Enzymatic enantioseparation of bicyclo[3.3.1]nonane-2,6-diones

Article abstract of DOI:10.1007/s007060050311

Preparative enantioseparation of the (+)-and (-)-enantiomers of bicyclo[3.3.1]nonane-2,6-dione was performed by means of a horse liver alcohol dehydrogenase catalyzed reduction, coupled with the regeneration of the coenzyme NAD by dithionite or ethanol.

Full text of DOI:10.1007/s007060050311

Monatshefte fuÈr Chemie 130, 1513±1517 (1999)
Enzymatic Enantioseparation
of Bicyclo[3.3.1]nonane-2,6-diones
1
2;Ã
Ç
Ç1
Jule Malinauskiene , Petras Kadziauskas , Albertas Malinauskas
,
and Juozas Kulys3
1
Department of Organic Chemistry, Vilnius University, LT-2734 Vilnius, Lithuania
Institute of Chemistry, LT-2600 Vilnius, Lithuania
2
3
Institute of Biochemistry, LT-2600 Vilnius, Lithuania
Summary. Preparative enantioseparation of the (‡)- and ( )-enantiomers of bicyclo[3.3.1]nonane-
2,6-dione was performed by means of a horse liver alcohol dehydrogenase catalyzed reduction,
coupled with the regeneration of the coenzyme NAD by dithionite or ethanol.
Keywords. Bicyclo[3.3.1]nonanes; Enantioseparation; Enzymatic reduction; Alcohol dehydrogen-
ase; Stereochemistry.
Enzymatische Enantiomerentrennung von Bicyclo[3.3.1]nonan-2,6-dionen
È
Zusammenfassung. Eine praparative enzymatische Trennung der (‡)- und ( )-Enantiomeren von
Bicyclo[3.3.1]nonan-2,6-dion unter Verwendung von Pferdeleber-Alkoholdehydrogenase, verbunden
mit In-situ-Regenerierung des Koenzymes NAD mit Dithionit und Ethanol, wurde durchgefuÈhrt.
Introduction
Bicyclo[3.3.1]nonanes have attracted considerable interest in recent years.
Carbonyl derivatives of bicyclo[3.3.1]nonane are considered as precursors in
organic synthesis and useful models in conformational analysis. The introduction
of a carbonyl group into de®nite positions of the bicyclo[3.3.1]nonane skeleton
induces chirality of the latter. Thus, the existence of enantiomers of carbonyl
derivatives of bicyclo[3.3.1]nonane is possible. Among these compounds,
bicyclo[3.3.1]nonane-2,6-dione (1) seems to be of great interest. Enantiomerically
enriched (‡)-1 was synthesized for the ®rst time by reductive desulfurization of 2-
thiaadamantane-4,8-dione, and was obtained in optically active form (ee ˆ 2%) by
means of preparative chromatography on acetylcellulose [1]. Later, (‡)-1 was
obtained by oxidation of the corresponding enantiomeric diols prepared by
chemical resolution [2]. The absolute con®guration of (‡)-1 was assigned as
(1S,5S) [1, 2]. On a preparative scale, the resolution of the enantiomers of 1 was
performed by stereoselective reduction using bakers yeast [3]. (‡)-(1S,5S)-1 was
Ã
Corresponding author

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J. Malinauskiene et al.
1514
obtained in high enantiomeric excess (60±93%) as the remaining enantiomer,
non-active in yeast-catalyzed reduction, whereas ( )-(1R,5R)-1 was prepared with
an ee of 58% by oxidation of the yeast-catalyzed reaction products [3].
Chromatography of 1 on chiral microcrystalline triacetylcellulose yielded
enantiomerically pure ( )- and (‡)-diones [4]. Their absolute con®gurations were
assigned as ( )-(1R,5R)-1 and (‡)-(1S,5S)-1 based on circular dichroism spectra
[4] and by means of computational methods [5, 6]. Up to now, the preparative
synthesis of enantiomeric bicyclo[3.3.1]nonane-2,6-diones still remains an
unsolved problem.
Biocatalysis is often considered as one of the most promising synthetic
techniques in synthesis and resolution of enantiomers [7]. One of the most
prominent biocatalysts in this respect is horse liver alcohol dehydrogenase
(HLADH) which is able to catalyze reversible redox reactions between coenzyme
NAD or NADH and a wide variety of alcohols and ketones [8]. The aim of the
present work was a preparative resolution of (Æ)-1 into its enantiomers using of
HLADH-catalyzed redox transformations.
Results and Discussion
Spectrokinetic investigations showed that 1 possessed activity as a substrate in
HLADH-catalyzed reduction by coenzyme NADH. The reaction rate (v) in these
experiments was recorded monitoring the disappearance of the NADH absorbance
band in the near UV region and plotted in Lineweaver-Burk coordinates (1/v vs.
1/c) as a function of the concentration of 1 (Fig. 1). A linear dependence thus
obtained could be treated in a usual way following Michaelis-Menten kinetics,
yielding a Michaelis constant value of KM ˆ 57 mM.
Since the expensive coenzyme NADH is consumed in the HLADH-catalyzed
reduction of 1 in equimolar quantity, the preparative synthesis is in need of
a coupled continuous regeneration of the coenzyme. In the present work,
Fig. 1. Lineweaver-Burk plot for HLADH-catalyzed reduction of 1; for details, see Experimental

