Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
131~2~
ENZYMATIC MEMBRANE METHOD FOR THE
SYNTHESIS AND SEPARATION OF PEPTIDES
DESCRIPTION
Technical Field
The present invention relates generally to an
enzymatic method for the synthesis and separation of
peptides employing a membrane permeable to uncharged
peptides but impermeable to charged molecules; and, more
particularly, to the simultaneous synthesis and
purification of L,L-dipeptides, and its application to the
preparation of L-aspartyl-L-phenylalanine methyl ester
5aspartame).
Back~round Art
The use of proteolytic enzymes as condensation
catalysts for the stereospecific coupling of two L-amino
acids to yield L, L-peptides i8 known since the early days
of protein chemistry. As early as 1909 Mohr and
Strohschein described the formation of the water-insoluble
dipeptide Bz-Leu-Leu-NH2 by reacting Bz-Leu-OH and
H-Leu-NH2 in the presence of the protein degrading enzyme
papain. E. Mohr and F. Strohschein, Ber 42, 2521 (1909).
The Mohr and Strohschein-type reaction is possible only
between those amino acids that form peptide bonds that are
susceptible to cleavage by the papain or other enzyme
used. Indeed, the condensing reaction' 8 equilibrium
between the amino acid reactants and peptide product is
largely displaced towards the reacting amino acids.
Nevertheless, the condensing reactant can be driven to
completion by mass action if, e.g., the dipeptide product
is poorly soluble and precipitates out of the reaction
phaR~ .
Due to the commercial importance of certain peptides
and the fact that enzyme~ are known to catalyze peptide
formation under mild conditions there has been a great
deal of research done on the enzymatic synthesis of
peptides particularly simple dipeptides. K. Oyama and
13142~
--2--
K. Kihara, Kaaaku Sosetsu 35, 195 (1982); K. Oyama an~
K. Kihara, ChemTech. 14, lOO (1984).
The process for enzymatic synthesis of the peptide
derivative aspartame, described in U.S. Patent 4,165,311,
hereinafter the '311 process, involves the
thermolysin-catalyzed condensation of N-carbobenzoxy-
L-aspartic acid with D,L-phenylalanine methyl ester and
precipitation of an intermediary complex, D-phenylalanine
methyl ester salt of N-carbobenzoxy-aspartame, to drive
the reaction to the peptide product side. Further
processing of this intermediary complex allows for the
recovery of D-phenylalanine methyl ester, that may be
recycled after racemization, and of the
N-carbobenzoxy-aspartame derivative which can be converted
to aspartame by elimination of the N-carbobenzoxy
protecting group. The '311 process must be practiced on a
batch basis which is cumbersome and complicates the
recovery of enzyme. Also see: K. Oyama, S. Irino, T.
Harada and N. Hagi, Ann. N.Y. Acad. Sci. 434, 95 (1985).
Willi Kullmann, Enzymatic Peptide Synthesis (1987).
The N-carbobenzoxy protecting group plays an
essential role in the '311 process by: fulfilling the
structural requirement imposed by the active site of
thermolysin; and by contributing to the insolubility of
the intermediary complex thereby increasing the yield of
the reaction. Elimination of the N-carbobenzoxy
protecting group from the aspartame derivative must be
effected under mild conditions, e.g., catalytic
hydrogenation, to prevent cleavage of the methyl ester
function. Catalytic hydrogenation involves the
inconvenience of handling hydrogen gas on a large scale.
Alternative approaches to driving enzymatic
condensation reactions to completion have also been
described in the chemical literature. For example, the
use of organic solvents as reaction media has been found
effective for increasing the peptide product yields;
although, the concomitant decrease in enzyme stability has
131425~
--3--
precluded its practice on a large scale. K. Oy~ma,
S. Nishimura, Y. Nona~a, K. Kihara and T. Hashimoto,
J. Orq. Chem. 46, 5241 (1981); H. Ooshima, H. Mori and Y.
Harano, Biotechnolo~Y Letters 7, 789 (1985); K. Nakanishi,
T. ~amikubo and R. Matsuno, BiotechnoloqY 3, 459 (1985).
In view of the above-noted difficulties in the
practice of prior art methods for enzymatic synthesis of
peptides, particularly, the requirements for precipitation
of an intermediary complex and handling of dangerous
reagents; it would be desirable to provide an improved
process that avoids these difficulties and that safely
provides effective yields without rapid deactivation of
the enzyme catalysts.
Disclosure of Invention
The present invention provides a method for the
synthesis and purification of a compound, comprising the
steps of coupling a first reactant with a second reactant
to produce a membrane transportable compound; transporting
the transportable compound across a membrane that will not
transport the reactants; and irreversibly converting the
transported compound to a form that cannot be
retransported across the membrane.
The present invention also provide~ a process for the
enzymatic synthesi~ and purification of compounds
comprising the 3teps of coupling a first compound,
including a protonated amino group (ammonium), and a
second compound, including a free carboxylate group, using
a condensation enzyme in an aqueous mixture to produce an
uncharged (or non-ionized) coupled compound; continuously
removing the uncharged coupled compound from the aqueous
mixture by diffusion acro~s a membrane that selectively
transport~ the uncharged coupled compound to the product
side of the membrane. Preferably, the transported coupled
compound is a peptide or derivative thereof that is
converted to a charged (or ionized) molecule so that it
does not back-diffuse acros~ the membrane. Thus, the
~3~ ~2~
--4--
formation of the coupled compound product is favored in
the reaction mixture because it is constantly being
removed therefrom.
The present invention also provides a process for the
enzymatic synthesis of peptide derivatives, such as
aspartame and their analogs, comprising the steps of
condensing a N-acyl-~-substituted-L- aspartic acid
including an a-carboxylate group with a phenylalanine
lower alkyl ester including an a-ammonium group in an
aqueous reaction mixture including a condensation enzyme,
to form N-formyl-L-aspartyl (~-substituted)
-L-phenylalanine lower alkyl ester (i.e. 1-6 carbons), an
uncharged peptide; and transporting the uncharged peptide
from the aqueous reaction mixture to a product mixture
acro~s a permselective membrane.
The present invention also provides a method for the
enzymatic synthe~is of peptides comprising the steps of
condensing first and second amino acid compounds in an
aqueous initial reaction mixture to form an uncharged
compound; transporting the uncharged compound into an
aqueous second reaction mixture across a membrane that
will not transport substantial amounts of the amino acid
compound~; and converting the transported uncharged
compound to a form that cannot be retransported across the
membrane to the initial reaction mixture. The transported
compound, converted to a form that is not retransported
acro~ the membrane, can be removed from the second
reaction mixture. Also, first and second amino acid
compounds copermeating the membrane with the uncharged
compound into the second reaction mixture can be separated
from the second reaction mixture and optionally returned
to the initial reaction mixture.
The present invention also provides a method for the
enzymatic resolution of amino acid compounds comprising
the steps of: hydrolyzing a D,L-amino acid compound in an
aqueous reaction mixture including a hydrolyzing enzyme to
131~25~
-5-
form a charged L-amino acid compound and an uncharged
D-amino acid compound in the aqueous reaction mixture; and
transporting the uncharged D-amino acid compound from the
aqueous reaction mixture across an ion rejection membrane
into a product mixture thus effecting an enantiomeric
resolution. The uncharged D-amino acid compound in the
product mixture can be accumulated and can be recovered by
conventional means. The method for the enzymatic
resolution of amino acid compounds can be combined with the
other methods discussed above.
