World

Journal

of Microbiology

& Biotechnology

12, 247-250

Hydrolysis of di-substituted hydantoins, by an enzyme preparation from lentil (Lens esculenta) seeds, for the synthesis of a,a-dialkylated amino acids with linear and cyclic substituents R. Rai, R. Balaji Rao and V. Taneja* Hydrolysis of 5,5’-di-substituted hydantoins with linear and cyclic side chains, to produce the corresponding U,Udialkylated amino acids and I-aminocycloalkane-I-carboxylic acids, using an enzyme preparation from lentil (Lens esculentu) seeds is reported for the first time. This method could be developed on a large scale and should be of interest to the chemical and pharmaceutical industries. Key words: Di-substituted

amino acids, di-substituted

hydantoins,

Enzymatic D- and L-selective hydantoin hydrolysis, for the synthesis of D- and L-amino acids, has been investigated and reviewed by several workers (Esaki et al. 1980; Jones 1986; Syldatk et al. 1990). Hydantoinases and dihydropyrimidinases (EC 3.5.2.2) have been reported in several bacteria, animals and jackbean (Bernheim & Bernheim 1946; Eadie et al. 1949; Wallach & Grisolia 1957; Yamada et al. 1978, 1980). The enzyme preparations from bacteria and animals have been used for 5-monosubstituted hydantoin hydrolysis to yield the AJ-carbamoyl derivative which, on further hydrolysis by NaNO,/HCl, alkali or N-carbamoylamino acid amidohydrolase, produces the corresponding Dor L-N amino acids (Cecere et al. 1975; Olivieri et al. 1979, 1981; Takahashi et al. 1979; Jones 1986; Syldatk et al. 1987; Moller ef al. 1988; Ishikawa et al. 1993). These hydantoinases and dihydropyrimidinases do not hydrolyse the 5,5’di-substituted hydantoins (Yamada et al. 1978; Esaki et al. 1980; Syldatk et al. 1990) and have not been reported to

R. Rai and V. Taneja are with the Department Science, Banaras Hindu University, Varanasi313965. R. Balaji Rae is with the Department Science, Banaras Hindu University, Varanasiing author. @ 7996 Rapid

Science

of Biochemistry,

Faculty of 221 005, India; fax: 91 542 of Chemistry, Faculty of 221 005, India. ‘Correspond-

Lens esculenfa.

CH,

W

i

““\ 7” b

a

Figure 1. Structures of a.a-dialkylated amino acids, showing (a) linear, a,a-di-n-butylglycine (DBG) and (b) the cycloalkyl side chains of I-aminocycloalkane-1-carboxylic acid (AC&). The number of carbon atoms in the cycloalkane ring (n) varies from four in AC& to six in AC& and 10 in AC&.

hydrolyse 5,5’-Spiro-cycloalkyl hydantoins. Recently, a,adi-substituted amino acids with linear and ring substituents have emerged as an active area of peptide research and of significance to the pharmaceutical industry (Valle et al. 1991; Prasad et al. 1994; Crisma et al. 19%). The present synthesis of a,a-dialkylated amino acids with linear (Figure la) and cyclic substituents (Figure lb) from the corresponding 5,5’-di-substituted hydantoins, using a lentil-seed enzyme preparation, is the first reported.

