Mechanism of the yeast mediated reduction of nitrostyrenes in light

petroleum

Anita F. McAnda, Kade D. Roberts, Andrew J. Smallridge,* Abilio Ten and

Maurie A. Trewhella
Department of Chemical Sciences, Victoria University of Technology, PO Box 14428, MCMC,
Melbourne, 8001, Australia

The yeast mediated reduction of a range of á- and â-deutero substituted nitrostyrenes has been conducted
in light petroleum in the presence of a small amount of water or D2O. NMR analysis of the products from
these reactions has allowed the determination of the mechanism of this yeast reduction reaction.
The results indicate that initially, a reversible non-stereoselective protonation occurs at the â-centre,
followed by stereoselective addition of hydride at the á-position.

Introduction petroleum) 3 gave 1-deutero-2-nitro-1-phenylethane 2 in 65%

isolated yield and with no loss of deuterium from the α-carbon
The yeast mediated reduction of β-nitrostyrenes † has been stud- (Scheme 2).
ied using yeast in both an aqueous 1,2 and an organic solvent
system.3 In all cases reported, the reduction of nitrostyrenes D D
with substituents in the β-position gave rise to the production Yeast
NO2 NO2
of racemic products, whilst stereoselective reduction was Ph Ph
observed for α-substituted nitrostyrenes. The lack of stereo- light petroleum, H2O
1 2
selectivity in the aqueous systems had been attributed to
Scheme 2 ‡
racemisation of the product under the mildly basic reaction
conditions,4 however it was shown that this racemisation was The product obtained had a specific rotation of 10.28 1021
not occurring in the organic solvent system.3 In order to explain deg cm2 g21 indicating that some stereoselectivity had occurred
this unusual lack of stereoselectivity associated with a yeast at the α-carbon. To determine the enantiomeric excess of the
reduction reaction, a clearer understanding of the reaction product the nitro group was reduced to the amine using LiAlH4
mechanism is required. and the Mosher’s amide formed. NMR analysis of the amide in
Even though yeast has been extensively used to effect chiral the presence of the shift reagent Eu(fod)3 indicated a 61% ee
reductions in organic synthesis 5 very little work has been based upon integration of the proton at the α-centre. The find-
undertaken to elucidate the mode of action of this reduction
ing that the reduction was partially stereoselective is not sur-
reaction. Fuganti and co-workers have studied the mechanism
prising since α-methyl-β-nitrostyrene is stereoselectively reduced
of the yeast mediated reduction of cinnamyl alcohol and cin-
by yeast in an aqueous reaction system;2 it is at the β-centre that
namaldehyde (Scheme 1) 6 and a number of α,β-unsaturated the lack of stereoselectivity has been observed.1,3
D D
H Reduction of â-deutero-â-nitrostyrene 3
Ph
R Yeast
Ph
R β-Deutero-β-nitrostyrene 3 was prepared from [2H3]nitrometh-
D H ane and benzaldehyde in good yield and with 95% incorpor-
D R = CH2OH, CHO
ation of deuterium at the β-carbon when the reaction was
Scheme 1 conducted in D2O. Reduction of this compound with yeast in
the organic solvent system gave a 59% yield of a mixture of
lactones 7–9 using an aqueous reaction system. Through the use 2-nitro-1-phenylethane 4 and 2-deutero-2-nitro-1-phenylethane
of deuterium labelling they have shown that yeast stereo- 5 in a ratio of 4 : 1 (Scheme 3).
selectivity delivers both the hydride and the proton in an anti
fashion. The yeast reduction of nitrostyrenes must involve a NO2 NO2 NO2
Ph Yeast Ph + Ph
somewhat different mechanism since a racemic product results.
D light petroleum, H2O D
3 4 5
Results and discussion
Scheme 3
Reduction of á-deutero-â-nitrostyrene 1
α-Deutero-β-nitrostyrene 1 was prepared from nitromethane The loss of deuterium is a clear indication that exchange is
and α-deuterobenzaldehyde in good yield with 100% deuterium occurring at some stage during the reaction. We have previously
incorporation at the α-carbon. Reduction with bakers yeast shown that deuterium is not incorporated at the β-centre when
using the previously reported reaction conditions (1 mmol 2-nitro-1-phenylethane 4 is stirred in light petroleum, D2O and
substrate, 11 g yeast, 0.8 ml water per g yeast, 50 ml light yeast, indicating that exchange does not occur between the
water present in the reaction and the reduced product 4.3 To
show that exchange does not occur between protons bound to
† Throughout the text the compounds are referred to as β-nitrostyrenes
with the substituents prefixed as α- or β- (e.g.: α-deutero-β-nitrostyrene)
to assist in the description of where hydride and proton attack is occur- ‡ It is assumed that the major isomer produced is the (S)-enantiomer as
ring. IUPAC nomenclature, (E)-2-nitro-1-phenyl[1-2H]propene, is used this is the case for reduction of α-methyl-β-nitrostyrene under similar
in the Experimental section. conditions.

