-2-Methyl-N-phenylalkylsuccinimides 13–16. A
mixture of (R)-(+)-methylsuccinic acid (5.0 mmol in water) 21
and phenylalkylamines (Ph–(CH2)n–NH2; n = 1–4) 22–25 was kept
at 170 °C for 2 h and then cooled to 20 °C. The aqueous solution
was extracted with Et2O, dried with Na2SO4, filtered, and the sol-
vent was evaporated.7 The mixtures were subjected to silica gel
column chromatography using a mixture of hexane and ethyl ace-
tate (9:1) as eluent. Spectroscopic data of compound 13 were iden-
tical to those previously reported.3
2-Methyl-N-phenethylmaleimide 6: White crystals. Mp: 54–
56 °C. Yield: 86%. IR
m
max/cmꢂ1 (KBr): 1712. 1H NMR (CDCl3;
300 MHz): d 2.06 (3H, d, J = 1.8 Hz, CH3); 2.90 (2H, t, J = 7.5 Hz,
ArCH2); 3.75 (2H, t, J = 7.5 Hz, NCH2); 6.29 (1H, q, J = 1.8 Hz, H-3);
7.21–7.57 (5H, m, HAr) ppm. 13C NMR (CDCl3, 75 MHz): 10.9.
(CH3); 34.6 (ArCH2); 39.2 (NCH2); 126.6 (C-40); 127.2 (C-3); 128.5
(C-30,50); 128.8 (C-20,60); 138.0 (C-10); 145.5 (C-2); 170.7 (C-4);
171.6 (C-1) ppm. MS: m/z = 215 (M+). Anal. Calcd for C13H13NO2:
C, 72.5; H, 6.0; N, 6.5. Found: C, 70.2; H, 6.1; N, 6.7.
2-Methyl-N-phenethylsuccinimide 14: White crystals. Mp: 52–
54 °C. Yield: 59%. IR
300 MHz): 1.27 (3H, d, J = 7.2 Hz, CH3); 2.24 (1H, dd,
m
max/cmꢂ1 (KBr): 1712. 1H NMR (CDCl3,
2-Methyl-N-propylphenylmaleimide 7: White crystals. Mp: 45–
d
46 °C. Yield: 65%. IR
m
max/cmꢂ1 (KBr): 1713. 1H NMR (CDCl3;
J = 17.2 Hz; 4.5, H-3a); 2.70–2.97 (2H, m, H-2 and H-3b); 2.91
(2H, t, J = 7.8 Hz, ArCH2); 3.77 (2H, quint, J = 7.8 Hz, NCH2); 7.18–
7.34 (5H, m, HAr) ppm. 13C NMR (CDCl3, 75 MHz): 16.8 (CH3);
33.4 (ArCH2); 34.5 (C-2); 36.3 (C-3); 39.8 (NCH2); 126.7 (C-40);
128.4 (C-20,60); 128.9 (C-30,50); 137.7 (C-10); 176.2 (C-1); 180.4 (C-
4) ppm. MS: m/z = 217 (M+). Anal. Calcd for C13H15NO2: C, 71.9;
300 MHz): d 1.95 (2H, quint, J = 7.5 Hz, CH2CH2CH2); 2.06 (3H, d,
J = 1.8 Hz, CH3); 2.64 (2H, t, J = 7.5 Hz, ArCH2); 3.56 (2H, t,
J = 7.5 Hz, NCH2); 6.28 (H, q, J = 1.8 Hz, H-3); 7.10–7.40 (5H, m,
HAr
)
ppm. 13C NMR (CDCl3, 75 MHz): 10.9 (CH3); 29.8
(CH2CH2CH2); 33.1 (ArCH2); 37.7 (NCH2); 125.9 (C-40); 127.2 (C-
3); 128.3 (C-30,50); 128.4 (C-20,60); 141.1 (C-10); 145.4 (C-2); 170.9
(C-4); 171.9 (C-1) ppm. MS: m/z = 229 (M+). Anal. Calcd for
C14H15NO2: C, 73.3; H, 6.5; N, 6.1. Found: C, 73.3; H, 6.5; N, 6.3.
2-Methyl-N-butylphenylmaleimide 8: White crystals. Mp: 46–
H, 6.9; N, 6.5. Found: C, 71.5; H, 7.1; N, 6.2. ½a D27
¼ þ5:3 ꢃ 1:2 (c
ꢀ
0.54, CHCl3).
