WO1990012883A1

WO1990012883A1 - Enzymatic membrane method for the synthesis and separation of peptides

Info

Publication number: WO1990012883A1
Application number: PCT/US1990/002188
Authority: WO
Inventors: Daniel J. Brose; Roderick J. Ray; Paul Van Eikeren; Guillermo Iacobucci
Original assignee: The Coca-Cola Company
Priority date: 1989-04-24
Filing date: 1990-04-23
Publication date: 1990-11-01
Also published as: KR920701461A; JPH04502558A; CA2054754A1; AU641161B2; AU5554790A; EP0470175A1; FI915012A0

Abstract

The present invention discloses a method for the enzymatic synthesis of a peptide. A protected peptide having a C-terminal carboxylate group or a protected N-acyl amino acid having an alpha carboxylate group is reacted with a protected peptide having an N-terminal ammonium group or a protected amino acid having an alpha ammonium group in the presence of a condensation enzyme under conditions in which the carboxylate group and the ammonium group condense to form a protected, uncharged, peptide product. This peptide product is transported across a water-immiscible hydrophobic phase disposed in a hollow fiber lumen into an aqueous product phase and is removed from the aqueous product phase to prevent back diffusion across the water-immiscible hydrophobic phase. Reverse osmosis and other separation techniques may be utilized to remove the peptide product from the product phase. The water-immiscible hydrophobic phase functions as an ion rejection membrane separating the aqueous reaction phase from the product phase creating oil/water interfaces with each of the aqueous phases. The present invention can be practised either in ILM's supported on microporous hollow fiber modules, or in oil/water contactors made of hydrophilic hollow fiber. An integrated process for the enzymatic resolution of D-, L-phenylalanine isopropyl ester and the racemization and recycling of D-phenylalanine isopropyl ester is also disclosed.

Description

ENZYMATIC MEMBRANE METHOD FOR THE

SYNTHESIS AND SEPARATION OF PEPTIDES

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of application Serial No.

06/897,679, filed August 18, 1986 and of application Serial No. 07/078,504 filed July 28, 1987.

DESCRIPTION TECHNICAL FIELD

The present invention relates generally to an enzymatic method for the synthesis and separation of peptides employing a membrane permeable to uncharged peptides but impermeable to charged molecules; and, more particularly, to the simultaneous synthesis and purification of peptides, L,L-dipeptides, and its application to the preparation of L-aspartyl-L-phenylalanine methyl ester (aspartame).

BACKGROUND ART

The use of proteolytic enzymes as condensation catalysts for the stereospecific coupling of two L-amino acids to yield L, L-peptides is known since the early days of protein chemistry. As early as 1938, Rergmann and Fraenkel-Conrat described the formation of the water-insoluble dipeptide Bz-Leu-Leu-NHPh by reacting Bz-Leu-OH and H-Leu-NHPh in the presence of the protein degrading enzyme papain. M. Bergmann and H. Fraenkel-Conrat, J. Biol. Chem. 124, 1 (1938). This reaction is possible only between those amino acids that form peptide bonds that are susceptible to cleavage by the papain or other enzyme used.

Indeed, the condensing reaction's equilibrium between the amino acid reactants and peptide product is largely displaced towards the reacting amino acids. Nevertheless, the condensing reaction can be driven to completion by mass action if, e.g., the

dipeptide product is poorly soluble and precipitates out of the reaction phase.

Due to the commercial importance of certain peptides and the fact that enzymes are known to catalyze peptide formation under mild conditions there has been a great deal of research done on the enzymatic synthesis of peptides particularly simple

dipeptides. K. Oyama and K. Kihara, Kaqaku Sosetsu 35, 195 (1982); K. Oyama and K. Kihara, ChemTech. 14, 100 (1984).

The process for enzymatic synthesis of the peptide

derivative aspartame, described in U.S. Patent 4,165,311,

hereinafter the '311 process, involves the thermolysin- catalyzed condensation of N-carbobenzoxy-L-aspartic acid with

D, L-phenylalanine methyl ester and precipitation of an

intermediary complex, D-phenylalanine methyl ester salt of

N-carbobenzoxy-aspartame, to drive the reaction to the peptide product side. Further processing of this intermediary complex allows for the recovery of D-phenylalanine methyl ester, that may be recycled after racemization, and of the

N-carbobenzoxy-aspartame derivative which can be converted to aspartame by elimination of the N-carbobertzoxy protecting group. The '311 process must be practiced on a batch basis which is cumbersome and complicates the recovery of enzyme. Also see: K. Oyama, S. Irino, T. Harada and N. Hagi, Ann. N.Y. Acad. Sci. 434, 95 (1985).

The N-carbobenzoxy protecting group plays an essential role in the '311 process by: fulfilling the structural requirement imposed by the active site of thermolysin; and by contributing to the insolubility of the intermediary complex, thereby increasing the yield of the reaction. Elimination of the N-carbobenzoxy protecting group from the aspartame derivative must be effected under mild conditions, e.g., catalytic hydrogenation, to prevent cleavage of the methyl ester function. Catalytic hydrogenation involves the inconvenience of handling hydrogen gas on a large scale.

Alternative approaches for driving enzymatic condensation reactions to completion have also been described in the chemical literature. For example, the use of organic solvents as reaction media has been found effective for increasing the peptide product yields; although, the concomitant decrease in enzyme stability has precluded its practice on a large scale. K. Oyama,

S. Nishimura, Y. Nonaka, K. Kihara and T. Hashimoto, J. Org.

Chem. 46, 5241 (1981); H. Ooshima, H. Mori and Y. Harano, Biotechnology Letters 7, 789 (1985); K. Nakanishi, T. Kamikubo and R. Matsuno, Biotechnology 3, 459 (1985).

In view of the above-noted difficulties in the practice of prior art methods for enzymatic synthesis of peptides,

particularly, the requirements for precipitation of an

intermediary complex and handling of dangerous reagents, it would be desirable to provide an improved process that avoids these difficulties and that safely provides effective yields without rapid deactivation of the enzyme catalysts.

DISCLOSURE OF INVENTION

The present invention provides a process for the enzymatic synthesis of peptides which provides for simultaneous synthesis and purification of the peptide product.

The present invention provides a process for the safe, economical and efficient synthesis and purification of peptides and derivatives thereof, particularly aspartame.

Another advantage of the present invention is to provide an economical process for the enzymatic synthesis of peptides that provides for the efficient use of enzyme and the means to effect the synthesis on a continuous basis.

Another advantage of the present invention is to provide a process particularly adapted to the enzymatic synthesis of aspartame and its derivatives with D, L-phenylalanine and

N-protected-β-substituted-L-aspartate in substantially

quantitative yield.

The present invention provides a method for the synthesis and purification of a compound, comprising the steps of coupling a first reactant with a second reactant to produce a membrane transportable, uncharged compound; transporting the transportable compound across a membrane that will not transport the reactants; and preventing the transported compound from back-diffusing across the membrane.

The present invention also provides a process for the enzymatic synthesis and purification of compounds comprising the steps of coupling a first compound, including a protonated amino group (ammonium), and a second compound, including a free carboxylate group, using a condensation enzyme in an aqueous mixture to produce an uncharged (or non-ionized) coupled

compound; continuously removing the uncharged coupled compound from the aqueous mixture by diffusion across a membrane that selectively transports the uncharged coupled compound to the product side of the membrane. Preferably, the transported coupled compound is a peptide or derivative thereof that is converted to a charged (or ionized) molecule so that it does not back-diffuse across the membrane. Thus, the formation of the coupled compound product is driven in the reaction mixture because it is constantly being removed therefrom.

The present invention also provides a method for the

enzymatic synthesis of peptides comprising the steps of

condensing first and second amino acid compounds in an aqueous initial reaction, mixture to form an uncharged compound;

transporting the uncharged compound into an aqueous second reaction mixture across a membrane that will not transport substantial amounts of the amino acid compounds; and converting the transported uncharged compound to a form that cannot be retransported across the membrane to the initial reaction

mixture. The transported compound, converted to a form that is not retransportable across the membrane, can be removed from the second reaction mixture. Also, small amounts of first and second amino acid compounds cupermeating the membrane with the uncharged compound into the second reaction mixture can be separated from the second reaction mixture and optionally returned to the initial reaction mixture.

The present invention also provides a process for the enzymatic synthesis of etspartame and its analogs, comprising the steps of condensing a N-acyl-β-substituted-L-aspartic acid including an α-carboxylate group with a phenylalanine lower alkyl ester including an α-ammonium group in an aqueous reaction mixture including a condensation enzyme, to form

N-acyl-L-aspartyl-(β-substituted)-L-phenylalanine lower alkyl ester (i.e. 1-6 carbons), an uncharged peptide; and transporting the unchanged peptide from the aqueous reaction mixture to a product mixture across a permselective membrane . In one embodiment, for proteosynthesis done at or above pH 5, the

preferred acyl group is formyl, the preferred beta substituent is methyl, the preferred lower alkyl ester is isopropyl, and the preferred condensation enzyme is thermolysin. In another

embodiment, for proteosynthesis done at or below pH 4, the

preferred acyl group is carbobenzoxy, the preferred beta

substituent is hydrogen (β-COOH), the preferred lower alkyl ester is methyl and the preferred enzyme is pepsin. In the latter case the permeable aspartame intermediate is N-CBZ-asp-phe-OMe, where the charge of the free β-carboxylate of the aspartic acid has been suppressed by protonation in order to make the peptide permeable.

The present invention also provides a method for the

enzymatic resolution of racemic alpha amino acid compounds comprising the steps of: hydrolyzing an uncharged D,L-alpha amino acid derivative carrying at least one hydrolyzable functional group attached to the chiral carbon, in an aqueous reaction mixture in the presence of a hydrolase enzyme capable of

hydrolyzing a sensitive functional group, to form a charged

L-amino acid compound and an uncharged D-amino acid derivative, and transporting the uncharged D-amino acid derivative from the aqueous reaction mixture across an ion rejection membrane into a product mixture. The uncharged D-aminα acid derivative in the product mixture can be converted to a species that cannot

back-diffuse across the membrane. The method for the enzymatic resolution of racemic alpha amino acid compounds can be practiced in combination with the peptide synthesis methods discussed above. An example of racemic alpha amino acid derivative is DL-phenylalanine methyl ester. An example of hydrolase enzyme is the esterase aminoacylase I.

The present invention also provides a method for the

enzymatic synthesis of a peptide, comprising the steps of:

reacting a protected, peptide first reactant having a C-terminal carboxylate group with a protected, peptide second reactant having a N-terminal ammonium group in the presence of a

condensation enzyme in an aqueous reaction phase under conditions in which the C-terminal carboxylate group and the N-terminal ammonium group condense forming a protected, uncharged, peptide product; transporting the protected, uncharged, peptide product across a water-immiscible hydrophobic phase into an aqueous product phase; and preventing the protected, uncharged, peptide product in the aqueous product phase from back-diffusing across the water-immiscible hydrophobic phase.

The present invention also provides a method for the

enzymatic synthesis of a peptide, comprising the steps of:

reacting a protected, N-acyl amino acid first reactant having an alpha carboxylate group with a protected, peptide second reactant having a N-terminal ammonium group in the presence of a

condensation enzyme in an aqueous reaction phase under conditions in which the alpha carboxylate group and the N-terminal ammonium group condense forming a protected, uncharged, peptide product; transporting the protected, uncharged, peptide product into a water-immiscible hydrophobic phase into an aqueous product phase; and preventing the protected, uncharged, peptide product from back-diffusing across the water-immiscible hydrophobic phase.

The present invention also provides a method for the

enzymatic synthesis of a peptide, comprising the steps of:

reacting a protected, peptide first reactant having a C-terminal carboxylate group with a protected, amino acid second reactant having an alpha ammonium group in the presence of a condensation enzyme in an aqueous reaction phase under conditions in which the C-terminal carboxylate group and the alpha ammonium group

condense forming a protected, uncharged, peptide product;

transporting the protected, uncharged, peptide product across a water-immiscible hydrophobic phase into an aqueous product phase; and preventing the protected, uncharged, peptide product from back diffusing across the water-immiscible hydrophobic phase.

The present invention also provides a method for the

enzymatic synthesis of a peptide, comprising the steps of:

combining a protected, N-acyl amino acid first reactant having an alpha carboxylate group with a protected, amino acid second reactant having an alpha ammonium group in the presence of a condensation enzyme in an aqueous reaction phase under conditions in which the alpha carboxylate group and the alpha ammonium group condense forming a protected, uncharged, peptide product;

transporting the protected, uncharged, peptide product across a water-immiscible hydrophobic phase into an aqueous product mixture; and preventing the protected, uncharged, peptide produc from back-diffusing across the water-immiscible hydrophobic phase.

The peptides of the present invention comprise a plurality of amino acid residues. The peptides of the present invention include, but are not limited to dipeptides. The peptides of the present invention include but are not limited to peptides comprising from three to eight amino acid residues . An example of protected, N-acyl amino acid first reactant is

N-formyl-(β-methyl)-aspartic acid. Examples of protected, amino acid second reactants are L-phenylalanine methyl ester and

L-phenylalanine isopropyl ester. An example of a condensing enzyme is thermolysin. Another example of a protected, peptide first reactant is N-formyl-(O-Bzl)-tyr-gly-OH. Another example of a protected, peptide second reactant is H-gly-phe-leu-OMe. Another example of a condensing enzyme is papain. Another example of a protected, peptide first reactant is

N-formyl-(β-methyl)-asp-phe-OH. Another example of a protected, amino acid second reactant is H-trp-OMe. Another example of a condensing enzyme is pepsin.

In another embodiment of the present invention

N-carbobenzoxy-aspartic acid having an alpha carboxylate group the first reactant and L-phenylalanine methyl ester having an alpha ammonium group is the second reactant. The alpha

carboxylate group of the first reactant and the alpha ammonium group of the second reactant condense in the presence of pepsin in an aqueous reaction phase to form a protected, uncharged, peptide product. This product is transported across a

water-immiscible hydrophobic phase and prevented from back diffusing across the same.

The present invention also provides a method for the enzymatic synthesis of a peptide, comprising the steps of:

reacting N-acyl-(β-substituted) aspartic acid first reactant having an alpha carboxylate group with L-phenylalanine lower alkyl ester second reactant derived from a secondary alcohol having 3 to 6 carbon atoms having an alpha ammonium group in the presence of a condensation enzyme in an aqueous reaction phase under conditions in which the alpha carboxylate group and the alpha ammonium group condense forming a protected, uncharged, peptide product; transporting the protected, uncharged, peptide product across a water-immiscible hydrophobic phase into an aqueous product phase; and preventing the protected, uncharged, peptide product from back-diffusing across the water-immiscible hydrophobic phase. The aqueous reaction phase can be maintained at a temperature range of from about 20°C to about 65°C. An example of a N-acyl-(β-substituted) aspartic acid first reactant is N-formyl-(β-methyl)-aspartic acid. Another example of a

L-phenylalanine lower alkyl ester second reactant is

L-phenylalanine isopropyl ester. An example of the condensing enzyme is thermolysin wherein the temperature of the aqueous reaction phase is about 50°C.

In one embodiment of the present invention an ILM module is utilized comprising a plurality of microporous hollow fibers made of polymeric materials. These hollow fibers can support

water-immiscible organic liquids immobilized by capillarity within the microporous walls. This immobilized water-immiscible organic liquid constitutes a hydrophobic phase that functions as an ion rejection membrane separating the aqueous reaction phase from the aqueous product phase. The aqueous reaction phase, also referred to herein as the "tube phase," is located within the lumen of hollow fibers. The aqueous product phase, also referred to herein as the "shell phase" is located in the shell spaces existing in the module between hollow fibers. Oil/water

interfaces are thus created with each of the two aqueous phases. A schematic representation of this ILM module is shown in Figures 2, 9, 25 and 26. In a preferred ILM configuration the ends of the hollow fibers are sealed or potted in resinous material so that aqueous solution being circulated through the lumens will not mix with aqueous solution being circulated through the shell spaces. Hollow fibers made of microporous polypropylene

exemplify a preferred material. While this embodiment describes the shell phase as the aqueous product phase and the tube phase as the aqueous reaction phase, alternatively, the shell phase can be the aqueous reaction phase and the tube phase can be the aqueous product phase.

Alternatively, the water-immiscible hydrophobic phase comprises an organic liquid located within the lumen defined by the walls of a hollow fiber comprising hydrophilic material. An example of the hydrophilic material is cellulose. In one

embodiment of the present invention, oil/water interfaces can be created by utilizing two membrane modules comprising hydrophilic hollow fibers arranged as shown in Figures 17, 27 and 28. In a preferred membrane module configuration the ends of the hollow fibers are sealed or potted in a resinous material so that organic liquid being circulated through the lumens will not mix with the aqueous solution (phase) being circulated through the shell spaces. Each module comprises a plurality of hydrophilic hollow fibers. The lumens of the hydrophilic hollow fibers are filled with a water immiscible organic liquid. The two membrane modules having a connecting means such as a common loop of circulating organic liquid comprise a membrane contactor. The water-immiscible organic liquid located within the lumens of the hydrophilic hollow fibers comprises the water-immiscible

hydrophobic phase of each membrane module, and the two isolated aqueous phases located outside of the walls of the hydrophilic hollow fibers respectively comprise the aqueous reaction and product phases. The water-immiscible hydrophobic phase functions as an ion rejection membrane separating the aqueous reaction phase in the first membrane module from the aqueous product phase in the second membrane module.

