part 1, section 3 applications of stenotrophomonas...

PART 1, SECTION 3

Applications of Stenotrophomonas maltophilia Lipase in the

Preparation of Enantiomerically Enriched Compounds

Part I, Section 3, Resolution of 2-arylpropanoates:Results

In the previous section, we have described the characterization of a new lipase

from Stenotrophomonas maltophilia. In this section, we describe the applications of

Stenotrophomonas maltophilia in the preparation of enantiomerically enriched

compounds on preparative scale. We decided to explore biocatalysis by whole cells of

Stenotrophomonas maltophilia for the following reasons:

(i) The lipase has been shown to be present on the surface of the cells of

Stenotrophomonas maltophilia (Section 1.2.5). The presence of an

enantioselective lipase on the surface of a cell is a highly desirable

property from practical point of view as it eliminates the need for

expensive isolation, purification and stabilization of the protein. Moreover,

the penetration of the substrate into the cell and excretion of the product

into the media is unlikely to be an issue when lipase is present on the

surface of the cell.

(ii) The disadvantages of using whole cell biocatalysis include reduction in

e. e. because of the presence of other intracellular enzymes of opposite or

relaxed specificity. But the lipase of Stenotrophomonas maltophilia has

been shown to be present on the surface of the cells, we, therefore,

envisaged that intracellular esterases, even if present, are unlikely to

compete with the surface bound enzyme.

Several publications in the recent past have described the expression of known

lipases on the surface of fusion proteins where several expression systems have been

developed to display lipases and other proteins on the surface of bacteria, fungi or

mammalian cells by appropriately fusing them to surface anchoring motifs. 132-139 The

outer membrane proteins have been widely used as anchoring motifs, because they

have unique membrane-spanning structures. Several membrane proteins including

OmpA, OprF, OmpS, invasin, LamB, PhoE, OmpC and FadL have been used as

anchoring motifs. 139'147

'148

Traditional substrates, 2-methyl and 2-hydroxy substituted aromatic esters

were selected for testing the substrate specificity of the enzyme. In addition, aliphatic

ester, ethyl2-chloropropanoate was also tested.

1.3.1 Stenotrophomonas maltophilia catalyzed kinetic resolution of 2-

arylpropanoates: formation of enantiomerically pure (S)-acids

We started our investigation by studying the biocatalyzed hydrolysis of methyl

2-( 4-isobutylphenyl)propanoate (ibuprofen methyl ester; 39). Stenotrophomonas

38

Part 1, Section 3, Resolution of2-arylpropanoates:Results

maltophilia was grown in medium comprising of peptone (1.0%) and beef extract

(0.5%) at 30 oc for I8-22 has described in experimental section. Cells were harvested

by centrifugation and washed with phosphate buffer (50 mM, pH 7.0). The washed

cells were resuspended in IOO mL of same buffer at a concentration of 0.1 g/mL. To

the cell suspension, racemic methyl 2-( 4-isobutylphenyl)propanoate (39) at 20 mM

concentration was added and the contents incubated at 37 °C on an orbital shaker at

200 rpm. The reaction was stopped at 48% conversion (28 h; Scheme I4). Progress of

the reaction was monitored by TLC. pH of the reaction mixture was adjusted to 8.0

with IN NaOH. The remaining ester was removed by extraction with ethyl acetate.

The aqueous layer was acidified to pH 2.0 with IN HCl and extracted with ethyl

acetate to recover acid formed during the reaction. The organic layer was separated,

dried over sodium sulphate and evaporated to give 40 in 45% yield, which was

purified by column chromatography over silica-gel (200-400 mesh; ethyl

acetate/hexane, 2:3).

Scheme 14

39 (S)-40, e. e. - 97% (R)-39

The structure of 40 was in agreement with its 1HNMR spectral data. The

absence of singlet for -OCH3 protons at 8 3.64 in the 1HNMR of the product

confirmed that ester hydrolysis has occurred. 2-methyl protons and single proton at C-

2 appeared as doublet (J=7.2 Hz) at 8 1.55 and quartet (J=7.2 Hz) at 8 3.73,

respectively. Single methine proton and two methyl groups of the isobutyl were

present as multiplet at 8 1.84-1.97 and doublet (J=7.2 Hz) at 8 0.96, respectively. The

aromatic protons appeared as multiplet at 8 7.03-7.43, whereas methylene protons

appeared as doublet (J=7.2 Hz) at 8 2.51. Except for methine proton of isobutyl

group, all protons resonated slightly downfield as compared to ester 39. The presence

of acid was also confirmed by IR which showed carbonyl of carboxylic acid at I7I 0

cm-1• IR and 1HNMR of the product and racemic 2-(4-isobutylphenyl)propanoic acid

were indistinguishable.

39


The enantiomeric excess and absolute configuration of the acid was

determined by NMR method using (1R,2R)-1 ,2--diphenylethane-1 ,2-diamine as chiral

solvating agent as described by Fullwood et a/. 140 When 1.5 equivalent chiral

solvating agent was added to racemic 2-(4-isobutylphenyl)propanoic acid, the doublet

for the -CH3 protons at C-2 for (R) and (S) enantiomers experienced unequal shift and

appeared as two separate doublets at 8 1.46 (J=7.2 Hz) and 1.43 (J=7.2 Hz) (Figure

13a). When shift reagent was added to the acid obtained from the biocatalyzed

hydrolysis, mainly one doublet at 8 1.43 (J=7.2 Hz) could be seen in 1HNMR

spectrum (Figure 13b ). This indicated predominant formation of one enantiomer of

the acid. To rule out any artifact, racemic acid along with 1.5 equivalent chiral

solvating agent was added to the sample obtained from biocatalyzed reaction and 1HNMR was recorded again. The two doublets were observed at 8 1.43 (J=7.2 Hz)

and 8 1.46 (J=7.2 Hz) in 2:1 ratio, which confirmed the enantiopurity of the product

formed in the biocatalyzed reaction.

1.4

(a) (b)

Figure 13: 1HNMR of(a) (R,S)-2-(4-isobutylphenyl)propanoic acid and (b) (S)-2-(4-isobutylphenyl)propanoic acid obtained from biocatalyzed reaction, in the presence of 1.5 equivalent of (1R,2R)-1 ,2-diphenylethane-1 ,2-diamine

It is known in the literature/ 40 that in the presence of chiral solvating agent the

doublet for the -CH3 protons at C-2 of (S)-enantiomer resonates consistently at lower

frequency than the doublet of the corresponding (R)-enantiomer of 2-( 4-

isobutylphenyl)propanoic acid. Accordingly, the absolute configuration of the

biocatalyzed product was assigned as (S). This was further confirmed by the +ve sign

of optical rotation of the sample.

The biocatalyzed hydrolysis of ethyl 2-(3-fluorobiphenyl-4-yl)propanoate

(flurbiprofen ethyl ester; 41) was studied next. Stenotrophomonas maltophilia was

grown in rich medium as described above. Cells were harvested by centrifugation and

40

Part I, Section 3, Resolution of2-arylpropanoates:Results

washed with phosphate buffer (50 mM, pH 7.0). The washed cells were resuspended

in 100 mL of same buffer at a concentration ofO.l g/mL. To the cell suspension, ethyl

2-(3-fluorobiphenyl-4-yl)propanoate (41) at 20 mM concentration was added and the

contents incubated at 37 °C in an orbital shaker at 200 rpm. The reaction was stopped

at 48% conversion after 29 h (Scheme 15). The desired acid 42 formed in the reaction

was separated from the ester according to the procedure described above for methyl 2-

( 4-isobutylphenyl)propanoate (39) and purified by column chromatography over

silica-gel (200-400 mesh; ethyl acetate/hexane, 1 :3).

Scheme 15

41 (S)-42, e.e. > 99% (R)-41

The structure of the product was in agreement with its 1 HNMR spectral data.