2-Methyl-N-propylphenylsuccinimide 15: White crystals. Mp:
53–55 °C. Yield: 49%. IR
max/cmꢂ1 (KBr): 1712. 1H NMR (CDCl3,
300 MHz): 1.30 (3H, d, J = 7.2 Hz, CH3); 1.94 (2H, quint,
m
47 °C. Yield: 71%. IR
m
max/cmꢂ1 (KBr): 1710. 1H NMR (CDCl3;
d
300 MHz): 1.50–1.75 (4H, m, CH2(CH2)2CH2); 2.08 (3H, d,
d
J = 7.2 Hz, CH2CH2CH2); 2.22 (1H, dd, J = 17.3 Hz; 3.6, H-3a); 2.65
(2H, t, J = 7.2 Hz, ArCH2); 2.70–2.86 (2H, m, H-2 and H-3a); 3.58
(2H, t, J = 7.2 Hz, NCH2); 7.12–7.32 (5H, m, HAr) ppm. 13C NMR
(CDCl3, 75 MHz): 16.7 (CH3); 28.7 (CH2CH2CH2); 33.2 (ArCH2);
34.6 (C-2); 36.4 (C-3); 38.7 (NCH2); 126.0 (C-40); 128.3 (C-20,60);
128.4 (C-30,50); 140.9 (C-10); 176.5 (C-1); 180.6 (C-4) ppm. MS:
m/z = 231 (M+). Anal. Calcd for C14H17NO2: C, 72.7; H, 7.4; N, 6.1.
J = 1.8 Hz, CH3); 2.64 (2H, t, J = 7.2 Hz, ArCH2); 3.54 (2H, t,
J = 7.2 Hz, NCH2); 6.30 (H, q, J = 1.8 Hz, H-3); 7.14–7.30 (5H, m,
HAr) ppm. 13C NMR (CDCl3, 75 MHz): 10.9 (CH3); 28.2 (ArCH2CH2);
28.5 (NCH2CH2); 35.3 (ArCH2); 37.7 (NCH2); 125.8 (C-40); 127.3 (C-
3); 128.3 (C-30,50); 128.4 (C-20,60); 142.0 (C-10); 145.5 (C-2); 170.9
(C-4); 171.9 (C-1) ppm. MS: m/z = 243 (M+). Anal. Calcd for
C15H17NO2: C, 74.0; H, 6.9; N, 5.8. Found: C, 73.6; H, 7.2; N, 5.8.
Found: C, 71.5; H, 7.4; N, 6.2. ½a D27
¼ þ1:3 ꢃ 0:9 (c 0.27, CHCl3).
ꢀ
2-Methyl-N-butylphenylsuccinimide 16: White crystals. Mp:
70–72 °C. Yield: 52%. IR mmax (KBr) 1697 cmꢂ1 (C@O). 1H NMR
(CDCl3, 300 MHz): d 1.34 (3H, d, J = 7.2 Hz, CH3); 1.57–1.69 (4H,
m, CH2(CH2)2CH2); 2.30 (1H, dd, J = 17.1 Hz, 3.6, H-3a); 2.65 (2H,
t, J = 7.2 Hz, ArCH2); 2.74–7.78 (2H, m, H-2 and H-3b); 3.53 (2H,
t, J = 6.9 Hz, NCH2); 7.14–7.33 (5H, m, HAr). 13C NMR (CDCl3,
75 MHz): 16.7 (CH3); 27.3 (ArCH2CH2); 28.5 (NCH2CH2); 34.6 (C-
2); 35.2 (CH2); 36.4 (C-3); 38.5 (NCH2); 125.8 (C-40); 128.3 (C-
20,60); 128.4 (C-30,50); 141.9 (C-10); 176.5 (C-1); 180.6 (C-4). MS:
m/z = 245 (M+). Anal. Calcd for C15H19NO2: C, 73.4; H, 7.7; N, 5.7.
4.1.2.2. 2,3-Dimethyl-N-phenylalkylmaleimides
9–12. Dime-
thylated maleimides were prepared from commercial 2,3-dimeth-
ylmaleic anhydride and phenylalkylamines [Ph–(CH2)n–NH2;
n = 1–4] 22–25 as described for monomethylated maleimides.
