Tetrahedron: Asymmetry 20 (2009) 2854–2860

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Tetrahedron: Asymmetry

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Lipase activity of Lecitase�

Ultra: characterization and applicationsin enantioselective reactions

Mithilesh Kumar Mishra, Thenkrishnan Kumaraguru, Gurrala Sheelu, Nitin W. Fadnavis *

Biotransformation Laboratory, Indian Institute of Chemical Technology, Uppal Road, Habsiguda, Hyderabad 500 007, India

a r t i c l e i n f o a b s t r a c t

�

Article history:Received 20 October 2009Accepted 13 November 2009Available online 6 January 2010

0957-4166/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.tetasy.2009.11.012

* Corresponding author. Tel.: +91 40 27191631; faxE-mail addresses: fadnavis@iict.res.in, fadnavisnw@

The general properties of Lecitase Ultra, a phospholipase manufactured and marketed by Novozymes,Denmark, have been studied after purification by ultrafiltration. The enzyme has a molecular mass of35 KD, pH-optimum of 8.5, and appears to possess a single active site which exhibits both the lipaseand phospholipase activities that increase in the presence of Ca2+ and Mg2+ ions. The enzyme is inhibitedby heavy metal ions and surfactants, and does not accept p-nitrophenyl acetate and glycerol triacetate.Substrates, such as glycerol tributyrate and p-nitrophenyl palmitate, esters of N-acetyl-a-amino acidsand a-hydroxy acids are readily accepted. Amino acids with aliphatic residues, such as alanine, isoleu-cine, and methionine, are hydrolyzed with high enantioselectivity for the L-enantiomer (E >100), butamino acids with aromatic residues such as phenylalanine and phenylglycine, and esters of a-hydroxyacids are hydrolyzed with low enantioselectivity (E = 1–5). Immobilization of the enzyme in a gelatinmatrix (gelozyme) leads to a marginal improvement in the enantioselectivity for these substrates.However, a dramatic improvement in enantioselectivity is observed for ethyl 2-hydroxy-4-oxo-4-phen-ylbutyrate (E value increases from 4.5 to 19.5 with S-selectivity). Similarly, glycidate esters, such as ethyltrans-(±)-3-phenyl glycidate and methyl trans-(±)-3-(4-methoxyphenyl) glycidate, are selectivelyhydrolyzed with a remarkable selectivity towards the (2S,3R)-enantiomer providing unreacted (2R,3S)-glycidate esters (ee >99%, conversion 52–55%) by the immobilized enzyme.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Lipases and phospholipases are well known for the exploitationof their lipolytic activity in the synthesis of a wide variety of prod-ucts ranging from pharmaceutical intermediates to neutraceuti-cals. Generally, lipases are employed for the preparation of chiralentities1 while phospholipases are used to produce 2-acyl lyso-phospholipids,2 and hydrolyzed lecithin products for food compo-sitions.3 Although it has been known that most commerciallyavailable phospholipase preparations are accompanied by lipaseactivity,4 applications of this lipase activity in organic synthesisare seldom studied.5 The case of Lecitase� Ultra, a protein-engi-neered phospholipase A1 introduced by Novozymes, Denmark,for the degumming of vegetable oils, is very interesting in this re-gard. Over the past few years, Guisan et al. have employed the li-pase activity of Lecitase Ultra� in the resolution of mandelateesters and the regioselective hydrolysis of carbohydrate esters.They have observed drastic changes in enzyme activity and evenenantioselectivity when the enzyme was immobilized on differentsupports.5 It was, however, unclear whether a single enzymedisplays both lipase and phospholipase activities, or the commer-

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: +91 40 27160512.yahoo.com (N.W. Fadnavis).

cial sample contains a mixture of different enzymes which getpreferentially immobilized displaying different enantioselectivi-ties. Herein, we report our data on the enzyme properties afterpurification via ultrafiltration. We have observed that the commer-cial preparation of Lecitase� Ultra consists mainly of a singleprotein with a molecular mass of 35 KD which displays bothphospholipase and lipase activities, and it is very useful for thepreparation of enantiomerically pure commercially importantintermediates such as (�)-ethyl (R)-2-hydroxy-4-phenyl butyrate(HPB ester), methyl trans-(2R,3S)-3-(4-methoxyphenyl) glycidateand ethyl trans-(2R,3S)-phenyl glycidate.