A membrane contactor comprises a first membrane module for transferring the protected, uncharged peptide product from the aqueous reaction into the water-immiscible hydrophobic phase; a second membrane module for transferring the protected, uncharged peptide product from the water-immiscible hydrophobic phase into the aqueous product phase; and a connecting means between the water-immiscible hydrophobic phase in the first membrane module and the water-immiscible hydrophobic phase in the second membran module of the membrane contactor. The aqueous reaction phase in the first membrane module is located outside of the hollow fibers and wets the walls of the hollow fibers creating an oil/water interface between the aqueous reaction phase and the

water-immiscible hydrophobic phase; and the aqueous product phase in the second membrane taodule is located outside of the hollow fibers and wets the walls of the hollow fibers creating an oil/water interface between the aqueous product phase and the water-immiscible hydrophobic phase. The circulation of liquids at the oil/water interfaces is countercurrent. The aqueous product phase may be processed repeatedly through a plurality of membrane contactors.

One example of the step of preventing the protected, uncharged, peptide product from back-diffusing across the water-immiscible hydrophobic phase comprises converting the protected, uncharged, peptide product to a charged species. The conversion can be chemical or enzymatic. Chemical means include pH dependent ionization of a prototropic functional group, ionization resulting from a dissociation of a carboxylic acid function and/or resulting from a protonation of a free amino group. An example of an enzymatic conversion is the hydrolysis of an ester function utilizing a protease having esterolytic activity. An example of the protease enzyme having esterolytic activity is aminoacylase I. The enzyme having esterolytic activity can be circulated against the membrane in the aqueous product phase. The enzyme having esterolytic activity can be immobilized on a water insoluble support and the aqueous product phase can be circulated over the enzyme.

The present invention also provides peptide compounds selected from the group consisting of N-formyl-(β-benzyl)-L- aspartyl-L-phenylalanine methyl ester, N-formyl-(β-benzyl)-L- . aspartyl-L-phenylalanine,

N-carbobenzoxy-(β-methyl)-L-aspartyl-L-phenylalanine methyl ester, N-carbobenzoxy-(β-methyl)-L-aspartyl-L-phenylalanine, N-formyl-(β-methyl)-aspartyl-phenylalanine methyl ester,

N-formyl-(β-methyl)-aspartyl-phenylalanine,

N-formyl-(β-methyl)-L-aspartyl-L-phenylalanine, N-formyl(β-methyl)-aspartyl-phenylalanyl-tryptophan methyl ester, N-formyl(β-methyl)-aspartyl-phenylalanyl-tryptophan,

N-carbobenzoxy-phenylalanyl-glycyl-glycyl-phenylalanine methyl ester, N-carbobenzoxy-phenylalanyl-glycyl-glycyl- phenylalanine, N-formyl-(O-benzyl-tyrosyl)-glycyl-glycyl- phenylalanyl-leucine methyl ester, N-formyl-(O-benzyl- tyrosyl)-glycyl-glycyl-phenylalanyl leucine, N-formyl- (β-methyl)-aspartyl-phenylalanine isopropyl ester.

The present invention provides a method for the enzymatic synthesis of a peptide, comprising the steps of: reacting a first compound selected from the group consisting of a protected peptide having a C-terminal carboxylate group and a protected, N-acyl amino acid having an alpha carboxylate group with a second compound selected from the group consisting of a protected peptide having a N-terminal ammonium group and a protected amino acid having an alpha ammonium group in the presence of a

condensation enzyme in an aqueous reaction phase under conditions in which the carboxylate group and ammonium group condense forming a protected, uncharged, peptide product; transporting the protected, uncharged, peptide product across a water-immrscible hydrophobic phase into an aqueous product phase; and separating the protected, uncharged, peptide product from the aqueous product mixture to prevent that product from back-diffusing across the water-immiscible hydrophobic phase.

The present invention also provides a method for the

enzymatic synthesis of a peptide, comprising the steps of:

reacting N-acyl-(β-substituted) aspartic acid first reactant having an alpha carboxylate group with L-phenylalanine-lower alkyl ester second reactant having an alpha ammonium group in the presence of a condensation enzyme in an aqueous reaction phase under conditions in which the alpha carboxylate group and the alpha ammonium group condense forming a protected, uncharged, peptide product; transporting the protected, uncharged, peptide product across a water-immiscible hydrophobic phase into an aqueous product phase; and separating the protected, uncharged, peptide product from the aqueous product phase to prevent that product from back-diffusing across the water-immiscible hydrophobic phase. The step of separating the protected,

uncharged, peptide product from the aqueous product phase may be carried out utilizing a trapping means such as reverse osmosis or the formation of specific molecular complexes. Specific cavities comprising zeolites and/or cyclodextrins may be utilized. The step of separating may also be carried out utilizing solvent extraction, adsorption on a matrix or by precipitation with a reagent. The aqueous reaction phase can be maintained at a temperature in a range of from about 20°C to about 65°C. An example of the phenylalanine reactant is a lower alkyl ester derived from a secondary alcohol having 3 to 6 carbon atoms. An example of the condensing enzyme is thermolysin wherein the temperature of the aqueous reaction phase is about 50°C. An example of the aspartic acid N-acyl-(β-substituted) first

reactant is N-formyl-(β-methyl)-asp-OH. An example of the phenylalanine lower alkyl ester second reactant is

L-phenylalanine isopropyl ester.

In general, the amino acids which can be utilized for peptide synthesis according to the present invention comprise the L-enantiomers of the 20 natural amino acids recognized by the genetic code as protein building blocks, plus their various protected derivatives available through standard procedures commonly used in the field of peptide synthesis. The preferred protecting groups will vary according with the choice of

condensing enzyme, nature of the hydrophobic phase, pH,

temperature and nature of the solvent for any particular

proteosynthesis. BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects and advantages are attained by the invention, as will be apparent from the following detailed description taken in conjunction with the accompanying drawings, wherein:

Figure 1 is schematic illustration of the enzymatic

synthesis of aspartame in accordance with the present invention.

Figure 2 is a schematic representation of an apparatus for practicing the process of the present invention. Figure 3 is a graph illustrating the quantity of product (aspartame derivative) formed over time in Examples 1 and 2.

Figure 4 is a graph illustrating the quantity of product (aspartame derivative) formed over time in Example 4.

Figure 5 is a graph illustrating the quantity of product (aspartame derivative) formed over time in

Example 5.

Figure 6 is a graph illustrating the quantity of product (aspartame derivative) formed over time in Example 6.

Figure 7 is a graph illustrating the quantity of product (aspartame derivative) formed over time in Example 7.

Figure 8 is a graph illustrating the enzymatic resolution of DL-phe-OMe described in Example 8.

Figure 9 is a schematic representation of an apparatus for practicing the present invention which illustrates the vessels on the product side as described in Example 9.

Figure 10 is a graph illustrating the quantity of product (aspartame derivative) formed over time in Example 9 with and without the utilization of an ion exchange resin.

Figure 11 describes the pepsin catalyzed proteosynthesis of N-formyl-(β-methyl)-asp-phe-trp-OH over time.

Figure 12 compares the V_syn and V_perm for the pepsin catalyzed proteosynthesis of N-formyl-(β-methyl)-asp-phe- trp-OMe.

Figure 13 describes the pepsin catalyzed proteosynthesis of N-formyl-(β-methyl)-asp-phe-trp-OMe over time.

Figure 14 describes the papain catalyzed proteosynthesis of N-CBZ-phe-gly-gly-phe-OH over time.

Figure 15 describes the rate of synthesis of

N-CBZ-phe-gly-gly-phe-OMe.

Figure 16 describes the papain-catalyzed proteosynthesis of N-formyl-(O-BzI)-tyr-gly-gly-phe-leu-OH.

Figure 17 describes the process in which RO is used to trap product on one side of the membrane contactor.

Figure 18 describes the concentration of C in the

thermolysin reactor during the long-term run. Figure 19 describes the thermolysin-catalyzed

proteosynthesis of N-formyl-(β-methyl)-asp-phe-OH.

Figure 20 describes the kinetics of synthesis of

N-formyl-(β-methyl)-asp-phe-O-<.

Figure 21 describes the effect of the enzyme aminoacylase on the rate of permeation of N-formyl-(β-methyl)-asp-phe-O-< across an ILM including N,N-diethyldodecanamide.

Figure 22 describes the thermolysin-catalyzed

proteosynthesis of N-formyl-(β-methyl)-asp-phe-O-< in a ILM module.

Figure 23 describes the synthesis of

N-formyl-(β-methyl)-L-asp-L-phe-O-< in a membrane contactor.

Figure 24 describes the pepsin-catalyzed proteosynthesis of N-CBZ-asp-phe-OMe in a ILM module.

Figure 25 is a partial view (enlarged) of a cross section of hollow fibers in an ILM module.

Figure 26 is a partial schematic view (enlarged) of an ILM module.

Figure 27 is a partial schematic view (enlarged) of a cross section of hollow fibers in a membrane contactor.

Figure 28 is a partial schematic view (enlarged) of a membrane contactor.

Figure 29 is a schematic presentation of an apparatus for integrated enzymatic resolution of D-, L-phenylalanine isopropyl ester and the racemization and recycling of D-phenylalanine isopropyl ester.

BEST MODEST FDR CARRYING OUT THE INVENTION

The invention disclosed herein provides a procedure for driving to completion the enzymatic synthesis of peptides in an aqueous reaction mixture at equilibrium, by separating the uncharged peptide intermediate, or derivative thereof, from the reaction mixture by means of a membrane that selectively

transports the uncharged peptide out of the reaction mixture into a product mixture. Because the membrane is substantially

impermeable to the reactants (charged molecules) removal of the peptide intermediate from the reaction mixture causes a decrease of peptide concentration at equilibrium that pushes the reaction toward completion by mass action.

The membranes most useful in the practice of the present invention are Immobilized Liquid Membranes (ILM) comprising a nonpolar liquid embedded in microporous support material

providing an oil-water interface that is substantially impervious to the enzymes, reactants and other charged products.

Hydrophobic polymers such as polypropylene are preferred support materials. ILM modules can be produced utilizing polypropylene hollow fibers. Celgard, a registered trademark of the Celanese Corporation, 1211 Avenue of the Americas, New York, N.Y. 10036 and sold by Celanese Fibers Marketing Corporation, Charlotte, N.C., is an example of commercially available hollow fibers comprising polypropylene. Potting compounds known in the art and polyvinyl chloride pipe or tubing may optionally be utilized in fabricating an ILM module. Another polymer for fabricating the microporous support material is TEFLON, a trademark of

E. I. DuPont de Nemours & Co. for fluorinated hydrocarbon

polymers. A typical microporous support is GORE-TEX, a trademark of W. C. Gore & Associates, Inc. Figure 25 is a partial view (enlarged) of a cross section of hollow fibers in an ILM module. The partial view which is enlarged shows hydrophobic hollow fibers 101 having a lumen (bore) 102, made from microporous polymeric material 105 which can support water-immiscible organic liquid by capillarity within microporous walls. A capillary 104 is shown in the microporous polymeric material 105; however, the microporous polymeric material 105 actually includes many such capillaries 104 extending from the lumen to the exterior of the hollow fiber 101. The lumen 102 comprises the tube phase (e.g., aqueous reaction phase). The space 103 between hollow fibers comprises the shell phase (e.g., aqueous product phase).

Figure 26 is partial schematic view (enlarged) of an an ILM module 115. The partial view which is enlarged shows hydrophobic hollow fibers 101. The ends of each hollow fiber are potted in a resinous material (potting compound) so that the tube phase being circulated through the lumen 102 through opening 107 on first wall 106 and returned through opening 111 on third wall 110 will not mix with the shell phase being circulated through space 103, through opening 109 on second wall 108 and returned through opening 113 on fourth wall 112. In ILM module 115 actually comprises many hydrophilic hollow fibers 101 although only three are shown in this figure.

The micropores pass through the support material and should be sized so that an immobilized liquid will be held therein by capillarity and will not escape therefrom when subjected to, e.g., pressure differentials across the membrane or other

ordinary reaction conditions. Subject to the foregoing

limitations is advantageous to maximize the contact area between the immobilized liquid and reaction mixture to maximize the rate of transfer (flux) of the uncharged peptide product across the membrane. It will be appreciated that the preferred pore size will vary depending on the properties of the particular

immobilized liquid, reactants employed, products produced, and like factors; and further, that the optimum pore size can be determined empirically by those skilled in the art. A useful discussion of pore size selection is found in U.S. Patent

No. 4,174,374 the text of which is incorporated herein by

reference. The use and preparation of immobilized liquid

membranes are described in the following references, the texts of which are also incorporated herein by reference, S. L. Matson, J. A. Quinn, Coupling Separation and Enrichment to Chemical

Conversion in a Membrane Reactor, paper presented at the AICHE Annual Meeting, New Orleans, Louisiana (November-8-12, 1981) and S. L. Matson and J. A. Quinn, Membrane Reactors in Bioprocessing, Ann. N.Y. Acad. Sci. 469, 152 (1986).

The immobilized liquid held in the microporous support by capillarity should be water immiscible and a good solvent for the uncharged peptide product which must be transported across the membrane (diffused) at a reasonable rate, i.e., good transport characteristics/high flux; while, charged or ionized molecules on both the reaction side and product side of the membrane are, for the most part, not transported across the membrane in either direction, i.e, good selectivity/ion rejection.

Selection of the best combination of support material and immobilized liquid for use in an enzymatic synthesis of a peptide in accordance with the present invention will depend, in part, on the nature of the particular reactants employed, products desired and solvents in the system.

The generally preferred immobilized liquids for the practice of the present invention include water-immiscible organic

solvents, such as alcohols of 6 to 20 carbons, branched and unbranched, for example, n-decanol, n-dodecanol, iso-hexadecanol and mixtures thereof. Also preferred are mixtures of water immiscible organic solvents including mixtures thereof. Such solvents include but are not limited to N,N-diethyl-dodecanamide, dodecane and 2-undecanone.

Another type of membrane useful in the practice of the invention comprises hydrophobic solid films made of organic polymers such as polyvinyl chloride or the like. The preparation of these polymer membranes is well described in the literature, for example, O. J. Sweeting, Editor, Science and Technology of Polymer Films, Interscience, New York (1968), while extensive application of such membranes to the separation of gases and liquids are discussed in S. T. Hwang and K. Kammermeyer,

Membranes in Separations, Techniques of Chemistry, Vol. VII.

(A. Weissberger, editor) John Wiley & Sons, Inc., New York

(1975).

A preferred embodiment of the invention employs a membrane reactor/separator system which provides an aqueous reaction mixture or phase circtil atirrgrin contact with one side of an ILM membrane and a product aqueous phase or mixture circulating countercurrently at the opposite surface of the membrane.

S. L. Matson and J. A. Quinn, Ann. N.Y. Acad. Sci. 469, 152

(1986). The pH and temperature of the reaction and product phases are maintained at a value that keeps the reactants in a form that minimizes their transport across the membrane at pH's between about 4.0 and 9.0. Transport of the uncharged peptide intermediate from the reaction phase to the product phase is driven by the concentration gradient across the membrane created by increasing uncharged peptide concentration in the reaction phase. The transport activity or flux across the membrane can be significantly enhanced by the simultaneous, irreversible

conversion of the transported peptide, in the product phase, to a species that cannot back-diffuse. For example, the latter conversion may result in the formation of a polar peptide that cannot back-diffuse and thus generating the driving force needed to achieve completion of the coupling reaction. An example of a membrane reactor/separator that could be adapted for the practice of the present invention is found in U.S. Patent No. 4,187,086.

Available alternative membrane reactor/separator

configurations that could be adapted to practice of the present invention include the hollow fiber modules described in U.K.

Patent Application 2,047,564 A, and conventional plate and frame-type filter devices well known in the art.

In addition to selective transport of the uncharged peptide the membrane provides a barrier between the reaction phase and product phase that prevents undesirable mixing of, and reactions between, the components of each phase.