Spectroscopic data of compound 9 were identical to those previ-
ously reported.8
2,3-Dimethyl-N-phenethylmaleimide 10: White crystals. Mp:
54–56 °C. Yield 78%. IR
m
max/cmꢂ1 (KBr): 1698. 1H NMR (CDCl3,
300 MHz): d 1.96 (6H, s, CH3); 2.90 (2H, t, J = 7.8 Hz, ArCH2); 3.74
(2H, t, J = 7.8 Hz, NCH2); 7.20–7.38 (5H, m, HAr) ppm. 13C NMR
(CDCl3, 75 MHz): d 8.6 (CH3); 34.8 (ArCH2); 39.1 (NCH2); 126.5
(C-40); 128.5 (C-20,60); 128.8 (C-30,50); 137.1 (C-10); 138.2 (C-2,3);
172.0 (C-1,4) ppm. MS: m/z = 229 (M+). Anal. Calcd for
C14H15NO2: C, 73.3; H, 6.6; N, 6.1. Found: C, 76.2; H, 6.8; N, 6.3.
2,3-Dimethyl-N-propylphenylmaleimide 11: Colorless oil. Yield
Found: C, 71.6; H, 7.9; N, 5.5. ½a D27
¼ þ2:7 ꢃ 1:25 (c 0.23, CHCl3).
ꢀ
4.1.3.2. rac-2-Methyl-N-phenylalkylsuccinimides 13–16. Each
monomethylated maleimide 5–8 (2 mmol) was dissolved in CH2Cl2
(2 mL) and 5% Pd/C (catalyst) was added. Then, the mixture was
submitted to an H2 atmosphere at room temperature for 2 h. The
crude reaction mixture was filtered, the solvent was evaporated,
and the product was purified by silica gel column chromatography
using a mixture of hexane and ethyl acetate (9:1) as eluent.
68%. IR m
max/cmꢂ1 (KBr): 1711. 1H NMR (CDCl3, 300 MHz): d 1.87–
2.10 (8H, m, CH2CH2CH2 and CH3); 2.45 (2H, t, J = 7.8 Hz, ArCH2);
3.36 (2H, t, J = 8.0 Hz, NCH2); 7.10–7.35 (5H, m, HAr) ppm. 13C
NMR (CDCl3, 75 MHz):
d 8.7 (CH3). 29.8 (CH2CH2CH2); 33.1
(ArCH2); 37.7 (NCH2); 125.9 (C-40); 128.3 (C-20,60); 128.3 (C-30,50);
137.0 (C-10); 141.1 (C-2,3); 172.3 (C-1,4) ppm. MS: m/z = 243
(M+). Anal. Calcd for C15H17NO2: C, 74.0; H, 6.9; N, 5.8. Found: C,
73.9; H, 7.0; N, 5.9.
4.1.3.3. rac-trans-2,3-Dimethyl-N-phenylalkylsuccinimides 17–
20. Dimethylated succinimides trans-( )-17–20 were prepared
from 2,3-dimethylsuccinic acid 26 (mixture of and meso) and
phenylalkylamines (Ph–(CH2)n–NH2; n = 1–4) 22–25 as described

M. Sortino, S. A. Zacchino / Tetrahedron: Asymmetry 21 (2010) 535–539
539
for (R)-(+)-2-methyl-N-phenylalkylsuccinimides. The mixtures
were subjected to silica gel column chromatography using a mix-
ture of hexane and ethyl acetate (9:1) as eluent to give rac-trans-
2,3-dimethyl-N-phenylalkylsuccinimides 17–20.
Suspensions of conidia (2–5 ꢁ 106 CFU/mL) were used to inoculate
2L erlenmeyer flasks containing Czapek broth medium (1 L). The
cultures were incubated at 30 °C for 72 h on an orbital shaker
(150 rpm; Innova 4000, New Jersey, USA).
trans-2,3-Dimethyl-N-bencylsuccinimide 17: White crystals.
Mp: 123–126 °C. Yield: 47%. IR
The substrates (125 mg) in DMSO (5 mL) were poured into
flasks containing the fungal biomass and the reaction mixtures
were incubated at 30 °C for 72 h on an orbital shaker (150 rpm).