2. Results and discussion

Lecitase� Ultra is supplied as an aqueous solution containingapproximately 6.5% protein. This commercial preparation is a pro-tein-engineered carboxylic ester hydrolase from Thermomyceslanuginosus/Fusarium oxysporum produced by the submerged fer-mentation of a genetically modified Aspergillus oryzae.6 Althoughit is known that Lecitase� Ultra possesses activity towards bothphospholipid and triglyceride structures,6a most of the efforts aredirected towards the application of phospholipase activity of thisenzyme for the degumming of vegetable oils6a,7 while very little ef-fort has been made in exploring applications of the lipase activity

3 4 5 6 7 8 9

0

5

10

15

20

108 v ob

sd M

-1 s

-1

pH

Figure 1. Effect of pH on the activity of the purified Lecitase Ultra. [PNPP] = 20 lM,[enzyme] = 0.3 lg/mL in 0.05 M citrate buffer containing 25 mM CaCl2 at 30 �C.

M. K. Mishra et al. / Tetrahedron: Asymmetry 20 (2009) 2854–2860 2855

in organic synthesis.5 Considering the ready availability and cost ofthe enzyme, we have purified the commercial sample by ultrafil-tration and studied its properties.

2.1. Purification by ultrafiltration

The commercial enzyme preparation (protein content 65 mg/mL estimated by Lowry’s method)8 shows several protein bandsat 35, 25 and 14–10 KD. The crude enzyme solution was thus puri-fied by membrane filtration through a Millipore stirred cell usingmembranes of different molecular weight cutoffs. The fractionswere then assayed for enzyme activity and purity. The highest pro-tein content and lipase activity was found in the fraction between30 KD retentate and 50 KD filtrate. The SDS–PAGE of this fractionshowed a single band with a molecular mass of 35 KD. This fractionwas concentrated with a 10 KD filter to a protein content of 60 mg/mL and was stored in a refrigerator. The enzyme activity in thesolution was found to be stable over a period of at least two weekswhen stored in cold.

2.2. Measurement of enzyme activity

2.2.1. Measurement of lipase activityThe lipase activity of the enzyme was measured by both titri-

metry and spectrophotometry. The purified enzyme did not acceptglycerol triacetate (triacetin) or p-nitrophenylacetate as a sub-strate. It did however accept glycerol tributyrate (tributyrin) andp-nitrophenyl palmitate as substrates. Thus, the lipolytic activitywas determined titrimetrically with glycerol tributyrate (tribu-tyrin) as the substrate in pH-stat mode. The activity of the com-mercial sample using tributyrin was found to be 2118 units/mL(32 units/mg protein) at 30 �C in 0.005 M Tris–HCl buffer contain-ing 25 mM CaCl2, pH 8.5 (one unit of lipase is the amount of en-zyme which releases 1 lmol of titratable free butyric acid perminute under the described conditions). This activity is somewhatlower than other commercially available lipases such as the lipasefrom Candida rugosa (98 units/mg), porcine pancreas (125 units/mg) and Amano Ps (177 units/mg), but the cost (approx. 50$/L) isat least 15 times lower. The purified enzyme showed an activityof 130 units/mg indicating a fourfold purification.

The enzyme activity was also measured using p-nitrophenyl pal-mitate (PNPP) as a typical lipase substrate in 0.05 M Tris–HCl buffercontaining 25 mM CaCl2 (pH 8.5) at 30 �C. Due to the limitation ofthe solubility of the substrate in a buffer, the apparent kinetic con-stants Km,app and kcat,app were determined from rate measurementsat low substrate concentrations varying from 2 to 20 lM at a fixedconcentration of enzyme (0.3 lg/mL). Based on the protein content,the values obtained from a non-linear curve fitting using GraphPadPrism 5 were kcat,app = 102 s�1 and Km,app = 0.3 mM for the purifiedenzyme under experimental conditions.

2.2.2. Measurement of phospholipase activityPhospholipase activity was measured by the hydrolysis of

soybean lecithin emulsion in Tris–HCl buffer (0.01 M, pH 8.5)containing calcium chloride (15%), sodium deoxycholate (3.5%)and soybean lecithin (15%).7 Upon hydrolysis at a fixed pH (e.g.,8.5), a long chain fatty acid is released which is then neutralizedby the addition of 0.2 N sodium hydroxide solution to maintain aconstant pH. The enzyme activity is expressed as micromoles ofNaOH consumed per minute. The activity of the enzyme was foundto be 1000 units/mL for the commercial sample.