The absence of a triplet (J=7.2 Hz) at 8 1.27 and quartet (J=7.2 Hz) at 8 4.2

corresponding to ester group of 41 confirmed that the hydrolysis had occurred. 2-

methyl protons and single proton at C-2 appeared as doublet (J=7.2 Hz) at 8 1.55 and

quartet (J=7.2 Hz) at 8 3.80, respectively. The aromatic protons appeared as multiplet

at 8 7.40-7.53. The presence of acid was also confirmed by IR which showed carboxyl

carbonyl group at 1698 cm-1•

The e.e. and the absolute configuration of acid was determined by NMR

method using (1R,2R)-1 ,2-diphenylethane-1 ,2-diamine as chiral solvating agent as

described. 140 When 1.5 equivalent shift reagent was added to racemic 2-(3-

fluorobiphenyl-4-yl)propanoic acid, the doublet for the -CH3 protons at C-2 for (R)

and (S) enantiomers experienced unequal shift and appeared as two separate doublets

at 8 1.46 (J=7.2 Hz) and 1.48 (J=7.2 Hz) (Figure 14a). When shift reagent was added

to the acid obtained from the biocatalyzed hydrolysis, only one doublet at 8 1.46

(J=7.2 Hz) could be seen in 1HNMR spectrum (Figure 14b). This indicated

predominant formation of one enantiomer of the acid. To rule out any artifact,

racemic acid along with 1.5 equivalent solvating reagent was added to the sample

obtained from biocatalyzed reaction and 1 HNMR was recorded again. The two

41


doublets were observed at o 1.46 (J=7.2 Hz) and o 1.48 (J=7.2 Hz) in 2:1 ratio, which

confirmed the enantiopurity of the product formed in the biocatalyzed reaction .

(a)

. s 1.4

(b)

Figure 14: 1HNMR of (a) (R,S)-2-(3-fluorobiphenyl-4-yl)propanoic acid and (b) (S)-2-(3-fluorobiphenyl-4-yl)propanoic acid obtained from biocatalyzed reaction, in the presence of 1.5 equivalent of (1R,2R)-1 ,2-diphenylethane-1 ,2-diamine

It has been established in the literature/40 that in the presence of shift reagent

the doublet for the -CH3 protons at C-2 of (S)-enantiomer resonates consistently at

lower frequency than the doublet of the corresponding (R)-enantiomer of 2-(3-

fluorobiphenyl-4-yl)propanoic acid. Accordingly, the absolute configuration of the

biocatalyzed product was assigned as (S). This was further confirmed by the +ve sign


The biocatalyzed hydrolysis of methyl 2-(3-benzoylphenyl)propanoate

(ketoprofen methyl ester; 43) was studied next under the reaction conditions described

above. The reaction was stopped at 48% conversion after 48 h (Scheme 16). The

product was isolated as described above and purified by column chromatography over

silica-gel (200-400 mesh, ethyl acetate-hexane, 1 :3).

Scheme 16

43

S. maltophilia Phosphate buffer, pi/7.o

conversion 48%

0 0

+

0

OH OCH3

(S)-44, e.e. > 99% (R)-43

Structure of the product was confirmed by 1 HNMR. The absence of singlet at

o 3.42 in the 1HNMR of the product confirmed that ester hydrolysis had occurred. 2-

methyl protons and single proton at C-2 appeared as doublet (J=7.2 Hz) at o 1.55 and

42


quartet (J=7.2 Hz) at 8 3.78, respectively. The aromatic protons appeared as multiplet

at 8 7.19-7. 81. The presence of acid was also confirmed by IR which showed carboxyl

carbonyl group at 1697 crn-1.

The e.e. and absolute configuration of the acid was determined by NMR

rnethod. 140 When 1.5 equivalent (1R,2R)-1 ,2-diphenylethane-1 ,2-diarnine was added

to racemic 2-(3-benzoylphenyl)propanoic acid, the doublet for the -CH3 protons at C-

2 for (R) and (S) enantiomers experienced unequal shift and appeared as two separate

doublets at 8 1.43 (J=7.2 Hz) and 8 1.47 (J=7.2 Hz) (Figure 15a). When shift reagent

was added to the acid obtained from the biocatalyzed hydrolysis, only one doublet at 8

1.43 (J=7.2 Hz) could be seen in 1HNMR spectrum (Figure 15b). This indicated

predominant formation of one enantiomer of the acid. To rule out any artifact,

racemic acid along with 1.5 equivalent of chiral solvating agent was added to the

sample obtained from biocatalyzed reaction and 1HNMR was recorded again. The two

doublets were observed at 8 1.43 (J=7.2 Hz) and 8 1.47 (J=7.2 Hz) in 2:1 ratio, which

confirmed the enantiopurity of the product formed in the biocatalyzed reaction.

(a) (b)

Figure 15: 1HNMR of (a) (R,S)-2-(3-benzoylphenyl)propanoic acid and (b) (S)-2-(3-benzoylphenyl)propanoic acid obtained from biocatalyzed reaction, in the presence of 1. 5 equivalent of ( 1R, 2R)-1 ,2-diphenylethane-1 ,2-diamine

It has been established, 140 that in the presence of shift reagent the doublet for

the -CH3 protons at C-2 of (S)-enantiomer resonates consistently at lower frequency

than the doublet of the corresponding (R)-enantiomer of 2-(3-

benzoylphenyl)propanoic acid. Thus, the absolute configuration of the biocatalyzed

product was assigned as (S). This was further confirmed by the +ve sign of optical

rotation of the sample.

43


1.3.2 Stenotrophomonas maltophilia catalyzed kinetic resolution of 2-

arylpropanoates: formation of enantiomerically pure (R)-acids

In the preceding section, we have described enantioselective formation of (S)

acids from the esters of 2-arylpropanoic acids. In this section, we describe the

formation of corresponding (R)-acids using the cells of same organism, but afte:r

treatment with acetone. Acetone treated cells were obtained by following method.

Stenotrophomonas maltophilia was grown as described in Section 1.3.1. The cells

were harvested by centrifugation and washed twice with phosphate buffer (pH 7.0, 50

mM). 10 g cells were then resuspended in 100 mL acetone and incubated at 30 °C for

30 min. Acetone was removed by filtration. Acetone treatment was repeated 4 times.

Finally, cells were dried under vacuum, lyophilized and stored at 4 °C.

As first example, biocatalyzed hydrolysis of methyl 2-( 4-isobutylphenyl)

propanoate (39) was studied. The acetone treated cells (1 g) were suspended in the

100 mL of phosphate buffer (pH 7.0, 50 mM). To the cell suspension, methyl 2-(4-

isobutylphenyl)propanoate (39) at 20 mM concentration was added and the contents

incubated at 37 °C in an orbital shaker at 200 rpm. The reaction was stopped at 48%

conversion after 22 h (Scheme 1 7). The desired acid was isolated, purified and

analyzed as described in Section 1.3 .1.

Scheme 17

Acetone treated S. maltophilij ~ IClf OCH3 Phosphate buffer, pH 7.0 : OH + OCH3

conversion 48% ~

39 (R)-40, e.e. > 99% (S)-39


method.140 As shown above (Section 1.3 .1 ), the 2-CH3 protons of racemic 40 in the

presence of 1.5 equivalent chiral solvating agent appeared as two doublets (J= 7.2

Hz) at 8 1.43 and 8 1.46 (Figure 16a) When shift reagent was added to the acid

obtained from the biocatalyzed hydrolysis, only one doublet (1=7.2 Hz) at 8 1.46

could be seen in 1HNMR spectrum (Figure 16b). This indicated predominant

formation of one enantiomer of the acid. To rule out any artifact, racemic acid along

with 1.5 equivalent chiral solvating agent was added to the sample obtained from

44


biocatalyzed reaction and 1HNMR was recorded agam. The two doublets were

observed at o 1.43 (1=7.2 Hz) and o 1.46 (1=7.2 Hz) in 1:2 ratio, which confirmed the

enantiopurity of the product formed in the biocatalyzed reaction.

qf )I I \ ~ I v ~ ! 1 I I V'' ) l_

(a)

1.4

(b)

Figure 16: 1HNMR of(a) (R,S)-2-(4-isobutylphenyl)propanoic acid and (b) (R)-2-(4-isobutylphenyl)propanoic acid obtained from biocatalyzed reaction, in the presence of 1.5 equivalent of (1R,2R)-1 ,2-diphenylethane-1 ,2-diamine

As stated earlier, it has been established!40 that in the presence of shift reagent

the doublet for the -CH3 protons at C-2 of (S)-enantiomer resonates consistently at

lower frequency than the doublet of the corresponding (R)-enantiomer of 2-( 4-

isobutylphenyl)propanoic acid. Accordingly, the absolute configuration of the

biocatalyzed product was assigned as (R). This was further confirmed by the -ve sign


Biocatalyzed hydrolysis of ethyl 2-(3-fluorobiphenyl-4-yl)propanoate (41)

with acetone treated cells was studied next under the reaction conditions described

above (Scheme 18). The reaction was stopped at 48% conversion after 48 h. The

desired acid 42 was isolated and purified as described in Section 1.3.1. The structure

of the product was confirmed by 1 HNMR as described before.