A flask with fungal biomass and 5 mL DMSO instead of substrate
was taken as fungal growth control. After incubation, the mixtures
were filtered, and the aqueous phases were bulked and extracted
with ethyl acetate (3 ꢁ 250 mL). The organic phases were dried
over Na2SO4 and the products were analyzed by TLC and GC. Con-
version rates of the products were determined by GC analysis of
the crude extracts, and determined by using the following equa-
tion: percentage of conversion: product TIC (total ion current)/
(product TIC + substrate) ꢁ 100.
m
max/cmꢂ1 (KBr): 1695. 1H NMR
(CDCl3, 300 MHz): d 1.32 (6H, d, J = 7.2 Hz, CH3); 2.36–2.47 (2H,
m, H-2,3); 4.65 (2H, s, CH2); 7.22–7.41 (5H, m, HAr) ppm. 13C
NMR (CDCl3, 75 MHz): 14.9 (CH3); 42.1 (CH2); 43.1 (C-2,3); 127.9
(C-40); 128.6 (C-20,60); 128.7 (C-30,50); 136.0 (C-10); 179.2 (C-1,4)
ppm. MS: m/z = 217 (M+). Anal. Calcd for C13H15NO2: C, 71.8; H,
6.9; N, 6.5. Found: C, 72.3; H, 6.8; N, 6.7.
trans-2,3-Dimethyl-N-phenethylsuccinimide 18: White crys-
tals. Mp: 136–138 °C. Yield: 28%. IR mmax (KBr) 1703 cmꢂ1 (C@O).
1H NMR (CDCl3, 300 MHz): d 1.27 (6H, d, J = 7.2 Hz, CH3); 2.25–
2.37 (2H, m, H-2,3); 2.90 (2H, t, J = 7.5 Hz, ArCH2); 3.75 (2H, t,
J = 7.5 Hz, NCH2); 7.16–7.34 (5H, m, HAr) ppm. 13C NMR (CDCl3,
75 MHz): 15.1 (CH3); 32.3 (ArCH2); 39.6 (NCH2); 42.9 (C-2,3);
126.6 (C-20,60); 128.4 (C-40); 128.9 (C-30,50); 137.7 (C-10); 179.3
(C-1,4) ppm. MS: m/z = 231 (M+). Anal. Calcd for C14H17NO2: C,
72.7; H, 7.4; N, 6.1. Found: C, 73.1; H, 7.8; N, 6.0.
The compounds were purified by silica gel column chromatog-
raphy using a mixture of hexane and ethyl acetate (9:1) as eluent.
The structure of each product was elucidated by NMR and MS anal-
ysis and the spectra were identical to the previously synthesized
ones.
trans-2,3-Dimethyl-N-propylphenylsuccinimide 19: White
crystals. Mp: 130–133 °C. Yield: 40%. IR mmax (KBr) 1706 cmꢂ1
(C@O). 1H NMR (CDCl3, 300 MHz): d 1.28 (6H, d, J = 7.2 Hz, CH3);
1.95 (2H, quint, J = 7.2 Hz, CH2CH2CH2); 2.25–2.36 (2H, m, H-2,3);
2.65 (2H, t, J = 7.2 Hz, ArCH2); 3.56 (2H, t, J = 7.2 Hz, NCH2); 7.15–
7.36 (5H, m, HAr) ppm. 13C NMR (CDCl3, 75 MHz): 14.9 (CH3);
28.8 (CH2CH2CH2); 33.2 (ArCH2); 38.6 (NCH2); 42.9 (C-2,3); 125.9
(C-20,60); 128.3 (C-40); 128.4 (C-30,50); 137.0 (C-10); 179.5 (C-1,4)
ppm. MS: m/z = 245 (M+). Anal. Calcd for C15H19NO2: C, 73.4; H,
7.7; N, 5.7. Found: C, 71.3; H, 7.8; N, 5.6.
The enantiomeric purity of the products was determined on
the basis of the peak analysis of the methyl proton signals of
the 1H NMR with Eu(hfc)3. This was first performed for racemic
mixtures and, then, in the same conditions, for biotransformation
products.
Acknowledgments
M.S. acknowledges CONICET for the doctoral fellowship. M.S.
and S.Z. are grateful to the Agencia Nacional de Promoción Cientí-
fica y Tecnológica de Argentina (ANPCyT, PICT 995) and the Na-
tional University of Rosario.
trans-2,3-Dimethyl-N-butylphenylsuccinimide 20: White crys-
tals. Mp: 122–125 °C. Yield: 67%. IR mmax (KBr) 1705 cmꢂ1 (C@O).
1H NMR (CDCl3, 300 MHz): d 1.32 (6H, d, J = 7.2 Hz, CH3); 1.55–
1.74 (4H, m, CH2(CH2)2CH2); 2.26–2.34 (2H, m, H-2,3); 2.45 (2H,
t, J = 7.2 Hz, ArCH2); 3.52 (2H, t, J = 7.2 Hz, NCH2); 7.16–7.24 (5H,
References
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A. fumigatus ATCC 26934 was grown on a plate with an agarized
Czapek culture medium for 3 days at 30 °C until well sporulated.