Publishers World Journal of Mrcmbiology

h Bmtecknofogy,

Vol 22, 1996

247

R. Rai, R.

Balaji Rao and V. Taneja

Materials

and Methods

5,5’-Di-n-butyland 5,5’-spirocycloalkyl hydantoins were prepared from the corresponding ketones (Henze & Speer 1942) and standard cc,a-di-n-butylglycine (DBG) and I-aminocycloalkane-Icarboxylic acid (AC&, where n is the number of carbon atoms in the cycloalkane ring) from the corresponding hydantoins (Jacobson 1946). All other chemicals (analytical grade) were purchased from Merck (BDH) except Dowex-50 W (hydrogen form, strongly acidic cation exchange resin, dry mesh 50 to 100, I% crosslinked), which was from Sigma. Lentil (Lens esculentu) seeds were procured from the local market. All experiments were replicated at least three times. Enzyme Preparation from Lentil Seeds Surface-sterilized lentil seeds were soaked in double-distilled water and homogenized in 0.02 M Tris/HCl buffer, pH 6.8, at 4°C. The homogenate was filtered through a double layer of muslin cloth and the filtrate centrifuged at 10,000 X g for 20 min at 4°C. The supematant was treated with an equal volume of 50% PEG 6000 in 0.02 M Tris/HCl buffer, pH 6.8, and the protein, collected by centrifugation, was suspended in a minimal amount of 0.02 M Tris/HCl buffer, pH 6.8, and designated as the lentil-seed enzyme preparation. Protein content was determined using the Bradford method with BSA as standard. Amino-acid Synthesis Reaction conditions were those used by Yamada et al. (1978) with the following modification: 5,5’-di-n-butylor 5,5’-spirocycloalkane hydantoin (0.12 M) and the lentil-seed enzyme preparation (20 mg protein) in 10 ml 0.1 M Tris/HCl buffer, pH 8.5, were incubated at 40°C for 24 h with moderate shaking. The reaction was terminated by adding 10% trichloroacetic acid (TCA) and centrifuged. The supematant was tested and processed for the isolation of the corresponding N-carbamoyl derivative. Two controls were run simultaneously; in one, the hydantoin was added after the TCA and in the other the enzyme preparation was added after the TCA.

Table 1. Characterization aminocyclohexane-lcarboxylic Compound AC& Melting

of

enzymatically synthesized acid (AC&) and its derivatives.

and characteristic

Value

point’

4 (TWt IR characteristic (cm-‘)

Decomposed

at 260°C

0.01 band

1614, 3030

AC&-OMe.HClS Melting point* 4 WW IR characteristics of ester (cm-‘)

l-

1525 (COO-); (NH,+)

210°C 0.60 1743

band

BOC-leu-AC&-OMe§ Melting

point*

17o’c 0.66

6 WW IR characteristic band of peptide (cm-‘) HPLC retention time (min) * Melting points are uncorrected. t R, is the ratio of the distance moved by the solvent.

1628, 2930 11.3

moved

$ AC&-methyl ester derivative. 5 t-Butyloxycarbonyl-leucine-AC&-OMe

1575 (N-H)

by the

dipeptide

(GO);

3329,

compound

to that

derivative.

carbamoyl derivatives and amino acids were detected, respectively, in an iodine chamber, with 1% p-dimethylaminobenzaldehyde solution in acetone/cone. HCl (91, v/v) and with 0.2% ninhydrin in acetone by heating at 60°C for 10 min. Analytical TLC on silica-gel plates using chloroform/methanol (95:5, v/v) at 32°C; AC,C and its methyl ester derivative were detected with ninhydrin reagent, and BOC-leu-AC&OMe dipeptide in an iodine chamber.

Isolation, Detection and Alkaline Hydrolysis of N-curbumoyl Derivatives The supematant from the above reaction was neutralized and developed on a Dowex-50 W column (1 X 20 cm) with water. The fractions containing N-carbamoyl derivative were collected and concentrated. The N-carbamoyl derivatives formed were then tested by the calorimetric method, using p-dimethylaminobenzaldehyde (Cecere et al. 1975). Th e d erivatives were hydrolysed in NaOH overnight at 110°C (Stark 1967) and the crystals obtained on concentration were collected and recrystallized in methanol/ water.

Fourier trunsforrn intru-red (FUR) spectroscopy. The FTIR spectra of various di-substituted hydantoins, spirocycloalkyl hydantoins, their corresponding N-carbamoyl derivatives, amino acids, A&COMe.HCl and BOC-leu-AC&-OMe dipeptide (KBr pellets, range 4600 to 400 cm-‘) were recorded using an FTIR spectrophotometer.

Other Derivatives The amino-acid methyl ester (AC&OMe.HCl) derivative was synthesized by the thionyl chloride/methanol method (Brenner & Huber 1953). The t-butyloxycarbonyl-leucine-AC,C-OMe (BOCleu-AC,C-OMe) dipeptide derivative, was synthesized, from BOC-leucine prepared by the method of Schnabel (1967), by a conventional, solution-phase procedure using dicyclohexylcarbodiimide-mediated coupling.