Enzymatic Enantioseparation of Bicyclo[3.3.1]nonane-2,6-diones
1515
regeneration of NADH was performed following two different ways, either by
addition of dithionite (method A) or by using an excess of ethanol (method B). In
‡
the latter case, the oxidized form of the coenzyme (NAD ) is regenerated to its
reduced form (NADH) in the enzymatic reaction catalyzed by HLADH according
to Eqs. (1) and (2).
‡
‡
1 ‡ NADH ‡ H ! Products ‡ NAD
ꢀ1†
ꢀ2†
‡
‡
NAD ‡ C2H5OH ! NADH ‡ H ‡ CH3CHO
Since both reactions are catalyzed by the same enzyme, a coupled substrate
recycling results in the reduction of 1 by ethanol (Eq. (3)).
1 ‡ C2H5OH ! Products ‡ CH3CHO
ꢀ3†
In case of a molar excess of the reducing agent (i.e. dithionite or ethanol) relative to
1, the equilibrium of the coupled process (3) should be strongly shifted towards the
products of enzymatic reduction of 1. In the present work, this was achieved by the
use of a 16-fold molar excess of dithionite (method A) or a 42-fold molar excess of
ethanol (method B). The performance of a coupled process with the regeneration of
coenzyme allows to use much lower amounts of NAD than demanded by the
stoichiometry of Eq. (1). In the present work, a molar ratio of NAD to 1 as low as
10:1 or 30:1 was used, permitting to perform a preparative synthesis in the range of
ca. 1 g of 1.
It was established by thin layer chromatography that, even at long incubation
times, the amount of converted 1 did not exceed 30 to 50%. After the conversion is
completed, 6-hydroxy-bicyclo[3.3.1]nonane-2-one (2) was found as the main
reaction product next to a small amount of bicyclo[3.3.1]nonane-2,6-diol (3). The
amount of 3, as compared to that of 2, was found to be higher upon using method
B. Also, a higher degree of conversion of 1 to 2 and 3 was observed in this case. It
therefore follows that HLADH-catalyzed regeneration of NAD by means of ethanol
is more ef®cient than the dithionite-assisted process.
After the enzymatic conversion was completed, unreacted 1 could be easily
extracted from the reaction mixture by means of organic solvents and puri®ed by
column liquid chromatography. Unreacted 1 obtained in this way displayed optical
activity which increased with increasing conversion degree of 1 up to 50%.
Optically active ( )-1 obtained in this way showed lower activity in an enzymatic
reaction when compared to racemic 1. ( )-1 obtained by method A showed an
[ꢀ]D value of 66ꢁ and a relative activity in the HLADH-catalyzed reaction of
59% as compared to racemic 1, whereas the corresponding values for ( )-1
obtained by method B were 149ꢁ and 37%.
It could be concluded from this result that only (‡)-1 undergoes an enzymatic
reduction in the HLADH-catalyzed reaction, whereas ( )-1 remains unconverted.
The extrapolation of [ꢀ]D and the relative activity in enzymatic reaction to zero for
the latter, provided for the samples of ( )-1 and obtained by varying of reaction
conditions, yield an [ꢀ]D value of 197ꢁ for enantiomerically pure ( )-1. The
value obtained is closely related to that reported for (‡)-1 ([ꢀ]D ˆ ‡191ꢁ [2]). The
oxidation of the main enzyme reaction product 2 by dichromate following the well-
known Jones procedure yielded (‡)-1 with [ꢀ]D ˆ ‡119ꢁ. The interconversions of
the bicyclic compounds studied are shown in Scheme 1.