In a preferred membrane reactor/separator configured in accordance with the present invention, the chemical equilibrium between the reactants is actually "pulled" across the membrane by conducting an irreversible conversion of the transported

uncharged peptide, to a membrane impermeable species, on the product side of the membrane. This type of membrane

reactor/separator employs a coupled two enzyme (E¹ and E²) process of the general type:

E¹ E²

A⁺ + B- → C → D^±

The-charged reactants A and B are amino acids and/or small peptides which are condensed with the aid of peptide forming enzyme E¹ to form the uncharged intermediary peptide C which is selectively transported across the membrane to the product side. It is understood that reactive functional groups in the reactants that do not participate in the desired reaction may be protected or blocked, where necessary, to prevent undesirable side

reactions and/or charges in the products. On the product side of the membrane uncharged peptide C is converted to charged peptide D which cannot back-diffuse across the membrane, causing the chemical equilibrium in the reaction mixture to shift toward the production of more C.

This concept is illustrated in Figure 1, for the specific case of the enzymatic condensation of D,L-phenylalanine methyl ester with (N-and β-protected) N-formyl-β-benzyl-L-aspartate, in the presence of thermolysin at about pH 5.5.

In the reaction scheme illustrated in Figure 1 the reactant A⁺ is D, L-phenylalanine methyl ester and B- is

N-formyl-β-benzyl-L-aspartate. The reactants are condensed on the reaction side of the membrane by the enzyme E¹ thermolysin forming the uncharged peptide C. The pH is selected to maintain the reactants in their charged state and thus minimize their diffusion across the membrane along with uncharged peptide C.

Although the chemical equilibrium for the condensation reaction largely favors the reactant A⁺ and B- species, diffusion of the uncharged peptide product C across the membrane to the product side requires the constant production of C to maintain the chemical equilibrium on the reaction side of the membrane.

In one embodiment, on the product side of the membrane an esterase enzyme E² quickly and irreversibly converts the

uncharged peptide C diffused across the membrane to charged peptide D which cannot back-diffuse to the reaction side. Thus the chemical equilibrium on the reaction side is effectively

"pulled" across the membrane and toward the production of

uncharged product C. Thereafter, the peptide D is converted to aspartame by acid hydrolysis, which removes the formyl and benzyl protecting groups, followed by C-terminal esterification with methanol. In another embodiment the esterase is not utilized and the uncharged product C is directly separated from the product mixture by physical means.

The foregoing method may be practiced in a countercurrent flow membrane reactor/separator 10, as shown in Figure 2, operating under controlled conditions of pH and temperature, so that a reaction mixture comprising a

N-acyl-β-substituted-L-aspartic acid, e.g.,

N-formyl-β-benzyl-L-aspartate, having a free α-carboxylate group

(electronegative species) which is allowed to condense with a reactant, e.g, D, L-phenylalanine methyl ester, having a

protonated free α-amino group (electropositive species), under the catalytic action of proteases active in the pH range of about 4.0-9.0, to yield a fully protected L-aspartyl-L-phenylalanine dipeptide bearing no ionized groups (electroneutral species). On the reaction side of the membrane, the reaction mixture is circulated from reactor tank 12 aided by pump 14, through feed-in conduit 16 through separator 18 to feed-out conduit 20 which returns to reactor tank 12. On the product side of the membrane a product mixture or sweep including fully protected uncharged peptide, e.g., N-formyl-β-benzyl- L-aspartyl-L- phenylalanine methyl ester transported across the membrane, is circulated from product reactor tank 22, aided by pump 24, through product sweep in-conduit 26, through the product side of separator 18, to product sweep out-conduit 30. This product mixture in product reactor tank 22 includes a second enzyme E², an esterase, or other suitable reagent, that can cleave at least one of the protected groups borne by the uncharged peptide, thus generating an electrocharged species that cannot escape the sweep stream by back-diffusing through the membrane. If an esterase is utilized, a preferred esterase will have a preferred pH range of from about 6.0 to 9.0. Aminoacylase I, α-chymotrypsin and subtilisin A are examples of esterases considered useful in the present invention.

The conversion to charged species that cannot back diffuse across the water-immiscible hydrophobic phase can be carried out utilizing chemical or enzymatic means. Chemical means include pH dependent ionization of a prototropic functional group,

ionization resulting from dissociation of a carboxylic acid function and/or resulting from a protonation of a free amino group or hydrolysis of an ester function. For example, the conversion of an uncharged product into a charged product that cannot back-diffuse can be achieved by an appropriate pH gradient between the two aqueous phases separated by the hydrophobic membrane. This is physically possible as the hydrophobic phase is impervious to ions, thus allowing the existence of two aqueous phases of different pH in equilibrium with respect to non-ionic solutes. For example, a diffusible free amine R-NH₂, bearing no charges in an aqueous phase at pH 8, can be transferred across a hydrophobic membrane into a second aqueous phase at pH 3 and be irreversibly trapped by protonation to form the

non-diffusible ammonium salt R-NH₃. ⁺Similarly, a diffusible acid R-COOH bearing no charges at pH 2 can be transferred at pH 2 and trapped at pH 6 through dissociation to the non-diffusible carboxylate R-COO-. The utilization of a pH gradient is

particularly useful in the synthesis and separation of peptides and other similar compounds bearing carboxyl and ammonium groups.

A similar way of practicing this invention but using a different liquid membrane configuration is illustrated in

Figure 17. The liquid membrane configuration of Figure 17 is preferred under conditions wherein the water-immiscible

hydrophobic phase may leak as a result of higher reacting

temperatures such as temperatures above 40°C. In this oil/water contactor the organic membrane is created at the inner walls of hydrophilic cellulose fibers, whose bores or lumens are filled with the desired hydrophobic. organic phase, and having the bulk aqueous phase located outside of the fibers and wetting the walls of said fibers. Packing of bundles of said fibers in a modular arrangement allows for the creation of two compartments,

separated by an oil/water interface. Circulation of said oil phase between two separated aqueous phases contained in two individual modules constituting a single contactor is shown in Figure 17. A plurality of contactors may be utilized. Each aqueous phase representing the reaction and product phases discussed above, allows for the transport of permeable product from one aqueous phase to the second one containing the esterase enzyme. Membrane separators of the general type shown in

Figure 17 are well known in the art. U.S. Patent No. 4,754,089. describes the utilization of such membranes in phase transfer catalysis. U.S. Patent No. 4,778,688 describes a similar membrane. U.S. Patent Nos. 4,572,824, 4,563,337 and 4,443,414 also describe such oil water contactors. Another example of similar membranes is described in U.S. Patent No. 4,664,808.

Membrane variations in multiphase assymetric reactor systems are described in U.S. Patent No. 4,795,704. The charged product may be periodically discharged and/or continuously removed from the product phase by conventional separation means such as ion exchange resins and other techniques including reverse osmosis and the like, and the remaining

effluent may be recycled through the system. Where this

separation is by ion-exchange the resulting product bound to the ion-exchange resin may be desorbed and recovered using

conventional procedures.

In the case of reverse osmosis separation, it may be first necessary to retain the esterase enzyme utilizing an

ultrafiltration membrane of adequate porosity that will produce an ultrafiltrate containing only the charged product. This ultrafiltrate can then be concentrated over a reverse osmosis membrane and the charged product isolated from the resulting retentate.

Alternatively, in another embodiment the uncharged peptide can be prevented from back-diffusing across the membrane

utilizing a trapping such as a reverse osmosis membrane. In this case the second enzyme may not be required to efficiently operate the process. Reverse osmosis is well-known in the art. James S. Johnson, Jr., "Reverse Osmosis," in Kirk-Othmer's Encyclopedia of Chemical Technology, Third Edition, Vol. 20, pp. 230-248 John Wiley & Sons, New York, N.Y. (1984) and U.S. Patent"

Ne. 4,643,902.

Other trapping means include the formation of specific molecular complexes. Specific cavities of zeolites and/or cyclodextrins may be utilized. In addition to trapping, solvent extraction, adsorption on a matrix or precipitation with a reagent may be utilized for the step of separating the protected, uncharged peptide product from the aqueous product phase. The use of membrane contactors in conjunction with reverse osmosis, as described in Examples 14 and 17, will cause the desired displacement of the proteosynthetic equilibrium without affecting the kinetics of peptide synthesis in the enzyme reactor. This approach is considered as a viable alternative to the use of a second enzyme for the purpose of driving the proteosynthesis to completion. Its usefulness, however, is dictated in practice by the relative affinity of the uncharged peptide intermediate towards the oil/water phase. The higher the partition towards the oil phase is, the lower the permeability of the peptide towards the aqueous product phase will become, making the

transfer from oil to water the rate-limiting step of the process. This phenomenon is illustrated in Example 16, and Figure 21, where the rate of release of the peptide

N-formyl-(β-methyl)-asp-phe-O-< from the oil phase into the aqueous product phase, called here V_perm, is increased by a factor of two when the aqueous phase contains the enzyme

aminoacylase able to convert that peptide into the more

hydrophilic N-formyl-(β-methyl)-asp -phe-OH.

The selection of appropriate deprotection reagent(s) is determined by the chemical nature of the protecting groups used on the reactants, such as, N-protected-L-aspartic acid, and as indicated above the choice of protecting groups is in turn dictated by structural constraints imposed by the active site of the condensing enzyme.

In general, the condensing enzymes useful in the practice of the present invention are proteolytic enzymes, sometimes called proteases, which may be divided into two groups. The more commonly used are endopeptidases, which only cleave inner

linkages, and exopeptidases, which preferably cleave terminal linkages. Useful enzymes include serine proteinases, (ETC 3.4.21) such as chymotrypsin, trypsin, subtilisin BNP' and Achromobacter protease; thiol proteinases (EC 3.4.22), such as, papain;

carboxyl proteinases (EC 3.4.23), such as, pepsin; and

metalloproteinases (EC 3.4.24), such as, thermolysin, prolisin, Tacynasen N (St. caespitosus) and Dispase. Binding of the enzymes to insoluble supports, following procedures well known to practitioners of the art, may be incorporated to the practice of this invention; and although binding the enzymes is not an essential step, it may be desirable in certain applications.

Among the many proteases potentially useful for the practice of this invention, thermolysin [E.C. 3.4.24] is the condensing enzyme most preferred because of its remarkable thermostability, wide availability, low cost and broad useful pH range between about 5.0 to 9.0. Other preferred proteases include pepsin and penicillopepsin [T. Hofmann and, R. Shaw, Biochim. Biophys. Acta 92 543 (1964)] and, the thermostable protease from Penicillium duponti [S. Emi, D. V. Myers and G. A. Iacobucci, Biochemistry 15, 842 (1976)]. They would be expected to function at about pH 4.5 or below, exhibit good stability at such pHs, and do not require the presence of Zn++ and Ca++ ions to maintain their activity.

The practical realization of enantiomeric selectivity that is, in the case described above, production of only the L, L isomer of the peptide C, is directly related to the enzyme selected, optimal functioning of the membrane, chemical nature of the support material and pH of the aqueous reaction phase.

One preferred membrane for practicing the above-described specific method is.a microporous polypropylene support material including a mixture of iso-hexadecanol and n-dodecane immobilized therein. This membrane is available from Bend Research, Inc., 64550 Research Road, Bend, Oregon 97701, U.S.A. under the

tradename/designation. "Type 1 Hollow Fiber Selective Dialysis Membrane" and is preferred with the reactions of Examples 1-4. Another preferred membrane utilizes Celgard Type 2400

polypropylene hollow fibers (Celgard is a registered trademark of the Celanese Corporation, 1211 Avenue of the Americas, New York, N.Y- 10036. Celgard can be.obtained from Celanese Fibers

Marketing Corporation, Charlotte, N.C.) as the microporous material supporting a mixture of 30% v/v N,N-diethyl-dodecanamide in dodecane as the water-immiscible organic liquid ILM. This membrane was obtained from Bend Research, Inc., 64550. Research Road, Bend, Oregon 97701, U.S.A. under the tradename/designation "Type 2 Hollow Fiber Selective Dialysis Membrane" and is

preferred with the reactions of Examples 5-9. Celgard Type 2400 polypropylene hollow fibers having a pore size of 0.025 - 0.050 μm and a wall thickness of 25 μm were utilized in the Type 2 Hollow Fiber Selective Dialysis Membrane of Bend Research, Inc. When operated at pHs of about 5.5, these membranes exhibit a high selectivity, for example, when practicing the process of

Figure 1, selectivity in the range of about 500:1 (w/w) in favor of the uncharged peptide species have been measured. That is,

500 milligrams of the uncharged peptide (C) are transported across the membrane for each milligram of charged reactant (A⁺ or

B-) transported.

As mentioned above, for the application of the present invention it will be necessary, or desirable, to block or protect various functional groups on the reactants and products to prevent undesirable side reactions that could prevent production of the desired product and/or reduce its yield and to suppress electrocharges in intermediate peptide C. Table I below lists a series of selected combinations of protecting groups and

deprotection conditions useful in connection with the practice of this invention.

.

As a result of the enantioselectivity of selected

condensation enzymes and the functional discrimination exerted by the membrane, the practice of the invention using

N-formyl-L-aspartyl-β-benzyl ester and D, L-phenylalanine methyl ester could achieve a 99.8% enantiomeric resolution of the racemic phenylalanine methyl ester reactant, the L-enantiomer appearing as N-formyl-L-aspartyl(β-benzyl)-L- phenylalanine methyl ester, an aspartame derivative, with the unreacted

D-enantiomer remaining in the reaction phase.

The D-phenylalanine methyl ester remaining in the reaction phase may be recovered therefrom, re-racemized to the D,L-stage, and recycled into the feedstock. Racemization is a necessary step for processes employing racemic amino acid reactants, e.g., the '311 process described above.

The economic advantages of the present invention derive, at least in part, from the use of a racemate feed reactant rather than a more expensive pre-resolved L-enantiomer. This advantage is made possible by the in situ optical resolution of the

D,L-phenylalanine methyl ester due to: (a) the

enantioselectivity of the condensing enzyme; and (b) the high selectivity of the membrane in favor of the uncharged species.

Preferred methods for the low-cost synthesis of racemic phenylalanine are those based on the utilization of benzaldehyde via 5-benzalhydantoin sometimes called the Grace process, or the catalytic carbonylation of benzyl chloride to phenylpyruvic acid, a procedure developed by Sagami Chemical Research Center," Tokyo, Japan (sometimes referred to as the Toyo Soda process of Toyo Soda Manufacturing Co., Ltd., Yamaguchi, Japan).

It will be appreciated by those skilled in the art that the practice of this invention is not necessarily restricted to the synthesis of peptide sweeteners, such as, aspartame or its analogs and derivatives. The invention may also be used for the synthesis of other useful peptides, di-, tri-, tetra- and

pentapeptides, or peptides of higher complexity, that are capable of diffusing through a permselective membrane at a reasonable rate. For example, considering the bond specificity of

thermolysin, and assuming the presence of only one thermolysin sensitive bond in the product (indicated by the arrow in the formula below), one could synthesize met-enkephalin ( 1 ) by following scheme: (Bzl)

(BOC) -tyr-gly-gly-OH + H-phe-met- (Oter-Bu)

↑↓ Thermolysin pH 5.5

(Bzl)

(BOC)-tyr-gly-gly-phe-met-(Oter-Bu)

↓ H₃O⁺

H-tyr-gly-gly-phe-met-OH (1)

The feasibility of a stepwise total enzymatic synthesis of met-enkephalin using α-chymotrypsin and papain has been

documented in the literature. See: W. Kullmann, J. Biol. Chem 255, 8234 (1980).

Another potentially useful application of the present invention is in the enzymatic synthesis of Gramicidin S (2) by the following scheme:

2 H-val-orn(CBZ ) -leu-D-phe-pro-OH

↓ ↑ thermolysin pH 5.5

leu

(CBZ)orn D-phe

val pro

→ ←

pro val

D-phe orn(CBZ)

leu

↓ Pd/H₂

leu

orn D-phe

val pro

pro val

D-phe orn

(2)

leu

Various other examples of the enzymatic synthesis of useful peptide products where the present invention may find application and described in the literature are the synthesis of angiotensin, substance P, eledoisin, caerulein and leu-enkephalin.

Another useful class of peptide compounds' amenable to proteosynthesis according to the present invention are the β-lactam antibiotics. This group comprises the well-known penicillins and cephalosporins, that are characterized by the presence of a β-lactam function in their chemical structure.

The high reactivity of the β-lactam function has made the chemical synthesis of those antibiotics difficult, and is responsible for their poor stability in aqueous solutions in the presence of nucleophiles. The enzyme β-lactamase specifically catalyzes the hydrolysis of the amide bond in the β-lactam ring of penicillins and cephalosporins. Nathan Citri, "Penicillinase and other β-Lactamases", in Paul D. Boyer (Ed.), "The Enzymes," Volume IV, 3rd Edition, Academic Press, New York 1971.

The presence of β-lactamases in the microbial world, particularly in Gram-negative bacteria, is considered a defensive mechanism against β-lactam antibiotics. The facile induction of this enzyme with penicillin has made possible the production of β-lactamase (penicillin β-lactam amidohydrolase, EC 3.5.2.6) by fermentation of resistant strains of Bacillus cereus, Bacillus licheniformis and Escherichia coli, E. J. Vandamme, "Penicillin acylases and β-lactamases," in A. H. Rose (Editor), "Microbial Enzymes and Bioconversions," Chapter 9, pp. 504-522, Academic Press, New York, 1980.

