Lipase catalysed amidation of diethylglutamate derivatives: peculiarities ofethyl S- and R-pyroglutamateSantiago Conde*, Paloma Lopez-Serrano and Ana MartınezInstituto de Quımica Medica (C.S.I.C.). Juan de la Cierva 3. 20006 Madrid. Spain

Candida antarctica lipase mediated amidation of both ethyl S- and R-pyroglutamate at 60°C takes place in less than 5minutes. Kinetic differences, S-enantiomer rate higher than that of R, appeared when the reaction was performed at45°C.

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IntroductionFollowing our studies (Chamorro et al., 1995) on theenantio and regioselective amidation of diethyl glutamatederivatives catalyzed by lipases, we have recently reported(Conde et al., 1997) the effect of the N-protecting group onthe reaction rate: small N-protecting group L-glutamatederivatives react faster than the larger ones and N-carbamates much faster than the corresponding amides. Onthe other hand, the regioselectivity resulting from theenantioselectivity was not affected and a-substitution wasexclusively observed in all cases. The behaviour of the N-free derivative (diethyl glutamate) have also been studiedunder the same experimental conditions, the amidationwith n-pentylamine catalyzed by the lipase B of Candidaantarctica (CAL) in anhydrous diisopropylether. Reports ofN-free amino acid esters as acyl donors in amidationreactions are unfrequent (Gerisch and Jakubke, 1997) andvery rare when they are catalysed by lipases (de Zoete et al.,1995). Different results from those obtained with N-blockderivatives can be expected: a free amino group is thesmallest possible “N-protecting” group (a hydrogen atom)and it can, and certainly does, form hydrogen bonds andelectronic interactions with organic solvents such as ethers,and also with polar groups located in the enzyme (pocket,active site, etc.). In the experimental conditions used, bothL- and L-substrates underwent a spontaneous cyclizationfollowed by the enzymatic amidation of the resultingethyl S- and R-pyroglutamates (2-pyrrolidone-5-carboxylicesters). In this communication, we present our initialresults on the CAL-catalyzed amidation of these cyclicglutamic derivatives that, as far as our knowledge, havenever been used as lipase substrates.

Materials and methodsMaterialsAll l (or S) and d (or R)-esters and acids were obtained fromcommercial sources (Sigma and Aldrich) and used without

© 1998 Chapman & Hall

further purification. n-Pentylamine was distilled and storedon KOH pellets. Diisopropylether was refluxed on sodiumwire, distilled and stored on molecular sieves 0.4 nm.Novozym 435, a Novo Nordisk commercial immobilizedpreparation of CAL was used as received. Its maximumactivity, occurring at 65–75°C, is 7000 PLU/g that falls toabout 4600 at 45°C (activity expressed in Propyl LaurateUnits synthesised from n-propanol and lauric acid in15 minutes per g of catalyst. Data supplied by NovoNordisk).

Analytical methods1H and 13C-NMR spectra were recorded in CDCl3 solutionusing a Varian-Gemini-200 spectrometer. Optical rotationswere determined on a Perkin-Elmer 241-C polarimeter.HPLC was performed by using a reverse phase C18 5 m(3.9 3 15 mm) column eluted with acetonitrile/water(25:75 v/v) at 1 ml/min; an UV detector was used at 200nm. Analytical TLC was performed on aluminium sheetscoated with a 0.2 mm layer of silicagel 60 F254 (Merck).

Amidation procedureThe reactions were carried out at an analytical scale inscrew-cap 2 ml vials. CAL (50 mg/ml) and molecular sieves0.4 nm (50 mg/ml) were added to a solution of theglutamate derivative (20 mM) and n-pentylamine (50 mM)in anhydrous diisopropylether. They initially were incu-bated at 60°C in an orbital shaker at 250 r.p.m. Aliquots(20 ml) were withdrawn after 5 min, evaporated to dryness,extracted with acetonitrile and analysed by HPLC.

Preparative reactions were performed at a 1 mmol and keptshaking at 60°C for 30 minutes. Afterwards, the enzymeand molecular sieves were filtered off and washed withacetonitrile, the combined organic extracts were evaporatedto dryness and the residue crystallized with a di-isopropylether/acetonitrile mixture and identified by NMRand comparison, if possible, with commercial samples.

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Scheme 1 Enzymatic amidation of ethyl pyroglutamate, chemically in situ afforded from ethyl glutamate (a) or directlyset to react (b).

S. Conde et al.

ResultsAmidation of diethyl glutamateUnder the above described conditions, the amidation ofdiethyl L-glutamate 1 took place in less than 5 minutesand afforded a single product which was identified as theN-pentylamide of the S-pyroglutamic acid 4. A blankreaction without enzyme also over 5 min yielded a uniqueproduct identified as the intermediate ester ethyl S-pyroglutamate 2. Identical results were obtained whendiethyl L-glutamate L-1 was used the corresponding R-4and R-2 were respectively obtained from the enzymatic andblank reactions. These results suggest a two high-rate stepsprocess (Scheme 1, Reaction a): a chemical intramolecularaminolysis of diethyl glutamate 1 followed by the enzy-matic amidation of the resulting ethyl pyroglutamate 2.The absence of the acyclic amidoester 3 points out thechemical cyclization of 1 as the first step; it is not anunknown reaction ( Caswell et al., 1981) although it wasnot expected to take place so fast in the mild experimentalconditions used.

