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When the degrees of acylation were. 35%, the modified chymotrypsin retained its activity of hydrolysis about 80% compared with native one. The best de...
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Chapter 11

Convenient Synthesis of Flavor Peptides Masaru Nakatani and Hideo Okai

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Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, Higashihiroshima, Hiroshima 724, Japan

For the purpose of synthesizing flavor peptides or proteins in large scale, we developed "protein recombination method" and "enzymatic synthesis using chemically modified enzyme". "Protein recombination method" was applied to the synthesis of C-terminal portion of β-casein and its analog. Chymotrypsin was chemically modified by Z-DSP in aqueous solution. It was stable for organic solvents. Using this modified enzyme, we succeeded in the synthesis of Inverted-Aspartame-Type Sweetener "Ac-Phe-Lys-OH" in one step.

Today, it is well-known that peptides or proteins exhibit various kinds of taste. Our group has been researching on the relationship between taste and structure of peptides, BPIa (Bitter peptide la, Arg-Gly-Pro-Pro-Phe-Ile-Val) (1) as a bitter peptide, Orn-βAla-HCl (OBA), Orn-Tau-HCl as salty peptides^), and "Inverted-Aspartame-Type Sweetener" (Ac-Phe-Lys-OH) as a sweet peptide^. The relationship between taste and chemical structure was partly made clear. Since commercial demand for these flavor peptides is increasing, we need to develop new synthetic methods which can prepare these peptides in large scale. We developed the following two methods: (1) protein recombination method as a chemical method, (2) enzymatic synthesis using chemically modified enzyme as a biochemical method. Protein Recombination Method For protein recombination method, starting materials arefreepeptides or proteins which are existing inexpensive, in large quantities in nature. We can also obtain peptides by solid-phase peptide synthesis or gene manipulation. By this method, we need to leave the functional group on the side chain unprotected as long as they don't make any problems. Another word, we only have to protect the amino group and the thiol group on the side chain. This protected peptide is coupled with another protected peptide, and product is next deblocked (Figure 1). This is an ideal route. But for realization of this method, there are following three problems: 1. selective blocking of side chain of basic amino acid in amine component

0097-6156/93/0528-0149$06.00/0 © 1993 American Chemical Society In Food Flavor and Safety; Spanier, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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F O O D FLAVOR A N D SAFETY

2. selective blocking or activation of acidic amino acids in acid component 3. selection of coupling method without racemization.

N»2 ÇOOH NH -{Peptide Fragment A ]-COOH j Blocking NH ÇOOH Ζ NH{ Peptide Fragment A J-COOH

NH COOH NH -{Peptide Fragment Β )-COOH 2

2

2

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Z

I Selective I Blocking

j Selective Blocking or Activation NHZ ÇOOH NHZ ÇOOH Ζ NH {Peptide Fragment A }-COX NH -{Peptide Fragment Β }-COOH 2

I Coupling NHZ ÇOOH Ο NHZ ÇOOH ZNH{Pept"ide Fragment A }-C-NH-{Peptide Fragment Β }-COOH 1 Deblocking NH ÇOOH 0 NH ÇOOH NH -{Peptide Fragment A }-C-NH{Peptide Fragment Β J-COOH T

2

2

2

Fig. 1.

Protein Recombination

Problem of Amine Component. If basic amino acids exist in amine component, selective blocking of amino group on the side chain is necessary. But it was impossible by conventional methods. We attempted to develop a new method. Selective Blocking of Amino Group on Side Chain of Basic Amino Acids. In 1987, we developed a new water-soluble active ester reagent, jp-hydroxyphenyldimethylsulfonium methyl sulfate (HODSP) (4-6), and acylating reagents, ZDSP {[/7-(benzyloxycarbonyloxy)phenyl]dimethylsulfonium methyl sulfate}, BocDSP{[p-(t-butoxycarbonyloxy)phenyl]dimethylsulfonium methyl sulfate}, FmocDSP{p-(9-fluorcnylmethyloxycarbonyloxy)phenyl]dimethylsulfonium methyl sulfate} and so on (7). These reagents can be used in aqueous solution because of high solubility due to the presence of a counter anion in the molecules. Making the best use of this character, Z-DSP was allowed to react with lysine by controlling the pH of the reaction mixture in aqueous media. The amino group on the side chain was selectively benzyloxycarbonylated in good yield when pH of thereactionmixture was 11.3. Application to Selective Blocking of Amino group on Side Chain of Peptides. We investigated to selectively protect the amino group on the side chain in peptides using Z-DSP. Phenylalanyllysine, ornithylphenylalanine and others were selected for this model. Z-DSP was allowed to react with these peptides in water in the same manners as described before. As shown in Table I, the result definitely shows that we succeeded in selective blocking of the amino group on the side chain of these peptides.