It is an object of an aspect of the present invention
to provide a process for the enzymatic synthesis of
peptides which provides for simultaneous synthesis and
purification of the peptide product.
It is an object of an aspect of the present invention
to provide a process for the safe, economical and efficient
synthesis and purification of peptides and derivatives
thereof, particularly aspartame.
It is an object of an aspect of the present invention
to provide an economical process for the enzymatic
synthesis of peptides that provides for the efficient use
of enzyme and the means to effect the synthesis on a
continuous basis.
It is an object of an aspect of the present invention
to provide a process particularly adapted to the enzymatic
synthesis of aspartame and its derivatives with D, L-
phenylalanine and N- protected-, B-substituted-L-aspartate
in substantially quantitative yield.
Other aspects of this invention are as follows:
In a method for the enzymatic synthesis of peptides,
comprising the steps of: condensing a N-acyl-
~
13l~2~
-substituted-L-aspartic acid having an d-carboxylate group
with a phenylalanine lower alkyl ester having an ~-ammonium
group, in an aqueous reaction mixture including a
condensation enzyme to form N-acyl-L aspartyl-(~
-substituted) -L-phenylalanine lower alkyl ester, an
uncharged peptide; the improvement comprising transporting
the uncharged peptide from the aqueous reaction mixture
across an ion rejection membrane into a product mixture;
and converting the uncharged peptide to a charged species
by cleavage with an esterase enzyme having a proteolytic
activity.
In a method for the enzymatic synthesis of peptides,
comprising the steps of: condensing a N-acyl-
~-substituted-L-aspartic acid having an ~-carboxylate group
with a phenylalanine lower alkyl ester having an a-ammonium
group, in an aqueous reaction mixture including a
condensation enzyme to form N-acyl-L-aspartyl-(~
-substituted) -L-phenylalanine lower alkyl ester, an
uncharged peptide; the improvements comprising transporting
the uncharged peptide from the aqueous reaction mixture
across an ion rejection membrane into a product mixture;
converting the uncharged peptide in the product mixture to
a species that cannot back-diffuse across the membrane; and
removing the species that cannot back-diffuse across the
membrane from the product mixture.
In a method for the enzymatic synthesis of peptides
comprising the steps of: enzymatically condensing first
and second amino acid compounds in an aqueous initial
reaction mixture to form an uncharged compound the
improvement comprising; transporting the uncharged compound
into an aqueous second reaction mixture across a membrane
that will not transport substantial amounts of the amino
~31~2~
5b
acid compounds; converting the transported uncharged
compound to a form that cannot be retransported across the
membrane to the initial reaction mixture; and removing the
compound form that cannot be retransported across the
membrane to the initial reaction mixture.
In a method for the enzymatic resolution of racemic
carboxylic acid compounds, comprising the steps of:
hydrolyzing a racemic carboxylic acid ester compound in an
aqueous reaction mixture including a hydrolyzing enzyme to
form a charged enantiomeric compound and an uncharged
enantiomeric ester compound in the aqueous reaction
mixture; the improvement comprising transporting the
uncharged enantiomeric ester compound from the aqueous
reaction mixture across an ion rejection membrane into a
product mixture.
A method for the enzymatic synthesis of peptides
comprising the steps of: condensing first and second amino
acid compounds in an aqueous initial reaction mixture to
form an uncharged compound; the improvement comprising
transporting the uncharged compound into an aqueous second
reaction mixture across a membrane that will not transport
substantial amounts of the amino acid compounds; and
converting the transported uncharged compound to a form
that cannot be retransported across the membrane to the
initial reaction mixture.
Brief Description of Drawings
The foregoing and other objects and advantages are
attained by the invention, as will be apparent from the
following detailed description taken in conjunction with
the accompanying drawings; wherein:
Figure 1 is a schematic illustration of the enzymatic
synthesis of aspartame in accordance with the present
invention.
13l~2~
--6--
Figure 2 is a schematic representation of an
apparatus for practicing the process of the present
invention.
Figure 3 is a graph illustrating the quantity of
product (aspartame derivative) formed over time in
Examples 1 and 2.
Figure 4 i~ a graph illustrating the quantity of
product (aspartame derivative) formed over time in
Example 4.
Figure 5 is a graph illustrating the quantity of
product (aspartame derivative) formed over time in
Example 5.
Figure 6 is a graph illustrating the quantity of
product (aspartame derivative) formed over time in
Example 6.
Figure 7 is a graph illustrating the quantity of
product ~aspartame derivative) formed over time in
Example 7.
Figure 8 is a graph illustrating the quantity of
product (aspartame derivative) formed over time in
Example 8.
Figure 9 is a schematic representation of an
apparatu~ for practicing the present invention which
illustrates the vessels on the product side as described
in Example 9.
Figure 10 is a graph illustrating the quantity of
product (aspartame derivative) formed over time in
Example 9 with and without the utilization of an ion
exchange resin.
Best Mode~ for Carrvina Out the Invention
The invention disclosed herein provides a procedure
for driving to completion the enzymatic synthesis of
peptides in an aqueous reaction mixture by separating the
unchargad peptide intermediate, or derivative thereof,
from the reaction mixture by means of a membrane that
selectively transport~ the uncharged peptide out of the
.
7 131~2~
reaction mixture into a product mixture. Because the
membrane is substantially impermeable to the reactants
(charged molecules) removal of the peptide intermediate
from the reaction mixture causes a decrease of peptide
concentration at equilibrium that pushes the reaction
toward completion by mass action.
The membranes most useful in the practice of the
present invention are Immobilized Liquid Membranes ~ILM)
comprising a nonpolar liquid embedded in microporous
support material preferably a polymeric sheet that is
substantially impervious to the enzymes, reactants and
products. Hydrophobic polymers such as polypropylene are
preferred support materials. ILM modules can be produced
utilizing polypropylene hollow fibers. Celgard, a
registered trademark of the Celanese Corporation, 1211
Avenue of the Americas, New York, N.Y. 10036 and sold by
Celanese Fibers Marketing Corporation, Charlotte, N.C., is
an example of commercially available hollow fibers of
polypropylene. Potting compounds known in the art and
polyvinyl chloride pipe or tubing may optionally be
utilized in fabricating an ILM module. Another polymer
for fabricating the microporous support material is
TEFLON, a trademark of E. I. DuPont de Nemours & Co. for
fluorinated hydrocarbon polymers. A typical microporous
support i8 GORE-TEX, a trademark of W. C. Gore &
Associates, Inc.
The micropores pass through the support material and
should be sized so that an immobilized liquid will be held
therein by capillarity and will not escape therefrom when
subjected to, e.g., pressure differentials across the
membrane or other ordinary reaction condition~. Subject
to the foregoing limitation~ is advantageous to maximize
the contact area between the immobilized liquid and
reaction mixture to maximize the rate of transfer (flux)
of the uncharged peptide product across the membrane. It
will be appreciated that the preferred pore size will vary
depending on the properties of the particular immobilized
131~25~
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liquid, reactants employed, products produced, and like
factors; and further, that the optimum pore size can be
determined empirically by those skilled in the art. A
useful discussion of pore size selection is found in U.S.