β-Lactamase may be used as a reverse hydrolase for the synthesis of a penicillin methyl ester B from the corresponding penicilloic acid A, according with the teachings of the present invention, the uncharged B being selectively transported over A across a hydrophobic membrane, and further made non-permeable by the action of an esterase to give the polar penicillin C.

β-lactamase

→

←

A (charged) B (uncharged)

esterase

C (charged)

This approach could facilitate the synthesis of penicillins from penicilloic acid precursors, that are per se amenable to total synthesis. J. C. Sheehan and K. R. Henery-Logan, J. Am. Chem. Soc. 84, 2983 (1962).

Those skilled in the art will appreciate that the present invention may also find application to compounds other than peptides, peptide like compounds and equivalents thereof.

For example, the use of ester hydrolases like pig liver esterase [EC 3.1.1.1] for the asymmetric resolution of prochiral dicarboxylic acid diesters into chiral monoesters is well known [C. J. Sih et al., Ann N.Y. Acad. Sci. 471, 239 (1986)], tlie text of which is incorporated herein by reference. The same enzyme can be used in the fashion described in this invention for the resolution of racemic carboxylic acid compounds, through the selective transport of a chiral ester through the membrane and the retention of the non-reactive enantiomeric acid in the reaction phase.

The present invention utilizes standard biochemical

terminology as follows: "H-val-" indicates that valine is the N-terminal amino acid residue of a peptide having the terminal amino group free; "-phe-OH" indicates that phenylalanine is the C-terminal amino acid residue of the peptide which has a

C-terminal free carboxy group. Also "V_syn" indicates the average rate of synthesis and "V_perm" indicates the average rate of permeation.

The following detailed Examples are presented to further illustrate the present invention.

Example 1.

An aspartame derivative was prepared in accordance with the present invention as follows:

To a solution containing 5.02g (20 mmoles)

N-formyl-L-aspartyl-β-benzylester, 4.31 g (20 mmoles) L- phenylalanine methyl ester in 100 mL in water, adjusted to pH 5.5, was added 500 mg thermolysin enzyme (Daiwa Chem. Co., Osaka, Japan) representing a total of 8 x 10⁵ proteolytic units.

The resulting clear reaction mixture was incubated for

15 hrs at 40°C, when the presence of insoluble dipeptide

N-formyl-β-benzyl-L-aspartyl-L-phenylalanine methyl ester became apparent. The resulting mixture was then placed in a 200 mL vessel, connected to the reaction side, in this case tube side, of an experimental hollow-fiber separator of a Bend Research, Inc. "Type 1 Hollow Fiber-Selective Dialysis Membrane" that provided 1 square foot of membrane area. The product side of the membrane (shell side of the separator) was connected to a source of aqueous (product) mixture (total volume = 200 mL) containing 500 mg of the enzyme Acylase I (EC 3.5.1.14) from Aspergillus Sp. (Sigma A 2156), at pH 7.5. This enzyme, usually described as an aminoacylase, was found to function as a C-terminal esterase, on both N-acetyl- and N-formyl-β-benzyl aspartame.

The reaction and product mixtures were circulated at room temperature through the hollow fiber separator countercurrently at the rate of 600 mL/min, with the assistance of peristaltic pumps. The configuration of this apparatus resembles that illustrated in Figure 2. Since the pH in both the reaction and product mixtures drops as the process progresses, constancy of pH was maintained through the use of pH stats.

The formation of N-formyl-β-benzyl-L-aspartyl-L- phenylalanine was monitored by HPLC. Chromatographic analysis was conducted on a Tracor Model 995 instrument along with a LDC Spectromonitor II detector set at 254 nm for the detection of the amino acids, fully protected product dipeptide, and dipeptide. The column used was a NOVA-PAK C₁₈ Radial-Pak cartridge, 8mm x 10cm, housed in a Millipore Waters RCM-100 Radial Compression Module.

The mobile phase used for the detection of the fully

protected dipeptide was a v/v mixture of 45% methanol (HPLC grade) 5% tetrahydrofuran (HPLC grade); and 50% of a 1% KH₂PO₄ buffer solution. For the detection of the product dipeptide, the mobile phase consisted of a v/v mixture of 40% methanol and 60% of a 1% KH₂PO₄ buffer solution (1 mL of triethylamine per liter solvent was added to minimize tailing and the pH was adjusted down to 4.3 using 80% phosphoric acid). The flow rate was kept at 1 mL/minute.

The HPLC data relating to formation of

N-formyl-β-benzyl-L-aspartyl-L-phenylalanine is summarized in Table II below, and is expressed as the total amount (mg) of product dipeptide accumulated in the product solution as a function of time.

The value of the uncharged dipeptide concentration at equilibrium which corresponds to its saturation point in water at pH 5.5, was found to be about 0.05% at 25°C. The amount of uncharged dipeptide transported per square foot of membrane per hour was found to be about 200 mg, indicating that maintenance of the equilibrium required dissolution of insoluble dipeptide and/or dipeptide synthesis de novo. Almost complete dissolution of the insoluble uncharged phase dipeptide in the reaction mixture was observed after about 5 hrs, when the reaction was stopped. A plot of the data of Table II is shown in Figure 3. The linear function indicates that the transfer of peptide across the membrane proceeds at a steady state. The observed rate of formation of product dipeptide of about 200 mg/ft²/hr (Table II) was confirmed similar to the flux of uncharged dipeptide across the membrane (190 mg/ft²/hr) measured at 0.05% in water as was anticipated on theoretical grounds.

At this point the described system would be expected to continue in steady state if continuous addition were made to the reaction mixture of the two amino acid reactants, at a rate of about 120 mg/hr each, to keep the system saturated in uncharged dipeptide.

In order to fully realize the membrane's selectivity the intercalation of a second membrane in series with the first membrane before the contact with the second enzyme may be

necessary because the selectivity across a single membrane is lower due to the high amino acid concentration in the reaction solution.

The product solution (200 mL) was recovered, adjusted to pH 2.5 and cooled at 4°C overnight. The precipitate collected was recovered and recrystallized from MeOH:H₂O to give 307 mg of

N-formyl-β-benzyl-L-aspartyl-L-phenylaIanine _D=

-5.6º(C=1.2; EtOH), identical (IR, 13C-NMR) to an authentic sample prepared by the batch hydrolysis of

N-formyl-β-benzyl-aspartame with Aspergillus esterase, [ ^° _D=

-5.3°(C=1.3; EtOH).

Example 2.

An experiment similar to that of Example 1 was conducted, except for the Use of 8.63g D, L-phenylalanine methyl ester instead of the L-enantiomer. The results are summarized in Table III. Isolation of the uncharged peptide from the 200 mL of product solution gave 310 mg of product, = -6.4°(C=1.4;

EtOH), identical in all respects (IR, 13C-NMR) to an authentic sample of N-formyl-β-benzyl-L-aspartyl-L-phenylalanine.

Table III.

A plot of the data summarized in Table III (Figure 3) again showed the existence of a steady state process when the reactor was operated with D,L-phenylalanine methyl ester. The

stereospecificity of thermolysin is demonstrated by the exclusive formation of the same L,L-dipeptide described in Example 1. The D-phenylalanine methyl ester retained in the tube phase (reaction mixture) did not inhibit the overall kinetics of peptide

formation.

Example 3.

A mixture of 1.0 g N-formyl-β-benzyl-L-aspartyl- L-phenylalanine, prepared in accordance with Example 1, 4.0 mL water, 4.0 mL tetrahydrofuran, and 1.0 mL conc. hydrochloric acid (12N) was heated at reflux for 9 hrs. The mixture was then cooled and the pH adjusted to 4.0 with 50% NaOH solution. The tetrahydrofuran was then removed by evaporation at < 35° and 20 mm Hg. Crystallization was completed by storage at 5°C for 1 hr, the sample then filtered, washed with 1 mL ice water, and dried in vacuo to give 367 mg of white solid. This material was identical to an authentic sample of aspartyl phenylalanine by HPLC and IR comparison. [α]^25º _D= +12° (C = 0.5; 0.1 N HCl in 50% MeOH).

Aspartyl phenylalanine has been converted to aspartame by treatment with methanol and hydrochloric acid, as described in G.L. Bachman and B.D. Vineyard, U.S. Patent No. 4,173,562, example #1. Example 4.

An experiment similar to that of Example 1 was conducted, except for the use 5.65g (20.1 mmoles) N-carbobenzoxy-L-aspartic acid β-methyl ester and 4.38g (20.3 mmoles) L-phenylalanine methyl ester as reactants. The amino acids were dissolved in

100 mL water, the pH of the solution adjusted to 5.5, and 500 mg of thermolysin Daiwa (8 x 10⁵ proteolytic units) was added. The solution was preincubated for 15 hours at 40°C, when a

substantial amount of N-CBZ- (β-methyl

ester)-L-asp-L-phenylalanine methyl ester was precipitated. The suspension was connected to the tube side of a "Type 1 Hollow

Fiber Selective Dialysis Membrane" (Bend Research Inc.)

containing 1 ft² of membrane surface, and the machine was

operated at room temperature for 5 hours against a shell side phase of 200 mL water containing 500 mg Acylase I (Sigma) at pH 7.5. The accumulation of peptide product in shell phase was monitored by HPLC, and the results are reproduced in Table IV and

Figure 4.

After 5 hours run the reaction was stopped, the shell side phase (200 mL) was recovered, adjusted to pH 2.5, and stored overnight at 4°C. The product precipitated was collected and recrystallized from CH₃OH:H₂O to yield 405 mg (86% recovery) of

N-CBZ-(β-methyl ester)-L-asp- L-phenylalanine (N-CBZ-iso-APM) [α]^25º _D= +6.0° (C = 1.1, EtOH), identical (¹³C-HMR) to an

authentic sample of N-CBZ-iso-APM, [α]^25º _D= +5.5° (C = 1.1, EtOH), prepared by chemical coupling and partial esterolysis with

Acylase I.

As observed before in the experiments of Examples 1 and 2, the plot of Figure 4 indicates that the accumulation of

N-CBZ-iso-APM proceeded at a steady rate of about 200 mg/hr.

The conversion of N-CBZ-iso-APM to APM can be practiced under the conditions described in Example 3.

Example 5.

The aspartame derivative, N-formyl-(β-methyl)-asp-phe-OH was prepared in accordance with the present invention as follows: To a solution containing 6.00 g (35 mmoles) of

N-formyl-(β-methyl)-L-aspartic acid, 10.00 g (48 mmoles) L-phenyl alanine methyl ester in 100 mL water, adjusted to pH 7.0, was added 770 mg thermolysin enzyme (Daiwa Chemical Co., Osaka,

Japan) representing a total of 1.2 x 10⁶ proteolytic units. The resulting clear solution was incubated for 1 hr at 40°C, when HPLC analysis indicated the presence of 433.8 mg of

N-formyl-(β-methyl)-asp-phe-OMe. The solution was cooled to 25°C, the pH adjusted to 5.0, and the solution placed in a 200 mL vessel connected to the tube side of hollow-fiber separator

("Type 2 Hollow Fiber Selective Dialysis Membrane", Bend Research Inc.) that provided 0.5 ft² (450 cm²) of a ILM made of 30% v/v

N,N-diethyl-dodecanamide in dodecane. The shell side of the separator was connected to the product vessel containing 500. mg of the enzyme Acylase I (EC 3.5.1.14) from Aspergillus Sp.

(Aminoacyiase AMANO, Nagoya, Japan), at pH 7.5.

The two phases were circulated at 25°C countercurrently, at the rates of 50 mL/min. (tube phase) and 500 mL/min. (shell phase), with the assistance of two peristaltic pumps using the configuration illustrated in Eigure 2. Constancy of pH in both phases was secured through the use: of pH stats.

The formation of N-formyl-(β-methyl)-asp-phe-OH was

monitored by HPLC, using a Tracor Model 995 instrument together with a LDC Spectromonitor II detector set at 254 nm. The column used was a NOVA-PAK C18 Radial-Pak cartridge, 8 mm x 100 mm, housed in a Millipore Waters RCM-100 Radial Compression Module. The mobile phases used for the analysis were:

(a) for N-formyl-(β-methyl)-asp-phe-OMe: 40% v/v methanol in 0.1% KH₂PO₄ buffer pH 4.6;

(b) for N-formyl (β-methyl)-asp-phe-OH: 20% v/v methanol in 0.1% KH₂PO₄ pH 4.6; flow rates used were 1 mL/min for both analyses.

The data relating to the formation of N-formyl-(β-methyl)- asp-phe-OH (product dipeptide) is reproduced in Table V below, and is expressed as the total amount (mg) accumulated in the 200 mL shell phase as a function of time.

A plot of the data of Table V is shown in Figure 5.

At the end of the run, HPLC analysis indicated the presence of 283 mg of N-formyl-(β-methyl)-asp-phe-OMe in the tube phase. These values permitted calculation of the amount of peptide synthesized during the operation of the reactor.

P_tr = peptide transferred into shell phase = 400. =

417.4 mg;

P_o = initial peptide in tube phase = 433.8 mg;

P_T = peptide remaining in tube phase at the end of the run

283 mg;

P_tr= (P_o - P_T) + P_syn;

P_syn = P_tr - (P_o - P_T) = 266.6 mg; and

V_syn = = 53.5 mg/hr. 100 mL.

The V_syn of 53.5 mg/hr. 100 mL coincides .with the rate of synthesis (500 mg/hr. L) measured for the forward velocity in equilibration studies done with N-formyl-(β-methyl)-L-aspartic acid and L-phenylalanine methyl ester in the presence of

thermolysin.

The product solution (200 mL) was recovered, adjusted to pH 2 with 1 N HCl and extracted twice with 200 mL EtOAc. The combined extracts left a white residue upon-evaporation, that after recrystallization from EtOAc/hexane yielded 100 mg of

N-formyl-(β-methyl)-L-asp-L-phe-OH, [α]^25º _D= +0.70°

(c, 0.29;MeOH), identical (IR, C-NMR) to an.authentic sample prepared by the batch hydrolysis of synthetic

N-formyI-(β-methyl)-L-asp-L-phe-OMe with Aspergillus

esterase, [α]^25º _D = +0.80° (c, 0.29; MeOH) .

Example 6.

The experiment described in Example 5 was scaled up in a

Type 2 Hollow fiber Dialysis Membrane (Bend Research Inc.), containing 1 ft² of liquid membrane (30% v/v N,N-diethyl- dodecanamide in dodecane). The tube phase contained 40 g

L-phe-OMe, 24 g N-formyl-(β-methyl)-L-asp and 3.08 g thermolysin Daiwa (a total of 5 x 10⁶ proteolytic units) in 400 ml water, adjusted to pH 7.0. After an incubation period of 1 hr at 40°C, the amount of 1,068 g (2.7 g/L) of

N-formyl-(β-methyl)-L-asp-L-phe-OMe was found to be present. The solution was cooled to 25°C, adjusted to pH 5.0 with 1 N HCl, and connected to the tube side of the hollow fiber separator. The shell phase was made of 400 mL water, pH 7.5, containing 2 g aminoacylase I (Amano). The two phases were circulated

countercurrently at 25°C for 5 hrs., as described in Example 5. The results are summarized in Table VI and Figure 6.

At the end of the run, the amount of N-formyl- (β-methyl)-asp-phe-OMe remaining in tube phase was 586 mg.

The highest transport value observed (404 mg/hr) during the first 30 min. is the result of the high initial peptide

concentration produced during the preincubation period.

Departure from equilibrium caused by the transport of peptide set in the synthesis of more peptide, thus establishing a steady state condition after the first hour into the run, at the

expected level of 200 mg/hr (Figure 6).

Example 7.

An experiment similar to that of Example 5 was conducted, except for the use of 10.00 g of D, L-phenylalanine methyl ester instead of the L-enantiomer. The results, presented in Table VII and Figure 7, clearly show that the observed rate of formation of N-formyl-(β-methyl)-L-asp-L-phe-OH was one-half of that seen with L-phe-OMe (Example 5, Table V), as expected from the enantioselectivity of thermolysin and the prior results of

Examples 1 and 2.

Example 8.

Membrane-assisted enzymatic resolution of D,L-phenylalanine methyl ester was utilized in accordance with the present

invention as follows: To a solution of 1.0 g (5.6 mmoles) of D,L-phenylalanine methyl ester in 100 mL water, pH 7.5, was added 500 mg of aminoacyiase I (Amano Pharmaceutical Co., Nagoya,

Japan). The mixture was allowed to react at 25°C for 30 min., at the end of which the presence of 266 mg L-phe-OH (1.6 mmoles) and 712 mg D, L-phe-OMe (4 mmoles) was observed by HPLC analysis.