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J. Malinauskiene et al.
1516
Scheme 1
Fig. 2. Projections of (1S,5S)- and (1R-5R)-enantiomers of 1 into a `diamond lattice' model of the
active center of HLADH; the arrows indicate the site of enzyme-catalyzed reduction of the keto
group; undesirable positions are marked by full circles
It was of great interest to assign the absolute con®guration of the enantiomers
obtained. Following a known assignment [1, 2] (‡)-1 can be de®ned as (‡)-
(1S,5S)-1. Thus, the non-active in enzymatic reaction enantiomer can be assigned
as ( )-(1R,5R)-1. The same conclusion can be drawn also following an
independent way, based on the `diamond lattice' model of the active site of
HLADH [10]. Figure 2 shows the projections of (1S,5S)-and (1R,5R)-enantiomers
into a `diamond lattice' of HLADH. It can be seen that the (1S,5S)-enantiomer can
be placed into the `diamond lattice' in such a way that no carbon atom of its
skeleton occupies an undesirable position. In contrast, placing (1R,5R)-1 into this
model requires the occupation of one of the undesirable positions (as indicated by
an asterisk in Fig. 2). Thus, from both enantiomers of 1, only the (1S,5S)-
enantiomer can be bound into an active center of HLADH enabling an enzyme-
catalyzed reaction to proceed.

Enzymatic Enantioseparation of Bicyclo[3.3.1]nonane-2,6-diones
1517
Experimental
Horse liver alcohol dehydrogenase (activity: 2 units/mg) in the form of a suspension in ammonium
sulfate solution containing 19 mg/cm3 of enzyme and oxidized and reduced forms of the coenzyme
nicotineamide adeninedinucleotide (NAD and NADH) were obtained from Reanal (Hungary) and
used as received. (Æ)-Bicyclo[3.3.1]nonan-2,6-dione (1) was synthesized according to a known
procedure [9].
The kinetics of the enzyme-catalyzed reduction of 1 was studied using spectrophotometer
Specord UV-Vis (Germany) equipped with a thermostatted cuvette holder. All kinetic experiments
were performed at 25ꢁC. For kinetic investigations, 1 cm3 of a solution of 1 in 0.1 M phosphate buffer
(pH ˆ 7.5) was mixed with 0.112 cm3 of 5 mM NADH solution in the same buffer. The total
concentration of 1 in the kinetic experiments was varied within the range of 4 to 40 mM. The reaction
was started by addition of 0.087 cm3 of HLADH solution containing 0.033 mg of the enzyme. The
rate of enzymatic oxidation of NADH was monitored at 370 nm. The optical activity of the solutions
of 1 in dioxane (c ˆ 0.2±0.3 g in 100 cm3 of the solvent) was recorded in a 10 cm path length cuvette
by means of a Perkin-Elmer model 141 polarimeter.
Preparative synthesis of optical active enantiomers of 1 was performed according to two different
methods as follows:
Method A. 456 mg (3 mmol) of 1 was dissolved in 250 cm3 of 0.1 M phosphate buffer (pH ˆ 7.5)
together with 210 mg (0.3 mmol) of NAD and 8.7 g (50 mmol) of sodium dithionite. Then, 2 cm3 of
HLADH suspension containing 38 mg of the enzyme were added. The reaction mixture was kept for
24 h at 30ꢁC, then evaporated in vacuo to a ®nal volume of ca. 50 cm3, and extracted exhaustively
with CHCl3. The extract was dried over MgSO4 and evaporated in vacuo. The rest was dissolved in a
minimum volume of CH2Cl2 and fractionated by liquid chromatography (solvent: CH2Cl2) on an
alumina (2nd grade of activity) column. The fraction containing 1 (Rf ˆ 0.69) was dried, evaporated,
and handled in the usual manner. Yield: 170 mg; [ꢀ]D ˆ 66ꢁ.
Method B. A similar procedure was used, except that a lower amount of NAD (70 mg; 0.1 mmol) and
7.2 cm3 (125 mmol) of ethanol instead of sodium dithionite were added to the reaction mixture. Yield
182 mg; [ꢀ]D ˆ 149ꢁ.
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Received April 19, 1998. Accepted (revised) June 28, 1999