Scheme 18

41

Acetone treated S. ma/tophilia._ Phosphate buffer, pH 7.0

conversion 48%

(R)-42, e. e. > 99% (S)-41


method. 140 As described above (Section 1.3 .1 ), the 2-CH3 protons of racemic 42 in the

presence of 1.5 equivalent chiral solvating agent appeared as two doublets (1=7.2 Hz)

at o 1.46 and o 1.48 (Figure 17a). When shift reagent was added to the acid obtained

45


from the biocatalyzed hydrolysis, only one doublet at 8 1.48 (J=7.2 Hz) could be seen

in 1HNMR spectrum (Figure 17b). To rule out any artifact, racemic acid along with

1.5 equivalent chiral solvating agent was added to the sample obtained from

biocatalyzed reaction and 1 HNMR was recorded again. The two doublets were

observed at 8 1.46 (J=7.2 Hz) and 8 1.48 (J=7.2 Hz) in 1:2 ratio, which confirmed the

enantiopurity of the product formed in the biocatalyzed reaction.

I '

r 0

' 1\

J 'J\ 1.5 .)

(a) (b)

Figure 17: 1HNMR of (a) (R,S)-2-(3-fluorobiphenyl-4-yl)propanoic acid and (b) (R)-2-(3-fluorobiphenyl-4-yl)propanoic acid obtained from biocatalyzed reaction, in the presence of 1.5 equivalent of (lR, 2R)-l ,2-diphenylethane-1 ,2-diamine

As already stated, it has been established, 140 that in the presence of shift

reagent the doublet for the -CH3 protons at C-2 of (S)-enantiomer resonates

consistently at lower frequency than the doublet of the corresponding (R)-enantiomer

of 2-(3-fluorobiphenyl-4-yl)propanoic acid. Accordingly, the absolute configuration

of the biocatalyzed product was assigned as (R). This was further confirmed by the -

ve sign of optical rotation of the sample.

The biocatalyzed hydrolysis of methyl 2-(3-benzoylphenyl)propanoate (43)

was studied next. Reaction conditions were same as described above, the reaction was

stopped at 48% conversion after 14 h (Scheme 19). The desired acid 44 was isolated

and purified as described in Section 1.3 .1. The structure of the product was confirmed

by 1 HNMR as described before.

Scheme 19

43

Acetone treated S. maltophilia,.

Phosphate buffer, pH 7.0 conversion 48%

46

+

0

H

(R)-44, e.e. > 99% (S)-43



method. 14° From the NMR spectral data (Figure 18b) it was clear that only one

enantiomer of the acid was formed. As mentioned in literature, 140 in the presence of

shift reagent the doublet for the -CH3 protons at C-2 of (S)-enantiomer resonates

consistently at lower frequency than the doublet of the corresponding (R)-enantiomer

of 2-(3-benzoylphenyl)propanoic acid. Accordingly, the absolute configuration of the

biocatalyzed product was assigned as (R). This was further confirmed by the -ve sign


1.4 (a) (b)

Figure 18: 1HNMR of (a) (R,S)-2-(3-benzoylphenyl)propanoic acid and (b) (R)-2-(3-benzoylphenyl)propanoic acid obtained from biocatalyzed reaction, in the presence of 1.5 equivalent of (1R,2R)-1 ,2-diphenylethane-1 ,2-diamine

1.3.3 Formation of (R)-2-arylpropanoic acids in >99% e.e. at 100% conversion

with acetone treated cells of Stenotrophomonas maltophilia: Dynamic

Kinetic Resolution

As described in the preceding section, untreated whole cells of

Stenotrophomonas maltophilia enantioselectively hydrolyzed (S)-enantiomers of

methyl 2-( 4-isobutylphenyl)propanoate (39), ethyl 2-(3-fluorobiphenyl-4-

yl)propanoate (41) and methyl 2-(3-benzoylphenyl)propanoate (43), whereas acetone

treated cells of Stenotrophomonas maltophilia enantioselectively hydrolyzed (R)

enantiomers of these esters. In both the cases, the reaction was deliberately stopped at

<50% conversion to obtain high e.e. of the products. However, we were pleasantly

surprised to note that (i) the hydrolysis reaction of methyl 2-( 4-

isobutylphenyl)propanoate (39) with acetone treated cells proceeded to 100%

conversion and (ii) at 100% conversion, the e.e. of the product was >99% and the

configuration was (R). The reaction was done under same conditions as described

above for acetone treated cells, only time-period of the reaction was increased to 80-

90 h (Scheme 20). The e.e. and configuration of the product was determined as

described above. The other two esters, ethyl 2-(3-fluorobiphenyl-4-yl)propanoate

47

l

Part I, Section 3, Resolution of2-arylpropanoates:Discussion

(41) and methyl 2-(3-benzoylphenyl)propanoate (43) also produced (R) enantiomers

of the acids in >99% e.e. at 100% conversion with acetone treated cells of

Stenotrophomonas maltophilia (Scheme 21 and Scheme 22).

Scheme 20

39

Scheme 21

41

Scheme 22

43

Acetone treated S. maltophilia• OCH3 Phosphate buffer, pH 7.0

conversion 100%

Acetone treated S. maltophilia.._


Acetone treated S. maltophilia•


IGlJ_ : OH s 5

(R)-40, e.e. > 99%

(R)-42, e.e. > 99%

OH

(R)-44, e.e. > 99%

1.3.4 Discussion: Stenotrophomonas maltophilia catalyzed resolution of 2-

arylpropanoates

1.3.4.1 Either enantiomer, one strain

Enantioselective formation of either (R)- or (S)-enantiomers of 2-

arylpropanoic acids in Stenotrophomonas maltophilia catalyzed hydrolysis

of corresponding esters

The effect of solvent on the activity and selectivity of lipases has been

described in Section 1.6.9. Two possibilities exist: (i) the change in enantioselectivity

is due to alteration of enantioselectivity of lipase on treatment with acetone and (ii)

48

Part I, Section 3, Resolution of 2-arylpropanoates:Discussion

more than one enzyme with opposite selectivity are present but with greatly different

rates of hydrolysis. Acetone treatment inactivates enzyme for major activity, but has

very little or no effect on the enzyme for minor activity. But none of these appears to

be operative in our case for following reasons (i) the lipase has been purified as

described in Section 1.2.3. The pure lipase behaved in a manner similar to acetone

untreated cells of Stenotrophomonas maltophilia and exhibited enantioselectivity for

(S)-enantiomer. Treatment of pure enzyme with acetone did not result in loss of

activity or change in selectivity and (ii) the rate of reaction after treatment of

Stenotrophomonas maltophilia cells with acetone remained almost similar, only the

enantioselectivity was reversed.

The most likely explanation for the result is as follows. In Section 1.2.5 above,

the presence of a 22 kDa lipase on the surface of the cells of Stenotrophomonas

maltophilia was established by immunogold labelling studies, carried out with ultra

thin sections of Stenotrophomonas maltophilia. We have also confirmed that the pure

enzyme is enantioselective for the (S)-enantiomer of all the three esters of 2-

arylpropanoic acids tested.

Repeated treatment with acetone removes the surface bound (S)-selective

lipase from the cells. The presence of 22 kDa lipase in acetone washing was

confirmed by SDS-PAGE and activity assay. This observation is consistent with the

fact that the lipase can be extracted from the cells with 1M NaCl (Section 1.2.3). In

addition, the acetone treatment increases the penetration of substrates into the cells by

increasing the permeability of the outer membrane. This allows the substrate to

experience the activity of other intracellular enzymes. The formation of (R)

enantiomer of 2-arylpropanonic acid, in all probability is due to the presence of an

(R)-selective esterase in the cells of Stenotrophomonas maltophilia. In brief, (i)

untreated cells of Stenotrophomonas maltophilia produced (S)-acids due to the

presence of an (S)-selective lipase on the surface of the cells, (ii) Acetone treatment

removes this surface bound enzyme and increases the permeability of the outer

membrane and (iii) acetone treated cells produced (R)-acids due to the presence of an

intracellular (R)-selective esterase.

49

Part I, Section 3, Resolution of 2-arylpropanoates:Discussion

1.3.4.2 Formation of (R)-enantiomers at 100% conversion in Stenotrophomonas

maltophilia catalyzed dynamic kinetic resolution

Kinetic resolution of racemates can proceed to a maxtmum of 50%

conversion. In case of acetone treated cells high e.e. of acids was obtained at 1 00%

conversion. The yield of isolated product was 80-90%, which rules out the selective

degradation of one enantiomer. Another process called Dyanamic Kinetic Resolution

(DKR) is known in literature, in which enzymatic kinetic resolution is combined with

in situ racemization of substrate with a base, a metal, metal catalyst or an enzyme.

