CA2054754A1 - Enzymatic membrane method for the synthesis and separation of peptides - Google Patents

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

WO ~/1~3 2~75~ PCT/US~/021~

ENZYMATIC MEMBRANE METHOD FOR THE
SYNTHESIS AND SEPARATION OF PEPTIDES

CROSS REFE~ENCE TO RELATED APPLICATIONS
This is a continuation-in-part of application Serial No.
06/897,679, filed Auqust 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 peptide~ employing a membrane permeable to uncharged peptides b~t impermeable to charged molecuies; and, more particularly, to the simultaneous synthesis and purification of peptides, L,L-dipeptides, and its application to.the preparation of L-asp~rtyl-L-phenylalanine ~ethyl ester (a~partame).

BACKGROUND AR~
The use of proteolytic enzyme as condensation ca~alyst~ for the stereospecific coupling of two L-amino acids to yield L, L-peptideq is known since the early days of protein chemistry.
As early as 19.38, Bergman~ and Fra~nkel-Conrat described the formation-of the ~ater iDJ~ble dipep*ld~ Bz~eu-Leu~Eoeh,~
reacting Bz-Lou-O~ and H-Leu-N~Ph in the presence of the protein degrading enzyme papain. M. Bergmann and H. Fraenkel-Conrat, J.
Biol. Chem. 124, 1 (1938~_ This re w tion is possible only betwaen th~-~ amino ac~ds that form peptid bcnd~ that are su~ceptihl~ to cleavage by the pap~in or other enzyme used.
Indeed, th~-condenJ$ng reaction' 8 eguilibrium between the amino acid reactant~ and peptide product i8 largely displaced towards the reacting amino acids. N~ertheless, the condensing reaction can be driven to completion by mass action if, o.g., the dipeptide product i~ poorly ~oluble and preci,oitate~ out of the reaction phase.
Due to the commercial importance of certain peptides and the fact that enzymos are known to catalyze peptide formation under mild conditions there has been a great deal of research done on .. . . . .
. : , : ,' . ' '' : ' : :: - . .
- . . . . . . . . .
. ! . ~ , .

WO ~/t~3 PCT/US90/021~ _ 2C~`~75/1~
the enzymatic syntheqi~ of peptides particularly ~imple dipeptides. K. Oya~a and K. Kihara, Kaqaku So~etsu 35, 195 (1982); K. Oyama and K. Kihara, ChemTech. 14, 100 (1984).
The process for enzymatic synthesiq of the peptide derivative aspartame, described in U.S. Patent 4,165,311, hereinafter the '311 proces~, involve~ the thermolysin- catalyzed condensation of N-carbobenzoxy-L-aspartic acid with D,L-phenylalanine methyl ester and precipitation of an intermediary complex, D-phenylalanin~ methyl ester salt of N-carbobenzoxy-a~partame, 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 tho N-carbobenzoxy protecting group.
The '311 pr~cesr ~ust be practtced on a batch basis which i~
cumbersome and complicates th~-rccover~ o~ cnzyme. Also ee: K.
Oyama, S. Irino, T. ~arada and N, ~gi, An~. N.Y. Acad. Sci'-43~f 95 (1985).
Tho N-carbobenzoxy protec~ing group play~ an e~sential role in the ' 311 procoss by: fulfilling thc structural requirement imposed by the active ~ite of thermolysin; and by contributing to the insolubility of the i~t~rmadiary compleL thereby increasi~g tho yield of tho r~ct~cr~ Elimination o~ th~ N-c~rbobonzoxy protecting group from the a~partame derlvative must be effected under mild conditions, e.g., catalytic hydrogenation, to prevent cleavage of the methyL eat~r function. CataLytic hydrogenation invol7a~ the incoDv~r~enco o~ handling hydrogen gas on a l~rge 5CaL~
~ It(erD-t~ve-approachea for dr~v~n~ enzymatic cond~naation reaction~ to completion havo also baen described in tho chemical literature. Eor example, the U8~ of organic Yolvents as reaction media has been found effective for increaQing the peptide product yield~; although, the concomitant decreaae in enzyme ~tability hag precluded it8 practice on a large scale. K. Oyama, S. Ni~himura, Y. Nona~a, R. Kihara and T. ~a~himoto, J. Ora.
Chem. 46, 52~1 (1981); H. Ooshima, H. Mori and Y. Harano, ~, ,: , . . ..
.. : . , ~ , , .

2~5~754

3 ~ ~ PCT/US~/021 BiotechnoloaY Letter-~ 7, 7ag (1985): K. Nakani~hi, T. Kamikubo and R. Matsuno, BiotechnoloaY 3, 459 (1985).
In view of the above-noted difficulties in the practice of prior art methods for enzymatic synthei~i~ of peptidec, particularly, the requirements for precipitation of an intermediary complex and handling of dangerous reagents, it would be desirable to provide an improved proces~ that avoids these difficulties and that safely provides effective yields without rapid deactivation of the enzyme catalyst~.

DISCLOSURE OF INVENTION
The present invention provide~ a process for the enzymatic synthesi~ of peptide~ which provides for cimultaneoui synthesis and purification of the peptide product.
ThQ pre~ent invention provides a process for the safe, economical.and efficient synthesis and purification of peptides and derivatives thereof, particularly a~p~irt~me.
Another advantage of the pre~ent invention i8 to provtdk an s econo~ical proces~ or the onzy~a ff c Jynthe~is of pepti~es th~t provide~i for the efficient use of cnzyme and th~ means ~o effect the synthesis on a continuous basis.
Another advantage of the pre~Qnt invention i 8 to provide a proceas particularLy adapted to the enzymatic synthesis of aspartane and ~tg deri~ative~ ~ith D, L~ph~nylalanine and N-protected-3-substituted-L-a3partate in substantially quantitativo yield.
The present inYentlon proYides a method for the ~ynthesis and purification of a compoun~,-comprising the step~ o-~coupling :: -a first reactant with a s~cond.. roactant.to produco a D~brane ,. transportabLer uncharged compound; tran~por.ting the transportable compound acro~s a membrane that will not transport the reactants;
and prevonting tho transported compound from back-diffusing acroi~s the membrane.
The present invention also provides a process for the enzymatic synthesis and purification of compounds comprising the steps of coupling a fir~t compound, including a protonated amlno group (ammonium), and a second compound, including a free : '; ' ' ', , WO90~ 3 PCT~US~/~188 2~ 75~
carboxylate group, using a condensation enzyme in an aqueou~
mixture to produce an uncharged (or non-ionized) coupled compound; continuously removing the uncharged coupled compound from the aqueous mixture by difusion acro~s a membrane that selectively transports the uncharged coupled compound to the product side of the membrane. Preferably, the tran~ported coupled compound is a peptide or derivative thereof that is converted to a charged (or ionized) molecule 80 that it doe~ not back-diffuse across the membrane. Thu~, the formation of the coup~ed compound product i~ driven in the reaction mixture because it i8 constantly beinq removed therefrom.
The present invention al80 provldes a method for the enzymatic synthesis of peptide~ comprising the steps of condensing first and second amino acid compounds in an a~ueous in~tial reaction mixture to form an uncharged compound;
tran~porting the uncharged compound into an a~ueoùs second reaction mixtur~ ~cross a memhr~ne that ~ill not transpart sub~tantial amoun~:of ths am~nn ac~ compounds; ~nd ccnvert~s the tran~ported uncharged compoun~ to a form that cannot b~ .-retransported acro~s the membrane to the initial reaction mixture. The tran~ported compound, converted to a form that is not retransportable acros~ the membrane, can be removed from the second reaction mixtuse. Also, small amount o first and seconl amino acid compound coperresting tho-~e~br~ne-w~th the unc~rg~d compound into th~ second reaction mixture can be ~eparated from the second reaction mixture and optionally returned to the initial reaction mixturR.
The proJent invention ~l~o pro~ldes a-proc~6:for;the !~
enzymatic. ~yn~he0i8 o aJp~rtum~- ~nd.itJ analog~, comprisi~ the step~ of coDd-na~ng a ~-acyl-~-~ubsff tu*ed-L-aspartic acid' includ$ng ~ carboxylato group with a phenylalanine lower alkyl ester including an ~-ammonium group in an aqueous reaction mixture including a conden~ation enzyme, to form N-acyl-L-a~partyl-(B-sub~titutedj-L-phenylalanine lower alkyl e~ter (i.e. 1-6 carbons), an uncharged peptide; an~ transporting the uncharged peptide from tho aqueou~ reaction mixture to a product mixture acros~ a permselective mombrane. In one ., ,,- ~

W090/t2~3 `` 2C~7S~ PCT/VS~/021~

embodiment, for proteo~ynthesis done at or above pH 5, the preferred acyl group is formyl, the preferred beta sub~tituent is methyl, the preferred lower alkyl ester is i30propyl, 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 (B-COOH), the preferred lower alkyl ester is methyl and the preferred enzyme is pepsin. In the latter ca~e the permeable aQpartame intermediate is N-CBZ-asp-phe-OMe, where the charge of the free B-carboxylate of the aspartic acid has been suppres~ed by protonation in order to make the peptide permeable.
The present invention al_o provides a method or the enzymatic resolution of racemic alpha amino acid compounds comprising the steps of: hydrolyzing an uncharged D,L-alpha amino acid derivative carrying at lea~t one hydrolyzable functional group attached to the chiral carbon, in an aqueo~s reaction-mixture in the pr~sence of a hydro~e enzyme capabLe of hydrolyzing a sen~itive functiona~L-group, to form a charged ~-am$~o acid compound and an uncharged D-amino acid derivative, and transporting the uncharged D-amino ac~d derivative from the aqueous reaction mixture across an ion rejection membrane into a product mixture. The l~chargad D-amino acid derivative L~ the product mixt~re can-}~ converte~;t~.~ ~pccteaLth~ c~nnot back-difuse acro~s the membrane. The method for the enzymatic resoLution of racemic alpha amino acid compounds can be practiced in comblnation~ith the peptid~ synthe~is methnds discuased above. An 08ample o r~c~m~c alph- amino ~cid derivat~ve is DL-phenylal~nino m~thyL e~ter. An example of hydrQlA~e enzyme i9 the e~ter~a~ aminoacyIa~o I.
The pr~-~nt invontion al~o provido~ ~ method for the enzymatic synthesis of a peptide, compriqing the ~teps 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 aquoous reaction phas~ under conditionQ
in which the C-terminal carboxylate group and the N-terminal ..
,.
, , . ~ , . - :
, ~
,- - : ' ., , .. ~ : .
:. , . , . . : . . ~ . :

, WO ~ ` PCT/US~/021~
2~ 54 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 acros~
the water-immi~cibls hydrophobic phase.
The present invention alRo provides a method for the enzymatic synthesi~ of a peptide, compri~ing the ~tep~ 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 yroup in the presence of a condensation enzyme in an aqueous reaction pha~e under conditions in which the alpha carboxylate group and the N-terminal ammonium group condense forming a protected, uncharged, peptide product;
tr~nsporting t~e protected, uncharged, peptide product into a water-immiscibLe hydrophobic ph~se into an aqueous product phase;
and preventin~ the protectedj.uncharg~d, peptide prod~ct fram back-diffusin~ scross th~ w~ter~ iscible hydrophobi~.p~a~e The pre~ent invention al~o provides a method fos.th~....
enzymatic ~ynthesis of a peptide, comprising the steps of.
reacting a prot~cted, peptide fir~t reactant having a C-terminal carboxylate group with a protected, amino acid ~econd reactant having an alpha ammonium group in the preaence of..a condensation enzy~e in an aqueous re~ction~phaoe under ccudl ff ons in which th~ -C-terminal carboxylate group and the alpha ammonium group condense forming a protected, uncharged, peptide product;
transporting tho prot~ctod, uncharged, peptide product acro~ a water-i~mi~c~ble hydr~phobic ph~e into an aqueou~ product phase; .
and prev^~t~ng the protectod, unchzrged, peptide product.from .
back-di~uu~nJ acros~:the water-imm~scible hydrophobic phase.
The pr-sent invention al80 provides a method for the enzymatic synthe~is of a peptide, compri~ing the ~teps of:
~omb$ning a protected, N-acyl amino acid f~rst reactant having an alpha carboxylate group with a protected, amino acid second reactant having an alpha ammonium group in the pre~ence of a condenJation enzyme in an agueou~ reaction phase under condition~
in which the alpha carboxylate group and the alpha ammonium group , ' .

Z1~75~
WO ~ 3 ~ PCT/US90/02~88 ~ } 7-.
condense forming a protected, uncharged, peptide product;
tranqporting the protected, uncharged, peptide product across a water-immiscible hydrophobic pha~e into an aqueou~ product mixture; and preventing the protected, uncharged, peptide product from back-diffusing across the water-immiscible hydrophobic phase.
The peptide~ of the pre~ent invention comprise a plurality of amino acid re~idues. The peptide~ of the pre~ent invention include, but are not limited to dipeptideq. 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-(~-methyl)-aspartic acid. Example~ of protected, amino acid second reactants are L-phenylalanine methyl e~ter and L-phenylalanine isopropyl ester. An example of a condensing enzyme is thermoly~in. Another exa~ple of a protected, peptide first reactant i~ N-formyl-(O-Bzl)-t~r-qly-OH. Another example of a protected, p~pt~de ~econd react~nt is H-gly-phe-leu-OMe.
Another exJ~pl~ of a condensing onz~n~ i~ papain. Anot~er example of a protected, peptide first reactant i5:
N-formyl-(~-methyl)-aqp-phe-OH. Another example of a protected, amino acid second r~actant i~ H-trp-OMe. Another example of a condensing enzym~ i~ pop ~n.
In another ombodim~nt of the~present invent~n N-carbobenzoxy-a~partic acid having an alpha carboxylate group i~
the fir~t roActant and L-phenylalanine methyl e~ter having an alpha ammonium group is the ~econd reacta~t. ~he alpha carboxylate group of tho firs~-reactant and th~ alpha ammonium group of the aecond reactant condense in the pres~nce of pepsin in an aqueour re~ction pha~e to form a protected, uncharged, peptide product.~Thi~ product is transported acros~ a water-immiscible hydrophobic phaso and prevented from bacX
diffuoing acroo3 the ~ame.
The present invention also provides a method for the enzymatic synthesis of a peptide, comprising the step~ of:
reacting N-acyl-(~-substitutod) a~partic acid first reactant having an alpha carboxylate group with L-phenylalanine lower WOgOtl~3 PCT/US~/021~
-8- 2C~75~ ~

alXyl ester ~econd reactant derived from a Recondary alcohol having 3 to 6 carbon atoms having an alpha ammonium group in the pre.cence of a condensation enzyme in an aqueouR reaction phase under conditions in which the alpha carhoxylate group and the alpha ammonium group condense forming a protected, uncharged, peptide product; transporting the prote~ted, uncharged, peptide product acrocs a water-immiccible hydrophobic pha~e into an aqueous product phase; and preventing the protected, uncharged, peptide product from b~ck-diffusing acro~s the water-immiscible hydrophobic phase. The aqueous reaction pha~e can be maintained at a temperature range of from about 20C to about 65C. An example of a N-acyl-(B-substituted) aspartic acid first reactant is N-formyl-(~-methyl)-aspartic acid. Another example of a L-phenylalanine lower alkyl ester ~econd reactant i~
L-phenylalanine.i~opropyl estcr. An example of the conden~ing e~nzyme is thermoly~in wherein the temperature of the aqueous reaction phase ia.~bout 50C.
I~ one embod~mont of the pre~knt invention an IL~ ~ad~le.is utiliz~ cempris~ng a plurality o n~croporou~ hollow. fiber1~de of polymeric materials. The~e hollow fibers can support water-immiscible organic liquids immobilized by capillarlty within the microporous wallo. ~his immobilized water-immiscible organic li~uid constitutes a hydrophobic ph~ that functions a~
an ion rejection me~hran~ ~op-ratin~ the ~ueo~-ro~ction pha~e from the aqueous product phase. Tho aqueous reaction phase, also referred to herein as the "tube pha~e, n is locat0d within the lumen of hollow fiber~. The ayueou~ product phase, also refarred to h~reir~a~ tho "nh~Ll phaae~ i~..located in the shel1 spacoa exi~ting~i~.the moduL~ betweon hoLlow fiber~. Oil~water....
interface~-.~r~.thus cre~tod with ~c~L-of the two aque~u~-ph~es.
A schemaff c representation of thi8 ILM modulo. i.~ shown in Eigures 2, 9, 25 and 26. In a preferred I~M configuration the ends of the hollow fibers are sealed or pottod in resinou~ material 90 that aqueous solution being circulated through the lumens will not mix with aqueou~ solution being circulated through the ~hell spaces. Hollow fibers made of microporous polypropylene exemplify a preferred material. While thi~ embodiment describes :. ., , : - . : , : , ~ ::
. .
.. :: , . . . :
. . :. . ' : :
:: : ' ', :
,: ' , .' , .. . : ' - ' WO ~/1~3 2~5~ PCT/US~/02188 g _ the ~hell phase as the aqueou~ product pha~e and the tube phase as the aqueou~ reaction phase, alternatively, the shell pha~e can be the aqueous reaction pha~e 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 compri~ing hydrophilic material. An example of the hydrophilic material i cellulose. In one embodiment of the present invention, oil/water interfaces can be created by utilizing two membrane modules compri~ing hydrophilic hollow fiber~ arranged a~ shown in Figures 17, 27 and 28. In a preferred membrane module confiquration the end~ of the hollow fibers are sealed or potted in a resinou~ material 80 that organic liquid being circulated through the lumens will not mix with the aqueou3 solution (phase) being circulated through the shell ~paces. Esch module comprLses a plurality of hydrophiIic hollow fibers. The lumens of the hydroph~lic hollow fibers are fill~d with a water immi~cible organic ~quid. The two membran~
modules having a connectlng mean~ such as a common loop o~
circulating organic liquid compri~e a mem~rano contactor. The water-immi~cible organic liquid located within the lumen~ of the hydrophilic hollow fiber3 comprises the water-immiscible hydrophDbic phsse of each membr~ne module, and the two iaolated agueou~ phase3 loc~ted out~id~ of tho wall~ of~thc hydrophilic hollow fibers re~p~ctively comprise the aquoou~ reaction and product phases. The water-immiscibl~ hydrophobic phase functions as an~on reject~on ~e~brane aeparating th~ aqueous reaction pha~e in the first me~brarxF~odhle frnm th~ aqueous product phase in the 8econ~ mombrano module.
A m~mhrano contactor comprisos a~flrst membrane mod~Ie for transferring tho protccted, uncharged peptide product from the aqueou~ reaction tnto the water-immi~cible hydrophobic pha~e; a sec3nd 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 ~econd membrane ., .. , . - - . . . . . . . .
.. . ........ . .