J. Chem. Soc., Perkin Trans. 1, 1998 501

the yeast and the reduced nitrostyrene, 2-deutero-2-nitro-1- β-carbon and hydride attack at the α-position. It is of course not
phenylethane 5, prepared by sodium borohydride reduction possible to directly observe the reversible nature of the proton-
of β-deutero-β-nitrostyrene 3, was stirred in light petroleum, ation in this case due to the presence of the methyl group.
D2O and yeast. No loss of deuterium was observed. Taken to- The fact that no deuterium loss occurs in the reduction of
gether, these two observations confirm that the hydrogen atoms α-deutero-β-nitrostyrene 1 and α-deutero-β-methyl-β-nitro-
bound to the β-carbon in 2-nitro-1-phenylethane 4 are non- styrene 7 indicates that the reversible protonation occurs
exchangeable under the reaction conditions; the exchange at exclusively at the β-carbon and hydride attack therefore occurs
the β-centre must therefore be occurring prior to product solely at the α-carbon. The electronic preference for hydride
formation. attack at the α-carbon was demonstrated by the reduction of
Reduction of β-deutero-β-nitrostyrene 3 using a small these nitrostyrenes using NaBD4, a reaction which delivers
amount of yeast (7 g mmol21) resulted in incomplete reduction deuteride exclusively to the α-carbon (Scheme 6).
and the recovered starting material 6 did not contain deuterium
(Scheme 4). The formation of reduced material, devoid of deu- D
NO2 NaBD4 NO2
Ph Ph
NO2 R = Me, H
Ph R
NO2 R
NO2 Yeast Ph D Scheme 6
+
Ph
4 5
light petroleum, H2O
D Reactions using D2O
3 NO2
Ph Other workers have concluded that in yeast mediated reduction
6 reactions conducted in aqueous systems, the yeast delivers the
Scheme 4
hydride but the proton comes from the water.7 If this mechan-
ism is reflected in the present system, replacing the water with
D2O should result in little or no loss of deuterium from the
terium, therefore arises via reduction of non-deuterated nitro-
β-centre. Reduction of β-deutero-β-nitrostyrene 3 with yeast
styrene, rather than by loss of deuterium from the reduced
(7 g mmol21) in light petroleum and using D2O, in place of
product.
the small amount of water usually employed, resulted in the
The production of both deuterated and non-deuterated
recovery of β-nitrostyrene 6 and 2-nitro-1-phenylethane 4 as
products and substrates in the reaction can best be explained if
well as the corresponding β-deuterated analogues, 3 and 5
the initial step in the reaction is a reversible protonation of the
(Scheme 7). The loss of deuterium in this system indicates that
nitrostyrene at the β-carbon followed by hydride attack at the
α-carbon. The protonation is yeast catalysed, as loss of deuter-
ium does not occur if β-deutero-β-nitrostyrene 3 is stirred in Ph
NO2
light petroleum and water in the absence of yeast. Previous NO2
NO2 Yeast Ph D
reports on the mechanism of yeast reduction of carbon– +
Ph
4 5