This solution was fed to the tube (reaction) side of a

hollow-fiber Celgard supported ILM separator 4 "Type 2 Hollow

Fiber Selective Dialysis Membrane", Bend Research Inc.)

containing 0.5 ft² of a 30% v/v N,N-diethyl-dodecanamide in dodecane liquid film in it. The product side of the membrane

(shell side of the separator) was filled with 200 mL water adjusted to pH 2.0 with diluted HCl. The two phases were circulated through the separator countercurrently at the rate of

200 mL/min., with the assistance of peristaltic pumps (Figure 2).

The separation proceeded through the continuous addition of

D, L-phe-OMe to the tube phase, done at a rate of about 1 g

D, L-phe-OMe per hour. A total of 30 mL of a 7% solution of

D, L-phe-OMe (2.1 g; 11.7 mmoles) in water pH 7.5 was added in a period of 2 hrs. The pH of both phases was kept constant by the use of pH stats. The course of the resolution was followed by HPLC, on samples taken from both phases every 30 min. The HPLC

instrumentation and procedures are those described in Example 5. The mobile phase used in this case was 20% v/v methanol in 0.1% KH₇PO₄ buffer pH 4.6; with a flow rate of 1 mL/min. The results, presented in Table VII and plotted in Figure 8, showed the accumulation of L-phe-OH in tube phase and of D-phe-OMe in shell phase. At the end of the experiment both phases were recovered and worked out as follows:

(a) Tube phase: The contents (130 mL) were adjusted to pH 8.5 with IN NaOH, then extracted with 2 x 50 mL EtOAc. The aqueous phase was then adjusted to pH 4.0 with IN HCl and the solution passed through a 2.5 x 20 cm Dowex 50 (NH₄) column. After washing with 200 mL water, the product was eluted'with 200 mL of 10% NH₄OH. The eluate was

concentrated to 50 mL in vacuo, and the solution

freeze-dried. Yield: 249 mg, white solid,

L-phenylaIanine, [α]^25º _D= -29.2° (c, 2; H₂O), lit.

(Aldrich) [α]^25º _D= -35.0° (c,2;H₂O), optical purity 92%.

(b) Shell phase: (200 mL) was adjusted to pH 8.5 with diluted NaOH and extracted with 2 x 50 mL EtOAc. The organic extract was dried over anh. Na₂SO₄, evaporated to dryness, dissolved in 50 mL water acidified to pH 3.0 with IN HCl and then freeze-dried, to yield 989 mg ( 4. 6 mmole ) of D-phe-OMe. HCl, white solid, [α]^25º _D = -21.0° (c, 2; EtOH), lit. (Aldrich) [α]^25º _D = -32.4° (c, 2; EtOH), optical purity 83%. Based on the permeability value of 32 mg/cm². min found for phe-OMe (water, pH 8, 25°C) on this membrane, the expected flux for a membrane area of 450 cm² (0.5ft²) was 880 mg/hr.

Table VIII shows that the amount of phe-OMe transferred into the shell phase at the end of the first hour was 860 mg, suggesting that the transport was operating under membrane-limiting

conditions.

Batch resolution of D, L-phe-OMe through the enantioselective hydrolysis of the methyl ester function catalyzed by subtilisin A (a serine-type alkaline protease) has been recently disclosed

[Shui-Tein Chen, Kung-Tsung Wang and Chi-Huey Wong, J. Chem. Soc. Chem. Commun. 1986, 1514]. A hydrolase is required for membrane resolution of racemic carboxylic acid ester compounds. A

hydrolase which is a protease having esterolytic activity such as aminoacyiase I, α-chymotrypsin and subtilisin A can be utilized to resolvte a D,L-amino acid compounds such as D, L-phenylalanine methyl ester. Also, the membrane-assisted process of the present invention can be practiced by substituting subtilisin A or α-chymotrypsin for the preferred aminoacyiase I. Aminoacyiase I is generally preferred for the esterolysis of peptides over other esterolytic enzymes such as subtilisin A and α-chymotrypsin also having endoproteolytic activities.

If the above described resolution of D,L-phenyIalanine methyl ester is utilized to produce a peptide as described in the present invention, the L-phenylalanine produced is converted to L-phenylalanine methyl ester by standard procedures known in the art.

This example may be adaptable for resolution of other racemic carboxylic acid esters. For example, a racemic

carboxylic acid ester compound in an aqueous reaction mixture including a hydrolyzing enzyme can be hydrolyzed to form a charged enantiomeric compound and an uncharged enantiomeric ester compound in the aqueous reaction mixture. The uncharged

enantiomeric ester compound is then transported from the aqueous reaction mixture across an ion rejection membrane of the present invention including Type 1 or Type 2 Hollow Fiber Selective Dialysis Membranes from Bend Research, Inc. In one type of example such as Example 8, the racemic carboxylic acid ester compound is a D,L-amino acid ester compound; the charged

enantiomeric compound is a L-amino acid compound and the

uncharged enantiomeric ester compound is a D-amino acid ester compound. It will be appreciated that the selection of enzymes and reaction conditions is within the understanding and knowledge of persons skilled in the art of the present invention.

Continuous or batch processing means are provided by this Example in that the desired enantiomer of the reactant can be produced and added to the reaction mixture.

Example 9.

Synthesis pf N-formyl-(β-methyl)-L-asp-L-phe-OH in

accordance with the present invention was conducted utilizing immobilized aminoacylase I and ion-exchange resins for the removal of permeable products as follows: To a solution of 10.0 g

(48 mmoles) L-phenylalanine methyl ester and 6.0 g (35 mmoles) of

N-formyl-(β-methyl)-L-aspartic acid in 100 L deionized water, adjusted to pH 7.0, was added 770 mg of thermolysin (E₁) (Daiwa Chemical Company, Osaka, Japan) representing a total of 1.2 x 10⁶ proteoiytic units. The resulting solution was incubated for 1 hr. at 40ºC, when HPLC analysis indicated the presence of 383 mg of N-formyl-(β-methyl)-asp-phe-OMe. The solution was cooled to 25°C, the pH adjusted to 5.0, and the solution was placed in a 200 mL vessel 40 connected to the tube side 46 of a hollow fiber separator 44 ("Type 2 Hollow Fiber Selective Dialysis Membrane",

Bend Research, Inc.) containing 900 cm² (1 ft²) of a hydrophobic liquid membrane made of 30% N,N-diethyl- dodecanamide in

dodecane. The shell side of the separator 48 was arranged as a closed circuit made of a series of connecting vessels illustrated in Figure 9. The solution returning to the separator 44 was adjusted to pH 4.0 in order to protonate the L-phe-OMe

copermeating with the dipeptide N-formyl-(β-methyl)-asp-phe-OMe. Circulation through a column of Dowex 50 (Na+) 50 removed the positively charged L-phe-OMe, leaving the uncharged dipeptide in solution. The effluent was adjusted to pH 7.0 and submitted to the action of the aminoacyiase I (E₂), immobilized over

DEAE-Sephadex 52 [T. Tosa, T. Mori and I. Chibata, Agr. Biol.

Chem. 33, 1053 (1969)]. The resulting dipeptide

N-formyl-(β-methyl)-asp-phe-OH was negatively charged at that pH, and was subsequently captured by the Dowex 1 (Cl-) resin 54. The column effluent 57 was returned to the membrane separator with prior adjustment to pH 4.0 58, thus closing the loop.

The tube 46 (100 mL) and shell 48 (500 mL) phases were circulated at 25ºC countercurrently through the membrane

separator, at the rates of 50 mL/min. (tube phase) and 120 mL/min. (shell phase).

Periodic sampling of the shell phase was done on the

effluent from the second enzyme (E₂), (Figure 9, sampling port 56, before entering the Dowex 1 (Cl-) column 54) and the samples monitored by HPLC following the procedures described in

Example 5. As expected, at this point of the circuit (Figure 9) no L-phe-OH that could result from the enzymatic hydrolysis of L-phe-OMe by E₂ (see Example 8) was detected; only a circulating steady-state level of N-formyl-(β-methyl)-asp-phe-OH (average concentration: 54 mg/L) was observed, reflecting the continuous transfer of the dipeptide N-formyl-(β-methyl)-APM across the membrane and its subsequent hydrolysis by E₂. The efficient trapping of the charged dipeptide by the Dowex-1 resin is indicated by the low concentration of it observed at the column effluent 57 throughout the run.

A similar parallel experiment performed in the absence of Dowex-1 showed the rapid accumulation of

N-formyl-(β-methyl)-asp-phe-OH in the shell phase, as could be anticipated from the results discussed above. Again, no L-phe-OH was found in the circulating shell phase. A comparison of both experiments is seen in Figure 10, and Table IX. Table IX

In addition to separating phenylalanine lower alkyl ester copermeating the ion rejection membrane into the product mixture utilizing ion exchange resins as described in this Example, aspartic acid copermeating the ion rejection membrane into the product mixture can be separated utilizing such resins. The species or product that cannot back-diffuse across the membrane from the product mixture can be removed utilizing such ion exchange resins. Also, other separation methods known in the art including but not limited to electrophoresis, electrodialysis and membrane separations which are equivalents of ion exchange resin separations can be utilized in the present invention.

Immobilizing the condensing enzyme allows the enzymatic reaction in the tube phase to be conducted at an initial reaction mixture pH preferred for optimum efficiency of the enzymatic reaction considering the reactants, product(s) and enzyme including the desired equilibrium of the enzymatic reaction.

Optionally, the initial reaction mixture pH in the tube phase can be readjusted to a second reaction mixture pH prior to contact with the membrane so that the second reaction mixture pH will maximize the membrane efficiency in transporting the uncharged product from the tube phase across the membrane into the shell phase. Figure 9 does not show adjusting the initial reaction mixture pH in the tube phase to a second reaction mixture pH.

Similarly, the esterase in the shell phase can be

immobilized and the pH of the product mixture in the shell phase can be adjusted and readjusted as necessary to effect the most efficient processing. Examples 8 and 9 provide additional means for efficient continuous or batch processing utilizing the present invention. In continuous processing the desired

enantiomer of reactants and any copermeating compounds can be returned to the tube phase or reaction mixture.

Example 10.

The dipeptide N-formyl-(β-methyl)-asp-phe-OH synthesized in

Example 5 was reacted with L-trp-OMe, in the presence of pepsin, to yield the protected tripeptide

N-formyl-(β-methyl)-asp-phe-trp-OMe. After permeation, the protected tripeptide was irreversibly hydrolyzed by the enzyme aminoacyiase, to yield N-formyl-(β-methyl)-asp-phe- trp-OH.

To a solution of 2.04g (8 mmoles) L-trp-OMe and 0.484g pepsin in 250 mL 0.1M citrate buffer pH 4.5, was added 0.642g (2.1 mmoles) N-formyl-(β-methyl)-asp-phe-OH dissolved in 25 mL absolute MeOH, and the clear 10% MeOH solution resulted was incubated at room temperature for one hour. HPLC analysis at this point indicated the presence of 144 mg/L of the tripeptide N-formyl-β-methyl)-asp-phe-trp-OMe. The solution was then connected to the tube side of an experimental hollow-fiber separator (Bend Research, Inc.), that provided 0.5 ft² (450 cm²) of a ILM made of 50% N,N-diethyldodecanamide in dodecane. The shell side of the separator was connected to the product vessel containing 0.160g of the enzyme aminoacyiase AMAN0 (AMANO

Pharmaceutical Co., Nagoya, Japan) dissolved in 250 mL 10% MeOH at pH 6.0. The two phases were circulated countercurrently at

25°C. at the rates of 50 mL/min. (tube phase) and 250 mL/min.

(shell phase) with the assistance of two peristaltic pumps

(Figure 2). The shell phase was kept at pH 6.0 constant through out the run by the use of a pH stat, with 0.5N NaOH as titrant.

Throughout the experiment, the concentration of

N-formyl-β-methyl)-asp-phe-OH in the reaction vessel (tube side) was kept approximately constant by the continuous addition of this reactant at the rate of 0.5 mmole/hr. (0.8g in 5 hrs.), to compensate for its rate of permeation at pH 4.5, in an effort to maintain constant the pepsin-catalyzed velocity of

proteosynthesis synthesis during the run.

The formation of the intermediate N-formyl-(β-methyl)- asp-phe-trp-OMe and its product of hydrolysis was monitored by HPLC, using a TRACOR 995 isochromatographic pump together with an LDC Spectro Monitor II (1202) detector, set at 254nm. The column used was a NOVA-PAK C₁₈ cartridge (8mm I.D. x 10cm) housed in a Millipore Waters RCM-100 Radial Compression Module. The mobile phases used for the analysis were:

A. N-formyl-(β-methyl)-asp-phe-trp-OMe. A v/v mixture of 40% CH₃CN and 60% 0.1% KH₂PO₄ buffer solution containing 0.1% triethylamine v/v, adjusted to pH 4.2, was utilized. Retention time was 12.6 minutes (1 mL/min. flow rate). B. N-formyl-(β-methyl)-asp-phe-trp-OH. A v/v mixture of 30% CH₃CN and 70% 0.1% KN₂PO₄ buffer solution containing 0.1% triethylamine v/v, adjusted to pH 4.2, was utilized. Retention time was 10.2 minutes (1 mL/min. flow rate). The data relating to the formation of the tripeptide.

N-formyl-(β-methyl)-asp-phe-trp-OH is reproduced in Table X below, and is expressed as mg tripeptide accumulated per liter shell phase as a function to time.

A plot of the data is shown in Eigure 11.

Peptide synthesized: 10.8 + 42.8 = 53.6 mg

The hourly averaged rate is = 10.7 mg/L. hr., a value

of the same order of magnitude as the average rate of a

pepsin-catalyzed batch proteosynthesis (Figure 12).

Identification of the product was done by the comparison of retention times in the above HPLC system, against an authentic sample of N-formyl-(β-methyl)- asp-phe-trp-OH prepared by

chemical synthesis as follows:

A. Synthesis of N-formyl-(β-methyl)-asp-phe-trp-OMe. To a solution of 406 mg (1.6 mmoles) L-trp-OMe.HCl (Aldrich) and 0.23 mL (1.7 mmoles) (Et)₃N in 50 mL dioxane was added 500 mg (1.6 mmoles) N-formyl(β-methyl)-asp-phe-OH, prepared as indicated in Example 5. The solution was immersed in a ice bath, and 350 mg dicyclohexylcarbodiimide (Aldrich) plus 275 mg N-hydroxy-5-norbornene-2,3-dicarboximide (Aldrich) was added to the cold dioxane solution. After stirring for 15 minutes in the ice bath, the solution was left overnight at room temperature. Next morning, the precipitated

dicyclohexyluxea was filtered off, the dioxane evaporated and the residue dissolved in 200 mL EtOAc, washed with 200 mL 4% citric acid, 5% NaHCO₃, water and dried over anh.

Na₂SO₄. Removal of the solvent gave a colorless residue, that was crystallized from EtOAc/hexane to give 630 mg (78% yield) of N-formyI-(β-methyl)-asp-phe-trp-OMe, m.p.

153-154°C.

[α]²² _D = -35.4° (c = 0.8, MeOH). Analysis: Calculated for C₂₇H₂₉N₄O₇ : C, 62-22; H, 5.56; N, 10.74. Found: C, 61.95; H, 5.85; N, 10.72. ¹³C-NMR spectrum confirmed the structure.

B. Synthesis of N-formyl-(β-methyl)-asp-phe-trp-OH.

One gram (1.9 mmoles) N-formyl-(β-methyl)-asp-phe- trp-OMe was dissolved in 150 mL MeOH, and the solution added to a solution of 203 mg aminoacyiase AMANO in 1350 mL water adjusted to pH 6.0. The solution was kept at pH 6.0 at room temperature for 6 hrs., using a pH stat and 0.2N NaOH as titrant. The course of hydrolysis of the C-terminal methyl ester was monitored by HPLC using the conditions described before.

The N-formyl-(β-methyl)-asp-phe-trp-OH formed was extracted with EtOAc at pH 2. Evaporation of the solvent gave a clear residue, that was crystallized from

EtOAc/hexane to yield 475 mg (49%) of a white crystalline solid, m.p. 155 - 158°C, [α]²² _D -30.2º

(c, 1.19; MeOH).

Analysis. Calculated for C₂₆H₂₇N₄O₇: C, 61.53; H, 5.36; N, 11.04. Found: C, 60.31; H, 5.62; N, 10.66. ¹³C-NMR spectrum confirmed the structure.

Under the HPLC conditions described above, the peptide N-formyl-(β-methyl)-asp-phe-trp-OH had a retention time of 10.2 min., identical (alone or in admixture) to the product farmed in the pepsin-catalyzed reaction.

Example 11.

Independent proof that the rate of accumulation of

N-formyl-(β-methyl)-asp-phe-trp-OH (V_perm) observed in Example 10 is solely determined by the rate of pepsin proteosynthesis

(V_syn), is given in the following experiment.