-, ,: ~ . . ' ' . ,, , ~' . . ,, ' . ' :

..

WO ~/l~3 PCT/US~/021~

ZCC;~75~
module of the membrane contactor. The aqueous reaction phase in the first membrane module is located outside of the hollow fiberq 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 aqueouq product pha~e in the qecond membrane module is located outside of the hollow fiberq and wets the walls of the hollow fibers creating an oil/water interface between the aqueous product phase and the water-immiscible hydrophobic pha~e. The circulation of liquid~
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 proventing the protected, uncharged, peptide prcduct from back-diffu3ing acro~s the wator-immi~cibLQ hydrophobic phase compri~es converting th~
protected, uncharged, peptide product to a charged ~pecies. The conver~ion can ~e chemical or ~n2ymatic. Chemical means includc p~ d~pen~ent i~n;~Jtion o~ ~ prot~tropic unctional g~oup,~. -ionization ronulting from a d~s~ociatton of a carbo~ylic~~cid function and/or re~ultln~ from a protonation of a ~ree amino group. An example of an enzymatic convorsion i8 the hydrolysis of an ester function utilizing a protoase having esterolytic actiyity. An exampLe o.thc.protoase enzym~ havin~ esterolytic acti~ity i~ aminoacyl~ r. T~a onzymc h~v~ns-~t~r~lytic activity c~n b~ c$rculated againQt the mombrane in the agueoua product phaso. The enzyme havlng ~torolytic activity can bo immob$1ized on a wator insolu~lo ~upport and t~e agueous product phas~ cfin bo circul-t~d over t~o onzym~; : ' ~ h^-pr~aent ~n~ention d80 provid-~ pepttde compound~ -.
select~..ro~ tho group conJisting of'N-formyl-(B-benzyl)~
aspartyl-1-phenylalan~ne mothyl e-~ter, N-formyl-(B-benzyl)-L-aspartyl-L-phenylalan$n~, N-carbobenzoxy-(B-methyl)-L-aspartyl-L-phonylalanine methyl eJter, N-carbobenzoxy-(B-methyl)-L-aspartyl-L-phenylalanine, N-formyl-(B-msthyl)-a3partyl-phenylalanine m~thyl éster, N-formyl-(B-methyl)-a~partyl-phenylalanlne, N-formyl-(B-methyl)-L-aspartyl-L-phenylalanine, ' ~ ' . . - . .', ' , . . .

WO ~tl~3 Z~75~ PCTtUS~/02188 i N-formyl(fl-methyl)-aspartyl-phenylalanyl-tryptophan methyl e~ter, N-formyl(~-~ethyl)-aspartyl-phenylalanYl-tryptophan, N-carbobenzoxy-phenylal~nyl-glycyl-glycyl-phenylalanine methyl ester, N-carbobenzoxy-phenylalanyl-glycyl-glycyl- phenylalanine, N-formyl-(0-benzyl-tyroRyl)-glycyl-glycyl- phenylalanyl-leucine methyl ester, N-formyl-(0-benzyl-tyrosyl)-glycyl-glycyl-phenylalanyl leucine, N-formyl-(B-methyl)-aspartyl-phenylalanine isopropyl ester.
The pre~ent invention provides a method for the enzymatic ~ynthesi~ of a peptide, comprising the ~teps of: reacting a first compound selected from the group con~isting of a protected t 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 consicting of a protected peptide having a N-terminal ammonium group and a protectod amino acid ha~ng an alpha ammonium group in the presence of a ~~
condensation enzyme in an aqueous react~on phas~ under conditions in whic~ the carboxylate group and am~onium group condensQ
ormin~.a protected, uncharged, peptide product; transport~ng the protected, uncharged, peptide praduct acro~s a water-immi~cible hydrophobic pha~e into an aqueous product phase; and soparating the protected, uncharged, poptide product from the aqueou~
product mixture to prevent that product L4m back-dlffusing across t~e water-tmmtscibL~ hydropbo~ic pha~e.~
~ho present lnvention also provideo a method for the enzymatic synthesis of a peptido, comprising the steps of:
reacting N-acyl-(~-~ubstituted) aspartic acid irst.reactant having an alpha carbc~ylate group with L-phenylalanine lower alkyl ester ~econd reactant having an alpha amm~nium ~roup in the presence of a conden~ation enzyme~ n aqueou~ re~ctlon p~a~e undor condit~oA~ in which tho ~lpha carboxylate group and the ¦
alpha ammoniu~ group conden~e forming a protect~d, uncharqed, peptide product; transporting the protected, uncharged, peptide product across a water-immiscible hydrophobic phase into an ¦
aqueou3 product pha8e; and Jeparatin~ the protect~d, uncharged, pept$de product from the aquoous product phaoe to prevent that product from back-diffusing across the water-immiscible 1' ., ', . ' ', ~ ', :
. . , . , ~ . .

WO90~ 3 PCT/US~/021~
12- Z~75~ -~

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 a~ reverse o~mosis 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, adcorption on a matrix or by precipitation with a reagent. The aqueou~ reaction phase can be maintained at a temperature in a range of from about 20C to about 65C. An example of the phenylalanine reactant is a lower alkyl ester derivsd from a secondary alcohol havin~ 3 to 6 carbon atoms. An example of the condensing enzyme i8 thermoly~in wherein the temperature of the aqueou~ reaction pha3e i 3 about 50C. An example of the a~partic acid N-acyl-(3-sub~tituted) first reactant i8 N-formyl-(3-methyl)-asp-OH. An example of the phenylalanine lower alkyl ester second reactant i 8 L-phenylalanine i~npropyl e~ter.
~ n general, the amino ~cidJ-~hich can be utilized or.~
peptide ~ynth ~t- according to th~ pre~ent $nventio~ compr~.the L-enantiom~rs of th~ 20 natural amino acidc recognized by the genetic code as protein building block~, plus their various protected derivatives available through standard procedures commonly used in t~e iRld of peptide sy~theai~. ~he preferred protecting groups w~ rry-accordin~-w~t~ t~e choice of condensing enzyme, nature of the hydrophobic phase, p~, temperature and nature of th~ ~olvent for any particulur proteosynthesi J .

BRIEF DESCRI2TION OF TEE DRAW~NGS :
The for~g~1ng and other ob~ect~-and advantages are attain~d by tho invon~o~, ~ will be apparent from the following detailod description tak~n in con~unction wlth the accompanying drawing~, wherein:
Figurs 1 i~ ~chematlc illu~tration of the enzymatic synthosi~ of a~partame in accordanco with t~e pre~ent invention.
Eigure 2 i~ a schematic repreJefitation of an apparatu~ for practicing the process of the pre~ent invention.

. . . - , , ~. ~ .-.: .

', . .
. , .

'17S~
WO ~12~ - PCT/US~/021 Figure 3 i~ 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 o product (aspartame derivative) formed over time in Example 5.
Figure 6 iR 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 i8 a graph illustrating the enzymatic resolution of DL-phe-OMe described in Example 8.
Figure 9 is a schematic repre~entation of an apparatus for practicing the present invention which illustrate3 the ve~sels on the product Jide as described in Examplo 9.
Figure 10 is a graph illustrating the quantity of product (aspartam~ derivative) ~ormed over time in Examp~e 9 with and without the utilization of an ion ~ch~Dge re~in.
Figure 11 describ~o the pepsin cat~lyzed proteosynthesi3 of N-formyl-(B-methyl)-asp-phe-trp-O~ over time.
Figure 12 compares the V8yn and Vperm for the pep5in catalyzed proteosynthesis of N-formyl-~-~ethyL)-asp-phQ-trp-OMe.
Figure 13 describos the pep d n catalyzed proteosynthesis of N-formyl-(~-methyl)-asp-phe-trp-OMe over time.
Figure 14 d~scribea t~e papain catalyzed proteosynthesis of N-CBZ-phe-gl~-gl~-phe-O~ over~timo_ .
Figure 15 d~-cr~be~ tho ratQ-~of synth sis of N-CBZ-phe-gly-g~y-ph~-OMe.
Figure 16 d~scribo~ tho papain-catalyzed proteosynthasis of N-formyl-(O-Bzl)-tyr-gly-gly-phe-lou-O~.
F~gure 17 describo~ the process in which RO ls u~ed to trap product on one sida of tho membrane contactor.
Figure 18 describe~ the concentration of C in the thermolysin reactor during the long-term run.

~ ,. . .
. . . -., ., . r; ' ~ ' " ', ' ., , , . : . . . . . .
. .
:.' . ;: . . . .

WO90/1288~ PCT~US~/021 2C'~;~75~
Figure 19 describes the thermolysin-catalyzed proteo~ynthesi 9 of N-formyl-(B-methyl)-asp-phe-OH.
Figure 20 describes the kinetics of ~ynthesis of N-formyl-(B-methyl)-asp-phe-O-<.
Figure 21 de3cri~es the effect of the enzyme aminoacylase on the rate of permeation of N-formyl-(~-methyl)-asp-phe-o-< across an ILM including N,N-diethyldodecanamide.
Figure 22 describes the thermolyMin-catalyzed proteosynthesis of N-formyl-(~-mothyl)-asp-phe-O-~ in a ILM
module.
Figure 23 describes the synthesi~ of N-formyl-(B-methyl)-L-asp-L-phe-O-~ in a membrane contactor.
Figure 24 describes the pep~in-catalyzed proteo~ynthesis of N-CBZ-asp-phe-oMe in a ILM module.
Figur~ 25 i8 a partial view (enlarged) of a crocs ~ection of hollow fibers in an ILM mo~ule.
Figure 26 ~ a partial schematic v~ (enlarqed) of an ILM
module.
Figure 27 i8 a partial schezatic view ~enlarg~d) of-~ cr~
section o~ hollow fiber~ in a membran~ contactor.
Figure 28 is a partlal schomatic view (enlarged) of a membrane contactor.
Figure 29 is a ~chematic presentatio~ of an apparatus for i~tegratod enzycatic ro~olution of D~ phenylalaninc i~opropyl ester and the racemization and recycling of D-phonylalanine isopropyl oster.
.

3EST MODES EOR CARRYING OUT T~E rNVENTIO~
Th~ invontion di~clo~ed h~r~n provid~s a procedure or --driving~tor-c~mpletio~ the onzymat$c:~ynth~s~ of poptide~ in a~ .
aquoou~:rerction mi~turo at equil~briu~, by separating t~e uncharged peptide intormediate, or derivative thereof, from tho reaction mixture by means of a membrane that ~electively transports the uncharged peptid~ out of the reaction mixture into a product mi~turs. Becau~e tho membr~ne i~ ~ubJtantially impermeable to the reactants (charqed molecules) removal of the peptide intermediate from the reaction mixture cau~es a decrea~e of peptide concentration at eguilibrium that pushes the reaction :
- ' :' ' ' ' ' .

2~ 75~
WO ~ 3 PCT~US90~02188 t~rd ccmpletion by mass action.
The membraneQ most u~eful 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 subetantially impervious to the enzymes, reactants and other charged products.
Hydrophobic polymers such as polypropylene are prefe~red support materials. ILM modules can be produced utilizing polypropylene hollow fibers. Celgard, a registered trademark of the Celanese Corporation, 1211 Avenue of the America~, New York, N.Y. 10036 and sold by Celaneee Fibers Marketing Corporation, Charlotte, .C., is an example of commercially available hollow ibers 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. -~. DuPont de Nemours & Co. for fluorinated hydrocarbon ~ polymer~. A typical microporouJ support i8 GORE-TEX, a trade~ark : of W. C. Gore & A~sociates, Inc. Ei~ure 25'~ a partial view - (enlarged) of a.cross section of hollow fib-rs in an ~LM module.
- The partial vieu which 18 enlarged' shows hy~rophobic hollow fibers 101 having a lumen (bore) 102, made from microporou~
polymeric material 105 which can support water-immiscible organic liquid by capiLlarity w~thin microporoun walL~. A capil~ary 104 i8 shown ~n th~ ~i~Gr~poroUs p~ymQric mn~eria~ 105;-~ow~ver, the microporous polymcric materiaL 105 actually ~nclude~ many such capillaries 104 extending from the lumen to tho exterior of the hollow fibor 101. The lumen 102 co pr$ses the tub~ phase (e.g., agueou~ r~action phac~). Th~qp~ce 103 bet~een hoL~ow f~bers compri~es tho.~hell phase (e.g., agueous product phase).
Figur~ 26 is partial sch~mat~c view (entarged) of ~n sn ILM
module 115. ~ho partial view whl d i8 enlarged show~ hydrophobic hollow fibers 101. The ends of each hollow fiber are potted in a resinous material (potting compound) 80 that the tube phase being circulated through the lumen 102 through opening 107 on first wall 106 and returned through openlng 111 on ~hird wall 110 will not mix with the shell pha~e being circulated through space 103, through opening 109 on second wall 108 and returned through -: - . : ~

WOgO~ 3 ~16- PCT/US~/021~
2~ 75~
opening 113 on fourth wall 112. In ILM module 115 actually compri~es many hydrophilic hollow fibers 101 although only three are shown in this fiaure.
The micropores pass through the support material and ~hould be ~ized so that an immobilized liquid will be held therein by capillarity and will not escape therefrom when subjected to, e.q., precsure differentials acros~ the membrane or other ordinary reaction conditions. Subject to the ~oregoing limitationq is advantageous to maximize the contact area between the immobilized liquid and reaction mixture to maximize the rate of transfer (flux) of the uncharaed peptide product acro~s 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.~urther, that the optimum pore size can be.
determined empirically by those Bkilled in the art; A uaefu~
discussion of pore size aelection is ound in U S. Pat~nt .
No. 4,174,374 the text of whic~ is incorporated herein by:
ref~r~nce. -The u~ and preparation ~f immobilized liquid :, membrano~ are doscribed in the followin~ reerences, the text~ of which are also lncorporated herein by reference, S. L. Matson, J. A. Quinn, CouDlina SeDaration and Enrichment to Chemical Conversion in a Membrano Reactor, paper preaented at the AIC~E
Annual Meoting, Ne~ Orl~n~, Loui~iann t~bYænker 8-12,' 1981) ~nd - .
S. ~. Mat~on and J. A. Quinn, Membrane Reactors in Bio~rocessina, Ann. N.Y. Acad. Sci. 469, 152 (19~6).' The immobilized liqyld held in the microporous support by capillarity should b~ ~ator in~$-cibl~ and a good ~olvent-for the unchàrg~d peptido pro uct ~hic~ mNst be transported w ross the membrane~-(diffu's~d) at a roa~onabl~ rDte, i.e., good tran~port characteri~t~c~/high flux; while, charged or ioniz~d molccules on both the reaction side and product side of the membrane are, for the mo t part, not transport~d across the membrane in either diroction, i.e, good ~electivity/ion rejection.
Selection of the bost combination of ~upport'material and ' ' immobilized llquid for u~e in an enzymatic synthesis of a pept~de in accordance with the present invention will depend, in part, on - . , , , ~
: ' '' . ' , ' . ~ ' : ~

WO ~/12883 ~ 75~ PCT/US~/~2 ~ 17-; ~
the nature of the particular reactant~ employed, products de~ired and solvents in the system.
The generally preferred immobilized liquid~ for the practice of the present invention include water-immiscible organic solvents, such aQ alcohol~ 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 immiQcible organic solvents including mixtures thereof. Such solventQ 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 de~cribed in the literature, for example, 0. J. Sweeting, Editor, SCiQnCe and Technoloav of PolYmer_Films, Interscience, New York (1963), while extensive f application of such membranes to the- soparation o~ gas~a and ; li~uids are discus~ed in S. T. Hw~ng and R. Xammermeyer, Membranes in SeDaration~r-Techn oues of Chemistrv, Vol. VII, ~A. Weissberger, editor) John Wiley & Sons, -Inc., New York (1975).
A preferred embodimont of the invention employs a membrane reactor/sepa~ator syste~ which provid~ an aqueous reac~ion mixture or pha~e circu~ating in contact with one s~de-of an ILM
membrane and a product aqueous phaso or mixture circulating countercurrently at the opposite surface of the membrane.
S. L. Mstson and ~. A. Quinn, Ann. ~.Y. Acad. Sci. 469, 152 ! (1986~.. Tho p~ and tomperats2~ of the reaction and product phaQea are ~intained at a v~ that keeps the reactants in a form th~t r~ntmizes their transport across the membrane at p~' 8 betwoon about 4.0 and 9.O. Transport of the uncharged peptide intermediate from the reaction ph~e to the product phase is drivon by the coneentration gradient acros~ the membrana created by increasing unchargod peptide concentration in the reaction phase. Tho transport activlty or flux across the membrane can be - significantly enhanced by the simultaneous, ~rrever~ible conversion of the transported peptide, in the product pha3e, to a ', ' ' ,- ' ' :' WO ~/1~3 PCT/US~021~
-18- 2C5~75~

species that cannot back-diffuse. For example, the latter conversion may resu~t 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 J
of the pre~ent invention is found in U.S. Patent No. 4,187,086.
Available alternative membrane reactor/separator configuration~ that could be adapted to practice of the present invention include the hollow fiber modules de~cribed 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 uncharqed peptide the membrane provides a barrier between the reaction pha3e and product phase that prevents undesirable mixing of, and reaction~
between, the components of each phase.
In a preferred membrane reactor/separator confiqur~d in accordance with.the present invention, the chemical equiLibrium be+~cen-+~he re~ct~ts i8 actual~y npull~dn across the ne~bran~ hy conducting~ rrover~ible con~rs~on a +~he transportel~
uncharged peptide, to a membrano impermeable species, on ~he product side of the membrane. This type of membrane reactor/separator employs a coupled two enzyme (E1 and E2 process of the general typ~:

El E2 A + B , C ~ D
The churged roac+~D~ A ~n~ B ~re amino ac~ds:aa~or sma~7 pepti~e- whlch are condonsod with the aid o peptide form~ng enzy~ xr form-the uncharged int-rmndiary peptide C whi~h i~-selectlvely transported across the m~mbrane to the product side.
It is understood that reactive functlonal group~ in the reactants that do not participate in the de~ired reaction may be protected or blocked, where necessary, to pre~ent undesirable aide reaction~ and/or charge 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 mem~brane, cau~ing the ,. - ' . . ~ .
.
.
.
.: .