Patent No. 4,174,374. The use and preparation of
immobilized liquid membranes are described in the
following references, S. L. Matson, J. A. Quinn, Cou~lina
Se~aration and Enrichment to Chemical Conversion in a
Membrane Reactor, paper presented at the AICHE Annual
Meeting, New Orleans, Louisiana (November 8-12, 1981) and
S. L. Matson and J. A. Quinn, Membrane Reactors in
Bioprocessin~, Ann. N.Y. Acad. Sci. 469, 152 (1986).
The immobilized liquid held in the microporous
support by capillarity should be water immiscible and a
good solvent for the uncharged peptide product which must
be transported across the membrane ~diffu~ed) at a
reasonable rate, i.e., good transport characteristics/high
flux; while, charged or ionized molecules on both the
reaction side and product side of the membrane are, for
the most part, not transported across the membrane in
either direction, i.e, good selectivity/ion rejection.
Selection of the best combination of support material
and immobilized liquid for use in an enzymatic synthesis
of a peptide in accordance with the present invention will
depend, in part, on the nature of the particular reactants
employed, products desired and solvents in the system.
rhe generally preferred immobilized liquids for the
practice of the present invention include water-immiscible
organic solvent~, such a~ alcohols of 6 to 20 carbons,
branched and unbranched, for example, n-decanol,
n-dodecanol, n-hexadecanol and mixture~ thereof. Also
preferred are mixtures of water immiscible organic
solvent~ including mixtures thereof. Such solvents
include but are not limited to N,N-diethyl-dodecanamide,
dodecane and 2-undecanone.
Another type of membrane useful in the practiGe of
the invention comprises hydrophobic solid films made of
~3~2~
g
organic polymers such as polyvinyl chloride or the like.
The preparation of these polymer membranes is well
described in the literature, for example, 0. J. ~weeting,
Editor, Science and TechnoloqY of Polvmer Films,
Interscience, New York (1968), while extensive application
of such membranes to the separation of gases and liquids
are discussed in S. T. Hwang and K. Kammermeyer, Membranes
in Separations, Techniques of Chemistrv, Vol. VII,
(A. Weissberger, editor) John Wiley & Sons, Inc., New York
~197S).
A preferred embodiment of the invention employs a
membrane reactor/separator system which provides an
aqueous reaction mixture or phase circulating in contact
with one side of an ILM membrane and a product aqueous
phase or mixture circulating countercurrently at the
opposite surface of the membrane. S. L. Matson and J. A.
Quinn, Ann. N.Y. Acad. Sci. 469, 152 (1986). The pH and
temperature of the reaction and product phases are
maintained at a value that keeps the reactants in a form
that minimizes their transport across the membrane at pH's
between about 4.0 and 9Ø Transport of the uncharged
peptide intermediate from the reaction phase to the
product phase i3 driven by the concentration gradient
across the membrane created by increasing neutral or
uncharged pep~ide concentration in the reaction phase.
The transport activity or flux across the membrane can be
significantly enhanced by the simultaneous, irreversible
conversion of the transported peptide, in the product
phase, to a species that cannot back-diffuse. For
example, the latter conversion may re ult in the formation
of a polar peptide that cannot back-diffuse and thus
supplement the driving force to achieve completion of the
coupling reaction. An example of a membrane
reactor/separator that could be adapted for the practice
of the present invention is found in U.S. Patent No.
4,187,086.
131~254
-10-
Available alternative membrane reactor/separator
configurations that could be adapted to practice of the
present invention include the hollow fiber modules
described in U.K. Patent Application 2,047,564 A, and
conventional plate and frame-type filter devices well
known in the art.
In addition to selective transport of the uncharged
peptide the membrane provides a barrier between the
reaction phase and product phase that prevents undesirable
mixing of, and reactions between, the component~ of each
phase.
In a preferred membrane reactor/separator configured
in accordance with the present invention~ the chemical
equilibrium between the reactants is actually "pulled"
across the membrane by conducting an irreversible
conversion of the transported uncharged peptide, to a
membrane impermeable species, on the product side of the
membrane. This type of membrane reactor/separator employs
a coupled two enzyme (E and E ) process of the general
type:
El E2
A ~ B ~ C D
The charged reactants A and B are amino acids and/or small
peptides which are condensed with the aid of peptide
forming enzyme El to form the uncharged intermediary
peptide C which i Q selectively transported across the
membrane to the product side. It i8 understood that
reactive functional groups in the reactants that do not
participate in the desired reaction may be protected or
blocked, where necessary, to prevent undesirable side
reaction~ and/or changes in the products. On the product
side of the membrane uncharged peptide C is converted to
charged peptide D which cannot back--diffuse across the
membrane, causing the chemical equilibrium in the reaction
mixture to shift toward the production of more C.
13142~
This concept is illustrated in Figure 1, for the
specific case of the enzymatic condensation of
D,L-phenylalanine methyl ester with (N-and ~-protected)
N-formyl-~-benzyl-L-aspartate, in the presence of
thermolysin at about pH 5.5.
In the reaction scheme illustrated in Figure 1 the
reactant A is D, L-phenylalanine methyl ester and B is
N-formyl-B-benzyl-L-aspartate. The reactants are
condensed on the reaction side of the membrane by the
enzyme E1 thermolysin forming the uncharged peptide C.
The pH is selected to maintain the reactants in their
charged state and thus minimize their diffusion across the
membrane along with uncharged peptide C.
Although the chemical equilibrium for the
condensation reaction largely favors the reactant A and
B species, diffusion of the uncharged peptide product C
across the membrane to the product side requires the
constant production of C to maintain the chemical
equilibrium on the reaction side of the membrane.
On the product side on the membrane an esterase
enzyme E2 quickly and irreversibly converts the uncharged
peptide C diffused across the membrane to charged peptide
D which cannot back-diffuse to the reaction side. Thus
the chemical eguilibrium on the reaction side i~
effectively "pulled" across the membrane and toward the
production of uncharged product C. Thereafter, the
peptide D i~ converted to aspartame by acid hydrolysis,
which removes the formyl and benzyl protecting groups,
followed by C-terminal esterification with methanol.
The foregoing method may be practiced in a
countercurrent flow membrane reactor/separator 10, as
shown in Figure ~, operating under controlled conditions
of pH and temperature, 80 that a reaction mixture
compri~ing a N-acyl-~-substituted-L-aspartic acid, e.g.,
N-formyl-~-benzyl-L-aspartate, having a free #-carboxylate
group ~(electronegative species) which is allowed to
condense with a reactant, e.g, D, L-phenylalanine methyl
.
131~2.5~
-12-
ester, having a protonated free -amino group
(electropositive species), under the catalytic action of
proteases active in the pH range of about 4.0-9.0, to
yield a fully protected L-aspartyl-L-phenylalanine
dipeptide bearing no ionized groups (electroneutral
species). On the reaction side of the me~brane, 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 mi~ture or sweep including fully protected
uncharged peptide, e.g., N-formyl-B-benzyl- L-aspartyl-L-
phenylalanine methyl ester transported across the
membrane, is circulated from product reactor tank 22,
aided by pump 24, through product sweep in-conduit 26,
through the product side of separator 18, to product sweep
out-conduit 30. This product mixture includes a second
enzyme E2 an esterase, or other ~uitable reagent, that can
cleave at least one of the protected groups borne by the
uncharged peptide, thus generating an electrocharged
species that cannot escape the sweep stream by
back-diffusing through the membrane. If an esterase is
utilized, a preferred esterase will have proteolytic
activity and a preferred pH range of from about 6.0 to
9Ø Aminoacylas~ I, a-chymotrypsin and subtilisin A are
examples of esterases considered useful in the present
invention.
