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SOLUBILIZATION OF KETONES IN DIMETHYLSULFOXIDE FOR
USING AS ALCOHOL DEHYDROGENASE FROM SACCHAROMYCES
CEREVISIAE SUBSTRATES
University of São Paulo, School of Pharmaceutical Sciences, Department of Biochemical and
Pharmaceutical Technology, Av. Prof. Lineu Prestes, 580, B.16, 05508-000, São Paulo, SP,
Brazil.
ABSTRACT
Alcohol dehydrogenases (ADH) are catalysts capable of converting
alcohols to ketones and vice-versa depending on the pH of the reaction
medium. Each enzyme has its specificity in reference to ketones and
alcohols, as well as requiring specific coenzymes (β-NAD, β-NADP,
β-NADH and β-NADPH). However, the most interesting pro-chiral
ketones (acetophenone, 2-hexanone, for example) to be converted into
chiral alcohols (1-phenylethanol, 2-hexanol) are poorly soluble in
water. In this paper, the use of dimethylsulfoxide (DMSO) mixed with
water was considered for improving the solubility of the substrate and
its influence on ADH from Saccharomyces cerevisiae (ADHsc).
Among other results, pure acetophenone and 2-hexanone at a dilution
of 1:40 and 1:20, respectively, were solubilized in a 60% (v/v)
DMSO/water solution. The -NAD/-NADH recycling was also
clearly observed through using a membrane reactor (MR) having
ADHsc (150U) and 1mM -NADH inside it and fed alternately with acetone and 2-propanol.
A similar behavior was observed operating the MR under the same conditions, but fed
alternately with acetophenone and 1-phenylethanol. Thereby, the use of MR, ADHsc and -
NADH for the attainment of chiral alcohols from pro-chiral ketones was adequate.
KEYWORDS: Alcohol dehydrogenase, dimethylsulfoxide, coenzymes, prochiral-ketones.
World Journal of Pharmaceutical Research SJIF Impact Factor 6.805
Volume 5, Issue 5, 148-159. Research Article ISSN 2277– 7105
*Corresponding Author
DOI: 10.20959/wjpr20165-6127
149
INTRODUCTION
Alcohol dehydrogenases are a class of enzymes which catalyses the oxidation and reduction
reactions, requiring coenzymes (NAD(H) and NADP(H), for instance) as cofactors in order to
convert carbonyl compounds into alcohols and vice-versa. The direction of the reaction
(alcohol carbonyl compound) depends on the pH of the reaction medium, i.e., at an acidic
pH (carbonyl compound alcohol) and at an alkaline pH (alcohol carbonyl compound).
Regardless of the direction of the reaction, the recycling of the coenzyme is an indispensable
step for the success of the reaction for the coenzyme is very expensive when compared to the
enzyme. The recycling of the coenzyme can be made by either using a coupled bi-enzymatic
system [1]
or by changing one of the reaction parameters (pH, temperature, ionic strength)
using one enzyme. [2]
In the case of microbial alcohol dehydrogenases, by alternating the pH
of the medium (acid/alkaline), the coenzyme recycling may be achieved because the reduced
or oxidized form of the coenzyme would be regenerated by setting the pH of the reaction
medium above or below 7.0, respectively. Alcohol dehydrogenases are important catalysts for
attaining useful intermediate compounds in organic synthesis, mainly when it is considered
the preparation of chiral alcohols, which are employed for synthesizing a large variety of
complex molecules, such as those used as medicines. [3]
Chiral alcohols are always derived directly from the reduction of a pro-chiral ketone by the
alcohol dehydrogenase (ADH). [4]
However, pro-chiral ketones of interest (acetophenone, 2-
hexanone, for instance) are not fully soluble in water, which, in turn, promotes an
inefficiency on the overall enzyme catalysis.
This work dealt with the solubilization of ketones in mixtures of dimethylsulfoxide (DMSO)
and water whose proportions ranged from 10% (DMSO/H2O) up to 60% (DMSO/H2O) as
well as with the potential interference of DMSO on the method for alcohol determination and
ADH catalysis.