WO9a/1~3 2~5~75~ PCT~US90/02188 chemical equilibrium in the reaction mixture to shift toward the production of ~ore C.
This concept is illustrated in Figure l, for the specific case of the enzymatic condensation of D,L-phenylalanine methyl ester with (N-and B-protected) N-formyl-B-benzyl-L-aspartate, in the pre3ence of thermolysin at about pH 5.5.
In the reaction scheme illu~trated in Figure l the reactant A is D, L-phenylalanine methyl ester and B is N-formyl-B-benzyl-L-aspartate. The reactant~ are condensed on the reaction side of the membrane by the enzyme ~l thermolysin forming the uncharged peptide C. The pH is selected to maintain the reactant~ in their charged ~tate and thus minimize their diffusion across the membrane along with uncharged peptide C.
Although the chemical equilibrium for the condensation reaction largely favor~ the reactant A and B species, dif~usion of the unc~arged peptide product C across th~ membrane to the product side reguire~ the constant productio~-of C to maintain the chem~cal equilibr~um on the reaction side of the membrane.
In Q~ embod~ent, on thc product -~de of the membranb~ an esterase enzyme E guickly and irreversibly cRnverts the uncharged peptide C diffused across the membrane to charged peptide D which cannot back-diffuse to the reaction side. Thus the chemical eguilibrium o~ the reaction side ia effectively "pulled~ acro~ the mcmbran~ and towar~ the production of-uncharged product C. Thoreafter, the peptide D i~ converted to aspartame by acid hydrolysis, which remove~ the formyl and benzyl protecting group~, folLQwed by C-terminal esterification with mothanol. In asothor ombadime~ the esterase is Dot utilized and the uncharged product C i~ directly separated from the product mixture by phyrica~ mean~. r The foregoing method may bo practiced in a countercurrent flow membrane r~ ctor/separator 10, as shown in Figure 2, operating under controlled condition~ of pH and temperature, 80 that a roaction mixture comprising a N-acyl-B-sub~tituted-L-aspartlc ac~d, e.g., N-formyl-~-b~nzyl-L-aspartate, ha~ing a free ~-carboxylate group (electronegative species) which i~ allowed .o condens~ with a .

, WO ~ 3 PCT/US~/021 reactant, e.g, D, L-phenylalanine methyl e~er, hav~ng a protonated free ~-amino group (electropositive ~pecies), under the catalytic action of protea~e~ 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 includlng fully protected uncharged peptide, e.g., N-formyl-3-benzyl- L-a~partyl-L- phenylalanine methyl ester transported across the membrane, is circulated from product rea~tor 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. mis product mixture in product reactor tank 22 includes a second enzyme E2, an e~tera~e; or other suitable reagent, that can c}eaYe at }east one of th~ -protected groups borne by the unchargcd peptide, thu~ generating an electroch~rged pocie~ that cannot:~cape the ~w~ep~str~a~ by back-diffusing through th~ ~ombrane. If an esterase is utilized, a preferred estera~e will have a preferred pH range of from about 6.0 to 9.O. Aminoacylase I, -ch~motrypsin and subtilisin A are exa~ples of esteras~a con8idered useful in th~ present invention The conversion to charged species t~at c~n~ot bac~ diffu~e across the water-lmmiscible hydrophobic phase can be carried out utilizing chemical or enzymatic m~ans. Chemical mean3 lnclude pH
dependent ionization o~ ~ prototropic functicnal group, '-ion$zation resulting ~rom di~ociation of a car~oxylic acid !-functior~ aDd~or re~ulting ro~ ~ prot2nation of a fre~ amt~
group or hydr~ysis of an ester functiQn. For e~ampl~, the .
convorsion-of ~n uncharged product into a charged product that cannot back-diffu~e can bo achieved by an appropriate pH gradient between the two aqueous phases s~parated by the hydrophobic membrane. This ic physically po~sible as the hydrophobic pha3e i8 impervious to ions, thus allowing the existence of two aqueous phases of different pH in eguilibrium with respect to non-ionic solutes. For example, a diffu31ble free amine ~-NH2, bearing no .
. . ~
.

WO ~/1~3 ~C5~75~ PCT/US~/021~

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 ~orm 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 u~eful 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 liguid mem~rane 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 t temperatures such as temperatures above 40C. In this oil~water ~ contactor ,the organic membrane is created at the inner walls of t hydrophilic cellulose fibers,~whose bores or lumens are filled with the desired hydrophobic organic ph~se, and having the bulk aqueous pha~e located outsid~ of the f~ber~ and wetting the wall3 of said fibers. Packing of bundles o~ said fibe~a in ~ modular arrangement allows for tho creation of two compartments, ~eparated by an oil/water interface. Circulation of said oil phase between two separa~e~ a~ueous pha~e~ c~t~irR~ ~n two individual ~dules co~JtituLI~g-a singl~rcontactor i~ Jhown in Figure 17. A plurality of contactors may be utilized. Each aqueous phas~ representing the reaction and product phases discu~s~d above, aLlo~s for the tranJport of pe~a~l~ product from one aqu~o~D pha~e to the second one containing the e~terase enzyme. Mo~brano _ep-rators of the gen2ral type show~ ~n Figure 17 are-~elI~known in the art. U.S. Patent No. 4,754~89 desc~ibes the utilization of ~uch mem~ranes in phase transfer catalysis. U.S. Patent No. 4,778,688 descrlbes a 3imilar membrane. U.S. Patent Noa. 4,572,824, 4,~63,337 and 4,443,414 also de~crib~ such oil water contactors. Another example of similar membranes i8 deacribed 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.

::, . ', ' : ' .

,.~-, ' : . ,. :
.. . ... . .

WO ~ 3 PCT/US~/021 Z~ 75~
The charged product may be periodically discharged and/or continuously removed from the product phaqe by conventional separation means such as ion exchange resin~ and other techniques including rever~e o~mo6iq and the like, and the remaining effluent may be recycled through the ~ystem. Where thiq separation is by ion~exchange the resulting product bound to the ion-exchange re~in may be desorbed and recovered using conventional procedures.
In the case of reverse osmosis separation, it may be fir~t 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 ro~ back-difusing across the membr~ne .
utilizin4.a trappiD~ such a8 a rev~rse osmosis membranJ. -In.th~ .
case th~ s~co~d enz~m~ may not be r~guir~d to offic~nt~y ~p~rate the proce~s. ReverY~ osmosis is well-kn~n in the art. James S.
JohnYon, Jr., "Reverse Osmosis," in Kirk-Othmer's Encyclopedia of Chemical Technology, Third Edition, Vol. 20, pp. 230-248 John Wiley ~ Son~, New Yo~, N.Y. (1984) and.U.S..Patent No. 4,643,902.
Other trapping means include tho formation of specific molecul~r complex~J. Specific cavities of zeolite~ and/or cyclodextrins ~ay bo utiLiz~d. In additio~.to.trappi~, solvent extraction, addorption:on a.matrix or precip~.tatio~ with a reagent m~y be utiliz~d for.the. 8tep of ~epar~tinq the protected, uncharged, p~p~d~ product.from t~è:.nqusous product phase. .~ho: -use of membr~or contactors in con~unct~on with reverse o~mosi~, a~ describod in ExAmples 14 and 17, will cause the desir~d displacement of the proteo~ynthetic equilibrium without affecting the Xinetics of peptide 3ynthesis in the enzymo reactor. Th~ 8 approach is considered as a viable alternative to ~he u~e of a ~econd enzyme for the purpo~e of driving the proteo~ynthe~i3 to completion. Its usefulnoss, however, is dictated in practice by - . . . , :

, WO ~/l~3 2C~75~ PCT/VS90/02~88 ` -23-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, ma~ing the transfer from oil to water the rate-limiting ~tep of the process.
This phenomenon is illustrated in Example 16, and Figure 21, where the rate of release of the peptide N-formyl-(~-methyl)-asp-phe-O-~ from the oil phase into the aqueous product pha~e, called here Vperm, i8 increased by a factor of two when the aqueou~ phase contains the enzyme aminoacylase able to convert that peptide into the more hydrophilic N-formyl-(~-methyl)-a~p -phe-OH.
The selection of appropriate deprotection reagent(s) is determined by the chemical nature of the protecting group~ used on the. renctants, such as, N-protected-L-aspartic acid, and as _ indicated above the choice of protectinq group~ i5 in turn dictated by structuraL constraints imposed by the active site o the condenaing enzyme;.
- In ge~eral, the condensing e~2yme~ us~ul in the practice of the prssent invention are proteolytic enzymes, sometimes called proteases, which may be divided lnto two groups. The more commonly used are endopeptidases, which only cleave inner linkages, and exopeptidaJ-~, which preferably cleave terminal linkagos. Useful ~nzy~es includo-~rine proteinase3, tFC 3.4.21) such a~ chymotrypsin, trypsln, subtili3in BNP' and Achromobacter proteaQe; thiol protelnases (EC 3.4.22), ~uch ac, papain;
carboxyl proteinases (EC 3.4 23), such as, pepsi~; and metalloproteina~es ~EC 3.4.24), 8UC~ ~, therm~lysin, prolisin, Tacynasen N ~St. cacaDito~u~) nd ~ispase. Binding of the enzym~ to ins~ubLe ~upports, foIlow~ng procedures well k~own to practitioners of the art, may be incorporated to the practice of this i~vention; and althouqh bindinq the enzymes is not an es~ential step, it may be tesirable in cortain applications.
Among the many protoases potontially useful for the practice of this invention, thormolysin IE.C. 3.4.24] i8 the condensinq enzyme most preferred because of its remarkable thermostability, wide availability, low cost and broad useful p~ range between .. .. . . .
~ : . , ~ - : . -' '' "' ; ~ , ,, .... , . . ~.. ,, . ,., . ,~ ,"

W090/1~3 PCT/USgO/02188 about S.O to 9Ø Other preferred pro~eases include pep~in and penicillopep~in [T. Hofmann and, ~. Shaw, Biochim. Bio~hys. A~ta 92 543 (1964)] and, the thermo~table protease from Penicillium dunontl lS. Emi, D. V. Myers and G. A. Iacobucci, Biochemistr~
15, 842 (1976)~. They would be expected to function at about pH
4.5 or below, exhibit good stability at ~uch pHs, and do not require the pre~ence of Zn++ and Ca++ ions to maintain their activity.
The practical realization of enantiomeric selectivity that is, in the ca~e de~cribed above, production of only the L,L
i30mer of the peptide C, i8 directly related to the enzyme ~elected, optimal functioning of the membrane, chemical nature of the support material and pH o~ the aguoous reaction phase.
One preferred membrane for practicing the above-deccribed specific method i~ a microporous polypropylene support material including a mixture of iso-hexadecanol and n-dodecane immobilized theroi~. Thio membrane is available from Bend Research, }nc., 64550 Re~earch Road, Bend, Oregon 9770l, U.S.A. under the tradename/de~gnatiQc ~Type 1 HolLo~:F~ber Selective Dialy~i~
Membrane" and i~ preferr~ with the re~ctions of Example3 1-4.
Another preferred mem~rane utilize~ Colgard Typ0 2400 polypropylene hollow fiber3 (Colgard is a regi~tered trademark of the Celanese Corpor~tion,. l2ll Avenue o.the Americas, New York, N.Y. 10036. Colgard ca~ b~ obtained fro~ C~l~nese Fiber~
Marketing Corporation, Charlotto, N.C.) a~ the microporous matorial-Jupporting a mixture of 30~ v/v N,N-diothyl-dodecanamide in dodecane a~ tho water-immiscible organlc.1lguid ILM. ~hi~ .
membrane wa~ obtained fro~ Bend:Rssearch, Inc., 64550 Re~e~rch Road, Bend, Oregon 97701, U.S.A..und~r the tradenRme/de d ~nation ~Typo 2 HollJY~Fib-r Selective DiaIy-~s Me~brane~ and i~ I
proferrod with th reaction~ of Exi~plo~ 5-9. Celgard Type 2400 ¦
polypropylene hollow fibers having a pore size of 0.025 - 0.050 ~m and a wall thickness of 2S ~m were utilized in the Type 2 Hollow Fiber Selective D~alysis Mom~rano of Bend ~eJearch, Inc.
When operated at pH~ of about 5.5, these membrane~ exhibit a high 3electivity, for example, whon practicing the proee~s of Figure l, selectivity in the rango of about 500:l (w/w) in favor 1 ... . . . .. .
. . ; .
. -. ' ' " ' .
. . ' WO ~/12~3 ~ 75~ PCT/US~/021 of the uncharged peptide specie~ have been measured. That i8, 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 li8t8 a series of selected combinations of protecting groups and deprotection conditions useful in connection with the practice of this invention.

. . .

' - ' '~ .

-26- ~

ABLE I~ PROTECTING GROUPS AND DEPROT~CTION REAGENTS
USEFUL IN THE SYNTHESIS OF ASPARTAME (APM).
I
I

Rl R2 R3 Group Removed/Rea~ent Product I H209cH2oco- C~30- Rl, R2/(Pd)H2 APM
~CH20 -CH = C~30 R2/H30 B-benzyl-APM
~CH20-CH = o CH30- R3/Fungal e~terase N-formyl-3-benzyl-L-aspar~yl-L
phenylalanine ter-BuO -CR = o CH30- Rl,R2/H30 APM
CH30- -CE = o CH30- R3/fungal o~terase N-formyl-isoAPM
C~30 -C~ = 0- l-Pro-O- R3/ungal ~terase . . ~-fD y l-isoAP~
CH30 -CH = C 3o R3/-chymotrypsin N-formyl-~ oAPM
NH2 ~CH20CO- CH30- Rl/asparaginase N-CBZ-APM
L-dihydroorotyl-L- Rl,R2/hydantoinaffe APM
phenylalanine , -methyl ester H ~CH20CO- ~ O- R /OH N-CBZ-APM
CH30-- 1 ~ OCO- ~ O R~/fungal e tera~e N-CBZ-iso APM
CH30- ~CR2CO- ~ O- R2/poD~ctllin acyl~se . AP~-B-methyle~t As a result of the enantio~elactivity o~ select~d condensatlon enzymos and the functional discrimination exorted by the membrane, the practice of the invention using N-formyl-L-aspartyl-B-bonzyl ester and D,L-phenylaianine methyl estor could achieve a 99.8% enantiomeric resolution of the racemic phenylalanine methyl ester reactant, the L-enantiomer .. ..

WO ~/1~3 -27- 2~5~75~ PCTtUS~/02188 appearing as N-formyl-L-aspartyl(B-benzyl)-L- phenylalanine methyl ester, an a~partame 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 nece~sary step for processes employing racemic amino acid reactants, e.g., the '311 process described above.
The economic advantagec 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 i5 made posible by the ln situ optical resolution of the D,L-phenylalanine methyl ester due to: (a) the enantio3electivity of the condensing enzyme; and (b) the hiqh selec ff ~ity of the membrane in favor- of the uncharged specieQ.
Preferred methods for the low-cost synthesis of racemic phenylalantne are those,based on the utiLization of benzaldehyde via 5-benzalhydantoin sometimes called ~he Grace procoss, or th~
catalytic carbonylation of benzyl chloride to phenylpyruvic acid,-a procedure developed by Sagami Chemical ReQearch Center, ToXyo, Japan (sometimes referred to as the Toyo Soda proce~s of Toyo Soda Manufacturing Co., Ltd., Yamaguchi, Japan~.
It will be appreciat~d by those skilled in th~ art that the practice of thi8 invention is not neces~arily restricted to the synthesi~ of peptide ~weetener~, ~uch a~, aspartamo or its analogs and dorivatives. The invention may also be used for the synthesis of othor useul peptides, di-, t~i~ tra- and pentapeptides, or peptides of hiqher complexity, that..~re capable of diffusinq through a permsel~ctive membrane at a re~sonable rate. For e~a~e, considerinq th~ bond specificity of thermoly~in, and ssuming the pres~n~e of only one thormolycin sensitive bond in the product (indicated by the arrow in t~e formula below), one could synthesize met-enkephalin (1) by following scheme: -WO90~ 3 . . . PCT/US90/021~
-2~- .
(Bzl) Z~7S~

(BOC)-tyr-gly-gly-OH ~ H-phe-met-(Oter-Bu~
~ Thermoly3in pH 5.5 (~zl ) (BOC)-tyr-gly-gly-phe-met-(Oter-Bu) ~ H30 H-tyr-gly-gly-phe-met-OH (1) The fea~ibility of a ~tepw~se total enzymatic synthesi~ of met-enkephalin using s-chymotrypsin and papain ha~ been documented in the literature. See: W. Kullmann, J. Biol. Chem 255, 8234 (1980~.
Another potentially u~eful application of the pre~ent in~ent~on i~ in th~-e~zymatic ~ynthe8i8 of Gr~mic~dL~ 5 ~2) by the follo~ing sch~mo:

. .. -. . .~ , ' : .

....
... . . . .