2.3. Effect of pH on enzyme activity in an aqueous buffer

Figure 1 shows the effect of pH on the lipase activity of puri-fied Lecitase� Ultra. It was observed that the enzyme shows a

pH-optimum at pH 8.5 which is on the higher side of pH 5.0 re-ported by Yang et al. for the crude enzyme6a and the pH-activityprofile is much narrower than the earlier preparation of Lecitasemanufactured by Novozymes.7

2.4. Effect of salts and surfactants

Monovalent salts such as LiCl, NaCl and KCl did not affect theenzyme activity at concentrations of up to 0.5 M. The enzymeactivity decreased at higher salt concentrations and was com-pletely lost in 1 M salt solutions. In contrast, divalent metal ionssuch as Ca2+ and Mg2+ activated the enzyme at low concentrations,that is, a threefold increase in enzyme activity was observed in thepresence of 25 mM of CaCl2, while a 1.5-fold activation was ob-served in the presence of 10 mM magnesium sulfate. Such salt ef-fects are commonly observed for phospholipases and lipoproteinlipases.9 Salts are well known to cause changes in enzyme activi-ties via different mechanisms. Simple monovalent salts usually af-fect the dissociation of functional groups at high concentrationsand alter the catalytic efficiency. Divalent cations such as Ca2+ playan important role in catalytic action and several phospholipasesare known to be calcium dependent.9 However, Lecitase� Ultradoes not seem to be completely dependent on Ca2+ as much asthe other metal ions such as magnesium which also activates theenzyme, albeit to a lesser extent, and is also able to function with-out these metal ions. Heavy metal ions such as Cu2+ and Ni2+ com-pletely inhibited the enzyme activity at concentrations of 1 mM,thus suggesting the involvement of an essential cysteine residue.Since the presence of dithiothreitol or 2-mercaptoethanol did notcause any increase in the enzyme activity, it appears that the cys-teine is not directly involved in catalysis. Further work on this as-pect using specific inhibitors of serine and cysteine proteases iscurrently in progress.

In aqueous solutions, the enzyme activity was quickly lost inthe presence of various surfactants such as positively charged cetyltrimethylammonium bromide (CTAB), negatively charged bis(2-ethylhexyl sulfosuccinate) sodium salt (AOT) and zwitterionic N-dodecyl N,N-dimethyl-3-ammino-1-propanesulfonate (DDAP) atvery low concentrations (<0.1 mM). Similarly, the enzyme activitywas completely lost in AOT–isooctane and DDAP–chloroform re-verse micelles. Surprisingly, the enzyme activity was not lost inthe presence of 0.1 mM lauric acid in an aqueous solution, andthe activity was retained in CTAB–chloroform reverse micelles.

0

20

40

60

80

100

Res

idua

l Enz

yme

Activ

ity (%

)

2856 M. K. Mishra et al. / Tetrahedron: Asymmetry 20 (2009) 2854–2860

These observations indicate that the loss of enzyme activity is dueto some specific interaction of the surfactant with the enzymesurface.

2.5. Lipase versus phospholipase activity

The enzyme displays both lipase and phospholipase properties,that is, it can hydrolyze the esters of carboxylic acids as well as thephosphate esters of fatty acids, that is, lecithin. It was important toknow whether the enzyme has a single active site where bothtypes of substrates bind, or the active sites of phospholipase activ-ity and lipase activity are different. To ascertain this, the lipaseactivity of the enzyme was measured at a fixed concentration ofp-nitrophenyl palmitate in the presence of varying amounts ofphospholipase substrate, phosphatidylcholine (see Fig. 2).

-10 0 10 20 30 40 50 60 70 800.5

1.0

1.5

2.0

2.5

3.0

107

V obsd

, lit

mol

e-1 s

-1

[Phophatidylcholine], µM

Figure 2. Inhibition of lipase activity of Lecitase Ultra by phosphatidylcholine.[PNPP] = 20 lM, [enzyme] = 0.3 lg/mL in 0.05 M Tris–HCl buffer, pH 8.5 containing25 mM CaCl2 at 30 �C.

30 40 50 60 70Temperature, ºC

Figure 3. Effect of temperature on the stability of Lecitase Ultra in 0.05 M Tris–HClbuffer containing 25 mM CaCl2, pH 8.5.

It was observed that the lipase activity decreased in the pres-ence of phosphatidylcholine and the plot of log [phosphatidylcho-line] versus lipase activity gave an excellent fit to one-sitecompetitive binding (IC50 = 17.8 lM, r2 = 0.9927). These resultsstrongly suggest that Lecitase Ultra has a single active site whichpossesses both lipase and phospholipase activities.

2.6. Thermal stability

The thermal stability of the purified enzyme was studied bymeasuring the residual activity of the enzyme after incubating ata given temperature for 1 h. Figure 3 shows that the enzyme is sta-ble up to 50 �C and quickly loses its activity above 60 �C.