The details of DKR have been discussed in Section 1.6.2.

Another technique called deracemization reaction, which provides with a

route to obtain the enantiomerically pure compounds, theoretically in 100% yield

starting from the racemic mixture has been reported. In this reaction, synthesis of a

racemate is almost equal to the synthesis of the optically active compound, and this

concept is entirely different from the commonly accepted one in the asymmetric

synthesis. In this deracemization process, the chirality of either enantiomer of a

racemate is converted to the other antipode via a single or plural reactions, resulting in

an optically active compound starting from a racemic mixture as a whole. In total,

successful application of this process means that the synthesis of a racemate is almost

equal to the synthesis of an optically active compound. Most of the reported

deracemization reactions utilize oxidation-reduction process. A representative

example is the deracemization of secondary alcohols, the key being the combination

of two enzymes that catalyze the oxidation of one enantiomer followed by

enantioselective reduction to the antipode.149 Turner and co-workers reported the

deracemization of a-amino acids and a-methylbenzylamine combining the

enantioselective enzyme-catalyzed oxidation of the amine to imine and the non

enantioselective chemical reduction.150'151 There are only a few reports on the

enzymatic deracemization of a-substituted carboxylic acids with microorganisms.

Cordyceps militaris and Verticillium lecanii are capable of inverting the chirality of

the (R)-enantiomer of 2-arylpropanoic acids to the (S)-antipode, whereas,

Rhodococcus and Nocardia diaphanozonaria have opposite selectivity.152-155

50

Part I, Section 3, Resolution of2-arylpropanoates:Discussion

In rat liver, the reaction mechanism has been proposed based on various

studies with enantiomerically pure compounds and deuterated derivatives in which

three enzymes take part in this biotransformation system. 156-159 The well established

reaction mechanism is as shown below (Scheme 23).

Scheme 23

'Y'OJl : OH £ §

(R)-40

Acyl- CoA Synthetase

.,. Hydrolase

1 Net Result = Deracemization

X • .,. Hydrolase

(S)-40

~Q I ~COSCoA

I 5

(R)-45

2-Arylpropionyl-CoA Epimerase

(S)-45

The initial step of this biotransformation is considered to be the

enantioselective formation of the coenzyme A (CoA) thioester of (R)-acid. The

thioester is subsequently racemized by the aid of an epimerase and cleaved by a

hydrolase to release the free (S)-acid. Among these three enzymes, only the acyl-CoA

synthatase is enantioselective. Thus, the enantiomeric ratio in the reaction mixture

shifts to the (S)-form with the repetition of the reactions. These enzymes as well as

genes were purified and identified.

In order to check if the same mechanism ts operative m our case,

Stenotrophomonas maltophilia was subjected to reaction with (R,S)-2-( 4-

isobutylphenyl)propanoic acid (40). Acetone treated cells of Stenotrophomonas

maltophilia failed to deracemize (R,S)-2-(4-isobutylphenyl)propanoic acid (Scheme

24) even after 20 days, suggesting the absence of this mechanism in

Stenotrophomonas maltophilia.

51

Scheme 24

Part 1, Section 3, Resolution of mandelates:Results

-~~~~~~--~~~!ClA~ I o Acetone treated S. maltophilia,

OH Phosphate buffer, pH 7.0 20 days ! OH

(R,S)-40 (R)-40

The synthatase catalyzed thioester formation acyl-CoA requires the presence

of the free acid. Although no mechanistic studies have been done, we propose an

oxidation-reduction process via exomethylene type intermediate 46 for the

deracemization of esters (Scheme 25).

Scheme 25

~H ~~ ... SCoA

(S)-45 l Epim•ri,.Uon

Y'OJt :-.... ~ SCoA

(R)-45

Transesterification ~ R ~~~OCH3

(S)-39

Hydrolase .. X Y'OJt

~ OH

(R)-40

Oxidation

Reduction ~OCH3 46 CH2

! R<dn<tloo

1111111( Hydrolase ~ Q

I ~OCH3 (R)-39

However, transesterification of the ester group of 2-aryl propionates with acyl

CoA followed by epimerization and hydrolysis of CoA ester cannot be ruled out. The

results can also be explained based on DKR mechanisms, whereby the (S)-ester gets

racemized by a combination of oxidation-reduction sequence (Scheme 25).

1.3.5 Stenotrophomonas maltophilia catalyzed enantioselective resolution of 2-

hydroxyarylacetates (mandelates): formation of enantiomerically pure

(R)-2-hydroxyarylacetic acids (mandelic acids)

We started our investigation by studying the hydrolysis of ethyl mandelate

(47). Stenotrophomonas maltophilia was grown in medium comprising of peptone

(1.0%) and beef extract (0.5%). Cells were harvested by centrifugation and washed

with phosphate buffer (50 mM, pH 7.0). The washed cells were resuspended in the

52

Part I, Section 3, Resolution mandelates: Results

100 mL of same buffer at a concentration of 0.1 g/mL. To the cell suspension, ethyl

mandelate at 20 mM concentration was added and the contents incubated at 3 7 °C in

an orbital shaker at 200 rpm. The reaction was stopped at 48% conversion (34 h;

Scheme 14). Progress of the reaction was monitored by TLC. pH of the reaction

mixture was adjusted to 8.0 with 1N NaOH. The remaining ester was removed by

extraction with ethyl acetate. The aqueous layer was acidified to pH 2.0 with 1N HCl

and extracted with ethyl acetate to recover acid formed during the reaction. The

organic layer was separated, dried over sodium sulphate and evaporated to give acid

48 (Scheme 26).

Scheme 26

~O ~ __ S_te_n_ot_r_:op_h_o_m_o_na_s_m_a_l_to:....1Jh_,_·u_,a..,..

~ g Phosphate buffer, pH 7.0 conversion 100%

OH

G('cooH 47 (R)-48, e.e. >99%


The absence of a triplet (J=7.2 Hz) at 8 1.20 and quartet (J=7.2 Hz) at 8 4.24

corresponding to ester group of 47 confirmed that the hydrolysis had occurred. The

proton at C-2 appeared at 8 5.24 as a singlet. The aromatic protons appeared as

multiplet at 8 7.35-7.43.

The e.e. of the product was determined using (1R,2R)-1 ,2-diphenylethane-1 ,2-

diamine as chiral solvating agent. To the best of our knowledge, the use of this chiral

solvating agent for determination of the absolute configuration and e.e. of mandelic

acid and its derivatives has never been reported in literature. When 1.5 equivalent

shift reagent was added to racemic mandelic acid, the methine singlet at 8 5.24 for the

(R) and (S) enantiomers experienced unequal shifts and appeared as well separated

singlets at 8 4.80 and 8 4.78 (Figure 19a). When shift reagent was added to the acid

obtained from the biocatalyzed hydrolysis, only one singlet at 8 4.80 could be seen in 1HNMR spectrum (Figure 19b ). This indicated exclusive formation of one enantiomer

of the acid.

53


r

5.0 5.0

(a) (b)

Figure 19: 1HNMR of (a) (R,S)-mandelic acid and (b) (R)-mandelic acid obtained from biocatalyzed reaction, in presence of 1.5 equivalent of (1R,2R)-1 ,2-diphenylethane-1 ,2-diamine

The absolute configuration of the acid was determined as follows: 1HNMR of

a commercial sample of (S)-mandelic acid was recorded in the presence of 1.5

equivalent chiral solvating agent. A singlet at 8 4. 78 appeared along with a minor

singlet at 8 4.80 as the commercial sample was not enantiomerically pure. Thus, the

singlet resonating at lower frequency can be assigned to (S)-enantiomer. To determine

the configuration of the acid obtained from the biocatalyzed hydrolysis, racemic

standard acid along with 1.5 equivalent chiral solvating agent was added to the sample

obtained from biotransformation and 1 HNMR of the sample was again recorded. The

singlets at 8 4.78 and 8 4.80 appeared in approximately 1:2 ratio, thus confirming the

formation of (R)-acid. In this case, the reaction did not stop at 50% conversion and

went on to complete conversion when allowed to run for longer time period. The acid

obtained after 100% conversion had (R)-configuration, with e.e. >99%.

The next substrate taken for the study was ethyl 4-chloromandelate (49).

Biocatalyzed reaction was performed under the conditions as described above. Acid

50 was obtained in 70% yield at 100% conversion (Scheme 27).

Scheme 27

(R)-50 (S)-50

R/S=9:1

54

Part I, Section 3, Resolution mandelaies: Results

The structure of the productwas in agreement with its 1HNMR spectral data.