',',''''"'''' ' ''"', ~ ' ' . ' ' .'''' '. ' WO ~/l2883 PCT/US~J021 ~_ -29-2~75~
, ` 2 H-val-orn(CBZ)-leu-D-Phe-pro-OH
~ t thermoly~in p~ 5.5 leu (CBZ)orn D-phe val pro , pro val D-phe orn(CBZ) leu Pd/E~2 leu orn D-phe val pro pr~o ~ val D-pho orn (2) leu Various other examples of the enzymatic synthesis of useful peptide products where the pre~ent invention may find application and describQd in ~h~ literature are the synthesiR of angiotensin, substance P, d edoi~in, casrulein and l~u-en~epha~in. t Ano,ther u~ful clasa of peptid~ compound~ amenable to-proteosynthesi~ ~çcording to the prcsent invention are the ~-lactam antibiotics. This group comprises the well-known penicillinR and cephalosporins, that are characterized by the presence of a ~-lactam function in their chemical ~tructure.
The high reactivity of the B-lactam function has made the chemical synthe0i~ of tho0e antibiotics difficult, and is re~pon~ible for their poor stabiltty in aqueous solutions in the presonce o nucleophiles. The enzyme B-lactamase specifically - - ., . , . - . . .- , . ........................... : .

:'" :.: ' , - : , . ~ ' ., ' : :,... : , WO ~t1~3 -30- ZC~`~7~ PCT/US~102]~

catalyzes the hydrolysis of the amide bond in the ~-lactam ring of penicillins and cephalosporins. Nathan Citri, "Penicillinase and other B-Lactamases", in Paul D. Boyer (Ed.), "The Enzymes,"
Volume IV, 3rd Edition, Academic Press, New York 1971.
The pre~ence of ~-lactamases in the microbial world, particularly in Gram-negative bacteria, is con~idered a defensive mechanism against B-lactam antibiotics. The facile induction of t this enzyme with penicillin haa made possible the production of ~-lactamase (penicillin 3-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 ~-lactamases," in A. H. Rose (Editor), "Microbial Enzymes and Bioconversions," Chapter 9, pp. 504-522, Academic Press, New York, 1980.
B-Lactamase may be used as a rever~e hydrolase for the synthesis af a penicillin methyl c~ter B from the corresponding penicilloic acid A, according with the teachin~ of the present invention, the unch~rged _ bein~ sel~ctively transporte~ o~er A
across a hydrophobic uentbrane, andl ~urther made non-permeab~ hy the action of an esteraRe to ~ive the polar penicillin C.

W090~12883 -31- 2~5~75~ PCTIUS90/021~

~-lactamase A (char~ed) B (uncharged) e~terase (charged) .
T~i approach couid facilita~ the synthesi~ of penicillins from penicilloic acid precursor~, ~bat are p~r se amenable to total ~ynthe~is. J. C. Shoehan and K. R. Henery-Logan, J. Am.
Chem. Soc. 84 ! 2983 (1962).
Tho~e skilled in thQ art wlll appreciate that the preaent invention nay a1JQ iD~L~pplicat~o~ t~r compo~nd other than peptide~, poptide like compounds nnd eguiv~lents thereof.
For example, the use of oster hydrolases liXe pig liver esterase lEC 3.1.1.1] for th~ asymmetr~c resolution of prochiral dlcarbo~ylic~acid diester~ o c~iral mon~cters-is w~ll known 1C.J. Sih et al., Ann N.Y. Acad. Sci. 471, 239 ~19~6)1, the text of which is incorporatod here~in by reference.- The s~me enzyme can be u~d in the fashion de~crlbed in this invention ~or-the resolution of racemic carboxylic acid compounds, through the selective tran~port of a chiral ester through the membrane and the ret~ntion of the non-reactive enantiomeric acid in the reQction phase.
Th- pr~sent invention utilizeJ ~tandard biochemical terminology as follows: "H-val-n indicates that valine is the :
~.

: ~

WO ~/1~3 z ~ ~ ~ ~ US~/O~t~
-32- ~

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 "Vsyn" indicates the average rate of synthesis and "Vperm" indicatec the average rate of permeation.
The following detailed Examples are pre~ented to further illustrate the pre~ent invention.

Example l.
An aspartame derivative was prepared in accordance with the present invention as follows:
To a solution containing 5.02g (20 mmoles) N-formyl-L-a~partyl-B-benzyle~ter, 4.3l g (20 mmole~) L-phenylalanine methyl ester in lOO ~ in water, adjusted to pH

5.5, wa~ added 500 mg thermolyri~ enzyme (Daiwa Che~. Co., OM~a, Japan) represen~; n~ a total o a x loS proteolytic unit~.
~ he resultin~-clear reaction mixtur~ ua~ incu~a~ed o~
15 hr~ at 40C, wh~n the pr~enc~ of in~olubl~ dip~ptido N-formyl-~-benzyl-L-a~partyl-L-phenylalan~ne mothyl e~ter be~am~
apparent. The resulting mixture was then placed in a 200 mL ~`
ve~sel, connected to the reaction ~ide, in this case tube side, of an exporimental hollow-~ibor 3eparator of a Bend Re~earch, Inc. "Type 1 ~ollow Eiher-Sd octivo Di d ysi~ M~2branen that - -~
provided l squar~ foot of m~mbrane area. The product ~ide of the membrane (shell sldo of th~ separator) was connected to a source of aqueous (product) mixt~e (total volume = 2~0 ~L) containlng 500 r4~ of the ~nzy~ Acy~ EC 3.5.I.14) fror AJD~r~illu~ SD.
(Sigma A 2156), at p~.7~5. Thia enzymo, usually d-scrib~d a~ an aminoacy~ac~,~ ~a8 found to functio~ a~-a C-t~rminal ~t~r~ce,-on both N-acotyl- and N-formyl-B-benzyl.-~partame.
The roaction and product mixture~ were circulated at room temperature through the hollow fiber soparator cou~tercurrently at the rato of 600 mL/min, with th~ assi~tanco o~ pori~talti~ , -pUmp8. The configuration of this apparatu3 resembles that illustrated in Figuro 2. Since th~ p~ in both the reaction and ." . ' .' ',., ,,, .' ,. " ~': ' ' ' . ' ' ' ' .', . ' ', . ' WO ~/l~3 PCT/US~/021~
7S~
product mixture~ drop~ as the process prosres~es, constancy of pH
was maintained through the use of pH stat~.
The formation of N-formyl-B-benzyl-L-a~partyl-L- ;
phenylalanine was monitored by HPLC. Chromatographic analysis was conducted on a Tracor Model 995 instrument along with a LDC
Spectromonitor II detector ~et at 254 nm for the detection of the amino acids, fully protected product dipeptide, and dipeptide.
The column u~ed was a NOVA-PAK C18 Radial-Pak cartridge, 8mm x lOcm, housed in a Millipore Waterc RCM-100 Radial Compression Module.
The mobile pha~e u~ed for the detection of the fully protected dipeptide wa3 a v/v mixture of 4S% methanol (HPLC
grade) 5% tetrahydrofuran (HPLC grade); and 50% of a 1% KH2P04 bufer solution. For the detection of the product dipeptide, the : mobil- phase consi~ted of a v/v mi~ture of 40X methanol and 60%
of a 1% KH2P04 buffer ~olution (1 mL of triethylamine per liter solvent was added to minimize ta$1ing and the p~ was adju~ted ~ down to 4.3 using 80X phosphoric ac~). The flQw rate was kept at 1 mL/minute_l The HPLC data r~lating to formation of N-formyl-3-benzyl-L-aspartyl-L-phenylalanine i~ summarized in Table I$ below, and iB exprossed a~ the total amount (mg) of product dipeptide accumulated $n the product ~olution as function of time.
The value o the uncharged dipeptide concontration at eguilibrium which corresponds to its saturation point in water at pH 5.5, wa~ found to be about 0.05X at 25C. The ~uount of uncharged dipoptide transported pffr square foot of membrane per hour was found to be about 200 ~qr indicating that maintenance of the equilibrium.r~quired di~solu ff on of ~nsoluble dipoptido and/or dipeptide ~ynthesis do novo. Almost complete dissoLutio~
of the in~oluble uncharged phase dipeptlde in the reaction mixture was ob~ervod after about 5 hr~, when the reaction was ~topped. A plot of the data of Table II i8 shown in Figure 3.
The linear function indic~tes that the transfer of peptide acro~s the membrane proceeds at a steady ~tate. The observed rate o formation of product dipeptide o about 200 mg/ft2/hr (Table II) .:: : . . . . :
: . . ~ .: :: , .
- : - . . . . . : . .
. .
.
- - . . . ~
. .. , . . .,: : -WO 90/12883 PCr/US90/0 Z~7S~
was confirmed similar to the 1ux of uncharged dlpeptlde acro~s the membrane (190 mg/ft2/hr) measured at 0.05% in water a~ was anticipated on theoretical grounds.
At thi~ point the described ~ystem would be expected to continue in ~teady 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'~ ~electivity the intercalation of a second membrane in ~eries with the first membrane before the contact with the second enzym~ may be necessary because the ~electivity acros~ a single membrane i8 lower due to the high amino acid concentration in the reaction solution.
The product ~olution (200 mL) was recovered, ad~ust2d to p~
2.5 and cooled at 4C overnight. The procipitate collecte~ was r~covered and recrystallized from MeOH: ~ O to g~vo 307 mq o N-for~yl-B-konzyL-L-a~partyl-L-phQnyl~lan~c ~125 =
-5.6~C=t.2; EtO~, identical (IR, 13C-N~R) to an auth A~C, sample prepared by the batch hydroly~is o~
N-formyl-B-benzyl-aspartamo with Asperqillus esterase, 1~]2 -5.3(C=1.3; EtO~).
Table I r .

Time (~in) Amount ~e~ t~eroduct solution (ma) 180 6~
240 88Z::

~xamDle 2.
An experiment ~imilar to that of Example 1 wa~ conducted, except for tho u~ of 8.63g D,L-phenylalanine methyl ester lnste~d of the L-enantiomer. The r0sults are summarized in Table III. Isolation of the uncharged peptide from the 200 mL of produ~t ~olution gave 310 mg of product, [al2D = -6.4(C=1.4;

-. . ~ ., .

: . .
... . .

Wo ~/1~3 PCT/US~021~
2~75q~
EtOH), identical in all re~pects (IR, 13C-NMR) to an authentic sample of N-formyl-~-benzyl-L-aspartyl-L-phenylalanine.

Table III.

Time (min) Amount ~e~tide/~roduct 301ution ~ma~

A plot of the data summarized in ~able III (Figure 3) again showed the exi~tence of a steady state proce~s when the reactor ~a~ ~perated with D,L-phenylalanine methyl ester. The stereospocificity of thermolysin i8 demonstrated by the exclu~ive formation o the sa~e L,L-dipeptide described ~n Example 1. T~e D-phenylalanine methyl e-~ter retained in the tube phase-~reaction mixture) did not inhibit the overall ~inetics of peptide-format~on. ~ -.
Exam~le 3.
A mixture of 1.0 g N-formyl-B-benzyl-L-aspartyl-L-phenylalanine, prepare~ in accordance with ED~rple-~L 4.0 mL
water, 4.0 mL tetrahydrofuran, and 1.0 mL conc. hydrochloric acid (12N) was heatod at re~lux for 9 hro. The mixture was then cooled and the pH adjusted to 4.0 wit~ ~0% NaO~ solution. The tetrahydrofur~n was then rcmoved by evaporation at < 35 ~nd 20~
mm ~g. Cryst~llization was compl~ted by ~torage at 5C for 1 hr, the ~ample thQ~ filtorod, wa~hed wtt~ l m~ i~n water, and dried in vacuo to give 367 mg of whlto solld. Thi5 material was identical to an n~thentic sample of aspartyl phenylalanine by HæLC and IR comparison. ~125 = l12 (C = 0.5; 0.1 ~ HCl in 50~ -MeO~).
AJpartyl phenylalanine has been converted to a~partame 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.
.

.
: : . - . . . .
- - , .: . ' ~ . - ' WO90/1~ PCT/USgO/021~
-36- Z~r~75~

Exam~le 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 B-methyl e~ter and 4.38g (20.3 mmoles) L-phenylalanine methyl ester a~ reactants. The amino acids were dissolved in 100 mL water, the pH of the solution adjusted to 5.5, and 500 mg of thermoly~in Daiwa (8 x 105 proteolytic units) was added. The solution was preincubated for 15 hours at 40C, when a 3ubstantial amount of N-CBZ-(B-methyl ester)-L-asp-L-phenylalanine methyl ester wa~ precipitated. The suspension was connected to the tube side of a "Type 1 Hollow Fiber Selective Dialysis Membrane" (Bend Research Inc.) containing 1 ~t of membrane surface, and the machine was operated at room temperature for S hours against a ~hell side pha~e-of 200 mL water containing 500 mg Acylase I (Sigma) at pH 7.5. The accumulation of peptide product in shell phase *a~
moD~tored by H~LC, und the resuLt~ are reproduced in Table IV an~
Fig~r~ 4.
After 5 hours run the reactio~ ~ ~t~pped, the ~hell ~d~
phase t200 mL) was:recovered, ad~usted to pR 2.5, and stored overnight at 4C. The product precipitated was collected and recrystallized from CH30H:H20 to yield 405 mg (86% recovery) of N-C Z-~-methyl eater)-L-aap- L-phenylal~n~n~ (N-CBZ-iso-APM) lal D = ~6.0 (C = l.I, EtO~, fdentica~ tl3c~ to an - ' authcntic sample o N-CB2-iso-APM, [~]2D = ~5-5 (C = 1.1, EtOH), prepared by chemical coupling and partial esterolysis with Acylas~ I.
... . . .
Table IV, Tim~_lm~n) Amount Destide/Droduct solution (~g) - , .
- :
. .

WO ~tl~3 PCT/US90/02t88 -37- 2 ~ 5 ~ 7 5 g As observed before in the experiments of Examples l and 2, the plot of Figure 4 indicates that the a~cumulation of jN-CBZ-lso-APM proceeded at a steady rate of about 200 mg/hr.
t '` The conversion of N-CBZ-iso-APM to APM can be practiced under the conditions described in Example 3.

ExamDle 5.
~ he aspartame derivative, N-formyl-(B-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-(~-methyl)-L-aspartic acid, lO.OO g (48 mmole~) L-phenyl alanine methyl ester in lOO mL water, adjusted to pH 7.0, was added 770 mg thermolysin enzyme (Daiwa Chemical Co., O~aka, Japan) representing a total of 1.2 x 106 proteolytic units. The resulting clear solution was incubated for 1 hr at 40C, wh~
HPLC analysis indicated the presence of 4~3.8 mg of ^ N-for~yl-(B-~thyl)-asp-phe-OMe. The solution was coo~od to 25C, the pX adjusted to 5.0, and the solution placed in a 200 mL
vessel connected to th~ tube side of hollow-fiber ~eparator ("Type 2 Hollow Fiber Selective Dialysi 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 ~eparator was co~nected to the product vessel containing 500 mg of the enzyme Acylas~ E~ 3.5.l.14) fro~ AsDerq~llus SD.
- (Aminoacylase AMANO, Nagoya, Japan), at pH 7.5.
The two phases were circulated at 25C countercurrently, at the rates of 50 mL/min..~tube phase) and 500 mL/~in. (shell pha~e), with the a~i~tance of t~ peristaltic pump~ using the configuration ~llu~tr~ted in Figur~ 2. Constancy of pH in both phases was &~cur~d through the u~e of pH sta~;
The formation of N-formyl-(B-m~thyl)-a~p-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 lOO mm, hou~ed in a Millipore Waters RCM-lOO Radial Compression Module.

,:

WOgn/~2883 PCT/US~/02~
-38- 2~75 The mobile phases used for the analy~iR were:

(a) for N-formyl-(B-methyl)-asp-phe-OMe: 40% v/v methanol in 0.1% KH2P04 buffe~ pH 4.6;

(b) for N-formyl (~-methyl)-a~p-phe-O~: 20% v/v methanol in 0.1% KH2P04 pH 4.6; flow rates used were 1 mL/min for both analy~es.

The data relating to the formation of N-formyl-(B-methyl)-asp-phe-OH (product dipeptide) is reproduced in Table V below, and iq expressed a~ the total amount (mg) accumulated in the 200 mL shell pha3e as a function of time.

Table V

Time /Min) Amount D~pt~do~product_solution (m~L

12~ 230 1~0 280 A plot of thc d~ta of ~bl~ V is shou~ ~ Fig~re 5.
At the end of tho run, ~PLC analy~is indicated the presenc~
of 283 mg of N-for~yl-(~-methyl)-asp-phe-OMe in the tube phase.
The~e valuo~ permitt~d calculation of the amount of peptide synth~J~z~d during thc-op~ratlon-of the reactor.

., , . .. .. - , ' :: :. : :,' WO ~/1~ PCT/US~tO21 ~5~5~ 1 Ptr = peptide transferred into shell phase - 400. 336 =
417.4 mg; 322 PO = initial peptide in tube phase = 433.8 mg;

PT = peptide remaining in tube phase at the end of the run =
283 mg;

Ptr (Po PT) P8yn;
syn Ptr (Po ~ PT~ = 266.6 mg; and V8yn = 266.6 = 53.5 mg/hr. 100 mL.

, The Vsyn of 53.5 mg/hr. 100 mL coincide~ with the rate of ~ynthe~is ~SOO mg/hr. L) measurc~ for the forward velocity in equilibration ~t~lA~ es done with N-for~yl-(~-methyl)-~-aspartic acid and L-phenylalanin~ m~thyl ester in the pre3ence of- -thermoly~in.
The product ~olution (200 mL) was recovered, adjusted to pH 2 with 1 N HCl and extracted twice w~th 20D.mL EtOAc. The combined e~tract~ b~ft a ~b~te rssidue upon evapDration; that -after recrystallization from EtOAc~hexane yielded 100 mg of N-formyl-(~-methyl)-L-a~p-L-phe-OR, la]25 = +0.70 c, 0.29;MeOH), identicaL ~IR, C-NMR) to an authentic ~ample prepared by the b tch hydroly~i~ of ~ynthet~c N-formyl-(B-methyl)-L-asp-L-phQ-QM~ with AsDerqillus esterase, [a] D = ~0.80 (c, 0.2~j MeOH).