2.7. Immobilization of the enzyme in gelatin

Over the past few years, we have successfully demonstrated theuse of lipases immobilized in gelatin organo-gels (gelozymes) foruse in organic solvents. The procedure provides a technique of en-zyme immobilization at low temperature and the gelatin matrixprovides a porous support. The water content of the matrix canbe controlled to a level sufficient enough for an enzyme to beactive.10 Our attempts to immobilize Lecitase� Ultra in sodiumbis(2-ethylhexyl dioctyl sulfosuccinate) (AOT)–isooctane–watersystem failed due to extensive deactivation of the enzyme byAOT. Based on the enzyme activity studies in reverse micelles,we were successful in immobilizing purified Lecitase� Ultra in

gelatin organo-gel prepared in a microemulsion of CTAB in 50%chloroform–isooctane.11 The gelozyme thus obtained was allowedto set overnight, crushed into small pieces, washed with chloro-form until the organic layer was free of CTAB as evidenced fromits colour reaction with Tropaeolin OO,11 and dried under vacuumat room temperature. Assay of the immobilized enzyme withtributyrin showed that 65% of the enzyme was active afterimmobilization.

3. Hydrolytic reactions catalyzed by Lecitase� Ultra

3.1. Resolution of 2-hydroxy acids

Enantiomerically pure 2-hydroxy carboxylic esters and acidsare of considerable synthetic interest since they are useful as majorbuilding blocks for a variety of drug intermediates.12 We havestudied the enantioselectivity of the purified Lecitase� Ultra to-wards the hydrolysis of esters of various 2-hydroxy acids (Table 1).

It was observed that Lecitase� Ultra hydrolyzed the substrates1–4 with very low enantioselectivity in an aqueous buffer at pH8.2 (E = 3–5) and did not hydrolyze ethyl 2-hydroxy-4-phenylbutyrate (HPB ester) 5. Hydrolysis of substrate 2 is noteworthy.Although the substrate possesses two reaction centres, the enzymeregioselectively hydrolyzed the carboxylic ester function giving 2-O-acetyl-2-phenylacetic acid 2C and prolonged contact with theenzyme did not result in further hydrolysis of the acetate 2C tomandelic acid 1. Our results with substrate 1 are in agreement withthose of Fernandez-Lorente et al.5b In the case of substrate 2, ourobservation that the acetate 2C does not further hydrolyze to (R)-mandelic acid while the corresponding butyrate does5b is consis-tent with the fact that Lecitase� Ultra does not accept triacetinbut readily hydrolyzes tributyrin.

3.2. Effect of enzyme immobilization

Over the past few years, several studies have been carried out inorder to assess the impact of an immobilization protocol on theactivity and enantioselectivity of an enzyme. Apart from the obvi-ous advantages such as improved stability and enzyme recycle, anincrease in enantioselectivity and sometimes, even its reversal,5b,c

has been observed on immobilization, especially of lipases. Theeffects vary and depend upon the enzyme as well as the immobi-lization matrix. It has been argued that the observed changesin activity/enantioselectivity after immobilization occur due to

Table 1Enantioselective hydrolysis of 2-hydroxy esters by Lecitase� Ultra*

S. no. Substrate Product Enantioselectivity E

Free enzyme Immobilized enzyme

1 COOEt

OH

1

COOEt

OH

(S)-1a

COOH

OH

(R)-1b

3.1 5.4

2 COOET

OAc

2

COOET

OAc

(S)-2a

COOH

OAc

(R)-2b

3.5 10.1

3 COOET

O OH

3

COOET

O OH

(R)-3a

COOH

O OH

(S)-3b

4.1 19.5

4 COOET

OH

4

COOH

OH

4b

No selectivity

5 COOEt

OH

5

No reaction

Reaction volume 20 mL. Reactions followed up to 30% conversion by reverse phase HPLC.* [substrate] = 50 mM in 0.05 M Tris–HCl buffer containing 25 mM CaCl2, pH 8.2, 30 �C; [enzyme] = 0.3 mL or 1 g immobilized enzyme.

M. K. Mishra et al. / Tetrahedron: Asymmetry 20 (2009) 2854–2860 2857

selective adsorption/attachment of the ‘open form’ on hydropho-bic supports. Additionally, the enzyme may undergo structuralchanges that might alter its selectivity.13–15 Considering the possi-bility of an increase in enantioselectivity, we immobilized the en-zyme in a gelatin matrix using the organo-gel technique11 andemployed the immobilized Lecitase Ultra for hydrolytic reactions.Although a small increase in enantioselectivity was observed withimmobilized enzyme in most cases, the effect was quite significantin the case of substrate 3. The enantioselectivity of the reaction in-creased from 4 to 19.5 and it was possible to obtain the unreacted(R)-ester with ee >99% at 60–65% conversion. This ester on hydro-genation leads to (�)-ethyl (R)-2-hydroxy-4-phenyl butyrate (HPBester), an important intermediate used in the production of severalangiotensin converting enzyme (ACE) inhibitors such as Cilazapril,Benazepril, Spirapril and Enalapril16