Amidation of ethyl pyroglutamateIn contrast with the diethyl N-blockeL-glutamate derivat-ives, the amidation of both substrates L- and L-1 exhibitedno kinetic differences at the current experimental condi-tions, with the consequent lack of enantioselectivity. It wasconfirmed when the actual substrates of the enzymaticreaction S- and R-2 were separately used: again a completeconversion into the amides S- and R-4 was achieved in 5min (Scheme 1, Reaction b). Kinetic differences betweenthese two enantiomers appeared by performing the reac-tions in lower-rate conditions, 45 instead of 60°C and 10instead of 50 mg of enzyme/ml. Under these new condi-

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tions, the amidation rate of S-2 was higher than that of theR-2 enantiomer, displaying a modest enantiomeric ratio(Chen et al., 1982) E 5 25 at a conversion of 53% in 5min. No changes were detected in blank reactions (60°C)without enzyme.

DiscussionThese results are in accordance with the recently reportedcrystal structure of CAL (Uppenberg et al., 1994 and1995). The very high reactivity of pyroglutamic esters maybe explained because of its size and shape: the small cyclicmolecule can readily pass through the active site pocket,described as a hydrophobic narrow channel of approx-imately 1 3 0.4 nm wide and 1.2 nm deep, while biggerand acyclic diethyl N-block-glutamates have to carry outprecise conformational changes to cross the tunnel andreach the active site. On the contrary, acyclic derivativesdisplay a remarkable enantioselectivity that dramaticallydecreases in the case of pyroglutamates. According to theabove mentioned structure, we can assume at least twosubsites in the CAL-active site, the hydrophobic channelmainly formed by Ile and Leu residues accupied by theglutamate side chain, and a polar region surrounding thecatalytic Ser105. In addition to the His224 of the triad,there are three residues (Thr40, Asp34 and Gln157) thathave their polar side chain atoms within 0.5 nm of the Ogof the catalytic serine. This polar subsite interacts with theN-blocking chain where the carbonyl or carbonyloxygroups of the protecting amides or carbamates would beinvolved in the hydrogen bond network created by thesepolar residues.

In agreement with our previous results (Conde et al., 1997)these electronic interactions would determine the enantio-

Lipase catalysed amidation of diethyl glutamate derivatives: peculiarities of ethyl S- and R-pyroglutamate

selectivity of the reaction. At 60°C, it can be assumed as ahypothesis that, due to its size, rigidity and electronicfeatures, the pyroglutamate lactamic ring does not fitprecisely in the polar subsite (probably, neither in thehydrophobic one) and, consequently, its electronic inter-actions are not as definite and strong as for acyclic N-blockglutamic derivatives and they are not significant enoughfor both S- and R-enantiomers with the corresponding lackof selectivity. When the reactions were performed at alower temperature the conformational mobility of theenzyme-substrate complex decreases, the electronic inter-actions start showing some importance and kinetic differ-ences arose between the two enantiomers.

AcknowledgementGenerous gift of Novozym 435 is gratefully acknowledgedto Novo Bioindustrial S.A. We also thank to C.I.C.Y.T.(project SAF-96-0107) for financial support.

ReferencesCaswell, M., Chaturvedi, R.K., Lane, S.M., Zvilichovsky, B. and

Schmir, G.L. (1981). J. Org. Chem. 46, 1585–1593.Chamorro, C., Gonzalez-Muniz, R. and Conde, S. (1995). Tetra-

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J. Am. Chem. Soc., 104, 7294–7299.Conde, S., Lopez-Serrano, P., Fierros, M., Biezma, M.I., Martınez,

A. and Rodrıguez-Franco, M.I. (1997). Tetrahedron, 53,11745–11752.

de Zoete, M.C., Ouwehand, A.A., van Rantwijk, F. and SheldonR.A. (1995). Recl. Trav. Chim. Pays-Bas., 114, 171–174.

Gerisch, S. and Jakubke, H.-D. (1997). J. Peptide Science, 3, 93–98and references therein.

Uppenberg, J., Hanse, M.T., Patkar, S. and Jones, T.A. (1994).Structure, 2, 293–308.

Uppenberg, J., Ohrner, N., Norin, M., Hult, K., Kleywegt, G.J.,Patkar, S., Waagen, V., Anthonsen, T. and Jones, T.A. (1995).Biochemistry, 34, 16838–16851.

Received: 18 December 1997Revisions requested: 14 January 1998

Revisions received: 26 January 1998Accepted: 27 January 1998

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