A. Batch proteosynthesis of N-formyl-(β-methyl)-asp- phe-trp-OMe. Comparison of V_syn and V_perm.

1.02 g L-trp-OMe (4 mmoles) and 242 mg pepsin were dissolved in 125 mL of 0.1M citrate buffer pH 4.5, and to this was added 324 mg (1 mmole) N-formyl-(β-methyl)asp-phe-OH

dissolved in 12 mL methanol. The resulting 10% MeOH

solution was incubated for 3 hours at room temperature, and the rate of synthesis of N-formyl-

(β-methyl)-asp-phe-trp-OMe was followed by HPLC, as

described in Example 10. After the 3 hours of reaction, the solution was circulated in a hollow fiber separator with 0.5ft² ILM of 50% N,N-diethyldodecanamide in dodecane, against 150 mL 10% MeOH in 0.1M citrate buffer pH 5.0. The course of permeation of N-formyl-(β-methyl)-asp-phe-trp-OMe was followed by HPLC, as described before. The data, shown in Table XI and Figure 12, indicates that the rate of

permeation of intermediate tripeptide methyl ester follows closely the rate of proteosynthesis catalyzed by pepsin, measured in a batch reaction without transport.

B. Rate of permeation of N-formyl-(β-methyl)-asp-phe- trp-OMe under synchronous proteosynthesis, in the absence of aminoacyiase. A confirmatory experiment to the one

described in Example 10 was done in the absence of

aminoacyiase, during which V_perm was measured under

synchronous synthesis of tripeptide methyl ester

intermediate.

2.04 g L-trp-OMe (8 mmoles) and 484 mg pepsin were dissolved in 225 mL of 0.1M citrate buffer pH 4.5, and to this was added 642 mg (2 mmoles) of

N-formyl-(β-methyl)-asp-phe-OH dissolved in 25 mL MeOH. The solution was left overnight at room temperature, after which HPLC analysis indicated the presence of 167.4 mg/L of

N-formyl-(β-methyl)-asp-phe-trp-OMe at equilibrium. The solution was circulated through the tube side of a hollow fiber separator with Celgard fibers containing 1 ft² ILM of

50% v/v N,N-diethyl dodecanamide in dodecane,

countercurrently against a shell phase of 250 mL 10% MeOH in

0.1M citrate buffer pH 5.0. The permeation of the

intermediate tripeptide methyl ester into the shell phase was followed by HPLC, as described in Example 10. The corresponding data is tabulated in Table XII, and plotted in

Figure 13.

Total peptide synthesized: 25.0 + 91.4 = 116.4 mg

Per hour rate: = 23.3 mg/L. hr.

From the comparison of the initial rates of permeation V_perm of N-formyl-(β-methyl)-asp-phe-trp-OMe shown in Figure 12 and Figure 13, it is obvious that the aminoacyiase plays no role in the kinetics of peptide transfer across the ILM, as both rates are similar at about 40 mg/L. hr. ft². Consequently, the

rate-determining step in the permeation experiments of

Examples 10 and 11 is the rate of pepsin proteosynthesis, while the role of the aminoacyiase is simply to secure the displacement of the equilibrium through the quantitative transfer of

intermediate peptide from the reaction to the product phase.

On the other hand, physical factors affecting the

permeability of the intermediate peptide through the ILM, like the relative solubility oil/water phase (partition coefficient), membrane composition and temperature, will determine the

steady-state concentration of peptide intermediate in the reactor during continuous operations.

Example 12.

This example demonstrates the feasibility of the

proteosynthesis of a tetrapeptide, achieved through the

papain-catalyzed coupling of two dipeptides.

To a solution of 1084 mg (4 mmoles) of H-gly-phe-OMe.HCl in 112 mL Mcllvaine buffer pH 6.0 was added a solution of 20 mg papain plus 0.5 mL 2-mercaptoethanol in 20 mL of the same buffer. After the addition of 356 mg (I rnmole) of N-CBZ-phe-gly-OH dissolved in 15 mL methanol, the resulting solution (10% MeOH, buffer pH 6.0) was incubated for 1 hour at room temperature.

HPLC analysis at this point indicated the presence of 78.3 mg/L N-CBZ-phe-glygly-phe-OMe. This solution was circulated through the tube side of a hollow fiber separator made of Celgard hollow fibers, containing 0.5 ft² of a ILM of 50% v/v

N,N-diethyldodecanamide in dodecane. The shell phase (150 mL, 10% MeOH, pH 6.0) was unbuffered and contained dissolved 80 mg aminoacyiase AMANO. It was circulated countercurrently through the separator at room temperature, and the pH was kept constant at 6.0 with the help of a pH-stat, using 0.5N NaOH as titrant. The synthesis of N-CBZ-phe-gly-gly-phe-OMe in the reaction phase (tube) and the accumulation of N-CBZ-phe-gly-gly-phe-OH in the product (shell) phase were followed by HPLC analysis, using a Perkin-Elmer System consisting of a 410 LC Pump, LC 235 Diode Array detector set at 210 nm and a LCI-100 Laboratory Computing Integrator for data analysis. The analytical column chosen was NOVA-PAK C₁₈ cartridge (8mm x 10cm, 4μ) housed in a Millipore/Waters RCM-100 radial compression unit. The mobile phase was a v/v mixture of 50% CH₃CN and 50% 0.1% KH₂PO₄ buffer solution containing 0.1% triethylamine v/v, adjusted to pH 4.2 - flow rate was 1 mL/min. Retention times for the

N-CBZ-phe-gly-gly-phe-OMe and N-CBZ-phe-gly-gly-phe-OH were 7.05 minutes and 4.16 minutes, respectively.

In an effort to compensate for the permeation losses of H-gly-phe-OMe at pH 6.0, this reactant was continuously added to the reaction phase during the 5-hour run, at the rate of 0.5 mmole/hr. (total 680 mg).

The results of this experiment are detailed in Table XIII and Figure 14.

Synthesized peptide: 306.8 mg Hourly rate: = 61.4 mg/L. hr.

This rateivalue corresponds to the value V_syn = 75 mg/L.hr. found in a batch papain-catalyzed proteolysis (Figure 15), proving again that the rate of accumulation of product reflects the rate of proteosynthesis in the reaction phase. This is quite evident in the Figure 14, where the

V_perm = V_syn = 40 mg/L/hr. Identification of the product accumulated in the shell phase was done by HPLC comparison against an authentic sample of

N-CBZ-phe-gly-gly-phe-OH prepared as follows:

A. Synthesis of N-CBZ-phe-gly-gly-phe-OMe. To a solution of 500 mg (1.8 mmoles) of H-gly-phe-OMe. HCl in 50 mL was added 0.4 mL (1.9 mmoles) Et₃N, the solution immersed in an ice bath, followed by the addition of 640 mg (1.8 mmoles) N-CBZ-phe-gly-OH, 280 mg of dicyclohexylcarbodiimide and 220 mg of N-hydroxy-5-norbornene-2,3-dicarboximide.

The mixture was allowed to react overnight at room temperature, after which the precipitated dicyclohexylurea was filtered off, the dioxane removed by evaporation to yield a colorless residue. The crude product was dissolved in 200 mL EtOAc, washed with 200 mL each of 5% citric acid, 5% NaHCO₃, prior to drying over anh. Na₂SO₄. Removal of the solvent gave 720 mg of a clear glassy residue, that

crystallized from EtOAc/hexane to yield 600 mg (58%) of N-CBZ-phe-gly-gly-phe-OMe, white crystals, m.p. 85 - 86°C, [α]²² _D = 0.0°(c, 1.0; MeOH). Analysis. Calculated for

C₃₁H₃₄N₄O₇: C, 64.79; H, 5.96; N, 9.75. Found: C, 64.23; H, 6.05; N, 9.66. ¹³C-NMR spectrum substantiated the structure.

B. Synthesis of N-CBZ-phe-gly-gly-phe-OH. To a solution of 100 mg aminoacyiase AMANO in 90 mL deionized water pH 6.0, was added a solution of 400 mg (0.7 mmoles) of

N-CBZ-phe-gly-gly-phe-OMe in 20 mL methanol. The

miIky-white mixture was rapidly stirred at room temperature, and began to clear almost immediately. The mixture was allowed to react for one hour, keeping pH 6.0 constant with the use of a pH-stat and 0.1 N NaOH as titrant. At this point, HPLC analysis indicated that the ester hydrolysis was completed. The reaction mixture was filtered, the filtrate stripped of methanol in vacuo, acidified to pH 2.0 and extracted with EtOAc (3 x 200 mL). The organic extract was dried over anh. Na₂SO₄, and the solvent evaporated to yield 300 mg of a clear residue. Crystallization from

EtOAc/hexane gave 220 mg (60%) of N-CBZ-phe-gly-gly-phe-OH, white crystals,

m.p. 134-136°C; [α]²² _D = + 6.8° (c, 1.74; MeOH). Analysis. Calc,d for C₃₀H₃₂N₄O₇: C, 64.27; H, 5.75; N, 9.99. Found: C, 63.83; H, 5.80; N, 9.88. ¹³C-NMR and ¹Η-NMR spectra substantiated the structure.

HPLC analysis under the conditions described above indicated the presence of only one compound of retention time 4.16 minutes, alone or mixed with the product of papain-catalyzed proteosynthesis.

Example 13.

Papain-catalyzed proteosynthesis of a derivative of the

pentapeptide [leu]⁵-enkephalin. To a solution of 21 mg papain in 160 mL McIlvaine buffer pH 6.0, containing 0.5 mL

2-mercaptoethanol, was added a solution of 363 mg (1 mmole)

N-formyl-(O-Bzl)-tyr-gly-OH and 361 mg (1 mmole) gly-phe-leu-OMe in 40 mL methanol. After a brief standing (15 minutes) at room temperature, HPLC analysis indicated the presence of 3.2 mg/L of N-formyl-(O-Bzl)-tyr-gly-gly- phe-leu-OMe already formed. The solution (reaction phase) was then connected to the shell side of a hollow-fiber separator (Bend Research, Inc.), fitted with

Celgard fibers containing a 1 ft² ILM of 50% v/v

N,N-diethyldodecanamide in dodecane. The product phase was a solution of 158 mg aminoacylase AMANO in 200 mL 20% MeOH,

adjusted to pH 6.0, that was connected to the tube side of the separator. The two phases were circulated countercurrently at 25°C, with the assistance of two peristaltic pumps. The product phase (tube side) was kept at pH 6.0 throughout the run with the help of a pH-stat, using 0.5N NaOH as titrant.

Throughout the experiment, the concentration of the reactant H-gly-phe-leu-OMe in the reaction phase was held approximately constant, by adding an additional 1.284 g (4 mmoles) dissolved in 12 mL methanol, at the rate of 1 mmole/hr to compensate for permeation losses. The formation of the intermediate

N-formyl-(O-Bzl)-tyr-gly-gly- phe-leu-OMe and its hydrolysis product N-formyl-(O-Bzl)-tyr- gly-gly-phe-leu-OH was monitored by HPLC, using the

instrumentation described in Example 12. The solvent systems used were as follows:

A. N-formyl-(O-Bzl)-tyr-qlv-gly-phe-leu-OMe: A v/v mixture of 60% CH₃CN and 40% 0.1% KH₂PO₄ buffer solution containing 0.1% v/v Et₃N, adjusted to pH 4.2. Retention time was 5.01 minutes.

B. N-formyl(O-Bzl)-tyr-gly-gly-phe-leu-OH: A v/v mixture of 30% CH₃CN and 70% 0.1% KH₂PO₄ buffer solution containing 0.1% v/v Et₃N, adjusted to pH 4.2 was utilized. Retention time was 7.07 minutes.

The data showing the build up of hydrolyzed pentapeptide in the product phase is shown in Table XIV, and plotted in

Figure 16.

As experienced in previous examples, the average hourly rate of pentapeptide formation (Figure 16, Vperm = 45 mg/L. hr) was in agreement with the rate of N-formyl-(O-Bzl)-tyr- gly-gly-phe-leu-OMe synthesis measured in a parallel batch incubation of N-formyl-(O-Bzl)-tyr-gly-OH and H-gly-phe-leu- OMe with papain at pH 6.0, and found to be V_syn = 34.2 mg/L/hr. at 25°C.

The product phase (200 mL) was adjusted to pH 2 and

extracted twice with 200 mL EtOAc each. The organic extract was evaporated to dryness and the residue, containing

N-formyl-(O-Bzl)-tyr-gly-gly-phe-leu-OH and

N-formyl-(O-Bzl)-tyr-gly-OH (HPLC), was redissolved in 10 mL

EtOAc and introduced into a Ito Multilayer Coil

Separator-Extractor, already filled with a stationary phase of

400 mL 0.01 M phosphate buffer pH 5.4. The separation by

countercurrent distribution was done with 400 ML of EtOAc

saturated with 0.01 M phosphate buffer pH 5.4; 20 fractions 20 mL each of the EtOAc phase were collected. HPLC analysis indicated the presence of the pentapeptide acid in fractions 3-10, and the dipeptide acid in fractions 16-18. The presence of

N-formyl-(O-Bzl)-[leu]⁵-enkephalin in the pooled 3-10 fractions was confirmed through deprotection with 1 NHCl at 50°C to remove the formyl group, followed by hydrogenation over Pd/charcoal to remove the benzol group. The product was found identical to

H-tyr-gly-gly-phe-leu-OH by HPLC comparison with an authentic sample of synthetic [leu]⁵-enkephalin acetate salt (Sigma

Chemical Catalog, 1988, catalog number L-9133, p. 280).

Example 14.

Use of cellulose hollow-fiber contactors to support hydrophobic liquids - Application to proteosynthesis. The

thermolysin-catalyzed proteosynthesis described in Example 5 was practiced with equal success in a membrane contactor embodiment, comprising packed cellulose hollow fibers having their lumens filled with a hydrophobic liquid and their walls embedded in water. Forced countercurrent circulation of the tube phase between two membrane modules in series provides a high surface contactor, able to transfer uncharged organic molecules between an aqueous reaction phase and an aqueous phase. This

arrangement, useful for the practice of proteosynthesis according to this invention, is sketched in Figure 17. Circulation by a first pump 64 of the aqueous reaction phase 63 from the thermolysin reactor 61 to the first membrane module 67 allows for the transfer of the permeable intermediate N-formyl-(β-methyl)- asp-phe-OMe into the hydrophobic phase (oil) reservoir 65 via descending oil line 81. This enriched oil is circulated from the hydrophobic phase reservoir 65 via transfer line 82 by a second pump 66 via pump line 83 into the second membrane module 68, where the intermediate dipeptide ester diffuses out into an aqueous product phase 80 in a product reservoir 69. The

intermediate dipeptide ester is circulated to a membrane

separator such as a reverse osmosis membrane RO 62 by a third pump 70. The intermediate dipeptide ester is irreversibly trapped by the reverse osmosis membrane RO 62 in the rententate 77. The filtrate 78 from the RO 62 is returned 73 to the product reservoir 69. The retentate 77 from the RO 62 is recycled 79 to the product reservoir 59. The pure water 71 leaving the RO unit is returned to the second membrane. module 68, while the spent oil leaving the second membrane module 68 is recycled via line 72 into the first membrane module 67, where it gets reloaded with fresh intermediate produced by proteosynthesis in the reaction vessel 61. The oil loop 84 (water-immiscible hydrophobic phase) comprises hydrophobic phase reservoir 65 and line 82, pump 66, oil line 83, line 72 and line 81 comprising first contactor 67, second contactor 68, RO 62, aqueous reaction phase 63, aqueous product phase 80 and oil loop 84, thus closing the cycle. A first membrane contactor 74 is shown. The process can be

enhanced by utilizing a second membrane contactor 75 (not shown, but identical to 74) utilizing a bleedjstream 76. Generally, a plurality of membrane contactors, can hte utilized repeatedly to enhance recovery of product.

Figure 27 is a partial schematic view (enlarged) of a cross section of hollow fibers in a membrane contactor. The partial view shows hydrophilic hollow fibers 121 in a first membrane module and hydrophilic hollow fibers 141 in a second membrane module. In the first membrane module hydrophilic hollow fibers 121 are shown having a lumen (bore) 122 and hydrophilic polymeric material 125. The lumen 122 is filled with water-immiscible organic liquid comprising the tube phase (e.g., hydrophobic phase). The space 123 between the hydrophilic hollow fibers 121 comprises the shell phase (e.g., aqueous reaction phase).

Similarly, in the second membrane module hydrophilic hollow fibers 141 are shown having a lumen (bore) 142 and hydrophilic polymeric material 145. The lumen 142 is filled with

water-immiscible organic liquid comprising the tube phase (e.g., hydrophobic phase). The space 143 between the hydrophilic hollow fibers 141 comprises the shell phase (e.g., aqueous reaction phase).