The charged product may be periodically discharged
and/or continuously removed from the product phase by
conventional means such as ion exchange resins, and the
remaining effluent may be recycled through the system.
The resulting product bound to the ion-exchange resin may
be desorbed and recovered using conventional procedures.
The selection of appropriate deprotection reagent(s)
is determined by the chemical nature of the protecting
groups used on the reactants, such as,
N-protected-L-aspartic acid, and a~ indicated above the
131~25~
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choice of protecting groups is in turn dictated by
structural constraints imposed by the active site of the
condensing enzyme.
In general, the enzymes useful in the practice of the
present invention are proteolytic enzymes, sometimes
called proteases, which may be divided into two groups.
The more commonly used are endopeptidases, which only
cleave inner linkages, and exopeptidases, which only
cleave terminal linkages. Useful enzymes include serine
proteinases, (EC 3.4.21) such as chymotrypsin, trypsin,
subtilisin BNP' and Achromobacter protease; thiol
proteinases ~C 3.4.22), such as, papain; carboxyl
proteinases (EC 3.4.23), such as, pepsin; and
metalloproteinases (EC 3.4.24), such as, thermolysin,
prolisin, Tacynasen N ~ St. caespitosus) and Dispase.
Binding of the enzymes to insoluble supports, following
procedures well known to practitioners of the art, may be
incorporated to the practice of this invention; and
although binding the enzymes is not an essential step, it
may be desirable in certain applications. Among the many
proteases potentially useful for the practice of this
invention, thermolysin [E.C. 3.4.24] is the condensing
enzyme most preferred because of its remarkable
thermostability, wide availability, low cost and broad
useful pH range between about 5.0 to 9Ø Other preferred
proteases include pepsin and penicillopepsin [T. Hofmann
and, R. Shaw, Biochim. Bio~hYs. Acta 92 543 (1964)] and,
the thermostable protease from Penicillium duponti [S.
Emi, D. V. Myers and G. A. Iacobucci, BiochemistrY 15, 842
(1976)]. They would be expected to function at about pH
5.5, exhibit good stability at such pHs, and do not
require the presence of Zn++ and Ca++ ions to maintain
their activity.
The practical realization of enantiomeric selectivity
that is, in the case described above, production of only
the L,L isomer of the peptide C, is directly related to
the enzyme selected, optimal functioning of the membrane,
131~2~
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chemical nature of the support material and pH of the
aqueous reaction phase.
One preferred membrane for practicing the
above-described specific method is a microporous
polypropylene support material including a mixture of
n-hexadecanol and n-dodecane immobilized therein. This
membrane is available from Bend Research, Inc.,
64550 Research Road, Bend, Oregon 97701, U.S.A. under the
tradename/designation "Type 1 Hollow Fiber Selective
Dialysis Membrane" and is preferred with the reactions of
Examples 1-4. Another preerred membrane utilizes Celgard
Type 2400 polypropylene hollow fibers (Celgard is a
registered trademark of the Celanese Corporation, 1211
Avenue of the Americas, New York, N.Y. 10036. Celgard can
be obtained from Celanese Fibers Marketing Corporation,
Charlotte, N.C.) as the microporous support material
including a mixture of 30% v/v N,N-diethyl-dodecanamide in
dodecane as the water-immiscible organic liquid
immobilized by capillarity in pores of a microporous sheet
of Celgard. This membrane wa~ obtained from Bend
Research, Inc., 64550 Research Road, Bend, Oregon 97701,
U.S.A. under the tradename/designation "Type 2 Hollow
Fiber Selective Dialysis Membrane" and is preferred with
the reactions of Examples 5-9. Celgard T,vpe 2400
polypropylene hollow fibers having a pore size of 0.025 -
0.050 ~m and a wall thickness of 25 ~m were utilized in
the Type 2 Hollow Fiber Selective Dialysis Membrane of
Bend Research, Inc. When operated at pHs of about 5.5,
these membra~e~ exhibit a high selectivity, for example,
when practicing the process of Figure 1, selectivity in
the range of about 500:1 (w/w) in favor of the uncharged
peptide species have been measured. That is, 500
milligram~ 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
131~25~
-15-
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 electro
charges in intermediate peptide. Table I below lists a
series of selected combinations of protecting groups and
deprotection conditions useful in connection with the
practice of this invention.
13l~2~ll
-16-
TABLE I. PROTECTING GROUPS AND DEPROTECTION REAGENTS
USEFUL IN THE SYNTHESIS OF ASPARTAME (APM).
Rl~R3
1 2 3 R Group
R R R Removed/Reaaent Product
~CH20 OCH20CO_ CH30- Rl, R2/(Pd)H2 APM
2- -CH = O CH30- R2/H30 ~-benzyl-APM
~CH20 -~H = O CH30- R3/Fungal esterase N-formyl-~-
benzyl-L-
aspartyl-L
phenyl-
alanine
ter-BuO -CH = CH30- Rl,R2/H30 APM
C~30 -CH = CH30 R3/fungal eQterase N-formyl-isoAPM
CH30 -CH = CH30 R3/~-chymotryp~in N-formyl-isoAPM
N~2 ~CH20CO-CH30- Rl/asparaginase N-CBZ-APM
L-dihydroorotyl-L- Rl,R2/hydan- APM
phenylalanine toinase
methyl ester
. ~,~CH3
H~j~H
(~ "
C~30 PCH20CO-CH30- R /fungal N-CBZ-isoAPM
esterase
H30 ~CH2CO- CH30 R /penicillin APM-~-methylester
acylase
131~2~
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As a result of the enantioselectivity of selected
condensation enzymes and the functional discrimination
exerted by the membrane, the practice of the invention
using N-formyl-L-aspartyl-~-benzyl ester and D,
L-phenylalanine methyl ester could achieve a 99.8%
enantiomeric resolution of the racemic phenylalanine
methyl ester reactant, the L-enantiomer appearing as
N-formyl-L-aspartyl(B-benzyl)-L-phenylalanine methyl
ester, an aspartame derivative, with the unreacted
D-enantiomer remaining in the reaction phase.
The D-phenylalanine methyl ester remaining in the
reaction phase may be recovered therefrom, re-racemized to
the D,L-stage, and recycled into the feedstock. This is
an advantage common to many processes employing racemic
amino acid reactant~, e.g., the '311 process described
above.
The economic advantages of the present invention
derive, at least in part, from the use of a racemate feed
reactant rather than a more expensive pre-resolved
L-enantiomer. This advantage i 9 made possible by the
in situ optical resolution of the D,L-phenylalanine methyl
ester due to: (a~ the enantioselectivity of the
condensing enzyme; and (b) the high selectivity of the
membrane in favor of the uncharged species.
Preferred methods for the low-cost synthesis of
racemic phenylalanine are those based on the utilization
of benzaldehyde via 5-benzalhydantoin sometimes called the
Grace process, or the catalytic carbonylation of benzyl
chloride to phenylpyruvic acid, a procedure developed by
Sagami Chemical Research Center, Tokyo, Japan (sometimes
referred to as the Toyo Soda process of Toyo Soda
Manufacturing Co., Ltd., Yamaguchi, Japan).