carbon double bonds have not determined whether hydride D
light petroleum, D2O
attack or protonation occurs first, but have assumed that the 3 NO2
mechanism is similar to metal hydride reduction where hydride Ph
6
attack is the first step. Clearly, in the case of nitrostyrenes, the
first step in the reduction is a yeast catalysed protonation rather Scheme 7
than a hydride attack.
the protonation at the β-centre is not only yeast catalysed but
Reduction of á-deutero-â-methyl-â-nitrostyrene 7 also that the proton is delivered by the yeast rather than coming
Whilst the foregoing reactions have provided considerable evi- directly from the water present.
dence regarding the mechanism of the yeast mediated reduction Reduction of β-nitrostyrene 6 using the same D2O system
of β-nitrostyrenes, of more interest was the mechanism of resulted in the production of the same four compounds 3, 4, 5
reduction of β-methyl-β-nitrostyrenes, since this reaction pro- and 6. The production of the deuterated compounds in this
duces a potentially useful chiral centre, as previously mentioned system indicates that (not surprisingly) ready exchange occurs
however, all attempts to date have produced racemic material. between the yeast and the D2O, with the result that the yeast
α-Deutero-β-methyl-β-nitrostyrene 7 was prepared from nitro- can deliver both protons and deuterons.
ethane and α-deuterobenzaldehyde in 50% yield with 100% In both of the previous reactions a small amount (<5%)
incorporation of deuterium at the α-carbon. The yeast medi- of the dideurated product 1,2-dideutero-1-phenyl-2-
ated reduction of this compound gave 1-deutero-2-nitro-1- nitroethane 9 was detected (Scheme 8). The identity of this
phenylpropane 8, as an equimolar mixture of diastereomers,
in 66% yield with no loss of deuterium from the α-position D
(Scheme 5). The retention of the deuterium could be clearly NO2 Yeast
NO2
Ph Ph
+ 3–6
light petroleum, D2O D
D D
6 9
NO2 Yeast NO2
Ph Ph
light petroleum, H2O Scheme 8
7 8
Scheme 5 dideuterated product was confirmed by 2H–2H TOCSY NMR
spectroscopy. A crosspeak was observed between the deuterium
seen in a 1H–1H COSY NMR experiment which showed no signals at the 1- and 2-position indicating that both signals
crosspeak between the two diastereotopic protons at the came from a single molecule. This result strongly suggests that
α-position indicating there was no material present containing some exchange is occuring between the D2O and the NADPH
two protons at this carbon atom and therefore complete reten- in the yeast, so that the yeast can deliver both a deuteride and a
tion of the deuterium had occurred. This retention of deuter- deuteron.
ium supports the mechanism proposed for the reduction of Further evidence for this exchange came from the reduction
β-nitrostyrenes, with reversible protonation occurring at the of β-methyl-β-nitrostyrene 10, using the D2O reaction system,