Alcohol dehydrogenase (NAD(H)-dependent; EC.1.1.1.11) from Saccharomyces cerevisiae
and the coenzymes (β-NAD and β-NADH) were purchased from Sigma-Aldrich (St. Louis,
MO, USA). The UF-membrane (cut off of 500 Da) was kindly donated by Bioengineering ®
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AG (Wald, Switzerland). All of the other chemicals used were also of the highest analytical
grade.
METHODS
Enzyme Assay
The enzyme activity was determined at 25 o C through a Beckman DU 640 spectrophotometer
by following the change in absorbance of β-NADH at 340nm. The standard assay for the
ketone reduction was carried out adding an amount of enzyme, enough to attain a linear
disappearance of the coenzyme for a 60s reaction, into a preheated assay mixture containing
200mM Bis-TRIS buffer (pH 6.5), 1mM β-NADH and 10 mM ketone (2-hexanone,
acetophenone or cyclohexanone).
One unit of ADHsc (U) represented 1mmol of β-NADH consumed per minute at 30 o C on a
basis of an absorption coefficient of 6.22 mM -1
.cm -1
for β-NADH at 340nm.
The enzyme activity (EA), expressed as U/mL, was calculated through the equation:
EA = [(Abs/min).V] (Vadh .6.22) (Eq. 1)
Where V = reaction volume and Vadh = volume of the enzyme solution.
Regeneration of Coenzyme
The process of coenzyme regeneration was based on applying the reduction and the oxidation
activities presented by ADHsc. The strategy consisted of taking a 3mL-cuvette (length path =
1cm) in which 200μL of 200mM Bis-TRIS buffer (pH 6.5), 400 μL of 10 mM ketone (2-
hexanone, acetophenone or cyclohexanone), 100μL of ADH (0.75U) and 100μL of 0.03 mM
β-NADH were added. The reaction was followed for 30s, when the β-NADH was exhausted.
Then, one drop of NaOH 1M was added and the formation of β-NADH occurred up to 40s,
followed by the addition of one drop of HCl 1M after which the β-NADH diminished again.
The cycle NaOH/HCl was repeated for five times in all.
Solubilization of Ketones in the Presence of Dimethylsulfoxide (DMSO)
Dimethylsulfoxide (DMSO) was mixed with water in order to attain solutions at
concentrations of 10%, 20%, 30%, 40%, 50% and 60% (v/v). Acetophenone (d = 0.812
g/mL), 2-hexanone (d = 0,812 g/mL) or cyclohexanone (d = 0.95 g/mL) were diluted in
aqueous DMSO solutions at proportions of 1:10 to 1:60, 1:10 to 1:50 and 1:10 to 1:20,
respectively. The criterion for considering the complete dissolution of the ketone was to
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observe the disappearance of the turbidity. Just for a comparison purpose, 1-phenylethanol
and 2-hexanol were also diluted in aqueous DMSO solutions both at the proportion of 1:10 to
1:30.
Membrane Rector
The membrane reactor used, BEMR (Bioengineering, AG, Wald, Germany) is a 10 mL
reactor, there being a 316L-stainless steel cylinder, on its bottom part, also an inlet and an
outlet for the external water bath for the temperature control. On the bottom part of the
reactor, there is also a port occluded with a silicone layer, from which samples from the
reactor content can be taken in order to measure the coenzyme form present (β-NAD and/or
β-NADH) at a given moment of the continuous process. The samples (20 µL) were taken by
using a syringe with a very small and fine needle. The diameter of the UF-membrane was
63mm. The reactor can be sterilized (autoclave up to 134 o C for 30 min) and resists high
temperatures (up to 150 o C) and also corrosion caused by most substances (except strong
acids, pH <1.0; and alkalies, pH > 12.0).