In Food Flavor and Safety; Spanier, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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Selection of Coupling Method. For protein recombination, coupling method must not cause racemization. Most suitable one seems to be azide method. If azide method is employed, the problem is esterification of peptide fragment. If esterification of peptide fragment is possible and it was applied to azide method, we will be get the object. A new reagent for the preparation of benzyl ester, p-hydroxyphenylbenzylmethylsulfonium chloride (HOBMC1), was introduced by our group (9). This reagent converts N-acyl amino acids into N-acyl amino acid benzyl esters under mild conditions. We thought that this reagent has a possibility of esterification of peptide fragment. Actually as shown in Table Π, many kinds of N-protected peptides were converted into benzyl esters in satisfactory yields. And we examined whether this esterification cause racemization. As a model peptide, Gly-Ala-Leu was synthesized by azide method via benzyl ester prepared by this reagent . We confirmed that no racemization occurred in this reaction (10). Application to the Synthesis of Flavor Peptide. As shown in figure 2, we applied this method to the synthesis of C-terminal portion of β-casein (Arg -Gly-ProPhe-Pro-Ile-Ile-Val ) and its analog (Arg-Gly-Pro-D-Phe-Pro-Ile-Ile-Val) (10). This synthetic peptide by this method possessed a bitter taste 250timesthat of caffeine, and it was the same result as which Kanehisa et al. had reported 8 years agof/7). And it is interesting that this analog produced the bitterness only about one twentieth as strong as that of original octapeptide. These results showed that protein recombination method is an effective strategy in synthesis of flavor peptides. 202

209

Enzymatic Synthesis Using Chemically Modified Enzyme It is advantageous if enzymatic synthesis could be carried out in organic solvents. In order to avoid denaturation, we thought to prepare more hydrophobic enzyme than native one. We made good use of water-soluble acylating reagents (Z-DSP, Boc-DSP) described above, and tried to modify the amino groups of enzyme. Preparation of Chemically Modified Chymotrypsin. Chemically modified chymotrypsin (12) was prepared by following methods: Z-DSP (2.5-20 equivalent to the amino groups of chymotrypsin) was added to the buffer solution (Na2B407-HCl, pH 8) of chymotrypsin (14mg). The solution was allowed to stand for 16 hours in refrigerator. And the solution was centrifuged (lO.OOOrpm, 10min). The solution was dialyzed, and obtained chemically modified chymotrypsin by lyophilization. Characterization of Modified Chymotrypsin. Measurement of the degree of modification of prepared chymotrypsin was determined by measuring the unreacted amino groups of the modified products with trinitrobenzenesulfonic acid (13). It was found that the degrees of benzyloxycarbonylation increased as the amount of the reagent increased (Table ΠΙ). Activities of the modified and unmodified chymotrypsin were determined by measuring the hydrolysis rates of Ac-Tyr-OEt, and methyl 2,3-di0-(L-phenylalanyl)-a-D-glucopyranoside, which was synthesized in our laboratory and found to be a suitable substrate for chymotrypsin. When the degrees of acylation were 35%, the modified chymotrypsin retained its activity of hydrolysis about 80% compared with native one. The best degree of modification was 35%. Less modified or more modified ones were inferior. We measured pH Dependence (Figure 3) and temperature dependence (Figure 4) of 35% modified chymotrypsin, and confirmed that those of modified chymotrypsin were not changed compared with those of native one.

In Food Flavor and Safety; Spanier, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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Table I. Selective Protection of Peptides Containing Lysine or Ornithine in Water

Z-DSP, pH controled Phe-Lys

Phe-Lys(Z)

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NaOH, in water

Reagents Z-DSP(1.6eq)

Peptides

PH

Phe-Lys Phe-Orn Lys-Phe Orn-Phe

11.3 11.3 11.3 11.3

Yield(%) 75 89 90 84

Table II. Preparation of N-Protected Peptide Benzyl Esters Using HOBMCI N-protected peptides Z-Phe-Gly-OH Z-Val-Tyr-OH Z-Pro-Leu-OH Z-Gly-Ala-OH Z-Lys(Z)-Pro-OH Boc-Ala-Glu-OH Boc-Gly-Gly-OH Boc-Ile-Val-OH Boc-Pro-Phe-OH Boc-Thr-Pro-OH Boc-Gln-Pro-OH Boc-Ser-Ile-OH Boc-Lys(Boc)-Val-OH Boc-Met-Pro-OH Boc-Phe-Asp-OH Boc-Pro-Pro-Pro-OH Boc-Val-Val-Val-Pro-Pro-OH