It will be appreciated by those skilled in the art
that the practice of this invention is not necessarily
restricted to the synthesis of peptide sweeteners, such
as, aspartame or its analogs and derivatives. The
invention may also be used for the ~ynthesis of other
131~L2~
-18-
useful peptides, di-, tri-, tetra- and pentapeptides, or
peptides of higher complexity, that are capable of
diffusing through a permselective membrane at a reasonable
rate. For example, considering the bond specificity of
thermolysin, and assuming the presence of only one
thermolysin sensitive bond in the product ~indicated by
the arrow in the formula below), one could synthesize
met-enkephalin (1) by following scheme:
(Bzl)
(tOCO)-Tyr-Gly-Gly-OH + H-Phe-Met-(Ot)-C(CH3)3
t~ Thermolysin pH 5.5
(Bzl)
(+OCO)-Tyr-Gly-Gly-Phe-Met- (t
~ H30
H-Tyr-Gly-Gly-Phe-Met-OH (1)
The feasibility of a stepwise total enzymatic
synthesis of met-enkephalin using ~-chymotrypsin and
papain has been documented in the literature. See:
W. Kullmann, J. Biol. Chem 255, 8234 (1980).
Another potentially useful application of the present
invention i8 in the enzymatic synthesis of
Gramicidin S (2) by the following scheme:
131~2~
--19--
2 H-Val-Orn(CBZ)-Leu-D-Phe-Pro-OH
~ ~ Thermolysin pH 5.5
Le ~
(CBZ) ~~-Phe
Val / ~ ro
Pro ~Val
Phe~ rn(CBZ)
\ Leu
Pd/H2
Leu
D-Phe \
~al Pro
/
-Phe ~
/ (2)
~ Leu
Various other examples of the enzymatic synthesis of
useful peptide products where the present invention may
find ap~lication and de~cribed in the literature are the
synthe~is of angiotensin, substance P, eledoisin,
caerulein and Leu-enkephalin.
Tho e skilled in the art will appreciate that the
present invention may also find application to other
system~ besides peptides.
For example, the u~e of e~ter hydrolases like pig
liver esterase lEC 3.1.1.1] for the asymmetric resolution
of prochiral dicarboxylic acid diester~ into chiral
monoesters is well known [C.J. Sih et al., Ann N.Yl Acad.
Sci. 471, 239 (1986)]. The same enzyme can be used in
~ 3 1 ~ 2 ~ L?~
-20-
the fashion described in this invention for the resolution
of racemic carboxylic acid compounds, through the
selective transport of a chiral ester through the membrane
and the retention of the non-reactive enantiomeric acid in
the reaction phase.
The following detailed Examples are presented to
further illustrate the present invention.
Example 1.
An aspartame derivative was prepared in accordance
with the present invention as follows:
To a solution containing 5.02g (20 ~mol~s)
N-formyl-L-aspartyl-~-benzylester, 4.31 g (20 mmoles) L-
phenylalanine methyl ester in 100 mL in water, adjusted to
pH 5.5, was added 500 mg thermolysin enzyme (Daiwa Chem.
Co., Osaka, Japan) representing a total of 8 x 105
proteolytic units.
The resulting clear reaction mixture was incubated
for 15 hrs at 40C, when the presence of insoluble
dipeptide N-formyl-B-benzyl-L-aspartyl-L-phenylalanine
methyl ester became apparent. The resulting mixture was
then placed in a 200 mL vessel, connected to the reaction
side, in thi~ ca~e tube side, of an experimental
hollow-fiber separator of a Bend Research, Inc. "Type 1
Hollow Fiber Selective Vialysis Membrane" that provided
1 squsre foot of membrane area. The product side of the
membrane ~shell 3ide of the separator) was connected to a
source of aqueous (product) mixture (total volume = 200
mL) containing 500 mg of the enzyme Acylase I (EC
3.5.1.14) from A~e~g i lu~ S~. (Sigma A 2156), at pH 7.5.
ThiC enzyme, usually described as an aminoacylase, was
found to function as a C-terminal esterase, on both
N-acetyl- and N-formyl-~-benzyl aspartame.
The reaction and product mixtures were circulated at
room temperature through the hollow fiber separator
countercurrently at the rate of 600 mL/min, with the
assistance of peristaltic pumps. The configuration of
2 ~ ~
-21-
this apparatus resembles that illustrated in Figure 2.
Since both reactions (condensation and the ester
hydrolysis) are protogenic, the pH in both the reaction
and product mixtures drops as the process progre~ses.
Constancy of pH, in both the reaction and product
mixtures, was maintained through the use of pH stats.
The formation of N-formyl-B-benzyl-L-aspartyl-L-
phenylalanine was monitored by HPLC. Chromatographic
analysis was conducted on a Tracor Model 995 instrument
along with a LDC Spectromonitor II detector set at 254 nm
for the detection of the amino acids, fully protected
product dipeptide, and dipeptide. The column used was a
NOVA-PAK C18 Radial-Pak cartridge, 8mm x lOcm, housed in a
Millipore Waters RCM-100 Radial Compression Module.
The mobile phase used for the detection of the fully
protected dipeptide was a v/v mixture of 45% methanol
(HPLC grade) 5% tetrahydrofuran (HPLC grade); and 50~ of a
1% KH2P04 buffer solution. For the detection of the
product dipeptide, the mobile phase consisted of a v/v
mixture of 40% methanol and 60% of a 1% KH2P04 buffer
solution (1 mL of triethylamine per liter solvent was
added to minimize tailing and the p~ was adjusted down to
4.3 using 80% phosphoric acid). The flow rate was kept at
1 mL/minute.
The HPLC data relating to formation of
N-formyl-~-benzyl-L-aspartyl-L-phenylalanine is summarized
in Table II below, and is expressed as the total amount
(mg) of product dipeptide accumulated in the product
solution as a function of time.
The value of the uncharged dipeptide concentration at
equilibrium which corresponds to its saturation point in
water at pH 5.5, was found to be about O.OS~ at 25C. The
amount of uncharged dipeptide transported per square foot
of membrane per hour was found to be about 200 mg,
indicating that maintenance of the equilibrium required
dissolution of insoluble dipeptide and/or dipeptide
synthesi~ de novo. Almost complete dissolution of the
,j,r .,
. .~
~1425~
-22-
insoluble uncharged phase dipeptide in the reaction
mixture was observed after about 5 hrs, when the reaction
was stopped. A plot of the data of Table II is shown in
Figure 3. The linear function indicates that the transfer
of peptide across the membrane proceeds at a steady state.
The observed rate of formation of product dipeptide of
about 200 mg/ft2/hr (Table II) was confirmed similar to
the flux of uncharged dipeptide across the membrane (190
mg/ft2/hr) measured at 0.05% in water as was anticipated
on theoretical grounds.
At this point the described system would be expected
to continue in steady state if continuous addition were
made to the reaction mixture of the two amino acid
reactants, at a rate of about 120 mg/hr each, to keep the
system saturated in uncharged dipeptide.
In order to fully realize the membrane's selectivity
the intercalation of a second membrane in series with the
first membrane before the contact with the second enzyme
may be neces~ary because the selectivity acro~s a single
membrane is lower due to the high amino acid concentration
in the reaction solution.
