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Enzyme-Catalyzed Synthesis of (S)-Cyanohydrins.

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cyclobutane partial structure, which was identified from the
typical CH coupling constants ('J = 140-145 Hz) in the
coupled 13C-NMR spectrum. In the C,H COLOC diagram,
the connectivity of all six carboxyl C atoms (6 = 164.7174.7) is clearly revealed by the two- and three-bond coupling constants 'JCH
and 3JcH,respectively. Accordingly,
tropane rings A and B are linked as mesaconic diester D.
Further cross signals in the C,H COLOC diagram show that
a 2-methyl-4-phenylcyclobutane-1,2,3-tricarboxylic
ester E
is present, whereby the carboxylic acid function on C 1 forms
an ester linkage with the third 3,6-dihydroxytropane C at its
3-hydroxyl group. Furthermore, the C,H COLOC diagram
reveals an angelic ester partial structure F, which is linked to
tropane ring C through the 6-hydroxyl group. Combination
of all partial structures gives the constitution of grahamine
(l),which accounts for the twenty double-bond equivalents.
The relative configuration of the 0-acyl groups on the
tropane rings may be derived from the HH coupling constants. All signals of protons in a 3-position are split into a
doublet of doublets (3JHH
= 4-5 Hz) owing to coupling with
the pseudo-axial protons 2-H and 4-H (interplanar angle ca.
60"). Signal broadening occurs as a result of unresolved HH
coupling to the pseudo-equatorial protons in 2- and 4-positions (interplanar angle ca. 90°). Accordingly, the 3-H protons are pseudo-equatorial and the 3-0-acyl groups pseudoaxial. The signals of the 6-H protons are each a doublet of
doublets with 3JHH
= 7.8-8.8 Hz to 7-H,,,, and 3.5-3.8 Hz
to 7-H,,,. Further couplings with 5-H are again unresolved
(interplanar angle nearly 90"); examination of the interplanar angle between the protons 5-H and 6-H in a Dreiding
model reveals that only 6-H in endo position allows an interplanar angle to 5-H of about 90". Thus, the 6-0-acyl groups
are exo.
The CH coupling constants of the carboxyl C atoms at
6 = 166.3 (D) and 167.7 (F) reflect the relative configuration
of the two C-C double bonds. A 3JCHcoupling of 15 Hz
between the carboxy signal at 6 = 167.7 and the proton signal at 6 = 5.87 is characteristic of the trans configuration of
the methyl group of angelic ester F. A correspondingly large
coupling with the proton signal at 6 = 6.92 is absent for the
carboxyl signal at 6 = 166.3, which confirms the E configuration of the mesaconic diester partial structure D.
0
D
H
v
0%cH3
F
Since the 3JHH
couplings of the cis and trans protons in
cyclobutane derivatives differ only slightly,['] the relative
configuration of this ring was determined by H,H NOE dif386
c', VCH Verlagsgesellschafi mbH. D-6940 Weinhelm. 1990
ference spectroscopy. Pronounced nuclear Overhauser effects corresponding to a cis relationship are found between
the protons at 6 = 4.45 and 4.96 as well as 4.13 and 2.03
(methyl). Saturation of the resonance at 6 = 2.03 also leads
to an NOE at the N-methyl proton of tropane ring B
(6 = 2.46). Accordingly, the partial structures may be joined
as shown by 1.
Presumably, the plants, which grow at sunny sites, form
the cyclobutane ring from mesaconic diester (left half in 1)
and cinnamic ester (right half) by [2 + 21 cycloaddition.
Received: November 30, 1989 [Z 3660 IE]
German version: Angew. Chem. 102 (1990) 441
CAS Registry number:
grahamine, 24583-56-0.
[l] C . Liebermdnn, Ber. Dtsch. Chem. Ges. 2 f (1888) 2342; ihid. 22 (1889) 672;
W. C. Evans, J Ethnopharmacol. 3 (1981) 265.
[2] I. Ribas, E. Riviera, An. R. Soc. ESP.Fis. Quim. Ser. 8 4 9 (1953) 707.
[3] E. Petersen: Praktisches Garrenlexikon, 3rd ed. Nymphenburger Verldgshandlung, Miinchen 1982.
[4] H. Ripperger, Phyrochemistry 18 (1979) 717; A. San-Martin, J Rovirose, V
Gambaro, M. Castillo, ihid. 19 (1980) 1007.
[5] A. San-Martin, C. Labbe. M. Castillo, Phvtochemistr.v 22 (1983) 1838; ibid.
26 (1987) 819.
[6] R. Hartmann, Dissertation, Universitit Bonn 1989 (detailed description of
the interpretation and documentation of the spectra).
[7] D. J. Pasto, S. H. Yang, J. Am. Chem. Soc. 106 (1984) 152.