502 J. Chem. Soc., Perkin Trans. 1, 1998

where a significant amount of the dideurated product 1,2- Preparation of nitrostyrenes
dideutro-2-nitro-1-phenylpropane 11 was detected using 2H–2H (E)-2-Nitro-1-phenylethene 6. A mixture of methanol (2 ml),
TOCSY NMR spectroscopy (Scheme 9). Since protonation has benzaldehyde (1.0 ml, 9.8 mmol) and nitromethane (0.63 ml,
11.6 mmol) was cooled to 210 8C. To this mixture, sodium
D hydroxide (464 mg, 11.6 mmol) in water (1 ml) was added with
NO2 NO2 NO2 stirring at a rate such that the temperature of the mixture did
Yeast
Ph Ph + Ph
not exceed 15 8C and the reaction mixture was allowed to stand
light petroleum, D2O D D
for 15 min. Water (7 ml) was added to dissolve the resultant
10 11 precipitate and the solution was added slowly to 4  HCl (4.7
Scheme 9 ml) with stirring. The yellow precipitate formed was filtered off
and washed with cold water. The precipitate was placed in a
been shown to occur exclusively at the β-carbon the incorpor- beaker immersed in hot water whereby the yellow precipitate
ation of deuterium at the α-position can only come from melted and two layers were formed. On cooling, crude product
exchange of deuterium between NADPH and the D2O. solidified and the remaining liquid was removed. The crude
Guenther et al. have demonstrated in vitro that the NADH solid was then recrystallised from ethanol to give 2-nitro-1-
specific diaphorase, lipoamide dehydrogenase, catalyses the phenylethene (0.799 g, 54%) mp 57–58 8C (lit.,12 57–58 8C);
exchange of hydride between NADH and water.10 Lipoamide δH(300 MHz) 7.53 (5H, m, Ph), 7.61 (1H, d, J 13.5, 2-H), 8.03
dehydrogenase is a subunit of the enzyme pyruvate dehydro- (1H, d, J 13.5, 1-H).
genase, an enzyme that is abundant in yeast. It is proposed that (E)-2-Nitro-1-phenyl[1-2H]ethene 1. The title compound was
the exchange between NADPH and D2O is catalysed by an obtained using [α-2H]benzaldehyde in place of benzaldehyde.
enzyme of this type present in the yeast. Yield: 0.874 g, 60% (100% deuterium incorporation at the α-
carbon), mp 57–58 8C; δH(300 MHz) 7.53 (5H, m, Ph), 7.61
(1H, t, J 1.8, 2-H); δD(46 MHz) 8.02 (1-D).
Conclusion
(E)-2-Nitro-1-phenyl[2-2H]ethene 3. The title compound was
The reduction of a number of deuterated nitrostyrenes in an obtained using [2H3]nitromethane in place of nitromethane
organic solvent system incorporating a small amount of either and the NaOH solution was made using D2O instead of water.
water or D2O has allowed us to determine that the yeast medi- Yield: 0.825 g, 57% (95% deuterium incorporation at the β-
ated reduction of these compounds proceeds via the following carbon), mp 58 8C (lit.,12 54–55 8C); δH(300 MHz) 7.52 (5H, m,
mechanism: a reversible non-stereoselective protonation at the Ph), 8.02 (1H, t, J 2.1, 1-H); δD(46 MHz) 7.61 (2-D).
β-carbon followed by a stereoselective addition of a hydride at (E)-2-Nitro-1-phenylprop-1-ene 10. Nitroethane (1.0 ml, 13.9
the α-carbon (Scheme 10). It is unclear at this stage whether the mmol) and cyclohexylamine (1.3 ml) were added to benzalde-
hyde (1.0 ml, 9.8 mmol) in glacial acetic acid (5.3 ml). The
R2 R2 R2 mixture was held at 100 8C for 6 h, cooled and diluted with
NO2 H+ NO2 H – NO2 water (1 ml). The reaction mixture was cooled overnight in a
Ph Ph Ph water bath. The crystals formed were filtered and air dried. The
R1 R1 R1 crude solid was recrystallised from ethanol to give (E)-2-nitro-