NMR.

Analyses Paper Chrmu~ogruphy formed on Whatman water (15:3.6:15, by

and TLC. Paper chromatography was perNo. 1 paper using butan-l-al/acetic acid/ vol.) at 32°C. Substituted hydantoins, N-

HPLC. HPLC analysis of the BOC-leu-AC&-OMe dipeptide was carried out, using a methanol-water linear gradient, a lichrosorb, reverse-phase C-18 column and an injection loop of 504, in an LKB instrument. BOC-leu-AC&-OMe

(400 MHz, CDCl Lucknow, India.

solvent)

was at the

central

analysed Drug

by

‘H-NMR

Research

Institute,

was

obtained

Results and Discussion N-Carbamoylcyclohexane-I-carboxylic as white hydantoin

crystals (yield 70%) following hydrolysis

acid

from 5,5’-spirocyclohexane using a lentil-seed enzyme

a,~-LX-substituted Table

2. Substrate

Hydantoin

specificity

of lentil-seed

substrate

5,5’-Di-n-butyl 5,5’-Spiro-cyclohexane 5,5’-Spiro-cyclooctane

hydantoin hydantoin

5,5’-Spiro-cyclododecane hydantoin Rt is the ratio MP-Uncorrected

l

enzyme. Corresponding

4’

hydantoin

of the distance moved melting points.

amino acid synthesis

N-carbamoyl

derivative

Corresponding

amino

acid

MP

Rf*

Acid

0.77 0.72 0.62

130°C 185°C 2oo’c

0.36 0.47

DBG AC&

Decomposed Decomposed

at 270% at 280°C

0.81 0.65

220°C

0.40 0.36

AC&

0.78

AC&

Decomposed Decomposed

at 270°C at 270°C

0.75 0.87

by the

compound

to that

moved

by the solvent

in paper

MP

Rf ’

chromatography.

derivatives. Similarly, the HPLC retention time and ‘HNMR spectrum of the presumed BOC-leu-AC,C-OMe are also similar with those of standard BOC-leu-AC,COMe.

I

14000

I

I

3000

2000 Wavenumber

Figure

2.

Fourier

carbamoylcyclohexane-I-carboxylic

transform

I

1000



(cm-‘) infra-red acid,

spectra (b) AC&

of and

(a)

N-

(c) BOC-

leu-AC&-OMe.

preparation. However, hydantoin was recovered unaltered in both control reactions. Alkaline hydrolysis of the Ncarbamoylcyclohexane-I-carboxylic acid resulted in the production of white crystals of l-aminocyclohexane-lcarboxylic acid (A&C), which decomposed at 26O”C, with an overall yield of 50% (yield optimization was not attempted). The melting point, TLC R( value and IR characteristic bands of the presumed A&C, AC,C-OMe.HCl and BOC-leu-AC,-C-OMe are given in Table I and are identical with those of the corresponding authentic compounds. The FTIR spectra of the presumed N-carbamoylcyclohexane-I-carboxylic acid, A&C and BOC-leu-AC,C-OMe (Figure 2) are also comparable with the corresponding spectra of standard A&C and its