The product solution (200 mL) was recovered, adjusted
to pH 2.5 and cooled at 4C overnight. The precipitate
_ollected was recovered and recrystallized from MeOH:H20
to give 307 mg of N-formyl-~-benzyl-L-aspartyl-
25
L-phenylalanine [~ D = -5.6(C=1.2; EtOH), identical
(IR, 13C-NMR) to an authentic sample prepared by the batch
hydrolysis of N-formyl-~-benzyl-aspartame with Asperaillus
esterase, 1125 = -5.3(C=1.3; EtOH)
-23- 131~25~
Table II.
Time (min) Amount Pe~tide/Product solution (ma)
124
228
120 412
180 617
240 882
300 lOlS
Exampl~ 2.
An experiment similar to that of Example 1 was
conducted, except for the use of 8.63g D,L-phenylalanine
methyl ester instead of the L-enantiomer. The results are
summarized in Table III. Isolation of the uncharged
peptide from the 200 mL of product ~olution gave 310 mg of
product, [~] 5 = -6.4(C=1.4; EtOH), identical in all
respects (IR, 13C-NMR) to an authentic sample of
N-formyl-~-benzyl-L-aspartyl-L-phenylalanine.
Table III.
Time (min) Amount PePtide/Droduct solution tma)
166
258
120 475
180 686
240 996
300 1073
A plot of the data summarized in Table III (Figure 3)
again showed the existence of a steady state process when
the reactor wa~ operated with D,L-phenylalanine methyl
ester. The stereospecificity of thermolysin is
demon~trated by the exclusive formation of the same
L,L-dipeptide described in Example 1. The D-phenylalanine
methyl ester retained in the tube phase (reaction mixture)
did not inhibit the overall ~inetics of peptide formation.
ExamPle 3.
A mixture of 1.0 g N-formyl-~-benzyl-L-aspa~tyl-
L-phenylalanine, prepared in accordance with Example 1,
2 ~ ~
-24-
4.0 mL water, 4.0 mL tetrahydrofuran, and 1.0 mL conc.
hydrochloric acid (12N) was heated at reflux for 9 hrs.
The mixture was then cooled and the pH adjusted to 4.0
with 50% NaOH solution. The tetrahydrofuran was then
removed by evaporation at < 35 and 20 mm Hg.
Crystallization was completed by storage at 5C for 1 hr,
the sample then filtered, washed with 1 mL ice water, and
dried in vacuo to give 367 mg of white solid. This
material was identical to an authentic sample of aspartyl
phenylalanine by HPLC and IR comparison. [a]2D = +12 (C
= 0.5; 0.1 N HCl in 50% MeOH).
Aspartyl phenylalanine has been converted to
aspartame by treatment with methanol and hydrochloric
acid, as described in G.L'. Bachman and B.D. Vineyard,
U.S. Patent No. 4,173,562, example #1.
Example 4.
An experiment similar to that of Example 1 was
conducted, except for the use 5.65g (20.1 mmoles)
N-carbobenzoxy-L-aspartic acid ~-methyl ester and 4.38g
(20.3 mmoles) L-phenylalanine methyl ester as reactants.
The amino ac$ds were dissolved in 100 mL water, the pH of
the solution adjusted to 5.5, and 500 mg of thermolysin
Daiwa (8 x 105 proteolytic unlts) was added. The solution
was preincubated for 15 hours at 40C, when a substantial
amount of N-CBZ-(~-methyl ester)-L-asp-L-phenylalanine
methyl e~ter was precipitated. The suspension was
connected to the tube side of a "Type 1 Hollow Fiber
Selective Dialysis Membrane" (Bend Research Inc.)
containing 1 ft2 of'membrane surface, and the machine was
operated at room temperature for 5 hours against a shell
side phase of 200 mL water containing 500 mg Acylase I
(Sigma) at pH 7.5. The accumulation of peptide product in
shell phase was monitored by HPLC, and the results are
reproduced in Table IV and Figure 4.
After 5 hours run the reaction was stopped, the shell
side phase (200 mL) was recovered, adjusted to pH 2.5, and
131~2~
-25-
stored overnight at 4C. The product precipitated was
collected and recrystallized from CH30H:H20 to yield
405 mg (86% recovery) of N-CBZ-(~-methyl
ester)-L-asp-L-phenylalanine (N-CBZ-lso-APM) I~] D = +6.0
(C = 1.1, EtOH), identical (13C-NMR) to an authentic
sample of N-CBZ-iso-APM, [~]2D = ~5-5 (C = 1.1, EtOH),
prepared by chemical coupling and partial esterolysis with
Acylase I.
Table IV.
Time (min) Amount ~e~tide/~roduct solution Imal
177
214
120 333
1~0 381
240 459
300 470
As observed before in the experiments of Examples 1
and 2, the plot of Figure 4 indicates that the
accumulation of N-CBZ-iso-APM proceeded at a steady rate
of about 200 mg/hr.
The conversion of N-CBZ- iso-APM to APM can be
practiced under the conditions described in Example 3.
ExamPle 5.
The aspartame derivative, N-formyl-(~-methyl)-asp-phe
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, 10.00 g (48 mmoles)
L-phenyl alanine methyl e~ter in 100 mL water, adjusted to
pH 7.0, was added 770 my thermoly~in enzyme (Daiwa
Chemical Co., Osaka, Japan) representing a total of 1.2 x
106 proteolytic units. The resulting clear solution was
incubated for 1 hr at 40C, when HPLC analysis indicated
the presence of 433.8 mg of
N-formyl-(~-methyl)-asp-phe-OMe. The solution was cooled
to 25C, the pH adjusted to 5.0, and the solution placed
.
13l~2~
-26-
in a 200 mL vessel connected to the tube side of
hollow-fiber separator ("Type 2 Hollow Fiber Selective
Dialysis Membrane", Bend Research Inc.) that provided 0.5
ft (450 cm ) of a ILM made of 30% v/v
N,N-diethyl-dodecanamide in dodecane. The shell side of
the separator was connected to the product vessel
containing 500 mg of the enzyme Acylase I (EC 3.5.1.143
from AsPeraillus Sp. (Aminoacylase AMANO, Nagoya, Japan),
at pH 7.5.
The two phases were circulated at 25C
countercurrently, at the rates of SO mL/min. ~tube phase)
and 500 mL/min. (shell phase), with the assistance of two
peristaltic pumps using the configuration illustrated in
Figure 2. Constancy of pH in both phases was secured
through the use of pH stats.
The formation of N-formyl-(~-methyl)-asp-phe 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, housed in a Millipore Waters
RCM-lOO Radial Compression Module.
The mobile phases used for the analysis were:
(a) for N-formyl-(~-methyl)-asp-phe-OMe: 40% v/v
methanol in 0.1% KH2P04 buffer pH 4.6;
(b) for N~formyl (~-methyl)-asp-phe: 20% v/v
methanol in 0.1% KH2P04 pH 4.6; flow rat~s used were
1 mL/min for both analyses.
The data relating to the formation of
N-formyl-(~-methyl)- asp-phe (product dipeptide) is
reproduced in Table V below, and is expressed as t~e total
amount (mg) accumulated in the 200 mL shell phase as a
function of time.
13142~
-27-
Table V
Time (min) Amount pePtide/product solution (ma)
130
120 230
180 280
240 360
300 400
A plot of the data of Table V ic shown in Figure 5.
At the end of the run, HPLC analysis indicated the
presence of 283 mg of N-formyl-(~-methyl)-asp-phe-OMe in
the tube phase. These value~ allowed to calculate the
amount of peptide synthesized during the operation of the
reactor.