solvent DCM DCM-DMF DCM DCM DCM DCM-DMF DCM DCM DCM DCM DCM-DMF DCM DCM-DMF DCM DCM-DMF DCM DCM-DMF

yield (%) 83 46 85 57

889 64 79 53 92 74 24 71 57 *) 80 57

*) Dibenzyl ester DCM - dichloromethane DMF -NJV- dimethylformamide

In Food Flavor and Safety; Spanier, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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Convenient Synthesis of Flavor Peptides

NAKATANI AND OKAI

Gly

Arg

Pro

Pro

Phe

He

He

Boc

J OH

Boc

[MA] +

Boc|OH 1

Boc

Η

[H ]

~ [H ] +

Boc Boc Z-KOH ,N02 ,|/

N Q

2

,N02

2

H-J-OBzl Boc

OH Boc+OH HOBMC11

ΓΝ Η 1 2

4

OBzlBoc^-™ [H ] NH Η [N3 +

2

•OBzl Boc

H

3

3

+

OH H[DCC]

[H ]

[H /Pd] 2

Fig. 2. The Synthetic Route of C-Terminal Portion of β-Casein (Arg-GIy-Pro-Phe-Pro-IIe-IIe-Val) and Its Analog

In Food Flavor and Safety; Spanier, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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100

80

Z:35% Native

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40

20

8

pH

Fig. 3. pH Dependence of Modified (Z:35%) and Native Chymotrypsin (Reproduced from ref. 8 with permission. Copyright 1992 Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

I

20

1

30 40 Temperature (°C)

I

50

Fig. 4. Temperature Dependence of Modified (Z:35%) and Native Chymotrypsin (Reproduced from ref. 8 with permission. Copyright 1992 Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

In Food Flavor and Safety; Spanier, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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Table III. Determination of Modified Amino Groups of Chymotrypsin Z-DSP (eq.)

number of free amino groups

0 42.5 85 175 340

17 15.4 11.1 9.6 6

degree of modification (%) 0 10 35 44 65

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SOURCE: Adopted from ref. 8.

Synthesis of Z-Tyr-Gly-NH2 in Aqueous-DMF Solvent Media. Using this modified enzyme, we carried out the synthesis of "Z-Tyr-Gly-NH2", which has never been formed in 100% aqueous system (14), and compared with native chymotrypsin on the effect of organic solvent. To a solution of Z-Tyr-OH (315mg) in Tris buffer (pH 6.7, 0.5ml), which contained Ν,Ν-dimethylformamide (DMF) (0-100%), was added a solution of H-Gly-NH2-HCl (1 lmg) and native or modified chymotrypsin (2mg) in the same Tris buffer. The mixture was incubated at 20°C for 24 hours and heated at 100°C for 15 minutes. The products were isolated by HPLC (ODS column, 278nm, 50% acetonitrile). Native chymotrypsin inactivated when concentration of DMF was 50%, while chemically modified chymotrypsin kept its activity even up to 80% (Table IV).

Table IV. Effect of DMF Concentration on Synthesis of Z-Tyr-Gly-NH by Native and Modified Chymotrypsin 2

DMF(%) 0 10 20 30 40 50 60 70 80 100

synthetic rate of Z-Tyr-Gly-NH? (%) native chymotrypsin modified chymotrypsin (Z:35%) 0 0 13 16 25 5 0 0 0 0

0 14 24 19 43 33 27 24 25 0

SOURCE: Adoptedfromref. 8.

In Food Flavor and Safety; Spanier, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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Table V. Synthetic Reactions of Ac-Phe-Lys-OH in Various Water-Organic Solvents

DMF

AcOEt

CH C1 2

2

Hexane

+ +

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n-BuOH

+(10%)

Acetone

+

+(1%)

+

+(5%)

SOURCE: Adopted from ref. 8. -: Ac-Phe-Lys-OH was not detected. +: Ac-Phe-Lys-OH was detected by thin layer chromatography (): Yields of Ac-Phe-Lys-OH were measured by high performa.nce liquid chromatography.