The absence of a triplet at 8 1.22 (1=7.2 Hz) and quartet at 8 4.30 (1=7.2 Hz)


me thine proton appeared as a singlet at 8 5.20 and the aromatic protons appeared as


The e.e. of the product was determined using (1R,2R)-1 ,2--diphenylethane-1 ,2-

diamine as chiral solvating agent as described above. When shift reagent (1.5

equivalent) was added to racemic 4-chloromandelic acid, the methine singlet at 8 5.20

for (R) and (S) enantiomers experienced unequal shift and appeared as two singlets at

8 4.65 and 4.73 (Figure 20a). When shift reagent was added to the acid obtained from

the biocatalyzed hydrolysis, singlets in unequal ratio at 8 4.73 and 8 4.65 could be

seen in 1 HNMR spectrum (Figure 20b ). The major enantiomer resonated at higher

frequency and was tentatively assigned (R)-configuration in analogy with the results

obtained with ethyl mandelate.

5.0 5.0

(a) (b)

Figure 20: 1HNMR of (a) (R,S)-4-chloromandelic acid and (b) (R)-4-chloromandelic acid obtained from biocatalyzed reaction, in the presence of 1.5 equivalent of (1R,2R)-1 ,2-diphenylethane-1 ,2-diamine

Biocatalyzed hydrolysis of ethyl 4-methoxymandelate was studied next under

the reaction conditions as described above. Acid 52 was obtained in 75% yield at

100% conversion (Scheme 28).

The structure of the product was in agreement with its 1HNMR spectral data.



methine proton appeared as a singlet at 8 5.19 and the aromatic protons appeared as a


55


Scheme 28

OHOOE_t __ ==~~~~==~==-- mOHOOH Stenotrophomonas maltophilia

Phosphate buffer, pH 7.0 Me conversion 100% MeO

+

51 (R)-52 (S)-52

RIS=6:1



equivalent) was added to racemic 4-methoxymandelic acid, the methine singlet at 8

5.19 for (R) and (S) enantiomers experienced unequal shift and appeared as two

singlets at 8 4.63 and 4.71 (Figure 21a). When shift reagent was added to the acid

obtained from the biocatalyzed hydrolysis, singlets in unequal ratio at 8 4.71 and 8

4.63 could be seen in 1HNMR spectrum (Figure 21b). The acid obtained after 100%

conversion was tentatively assigned (R)-configuration in analogy with the results of

ethyl mandelate.

115.0 I !i

(a) (b)

Figure 21: 1HNMR of (a) (R,S)-4-methoxymandelic acid and (b) (R)-4 methoxymandelic acid obtained from biocatalyzed reaction, in the presence of 1.5 equivalent of (1R,2R)-1 ,2-diphenylethane-1 ,2-diamine

Biotransformation of ethyl 2-chloromandelate with the whole cells of

Stenotrophomonas maltophilia was studied next under the reaction conditions described

above. Acid 54 was obtained in 77% yield at 100% conversion (Scheme 29).

Scheme 29

~OEt VH

53

Stenotrophomonas maltophilia • Phosphate buffer, pH 7.0

conversion 100%

56

~OH VH

+

(R)-54 R/S=4:1 (S)-54





methine proton appeared as a singlet at 8 5.63 in the spectrum of the acid. The

aromatic protons appeared as a multiplet at 8 7.27-7.82.

The e.e. of the product was determined using (1R,2R)-1,2-diphenylethane-1,2-


equivalent) was added to racemic 2-chloromandelic acid, the methine singlet at 8 5.63



the biocatalyzed hydrolysis, singlets in ratio of 1:4 at 8 5.15 and 8 5.21 could be seen

in 1HNMR spectrum (Figure 22b). The acid obtained after 100% conversion was

tentatively assigned (R)-configuration in analogy with the results of ethyl mandelate.

: I s.o 5.0

(a) (b)

Figure 22: 1HNMR of (a) (R,S)-2-chloromandelic acid and (b) (R)-2-chloromandelic acid obtained from biocatalyzed reaction, in the presence of 1.5 equivalent of ( 1R, 2R)-1 ,2-diphenylethane-1 ,2-diamine

Biotransformation of ethyl 4-bromomandelate with the whole cells of

Stenotrophomonas maltophilia was studied next under the reaction conditions

described above. Acid 56 was obtained in 72% yield at 100% conversion (Scheme

30).

Scheme 30

~Et Stenotrophomonas maltophilia ~ B» B Phosphate buffer (pH 7.0, 50 mM)

conversion 100%

OH

+ ~H BM tl 55 (S)-56

RIS=1:12 (R)-56

57


l-'"

( .I

____) '.'---

.0

(a) (b)

Figure 23: 1HNMR of (a) (R,S)-4-bromomandelic acid and (b) (S)-4-bromomandelic acid obtained from biocatalyzed reaction, in the presence of 1.5 equivalent of (lR,2R)-1 ,2-diphenylethane-1 ,2-diamine




methine proton appeared as a singlet at 8 5.18 in the spectrum of the acid. The

aromatic protons appeared as multiplet at 8 7.23-7 .50.



equivalent) was added to racemic 4-bromomandelic acid, the methine singlet at 8 5.1



the biocatalyzed hydrolysis, singlets in ratio of 1:12 at 8 4. 75 and 8 4.68 could be seen

in 1HNMR spectrum (Figure 23b). (S)-configuration was tentatively assigned to the

product ofbiocatalyzed reaction in analogy with the results of ethyl mandelate.

1.3.6 Acetone treated Stenotrophomonas maltophilia catalyzed resolution of 2-

hydroxyarylacetates (mandelates): formation of enantiomerically pure

(R)-2-hydroxyarylacetic acids (mandelic acids)

In 2-arylpropanoate series, untreated and acetone treated cells of

Stenotrophomonas maltophilia showed opposite enantioselectivity. Biocatalyzed

hydrolysis of mandelates 47, 49, 51, 53 and 55 with acetone treated cells was also

studied. The biocatalyzed reactions were performed as described in Section 1.3.5,

except that the acetone treated cells of Stenotrophomonas maltophilia, prepared as

described in Section 1.3.2 were used as biocatalyst in place of untreated cells. The

products were isolated, purified and analyzed as described in Section 1.3.5. The

results are summarized in Schemes 31-35 and Figures 24-28.

58

Scheme 31

~0~ VH

47

5.0

(a)


OH

Acetone treated S. maltophilia cfcoon Phosphate buffer, pH 7.0

conversion 100%

(R)-48,e.e. >99%

___l_ .,....'T""""-,--

5.0 I!.

(b)

Figure 24: 1HNMR of (a) (R,S)-mandelic acid and (b) (R)-mandelic acid obtained from biocatalyzed reaction, in presence of 1.5 equivalent of (1R,2R)-1,2-diphenylethane-1 ,2-diamine

Scheme 32

OH OH

~Et Acetone treated S. maltophilia , ~H + Cl~ H Phosphate buffer, pH 7.0 Cl~ H

~H ~ H

conversion 100% Cl

49 (R)-50 (S)-50 R/S=8.5: 1.5

L 5.0

(a) (b)

Figure 25: 1HNMR of (a) (R,S)-4-chloromandelic acid and (b) (R)-4-chloromandelic acid obtained from biocatalyzed reaction, in the presence of 1.5 equivalent of ( 1 R, 2R)-I ,2-diphenylethane-1 ,2-diamine

59

Scheme 33

Me mOH Et

.....:

51

Acetone treated S. maltophili(j. Phosphate buffer, pH 7.0

conversion 100%

(a)

Me


mOHH

0

(R)-52 R/S=8:2

(b)

Figure 26: 1HNMR of (a) (R,S)-4-methoxymandelic acid and (b) (R)-4 methoxymandelic acid obtained from biocatalyzed reaction, in the presence of 1.5 equivalent of ( 1R, 2R)-1 ,2-diphenylethane-1 ,2-diamine

Scheme 34

53

Acetone treated S. maltophilia


JL . I S,O

(a)

(R)-54

+

(S)-54

R/S=8.3:1.7

I

5.0

(b)

Figure 27: 1HNMR of (a) (R,S)-2-chloromandelic acid and (b) (R)-2-chloromandelic acid obtained from biocatalyzed reaction, in the presence of 1.5 equivalent of (1R,2R)-1 ,2-diphenylethane-1 ,2-diamine

60

Scheme 35

Acetone treated S. maltophilia. Phosphate buffer, pH 7.0

conversion 100%

.0

Part 1, Section 3, Resolution mandelates: Discussion

(R)-56 RIS=1:6

(S)-56

(a) (b) Figure 28: 1HNMR of (a) (R,S)-4-bromomandelic acid and (b) (S)-4-bromomandelic acid obtained from biocatalyzed reaction, in the presence of 1.5 equivalent of ( 1 R, 2R)-1 ,2-diphenylethane-1 ,2-diamine

In the mandelate series, untreated and acetone treated cells of

Stenotrophomonas maltophilia gave essentially similar results. In both the cases, the

biocatalyst showed selectivity for (R)-enantiomer, except in the case of 55, which

gave (S)-acid as the major product with treated as well as untreated cells. At 100%

conversion, (R)-enantiomer was produced exclusively (48) or predominantly (50, 52,

54). Only in case of ethyl mandelate (47) e.e. of more than 99% was obtained.