In the Solvias technology for HPB ester, the key step involvesthe enantioselective hydrogenation of ethyl 2,4-dioxo-4-phenylbu-tyrate with a Pt–dihydrocinchonidine/Al2O3 catalyst to obtain (R)-3 with 85–88% ee and 98% yield, which is then brought to >99% eevia enrichment and recrystallization in 65% yield.16 It is our con-tention that this enrichment step can be replaced with the enantio-selective hydrolysis of the (S)-ester with immobilized Lecitase�

Ultra. Since the ester is already enriched to a level of 85%, a simplecalculation shows that enzymatic hydrolysis up to 10–12% conver-sion would remove almost all the (S)-ester bringing the ee to >99%and overall isolated yields would improve substantially from 65%to 80–85%.

Lecitase Ultra

Tris buffer , 0.05M,pH 8.5

AcHN COOMe

Me

6

A

Scheme 1. Hydrolysis of N-acetyl-(DL)-ala

3.3. Resolution of a-amino acids

Lipases are known for their activity towards amino acids and inmany cases, lipases have been successfully used for their resolu-tion.17 In order to explore the substrate selectivity of Lecitase� Ul-tra, we have studied the hydrolysis of some N-acetyl-a-amino acidmethyl esters (Scheme 1, Table 2).

It was observed that the enzyme readily accepted most aminoacid derivatives. Although their rates of hydrolysis were not dra-matically different, esters of a-amino acids with aliphatic residuessuch as alanine 6, isoleucine 7 and methionine 8 hydrolyzed withexcellent enantioselectivity with a preference for the L-enantiomer(E >100), esters of aromatic amino acids such as phenylalanine 9and phenylglycine 10 were hydrolyzed with little enantioslectivity.

3.4. Resolution of glycidate esters

Enantiomerically pure trans-ethyl phenyl glycidate 11a andtrans-methyl (4-methoxyphenyl) glycidate 12a are important drugintermediates. While the (2R,3S)-enantiomer of ethyl phenyl glyci-date 11a is useful in the synthesis of the Taxol side chain, N-ben-zoyl-(2R,3S)-3-phenylisoserine,18 the (2R,3S)-enantiomer of 12ais used in the synthesis of Diltiazem, a drug used to treat hyperten-sion.19 The synthesis of these enantiomers via the resolution ofracemic glycidate esters by an esterase/lipase-catalyzed enantiose-lective hydrolysis is well known.19 Several lipases have been re-ported to hydrolyze the (2S,3R)-enantiomer selectively to provide

+

6a : e.e. > 99%

H Me

cHN COOMe

6b : e.e 91%

Me H

AcHN COOH

nine methyl ester by Lecitase� Ultra.

Table 2Stereoselective hydrolysis of methyl esters of N-acetyl amino acids by Lecitase� Ultra

Substrate Conversion(%) in 12 h

Stereochemicalpreference

Enantioselectivity E

N-Ac-Ala–OMe 6 33 L >100N-Ac-Ile–OMe 7 33 L >100N-Ac-Met–OMe 8 13 L >100N-Ac-Phe–OMe 9 10 L 2.2N-Ac-phenylglycine–

OMe 1028 L 1.3

[Substrate] = 1 mM in 0.05 M Tris–HCl buffer containing 25 mM CaCl2, pH 8.2 at30 �C; [enzyme] = 1 mL, reaction volume 20 mL. Reaction period 12 h.

2858 M. K. Mishra et al. / Tetrahedron: Asymmetry 20 (2009) 2854–2860

the required (2R,3S)-enantiomer as the unreacted glycidate esterwith enantiomeric purities ranging from 20% to >99% dependingupon the conversion and the biocatalyst. However, the glycidicacid 13, which forms as the hydrolysis product is unstable andquickly decomposes to aldehyde 14. This aldehyde acts as aninhibitor of the enzyme and it is necessary to design the biore-actor, which allows continuous removal of the aldehyde as abisulfite adduct.20 Recently, we have reported the resolution ofglycidate esters 11 and 12 using mung bean epoxide hydrolase.21

Unfortunately, the stereoselectivity of the enzyme providesthe (2S,3R)-enantiomers 11b and 12b instead of the required(2R,3S)-enantiomers. Thus, our search for the commercially avail-able lipase/esterase which provides the (2R,3S)-enantiomers ofglycidates 11a and 12a and which is not strongly inhibited bythe presence of the aldehyde produced during the hydrolyticreaction, led us to investigate the possibility of employingLecitase� Ultra for the purpose. To avoid spontaneous epoxide ringopening of 12 by excess water, the hydrolytic reaction was carriedout with gelatin immobilized enzyme (gelozyme) in toluene(Scheme 2).