Scheme 10 1-phenylprop-1-ene (0.99 g, 62%), mp 64–65 8C (lit.,13 64–65 8C)
δH(300 MHz) 2.48 (3H, s, CH3), 7.47 (5H, m, Ph), 8.11 (1H,
reversible protonation is catalysed in a non-stereoselective s, 1-H).
manner or whether two (or more) enzymes with opposite enan- (E)-2-Nitro-1-phenyl[1-2H]prop-1-ene 7. The title compound
tioselectivities are involved. This mechanism accounts for the was obtained using [α-2H]benzaldehyde instead of benzalde-
finding that stereoselective reduction occurs at the α-centre but hyde. Yield: 0.780 g, 50% (100% deuterium incorporation at the
not at the β-centre, in yeast mediated reductions of this type. α-carbon), mp 62–63 8C; δH(300 MHz) 2.48 (3H, s, CH3), 7.47
This work complements other studies concerned with the mode (5H, m, Ph); δD(46 MHz) 8.11 (1-D).
of action of yeast mediated reduction reactions, however the
use of an organic solvent system has enabled a more detailed Yeast reactions
determination of the mechanism to be achieved. Reduction of (E)-2-nitro-1-phenylethene 6. (E)-2-Nitro-1-
phenylethene 6 (0.3 g, 2.0 mmol), yeast (22.13 g, 11 g mmol21 of
Experimental 6), H2O (17.7 ml) (0.8 ml per g of yeast) and light petroleum
(200 ml) were stirred at room temperature for 24 h. The solvent
General was removed by filtration and the yeast was washed with
NMR spectra were recorded using Bruker DPX300 and dichloromethane (3 × 50 ml). The filtrates were combined and
DRX500 spectrometers at 300 and 500 MHz (1H) and 46 MHz evaporated in vacuo to give a residue which was bulb-to-bulb
(2H). 1H NMR and 1H–1H COSY NMR spectra were recorded distilled to give 2-nitro-1-phenylethane 4 (0.216 g, 72%), bp
as deuterochloroform solutions using tetramethylsilane (δ = 0.0 150 8C/0.5 mmHg; δH(300 MHz) 3.34 (2H, t, J 7.5, 1-H), 4.63
ppm) as an internal reference. J Values are given in Hz. 2H (2H, t, J 7.5, 2-H), 7.3 (5H, m, Ph).
NMR and 2H–2H TOCSY NMR spectra were recorded in Reduction of (E)-2-nitro-1-phenyl[1-2H]ethene 1. (E)-2-Nitro-
chloroform solutions using deuterochloroform (δ = 7.26 ppm) 1-phenyl[1-2H]ethene 1 (0.3 g, 2.0 mmol) was stirred with yeast
as an internal reference. 2H–2H TOCSY NMR spectra were (21.98 g, 11 g mmol21 of 1), H2O (17.5 ml) and light petroleum
recorded using a similar method to that described by Chandra- (150 ml) for 24 h to give 2-nitro-1-phenyl[1-2H]ethane 2 (0.198
kumar and Ramamoorthy.11 Bulb-to-bulb distillations were g, 65%) bp 150 8C/0.5 mmHg; [α]D 10.28 (c 5 in CHCl3); δH(300
carried out using a Buchi-GKR 50 distillation unit. [α]D Values MHz) 3.33 (1H, tt, J 9.6, 2.1, 1-H), 4.63 (2H, dt, J 7.5, 0.9, 2-
are given in units of 1021 deg cm2 g21. H), 7.3 (m, 5H, Ph); δD(46 MHz) 3.33 (1-D). A sample of this
[α-2H]Benzaldehyde was purchased from ISOTEC Inc. nitroalkane was reduced to the amine using LiAlH4 in diethyl
[2H3]Nitromethane and D2O were purchased from Cambridge ether and then converted into the MTPA amide. An NMR spec-
Isotopic Laboratories. All other chemicals were purchased from trum was taken in the presence of 0.2  Eu(fod)3 shift reagent.
the Aldrich Chemical Company. AR grade light petroleum (40– Integration of the methine proton in the 1-position indicated a
60 8C) was used without further purification. Mauripan Instant 61% ee.
Dry Yeast was generously supplied by Mauri Foods Ltd, Reduction of (E)-2-nitro-1-phenyl[2-2H]ethene 3. (E)-2-Nitro-
Australia. 1-phenyl[2-2H]ethene 3 (0.4 g, 2.7 mmol) was stirred with yeast