Substrate Specificity Besides hydrolysing A&C hydantoin, the lentil-seed enzyme preparation also acted on 5,5’-di-n-butyland several 5,5’-spirocycloalkyl hydantoins. The melting point and paper chromatography R( values of di-substituted hydantoins, their corresponding N-carbamoyl derivatives and amino acids are given in Table 2. The values for DBG, A&C and AC,C are identical to those of the corresponding, standard amino acids. The FTIR spectra of DBG, AC,C, A&C and A&C show the characteristic IR bands at 1610 to 1640 cm-’ (COOgroup) and 3060 to 309Ocm-’ (NH+,) which agree well with those of the standard amino acids. Characteristic carbonyl-stretching bands in the IR spectra of the hydantoins are at 1710 to 1740 and 1760 to 1780 cm-‘. Synthesized A&C could not be compared with a standard as AC,,C and higher analogues could not be prepared by the chemical method. Recently, it has been shown that a,a-di-substituted amino acids with linear and ring substituents produce constraint in the peptide backbone and are important in peptide-structure conformation studies, peptide de n~uo design and protein engineering (Hruby et al. 1990; Karle et al. 1991, 1994; Valle et al. 1991; Prasad et al. 1994, 1995; Crisma et al. 1995). Also, chemotactic peptide analogues, N-formyl-MetXxx-Phe-OY (where Xxx is the a,a-di-substituted glycine/ AC,C and Y is CH, or H), exhibit enhanced chemotactic activity which is dependent on the chain length/ring size of the substituents (Prasad 1992). In addition to providing vital insight on the binding of physiological peptides to their receptors and on the mechanism of action involved, similar analogues of physiological peptides can be of use as agonisWantagonists. Use of lentil-seed enzyme preparation provides a simple procedure for the production of such cr,cr-di-substituted amino acids which, when scaled up, should be of significance to the chemical and pharmaceutical industries. World ]oumai

of Minobrology

6 Biotechnology,

Vol 12. 1996

249

R.

Rai, R. Balaji Rao and V. Taneja

Acknowledgements RR

is grateful

Delhi,

for

a Junior

to

University Research

Grants

Commission,

New

Fellowship.

References Bemheim, F. & Bemheim, M.L.C. 1946 The hydrolysis of hydantoin by various tissues. Journal of Biological Chemistry 163,

683-685. Brenner, M. & Huber, W. 1953 Herstellung von a-Aminosiureestem durch Alkoholyse der Methylester. Helveticu Chimica A& 36,1109-1115. Cecere, F., Galli, G. & Morisi, F. 1975 Substrate and steric specificity of hydropyrimidine hydrase. FEBS Lefters 57, 192194. Crisma, M., Valle, G., Toniolo, C., Prasad, S., Balaji Rao, R. & Balaram, P. 1995 P-Turn conformations in crystal structures of model peptides containing a,a-di-n-propylglycine and a,a-di-nbutylglycine. Biopolymers 35, l-9. Eadie, G.S., Bemheim, F. & Bemheim, M.L.C. 1949 The partial purification and properties of animal and plant hydantoinases. ]ournuJ of Biological Chemistry 181, 449-458. Esaki, N., Soda, K., Kumagai, H. & Yamada, H. 1980 Recent advances in enzymatic synthesis of amino acids. Biotechnology and Biaengineering 22, Suppl. 1, 127-141. Henze, H.R. & Speer, RJ. 1942 Identification of carbonyl compounds through conversion into hydantoins. ]otrmal of the American Chemical Society 64, 522-523. Hruby, VJ., Al-Obeidi, F. & Kazmierski, W.M. 1990 Emerging approaches in the molecular design of receptor-selective peptide ligands: conformational, topological and dynamic cansiderations. Biochemical ]ournuJ 268, 249-262. Ishikawa, T., Watabe, K., Mukohara, Y., Kobayashi, S. & Nakamura, H. 1993 Microbial conversion of oL-5-substituted hydantoins to the corresponding r-amino acids by Pseudomonas sp. strain NS671. Bioscience, Biotechnology and Biochemistry 57, 982-986. Jacobson, R.A. 1946 Esters of a-aminoisobutyric acid. Journal of the American Chemical Society 68, 2628-2630. Jones, J.B. 1986 Enzymes in organic synthesis. Tetrahedron 42,

3351-3403. Karle, I.L., Balaji Rao, R., Prasad, S., Kaul, R. & Balram, P. 1994 Nonstandard amino acids in conformational design of peptides. Helical structure in crystals of S-10 residue peptides containing dipropylglycine and dibutylglycine. ]ownuJ of the American Chemical Society 116, 10355-10361. Karle, I.L., Flippen-Anderson, J.L., Sukumar, M., Uma, K. & Balram, P. 1991 Modular design of synthetic protein mimics. Crystal structure of two seven-residue helical peptide segments linked by &-aminocaproic acid. ]ownaJ of the American Chemical Society