Membrane Reactor Tests
In the continuous bioconversion tests, the soluble coenzyme (β-NAD/ β-NADH; 1mM) and
ADH (150U) were poured into the BEMR which was coupled to a 500 Da-ultrafiltration
membrane. In exploratory tests, the reactor was fed continuously and alternately with a
solution of 2-propanol (20mM) and acetone (20mM). Each substrate was prepared with the
correspondent phosphate buffer, i.e., 10mM phosphate buffer (pH 8.8) and 50mM phosphate
buffer (pH 6.5), respectively, for 2-propanol and acetone. The main tests were made by
continuously and alternately feeding the reactor with acetophenone (10mM) and 1-
phenylethanol (10mM). The test conditions were maintained constant at 30 o C, 120 rpm and
feeding rate of 2 mL/h.
The concentration of the substrate or the product was measured from the outlet samples
collected every 2h. The β-NAD and β-NADH amounts were determined by taking samples
from the reactor alternately fed with acetone and 2-propanol at every five residence times
(equivalent to 25h), when the amount of the alcohol reached approximately the maximal and
minimal values, points in which the β-NAD and β-NADH amounts were high and low,
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Analytical Techniques
was determined by the dichromate method previously described by Isarankura-Na-Ayudhya
et al. and modified by Vitolo and Yoriyaz. [5-6]
Determination of ketones
and modified by Vitolo and Yoriyaz. [6-7]
Coenzyme determination
Both coenzymes (β-NAD and β-NADH) in the MR were determined by taking samples from
the solution inside the reactor and reading their absorbance at 260nm and 340nm through a
spectrophotometer.
RESULTS
Table 1 shows the solubility in aqueous dimethylsulfoxide (DMSO) of alcohols and ketones
at different concentrations. This evaluation was important because those compounds are not
fully miscible in pure water, thus the ADHsc activity could be affected.
As the compounds (ketones and alcohols) used in this work might be diluted in a mixture of
DMSO and water. Considering that the dimethylsulfoxide is an organic solvent, making the
standard curve for their measurement is peremptory (alcohols were determined by the
dichromate method, whereas ketones were made by the 2,4-dinitrophenylhydrazine method)
in the presence of the minimal amount of DMSO in which they become soluble (Figure 1).
Table 1. Solubility of ketones and alcohols at different concentrations and DMSO/water
mixtures (expressed as v/v percent). The symbols (-), (+*) and (+) mean insoluble,
partially soluble and soluble, respectively.
Compound Compound DMSO/water (v/v)
Acetophenone
153
1:60 + + + + + +
2-Hexanone
1:10 - - +* +* + +
1:20 +* + + + + +
1:30 + + + + + +
Figure 1. Standard curves in the presence of DMSO for alcohols and ketones measured
through the dichromate and 2,4-dinitrophenylhydrazine methods, respectively. 2-
hexanol [(); dil. 1:10 in 50% DMSO/water], 1-phenylethanol [(); dil. 1:10 in 60%
DMSO/water], cyclohexanone [(); dil. 1:10 in 50% DMSO/water]; 2-hexanone [(o);
dil. 1:20 in 60% DMSO/water] and acetophenone [(); dil. 1:40 in 60% DMSO/water].
The numbers of the concentration scale related to 2-hexanol, 1-phenylethanol and 2-
hexanone must be divided by ten.
After establishing the standard curves related to the analytical methods for measuring
alcohols and ketones in the presence of DMSO, thus to verify if DMSO could interfere on the
ADHsc activity is also peremptory.
The ADHsc activity on the conversion of 1-phenylethanol, 2-hexanol, acetophenone, 2-
hexanone and cyclohexanone was determined by preparing a 10 mM solution of each
compound in 10% and 60% of the DMSO/water mixture (Table 2).
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Alcohol dehydrogenases, as already enhanced, have the capability of catalyzing both
oxidation and reduction reactions, depending on the pH of the medium. However, this
capability is inherently related to the β-NAD/β-NADH interconversion, and it becomes
important to verify whether the selected alcohol dehydrogenase for the catalysis has or not
this behavior. The alcohol dehydrogenase from Saccharomyces cerevisiae (ADHsc) employed
in this work presented this capability, as can be visualized from the oscillatory profile shown
in Figure 2.
Figure 2. Alternate conversion of β-NADH and β-NAD during the oxi-red reaction
catalyzed by alcohol dehydrogenase from Saccharomyces cerevisiae using acetophenone
(), cyclohexanone () and 2-hexanone () as substrates.