Substitution at ortho, meta or para position resulted in decrease in the e. e. to 66-70%.

1.3. 7 Discussion: Stenotrophomonas maltophilia catalyzed resolution of

mandelates

Stenotrophomonas maltophilia catalyzed hydrolysis of a senes of ethyl

mandelate derivatives was studied using intact cells and acetone treated cells (Table

3). In all the cases studied, hydrolysis of mandelates resulted in the formation of (R)

acids, except in the case of ethyl 4-bromomandelate (Entry 6, Table 3). This is in

contrast to 2-arylpropanoates, where intact cells of Stenotrophomonas maltophilia

gave (S)-acids and acetone treated cells gave (R)-acids. Two possibilities exist, (i)

mandelates are not the substrates for surface bound lipase; all the activity for

mandelates is because of intracellular esterase and (ii) surface bound lipase has

opposite selectivity for mandelates. The pure lipase did not show any activity for ethyl

mandelate indicating that the activity is due to intracellular esterase.

61

Part I, Section 3, Resolution mandelates: Discussion

The e.e. of the product formed in most cases range between 60-70%,

except in the case of mandelic acid, which was obtained in more than 99% e.e. The

lower e.e. may be due to (i) relaxed selectivity of the enzyme or (ii) presence of a

competing (S)-selective enzyme or (iii) partial conversion of (R)-mandelic acid to (S)

mandelic acid by the activity of deracemase. At least a part of (S)-enantiomer is

formed by deracemase as shown below (Section 1.3.9). Other two possibilities appear

less likely as entry 2 and 3 (Table 3) clearly show that (R)-mandelate is the preferred

substrate; the hydrolysis of (S)-ester did not proceed to an appreciable level after 24 h.

Table 3: Stenotrophomonas maltophilia catalyzed hydrolysis of a series of 2-hydroxyarylacetates using untreated and acetone treated cells

Entry Substrate Time Untreated cell& Acetone washed cells

(h) 0/oconversion * 0/oconversion * e.e. e.e.

OH 24 18 72 34 68 1 cnro~ 48 40 88 60 90

0 72 100 >99 100 >99

OH 24 40 100 100 68 2 ~0~ 48 70 100 - -

0 72 100 100 - -OH 24 n.d. - n.d. -

3 0/ro~ 48 n.d. - n.d. -0 72 degradation - degradation -

OH 24 21 77 45 85 4 __o-)_t~ 48 88 76 100 70

0 72 100 80 - -Cl

Mo~ 24 35 0 50 53 5 48 50 54 76 60

0 72 70 60 86 66

OH 24 45 60 65 66 6 __o~~t~ 48 100 85(S) 85 72 (S)

0 72 - - - -Br

OH 24 0 12

d't~ - -

7 48 28 82 22 58 0 72 35 72 34 62 H3CO

OH 24 24 100 24 100

8 c•mo~ 48 75 100 75 100

I h o -72 100 100

* unless stated otherw1se, configuratiOn of the ac1d produced IS R.

62

Part 1, Section 3, Resolution mandelates: Discussion

The e.e. ofthe mandelic acid formed at 24 h (18% conversion) was 72% but

increased to >99% after 72 h (100% conversion) (Entry1, Table 3). Thus, e.e. of the

product increased with increase in conversion. This is possible if either (i) the (S)-acid

formed is deracemized to (R)-acid or (ii) (S)-acid is selectively degraded. When (R,S)

mandelic acids were incubated with cells of Stenotrophomonas maltophilia, a slow

degradation to benzoic acid and keto acid could be seen, when the crude product of

the reaction was analyzed by NMR. However, (R)-acid showed no degradation

product even after 1 00 h of incubation. This observation suggests that the increase in

e.e. may have occurred due to selective oxidation and degradation of (S)-acid.

The presence of chloro group at ortho, meta or para position of aromatic

ring of ethyl mandelate does not appear to have any significant effect on the rate of

the reaction but a decreased enantioselectivity was observed. The substrate with 4-

methoxy, however, resulted in greatly reduced rate of reaction (Entry 7, Table 3).

Substitution by a bromo group at para position showed very interesting behaviour.

Not only the rate ofreation was enhanced, the configuration ofthe product obtained at

100% conversion was (S) (Entry 6, Table 3).

The reaction with ethyl mandelate proceeded to 100% conversion, but the

e.e. of product remained very high. This is possible if either (S)-ester or (S)-acid is

selectively transformed to corresponding (R)-enantiomer. As described below

(Section 1.3.9), deracemization of racemic acid occurs, but very slowly compared to

rate of ester hydrolysis. Therefore, deracemization must be occurring at the stage of

ester. This was confirmed by HPLC analysis of the reaction on chiral column (Diacel

chiracel OB-H) (Figure 29). As the reaction proceeded the (R)-ester started depleting

till whole of it disappears, leaving behind only the (S)-ester (Figure 29b ). As the

reaction is continued further, conversion of (S)-ester to (R)-ester could be clearly seen.

The proposed mechanism for deracemization is shown in Scheme 36. The enzyme

responsible for deracemization of ethyl mandelate and 2-arylpropanoates may be

similar.

63

svt!OXldo211 ETHYL MANDALATE CONn AU

,,. ~ ! '

15<><>

m-

l"o I !

I I•"' I

i A ~ \ ' o'o 25.0

(a)

(c)

uv"" W\11..:217nm

I

I

J5a

Part I, Section 3, Presence of mandelate dehydrogenase inS. maltophilia

6 ~undo ESd ~thvtmandelale19hr

•2

o'o s.o 1oo 1s.o 20.o 2so 30o JS.o 4D.o 4s.o

(b) S-ethylmend•

·""'

00 5.0 10.0

(d)

WVL217nnj

•

I

-! '""

Figure 29: HPLC profiles of (a) (R,S)-ethyl mandelate (b) ethyl mandelate recovered from biocatalyzed reaction after 19 h (c) ethyl mandelate recovered from biocatalyzed reaction after >72 h and (d) (S)-ethyl mandelate prepared from (S)mandelic acid

Scheme 36

Reductase .. lf

Oxidase

(S)-47 57 (R)-47

1.3.8 Demonstration of the presence of mandelate dehydrogenase in the cells of

Stenotrophomonas maltophilia

1.3.8.1 Activity Staining (Zymography)

The presence of mandelate dehydrogenase has been implicated in the

deracemization of ethyl mandelate. To confirm the presence of a dehydrogenase in the

system, cells of Stenotrophomonas maltophilia were grown and proteins extracted

according to the procedure described in the experimental section. Polyacrylamide gel

electrophoresis, under non denaturing conditions at pH 7.5 was performed in 10%

(w/v) polyacrylamide gels according to Maurer (1968). 169 NADP dependent

dehydrogenase activity was detected in the gel using staining solution consisting of

64

Part I, Section 3, Mandelate dehydrogenase inS. maltophilia

phenazine methosulfate (0.05 mg/mL), nitroblue tetrazolium (0.3 mg/mL), NADP

(0.5 mM) and ethyl mandelate (20 mM).170 The gel was kept in the staining solution

overnight, when white band appeared on blue background. The zymogram clearly

shows the presence of a NADP dependent dehydrogenase in the protein (Figure 30).

Figure 30: Activity staining of the dehydrogenase: Stenotrophomonas maltophilia was grown in peptone (1 %) and beef extract (0.5%). Proteins were extracted by incubating cells with 1M NaCl and incubated overnight in a solution consisting of phenazine methosulfate (0.05 mg/mL), nitroblue tetrazolium (0.3 mg/mL), NADP (0.5 mM) and ethyl mandelate (20 mM). White band appeared on blue background.