Exam~le 6.
The e~periment described in Example 5 was scaled up in a Type 2 Hollow fiber Dialysi~ Membrane (Bend Research Inc.3, containing 1 ft2 of liquid mambrane (30% v/v N,N-diethyl-dode,canamide in dodecane). The tube pha~e contained 40 g L-phe-OMe, 24 g N-formyl-(~-methylj-L-a-~p and 3.08 g thermolysin WO90/~3 PCT/US~02 2~ _ A 75 Daiwa (a total of 5 x lO proteolytic units) in 400 ml water, adjusted to pH 7Ø After an incubation period of 1 hr at 40C, the amount of 1,068 g ~2.7 g/L) of N-formyl-(B-methyl)-L-asp-L-phe-OMe was found to be present. The solution was cooled to 25C, adjusted to pH 5.0 with l N HCl, and connected to the tube side of the hollow fiber separator. The shell pha~e was made of 400 mL water, p~ 7.5, containing 2 g aminoacylase I (Amano). The two phases were circulated countercurrently at 25C for 5 hrs., as de~cribed in Example 5.
The results are summarized in Table VI and Fi~ure 6.

Table VI

Time (min) Amount De~tide~Droduct ~olution (mq) 300 a43 .
At the end of thc run, the amount-of N-formyl- :
(B-methyl)-asp-phe-OMe remaining in tube phaqe was 586 mg.
The highe~t transport value observed (404 mg/hr) during the first 30 mi~. ia the re~ult of the hi~ i~itial peptide concentrat~on produc~d d~r~n~ the pr~incub~t~on period.
Departure from equilibrium cau~ed by the transport of peptide set in tho synthesis of mor~ peptide, thu3 establishing a steady state condition after the irat hour into the run, at the exp~cted level of 200 m~hr ~Figuro 6).

ExamDle 7.
An exp~rimDnt ~imilar to that of Exa~ple 5 was conducted, except for the use of 10.00 g of D,L-phenylalanine mothyl estor instoad of the L-enantiomer. The re~ults, pro~entod in Table VII
and Figure 7, clearly show that the ob~erv0d rate of formation of N-formyl-(B-methyl)-L-asp-L-phe-OH was one-half of that seen with L-phe-OM~ (Example 5, Table ~ 9 expected from the - . .

WO ~/1~3 -41- 2 ~ ~ ~ 7 5 ~PCT/US~/o21 enantioselectivity of thermoly~in and the prior result~ of Examples 1 and 2.

Table VII

Time (min) Amount ~e~tide/Product_aolution (ma) 10.6 25.5 120 33.2 180 55.3 240 59.6 300 69.1 Exam~le a .
Membrane-a~si~ted enzymatic resolution of D,L-phenylalanine methyL e~ter w-s utilized in accordance with the preYent invention as follows: To a solutlon of 1.O g (5.6 mmoles) of D,L-phenylaL~nine methyl ester in loO mL water, pH 7 5, was added..
500 mg of a~inoacylase I (Amano Ph~rrnceutic~l Co., Nagoya,:
~ Japan). The mixture was aLlowed to r~act at 25C for 30 m~n., at the end of which the presence of 266 mg L-phe-O~ (1.6 mmole~) and 712 m~ D,L-phe-OMe (4 mmoles) was ob~erved by HPLC analy~is.
Thi8 solution was fed to the tube (reaction) side of a hollow-fiber Colgard suppQctod ILM s~parator (nType 2 Hollow Fiber Selective D~a~ysi.~.Membrane", ~end R~carc~ lnc.) containing 0.5 ft of a 30% v/v N,N-diethyl-dodocanamide in dodecane liguid film in it. The product side of tho mombrane (~hell side of the ~ep~rator) was illed with 20C..mL water ad~usted to pH 2.0 with d~luted~ HCl. The two phases were clrculated t~rou~h the separator countercurrently at the rate of :
200 mL/min., w~th the assistance of peristaltic pumps (Figure 2).
The 3eparati~n proceeded through the continuous addition o~ --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 m~ of a 7% ~olution of D,L-phe-OMe (2.1 9; 11.7 mmole~) i~ water pH 7.5 was added in a period of 2 hr#. The pH of both pha~e~ wa~ kept con3tant by the use of pH otats.

'':. , : . ,, ,. . :' ~. :'.................. ':' .:, : .: . :' .. : ., : .: .,: . . ............................ ......: - .:
- . .. . : : . : .

WO ~/1~3 ~ PCTt~S~/02~
Z~ 75~
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 pha~e used in this case was 20% v/v methanol in 0.1%
KH2P04 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 o L-phe-OH in tube phase and of D-phe-OMe in shell pha~e. At the end of the experiment both phases were recovered and worked out a3 follows:

(a) Tube phase: The contents (130 mL) were adjusted to pH 8.5 with lN NaOH, then extracted with 2 x 50 mL EtOAc.
The aqueous phase was then adjusted to pH 4.0 with lN HCl and the ~olution pa3sed throu~h a 2.5 x 20 cm Dowex 50 (NH4) coll~mn. Aftor washing with 200 mL water, the product was eluted with 200 mL of 10% M~40~. Th~ eluate was - concentratod to 50 mL in ~acuo, an~ the ~olution fre~ze-dri~d. Yield: 249 ~y, white aolid, -:
¦ ]25= _2g.2- (c, 2; ~2)~ lit.
(Aldrich) 1~]~5 = -35.0 (c,2;~20), opticàl purity 92~.

(b) Shell Dha~e: (200 mL) was adjusted to pX 8.5 with dilut~d NaOH and extracted with 2 x ~0 mL EtOAc. The organic e~tract ~a~ dried ovor nnh; Na2S04; ~v~porated to dryno~s, di~solved in 50 mL water acidified to pH 3.0 w~th lN ~Cl and then freeze-dried, to yield 989 mg (4.6 mmole) of D-phe-OMo. HCl, whlto solid, ¦a] 25 = -21 0 (c 2 EtOH) lit. (Aldrich) l-l Zs = -32.4 (c, 2; EtO~, opff cal purity 83Z.

Basod on tho pormeability v~lue of 32 mg/cm2. min found for phe-OMe (water, pH 8, 25C) on this mcmbrane, the expected flux for a membrane area of 450 cm2 (0.5ft2) was 880 mg/hr.
Table VIII show3 that the amount of phe-OMe transferred into the shell pha~e at the end of the first hour wa~ 860 mg; ~ugges~ng that the transport was operating under membrane-limiting conditions.

, ', '- : ' ' ' ', -.

, .
:: .

WO ~/12883 PCT/US~/021 Table VIII 2~75~
. _ D,L-phe-OMe Tube Phaqe Shell Phase _ added (~) L-~he-O~(mq) D,L-phe-OMe (ma) D-Phe-OMe (m~) 1 0 266 617 (O min) 2.4 615 531 860 (60 min) 3.1 743 628 1154 (90 min) Batch resolution of D,L-phe-OMe through the enantio~elective hydrolysis of the methyl ester function catalyzed by subtilisin A
(a serine-type alkaline protease) has been recently disclosed IShui-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
hydrol~e which is a protease having esterolytic activity ~uch as aminoacylase I, ~-chymotrypsin and subtilisin A can be utiliz~d to resolve a D,L-amino acid compound~ such as D,L-phenylalanine methyl e~ter. Al~o, the membrane--noi~ted proces~ of the preaent inventlon-can be practiced by sub~tituting 3ubtilisin A or : -3-chymotrypsin ~or the preferred aminoacylase I. Aminoacylase I
is generally preferred for the eJterolysis of peptide~ over other esterolytic enzyme~ such as subtilisin A and ~-chymotrypsin also having endDproteo~ytic act~vities.
If the above de~cribed resolution o D,L-phenylalanino methyl eJter 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 reso~ution of oth~r racemic carbosylic~acid esters. Eor oxamp~e, a racemic carboxylic acid o-ter compound in.an a~ueous reaction mixture including a hydrolyzing enzyme can be hydrolyzed to form a charged enantiomeric compound and an uncharged enantiomeric ester compound in the aqueous roaction mixture. The uncharged enantiomeric e~ter compound i~ then transported from the aqueous reaction mixture acro~s an ion re~ection membrane of the present invention including Type 1 or Type 2 Hollow Fiber Selective I', .. . . . . . .
- . , . . . . :, . . .. . . , , . :

,: , - - ' . ... ,' . ,' , ' : ., ,., . , ; , : , , - . , . ,, ' : . : ~: , . .. . . .
' . . , ~ ' :
.. . . ~ . : . : .

WO90/l~3 PCT/US~/021 Z~7S~
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 i~ 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 3killed in the art of the present invention.
Continuous or `oatch processing means are provided by this Example in that the desired enantiomer of the reactant can be produced and added to the reaction mixture.

ExamDle 9 .
Synthesis of N-formyl-(B-methyl)-L-asp-L-phe-OH in accçrdnnce with the present invention was ccnducted utilizing immobilized aminoacyIase I and ion-e~change resin~ for the removal of permeable products ais follows: To a solution o 10.0 g (48 mmole,s~, L-pheny~alanine methyl eater ~nd 6.0 g ~35 mmoL~s~ ~
N-formyl-(B-methyl~-L-aspartic acid ln lQO L deionized wator~
adi,iuJted to pH 7.0, was added 770 mg of thermolysin (E1) (Daiwa Chemical Company, Osaka, Japan) representing a total of 1.2 x 106 proteolytic units. The resulting solution was incubated for 1 hr. at 40 C, when HPLC unalysis 1ndicated th~ pres~nce c,f 383 mg of N-formyl-(3-methyl)-a~p-phe-OMe. Th~ ~olut~oc u~ c~,oled to 25C, the pH ad~usted to 5.0, and the solution was placed in a 200 mL ves-el 40 connected to the tube ~ide 46 of a hollow fiber 3eparator 44 ("Type 2 ~ollow Eiber Selective, Di~lyais Membrane~, 3end Res~ rch, Inc.) contain~ng 900 cm2 (1 ft2) of a hydrophobic liquid memhr~n~-~ade of 30X N,N-di~,t~yl- dodecanamlde in dodecane. T~ lI side of the separ~tor ~8 was arranged as a closs,d circuit mado of a ~-rie~ of connectin~.ve,~sels illustrated in Figuro 9. The solution returning to the ~eparator 44 wa3 adjusted to pH 4.0 in order to protonate the L-phe-OMe copermeating with the dipoptide N-formyl-(~-methyl)-a~p-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 adt,usted to pH 7.0 and submitted to i .
. . . .

WO ~/12~3 PCT/US~/021 ~45~ 2 ~ ~ ~ 7 S ~
the action of the aminoacyla~e I (E2), immobilized over DEAE-Sephadex 52 IT. To~a, T. Mori and I. Chibata, Ar. 8iol.
Chem.~`33,~rlO53 t1969)]. The re~ulting dipeptide N-formyl-(B-methyl)-asp-phe-OH wa negatively charged at that pH, and was subcequently 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 glOO mL) and 3hell 48 (500 mL) phases were circulated at 25 C countercurrently through the membrane separator, at the rates of 50 mL/min . ( tube phase~ and 120 mL/min. (shell phase).
Periodic ~ampling of the shell phase was done on the effluent from the second enzyme (E2), (Fi~ure 9, sampling port 56, before entering the Dowex l (Cl-) column 54) and the samples monitored by HPLC following the procedures described in Example 5. As expected, at this point of the circuit tFig~r~ g) no L-phe-OH that could result from t~o enzymatic hydrolysis of L-phe-Q~ by E2 (see Exa~ple 8) was det te`~; only a circulating steady-~tate level of N-formyl-~-methyr)---p-phe-OR (aver~
concentration: 54 mg/L~ was ob~crved, reflecting ~he continuous transfer of the dipeptide N-formyl-(B-methyl)-APM across the membrane and its subsequent hydrolysis by E2. The efficient trapping of the charged dipeptide by the Dowex-l resin is indicated by the.lQw c~nsentratiQn of ~t:observed at the'~column effluent 57 throughout the run.
A similar parallel experiment performed in the absence of Dowex-l ~howed the rapid accumulation of N-formyl-(~-m~thyl)-aJp-phe-OH.~ the dbeIl phaoe, ~ could be anticipated from the results disc~sed ~bove. Ag~in, no L-phe-OH--_ was found in t~r.c~rculating ~hell pha~e: A comparison-of both experiment~ i~ se~n in Figure lO, and Table IX.

, ' , ': , '. ,' ' ' . .

.

WO ~/~3 ~ ~ PCT/US~/021~ ,~

Table IX
2~S~75~
ma N-formYl-~B-methvl)-ass~ e-OH/shell ~hase Time rmin.) EXD. 1 . Dowex l DreYent Ex~.2.without Dowex l.

3~ 31.6 30.2 120 26.0 7l.8 2~0 45'4 83.l 300 51.2 --In addition to ~eparating phenylalanine lower alkyl ester copermeating the ion rejoction membrane into the product mixture utilizing ion exchange reoins as de~cribed in this Example, aspartic acid copermeating the ion rejection membrane into the product mixtur~,can be separat d utilizin4 ~uch resins. The-~pec~s or product:that cannot back-d~ffu~ ~cross the me~hruss from the product-~turo ~an bo r,~oved utillzing suc~ io~
exchang~ resins. Also, other Jqparation mothods known in the art including but not limited to electrophoresi~, electrodialysis and membrano separations which are equivalents of ion exchange resin separations can bo utilized ln the prosent invention.
Immobilizing the cnDdbn4iDg enzy~o alIow~ th~ enzymatic '~ -reaction in the tube phase to be conducted at an initial reaction mixture p~ preferred for optimum efficiency of tho enzymatic reactio~ considering the reactants, product(s) ~nd enzyme inc~uding the de d r~d ~qutlibr~um of the enzy~ti~ react~on.
Option~lly, bhe initial reaction ~$sture p~ in t~e tub~ phaaQ can be rcr~fu~t-d-t~ a soGond reaction ~ture p~ prior to contact' with th~ D~abrane ~o that the ~-cond~reaction mixture pH will maximize the membrano efflciency in transporting the uncharg-d product from the tubo phaso across the membrane into tho ~hell phaJe. Figure 9 doos not show ad~usting the initial reaction mixture pH ln the tl~e phaae to a ~econd reaction mixture p~.
Similarly, the esteraso in the sholl phase can be immobillzod and the pH of the product mixture in the shell phase -: , . ., . . :
,, WO ~12883 PCTtVS90/02188 can be adjuqted and read~usted as nece sary to effect the mo~t efficient processing. Examples 8 and 9 provide additional means for efficient continuous or batch processing utilizing the present invention. In continuouq processing the desired enantiomer of reactants and any copermeating compounds can be returned to the tube phase or reaction mixture.

Exam~le 10.
The dipeptide N-formyl-(~-methyl)-a~p-phe-OH qynthesized in Ex~mple 5 was reacted with L-trp-OMe, in the pre~ence of pepsin, to yield the protected tripeptide N-formyl-(B-methyl)-asp-phe-trp-OMe. After permeation, the protected tripeptide was irreversibly hydrolyzed by the enzyme aminoacylase, to yield N-formyl-(~-methyl)-asp-phe-trp-o~.
To a solution of 2.04g (8 mmoles) L-trp-OMe and 0.484g pepsin in 250 mL O.lM citrate buffer p~ 4 5, was added 0.642g (2.1 mmoles~ N-formyl-(B-methyl)-asp-Fbe-OH dissolved in 25 mL
absolute MaO~, an~ the clear lDX Mk~ solution resulted WaB
incubated at room tomp~r~tur~ for one hour. HPLC analysis at this point indicated the presence of 144 mg/L of the tripeptide N-formyl-~-methyl)-asp-phe-trp-OMe. The solution was then connected to the tub~ side of an experimental hollow-fiber ~eparator (Bend Rese~rch, Inc_~, that proYided 0.5 ft2 (450 cm2) of a ILM made of 50X N,N-diethyldodecanamide in dodecane. The shell side of the soparator was connected to the product vesael containing 0.160g of the. en2yme aminoacyLaae AMANO (AMANO
Pharmaceutical Co., Nagoya, Japan) dis~olv~d in 250 mL 10X MeO~
at pH 6Ø The.two phases were circulated countercurrently at 25C. at the rat~ of 50 mL/min. (tube phase) and 250 mL/~in.
(shell phase) w~th the as~istance of two periotaltic pumps (Figure 2). The shell phase wa~ 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-~-methyl)-asp-phe-O~ in the reaction ves~el (tube side) was kept approximately constant by the continuous addition of this reactant at the rate of O.5 mmole/hr. (0.8g in 5 hrs.), to ... . .

.. . , ~ .. .. ~ . ' . .