Figure 28 is a partial schematic view (enlarged) of a membrane contactor 160 comprising a first membrane module 131, a second membrane module 151 and a connecting means (tube) 130. In the first membrane module 131 hydrophilic hollow fibers 121 are potted in a resinous material (potting compound). The aqueous reaction phase is circulated through space 123, through opening 129 on first wall 128 and returned through opening 135 on third wall 134. The aqueous reaction phase does not mix with the water-immiscible liquid. The water-immiscible liquid is

circulated through lumen 121, through opening 127 on the second wall 126 to the second membrane module 151 through connecting tube 130 and returned to the first membrane module 131 through opening 133 on the fourth wall 132. Similarly, in the second membrane 151 hydrophilic hollow fibers 141 are potted in a resinous material (potting compound). The aqueous product phase is circulated through space 143, through opening 149 on first wall 148 and returned through opening 154 on third wall 153.

The aqueous product phase does not mix with the water-immiscible liquids. The water immiscible liquid is circulated from

connecting tube 130 through opening 147 on fourth wall 146

through lumen 142 and returned to the first membrane module 131 through opening 152 on second wall 150. The first and second membrane modules, 131 and 151, of the membrane contactor 160 each comprise many hydrophilic hollow fibers, 121 and 141,

respectively, although only three are shown in each such membrane module. The use of membrane contactors in conjunction with reverse osmosis as described will cause the desired displacement of the proteosynthetic equilibrium, without affecting the kinetics of peptide synthesis in the thermolysin reactor. This approach is considered as a viable alternative to the use of a second enzyme for the purpose of driving the proteosynthesis to completion.

Using this membrane arrangement, an experiment was conducted in which the enzyme thermolysin catalyzed the coupling of

N-formyl-(β-methyl)- H-phe-OMe(A) and asp-OH (B), and the

resulting peptide N-formyl-(β-methyl)-asp-phe-OMe(C) was

continually trapped in the RO retentate (Figure 17). The

conditions used in this experiment were as follows:

A. Thermolysin reactor. 300 mL, pH 5.0, 50°C, [B] = 340 mM, [A] = 627 mM, [ thermolysin] = 21 g/L, [CaCl₂] = 10 mM. The initial rate of C synthesis under these conditions was V_syn = 13 9/L-hr (37-5 mM/hr).

B. Membrane contactor. 0.8ft², loaded with 2-undecanone.

C. RO Membrane. 2ft² TW-30 spiral-wound module.

Manufactured by FilmTec Corporation, Minnetonka, Minnesota, (polyamide type membranes as described in U.S. Patent No. 4,277,344)

D. Product reservoir. 700 mL, pH 5.0, 50°C. The results from the operation of the first stage of this run are shown in Figure 18, which plots the concentration of C in the thermolysin reactor as a function of time. The data clearly shows the continuous removal of C from the reactor by the

membrane contactor, and the recovery of the chemical equilibrium upon stopping the flow of undecanone and the simultaneous addition of enough A and B to restore the original reactant concentrations. The system was run for 15 hours, at which time the concentrations of A and C in the product reservoir were 40 and 15 mM, respectively. (A:C molar ratio = 3:1). This product solution was concentrated about 10-fold using a combination of RO and evaporation. The concentrate (100 mL), containing 5.0g A and 3.5g C in the solution at pH 4.0, was then placed in the feed reservoir of the membrane-contactor system, and the system was operated at 25°C against 100 mL water, pH 4.0 placed in the product reservoir (Figure 17), until approximately 50% of C had been removed from the feed solution. The product from this second stage, which contained about 0.6g A and 1.8g C (A:C molar ratio = 2:3), was evaporated down to 25 mL and chilled, to yield 1.64g (81%) pure C, white crystals, m.p. 105-107°C. [α]²² _D =

-32.3° (c, 0.65; MeOH). An authentic sample of N-formyl- (β-methyl)-asp-phe-OMe had m.p. 108-109°C; [α]²² _D = -33.9° (c, 0.59; MeOH). Based on the optical rotation, the purity of the recovered dipeptide C was 95%.

Example 15.

This example describes the thermolysin-catalyzed

proteosynthesis of the dipeptide N-formyl-(β-methyl)-asp- phe-O-< , and its synchronous hydrolysis to the acidic dipeptide N-formyl-(β-methyl)-asp-phe-OH, across an ILM including

N,N-diethyldodecanamide. The symbol "-<" is utilized herein to indicate isopropyl.

To a solution of 13.09 g (54 mmoles) of L-phe-O-<.HCl and 6.64 g (38 mmoles) of N-formyl-(β-methyl)-asp-OH in 200 mL water at pH 5.0, was added 385 mg of thermolysin (Thermoase, Daiwa, 40,000 PU/g) and 150 mg CaCl₂. The solution was incubated at 25ºC for one hour air which time it was found by HPLC analysis to contain 148.9 mg/L of the dipeptide

N-formyl-(β-methyI)-asp-phe-O-<. The solution was then connected to the shell side of an experimental hollow-fiber separator (Bend Research, Inc.), that provided 1 ft² (900 cm2) surface of an ILM of N,N-diethyldodecanamide. The tube side of the separator was connected to the product vessel containing a solution of 0.162 g of the enzyme aminoacyiase (AMANO Pharmaceutical Co., Nagoya,

Japan) dissolved in 200 mL water at pH 6.0. The two phases were circulated countercurrently at 25°C at the rates of 100 mL/min (tube phase) and 250 mL/min (shell phase), with the assistance of two centrifugal pumps (Figure 2). The product (tube) phase was kept at pH 6.0 constant, by using a pH stat with 0.5N NaOH as titrant.

The formation of the intermediate and product peptides was monitored by HPLC, using a Perkin-Elmer System consisting of a 410 LC Pump, LC 235 Diode Array detector set at 210 nm, and a LCI-100 Laboratory Computing Integrator for data analysis. The analytical column chosen was a NOVA-PAK C₁₈ cartridge (8mm x 10cm, 4μ) housed in a Millipore/Waters RCM-100 radial compression unit. The mobile phase for the N-formyl-(β-methyl)-asp-phe-O-< was a v/v mixture of 50% CH₃CN and 50% 0.1% KH₂PO₄ buffer solution containing 0.1% v/v triethylamine, adjusted to pH 4.2. Flow rate was 1 mL/min. Retention time was 5.65 minutes. The mobile phase for the N-formyl-(β-methyl)-asp-phe-OH was a v/v mixture of 30% CH₃CN and 70% 0.1% KH₂PO₄ buffer solution

containing 0.1% v/v triethylamine, adjusted to pH 4.2. Flow. rate: 1 mL/min. Retention time: 3.52 minutes.

The results of this experiment are shown in Table XV and Figure 19.

Synthesized dipeptide: 358.6 mg/L.

Hourly rate = 358.6 = 72 mg/L/hr. This overall hourly rate is in line with the initial

permeation rate of 150 mg/L. hr measured at the first hour

(Figure 19), and with the initial synthesis rate of 220 mg/L/hr. measured independently for a batch incubation (Figure 20) run at the same conditions of pH, temperature and reagent

concentrations.

The dipeptide acid N-formyl-(β-methyl)-asp-phe-OH present in the 200 mL product phase (63 mg) was isolated by concentrating the solution in vacuo to 50 mL, removal of the enzyme by

ultrafiltration over an Amicom PM-10 membrane (molecular weight cut-off: 10 Kdalton), acidification of the ultrafiltrate to pH 2.0 and extraction with EtOAc (3 x 50 mL). The organic extract was evaporated to dryness, the residue dissolved in 2 mL water pH 6.0, and the dipeptide was crystallized by acidification to pH 2.0. Yield: 11 mg (17%), identical by HPLC comparison to an authentic sample of N-formyl-(β-methyl)-asp-phe-OH prepared according to the following procedure.

A. Synthesis of N-formyl-(β-methyl)-asp-phe-OMe. To 21.0 g (97.3 mmoles) L-phe-OMe.HCl dissolved in 300 mL dioxane was added 14.9 mL (107 mmoles) Et₃N. To this mixture was added 16.0g (98 mmoles) N-formyl-(β-methyl)- asp-OH, the, solution was cooled to 0°C, and 20.0 g (97 mmoles)

dicyclohexylcarbodiimide were added, followed by 16.0g

N-hydroxy-5-norbornene-2,3-dicarboximide. After stirring overnight at room temperature, the dicyclohexylurea

precipitated was filtered off, and the filtrate concentrated to a syrup by evaporation. It was dissolved in 500 mL

EtOAc and washed sequentially with 5% citric acid, 5%

NaHCO₃, water, and dried over anhydrous Na₂SO₄. Removal of the solvent gave a white solid, which was recrystallized twice from EtOAc/hexane to yield 17.0 g (53%) of

N-formyl-(β-methyl)-asp-phe-OMe, m.p. 108-109°C. [α] ^25º _D=

-36.9° (c=0.6, MeOH) ¹H-NMR (CD₃OD): 7.6 δ (s) formyl; 6.8 δ (s) phenyl; 4.0-4.4 δ (M) 2 CH; 3.3 δ (s) 2 CH₃ (6 protons, superimposed);

2.3-2.8 δ (M) 2 CH₂ (4 protons).

¹³C-NMR (CDCl₃): 35.52, 37.56 ppm (-CH₂-, t); 47.59, 53.54 ppm (-CH- , d); 52.09, 52.28 ppm (CH₃-, q);

127.08, 128.51, 129.19 ppm (phenyl, d); 135.62 ppm (phenyl, s); 160.93 ppm (H-C- ,d); 169.60, 171.34,

o

172.09 ppm (-C-O(NH),s) .

Analysis: Calculated for C₁₆H₂₀O₆N₂: C, 57.17; H, 5.95; N, 8.33. Found: C, 57.29; H, 6.02; N, 8.32.

B. Synthesis of N-formyl-(β-methyl)-asp-phe-OH. A

solution of 2.0 g (5.9 mmoles) of N-formyl-(β-methyl)

-asp-phe-OMe in 50 mL MeOH was added to a solution of 0.2 g aminoacyiase AMANO in 450 mL 0.05% KH₂PO₄ buffer pH 7.5, contained in a beaker fitted with a mechanical stirrer. The solution was stirred overnight at room temperature, and the pH was kept constant at 7.5 with the assistance of a pH stat, using IN NaOH as titrant. After filtration of any unreacted starting material, the solution was washed with EtOAc (2 x 100 mL), then adjusted to pH 2.0 and the

hydrolyzed dipeptide was extracted with EtOAc (3 x 200 mL). The combined extracts were dried over anhydrous Na₂SO₄ and evaporated to dryness. The yield of

N-formyl-(β-methyl)-asp-phe-OH was 1.3 g (68%), m.p.

185-186°C, [α]^25º _D = -14.3° (c=1.0; MeOH).

¹³C-NMR (CD₃OD): 37.37, 38.76 ppm (-CH₂-, t); 50.21, 55.68 ppm (-CH- , d); 52.21 ppm (CH₃-, q); 128.31, 129.90, 130.90 ppm (phenyl, d); 138.63 ppm (phenyl, s); 163.99 ppm (H-C-, d); 172,38, 172.83, 174.77 ppm

(-C-O(NH), s).

Analysis: Calculated for C₁₅H₁₈O₆N₂: C,55.93; H, 5.59; N, 8.69. Found: C,55.22; H, 5.71; N, 8.51.

Example 16.

This example shows the rate effect caused by the hydrolytic enzyme upon the permeation V_perm of the intermediary peptide during synchronous proteosynthesis. The measured rate increase is particularly important for those highly hydrophobic peptides showing high partition coefficients towards the hydrophobic membrane. The chemical transformation catalyzed by the

aminoacylase, that is the conversion of a C-terminal isopropyl ester into the free carboxylate, occurs at the interface

oil/water and assists positively in the transport of dipeptide across the boundary. The increase in permeation of the

intermediary peptide caused by the countercurrent sweeping of the oil surface with the aminoacyiase is an additional effect to the equilibrium displacement resulting from the chemical.

transformation to a non-permeable product. To a solution of 13.7 g (56 mmoles) of L-phe-O-<.HCl and 6.6 g (38 mmoles) of N-formyl-(β-methyl)-asp-OH in 200 mL water at pH

5.0, was added 397 mg Thermoase Daiwa (40,000 PU/g) and 150 mg

CaCl₂. The solution was incubated at 25°C for one hour, at that time it was found by HPLC analysis to contain 347.0 mg/L of the dipeptide N-formyl-(β-methyl)-asp- phe-O-<. The solution was circulated at 25ºC through the shell side of an hollow-fiber separator, containing 0.5ft² ILM of N,N-diethyldodecanamide, against 200 mL water pH 6.0 circulating countercurrently through the tube side (Figure 2). The rate of permeation across the ILM was measured for the dipeptide-O-< and L-phe-O-< by HPLC, following the details discussed in Example 15.

After two hours the circulation was interrupted, the product

(tube) phase was removed, and replaced by 200 mL water pH 6.0 containing 160 mg aminoacyiase AMANO in solution. The

countercurrent circulation of both aqueous phases was

reinitiated, and the rate of permeation V_perm of the dipeptide

N-formyl-(β-methyl)-asp-phe-OH was measured by HPLC again, together with L-phe-OH and L-phe-O-<. The results appear in Table XVI and Figure 21.

Figure 21 shows that the peptide permeation in the presence of aminoacylase (V_perm = 440 mg/L/hr.) is 2.3 times faster than in water (V_perm = 190 mg/L/hr.). This rate effect caused by the direct contact of the ILM with the soluble enzyme would be lost if the same enzyme is used in the immobilized form.

The data also show that only under the "enzyme sweeping" conditions indicated in Part II the proteosynthetic process becomes synchronous, that is, the dipeptide V_perm (440 mg/L.hr) reaches the V_syn value (340 mg/L.hr) measured during the batch incubation period (Figure 22), under identical conditions of pH, temperature and reactant concentrations.

It is also shown in Table XVI that the rate effect exerted by the aminoacyiase is resulting from the hydrolysis of the dipeptide isopropyl ester. The rate of permeation of L-phe-O-<, a poor substrate for the aminoacylase, is insensitive to the presence of the enzyme in the product phase.

Example 17.

This Example 17 illustrates the effect of operating the process of this invention at elevated temperatures and

effectiveness at higher temperatures in a range of from 20°C to 65°C of lower alkyl ester reactants [B'] wherein the alkyl group is selected to enhance stability of that reactant, e.g., esters derived from secondary alcohols having 3 to 6 carbon atoms.

The thermolysin-catalyzed proteosynthesis of the dipeptide N-formyl-(β-methyl)-asp-phe-O-< (C') described in Example 15, was successfully practiced, at 50°C utilizing- the cellulose

hollow-fiber contactor described in Example 14, in place of the ILM module used in Example 15.

The set-up used in this experiment is outlined in Figure 17, and the conditions for achieving the condensation equilibrium between N-formyl-(β-methyl)-asp-OH (B'), L-phe-O-< (A') and the resulting dipeptide C' were as follows: A. Thermolysin Reactor. 500 ml, pH 5.0, 50°C, containing 42 g/l thermolysin, 10mM CaCl₂, 230mM B' 450mM A'.

B. Membrane Contactors. 0.4 ft² of membrane area in each membrane contactor module, with 75 mL of

N,N-diethyldodecanamide as the circulating organic oil.

C. RO Membrane. 2 ft² TW-30 spiral-wound module

previously described in Example 14.

D. product Reservoir. 750 mL water, pH 5.0, 50°C,

circulating throught the RO module, the membrane contactor and the reservoir.

The accumulation of product C' in the reservoir as a function of time is shown in Figure 23. The data shows that the concentration of C' in the thermolysin-driven

equilibrium is maintained at 15mM upon addition of A' to keep [A'] constant, while the [C^,] in the product reservoir increases steadily as the dipeptide is trapped by the RO membrane, thus providing the constant driving force needed to displace the chemical equilibrium A'+B'

C'.

After 15 hours operation the reactor was stopped, and the product solution (20mM C', 160 mM A' ) was collected, adjusted to pH2, concentrated 10x by evaporation and cooled, to yield 3.9g (70% yield) of N-formyl-(β-methyl)-asp-phe-O<, white crystals, m.p. 68-69°C, [α]^{25 º} _D= -22.7° (C,3.6; MeOH).

Analysis: Calculated for C₁₈H₂₄O₆N₂; C, 59.37; H,6.59; N, 7.69. Found: C, 59.31; H, 6.66; N, 7.65.

Purity: (HPLC): 97%. After purification by chromatography on a C₁₈-reverse phase column, the pure dipeptide had m.p.

101-102°C, [α]^{25 º} _D= -32.6° (C,0.9; MeOH), and was identical in all respects (IR, ¹³C-NMR) to an authentic sample of

N-formyl-(β-methyl)-asp-phe-O-<, m.p. 100-101°C, [α]^{25 º} _D= -31.3° (C, 3.0; MeOH), prepared by the DCC coupling of

N-formyl-(β-methyl)-asp-OH and L-phe-O-< in dioxane. The chemical stability of L-phe-O-< over L-phe-OMe measured seven times higher at 50°C, in water pH 5.0, which is an

important advantage for the practice of this invention. Economic benefits deriving from operating at about 50°C, in preference over processing at lower temperatures are: a) the shift of the chemical equilibrium towards peptide formation that occurs with increasing temperature; and b) the increase of peptide

permeability across hydrophobic membranes with increasing

temperature.