J. Chem. Soc., Perkin Trans. 1, 1998 503

(29.3 g, 11 g mmol21 of 3), H2O (23.4 ml) and light petroleum 2-Nitro-1-phenyl[1-2H]propane 8. Using a similar procedure
(200 ml) for 24 h to give a mixture of 2-nitro-1-phenylethane (E)-2-nitro-1-phenylprop-1-ene 7 gave the title compound 8 as
and 2-nitro-1-phenyl[2-2H]ethane 5 (0.176 g), bp 150 8C/0.5 a mixture of diastereomers (0.096 g, 57%); δH(300 MHz) 1.57
mmHg; δH(300 MHz) 4: 3.34 (2H, t, J 7.5, 1-H), 4.63 (2H, t, J (3H, dd, J 6.9, 1.2, CH3), 3.02 (1H, dt, J 6.9, 1.8, 1-H), 3.33 (1H,
7.5, 2-H), 7.3 (5H, m, Ph); 5: 3.34 (2H, d, J 7.4, 1-H), 4.62 (1H, dt, J 6.9, 2.1, 1-H), 4.81 (1H, quintet, J 6.6, 2-H), 7.28 (m, 5H,
tt, J 7.4, 2.1, 2-H), 7.3 (5H, m, Ph); δD(46 MHz) 4.60 (2-D). Ph); δD(46 MHz) 3.03 (1-D), 3.36 (1-D).
Reduction of (E)-2-nitro-1-phenyl[1-2H]prop-1-ene 7. (E)-2-
Nitro-1-phenyl[1-2H]prop-1-ene 7 (0.3 g, 1.2 mmol) was stirred
with yeast (20.10 g, 11 g mmol21 of 7), H2O (16.1 ml) and light
References
petroleum (200 ml) for 24 h, to give 2-nitro-1-phenyl[1- 1 M. Takeshita, S. Yoshida and Y. Kohno, Heterocycles, 1994, 37, 553.
2
H]propane 8 as a mixture of diastereomers (0.198 g, 66%), bp 2 H. Ohta, N. Kobayashi and K. Ozaki, J. Org. Chem., 1989, 54, 1802.
150 8C/0.5 mmHg; δH(300 MHz) 1.57 (3H, dd, J 6.9, 1.2, CH3), 3 R. A. Bak, A. F. McAnda, A. J. Smallridge and M. A. Trewhella,
Aust. J. Chem., 1996, 49, 1257.
3.02 (1H, dt, J 6.9, 1.8, 1-H), 3.33 (1H, dt, J 6.9, 2.1, 1-H), 4.81 4 K. Sakai, A. Nakazawa, K. Kondo and H. Ohta, Agric. Biol. Chem.,
(1H, quintet, J 6.6, 2-H), 7.28 (m, 5H, Ph). 1985, 49, 2331.
Reduction of (E)-2-nitro-1-phenylprop-1-ene 10. (E)-2-Nitro- 5 S. Servi, Synthesis, 1990, 50, 1; R. Csuk and B. I. Glanzer, Chem.
1-phenylprop-1-ene 10 (0.3 g, 1.8 mmol) was stirred with yeast Rev., 1991, 91, 49.
(12.8 g, 7 g mmol21 of 10), D2O (10.2 ml) and light petroleum 6 C. Fuganti, D. Ghiringhelli and P. Grasselli, J. Chem. Soc., Chem.
(150 ml) for 24 h to give a mixture of starting material Commun., 1975, 846.
7 G. Fronza, C. Fuganti and P. Grasselli, Tetrahedron Lett., 1992, 33,
and reduced products (0.201 g, 67%) bp 150 8C/0.5 mmHg; 6375.
δD(46 MHz) 2.99 (1-D), 3.31 (1-D, 4.77 (2-D). 8 G. Fronza, C. Fuganti, P. Grasselli, A. Meles and A. Sarra,
Tetrahedron Lett., 1993, 34, 6467.
Sodium borodeuteride reductions 9 G. Fronza, C. Fuganti, P. Grasselli, S. Lanati and R. Rallo, J. Chem.
2-Nitro-1-phenyl[1-2H]ethane. (E)-2-Nitro-1-phenylnitro- Soc., Perkin Trans. 1, 1994, 2927.
ethene (0.15 g, 1 mmol) was dissolved in a mixture of 12 ml 10 H. Guenther, F. Biller, M. Kellner and H. Simon, Angew. Chem.,
Int. Ed. Engl., 1973, 12, 146.
chloroform (8 ml per g silica gel) and 2.3 ml isopropanol (1.5 ml 11 N. Chandrakumar and A. Ramamoorthy, J. Am. Chem. Soc., 1992,
per g silica gel) and stirred with 1 g silica gel. NaBD4 (0.16 g, 4.1 114, 1123.
mmol) was added in small amounts, until the yellow colour due 12 D. E. Worral, in Org. Synth., 1941, Coll. Vol. I, 413.
to the nitrostyrene disappeared. The mixture was then filtered, 13 B. C. Gairaud and G. R. Lappin, J. Org. Chem., 1953, 18, 1.
washed with brine (15 ml), extracted with dichloromethane (20
ml), dried over NaSO4 and the dichloromethane evaporated in
vacuo to give the title compound (0.11 g, 73%); δH(300 MHz) Paper 7/06353I
3.33 (1H, tt, J 9.6, 2.1, 1-H), 4.63 (2H, dt, J 7.5, 0.9, 2-H), 7.3 Received 1st September 1997
(m, 5H, Ph); δD(46 MHz) 3.33 (1-D). Accepted 21st October 1997

504 J. Chem. Soc., Perkin Trans. 1, 1998