WO90~ 3 PCT/US~/021 compen~ate for its rate of permeation at pH 4.5, in an effort to maintain constant the pepsin-catalyzed velocity of proteosynthesis ~ynthesi~ during the run.
The formation of the intermediate N-formyl-(3-methyl)-asp-phe-trp-OMe and it~ product of hydrolysis wa~ monitored by HPLC, using a TRACOR 995 i~ochromatographic pump together with an LDC Spectro Monitor II (1202) detector, set at 254nm. The column used was a NOVA-PAK C18 cartridgQ (8mm I.D. x lOcm) housed in a Millipore Waters ~CM-100 Radial Compression Module. The mobile phases used for the analy i8 were:
A. N-formvl-(~-meth~l)-a~D-Dhe-trD-OMe. A v/v mixture of 40% CH3CN and 60% 0.1% KH2P04 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-~B-methYl)-asD-~he-trD-OH. A v/v mixture of 30% C~3CN and 70% 0.1~ KN2P04 ~u~fer solution conta~n~ng 0.1% triethylamine v/v, ad~usted t~ p~ 4.2, was utilized.
RetQntion: ff m~ wa~ 10.2 mi~ut~r.(l ~L~min. flow rate~ h~
dat~.rolatlnq:to the format~o~ o~ the trip~ptid~
N-formyl-(B-methyl)-a~p-phe-trp-O~ i~ reproduced in Table X
below, and is expre~sed as mg tripeptide accumulated per liter shell pha~e as a function to time.
A plot of thQ data $8 shown i~ Eigur~

Table X
Time N-formyl-(3-methYl)- N-formvl-(3-methYl~-~mi~) asD-Dh~-trD-OMo as~-Dhe-tr~-OH
(mg/L, tub~.ph~e~ ~mg/L, ~hell pha~e) 0 39.6 0.0 2.8 ~ 5.8 120 13.7 180 31.3 240 40.7 300 50.4 42.8 ~ ~ = 10.8 mg ~~ = 42.8 P~ptide synthesized: 10.8 ~ 42.8 = 53.6 mg The hourly averaged rate is 53.6 = 10.7 mg/L. hr., a value - ' '. :

~49~ 2~5~75~

of the same order of magnitude as the average rate of a pep~in-~atalyzed 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-(B-methyl)- asp-phe-trp-OH prepared by chemical synthesis as follows:

A. SYnthesis of N-formYl-(B-methYl)-asp-Dhe-trD-OMe. To a solution of 406 mg (1.6 mmolesj 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(B-methyl)-asp-phe-OH, prepared as indicated in Example 5. The solution wa~ immer~ed 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 b~th, the sQlution wae left overni~ht at room temperature. Ne~t morning, the precipitated dicyclQhexylurea was filtered off, the dioxane evaporatad and the residue dissolved in 200 mL EtOAc, wa~hed with 200 mL 4% citric acid, 5% NaHC03, water and dried over anh.
Na2S04. Removal of the solvent ~ave a colorless residue, that was crystallized from EtOAc/hexane to give 63~ mg (78%
yield~ of Nkormyl-~othyl~-asp-phe-trp-Ohe, m.p.
153-154C.
1~¦22 = -35.4 (c = 0.8, MeOH). Analysis: Calculated for C27H29N407: C, 62.22: ~, 5.5~; ~, 10_7~. Found: C, 61.95; H, 5.85; N, 10.72. 13C-NMR spectrum con~irmed the ~tructure.

B. SYnthesis of N-formYl-lB-methYl)-as~-Dhe-tr~ OH.
One gram (1.9 mmoles) N-formyl-(B-methyl)-asp-phe- trp-OMe was dissolved in 150 mL MeOH, and the eolution added to a solution of 203 mg aminoacylase AMANO in 1350 mL water adjusted to pH 6Ø The solution was kept at pH 6.0 at room temperature for 6 hrs., us~ng a pH stat and 0.2N NaO~ as WO ~/l2883 PCT/US~/021~
-50- 2C~75~ ~

titrant. The course of hydroly~i~ of the C-terminal methyl ester was monitored by HPLC using the conditions deqcribed before.

The N-formyl-(~-methyl)-asp-phe-trp-OH formed was extracted with EtOAc at p~ 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. lS5 - 158C, ¦a¦22 = -30.2 (c, 1.19; MeOH).

Analysi~. Cal~ulated for C26H27N407: C, 61.53; H, 5.36;
N, 11.04. Found: C, 60.31; H, ~.62; N, 10.66. 13C-NMR
sp~ctrum confirmed the structure.

Undor the EPLC condition~ do~cribed above, the peptide N-formyl-(B-m~thyl)-a~p-phe-trD-O~ h~ a ret~ntion time of 10.2` min., idenff cal ~alone-or i~ adm~2ture) to the product form~d in~th~pop dn-catalyz~h reactton. : -:
ExamDle 11.Independent proof that the rate o accumulation of N-formyl-(~-mothyl)-a~p-pho-trp-OH (Vperm) Qbae~ved in Example 10 i8 solely determined by th~ rat~ of p-p~n prnteo~ynthesis --(V~yn), is given in the following exporiment.

A. Batch proteo~vnthesi of N-formYl-(B-methYl~-asD- _.
Dhe-t~-OM~. Compariron,of V8yn and Vpenn.
1.02 g L-trp-OMe (4-~mole~ n~ 242 ~g p~p~ln w~re dissoLv~d ,.
in 12S ~G-o-O.lM c~trate buffe~- ~ 4.5, and to thl~ wa~:
added 324 ~g (1 mmole) N-formyl-(B-mothyl)asp-phe-O~
dissolved ln 12 mL mot~anol. The resulting 10X MeOE
solution was incubated for 3 hours at room temperature, and tho rato of 8ynthe#i8 oS N-formyl-(B-mothyl)-aJp-pho-trp-OMe wa~ followed by HPLC, as de~cribed in Example 10. After the 3 hours of reaction, the Yolution was circulated in a hollow fiber separator with : , : . ' . ,: . .. . .. . .

:- . . ........................ : . '~ ' ~ :
, , : . ' :

WO90/l~83 ~ PCT/US90/02~88 51- 2 ~ ~ ~ 7 S ~

0 5ft2 ILM of 50~ N,N-diethyldodecanamide in dodecane, against 150 mL 10% MeOH in O.lM citrate buffer pH 5Ø The cour~e of permeation of N-formyl-(B-methyl)-asp-phe-trp-oMe was ollowed by ~PLC, as de~cribed 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 proteo~ynthesis catalyzed by pep~in, measured in a batch reaction without transport.

Table XI
Time N-form~l-(B-meth~ as - he-trp-OMe 33.5 39.6 48.0 ~ 120 54 5 ;- 180 61 0 Start permeation across IL~L50X N,~-diethyldod~canamide in dodecane 9.2 14.3 18.4 22.1 `
25.8 ~2~ 29.5 8. Rato of Dermeation of N-formvl-(B-meth~ as~-~he-trD-OMe und~r svnchronous ~rote~ynthesi~, in the absence of aminoac~la~. A ~onfirmatory e~perimont to the one-described in E~mple 10 ~a~-d~one in the absenc~ o~
ami~no~cyLa~e, during whtch Vperm was meAJured under synchronous synthe~is of trip-ptido methyl ester intermediate.

2.04 g L-trp-OMe (8 mmole~) and 484 mg pepsin were d~ssolved in 225 mL o~ O.lM cltrate buffer pH 4.5, and to this was added 642 mg (2 mmoles) of N-formyl-(B-methyl)-a~p-phe-OH di~olved in 25 mL MoOH. The - .: ~ ' : . : .. , . .,. .. , : -., WO ~t12883 , PCT/US90/021~
~ -52- ~C ~ 7~

~olution wa~ left overnight at room temperature, ater which HPLC analysis indicated the pre~ence of 167.4 mg/L of N-ormyl-(~-methyl)-asp-phe-trp-OMe at equilibrium. The ~olution 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 o 250 mL 10~ MeOH in O.lM citrate buffer pH 5Ø The permeation of the intermediate tripeptide methyl e~ter into the Qhell phase wa~ followed by HPLC, as described in Example 10. The correYponding data i8 tabulated in Table XII, and plotted in Figure 13.

Table XII

N-formvl-tB-methYl)-a~-Dhe-trD-OMe Time Reaction Dhaso Product pha~e ~in) (mg/L) ~m~L) O 167.4 0.~
204.9 8.6 192.4 22.3 183.0 29.8 204.2 37.6 120 182.6 59.1 180 177.3 73.1 240 142.8 85.4 300 l~Z.4 91.4 + ~=25.0 mg + ~=91.4 mg Total peptide synthesized: 25.0 + 91.4 = 116.4 mg P~r hour rate: 116.4 - 23.3 mg/L. hr.
Er~ th~ compari30n of the im~tlal rates o~ p~r~at1o~ Vperm of N-formyL~ mothyl)-a~p-phe-trp-o~e ~hown in Figure 12 and Figure 13, ~t is obviou~ that the aminoncylase.play~ no role in the kinetics of peptide tran~fer across the ILM, as both rates are similar at about 40 mg/L. hr. ft2. Consequently, the rate-determining step in the permeatlon experiments of Example~ 10 and 11 i8 the rate of pep~in proteo~ynthe~is, while the role of the aminoacylase is simply to secure the displacement ., -,. : ,. ~ :
- . : - .. . .
,................ . , ~ - , , '' ' : ~
. ~ . . .
: ' . ~

WO ~/12~ PCT/US~/021~
` ~ ~53~ 2~75~
of the equilibrium through the quantitative tran~fer of intermediate peptide from the reaction to the product phase.

On the other hand, physical factor~ 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 ~teady-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 dipeptide~

To a solution of 1084 mg (4 mmoles) of H-gly-phe-OMe.HCl in 112 mL McIlvaine buf~er pH 6.0 was added a ~olution of 20 mg papain plus 0.5 mL 2--ercaptoethanol in 20 mL of the ~ame buffer After th~-addition of 356 mg ll mmol-~ of N-CBZ-phe-gly-O~
dissolved in 15 mL methanol, the rosultin~ ~olution (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 circ~lated through the t~be side of ~ hollow fi~er separator ~ade of Colg~rd hollo~
fibers, containing 0.5 ft2 of a ILM of 50% v/v N,N-diethyldodecanamide in dodecane. The shell phase (150 mL, 10% MeO~, p~ 6.0~ was uDbuffered and contained dis~olved 80 mg aminoacylaoe AMANO. It was circulated counterc~rrently through tho separator at room temperature, a~d the pH wa~ kept constant at 6.0 with th~ h lp of a pH-st~t, uF~ng O.SN NaOH as titrant.
The synth~si~ of N-CBZ-phe-gly-gly-phe-OMe in the reaction p~ase (tube) and the accumulation of N-CBZ-phe-gly-gly-phe-OH in the product (~hell) pha~e were followed by HPLC analysi3, u~ing a Perkin-Elmer System consi~ting of a 410 LC Pump, LC 235 Diode Array detector set at 210 nm and a LCI-100 Laborat~ry Computing Integrator for data analysis. The analytical column cho3en was a NOVA-PAK C18 cartridge (8mm x lOcm, 4~) housed in a . . .
:' :................... ~ ' ~

.. .. ..

W090/12883 2C ~" 75~ PCrJUS90~02188 .

Millipore/Waters RCM-lOO radial compre~Qion unit. The mobile pha~e was a v/v mixture of 50% C~3CN and 50% 0.1% K~2P04 buffer Qolution containing 0.1% triethylamine v/v, adjusted to pH 4.2 -flow rate was l mL/min. Retention times for the N-CBZ-phe-gly-gly-phe-OMe and N-CBZ-phe-gly-gly-phe-OH were 7.05 minutec and 4.16 minute~, re~pectively.

In an effort to compensate for the permeation los~es of ~-gly-phe-ONe at pH 6.0, this reactant was continuously added to the reaction pha~e during the 5-hour run, at the rate of 0.5 mmole/hr. (total 680 mg).

The results of thi8 experiment are detailed in Table XIII
and Figure 14.

Table X~II

Time N-C~Z-Dho-alv-al~-~he-OMe N-C3Z-Dhe-alY-alY-~he-O~
(min) Reaction DhaJe - . (mg/L) 0 79.3 0.0 64.3 l9.0 69.0 41.l 120 133.0 72 8 280 174.4 l20.2 300 252.2 132.9 ~ ~ = 173.9 mq ~ ~ = 132.9 mg Sy~the~izod p~ptide: 306.8 mg :
306.
Hourly r~t~: S- = 61.4 mg~L_ hr~

Th~ r~to valu~ corresponds to the valuo V8yn = 75 mg/L.~r.
found in a batch papain-catalyzed proteolysi~ (Figure 15), proving again that tho rate of accumulation of product reflects the rate of proteosynthesi~ in tho reaction phase. This is quite evidcnt in tho Figure 14, where the Vporm = V8yn = 40 mg/L/hr.

.

- - . - . . , - . . . .. -. , : , . . .

.
, WO ~J~2~3 PCT/US~/02188 ~ ~55~ 2~ 5~

Identification of the product accumulated in the ~hell pha~e was done by HPLC comparison against an authentic ~ample of N-CBZ-phe-gly-gly-phe-OH prepared as follows:

A. ~vnthe~is o N-CBZ-Phe-~lY-al~-~he-OMe. To a solution of 500 mg (1.8 mmoles) of ~-gly-phe-OMe. HCl in 50 mL was added 0.4 mL (1.9 mmoles) Et3N, the ~olution immersed in an ice bath, followed by the addition o 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 pxecipitated dicyclohexylurea was filtered off, the dioxane removed by evaporation to yield a colorle~s residue. The crude product was dissolved - in 200 mL EtOAc, washed with 200 mL each of 5% citric acid, 5X NaHC03, prior to drying over anh. Na2S04. Removal of t~e - solvent gave 72~-~5 of a clear glassy re3idue, that cry~t2llized fro~ EtOAc/hexan~ to yiel~ 600 mg (58X) of : N-CBZ-phe-gly-gly-phe-OMe, whito cry~ta~J, m.p. 85 - 86~C, [al22 = 0.0(c, 1.0; MeOH). Analysis. Calculated for C31H34N407: C, 64.79; ~, 5.96; N, 9.75. Found: C, 64.23;
~, 6.05; N, 9.66. 13C-NMR apectrum subatantiated the t structure.

B. Svnthosis of N-CBZ-E~-aly-~lY-Dhe-OH. To a solution of 100 mg a~inoacyLase A~ANO in 90 mL delo~ized water pH

6.0, wa~ add~ ~.colution o 400 mg (~.7 mmole~) of N-CBZ-ph~-gLy-gly-phe-OMb in 20 mL methanol. The - milky-~h~t~ mi~turo was rapidly ~ti~nn~ at room temperature, and beg~n to clear almost imm~diately. The mixturo was allowed to react for one hour, keeping pH 6.0 constant w~th the use of a pH-stat and 0.1 N NaOH as titrant. At this point, HPLC analysis indicated that the ester hydroly i8 was comploted. The reaction mixturo was filtered,' the filtrate - strippod of methanol in vacuo, acldlfied to p~ 2.0 and extracted with EtOAc (3 x 200 m~). The organic extract wa~
, .
:.~. : , . . .
' '.', . ~ ~ ' ' ' , . , - , WOgO~ 3 PCT/US~/02188 2~75~
dried over anh. Na2S04, and the solvent evaporated to yield 300 mg of a clear residue. Crystallization from EtOAo/hexane gave 220 mg (60X) of N-CBZ-phe-gly-gly-phe-OH, white crystals, m.p. 134-136C; l~12D = + 6.~3 (c, 1.74; MeOH). Analysis.
lc~d for C30H32N47 C, 64.27; H, 5.75; N g 99 Found C, 63.83; H, 5.80; N, 9.88. 13C-NMR and lH-NMR spectra substantiated the structuro.

HPLC analysi~ under the conditions described above indicated the presence of only one compound of retention time 4.16 minutes, alone or mixod with the product of papain-catalyzed proteosynthe~is.

Exam~le_13.
PaDain-catal~ze~ Droteosvnthesis of a derivativo of the penta~eptide lleul -cnkeDhalin. To a 80~utl~n of 21 mg papai~Lin 160 mL McTl~aino buffer pH 6.0, contai~ing 0.5.mL - .
2-mercaptoethnDol, ~-~ added a soluttcn of 363 mg ~1 mmole) N-formyl-(O-Bzl)-tyr-gly-O~ and 361 mg (1 mmole) gly-phe-leu-OMe in 40 mL methanol. After a brief Jtanding (15 minutes) at room temperature, HPLC analy~is indicatod tho presence of 3.2 mg/L of N-formyl-(O-Bzl)-tyr-gly-gly- ph~-leu-OMe aLready ~ormed. The ~olution (reactio~ ph~e) w ~ then conne~be~ tD the ffhell side o . , a hollow-fiber separator (Bend Rosearch, Inc.), fitted with Celgard fiber~ containing a 1 ft2 ILM o~ 50~ v/v N,N-diothyldodecanamld~ i~ dodocan~. The product phase wa~ a solutic~Lof 158 mg m~noacyla~o AMANO in 200 mL 20% MeO~, ad~u~te~ to p~ 6.0~ that was connected to the tube aide o the ~eparatoc: Tho:two pha~e~ were cirG~ted countercurrent~y at 25C, w~th th a~ai~tanco of two per~-taltic pUmp8. The product phase (tube ~ido) wa~ k~pt at pH 6.0 throughout the run with the halp of a pH-stat, uslng 0.5N NaOH as titrant.

Throughout the experimont, ths concentration o the reactant H-gly-phe-leu-OMe in the reaction phase wa~ held approximately con~tant, by adding an additional 1.284 g (4 mmoles) dissolved in . ' '~ ' - ' ~ . ,. -- . . .: . ~ :

.

WO ~/12~3 PCT/US90/021 ~ 57- 2 ~ ~ ~ 7 S

12 mL methanol, at the rate of 1 mmole/hr to compensate for permeation losqes. 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-O~ was monitored by HPLC, using the instrumentation described in Example 12. The solvent systems used were as followq:
.1 A. N-formvl-(O-Bzl)-tYr-a~y-~ly-~he-leu--oMe: A v/v mixture of 60% CH3CN and 40% 0.1% XH2~04 buffer ~olution containing 0.1% v/v ~t3N, adjusted to pH 4.2. Retention time was 5.01 minutes.

B. N-formYl(O-Bzl)-tvr-alY-~ ~he-leu-OH: A v/v mixture . of 30% CH3CN and 70% 0.1~ K~2P04 buffer solution containing - O.1% v~v Et3N, adjusted to pH 4.2 was utilized. Retention - time wa~ 7.07 minutes.

The data ~howing-the build uFr.o~ hydrolyz~ pontapeptide in the product phase i8 shown in Table XIV, and plottqd in Figure 16.

Table XIV
N-form~ O-Bzl)-tyr- N-formvl-(O-Bzl)-tvr-Time aly-al~-Dhe-leu-OMe alY-alv-~he-leu OH
(min) (Reaction phase, mg/L) (~roduct pha~e, mg/L) 0 3.2 0.0 5.4 30.7 0.7 39 0 120 1.O 101_4 180 0.9 136.9 240 1.0 174 4 300 - 1.2 227 0 A~ experienced in previou~ examples, the average hourly rate of pentapeptide formation (Figure 16, Vperm = 45 mg/B. hr) was in.
agreement with the rate of N-formyl-(O-Bzl)-tyr-gly-gly-phe-leu-OMé ~ynthesi3 measured in a parallel batch incubation of N-formyl-(O-Bzl)-tyr-gly-OH and H-gly-phe-leu- OMe - - .