Enzyme-Catalyzed Synthesis
of (S)-Cyanohydrins **
By Uwe Niedermeyer and Maria-Regina Kula *
The enzyme-catalyzed addition of hydrogen cyanide to
aldehydes leads to optically active cyanohydrins.['] These
compounds open up a simple route to chiral j3-amino alcohols,[21a-hydroxy acids, and pyrethroid insecticide^.'^] However, obtaining cyanohydrins with high optical purity is fundamentally difficult, since the optical yield of the enzymecatalyzed asymmetric synthesis is decreased by the concomitant nonselective chemical addition of hydrogen cyanide.
This reaction cannot be completely suppressed in most solvent systems.r41The importance of optically active cyanohydrins, on the one hand, and the lack of methods for asymmetric synthesis of cyanohydrins, on the other, prompted
us to investigate in more detail the potential of enzymecatalyzed cyanohydrin formation.
The synthesis of (R)-cyanohydrins is documented by numerous examples. One of the most effective catalysts is an
oxynitrilase (4.1.2.10) from Prunus amygdalus (bitter almond).['. 5 , 61 By contrast, the enzymatic synthesis of ( S ) cyanohydrins has not yet been studied!***] We have now
found that an oxynitrilase from Sorghum bicolor (4.1.2.1 I),
which was described mainly from a botanical viewpoint in
the 1960~,[~"-allows the preparative synthesis of ( S ) cyanohydrins with high optical purity.'']
[*] Prof. Dr. M.-R. Kula. Dr. U. Niedermeyer
lnstitut fur Enzymtechnologie der Universitit Dusseldorf
in der KFA Julich
Postfach 2050. D-5170 Julich (FRG)
[**I This work was supported by the Deutsche Forschungsgemeinschaft and
the Fonds der Chemischen Industrie.
[***I Editorial note (March 16, 1990): A communication concerning the enzyme-catalyzed synthesis of (S)-cyanohydrinshas been published in issue
No. 911990 of Tetrahedron Lett: F. Effenberger, B Horsch, S. Forster, T.
Ziegler, Tetrahedron Lett. 31 (1990) 1249 (Received: December 22.1989).
0570-0833I90j0404-0386 3 02.SOj0
Angew. Chem. Inr. Ed. Engl. 29 (1990) No. 4
By detailed investigations of the reaction kinetics of enzymatic and nonenzymatic cyanohydrin formation, we were
able to show that the enantiomeric excess of the product
could be controlled, especially through the pH value. Figure
1 shows, for example, the pH dependence of the enzymatic
reaction of 4-hydroxybenzaldehyde. Under otherwise identical conditions, lower pH values clearly result in higher optical rotations, that is, higher enantiomeric purity, but lead to
a decrease in reaction rate.
In a continuous run the (S)-oxynitrilase, immobilized on
Eupergit C,"O1 was employed in a stirred tank reactor
(10 mL); 4-hydroxybenzaldehyde (21 mM) was converted
under identical conditions as described above into the corresponding (S)-cyanohydrin (re value 98%). For a residence
time of 66 min and a conversion of 85 YO,a space-time yield
of 58 g L-' d - ' was obtained. The productivity, as well as
the enantiomeric excess, can be further improved by using
higher concentrations of catalyst.["'
To determine the ee values, the cyanohydrins were reduced
to the corresponding aminoethanols.['21These compounds
were perfluoropropionylated and then analyzed directly by
gas chromatography on a chiral c ~ l u r n n . " ~ ]
Experimental Procedure
'I
000-c. ,
0
10
,
I
20
,
I
1
30
t Iminl
LO
-
I
I
50
60
'
I
70
Fig. I , Effect ofthe pH value on the asymmetric cydnohydrin synthesis at 25 "C
(determined from the change in optical rotation A[oL]~,~).
Starting concentrations: 27.5 mM 4-hydroxybenzaldehyde, 500.0 mM HCN, 1.7 U mL-' ( S ) oxynitrilase.
Carbonyl compounds react with hydrogen cyanide at different rates in the nonenzymatic reaction.[g1 This differing
reactivity of the starting materials in the spontaneous formation of cyanohydrins, as well as the affinity of the enzyme for
the substrates, governs the conditions under which almost
exclusive enzyme-catalyzed cyanohydrin formation occurs.
Although the rate of the reaction catalyzed by the ( S ) oxynitrilase decreases with decreasing pH value, reactions
can be carried out down to a pH value of 3.0. Thus, a relatively broad pH range is available for optimizing the reaction. The results obtained in batch runs with a number of
aldehydes under optimized conditions are summarized in
Table 1. In contrast to the (R)-oxynitrilase from Prunus
Table 1. Enzymatic synthesis of (S)-cyanohydrins in sodium citrate buffer at
20°C. The starting material (0.5 mmol) was treated with excess hydrogen
cyanide and the (S)-cyanohydrin was isolated after the reaction times indicated
(batch runs).
Carbonyl compound
Benzaldehyde
3-Hydroxybenzaldehyde
4-H ydroxybenzaldehyde
3-Methylbenzaldehyde
Furfural
Acetaldehyde
(R/S)-3-Methylcyclohexanone
r [min]
pH
45
40
25
90
60
60
60
3.25
3.20
3.75
3.25
4.00
3.25
4.50
Yield [%]
ee value
["A]
80
96
YO
98
87
99
80
96
no conversion
no conversion
no conversion
amygdalus, the (S)-oxynitrilase from Sorghum bicolor is apparently unable to catalyze the reaction of aliphatic aldehydes. The procedure presented here affords homocyclic aromatic (S)-cyanohydrins with very high optical purity.