One Step Synthesis of Ac-Phe-Lys-OH. Aso reported that Inverted-AspartameType Sweetener "Ac-Phe-Lys-OH", chemically synthesized by our group (3), was prepared using native chymotrypsin (14). This method demands five steps, and has no merit compared with chemical synthesis that we had reported. Therefore we investigated more effective synthesis of Ac-Phe-Lys-OH using chemically modified chymotrypsin. It is obvious that amino acids, especially lysine, are not suitable for nuceophiles because the amino groups of amino acids are ionized in aqueous solution. If in organic solvent, this ionization decreases extremely. So we attempted this one step synthesis of Ac-Phe-Lys-OH using this enzyme in the organic-aqueous solvent system. To a solution of Ac-Phe-OH (2.07mg), and Lys-HCl (5-100 equivalent to Ac-Phe-OH) in phosphate buffer (pH=6.7), organic solvents (DMF, n-butanol, benzene, acetone, dichloromethane, hexane and ethyl acetate) was added. And a solution of modified chymotrypsin (2mg) in O.lmM HC1 was added to the solution. The mixture was incubated at 37°C for 24 hours and the enzyme was filtered off by MOLCUT L (MILLIPORE). Synthesis of Ac-Phe-Lys-OH was followed by thin layer chromatography. In the case of DMF or n-butanol, Ac-Phe-Lys-OH was not detected, contrary to our expectation. But in the case of benzene, acetone, dichloromethane, hexane and ethyl acetate, synthesis of the objects was detected by thin layer chromatography in some cases (Table V). And quantitative analysis was carried out by ion exchange high performance liquid chromatography. Through a series of experiment, maximum yield was 10% in the case of 90% ethyl acetate-10% water solvent system. Conclusions For the purpose of synthesis of flavor peptides or proteins in large scale, we developed "protein recombination" as a chemical method. C-terminal portion of β-casein and analog were synthesized by using this method effectively. We also developed "enzymatic synthesis using chemically modified enzyme" as a biochemical method.

In Food Flavor and Safety; Spanier, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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Inverted-Aspartame-Type Sweetener, A c - P h e - L y s - O H , was synthesized i n one step i n the organic-aqueous solvent media. M a x i m u m y i e l d was 10%. B u t considering collection o f enzyme and raw materials, we think that this synthesis is able to be industrialized. W e also think that this modified technique can be applied to the preparation o f another modified enzyme. There are a few small problems i n these two methods. B u t we hope that these two methods w i l l be made full use as synthetic strategies of flavor peptides or the other functional peptides.

Literature Cited 1 Fukui, H.; Kanehisa, H; Ishibashi, N.; Miyake, I.; Okai, H. Bull. Chem. Soc. Jpn., 1983, 56, 766-769. 2 Tada, M.; Shinoda, I.; Okai, H. J. Agric. Food Chem., 1984, 32, 992-996. 3 Nosho, Y.; Seki, T.; Kondo, M.; Ohfuji, T.; Tamura, M.; Okai, H. J. Agric.Food Chem., 1990, 38, 1368-1373. 4 Kouge, K.; Koizumi, T.; Okai, H.; Kato, T. Bull. Chem. Soc. Jpn., 1987, 60, 2409-2418. 5 Kouge, K.; Soma, H.; Katakai, Y.; Okai, H.; Takemoto, M.; Muneoka, Y. Bull. Chem. Soc. Jpn., 1987, 60, 4343-4349. 6 Kouge, K.; Katakai, Y.; Soma, H.; Kondo, M.; Okai, H.; Takemoto, M.; Muneoka, Y. Bull. Chem. Soc. Jpn., 1987, 60, 4351-4356. 7 Azuse, I.; Tamura, M.; Kinomura, K.; Okai, H.; Kouge, K.; Hamatsu, F.; Koizumi, T. Bull. Chem. Soc. Jpn., 1989, 62, 3103-3108. 8 Kawasaki, Y; Murakami, M; Dosako, S; Azuse, I; Nakamura, T; Okai, H. Biosci. Biotech. Biochem., 1992, 56, 441. 9 Mukaiyama, N.; Kouge, K.; Nakatani, M.; Okai, H. Chem. Express, 1991, 6, 985-988. 10 Nakatani, M. Hiroshima University, unpublished data. 11 Kanehisa, H.; Miyake, I.; Okai, H.; Aoyagi, H.; Izumiya, N. Bull. Chem. Soc. Jpn., 1984, 57, 819-822. 12 Azuse, I.; Tamura, M.; Kinomura, K.; Kouge, K.; Kawasaki, Y.; Okai, H. Chem. Express, 1989, 4, 539-542. 13 Habeeb, A. F. S. Α., Anal. Biochem., 1966, 14, 328. 14 Aso, K. Agric. Biol. Chem., 1989, 53, 729-733. Received January 21, 1993

In Food Flavor and Safety; Spanier, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.