Ptr = peptide transferred into shell phase = 400. 336 =
417.4 mg;
PO = initial peptide in tube phase = 433.8 mg;
PT = peptide remaining in tube phase at the end of the run =
273 mg;
Ptr (Po PT) Psyn;
syn Ptr (PO ~ PT~ = 266.6 mg; and
Vsyn - 266.6 = 53.5 mg/hr. 100 mL.
The Vsyn of 53.5 mg/hr. 100 mL coincides with the
rate of synthesis (500 mg/hr. L) measured for the forward
velocity in equilibration studie~ done with
N-formyl-(~-methyl)-L-aspartic acid and L-phenylalanine
methyl ester in the presence of thermolysin.
131~25~
-28-
The product solution (200 mL) was recovered, adjusted
to pH 2 with 1 N HCl and extracted twice with 200 mL
EtOAc. The combined extracts left a white residue upon
evaporation, that after recrystallization from
EtOAc/hexane yielded 100 mg of
N-formyl-(~-methyl)-L-asp-L-phe, [a]25 = +0 700
(c, 0.29;MeOH), identical (IR, 13C-NMR) to an authentic
sample prepared by the batch hydrolysis of synthetic
N-formyl-(~-methyl)-L-asp-L-phe-OMe with AsPerqillus
esterase, [12D = +0.80 (C, 0.29; MeOH~.
Example 6.
The experiment described in Example 5 was scaled up
~n a Ben~d T~ 2 Hollow firber Dialysis Membrane (Bend Research
Inc.), containing 1 ft2 of liquid membrane (30% v/v
N,N-diethyl- dodecanamide in dodecane). The tube phase
contained 40 g L-phe-OMe, 24 g N-formyl-(~-methyl)-L-asp
and 3.08 g thermolysin Daiwa (a total of 5 x 106
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-(~-methyl)-L-asp-L-phe-OMe
was found to be present. The solution was cooled to 25C,
adjusted to pH 5.0 with 1 N HCl, and connected to the tube
side of the hollow fiber separator. The shell phase was
made of 400 mL water, pH 7.S, containing 2 g aminoacylase
I (Amano). The two phaseR were circulated
countercurrently at 25C for 5 hrs., as described in
Example 5. The results are summarized in Table VI and
Figure 6.
Table VI
Time (min) Amount pe~tide/product solution (m~)
204
251
120
180 634
240 748
300 843
131~2~
-29-
At the end o the run, the amount of
N-formyl-(~-methyl)-asp-phe-OMe remaining in tube phase
was 586 mg.
The highest transport value observed (404 mg/hr)
during the first 30 min. is the result of the high initial
peptide concentration produced during the preincubation
period. Departure from equilibrium caused by the
transport of peptide set in the synthesis of more peptide,
thus establishing a steady state condition after the first
hour into the run, at the expected level of 200 mg/hr
(Figure 6).
Example 7.
An experiment similar to that of Example 5 was
conducted, except for the use of 10.00 g of
D,L-phenylalanine methyl ester instead of the
L-enantiomer. The results, presented in Table VII and
Figure 7, clearly show that the observed rate of formation
of N-formyl~ methyl)-L-asp-L-phe was one-half of that
seen with L-phe-OMe (Example 5, Table V), as expected from
the enantioselectivity of thermolysin and the prior
results of Examples 1 and 2.
Table VII
Time (min) Amount pePtide/product solution (ma)
10.6
25.5
120 33.2
180 55-3
2~0 59.6
300 69.1
Example 8.
Membrane-assisted enzymatic resolution of
D,L-phenylalanine methyl ester was utilized in accordance
with the present invention as follows: To a solution of
1.0 g (5.6 mmoles~ of D,L-phenylalanine methyl ester in
13~2~
-30-
100 mL water, pH 7.5, was added 500 mg of aminoacylase
I (Amano Pharmaceutical Co., Nagoya, Japan). The mixture
was allowed to react at 25C for 30 min., at the end of
which the presence of 266 mg L-phe (1.6 mmoles) and 712 mg
D,L-Phe-OMe (4 mmoles~ was observed by HPLC analysis.
This solution was fed to the tube (reaction) side of a
hollow-fiber Celgard supported ILM separator ("Type 2
Hollow Fiber Selective Dialysis Membrane", Bend Research
Inc.) containing 0 5 f~2 of a 30% v/v
N,N-diethyl-dodecanamide in dodecane liquid film in it.
The product side of the membrane (shell side of the
separator) was filled with 200 mL water adjusted to pH 2.0
with diluted HCl. The two phases were circulated through
the separator countercurrently at the rate of 200 mL/min.,
with the assistance of peristaltic pumps (Figure 2). The
separation pro~eeded through the continuous addition of
D,L-Phe-OMe to tho tube phase, done at a rate of about 1 g
D,L-Phe-OMe per hour. A total of 30 mL of a 7% solution
of D,L-Phe-OMe (2.1 g; 11.7 mmoles) in water pH 7.5 was
added in a period of 2 hrs. The pH of both phases was
kept constant by the use of pH stats.
The course of the re~olution was followed by HPLC, on
samples taken from koth phases every 30 min. The HPLC
instrumentation and procedures are those described in
Example 5. The mobile phase used in this case was 20% v/v
methanol in 0.1% KH2P04 buffer pH 4.6; with a flow rate of
1 mL/min. The results, presented in Table VII and plotted
in Figur~ 8, showed the accumulation of L-Phe in tube
phase and of D-Phe-OMe in shell phase. At the end of the
experiment both phases were recovered and worked out a~
follows:
(a~ Tube phase: The contents (130 mL) were adjusted
to p~ 8.~ 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 solution passed through a
2.5 x 2~ cm Dowex 50 (NH4) column. After washing
13142~
-31-
with 200 mL water, the product was eluted with 200 mL
of 10~ NH40H. The eluate was concentrated to 50 mL
in vacuo, and the solution freeze-dried. Yield: 249
mg, white solid,
L-phenylalanine, [a]25 ~ -29.2 (c, 2; H20), lit.
(Aldrich) la] D = ~35 0 ~c,2;H20), optical purity
92%.
(b) Shell Phase: (200 mL) was adjusted to pH 8.5
with diluted NaOH and extracted with 2 x 50 mL EtOAc.
The organic extract was dried over anh. Na2S04,
evaporated to dryne~s, dissolved in 50 mL water
acidified to pH 3.0 with lN HCl and then
freeze-dried, to yield 989 mg (4.6 mmole) of
D-Phe-OMe HCl, white solid, [a] 25 = -21.0
(c, 2; EtOH), lit. (Aldrich) [a] ~5 = -32 4
(c, 2; EtOH), optical purity 83%.
Based on the permeability value of 32 mg/cm2. min
found for Phe-OMe (water, pH 8, 25C) on this membrane,
the expected flux for a membrane area of 450 cm2 (0.5ft2)
was 880 mg/hr. Table VIII shows that the amount of
Phe-OMe transferred into the shell phase at the end of the
first hour was 860 mg, suggesting that the transport was
operating under membrane-limiting conditions.
Table VIII
.
D,L-Phe-OMe Tube Phase Shell Phase_
1.0 266 712 0(0 min)
1.7 473 617 537(30 min)
2.4 615 531 860(60 min)
3.1 743 6281154(90 min)
.
Batch resolution of D,L-Phe-OMe through the
enantioselective hydroly~is of the methyl ester function
13142~
-32-
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. ~hem.