Thus, the racemic glycidate esters 11 in an aqueous buffer or 12dissolved in toluene (1% solution) were stirred with gelozymepowder at room temperature on a magnetic stirrer. The reactionwas followed by HPLC analysis on a chiral stationary phase. The(2S,3R)-ester was found to disappear slowly, accompanied by theappearance of the aldehyde peak. Additionally, we have also ob-served that the peak area for the unreacted (2R,3S)-ester did notdecrease with time, thus indicating the almost exclusive selectivity

O

COOR1

X

11 : X = H, R1 = Et

12 : X = OMe, R1 = Me

X

ImmobilizedLecitase

Toluene

PTRef

X

N

S

H3CO

H HOAc

O

N.HCl

Diltiazem

Scheme 2. Enzymatic resolution of glycidat

of the enzyme towards the (2S,3R)-ester. The ee of the unreactedester reached >99% in 72 h for 11 and five days for 12 (52–55% con-version). These reaction rates can be further optimized by a judi-cious selection of a bioreactor and reaction conditions such assolvent enzyme substrate ratio and residence time. The fact thatthe reaction goes to completion shows that the enzyme is not seri-ously inhibited by the aldehyde. A control reaction with immobi-lized C. rugosa lipase under similar conditions was found to stopafter only 10% conversion due to inhibition by aldehyde.

In the case of glycidate ester 12, the reaction mixture after theenzymatic reaction was used directly for the conversion of 12a tothe cis-lactam 15 without a purification step. The product is insol-uble in toluene and precipitates out upon the completion of thereaction in near quantitative yields and ee >99%.

4. Conclusion

Recently available phospholipase preparation, Lecitase� Ultraexhibits significant lipase activity. The enzyme accepts glyceroltributyrate and p-nitrophenyl palmitate, esters of N-acetyl-a-ami-no acids and a-hydroxy acids as substrates. Amino acids with ali-phatic residues such as alanine, isoleucine and methionine arehydrolyzed with high enantioselectivity for the L-enantiomer (E>100). On immobilization in a gelatin matrix, a dramatic improve-ment in enantioselectivity was observed for the hydrolysis of ethyl2-hydroxy-4-oxo-4-phenylbutyrate [E value increases from 4.5 to19.5 with (S)-selectivity]. Glycidate esters such as ethyl trans-(±)-3-phenyl glycidate and methyl trans-(±)-3-(4-methoxyphenyl) gly-cidate can be resolved in a two-phase system by the immobilizedenzyme in toluene, providing unreacted (2R,3S)-glycidate esters(ee >99%, conversion 52–55%). It appears that the enzyme Leci-tase� Ultra has excellent potential for biocatalytic conversions inorganic synthesis as well as the pharmaceutical industry.

5. Experimental

5.1. General

Lecitase� Ultra was a gift from Novozymes, Denmark. All otherreagents were purchased from Sigma–Aldrich. IR spectra were re-corded on a Perkin–Elmer RX-1 FT-IR system. 1H NMR (300 MHz)

O

COOR1

11a : X = H, R1 = Et

12a : X = OMe, R1 = Me

O

COOH

X

CHO

X

(2R,3S) (2S,3R)

13

14

HS

H2N

SAlux

N

S

H HOH

H

O

15: X = OCH3

+

e esters and synthesis of cis-lactam 15.

M. K. Mishra et al. / Tetrahedron: Asymmetry 20 (2009) 2854–2860 2859

and 13C NMR (75 MHz) spectra were recorded on Bruker Avance-300 MHz spectrometer. Optical rotations were measured with Hor-iba-SEPA-300 digital polarimeter. Mass spectra were recorded on aQ STAR mass spectrometer (Applied Biosystems, USA). HPLC anal-yses were carried out on Hewlett Packard HP1090 unit with diodearray detector and HP Chem Station software. Chiral HPLC columnswere obtained from Daicel, Japan. UV–vis spectrophotometricmeasurements were performed on a Perkin–Elmer Lambda 2 spec-trophotometer equipped with temperature control and PECSS soft-ware. All enzyme assays were performed at 30 �C. All kineticexperiments were repeated three times and were reproduciblewithin ±5%. Compounds 1, 1a, 1b, 3a, 3b, 5a and 5b were obtainedfrom Aldrich. Racemic mixtures were prepared by mixing pureenantiomers when only the enantiopure compounds were com-mercially available. Acetylation of 1 provided 2. Methyl esters ofN-acetyl-a-amino acids were prepared by standard procedures.22

Compound 4 was prepared by a literature method using racemiccyanohydrin derivative.10c All compounds were isolated and char-acterized by IR, NMR, Mass spectrometry and optical rotation. Incase of compound 3, reaction rates were measured individuallyfor the (R)- and (S)-enantiomers to calculate the E value since wecould not separate the corresponding acids on chiral HPLC column.