Table 2. Activity of ADHsc (U/mLenzima) measured by using as a substrate, a 10 mM
solution of 1-phenylethanol, 2-hexanol, acetophenone, 2-hexanone and cyclohexanone in
10% and 60% DMSO/water mixture.
In order to enhance the β-NAD/β-NADH interconversion through a real process, a test in
which both substrates (2-propanol and acetone, used as models) for the ADHsc were fully
soluble in water and were added alternately into a membrane reactor as planned. Under this
condition, an oscillatory behavior is observed insofar as β-NADH converts to β-NAD and
Substrate Activity of ADHsc, (U/mLenzyme)
Water DMSO 10% DMSO 60%
1-Phenylethanol (dilution 1:10) - - 3.21x10 -2
2-Hexanol (dilution 1:30) 1.44x10 -2
2.06x10 -2
0.78x10 -2
4.24x10 -2
13.2x10 -2
16.2x10 -2
25.1x10 -2
14.2x10 -2
5.05x10 -2
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vice-versa. The data are shown in Figure 3. At every 25h, a sample was taken from the
reaction medium in order to determine the concentration of each coenzyme (Table 3).
Figure 3. The profile on the continuous conversion in the membrane reactor fed with 20
mM acetone and 20 mM 2-propanol, which were added alternately. Conditions of
reaction: 30 o C, feeding rate = 2 mL/h, 120 rpm, UF-membrane = 500 Da and 50 mM
phosphate buffer [pH 6.5 (acetone solution)] and 10 mM phosphate buffer [pH 8.8 (2-
propanol solution)]. The up-directed arrows indicate a 2-propanol addition whereas the
down-directed arrows indicate an addition of acetone.
TABLE 3. β-NAD and β-NADH concentrations every 25 h of continuous feeding of the
membrane reactor with acetone and 2-propanol alternately.
T Coenzyme (mM)
(h) β-NAD β-NADH
0 0 1.0
25 0.92 0.080
50 0.17 0.83
75 0.88 0.12
100 0.20 0.80
Finally, in order to demonstrate that a similar behavior may be achieved, a test has been
envisaged in which the pro-chiral ketone (acetophenone) and its correspondent chiral alcohol
(1-phenylethanol) were continuously and alternately added into a membrane reactor under
similar operational conditions used for the acetone/2-propanol case described above
(Figure 4).
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Figure 4. The profile on the continuous conversion in the membrane reactor fed with 10
mM acetophenone and 10 mM 1-phenylethanol, which were added alternately.
Conditions of reaction: 30 o C, feeding rate = 2 mL/h, 120 rpm, UF-membrane = 500 Da
and 50 mM phosphate buffer [pH 6.5 (acetophenone solution)] and 10 mM phosphate
buffer [pH 8.8 (1-phenylethanol solution)]. The up-directed arrows indicate 1-
phenylethanol addition whereas down-directed arrows indicate an addition of
acetophenone.
Table 4. Solubility of alcohols and ketones taking into account both the minimum
dilution and DMSO/water ratio.
Acetophenone 1:40 60
2-hexanone 1:50 10
Cyclohexanone 1:20 20
2-hexanol 1:20 20
1-phenylethanol 1:20 40
From Figure 1, in all conditions considered, a linear relation between absorbance (Abs) and
concentrations (S) of alcohols and ketones occurred. The correspondent minimal linear
square regression equations are:
.S + 0.016 r = 0.9995
[1-phenylethanol] Abs590nm = 89x10 -5
.S + 0.194 r = 0.9991
[Cyclohexanone] Abs480nm = 243x10 -5
.S + 0.0757 r = 0.9996
[2-hexanone] Abs480nm = 60.5x10 -5
.S + 0.117 r = 0.9993
[Acetophenone] Abs480nm = 111x10 -5
.S + 0.321 r = 0.9995
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DISCUSSION
Table 1 shows that the solubility of ketones and alcohols varied according to both
DMSO/water ratio and concentration of the compound. The pairs of values, in which the
lowest dilution of the compound is related to the lowest percentage of DMSO/water ratio, are
the data presented in Table 4. Such a result is important because the amount of DMSO may
be reduced to a minimal to be used for diluting ketones and alcohols, which, in turn,
interferes minimally on the enzyme activity and on the analytical technique for evaluating
ketones or alcohols.
