Biotransformations of benzoylacetonitrile with the fungus Curvularia lunata: Highly diastereo- and enantioselective synthesis of α-alkyl β-hydroxy nitriles and γ-amino alcohols

Article abstract of DOI:10.1039/a909721j

Incubation of alcoholic solutions of benzoylacetonitrile, 1, with a suitable aqueous culture of C. lunata results in highly diastereo- and enantioselective reactions leading to α-alkyl β-hydroxy nitriles 3, which are easily reduced to the corresponding γ-

Full text of DOI:10.1039/a909721j

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Biotransformations of benzoylacetonitrile with the fungus
Curvularia lunata: highly diastereo- and enantioselective synthesis
of ꢀ-alkyl ꢁ-hydroxy nitriles and ꢂ-amino alcohols
1
Vicente Gotor,* Juan R. Dehli and Francisca Rebolledo
Departamento de Química Orgánica e Inorgánica, Facultad de Química, Universidad de Oviedo,
33071-Oviedo, Spain. Fax: ϩ34 985 103448; E-mail: vgs@sauron.quimica.uniovi.es
Received (in Cambridge, UK) 20th October 1999, Accepted 10th December 1999
Incubation of alcoholic solutions of benzoylacetonitrile, 1,
with a suitable aqueous culture of C. lunata results in
highly diastereo- and enantioselective reactions leading to
ꢀ-alkyl ꢁ-hydroxy nitriles 3, which are easily reduced to the
corresponding ꢂ-amino alcohols 6.
other formal α-ethylation, non-reductive processes have also
been reported during the exposure of BY to ethyl cyanoacetate
(aqueous medium; Fuganti et al.9), and to 1 (in petrol–water
10:1, and in the presence of two equivalents of acetaldehyde).10
No formal α-alkylation other than ethylation has been
described. A common feature of our work and those of Itoh7c
and Fuganti9 is the use of ethanol as cosolvent in aqueous
media.
The importance of optically active β-hydroxy acids and their
derivatives (especially β-hydroxy esters, α-substituted or not) as
building blocks for the synthesis of biologically active com-
pounds, agricultural chemicals, and natural products is well
established.1 This has led to the development of bioreduction
processes of β-keto acids and their derivatives as a powerful
methodology for obtaining the above mentioned optically
active building blocks.2
With our ongoing interest in preparing optically active β- or
γ-amino alcohols,3 useful as chiral auxiliaries in asymmetric
syntheses,4 and present as structural elements in biologically
active compounds,1e,1f,5 we envisaged that the bioreduction
of β-keto amides or -nitriles (much less studied than other
β-keto acid derivatives’ bioreductions6,7) could be an adequate
approach to this class of product. Indeed, we have recently
reported our first results on the highly stereoselective reduction
of 2-oxocyclopentanecarboxamides with the fungus Mortierella
isabellina.3a Herein we describe our somewhat unexpected
initial findings in the microbial reductions of benzoylaceto-
nitrile (3-oxo-3-phenylpropanenitrile, 1).
As product 3a can be seen as a precursor of interesting amino
alcohols bearing two stereogenic centres, our efforts were
immediately addressed to optimize its chemical and optical
yields.11 Thus, the best chemical yield for this product was
reached after the study of the effects of the C. lunata culture age
on the product distribution. In fact, using cells grown during
four days (when glucose from the culture medium has just been
consumed), we observed a maximum 3a–2 molar ratio of 7:1,12
compound 3a being obtained with very high de and ee (see
Table 1). From these results we assume that, at this time,
enzymes able to metabolize molecules smaller than glucose have
to be biosynthesized by the fungus, and that such enzymes
have also an enhanced ability to oxidize the ethanol added as
cosolvent to acetaldehyde, which is involved in the formation of
3a (see below).
Fuganti9 has supplied some evidence for a reasonable
α-ethylation pathway in the above mentioned BY biotrans-
formations of ethyl cyanoacetate and 3-oxobutanenitrile. This
pathway consists of three steps: (1) BY-mediated oxidation of
ethanol to acetaldehyde; (2) non-enzymatic aldol-type conden-
sation between the aldehyde and the active methylene com-
pound; (3) yeast reduction of the new C᎐C double bond13
᎐
(prior to the final bioreduction of the keto group).
In order to check the validity of this pathway for our
biotransformation with C. lunata, we performed several
experiments. (i) Since the step (2) should be favoured with
increasing pH, we carried out incubations with cells grown dur-
ing two days at pH values ranging from 6 to 10; as expected, the
3a–2 products molar ratio rose from 0.8:1 to 2.3:1. (ii) We
prepared, by Knoevenagel condensation between 1 and acet-
aldehyde, the supposed intermediate 2-benzoylbut-2-enenitrile,
4a, and subjected it to C. lunata under our standard conditions;
in this way, product 3a showing identical de and ee as that
obtained during the conventional biotransformation was
reached. (iii) Finally, in order to determine the source of the
ethyl group in 3a, we substituted hexadeuteroethanol for
ethanol as cosolvent, which led to the tetradeuterated product
03a;14 thus, it is clear that ethanol is the ethyl source. As a
whole, these three experiments also support the above three-
step pathway.
Bearing this in mind, we decided to test the potential of C.
lunata to promote other unknown formal α-alkylations.15 We
therefore used other alcohols instead of ethanol under the same
previously optimized conditions. The results summarized in
Table 1 show that, indeed, C. lunata is able to perform this task,
although with decreasing effectiveness as the alcohol chain
increases; this can probably be related to the substrate speci-
ficity of the enzymes responsible for the oxidation of alcohols
At first, we assayed several yeasts and fungi useful in the
microbial reduction of β-keto acid derivatives, but only two
strains were able to give significant amounts of the correspond-
ing 3-hydroxy-3-phenylpropanenitrile, 2. Thus, Mortierella isa-
bellina NRRL 1757 gave a 90% chemical yield of 2, but with a
moderate ee (70%), whereas a first experiment with Curvularia
lunata CECT 21308 (cells grown during two days) affords an
almost equimolar mixture of 2 and the unexpected 2-(1-
hydroxy-1-phenylmethyl)butanenitrile, 3a. To our knowledge,
only one example of carbonyl reduction with concomitant
formal α-ethylation has been described during the incubation
of 3-oxobutanenitrile with baker’s yeast (BY; Itoh et al.7c). Two
J. Chem. Soc., Perkin Trans. 1, 2000, 307–309
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307