1.3.8.2 Peptide Mass Fingerprinting (PMF) and bioinformatics studies

Non-denaturing PAGE of the purified protein was performed and the gel

was stained for dehydrogenase activity as described in detail above. White band

obtained from the zymogram was excised thoroughly and collected in eppendorf. The

gel pieces were dehydrated in 100% acetontrile ( 40 flL) for 5 min. After removing

acetonitrile, the gel pieces were destained with ammonium bicarbonate (20 mM in

30% acetonitrile) at 30 °C for 30 min. This destaining procedure was repeated four

times. Gel pieces were again dehydrated in 100% acetonitrile for 5 min and then dried

in speed-vac for 30 min. MS-grade trypsin solution (25 flL) was added to the finely

dried gel pieces and incubated at 3 7 °C for 36 h. After complete incubation, the

trypsinised sample was centrifuged and supernatant was collected. The proteolyzed

fragments obtained from the gel pieces were extracted twice with 60% acetonitrile in

0.1% trifluoroacetic acid and centrifuged again. This was combined with the

supernatant obtained above and concentrated to a final volume of 10 flL in speed-vac.

The sample so obtained was subjected for peptide mass fingerprinting. The peptide

masses obtained from peptide mass fingerprinting were searched against the proteins

65

Part I, Section 3, Mandelic acid deracemase

in MASCOT server at http//www.matrixscience.com. The database used was MSDB

20060831 (3239079 sequences; 1079594 700 residues). The search parameters used

were: Type of search: PMF, Enzyme: trypsin, Mass values: monoisotopic, Protein

mass: unrestricted, Peptide mass tolerance: ±1.2 Da, Peptide charge state: ±1.

Extensive search in MASCOT showed the masses matched with a NADP dependent

dehydrogenase from bacteria, Blastopireuella marina.

1.3.9 Stenotrophomonas maltophilia catalyzed deracemization of 2-

hydroxyarylacetic acids

Racemic mandelic acid (48), 4-chloromandelic acid (50) and 4-

methoxymandelic acid (52) were subjected to reaction with the acetone treated cells

of Stenotrophomonas maltophilia under the reaction conditions described in Section

1.3.6. The products were isolated, purified and analyzed as described in Section 1.3.5.

The results are summarized in Schemes 3 7-3 9 and Figures 31-3 3. In all the three case,

a conversion of (R)-enantiomer to (S)-enantiomer could be seen. The e.e. for the (S)

enantiomer was 60-65%. The configuration of the acid present in major amount after

7 days was found to be (S) in all the examples studied.

Scheme 37

~OH VH

(R,S)-48

5.0

(a)


Phosphate buffer, pH 7.0

OH

~OH ~ 0

(S)-48, e.e. 65%

4.t4o 4.7 4.()

(b)

Figure 31: 1HNMR of (a) (R,S)-mandelic acid and (b) Mandelic acid from biocatalyzed reaction, in the presence of 1.5 equivalent of (1R,2R)-1 ,2·diphenylethane-1 ,2-diamine

66

Scheme 38

Cl

~OH N H

(R,S)-50

(a)

Part I, Section 3, Resolution of 2-aryloxypropanoates: R1:sults


Phosphate buffer, pH 7.0 Cl

~OH ~ 0

(S)-50, e.e. 60%

(b)

Figure 32: 1HNMR of(a) (R,S)-4-chloromandelic acid and (b) 4-chloromandelic acid obtained from biocatalyzed reaction, in the presence of 1.5 equivalent of (lR, 2R)-l ,2-diphenylethane-1 ,2-diamine

Scheme 39

~OH N g

MeO

(R,S)-52

(a)


Phosphate buffer, pH 7.0

~OH ~ 0

MeO

(S)-52, e.e. 61%

5.0 II

(b)

Figure 33: 1HNMR of(a) (R,S)-4-methoxymandelic acid and (b) 4 methoxymandelic acid obtained from biocatalyzed reaction, in the presence of 1.5 equivalent of ( 1R, 2R)-1 ,2-diphenylethane-1 ,2-diamine

67

Part I, Section 3, Resolution of2-aryloxypropanoates: Results

Both (R) and (S) mandelic acid dehydrogenases are known in literature.160

The mechanism of deracemization is shown in Scheme 40. The formation of keto acid

and benzoic acid in the reaction was confirmed when crude product obtained from the

reaction mixture was analyzed by 1HNMR. In addition to mandelic acid, NMR

showed the presence of keto acid, 58 and benzoic acid, 66. Aromatic protons

corresponding to keto acid were present at 8 7.43-7.73 and 8 8.24-8.28 and that of

benzoic acid at 8 7.51-7.65 and 8 8.08-8.11.

Scheme 40

OH

cfcoou -.=::::O::xi::da::s::e ~ Reductase

(R)-48

0

c{coon 58

! ~OOH

v 66

Reductase 4 X

Oxidase

1.3.10 Stenotrophomonas

aryloxypropanoates

maltophilia catalyzed

OH

cfcoon S-48

hydrolysis of 2-

We started our investigation in this series by studying the hydrolysis of ethyl

2-(3-chlorophenoxy)propanoate (59). Stenotrophomonas maltophilia was grown in

medium comprising of peptone (1.0%) and beef extract (0.5%). Cells were harvested

by centrifugation and washed with phosphate buffer (50 mM, pH 7.0). The washed

cells were resuspended in the 100 mL of same buffer at a concentration of 0.1 g/ mL.

To the cell suspension, ethyl 2-(3-chlorophenoxy)propanoate (59) at 20 mM

concentration was added and the contents incubated at 3 7 °C in an orbital shaker at

200 rpm (Scheme 41). The product was isolated and purified as described in Section

1.3 .1. The biotransformation with the untreated cells of Stenotrophomonas

maltophilia proceeded very fast with 100% conversion of the substrate taking place in

8 h.

68


The formation of acid 60 was confirmed by 1 HNMR_ The absence of triplet at

8 1.23 (1=7.2 Hz) and quartet at 8 4.21 (1=7.2 Hz) confirmed that the hydrolysis had

occurred. The -CH3 protons at C-2 appeared at 8 1.64 (1=7.2 Hz) as a doublet,

methine proton as a quartet (1=7.2 Hz) at 8 4.75 and aromatic protons as a multiplet at

8 6.89-7.26.

Scheme 41

Ck ,::-1

..0. Jl cn,cn, S. maltophilia Ck "'' ..0.. Jl.,H ~k "'1..0.. Jl H U -T U Phosphate buffer, pti 7.0 U T l U T U

59 conversion 100% (S)-60 (R)-60

SIR= 1:0.9


method using (1R,2R)-1,2-diphenylethane-1,2-diamine as chiral solvating agent as

described. 140 When 1.5 equivalent solvating reagent was added to racemic 2-(3-

chlorophenoxy)propanoic acid (60), the quartet for the methine proton for (R) and (S)

enantiomers experienced unequal shift and appeared as two quartets at 8 4.32 (J=7.2

Hz) and 4.4 (1=7.2 Hz) (Figure 34a). When shift reagent was added to the acid

obtained from the biocatalyzed hydrolysis, quartets at 8 4.32 (1=7.2 Hz) and 4.4

(1=7.2 Hz) in 1HNMR spectrum were observed in the ratio 1:0.9 (Figure 34b),

indicating that no resolution has occurred in this case.

4.0

(a) (b)

Figure 34: 1HNMR of (a) (R,S)-2-(3-chlorophenoxy)propanoic acid and (b) 2-(3-chlorophenoxy)propanoic acid obtained from biocatalyzed reaction, in the presence of 1.5 equivalent of (lR, 2R)-I ,2-diphenylethane-1 ,2-diamine

The next substrate chosen for the biocatalyzed hydrolysis in this category of

compounds was ethyl 2-(2,4-dichlorophenoxy)propanoate (61). The reaction was

69


performed under the same reaction conditions described above. The product was

isolated and purified as described in Section 1.3 .1.

Scheme 42

(S)-62 SIR= 2:1


The absence of triplet at 8 1.23 (J=7.2 Hz) and quartet at 8 4.17 (J=7.2 Hz) confirmed

that the hydrolysis had occurred. The -CH3 protons appeared at 8 1.69 as a doublet

(J=7.2 Hz), the methine proton appeared as a quartet at 8 4.68 (J=7.2 Hz) and

aromatic protons appeared as a multiplet at 8 6.65-7.39.