,: ........ .
. , . : . :
.. . . .

. . ; ` ' '. ~:

WO ~ 3 PCT/US~tO21~ ~
-58 2~75~ ~

with papain at pH 6.0, and found to be Vsyn = 34.2 mg/L/hr. at 25C.

The product phase (200 mL) was adju~ted to p~ 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-10-Bzl)-tyr-gly-OH (HPLC), was redissolved in 10 mL
EtOAc and ~ntroduced into a Ito Multilayer Coil Separator-Extractor, already filled with a stationary pha~e of 400 mL 0.01 M pho~phate buffer pH S.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. HP~C analysis indicated the presence of the pentapeptide acid in fraction~ 3-10, and tho dipeptide ac~d in fraction3 ~6-18; Tho presence of N-formyl-(0-Bzl)-lleu]5-enkcphalin in th~ pooled 3-1~ ractlon~
wa~ conir~e~ t~r~ugh deprotect~o~ wtth l N~Cl at 50C to re~Dv~
the formyt gr~up, followed by hydr~genatiQn o~er Pd/charcoa}~t~-remove the benzyl group. The product wa~ found identical to H-tyr-gly-gly-phe-leu-OH by HPLC ccmparison with an authentic samplo of synthetic lleu]5-enkephalin acetate salt (Sigma Chemical Catalog, 1988, catalog number L-9133, p. 280).

Exam~le 14.
Use of C~ 108e hollow-fiber contactors to suPport hvdro~hobic liouid~ - A lication to~roteo~vnthe~is. The thermo~y~in-catalyz~ proteosynthe~is dcscrib~ in E~a~ple 5 waa practic~i.with eguaL ~ccess in a ~e~brane con~actor embod~ent, compri~tn4r.p~cksd cellulo~e hollow fiber~.huv~ng their lu~nu~
filled w~t~ a hydrophobic liquid and their walls embedded in wator. Forced countorcurrent circulation of the tube pha~e between two membrano modules in ~erie~ provide~ a high surface contactor, able to transfer uncharged organic moleculoJ between an aqueou~ reaction phase and an agUeouJ phase. ThiB
arrangement, useul for the practice of proteo~ynthe~ia according to this invention, is ~ketched in Figure 17. Circulation by a ~, .~. : : , :, :
, O ~/12883 2~ ,5~ PCT/US~/02188 sg ~ ..... .. , i first pump 64 of the aqueous reaction phase 63 from the thermolysin rea~tor 61 to the first membrane module 67 allows for the transfer of the permeable intermediate N-formyl-(B-methyl) asp-phe-OMe into the hydrophobic phase (oil) reservoir 65 via descendin~ oil line 81. This enriched oil i8 circulated from the hydrophobic phase reservoir 65 via transfer line 82 by a second pump 66 via pump line a3 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 me~brane separator such as a reverse osmosis membrane R~ 62 by a third pump 70. The intermediate dipeptide ester is irreversibly trapped by the reverse osmosi 9 membrane RO 62 in the rententate 77. The filtrate 78 from the RO 62 i-~ returned 73 to the product reservoir 69. The retentate 77 from the RO 62 is recycled 79 to the product reservoir 69. The pure water 71 leaving the RO unit is returned to the ~econd membr~ne ~odule 68, while the spent oil leaving the second me~brane module 6e is rocycled via line 72 into th~ f~rat membrane module 67,.-w~ere it gets re~oaded ~ith, fresh intermediate produced by protcoJynthe~is in the r~action vessel 61. The oil loop 84 (water-immiscible hydrophobic pha~e) comprises hydrophobic phase reservoir 65 and line 82, pump 66, oil line 83, line 72 ~ line 81 comprising fi~st contactor 67, second contact~r 68, R~ 62, aqueouQ roaction ph~se 63, aqueous-product phase 80 and oll loop 84, thus closing the cycle. A
first membrane contactor 74 is ~hown. The process can be enhanced by utilizing a second membrane contactor 75 (not shown, but identical to 74) utilizins a bleedstrean 76, Generally, a plurality of membrane contactorn can be uti}ized repeatedly to enhance reccv~rr of product. . -,~
Figure 27 i~ a partial schemat~c view ~enlarged) o~ a cross section o hollow fibers in a membrane contactor. The partia~
view shows hydrophilic hollow fibers 121 in a fir~t membrane module and hydrophilic hollow fibera 141 in a second membrane module. In the first membrane module hydrophilic hollow fibers L
121 are shown having a lumen (bore) 122 and hydrophilic polymeric material 125. The lumen 122 i8 filled with water-immiscible I

. . ~ . . .
. ~ . ': ,' .. . . ~

: .
.. .. ..
..

12C~`~75~
WO ~t~83 PCT/US90/021 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 pha~e).
Figure 28 i~ a partial schematic view (enlarged) of a membrane contactor 160 comprising a fir~t 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 i9 circulated through space 123, through oponing 129 on firct wall 28 and roturned through opening 135 on.third wall 134.. The aqyeQ~s reaction phn~o doe~ not mix w~th the -water-i~miscible L~quid. The ~te~-$-hi~cib}e liquid i~
circulated through }u~en S21, through opening 127 on the second wall 126 to the second membrane module 151 through connecting tube 130 and returned to tho first membrane module 131 through opening 133 on the fourth.~ll 132. Si~ilarLy, in the second membrane 151 hydrophi}ic hollow fibers 141 ar~ potted in a resinous material (potting compound). The ag~eous product phase is circulated through ~pace 143, through opening 149 on first wall 148 and returned th~ough opening 154 on third wall 153.
The agueoua product phn~e do~J not mlx with t~ ~ater-immiscible }iguid.- The Yater im~cibl~ ligu~d is circulated.fro~
connecting t~b~-l30 thr~uqh open~ng r4~ on fourth wall 14~
through lum~n 142 and returnod to ths first membrane module 131 through opening 152 on second wall 150. The first and second membrane modules, 131 and 151, of the membrano contactor 160 each compri~ many hydrophilic hollow fibors, 121 and 141, re~pecti~ely, although only thre~ are ~hown in each such membrane module.

: , .. -: - . . . :
. . . :. ~ . .
:: : - .

. .: :
. . . . . . . . . .

WO ~ 3 Z~ 75~ PCT/US~/021~

The use of membrane contactorq in conjunction with reverqe osmosi~ 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 thi3 membrane arrangement, an experiment was conducted in which the enzyme thermolysin catalyzed the coupling of N-formyl-(~-methyl)- H-phe-OMe(A) and asp-OH (B), and the resulting peptide N-formyl-~B-methyl)-asp-phe-oMe(c) was continually trapped in the RO retentate (Figure 17). The conditions used in this experiment were as follows:

A. Thermolv~in reactor. 300 mL, p~ 5.0, 50C, [B] = 340 mM, IA] = 627 mM, [thermolysin] = 21 g/L, lCaC12] = 10 mM.
The initial rate of C qynthesi~ under these conditions wa~
Vsyn = 13 g/L~hr (37.5 mM/hr) B. Membrane contactor. 0.8~2, loaded with 2-undecanor~.

C. RO Nembrane. 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 re~ervoir. 700 mL, pH 5.0, 50C.

The results from the operation of the irst stag~ of this run are ~hown in Eigure 18, which plots the conce~tration of C in the ther ~g~ re~ctor as a fuDct~o~ o time. T~e data clearly showq the continuou~ removal of C from the reactor by the membrane contactor, and the recovory of the chemical equilibrium upon stopping the flow of undecanone and the ~im~ltaneouq 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 mM
and 15 mM, respectively. (A:C molar ratio = 3:1). Thi~ product .: , :.

W090/1~ 62- ~5~75~ PCT~uS~/o21~

solution wa~ concentrated about 10-old 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 eed reservoir of the membrane-contactor sy~tem, and the system wa4 operated at 25C against 100 mL water, pH 4.0 placed in the product reservoir (Eigure 17), until approximately 50% of C had been removed from the feed solution. The product from this ~econd 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. 105-107C. [¦ D ~
-32.3 (c, 0.65; MeO~). An authentic sample of N-formyl-(B-methyl)-asp-phe-OMe had m.p. 108-109C; [~12D = -33.9 (c, 0.59; MeOH). Based on the optical rotation, the purity of the recovered dipeptide C wa~ 95~.

ExamDle 15.
This exa~plo describcs t~e thermoly~tn-catalyzed .
protcoaynthe~i~ Q.the dipeptide N-for~yl-~-methyl)-~sp-phe-O-< , ~nd it~ ~ynchronous hydrolysi~ to tho acidic-d~pept~do .
N-formyl-(B-mothyl)-asp-phe-O~, across an rLM including N,N-diethyldodecanamide. The symbol ~ n is utilized herein to indicate isopropyl.

To a ~olution of 13.C9 g (54 mmole~ of ~-ph~-O-<.HCl and 6.64 g (38 mmoles) of N-formyl-(~-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 CaC12. The solution ~a~ incubated at 25C for one hour at ~h~k timo it was f~un~ by EPLC analysis to cont~ 14R.9 mg/~ o th~ dipcptlde.
N-fonnyl~ nQthyl)-aqp-phe-O-<.- Tho ~olut~on W~B then connectod: :.
to the ~helI ~ do of an experimental hollow-fiber separator (8end Research, Inc.), that provided 1 ft2 (soo cm23 surface o an T~M
of N,N-diethyldodecanamide. Th9 tube ~ido of the separator wao connected to the product ve~sel containing a ~olution of 0.162 g of the enzymo aminoacyla~e (AMANO Pharmaceutical Co., Nagoya, Japan) dis~olved in 200 mL water at pH 6Ø The two phases were circulated countercurrently at 2~C at the rates of 100 m~/min . .

W090~12~3 2~7~ PCT/US90/02188 (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 con~isting 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 cho~en was a NOVA-PAK C18 cartridge (8mm x lOcm, 4~) housed in a Millipore/Water~ ~CM-100 radial compression unit. The mobile phase for the N-formyl-(3-methyl)-asp-phe-0-<
was a v/v mixture of 50% CH3CN and 50% 0.1% KH2P04 buffer solution containing 0.1% v/v triethylamine, adjusted to pH 4.2.
Flow rat~ wa~ 1 mL/min. Retention time was 5.65 minutes. The mobile phase for t~s N-formyl-(~-methyl)-asp-phe-OH waq a v/v mixture of 30% CH3CN and 70~ 0.1% K~2P04 buffer 801~t~on . -containing 0.1% v/~ triethylamine; adjusted to p~ 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 .

Ta~le ~

TimeN-formvl-(~-methYl)-N-formvl-(~-methvl)-(min) a~D-Dhe-O-< as~-Dhe-OH
(R~action phase,mq~L)~Product phase,mg/L) phase,mg/L~ . !
0 1~8.9 0 ! 30 41.1 75.8 228.9 115.7 120 172.~ 163.3 180 246.2 199.1 240 172.8 262.1 300 192.7 314.8 + ~ =43.8 mg + ~ =314.8 mg Synthesized dipeptide: 3~8.6 mg/L.
Hourly rate = 358.6 = 72 mg/L/hr.

.

: , ''. :, . " . '~, , WO90/1~3 PCTtUS~/02t88 -64- ZC_~75~

This overall hourly rate i~ in line with the initial permeation rate of 150 mg/L. hr measured at the first hour (Figure 19), and with the initial ~ynthesis rate of 220 mg/L/hr.
mea~ured independently for a batch incubation (Figure 20) run at the same conditions of pH, temperature and reagent concentrations.

The dipeptide acid N-formyl-(3-methyl)-a~p-phe-OH present in the 200 mL product pha~e (63 mg) was i~olated by concentrating the solution in vacuo to 50 mL, removal of ~he enzyme by ultrafiltration over an Amicom PM-10 membrane (molecular weight cut-off: 10 Rdalton), acidification o the ultrafiltrate to pH
2.0 and extraction with EtOAc (3 x 50 mL). The organic extract was evaporated to dryness, the resid~e dissolved in 2 mL water pH
6.0, and the dipeptide was crystallized by acidification to pH
2Ø Yield: 11 mg (17%), idontical by HPLC comparison to an authentic samp~c of N-formyl-~-methyl)-a~p-phe-OH prep~red-according to th~;ollowing proc~dur~_e A. SYnthe~i~ cf N-formvl-~-methvl)-asp-~he-OM~. 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 thi~ mixture was added 16.0g (98 mmoles) N-formyl-(B-methyl)- asp-OH, the solution was cooled t~-0C, and 20.0 5 (9~ mmole~
dicyclohexylcarbodilmide were added, followed by 16.0g N-hydro~y-5-norborn~ne-2,3-dicarboximide. After stirring ov~rnight at room temperature, the dicyclohexyluroa procipitated wa~ flltor~ off, and the fi~trate concentr~ted to a syrup by ovaporation. It was dissolved in 500 ~L
E~ , rnd wa~h~KC~-quentiall~ ~ith 5X citric acid, 5X -NaDC~; water, and drl~d ov~r x~hydrous Na2S04. Removal of the ~olvont gave a white solid, which was recry~tallized twice from EtOAc/hexane to yield 17.0 g (53~) of N-formyl-(~-methyl)-asp-phe-OMQ, m.p. 108-109C. [~] 25 =
-36.9 (c=0.6, MeOH).

.

2C`~:~75~ ~
WO90/1~o3 PCT/US90/021 H-NM~ (CD30D): 7.6 ~ (~) formyl; 6.8 ~ (s) phenyl; 4.0-4.4 ~ tM) 2 CH; 3-3~ (s) 2 C~3 (6 protons, superimposed~;
2. 3-2. 8 6 (M) 2 C~2 (4 proton9).

3C-NMR ~CDC13): 35.52, 37.56 ppm (-CH2-, t); 47.59, 53.54 ppm (-CH- , d); 52.09, 52.28 ppm (CH3-, q);
127. 08, l128.51, 129.19 ppm (phenyl, d); 135.62 ppm (phenyl, 8); 160.93 ppm (H-C- ,d); 169.60, 171.34, R
o 172.09 ppm (-C-O(MH), 8).
o .Analv~is: Calculated for C16~2006N2 C, 57 I7; ~
5.95; N, 8.33. Found: C, 57.29; H, 6.02; N, 8.32.

B. SYnth~sis ~ ~-formvl~ ~othvI~-an~-Phe-O~. A
~olution of 2.0 g (5.9 mmol~s) of N-formyl-(~-methyl) -asp-phe-OMo in 50 mL MeOH was added to a solution of 0. 2 g aminoacylase AMANO in 450 mL 0.05% KH2P04 buffer pH 7.5, contained in a beaker ~itted with a mechanical stirrer. ThQ
solUtion wa~ stirred overD~ght at roo~ temperature, and the pH was kept con~tant at 7.5 with the as~istance of a pH
3tat, u~i~g lN NaOH a~ titrant. Aft~r filtration of any unreacted ~tartin~.matQrial, the ~olution .W-3 wa~hed with EtOAc (2 x 100 ~L), then adju~ted to pH 2.U iln~ the .
hydrolyzed dipeptide wa~ extracted with EtOAc (3 x 200..~L).
The combined ~xtr~ct~ were drie~ ove~i~nhydrous Na2S04'and.-.
eviqporated to dryn~s. Tho yiold of N-formyI-(~-methyl)-asp-ph~-OH was 1.3 g (68%), m.p.
185-186C, 1~j]2D = -14.3 (c=l.O; MYO~).

13C-NMR (CD30D): 37.37, 38.76 ppm (-C~2-, t)j 50.21, 55.68 ppm (-CH- , d); 52.21 ppm (CH3-, q); 128.31, 129.90, 130.90 WOgO/~3 PCTtUS~/021~
-66- 2~75~ `

ppm (phenyl, d); 138.63 ppm tphenyl, 8); 163.99 ppm (H-C-, d); 172.38, 172.~3, 174.77 ppm O

(-C-O(N~), 8).
o Anal~sis: Calculated for C15H1806N2: C,55.93; H, 5.59; N, 8.69. Found: C,55.22; H, 5.71; N, 8.51.

Exam~le 16.
Thi~ exa~pL~ ~hows the rate effect caused by the hydrolytio enzyme upon the pormeation Vperm of the intermediary peptide during ~ynchronou~ proteo~yntheoi~. Th~ ~easured rate increa~e is p~rticularly importaot for thoso hiqhly hydrophob~c.pe~tides showing h~gh part~tion coe~fic~ontr tow~rd~ th~ hydrophobic:
membran~. The chomical tran~formation catalyzed by the aminoacylase, that iJ tho converJion of a C-terminal isopropyl ester into the froo carboxylate, occurs at the interface oil/water and as~i~ts poJitively in the tran~po~t of dipeptide .:
across th~ bound~ry. Th - . incr~a80 in pormeat1on of t~e intermediary p-ptide caused by th~ countercurrent 3weeping of the oil surface with th~ aminoacyla~e is an additional effect to the equillbrlum displacomo~t r~ulting ~rom the che~icaL :
tras~ormatlon to.~ no~-por~n~bl- product. ' To ~: wlution of513;7 g (56 ~o~r) o ~-phJ-O~.~Cl and 6.6 :.~. ~.
g (38 ~ol~) of N-formyl-(B-methyl~-a-p-0~ in 200 mL water a~ p~ ~
5.0, wa~ added 397 mg Th-rmoaae Daiwa (40,000 PU/g) and 150 mg .~.
CaC12. Th~ ~olution was incubated at 25C for on~ hour, at that tima it was found by EPLC analy~i~ to contain 347.0 mg/L of the dlpeptlde N-formyl-~B-~othyl)-aJp- phe-O-c. The solution wa~
circulatod at 25C through the ~hell side o an hollow-fiber separator, containing 0.5ft2 ILM of N,N-diethyldodecanamide, .
: : ., , . :

.. . . .

WO ~/1~3 2~75~ PCT/US90/0~188 again~t 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 aminoacyla~e AMANO in solution. The countercurrent circulation of both aqueous phases was reinitiated, and the rate of permeation Vperm of the dipeptide N-formyl-(3-methyl)-asp-phe-OH was mea6ured by HPLC again, together with L-phe-OH and L-phe-O-<. The resultq appear in Table XVI and Figure 21.