Commun. 1986, 1514]. A hydrolase is required for membrane
resolution of racemic carboxylic acid compounds. A
hydrolase which is an esterase which is an esterase such
as aminoacylase I, ~-chymotrypsin and subtilisin A can be
utilized to resolve a D,L-amino acid compound such as
D,L-phenylalanine methyl ester. The Aminoacylase I is
generally preferred for the demethylation of peptides over
other esterolytic enzymes such as subtilisin A and
a-chymotrypsin having strong proteolytic activities.
If the above described resolution of
D,L-phenylalanine methyl ester is utili~ed to produce a
peptide as described in the present invention, the
L-phenylalanine produced i5 methylated by standard
methylation means known in the art.
This example may ~e adaptable for resolution of other
racemic carboxylic acids. For example, a racemic
carboxylic acid ester compound in an aqueous reaction
mixture including a hydrolyzing enzyme can be hydrolyzed
to form a charged enantiomeric compound and an uncharged
enantiomeric ester compound in the aqueous reaction
mixture. The uncharged enantiomeric ester compound is
then transported from the aqueous reaction mixture across
an ion rejection membrane of the pre~ent invention
-~including Type 1 or Type 2 Hollow Fiber Selective Dialysis
Membrane~ from Bend Research, Inc. In one type of example
such as Example 8, the racemic carboxylic acid ester
compound is a D,L-amino acid ester compound; the charged
enantiomeric compound is a L-amino acid compound and the
uncharged enantiomeric ester compound is a D-amino acid
ester compound. It will be appreciated that the selection
of enz~nes and reaction condition~ is within the
understanding and knowledge of persons skilled in the art
of the present invention.
131~25~
-33-
- Continuous or batch processing means are provided by
this Example in that the desired enantiomer of the
reactant can be produced and added to the reaction
mixture.
Example 9.
Synthesis of N-formyl-(~-methyl)-L-asp-L-phe in
accordance with the present invention was conducted
utilizing immobilized aminoacylase I and ion-exchange
resins for the removal of permeable products as follows:
To a solution of 10.0 g (48 mmoles) L-phenylalanine methyl
ester and 6.0 g (35 mmoles) of
N-formyl-(~-methyl)-L-aspartic acid in 100 L deionized
water, adjusted to pH 7.0, was added 770 mg of thermolysin
(Daiwa Chemical Company, Osaka, Japan) representing a
total of 1.2 x 106 proteolytic units. The resulting
sol.ution was incubated for 1 hr. at 40 C, when HPLC
analysis indicated the presence of 383 mg of
N-formyl-(~-methyl)-asp-phe-OMe. The solution was cooled
to 25 C, the pH adjusted to 5.0, and the solution was
placed in a 200 mL vessel 40 connected to the tube side 46
of a hollow fiber separator 44 ("Type 2 Hollow Fiber
Selective Dialysis Membrane", Bend Research, Inc.)
containing 900 cm2 (1 ft2) of a hydrophobic liquid
membrane made of 30% N,N-diethyl- dodecanamide in
dodecane. The shell side of the separator 48 was arranged
as a closed circuit made of a series of connecting vessels
illustrated in Figure 9. The solution returning to the
separator 44 was adjusted to pH 4.0 in order to protonate
the L-phe-OMe copermeating with the dipeptide
N-formyl-(~-methyl)-asp-phe-OMe. Circulation through a
column of Dowex 50 (Na+) 50 removed the positively charged
L-phe-OMe, leaving the uncharged dipeptide in solution.
The effluent was adjusted to pH 7.0 and submitted to the
action of the aminoacylase I, immobilized over
DEAE-Sephadex 52 ~ T. Tosa, T. Mori and I. Chibata,-Aqr.
Biol. Chem. 33, 1053 (1969)]. The resulting dipeptide
~, .~- ,.
,~. ,,
131~25~
-34-
N-formyl-(~-methyl)-asp-phe was negatively charged at that
pH, and was subsequently captured by the Dowex 1 (Cl-)
resin 54. The effluent was returned to the membrane
separator prior adjustment to pH 4.0, thus closing the
loop.
The tube 46 (100 mL) and shell 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 sampling of the shell phase was done on the
effluent from the second enzyme (E2), (Figure 9, sampling
port 56, before entering the Dowex 1 (Cl-) column 54) and
the samples monitored by HPLC following the procedures
described in Example 5. As expected, at this point of the
circuit (Figure 9) no L-phe that could result from the
enzymatic hydrolysis of L-phe-OMe by E2 (see Example 8)
was detected; only a circulating steady-state level of
N-formyl-(~-methyl)-asp-phe (average concentration:
54 mg/L) was observed, reflecting the continuous transfer
of the dipeptide N-formyl-(~-methyl~-APM across the
membrane and its subsequent hydrolysis by E2. The
efficient trapping of the charged dipeptide by the Dowex-l
resin i 8 indicated by the low concentration o f it observed
at point A throughout the run. A similar parallel
experiment performed in the absence of Dowex-1 peptide in
the shell phase, as could be anticipated from the results
discussed above. Again, no L-phe was found in the
circulating shell phase. A comparison of both experiments
is seen in Figure 10, and Table IX.
131~2~
-35-
Table IX
mq N-formYl-(~-methyl)-asD-phe/shell Dhase
Time (min.) Ex~.l. Dowex 1 present Exp.2 .without Dowex 1.
31.6 30. 2
-- 37.8
120 26.0 71.8
180 3~.4 83.1
240 45.6 --
300 51.2 --
In addition to separating phenylalanine lower alkyl
ester copermeating the ion rejection membrane into the
product mixture utilizing ion exchange resins as described
in this Example, a~partic acid copermeating the ion
rejection membrane into the product mixture can be
~eparated utilizing such resins. The species or product
that cannot back-diffuse across the membrane from the
product mixture can be removed utilizing such ion exchange
resins. Also, other separation methods known in the art
including but not limited to electrophoresi~,
electrodialysis and membrane separations which are
equivalents of ion exchange resin separations can be
utilized in the present invention.
Immobilizing the condensing enzyme allows the
enzymatic reaction in the tube phase to be conducted at an
initial reaction mixture pH preferred for optimum
efficiency of the enzymatic reaction considering the
reactants, product(s) and enzyme including the desired
equilibrium of the enzymatic reaction. Optionally, the
initial reaction mixture pH in tha tube phase can be
readjusted to a second reaction mixture pH prior to
contact with the membrane so that the second reaction
mixture pH will maximize the membrane efficiency in
transporting the uncharged product from the tube phasse
across the membrane into the 3hell phase. Figure 9 does
131~2~1
-36-
not show adjusting the initial reaction mixture pH in the
tube phase to a second reaction mixture pH.
Similarly, the esterase in the shell phase can be
immobilized and the pH of the product mixture in the shell
phase can be adjusted and readjusted as necessary to
effect the most efficient processing. Example 8 and the
above Example provide additional means for efficient
continuous or batch processing utilizing the present
invention. In continuous processing the desired
enantiomer of reactants and any copermeating compounds can
be returned to the tube phase or reaction mixture.
Industrial ApPlicabilitY
The present invention can be utilized to produce
a~partame as well as other peptides including peptides
which are pharmacologically active.
While specific embodiments of the invention have been
shown and described in detail to illustrate the
application of the inventive principles, it will be
understood that the invention may be embodied otherwise
without departing from such principle~.