5.2. Enzyme assay

5.2.1. Spectrophotometric assayLipase activity in the solution was measured at 30 �C using p-

nitrophenyl palmitate as a substrate (2–20 lM) in Tris–HCl buffer(50 mM, 25 mM CaCl2, pH 8.5). The assay solution (2.0 mL) wasplaced in a cuvette, and the enzyme solution (10 lL) was added (fi-nal enzyme concentration was 0.5–1 lg in the cuvette). A changein absorbance was monitored at 348 nm (molar extinction coeffi-cient e 5150 M�1 cm�1) for 3–5 min.

5.2.2. Titrimetric assayLipase activity was determined by following the hydrolysis of

tributyrin. Upon hydrolysis at a fixed pH (e.g., 8.5), butyric acid isreleased, which is then neutralized by the addition of 0.2 M sodiumhydroxide solution to maintain a constant pH of 8.5. Typically, anassay was performed with 10 lL of free enzyme solution in25 mL of well sonicated assay solution consisting of 0.01 M Tris–HCl buffer, 25 mM CaCl2 and 100 lL of tributyrin at 30 �C for20 min. The enzyme activity in units is expressed as micromolesof NaOH consumed per minute. The enzyme activity was foundto be 32 units/mg for the commercial sample and 130 units/mgfor the purified enzyme (7800 units/mL).

The phospholipase activity was determined by following thehydrolysis of soybean lecithin. The assay buffer was prepared bydissolving calcium chloride (15 g) in a solution of Tris–HCl buffer(100 mL, 0.01 M, pH 8.5). Sodium deoxycholate (3.5 g) and soybeanlecithin (15 g) were added to the buffer. The mixture was warmedat 60 �C for 10 min and sonicated for 15 min. The emulsion was fil-tered through a cotton plug to remove any lumps and stored atroom temperature. Typically, the assay was performed with20 lL of free enzyme solution or 2.3 g (wet) gel in 25 mL of assaybuffer at 30 �C for 20 min. The enzyme activity is expressed asmicromoles of NaOH consumed per minute. The activity of the en-zyme was found to be 1000 units/mL for the commercial sample.

5.3. Immobilization of Lecitase� Ultra in gelatin

Gelatin (5 g) was heated with distilled water (8.5 mL) at 60 �Cfor 15 min to complete gelation. CTAB solution (35 mL, 0.3 M in1:1 chloroform–isooctane) was added to a gelatin solution withvigorous stirring. The viscous and turbid gel thus obtained wascooled in ice with shaking for 10 min to give a transparent free

flowing liquid. The enzyme solution of purified Lecitase� Ultra(5.0 mL, 40,000 units) was slowly added to the cold solution withvigorous stirring to achieve a uniform distribution of the enzyme.The cooling bath was removed and glutaraldehyde (1 mL, 25% solu-tion) was added. The contents were stirred at room temperaturewith a glass rod until the contents started to become viscous, atwhich point it was poured into a Petri dish and left at room tem-perature overnight. The dry gel was cut into small pieces andwashed several times with chloroform. The gel was then vacuumdried at room temperature for 12 h to obtain immobilized enzyme(10 g). Assay of enzyme activity by tributyrin showed lipase activ-ity corresponding to 2400 units/g indicating 60% of enzyme activ-ity after immobilization.

5.4. Enzymatic hydrolysis

Typically, racemic substrates 1 to 10 (50–200 mg) were sus-pended in 20 mL of Tris–HCl buffer (0.05 M, pH 8.5 containing25 mM CaCl2) with sonication. The enzyme was added either as asolution (100–300 lL, 800–2000 units) or immobilized enzymepowder (0.3–1.0 g, 700–2000 units) and the contents were stirredat room temperature on a magnetic stirrer. The reaction was fol-lowed by HPLC. After 30–35% conversion, the reaction mixturewas extracted with ethyl acetate to recover the unreacted ester.The aqueous phase was acidified and extracted again with ethylacetate to recover the hydrolyzed product. Analysis of the recov-ered products on the chiral stationary phase provided the valuesfor the enantiomeric excess of substrate (ees) and products (eep).The enantioselectivity E was calculated according to Sih et al.23

Product configurations were assigned on the basis of their specificrotation and retention times on chiral HPLC columns and compar-ison with authentic samples which were either commerciallyavailable or prepared in the laboratory by standard procedures(Table 3).