Successful operation of the process of this invention at elevated temperatures depends at least in part on the thermal stability of the lower alkyl ester reactant [B'] and minimizing or avoiding undesirable side reactions, such as hydrolysis of the ester to produce the corresponding acid. It is not possible to efficiently operate the process described in this Example when using L-phenylalanine methyl ester. because under the described process conditions at 50°C that ester is not very stable, i.e., it slowly hydrolizes to L-phenylalanine. The stable isopropyl ester, quickly reacts at 50°C to provide an effective yield of the desired peptide product and avoids or minimizes undesirable side reactions.

Other L-phenylalanine esters with branched aliphatic

alcohols, e.g., 2-butanol and 3-methyl-2-butanol are also

expected to have high thermal stability, and to show minimal formation of L-phe-OH by ester hydrolysis in water at pH 5.0 and 50°C.

Example 18. Pepsin-catalyzed proteosynthesis of

N-CBZ-asp-phe-OMe.

To a solution of 85.46 g (320 mmoles) N-CBZ-L-asp-OH and 19.45 g (90 mmoles) L-phe-OMe.HCL in 400 mL water pH 4.0 was added 9.63 g crystalline pepsin (Sigma) dissolved in 100 mL water pH 4.0. The resulting solution (500 mL) was incubated at 38°C for 48 hrs, at the end of which it was shown by HPLC to contain 3208 mg/L (7.5 mM) of N-CBZ-asp-phe-OMe. The HPLC analyses were done on a Perkin-Elmer System consisting of a 410 LC pump, LC 235 Diode Array detector set at 210 nm, and a LCI-100 Laboratory Computing Integrator for data analysis. The analytical column chosen was a NOVA-PAK C₁₈ cartridge (8 mm x 10 cm, 4 μ bore) housed in a Millipore/Waters RCM-100 radial compression unit.

The mobile phase was a v/v mixture of 40% CH₃CN and 60% 0.1% KH₂PO₄ buffer containing 0.1% v/v triethylamine, adjusted to pH 4.2. Flow rate was 1 mL/min. Under these conditions,

N-CBZ-asp-phe-OMe had a retention time of 7.78 min.

The above reaction mixture (500 mL) was placed in a jacketed vessel kept at 38°C, and it was circulated through the shell side of a hollow fiber separator made of Celgard fibers, containing 1 ft² (900 cm²) ILM of 50% v/v isohexadecanol (Hoechst) in dodecane. The product phase (500 mL) was deionized water pH 7.0, that was circulated countercurrently through the tube side of the module while keeping the pH constant at 7.0 with the assistance of a pH-stat unit, using 0.5N NaOH as titrant.

The synthesis of N-CBZ-asp-phe-OMe in the reaction phase (shell) and the accumulation of the same dipeptide in the product phase (trapped at pH 7.0 as the ionized β-carboxylate) were measured by HPLC, as described above. After a 5 hr run the circulation of the two phases was interrupted, the product solution was removed and replaced with 500 mL of fresh water pH 7.0 solution. Following a. second incubation at 38°C for 24 hrs, the reaction mixture contained 4170 mg/L of N-CBZ-asp-phe-OMe (HPLC). The countercurrent circulation was reassumed, and the permeation of peptide was monitored for another 5 hr period.

The results of this experiment are shown in Table XVII and Figure 24. It is evident from Table XVII that active

proteosynthesis has taken place in the enzyme reaction phase, while newly synthesized peptide was being transported across the membrane. Computation of the total N-CBZ-asp-phe-OMe accumulated in the product phases (542 mg in 10 hr) indicates an average permeation rate of 54 mg/L hr, in agreement with the individual Vperm rates shown in Figure 24 for part I and part II.

The isolated peptide product, 220 mg, m.p. 120-125°C, [α]²² _D

= -12.0° (c, 1.0; MeOH) was identified as N-CBZ-asp-phe- OMe by chromatographic (HPLC, TLC) and H-NMR comparisons with an

authentic sample prepared by the Schotten-Baumann acylation

(water, pH 7.5) of aspartame with benzyloxycarbonyl chloride, to yield crystals m.p. 122-124°C (from EtOAc/hexane), [α]²² _D = -13.0º

(c, 0.8; MeOH). Literature [D. D. Petkov and I. B. Stoineva,

Tetrahedron Lett. 25, 3754 (1984)], reported m.p. 120-124°C,

[α]²² _D = -14.4°; (c, 1.0; MeOH).

While specific embodiments of the invention have been shown and described in detail to illustrate the application of the inventive principles, it will be understood that the invention may be embodied otherwise without departing from such principles. Example 19. Integrated process combining the

enzymatic resolution of D-,L-Phe-O-< and the racemization and recycling D-Phe-O-<.

Example 8 demonstrates the preparation of L-Phe-OH from D-L, -Phe-OMe, by stereoselective hydrolysis of Phe-L-ester with aminoacyiase enzyme in water at pH 7.5, utilizing a SLM having a 30% N,N-diethyldodecanamide/70% dodecane organic phase disposed in a hollow fiber module. Enantiomeric resolution is attained when the D-Phe-Ome formed is separated from L-Phe-OH by selective membrane permeation, the latter enantiomer remaining in the reaction media as carboxylate anion:

D-,L-Phe-Ome + OH D-Phe-Ome + L-Phe-O + MeOH

(permeable) (non-permeable)

The measured flux of D-Phe-Ome across the SLM, was 0.5g/ft² hr, representing the limiting rate of the system for resolution under test conditions.

The L-Phe-OH separated by this method can be converted to the corresponding isopropyl ester by conventional esterification procedures using isopropanol and HCl. The resulting L-Phe-O-< is a preferred starting material for the synthesis of the dipeptide N-formyl-(beta-methyl)-Asp-Phe-)-<, described in Examples 15 and 17.

The economics of the overall-conversion D-,L-Phe-Ome

L-Phe < would be improved by fast and efficient racemization of the by product D-Phe-Ome, that does not destroy the methyl ester function. The racemization process described in U.S.

Patent No. 4,713,470 (Dec. 15, 1987); to Stauffer Chemical Co.), which involves heating of D-Phe-Ome in refluxing toluene (110°C) for one hour in the presence of a salicylaldehyde catalyst in such a racemization. The following experiment demonstrates the enzymatic

resolution of D-,L-Phe-Ome at an interface of a toluene/water biphasic system.

A solution of 8.0g D-,L-Phe-Ome in 200 mL toluene was added to a 500 mL glass beaker, containing a solution of 2g

aminoacyiase (crude soluble solids) in 200 mL of aqueous 1.5 mM CoCl₂, adjusted to pH 8.5. The resulting two phases were

thoroughly mixed by magnetic stirring, and as the reaction

proceeded, the pH of the water phase was maintained at 8.5 using a pH state and 0.2N NaOH, as titrant. After 30 minutes of mixing, the addition of NaOH, required to maintain the pH, had subsided considerably. HPLC analysis of the water phase showed the presence of 3.2g L-Phe-OH, confirming that the reaction was near completion. After an additional 30 minutes of mixing, the two phases were recovered and analyzed separately.

A. The aqueous layer (215 ML) was placed in a

diafiltration cell fitted with an Amicon YM5 membrand (5 K daltons cut-off), diafiltered with three volumes (650 mL) of 1.5 mM CoCl₂, pH 8.5 solution, and finally concentrated down to 200 mL. The combined filtrate (650 mL) was adjusted to pH 5.0 with diluted HCl, concentrated in vacuo to 50 mL and cooled at

4°C to yield 2.2g L-Phe-OH, [α]^{25 º} _D= -28.6°

(c,2; H₂O).

Optical purity: 100% with reference to a D-

Phe-OMe.HCl standard, [α]^{25 º} _D= -32.4º

c,2: EtOH).

The polarimetric results indicate that it is possible to achieve effective enantiomeric resolution by this procedure by feeding D-,L-Phe-Ome at the rate of about 40 g/L.hr, resulting in a L-Phe-OH output of 20 g/L.hr. Notably this productivity is, as much as, 50 times higher than those recorded in the literature for batch fermentations with Bacillus lactofermentum AJ3437, where the saturation of the culture medium in L-Phe-OH (about 30 g/L at 25°C) required a cultivation time of 72 hrs, or 0.4 g/L. hr average productivity [I. Shilo, "L-phenylalanine

Fermentation" in K.Aida, I. Chibata, K. Nakayama, K. Takinami and H. Yamada (Editors), "Biotechnology of Amino Acid Production", Kodansha, Tokyo, 1986, pp. 188-206].

Example 20. ENZYME ACTIVITY

The following experiment compares the effectiveness of aminoacylase, subtilisin and alpha-chymotrypsin for enzymatic resolution processes, in terms of the ratio of their rate constants for the hydrolysis of L- and D- enantiomers of the methyl and isopropyl esters of phenylalanine (L/D Kinetic ration). The rate constants for the hydrolysis of each ester by each enzyme were measures, under identical conditions of pH, S/E ratio and temperature, in accordance with the above-described methods and conventional practice. The results of those

measurements are summarized in Table XVIII below.

TABLE XVIII

L/D kinetic ratios in 0.1M phosphate buffer pH 8.0, S/E = 10, 25ºC, for phenylalanine esters. Reaction time: 2 hrs.

*Llterature Values, Dahod and Empie, supra. Alpha-Chymotrypsin would be the preferred enzyme for the resolution of D-, L-Phe-Ome.

Surprisingly, both subtilisin and alpha-chymotrypsin are able to hydrolyze L-Phe-O < very rapidly, but are unable to catalyze the hydrolysis of the D-Phe-O < . The aminoacyiase did not hydrolyze either D- or L- phenylalanine isopropylester. The discrimination towards L-Phe-O < shown by subtilisin and alpha-chymotrypsin is a highly advantageous in the integrated resolution process described in this Example.

The integrated resolution process, may be advantageously practiced as a batch or continuous operation because it enables recovery and reuse of both the enzyme and racemization catalysts without having deleterious effects of toluene on their activity. A cellulose hollow fiber contactor of the type described in

Examples 14 and 17 could be used so that the toluene phase circulates within the lumen of the fibers countercurrently to an aqueous enzyme phase embedded the walls of the fibers. The toluene phase leaving the contactor would carry D-Phe-O < that, after being racemized over an immobilized salicylaldehyde catalyst, would be recycled as D-,L-Phe-O into the contactor. The high interface surface existing in the contactor facilitates the enzymatic transfer of L-Phe-OH into the product/water phase. On leaving the contactor, the product/water phase would be continuously bled into a reverse osmosis (RO) unit, operating downstream of an ultrafiltration device (UF) that retains the enzyme but allows the free flow of L-Phe-OH.

Figure 29 schematically illustrates the above-described continuous process. The transfer rate of 20 g/L.hr measured above for L-Phe-OH may be attained, in a hollow fiber contactor providing about one ft² of interface area per liter of reaction mixture, and permeability of about 20 g/ft². hr or 386 1b/ft². yr.

Claims

CLAIMS :

1. A method for the enzymatic synthesis of a peptide, comprising the steps of:

reacting a first compound selected from the group consisting of a protected peptide having a C-terminal carboxylate group and a protected, N-acyl amino acid having an alpha carboxylate group with a second compound selected from the group consisting of a protected peptide having a N-terminal ammonium group and a protected amino acid having an alpha ammonium group in the presence of a condensation enzyme in an aqueous reaction phase under conditions in which the carboxylate group and ammonium group condense forming a protected, uncharged, peptide product;

transporting the protected, uncharged, peptide product across a water-immiscible hydrophobic phase into an aqueous product phase; and

separating the protected, uncharged, peptide product from the aqueous product phase to prevent that product from back-diffusing across the water-immiscible hydrophobic phase.

2. The method recited in claim 1, wherein the protected, uncharged, peptide product is separated frant the aqueous product phase by reverse osmosis.

3. The method recited in claim 1, wherein the second compound is a lower alkyl ester of L-phenylalanine derived from a secondary alcohol having 3 to 6 carbon atoms.

4. The ptetbod recited in claim 3, wherein the condensing enzyme is thermolysin and the temperature of the aqueous reaction phase is about 50°C.

5. The method recited in claim 4, wherein the first compound is N-formyl-(beta-methyl)-asp-OH and wherein the second compound is L-phenylalanine isopropyl ester.

6. The method recited in claim 1, wherein the water- immiscible hydrophobic phase functions as an ion rejection

membrane separating the aqueous reaction phase from the aqueous product phase creating oil/water interfaces with each of the aqueous phases.

7. The method recited in claim 6, wherein the water- immiscible hydrophobic phase is an organic liquid immobilized by capillarity in pores in a microporous support.

8. The method recited in claim 7, wherein the microporous support comprises polypropylene hollow fibers.

9. The method recited in claim 6, wherein the water- immiscible hydrophobic phase comprises an organic liquid located within a lumen defined by the walls of a hollow fiber comprising hydrophilic material.

10. The method recited in claim 9, wherein the hydrophilic material is cellulose.

11. The method recited in claim 9, wherein a plurality of hollow fibers comprise a membrane module and wherein the

water-immiscible hydrophobic phase is located within lumens of the hollow fibers.

12. The method recited in claim 9, wherein a plurality of hollow fibers comprise a membrane module.

13. The method recited in claim 12, further comprising a membrane contactor which comprises a plurality of membrane modules including: a first membrane module for transferring the protected, uncharged, peptide product from the aqueous phase into the water-immiscible hydrophobic phase; a second membrane module for transferring the

protected, uncharged, peptide product from the

water-immiscible hydrophobic phase into the aqueous product phase; and a connecting means between the water-immiscible hydrophobic phase in the first membrane module and the water-immiscible hydrophobic phase in the second membrane module.

14. The method recited in claim 13, wherein the aqueous reaction phase in the first membrane module is located outside of the hollow fibers and wets the walls of the hollow fibers

creating an oil/water interface between the aqueous reaction phase and the water-immiscible hydrophobic phase; and wherein the aqueous product phase in the second membrane module is located outside of the hollow fibers and wets the walls of the hollow fibers creating an oil/water interface between the aqueous product phase and the water-immiscible hydrophobic phase.

15. The method recited in claim 14, wherein circulation of liquids at the oil/water interfaces is countercurrent.

16. The method recited in claim 14, further comprising the steps of processing the aqueous product phase through a plurality of membrane contactors.

17. The method recited in claim 1, wherein the peptide product comprises from three to eight amino acid residues.

18. The method recited in claim 1 wherein the second compound is selected from the group consisting of L-phenylalanine isopropyl ester and L-phenylalanine methyl ester.

19. The method recited in claim 2, wherein the condensing enzyme is thermolysin.

20. The method recited in claim 1, wherein the first compound is N-formyl-(O-Bzl)-tyr-gly-OH and the second compound is H-gly-phe-leu-OMe.

21. The method recited in claim 20, wherein the condensing enzyme is papain.

22. The method recited in claim 1, wherein the first compound is N-formyl-(beta-methyl)-asp-phe-OH and the protected, amino acid second compound is H-trp-OMe and wherein the

condensing enzyme is pepsin.

23. A peptide compound selected from the group consisting of N-formyl-(beta-benzyl)-L-aspartyl-L-phenylalanine methyl ester, N-formyl-(beta-benzyl)-L-aspartyl-L-phenylalanine,

N-carbobenzoxy-(beta-methyl)-L-aspartyl-L-phenylalanine methyl ester, N-carbobenzoxy-(beta-methyl)-L-aspartyl-L-phenylalanine, N-formyl-(beta-methyl)-aspartyl-phenylalanine methyl ester, N-formyl-(beta-methyl)-aspartyl-phenylalanine, N-formyl- (beta-methyl)-L-aspartyl-L-phenylalanine, N-formyl

(beta-methyl)-aspartyl-phenylalanyl-tryptophan methyl ester, N-formyl (beta-methyl)-aspartyl-phenylalanyl-tryptophan,

N-carbobenzoxy-phenylalanyl-glycyl-glycyl-phenylalanine methyl ester, N-carbobenzoxy-phenyl-alanyl-glycyl-glycyl-phenylalanine, N-formyl-(O-benzyl-tyrosyl)-glycyl-glycyl-phenylalanyl-leucine methyl ester, N-formyl-(O-benzyl-tyrosyl)-glycyl-glycyl- phenylalanyl-leucine, N-formyl-(beta-methyl)-aspartyl- phenylalanine isopropyl ester.

24. An integrated process for enzymatic resolution of

D-,L-phenylalanine isopropyl ester and racemization and recycling of D-,phenylalanine isopropyl ester, comprising the steps of:

(a) treating a mixture of D-,L-phenylalanine isopropyl ester with an enzyme that selectively hydrolyzes the L- ester in an aqueous phase;

(b) extracting D- ester into an organic phase;

(c) racemization of the extracted D- ester; and

(d) returning the racemate to step (a).