Angew Chem. I n l . Ed. Engl. 29 (1990) No. 4
0 VCH
The enzyme preparation was obtained from five-day-old shoots of Sorghum
bicolor by standard biochemical methods of protein isolation. The (S)-oxynitrilase stock solution had an activity of 83 U mL-' (53 U mg- I ) , whereby I U
catalyzes the formation of one micromole of 4-hydroxymandelonitrile per
minute at 2 0 Y at pH 3.75.
Example: 4-Hydroxybenzaldehyde (61 mg, 0.5 mmol) was dissolved in 9.4 mL
of 50 mM sodium citrate buffer (pH 3.75) and incubated at 20'C. After
addition of 500 pL of oxynitrilase solution and 800 pL of 4.2 M aqueous HCN
solution, the reaction was monitored polarimetrically. The reaction is complete
after 15-30 min (constant optical rotation). The reaction mixture was extracted
with 4 x 10 mL of diethyl ether and the combined organic phases were dried
over Na,SO,. The solvent was then removed with a rotary evaporator, the
residue washed with 3 x 10 mL of pentane. and the product dried in vacuo.
Yield: 64.8 mg (83%).
To determine the optical purity, 1- 2 mg of the cyanohydrin was reduced by
treatment with 250 pL of a 1 M solution of diborane (Aldrich Chemie. Steinheim) in tetrahydrofuran at room temperature for 30 min. After hydrolysis of
the excess diborane with a few drops of ethanol and removal of the solvent, the
resulting amino alcohol was acylated directly by treatment with 20pL of
pentafluoropropionic anhydride at room temperature in dichloromeihane. The
excess anhydride was removed in a rotary evaporator and the residue was
dissolved in dichloromethane and then analyzed by gas chromatography (FSChirasil-Val, Macherey-Nagel, Duren).
Received: November 15, 1989 [Z3635 IE]
German version: Angew. Chem. 102 (1990) 423
CAS Registry numbers:
benzaldehyde, 100-52-7; 3-hydroxybenzaldehyde, 100-83-4; 4-hydroxybenzaldehyde, 123-08-0: 3-methylbenzaldehyde. 620-23-5: furfural, 98-01-1 ; acetaldehyde, 75-07-0; (R/5'-3-methylcyclohexanone, 625-96-7; (9-benzaldehydecyanohydrin, 28549.12-4: (S)-3-hydroxybenzaldehydecyanohydrin, 12578171807-09-5; (S)-3-methylbenz60-4; (S)-4-hydroxybenzaldehydecyanohydrin,
aldehydecyanohydrin, 105367-21-3; (9-oxynitrilase, 9024-43-5.
[I] W. Becker, U. Benthin, E. Eschenhof, E. Pfeil, Biochem. Z. 337(1963) 156.
121 W. Becker, H. Freund, E. Pfeil, Angew. Chrm 77 (1965) 1139; Angew.
Chem. Inr. Ed. Engl. 4 (1965) 1079.
[3] T. Matsuo, T.Nishioka, M. Hirano, Y . Suzuki, K. Tsushima, N . Itaya,
H. Yoshioka, Pestic. Sci. 1980, 202.
[4] H.-H. Hustedt, E. Pfeil, Jusrus Liebigs Ann. Chem 640 (1961) 15.
[5] F. Effenberger, T. Ziegler, S . Forster, Angew. Chem. 99 (1987) 491 : Angeu,
Chem. Int. Ed. Engl. 26 (1987) 458.
[6] J. Brussee, E. C. Roos, A. van der Gen, Tetrahedron Leu. 29 (1988) 4485.
I
B i d . Chem. 236(1961) 207; b) M. K. Seely, R. S .
[7] a) C. Bovt, E. E. Conn, .
Criddle, E. E. Conn, ibid. 241 (1966) 4457.
[XI U. Niedermeyer, M.-R., Kula. Patentanmeldung P 3823866 (July 14,
1988).
191 J. W. Baker, W. L. Bright, J. Am. Chem. Sac. 71 (1949) 1089.
[lo] Rohm Pharma: Produktinformation und DotenblLtter zu Eupergit C and
Eupergit C 250L. Darmstadt 1987.
I l l ] U. Kragl, U. Niedermeyer. C. Wandrey, M -R. Kula, Enzvmr Eng. f0.in
press.
[12] M:L. Anhoury. P. Crooy, R. De Neys, J. Eliars, J. Chem. Sor. Chem.
Commun. 1974, 1015.
[l3] W. Konig, I. Benecke, S. Severs, J: Chromurogr. 238 (1982) 427.
Verlugsgesellschafi mbH. D-6940 Weinheim, I990
OS70-0833/90~0404-0387
S 02 50/0
381
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