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Table 1 Products 3 obtained in biotransformations of benzoylaceto-
nitrile 1 with the fungus Curvularia lunataa
β-keto nitriles, with concomitant carbonyl group reduction, has
been shown with the fungus C. lunata. The alkyl group origin-
ates from the primary alcohol used as cosolvent. The process
is highly diastereo- and enantioselective, and furnishes α-alkyl
β-hydroxy nitriles easily transformable into almost homochiral
γ-amino alcohols. Further study concerning the scope and
applicability of this methodology is currently under way.
[α]D25 (EtOH)/
Product
Rb
Yield (%)c De (%) Ee (%)
10Ϫ1 deg cm2 gϪ1
3a
3b
3c
Et
Pr
Bu
69
38
13
96
97
86
98
98
86
ϩ16.8 (c 1.1)
ϩ12.8 (c 1.7)
ϩ7.3 (c 2.8)
Experimental
General procedures for growing C. lunata and for
biotransformations
a In the presence of the corresponding alcohol ROH as cosolvent (see
Experimental). b See 3 in Scheme 1. c After purification by column
chromatography.
A loop of a solid culture of C. lunata, from an agar plate, was
sowed on to a test tube containing 3 ml of sterilized Sabour-
aud’s liquid medium [composed of bactopeptone (10 g),
-glucose (20 g) in distilled water (1.0 l); pH adjusted to 5.8].
After growing over 72 h (rotary shaker, 200 rpm, 28 ЊC), this
initial culture (0.5 ml) was used to inoculate another sterilized
medium (75 ml, in a 250 ml Erlenmeyer flask), identical to that
used for fungi in ref. 22. After 96 h of incubation (same condi-
tions as above), a solution of 1 (75.0 mg, 0.517 mmol) in the
corresponding alcohol (750 µl) was added. Biotransformation
was continued until disappearance of the substrate (ca. 12 h,
TLC monitoring). The mycelia were then filtered off, washed
with aqueous 0.8% NaCl, and the combined aqueous phases
continuously extracted with ethyl acetate (24 h). After drying,
the solvent was eliminated and the crude residue purified by
silica gel flash column chromatography (eluent: hexane–ethyl
acetate 5:1) to obtain the corresponding 2-alkyl-3-hydroxy-
nitrile 3.
to aldehydes. The isobutyl group is also introduced (14% yield),
but the de and ee of the product have not been determined yet.
Interestingly, the fungus fails to insert methyl and isopropyl
substituents. The latter could be seen as an indirect support of
the Fuganti’s pathway, since it is well known that aldol-type
equilibria with ketones are strongly shifted towards their left-
hand sides.
As shown in Table 1, the de and ee of the β-hydroxy nitriles,
especially those of 3a and 3b, are very high. The des and ees
were determined by treatment of products 3 with both (R)-
and (S)-3,3,3-trifluoro-2-methoxy-2-phenylpropanoyl chloride
(MTPA-Cl), and the resulting (S)- and (R)-MTPA esters16 5,
respectively were analysed by GC.17 Through a compar-