113,3952-3956. MBller, A., Syldatk, C., Schulze, M. & Wagner, F. 1988 Stereoand substrate-specificity of a o-hydantoinase and a o-Kcarbamyl-

amino acid amidohydrolase of Arthrobacter crystcrilopoietes AM2. Enzyme and Microbial Technology 10, 618-625. Olivieri, R., Fascetti, E., Angelini, L. & Degen, L. 1979 Enzymatic conversion of N-carbamoyl-o-amino acids to o-amino acids. Enzyme and MicrobiaJ Technology 1, 201-204. Olivieri, R., Fascetti, E., Angelini, L. & Degen. L. 1981 Microbial transformation of racemic hydantoins to o-amino acids, Biotechnology and Bioengineering 23, 2173-2183. Prasad, S. 1992 Synthesis, conformation and chemotactic activity studies on peptides containing cyclic and acyclic a,a-dialkylated glycines. PhD Thesis. Banaras Hindu University, Varanasi, India. Prasad, S., Balaji Rao, R. & Balram, P. 1995 Contrasting solution conformations of peptides containing a,cr-dialkylated residues with linear and cyclic side chains. Biopolymers 35, 11-20. Prasad, S., Mitra, S., Subramanian, E., Velmurugan, D., Balaji Rao, R. & Balram, P. 1994 Coexistence of folded and extended conformations of a tripeptide containing a,a-di-n-propylglycine in crystals. Biochemical and BiophysicuJ Reseurch Commttnicutions

198,424-430. Schnabel, E. 1967 Verbesserte synthese von tert.- Butyloxycarbonyl-aminosiuren durch pH-stat-reaktion. Liebigs Annalen der Chemie 702, 188-196. Stark, G.R. 1967 Modification of proteins with cyanate. Methods in Enzymology 11, 59&594. Syldatk, C., Cotoras, D., Dombach, G., Groa, C., Kallwafi, H. & Wagner, F. 1987 Substrateand stereospecificity, induction and metallodependence of a microbial hydantoinase. Biafechnoiogy Letters 9, 25-30. Syldatk, C., Liufer, A., Miller, R. & Hijke, H. 1990 Production of optically pure D- and L-a-amino acids by bioconversion of D,L5-monosubstituted hydantoin derivatives. Advances in Biochemical Engineering/Biotechnology 41, 29-75. Takahashi, S., Ohashi, T., Kii, Y., Kumagai, H. & Yamada, H. 1979 Microbial transformation of hydantoins to iV-carbamyl-o-amino acids. ]otrmal of Fermentution Technology 5 7, 328-332. Valle, G., Crisma, M., Toniolo, C., Sudhanand, Balaji Rao, R., Sukumar, M. & Balram, P. 1991 Stereochemistry of peptides containing I-aminocycloheptane-I-carboxylic acid (AC,0 crystal structures of model peptides. International ]o~rnul of Peptide and Profein Research 38, 5 II-5 18. Wallach, UP. & Grisoiia, S. 1957 The purification and properties of hydropyrimidine hydrase. Journal of Biological Chemistry 226,

277-288. Yamada, H., Shimizu, S., Shimada, H., Yoshiki, T., Tani, Y.. Takahashi, S. & Ohashi, T. 1980 Production of o-phenylglycine-related amino acids by immobilized microbial cells. Biochimie 62, 395-399. Yamada, H., Takahashi, S., Kii, Y. & Kumagai, H. 1978 Distribution of hydantoin hydrolyzing activity in microorganisms. Journal of Fermentation Technology 56, 484-491. (Received 1995)

in revised form

8 December

1995; accepted

9 December

Hydrolysis of di-substituted hydantoins, by an enzyme preparation from lentil (Lens esculenta) seeds, for the synthesis of α,α-dialkylated amino acids with linear and cyclic substituents.

Hydrolysis of 5,5'-di-substituted hydantoins with linear and cyclic side chains, to produce the corresponding α,α-dialkylated amino acids and 1-aminoc...
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