The e.e. and absolute configuration of the acid was analyzed by NMR method

using (1R,2R)-1 ,2-diphenylethane-1 ,2-diamine as chiral solvating agent as

described. 140 When 1.5 equivalent solvating reagent was added to racemic 2-(2,4-

dichlorophenoxy)propanoic acid, the quartet for the methine proton for (R) and (S)

enantiomers experienced unequal shift and appeared as two separate quartets at 8 4.42

(J=7.2 Hz) and 4.51 (J=7.2 Hz) (Figure 35a). When shift reagent was added to the

acid obtained from the biocatalyzed hydrolysis, quartets at 8 4.42 (J=7 .2 Hz) and 4.51

(J=7.2 Hz) in 1HNMR spectrum were observed in the ratio 2:1 (Figure 35b). The

quartet resonating at lower frequency in presence of chiral shift reagent has been

assigned to (S)-enantiomer.140

4.0 4.0

(a) (b)

Figure 35: 1HNMR of (a) (R,S)-2-(2,4-dichlorophenoxy)propanoic acid and (b) 2-(2,4-dichlorophenoxy)propanoic acid obtained from biocatalyzed reaction, in the presence of 1.5 equivalent of (1R, 2R)-1 ,2-diphenylethane-1 ,2-diamine

70


1.3.11 Hydrolysis of 2-aryloxypropanoates with acetone treated cells of


The biocatalyzed hydrolysis of ethyl 2-(3-chlorophenoxy)propanoate (59) and

ethyl 2-(2,4-dichlorophenoxy)propanoate (61) with acetone treated cells was also

studied. The acetone treated cells were suspended in the 1 00 mL of phosphate buffer

(pH 7.0, 50 mM) at a concentration of 0.01 g/mL. To the cell suspension, substrate

(20 mM) was added and the contents incubated at 3 7 °C in an orbital shaker at 200

rpm. Acid was isolated as described above. Structure of the products was confirmed

by 1HNMR as explained above. The results are summarized in Scheme 43 and 44 and

Figures 36 and 37.

Scheme 43

ct ..... -..... ...o... ~ u T UEt

59

~



~ "'

!li

-

Ct.... --.. ...0... ~ Cl...._ -..... ...0.... ~. UTUH+ UTUH (S)-60 (R)-60

SIR= 3:1

- ~~~ ~JlJ ·.__ _l~ 4.0

(a) (b) (c)

Figure 36: 1HNMR of (a) (R,S)-2-(3-chlorophenoxy)propanoic acid, (b) 2-(3-chlorophenoxy)propanoic acid obtained from biocatalyzed reaction, and (c) (R,S) Ethyl-2-(3-chlorophenoxy)propanoate; a- acid; h-ester resonance peaks in the presence of 1.5 equivalent of (1R,2R)-1,2-diphenylethane-1,2-diamine

Scheme 44

~Et CI

Acetone treated S. maltophilj: l...o... .1 H + ~I H Phosphate buffer, pH 7.0 ~ T U ~ I

conversion 100% Cl Cl 61 (S)-62

S/R=l:0.7 (R)-62

71

Part I, Section 3, Resolution of2-aryloxypropanoates: Discussion

4.0 "I 4.0

Figure 37: 1HNMR of (a) (R,S)-2-(2,4-dichlorophenoxy)propanoic acid and (b) 2-(2,4-dichlorophenoxy)propanoic acid obtained from biocatalyzed reaction, in the presence of 1.5 equivalent of (1R,2R)-1,2-diphenylethane-1,2-diamine

1.3.12 Discussion: Stenotrophomonas maltophilia catalyzed hydrolysis of 2-

aryloxypropanoates

High enantioselectivity was obtained in Stenotrophomonas maltophilia

catalyzed hydrolysis of 2-arylpropanoates, whereas 2-aryloxypropanoates gave poor

e.e. Acetone treated cells were (R)-selective for 2-arylpropanoates, but exhibited

preference for (S)-enantiomer of 2-aryloxypropanoates. The rates of reaction were

much higher with 2-aryloxypropanoates than 2-arylpropanoates. Theses results show

the marked influence of inserting an oxygen atom between aryl moiety and chiral

carbon on the rate of reaction as well as enantioselectivity.

Providing an explanation of results with whole cell system containing multiple

enzyme activities at best could be speculative. A possible explanation of results could

be as follows (i) acetone treated cells have a deracemase and a hydrolase activity, (ii)

deracemase has high selectivity for (S)-enantiomer in case of arylpropanoates but has

low or poor selecitivity for (R/S) enantiomer of aryloxypropanoates, (iii) an (S)

selective esterase, but with low or poor selectivity for (R)-enantiomers is present.

Alternatively, two esterases one selective for (S) and other selective for (R) may be

present, the rate of hydrolysis being higher for the (S)-selective than the (R)-selective

esterase. Thus, in case of 2-arylpropanoic acids (Scheme 45) (S)-ester is rapidly

transformed to (R)-ester by deracemase before any hydrolysis could occur. The ester

that remains at 48% conversion in acetone treated Stenotrophomonas maltophilia

catalyzed hydrolysis of 39 had (R)-configuration (data not shown), which confirms

much faster rate of deracemization compared to hydrolysis. (S)-selective esterase of

relaxed selectivity then hydrolyses (R)-ester at a slow rate. In case of 2-

aryloxypropanoates, the deracemase may have relaxed selectivity; therefore the

equilibrium is driven towards (S)-acid because of faster rate of reaction with (S)-ester

than (R)-ester (Scheme 46)

72

Part I, Section 3, Resolution of ethyl 2-chloropropanoate

Scheme 45

2-Arylpropanoic acid (overall (R)-acid with high e.e.)

(S)-ester kt

(R)-ester

l k, k2

k,l slow

(S)-acid (R)-acid

k1>>k2 and k3

Scheme46

2-Aryloxypropanoic acid (overall (S)-acid with poor e.e.)

(S)-ester kt (R)-ester

j ., k2

(S)-acid (R)-acid

kt::k2::k3, k3>k4

1.3.13 Stenotrophomonas maltophilia catalyzed hydrolysis of ethyl 2-chloro

propanoate

An aliphatic ester was taken as substrate to study substrate specificity of the

biocatalyst. Stenotrophomonas maltophilia was grown in medium comprising of

peptone (1.0%) and beef extract (0.5%). Cells were harvested by centrifugation and

washed with phosphate buffer (50 mM, pH 7.0). The washed cells were suspended in

the 100 mL of same buffer at a concentration of 0.1 g/ mL. To the cell suspension,

ethyl 2-ch1oropropanoate at 20 mM concentration was added and the contents

incubated at 37 °C for 2 h in an orbital shaker at 200 rpm (Scheme 47). The product

was isolated, purified and analyzed as described previously (Section 1.3 .1 ).

The e.e. and absolute configuration of the acid was analyzed by NMR method

usmg (1R,2R)-1,2-diphenylethane-1,2-diamine as chira1 solvating agent as

described. 140 When 1.5 equivalent chira1 solvating agent was added to racemic 2-

chloropropanoic acid, the quartet for the -CH proton at C-2 for (R) and (S)

enantiomers experienced unequal shift and appeared as two separate quartets at 8 3.95

(1=7.2 Hz) and 3.80 (1=7.2 Hz) (Figure 38a). When shift reagent was added to the

73

Part I, Section 3, Conclusions

acid obtained from the biocatalyzed hydrolysis, quartets at 8 3.95 (1=7.2 Hz) and 3.80

(1=7.2 Hz) in 1HNMR spectrum were observed in the ratio 1:0.95 (Figure 38b),

indicating that no resolution has occurred in this case.

Scheme 47

Cl

~~ 0

63

v u L fl. II

(a)



4.0

(b)

Cl

~OH 0

64, e.e. 0%

Figure 38: 1HNMR of (a) (R,S)-2-chloropropanoic acid and (b) 2-chloropropanoic acid obtained from biocatalyzed reaction, in the presence of 1.5 equivalent of (lR, 2R)-1 ,2-diphenylethane-1 ,2-diamine

1.3.14 Conclusions

In conclusion, we have demonstrated the preparation of either (R) or (S)

enantiomer in >99% e.e. of 2-arylpropanoic acids by kinetic resolution of their esters

using the same strain of microorganism, but used under different set of conditions.

Detailed studies revealed that the result is due to presence of two different enzymes

within the organism. We have also demonstrated Dynamic Kinetic Resolution of 2-

arylpropanoates to (R)-acids in >99% e.e. Mandelates were also resolved by dynamic

kinetic resolution process, but the e.e. of the acids produced was in the range of 62-

99%. 2-Aryloxypropanoates and ethyl 2-chloropropanoate were hydrolyzed, but

without any selectivity for enantiomers. In addition, we have demonstrated the

deracemization of mandelic acids, in which biocatalyst converted racemic mandelic

acids to (S)-mandelic acids in 60-65%. Overall, the results achieve significance due to

the fact that by using appropriate reaction conditions, we have been able to harness

multiple enzyme activities of one organism for the selective production of either

enantiomer.

74

part 1, section 3 applications of stenotrophomonas...

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