ative analysis, de and ee values of 3 can be unambiguously
established.
The R absolute configuration of the benzyl carbon atom
(C-1Ј) in the products 3 was determined from the ∆δ values
18
1
(δS-MTPA Ϫ δR-MTPA
)
measured in the H-NMR spectra of the
MTPA esters 5 (the average magnitudes in Hz are placed beside
Selected analytical data
1
the corresponding H nuclei in structure 5). Furthermore, a
(2R,1ЈR)-2-(1-Hydroxy-1-phenylmethyl)butanenitrile, 3a. δH
(200 MHz, CDCl3) 1.05 (t, J 7.4, 3H), 1.42–1.66 (m, 2H), 2.66–
2.76 (m, 1H), 3.34 (br s, 1H), 4.73 (d, J 6.4, 1H), 7.36 (br s, 5H);
δC (75 MHz, CDCl3) 11.3, 22.0, 42.3, 73.0, 120.0, 125.9, 128.1,
128.3, 140.3; m/z (EI) 175 (Mϩ, <1%), 107 (100%), 79 (39%);
HRMS calc. for C11H13NO 175.0997, found 175.0999.
∆δ of ϩ50 Hz observed for the methoxy protons of the MTPA
moiety also corroborates this assignment.19
3,4,4,4-Tetradeutero-2-(1-hydroxy-1-phenylmethyl)butane-
nitrile, 03a. δH (200 MHz, CDCl3) 1.54 (d, J 10.0, 1H), 2.72 (dd,
J 10.0, 6.6, 1H), 3.00 (br s, 1H), 4.75 (d, J 6.6, 1H), 7.37 (br s,
5H); δC (75 MHz, CDCl3) 10.5 (m, CD3), 21.7 (t, CHD), 42.4,
73.5, 120.2, 126.1, 128.4, 128.6, 140.3; m/z (EI) 179 (Mϩ, 1%),
107 (100%), 79 (92%).
(1R,2R)-2-(Aminomethyl)-1-phenylbutan-1-ol, 6a. δH (300
MHz, CDCl3) 0.86 (t, J 7.4, 3H), 1.15–1.55 (m, 3H), 2.77 (dd,
J 12.3, 7.0, 1H), 3.00 (dd, J 12.3, 2.8, 1H), 3.28 (br s, 3H), 4.69
(d, J 6.5, 1H), 7.20–7.45 (m, 5H); δC (75 MHz, CDCl3) 11.4,
21.9, 43.0, 46.6, 79.0, 126.3, 126.7, 127.9, 144.7; m/z (EI) 179
(Mϩ, 4%), 132 (80%), 117 (100%); HRMS calc. for C11H17NO
179.1310, found 179.1307.
Scheme 1 Reagents and conditions: i, LiAlH4, Et2O, rt (86–90%); ii,
CDI, CH2Cl2, rt (63–75%).
Acknowledgements
In order to establish the absolute configuration of C-2 (C-α),
it was necessary to perform the transformations outlined in
Scheme 1. LAH reduction20 of 3 gave very good yields of
γ-amino alcohols 6, which, in turn, were treated with N,NЈ-
carbonyldiimidazole21 and thus easily converted into 1,3-
oxazinan-2-ones 7. From the 1H-NMR spectra of these
heterocycles, coupling constants of ca. 8.7 Hz are observed
between protons Hc and Hd, which implies an anti arrange-
ment and, consequently, the R configuration for the alkyl
substituted chiral centre. This geometry is consistent with the
coupling constants of proton Hc with Ha (ca. 9.5 Hz) and
with Hb (ca. 5.2 Hz). As it can be derived from the Scheme 1
reactions, both chiral centres on the β-hydroxy nitriles 3 and the
γ-amino alcohols 6 also show R absolute configurations.
This work was supported by the Comisión Interministerial de
Ciencia y Tecnología (CICYT, project BIO 98-0770). J. R. D.
thanks the Spanish Ministerio de Educación y Cultura for a
predoctoral grant.
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