5.5. Enzymatic resolution of glycidate esters 11 and 12

The racemic glycidate ester dissolved in toluene (1% solution,(w/v), 20 mL) was stirred with immobilized enzyme (2.5 g) at roomtemperature. The reaction was followed by analysis on chiral sta-tionary phase. The ee of unreacted ester reached >99% in 72 h for12 and five days for 11. The organic layer was then decanted, theenzyme was washed with toluene (2 � 5 mL). For the preparationof cis-lactam 15, the combined organic layer was used withoutfurther purification. For analytical purposes, the oily residue waschromatographed over silica gel (ethyl acetate/hexane, 5:95) torecover unreacted (2R,3S)-11b and 12b (90–92 mg, 90% oftheoretical).

5.6. (+)-cis-(2S,3S)-3-Hydroxy-2-(4-methoxyphenyl)-2,3-dihydrobenzo[b][1,4] thiazepin-4(5H)-one (cis-lactam) 15

The combined toluene layer after enzymatic reaction containingenantiomerically pure 12b along with the aldehyde 14 was mixedwith 2-aminothiophenol (54 mg, 0.05 mmol) and p-toluene sul-fonic acid (10 mg), and the contents were refluxed for 6 h. On cool-ing, the cis-lactam 15 separated as a pale yellow powder which wascollected by centrifugation, washed with toluene and dried undervacuum (130 mg, 90%). In 1H NMR, doublets at d 3.6 and 5.0with J = 15 Hz confirmed the cis-stereochemistry of the lactam.Mp 208–210 �C, ½a�25

D ¼ þ115 (c 0.5, DMF); lit.24 ½a�20D ¼ þ114:4

(c 0.5, DMF); ee >99%, chiral HPLC. 1H NMR (CDCl3 + DMSO-d6;200 MHz): d 3.6 (d, 1H, C(2)H, J = 15 Hz), 3.8 (s, 3H, OCH3), 4.4. (t,1H, OH), 5.0 (d, 1H, C(3)H, J = 15 Hz), 6.8–7.8 (m, 8H, Ar), 10.3(br, 1H, NH). MS (EI, M+): m/z = 301.

Table 3HPLC analysis conditions on a chiral stationary phase for various substrates and products

Column Mobile phase Flow rate (mL/min) Detection wavelength (nm) Retention times (min)

Chiralpak AD-H 3% 2-propanol in hexane + 0.1% trifluoroacetic acid 0.7 220 (R)-7b: 33.7, (S)-7b: 36.85% 2-propanol in hexane + 0.1% trifluoroacetic acid 0.5 230 (R)-1a: 22.0, (S)-1a: 23.9

220 (R)-1b: 47.9; (S)-1b: 54.7(R)-9a: 24.7, (S)-9a: 32.8

0.7 220 (R)-6b: 14.7, (S)-6b: 16.9220 (R)-8b: 30.6, (S)-8b: 34.3

7.5% 2-propanol in hexane 0.5 230 (2S, 3R)-11: 12.7(2R,3S)-11: 13.7

10% 2-propanol in hexane + 0.1% trifluoroacetic acid 1.0 254 (S)-4a: 11.2, (R)-4a: 12.7220 (S)-4b: 15.5, (R)-4b: 17.5220 (R)-9b: 8.0, (S)-9b: 9.5

(R)-10b: 22.8, (S)-10b: 29.615% 2-propanol in hexane 0.7 230 (2S, 3R)-12: 9.5

(2R,3S)-12: 10.6

Chiracel OD 1% 2-propanol in hexane + 0.1% trifluoroacetic acid 0.5 230 (R)-2a: 16.5, (S)-2a: 18.15% 2-propanol in hexane + 0.1% trifluoroacetic acid 0.5 230 (R)-2b: 17.8, (S)-2b: 20.110% 2-propanol in hexane + 0.1% trifluoroacetic acid 0.7 246 (S)-3a: 15.3, (R)-3a: 16.9

254 (S)-5a: 9.5, (R)-5a: 10.820% 2-propanol in hexane 0.7 230 (2R,3R)-15: 11.0,

(2S,3S)-15: 15.6

Chiralcel OD-H 3% 2-propanol in hexane + 0.1% trifluoroacetic acid 1 220 (R)-6a: 17.0, (S)-6a: 19.1(R)-10a: 23.6, (S)-10a: 25.2

5% 2-propanol in hexane + 0.1% trifluoroacetic acid 0.7 220 (R)-7a: 14.0, (S)-7a: 17.1(R)-8a: 23.4, (S)-8a: 27.5

2860 M. K. Mishra et al. / Tetrahedron: Asymmetry 20 (2009) 2854–2860

Acknowledgements

We are thankful to CSIR, New Delhi and DST, New Delhi, forfinancial assistance and Novozymes, Denmark, for gift of Lecitase�

Ultra.

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