It must be borne out that the compound solubility depends on the dilution made (1:10, 1:20,
etc.) and the percentage of DMSO/water mixture (Tables 1 and 2). Thereby, for each pair
(dilution; percent of DMSO in water), the correspondent standard curve must be made.
From Figure 2, the ADHsc fulfills the criterion on which by changing the pH of the medium,
the enzyme alternates itself between oxidative and reductive mechanisms, which represent
indirectly the exchange of β-NAD/β-NADH. It is also clear that the oscillatory behavior
depends on the type of ketone employed.
From Table 2, the ADHsc activity is affected by the presence of DMSO. Regarding 2-hexanol
in 10% DMSO/water, the activity was 30% and 62% higher than in water and in 60%
DMSO/water, respectively. Probably, in this case, the DMSO acted as an inhibitor of the
ADHsc. However, the ADHsc always increased when 60% DMSO/water was used as the
diluents for acetophenone and 2-hexanone, whereas an activity diminution occurred when the
substrate was cyclohexanone. Besides, for this ketone, the highest activity occurred in the
water instead of in the presence of DMSO at any concentration. Thereby, the DMSO reduced,
to some extension, the affinity of the enzyme by cyclohexanone. It must be borne out that the
more immiscible ketone in water, the more DMSO might be added with water in order to
attain a complete dissolution - the ADHsc activity was 3.2 and 1.6 times higher for
acetophenone and 2-hexanone, respectively.
The oscillatory profile of 2-propanol concentration, observed from Figure 3, along 100h-
continuous feeding of the membrane reactor, in which acetone and 2-propanol were
alternately added, is a clear confirmation of the β-NAD/β-NADH recycling during the
process, as already predicted from the previous data (Figure 2). Table 3, in turn, shows the
concentration of both forms of coenzyme every 25h. A clear coincidence occurred between
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the high amount of β-NAD present inside the reaction medium and the moment from which
the 2-propanol began to be added. Contrarily, the high amount of β-NADH coincided with
the beginning of the acetone addition. The average yield for acetone/2-propanol conversion
was about 87%.
The oscillatory profile observed in Figure 4 (alternate feeding of acetophenone and 1-
phenylethanol) is quite similar to that shown in Figure 3 (alternate feeding of acetone and 2-
propanol), which showed the ADHsc capability for catalyzing indistinctly the conversion of
ketones to alcohols and vice-versa. The necessary and sufficient condition for shifting the
conversion (ketone alcohol) is setting the pH of the reaction medium below or above 7.0,
depending on the substrate being a ketone or an alcohol, respectively. The average yield for
acetophenone/1-phenylethanol conversion was about 79%.
CONCLUSION
The data presented allow concluding that ketones such as cyclohexanone, acetophenone and
2-hexanone and alcohols as 2-hexanol and 1-phenylethanol became fully soluble when
diluted from 1:10 to 1:60 in DMSO/water solution, in which the proportion DMSO:water
ranged from 10% to 60% (v/v). The presence of DMSO seemed not to affect the analytical
measurement of ketones and alcohols. However, the ADHsc varied according to the
DMSO/water proportion as well as its depending on the type of substrate employed. The
ADHsc showed plentiful capability on shifting (2-propanol acetone) and (acetophenone
1-phenylethanol) according to the pH of the medium, besides coupling the conversion with
the recycling of the coenzyme forms (-NAD -NADH). By feeding the membrane
reactor alternately with acetone/2-propanol or acetophenone/1-phenylethanol, the average
conversion yields attained have been 87% and 79%, respectively.
ACKNOWLEDGEMENTS
This work was financially supported by CNPq (Conselho Nacional de Desenvolvimento
Científico e Tecnológico), Brazil. The authors thank Philip Barsanti for his invaluable and
constructive assessment of the manuscript.
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