..~. _ : ~ , .:

WO 90/1288~ PCr/USgO~02188 2~5~75~
~_ t`~
~ _ . ,~

~:

~ z n ~ _ _ ~ ~ 3 ~
~ V ~ ~ ~ N O ~
~ - ~ ~ ~ o O ~ ~ 0 Pl - fi. ~ o , ~ .` ID o o ~1 ~
g 3v Z~
.. --. . _ .
. ~
~ e @ ~ V ~ ~I ~ CD 3 N 1` N
~1 Cl~ ~ N ~ ~ t~ ~
~1 O I ~ N N t~ N N 111 a~ 1/) `
...
~ z~ , . ....
. ~ .....
IC I~OON ~

; ~
. . . . . I . ... .. ~ .......

WO90/12883 2 ~ ~ 7 S ~ PCT/US~/02t~

Figure 21 shows that the peptide permeation in the presence of aminoacyla5e (Vperm = 440 mg/L/hr-) is 2.~ times faster than in water (Vperm = 190 mg/L/hr.). Thi~ rate effect caused by the direct contact o the ILM with the soluble enzyme would be ~ost if the same enzyme is uQed in the immobilized form.

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

It is also shown in Table XVI that the rate effect exerted by the ~inoacylase is reQulting from the hydrolysis of th~
dipeptid~ isopropyl ester. The r~te of permeation of L-phe-O-<, a poor substrate for the aminoacyL~e, i8 insensitive to the preoence of the enzyme in the produrt phase. ~

Exam~lo 17.
This Example 17 illustrates the efect of operating the process of this inventlon at elovated temperatures and effectivenecs at higher temperatures in a rang~ of from 20C to 65C of lowar d kyl ~st~r roactant~ IB'~ wherei~ the alkyl group is selected to enhance ~tability of that reactant, e.g., esters derived from secondary alcohols having 3 to 6 carbon atoms.

m o th~rnolyain-catalyzed proteosynthesis of the dipeptide N-formyl-(B-mothyl)-a~p-phe-O-< (C') described in E~ample 15, was uccesJfully pr~cticed, at 50C utilizin~r the celluloso hollow-fiber contactor do~cribed in Example 14, in place of the ILM module u~ed in Example lS.

Tho 3et-up used in this o~periment is outlined in Figure 17, and the conditions for achieving the conden~ation oquilibrium botweon ~-formyl-(B-methyl)-a~p-OH (B'), L-phe-O-~ (A') and the resulting dipeptide C' were as follow~:

- . . --- , . , . ~ :

.:: , , , : . : . . .:

WO ~/1~3 PCT/VS~021 -70~

A. ThermolY in Reactor. 500 ml, pH 5.0, 50C, containin~
42 g/l thermolysin, lOmM CaC12, 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 _ mbrane. 2 ft2 TW-30 spiral-wound module previou~ly de~cribed in Example 14.

D. Product Reservoir. 750 mL water, pH 5.0, 50C, circulating throught the RO module, the membrane contactor and the re 8 ervoir.

The accumulation of product C' in the reservoir as a function of ti~o 15 shown in Figure 23. The data shows that the concentration of _' in the ther~ysin-drivon equilibrium is m~intained at 15~K upon-addition of A' to -keep:~A'] const~nt, while the tC'~ in the product rese~vvir -increase~ steadily as the dipeptide i8 trapped by the:RO
membrane, thus providing the constant driving force needed to displace the chemical equilibrium A'+B'~ C'.

After 15 ho~r~ op~rstlon th~ reactor w~ stopped, and -' the product solution ~20mM _', 160 mM A') was collected, adju~ted to pH2, concentrated lOx by evaporation and cooled, to yield 3.9g (70X yield) of N-formyl-(B-methyl)-a3p-phe-O~, .
white ~ry~tals, m.p, 68-69C, lu]25 = --22.7 (C,3.6: MeOH). :' AnalYs~~ C~lculated for C1 ~ 406Ni: C, 59.37; ~,6;5~, N,

7.69. Found: C, 59.31; ~, 6.66; N, 7.65.

Puritv: (HPLC): 97%. After purification by chromatography on a C18-reverse phase column, the pure dipep~ide had m.p.
101-102C, [~]25 = -32.6 (C,O.9; MeO~), and ~as identical in all resp~cts (IR, 13C-NNR) to an authentic sample of N-formyl-(~-methyl)-asp-phe-O-<, m.p. 100-101C, [~]2S =

:'"', - ', - ,- ' ' ' . '' ,' ,. ~ '. .
- ,,: , , . . - . .
.. . . . . .
.. ,: ' , - .. : ' ' '. , :' , ~ ' ... . . ..

WO ~/I~W3 -71- 2~ ~7S~ PCT/US~/02188 -31.3 ~C, 3.0; MeOH), prepared by the DCC coupling of N-formyl-(B-methyl~-asp-OH and L-phe-O-< in dioxane.

The chemical ~tability of L-phe-O-~ over L-phe-OMe meaRured seven times higher at 50C, in water pH 5.0, which i8 an 1.
important advantage for the practice of this invention. Economic benefitq deriving from operating at about 50C, in preference over processing at lower temperatures are: a) the ~hift of the chemical equilibrium towards peptide formation that occurq with increasing temperature: and b) the increase of peptide permeability across hydrophobic membranes with increasing temperature.
Successful operation of the process of thi~ invention at elevated temperature~ depends at least in part on the thermal - stability of the lower alkyl ester reactant [B'] and mini~izing .. or avoiding unded rable side reacti-on~, such as hydrolysis of the e.qter to produce.-~h~ corresponding acid. It i8 not po~sible to efficiently operste the procoss d~cribed in this ~xample when usins L-phenylal~nin~ methyl e~t~r becauJ~ under the dèscribed proce~s conditions at 50C that ester is hot very-stable, i.e., it slowly hydrolizes to L-phenylalanine. The stable isopropyl ester, quickly reacts at 50C 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, o.g., 2-butan31.and 3-methyl-2-butanoL are also L expect~ to have h~gh~.~her~aL rtabi~ity, and to show ~inimal formationLof L-ph--O~ by ester.hydrolysis in w~ter at pH 5.0 and 50C.

Exam~le 18. Peosin-catalYzed Droteo~vnthe4is of N-CBZ-a8D-Dhe-OMe.
To a solution of 85.46 g (320 mmoles) N-CBZ-~-asp-OH and 19.45 g (90 mmolo~) L-ph~-OMe.HCL in 400 mL water p~ 4.0 was added 9.63 g crystalline pepsln (Sig~a) di~solved in 100 mL water pH 4Ø The resulting solution (500 mL) wa~ incubated at 38C

;: ,' . ., , : - :: ~ .
- -.. . . . .

.-: ,: : , ,: . .:
:

WO90/1~3 ~ PCT/US~/02188 ~
-72- 2~5~5~

for 48 hrs, at the end of which it was shown by HPLC to contain 3208 mg/L (7.5 mM) of N~CBZ-a~p-phe-OMe. The ~oeLC analyses were done on a Perkin-Elmer System con~isting 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 wa~ a NOVA-PAK Cl~ cartridge (8 mm x 10 cm, 4 ~ bore) housed in a Millipore/Waters ~CM-100 radial compre~sion unit.
The mobile phase wa~ a v/v mixture of 40% CH3CN and 60% 0.1%
KH2P04 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 ve~sel kept at 38C, and it wa~ circulated through the shell side of a hollow fiber separator made of Celgard fibers, containing 1 ft (900 cm ) ILM of 50% v~ isoh~adecanol (Hoechst) dn dodocane. The product ph c (500 ~L~ was deionized water pH 7.0, .
that was circ~lat~d countercurrently through the tub~ ~d~of the mo~u7~ wh~l~.koeping tho p~ con~t~nt ~t 7.0 wi~h the ~ tance of a p~-stat UD~t, using 0.5N NaOH ~ t~trant.

The ~ynthe~ of N-CBZ-asp-phe-OMe in the reaction phase (~hell) and the accumulation of the same dipeptide in the product phase (trapped at p~ 7.0 a~ the ionized ~-carbo~yl~te) wore s measured by ~PLC, as de~cribed above. After a S hr run the circulation of th~ two pha~e~ was interrupted, tho product solution was removod and replacod w1th 500 mL of fre~h water pH
7.0 ~olution. Eollov~ng a s~cond incubation at 38C for 24 hr~, tho reaction mixture contained 4170 mq~L o N-CBZ-asp-phe-OMe (HPLC). The count~rcurrent c~rculat~an wa~ rea~Fumed, an~ the permeation of peptido wa~ monitor~d for another 5 hr period.

The results of this experimant are shown in Table XVII and Figure 24. It is evident from T~bl~ XVII that active protsosy~the~is has taken place in the enzyme réaction phase, while newly synthesized peptide was being tran~ported acros~ the membrane. Computation o the total N-CBZ-asp-phe-oMe accumulated . , ...... :-,: . ...... : ' . . . ' - . . ,. -: . :: . .:: :.

.

WO90/1~3 _73_ 2 ~ ~ ~ 7 S ~ PCT/US~021 in the product pha~es (542 mg in 10 hr) indicateq 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, [a]22 = -12.0 (c, 1.0; MeOH) was identified as N-CBZ-asp-phe- OMe by ~, chromatographic (HPLC, TLC) and lH-NMR compariRon~ with an authentic sample prepared by the Schotten-Baumann acylation (water, pH 7.5) of aspartame with benzyloxycarbonyl chloride, to yield crystals m.p. 122-124C (from EtOAc/hexane), [a]22 = -13.0 (c, 0.8; MeOH). Literature ¦D. D. Petkov and I. B. Stoineva, Tetrahedron Lett. 25, 3754 (1984)~, reported m.p. 120-124C, []25 = -14.4; (c, 1.0; MeOH).

Table XVII

Time Reaction Pha~e Product ~hase (Hr)N-CBZ-as~-Dhe-OMe N-CBZ a~o-~he-O~e (mq~L) (mc/L) PART I
0 3208.0 1 2726.8 42.5 2 -2a87.2 99.5 3 - 15~.8 4 - 176.4 2887.2 243.8 .
PART II
0 4170.4 1 - 75.4 3 - 170.0 4 ~ 181.2 3528.8 298.3 While sp~cific embodiment3 of the invention have been shown and de~cribed in detail to illustrate the application of the inventive principle~, it will bo understood that the invention may be embodied otherwi~e without departing from ~uch principles.

,. . . . .

.: - - - : ~ . . : :

: ' ' ~ '. -' : '"' . .' '~' ' ' ~: ,''," ', ' ' WO90/12~3 : PCl`/US~/02188 2~75~
ExamDle 19. Inteqrated Droces~ combinina the enzvmatic resolution of D- L-Phe-O-~ and the racemization and recyclin~ D-Phe-O-C.

Example 8 demonstrates the preparation of L-Phe-OH from D-L,-Phe-OMe, by ~tereo~elective hydrolysis of Phe-L-ester with aminoacyla~e enzyme in water at pH 7.5, utilizing a SLM having a 30% N,N-diethyldodecanamide/70% dodecane organic pha~e dispo~ed in a hollow fiber module. Enantiomeric resolution i~ 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 + 0~ D-Phe-Ome ~ L-Phe-O ~ MeO~
(permcable) (non-permeable) ~ he measured flu~ of D-Pne~4~e acro~-the SLM, wa~ O 5g~ft2 hr, rqpresenttng-the limitinq rat~ o~ the sy~te~ for r~ w~utiu~ -under test conditions.

The L-Phe-OH separated by this method can be converted to the corresponding isopropyl ester by conventional e~teriSication procedures using isoprop~nwl and HCl. The resulting L-Phe-O-~ i8 a preferred starting material for the synthesis of the dipeptide N-formyl-(b~ta-methyl)-Asp-Phe~ , described in Examples 15 and 17.

The cconomics of the overall conver~ion D-,L-~hc-o~e ~-Phe < w~uld bo i~proved b~-fastraad e~ficient race~zation:-of the by-product D-Phe-Ome, that do~s 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 (110C) for one hour in the pre~ence of a salicylaldehyde catalyst in such a racemization.

. . .

,, ~ . - ,:, W090~l2883 _75_ 2 ~ ~ 1 7 S ~ PCT/US90/02188 The following experiment demon~trates the enzymatic resolution of D-,L-Phe-Ome at an interface of a toluene/water biphasic sy~tem.

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 aminoacylase (crude soluble solids) in 200 mL of aqueous 1.5 mM
CoC12, adju~ted to pH 8.5. The resulting two phaces were thoroughly mixed by magnetic stirring, and as the reaction proceeded, the p~ of the water phase wa~ maintained at 8.5 u~ing 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 analysi~ of the water phase showed the presence of 3.2g L-Phe-OH, confirmin~ that the reaction was s near completion. After an additional 30 minutes of mixing, the two pbase~s were recovered and analyz~d separately.

A The aqueous layer (215 M~ was p}aced i~ a diailtration cell fitted witb n~ Amicon YM5 ~e~brand (5 R daltons cut off~, diafiltered with three volumes (650 mL) of 1.5 mM CoC12, pH 8.5 solution, and finally concentrat~d down to 200 mL. The combined filtrate (650 mL) w~a ad~u~ted to pH 5.0 with diluted HC1, concentrat~ in vacuo to 50 mL and co~led at 4C to yield 2.2g L-Phe-OH, I a ] 25 _ -28 . 6 ( C ~ 2; EI2 ) Optical purity: 100% with reerence to a D-Ph~-OMe.~Cl standard, l~125 = -32.4 c,2: EtO~.

The polarimetric results indicate that it is possible to achleve effective enantiomeric resolution by this procedure by feeding D-,L-Phe-Oma at the rate of about 40 g/L.h~, resulting in a L-Phe-OH output of 20 g/L.hr. Notably this productivity i8, a8 much as, 50 times higher than tho~e recorded in ~he literature ~or batch fermentations with Bacillus lactofermentum AJ3437, :, . . , . :

, WO 90/12883 ` 2~ 5~crtus9o/o2188 where the saturation of the culture medium in L-Phe-OH (about 30 g/L at 25C) required a cultivation time of 72 hrs, or 0.4 g/L. hr average productivity [I. Shilo, "L-phenylalanine Eermentation" in K.Aida, I. Chibata, K. Nakayama, K. Takinami and H. Yamada (Editors), "Blotechnology of Amino Acid Production", Kodansha, Tokyo, 1986, pp. 188-206].

Example 20. ENZYME ACTIVITY

The following experiment compare3 the effectiven~ss of aminoacyla~e, subtilisin and alpha-~hymotryp~in for enzymatic resolution proce~ses, in terms of the ratio of their rate constants for the hydrolysis of L- and D- enantiomers of the mothyl and i~opropyl esters of phenylalanine (L/D Kinetic ratio~. The rate conRtants for the hydrolysis of ~ach ester by oach enzyme were measur~, under identical conditions of pH, S/E .:
ratio.and temperature~-in ac~ordance with ~he above-doscribe~. ..-methnd~:aD~ convent~onal practice... The r~nults o .thoae.~
measurenunsts:-re s~r~rizod in T~b~-~XYIII below. . .

TABLE XVIII
L/D kinetic ratios in.O..l~ phosphate bu~r..p~ a.o,. s/~ = lo, 2SC, for phenylalan*n~ e~t~rs. Reactio~ ffm~ 2 hrs. ~.

Enz~mo L/D Kinetic Ratio Phe-OMe Phe-O ~ -Aminoacyl~s~ (A uno) 47~ 0 ~o hydsolysi~) Subtilisin, C~rL h rg (Slgma) 55~ 00 alpha-Chymotrypsin ~Sigma) 825* 00 ~Literature Value~, Dahod and Empie, ~u~ra.

~ ',, ,- . ,, ,- ' ' . .
. .-,., , . :' .: , .: ,: -:-: :

WO ~/1~ PCT~S~02188 ~77~

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 aminoacylase did not hydrolyze either D- or L- phenylalanine isopropyle~ter.
The discrimination towards L-Phe-O ~ shown by subtilisin and alpha-chymotrypsin i~ a highly advanta~eoua in the integrated resolution proce3s described in this Example.

The integrated re~olution process, may be advantageously practiced as a batch or continuouff operation becau~e it enable~
recovery and r~u~e of both the enzyme and ra~emization cataly~ts without having delet rious effects of toluene on their activit~.
: A cellulose hollow fiber contactor of the type doscrib~ in Examples 14 and 17 could be used ~o that the toluene ph~e : cilx~Lk~tes within th~ lumen of the ~ber~ countercurr~ntly to Yn aqueou~ ~nzyme phcr~ cmbodd-d t~e wa-llJ o~the fiber~. The toluene--p~ase lea~ing t~e contactor would carry D-~he-O <
that, after being racemized over an immobilized sali~ylaldehyde catalyst, would be recycled ac D-,L-Phe-O into the contac~r.
The hi4h interfac~ ~urace existing in the contactor facilitates .
the enzymatlc tran~fer of L-Ph~-O~ into t~e prcd~ct~w~ter phase.
On leavinq the contactor, the product/water phaso would be continuou~ly blod into a rever~e o~mouis (RO) un~t, operatlng downstream of an ultraf~ltration dev~ce (UF) ~al retains the onzymo but a~Iowr the froe f}owr.of L-Phe-OH.

Flgurc-29 ~chem~tically ~IluJtrates the a~ de~cribed .-continuous prQc-~s. The tran~for rato of 20 g/L.hr mea~ured above for ~-Phe-OH may be attained, in a hollow fiber contactor providing about on~ ft2 of interface area per liter of reaction mixture, and permeability of about 20 g/ft2. hr or 396 lb/ft2.
yr.

: . -: ~ , :
, .: , , .

.: .. : .:
. , ~ . - .. . ~: . , :

.. . ..

Claims (24)

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 l, wherein the protected, uncharged, peptide product is separated from the aqueous product phase by reverse osmosis.

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

4. The method 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-qlycyl-glycyl-phenylalanine methyl ester, N-carbobenzoxy-phenyl-alanyl-qlycyl-glycyl-phonylalanine, 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).