EP1180115A1 - Procede de synthese peptidique a isolement minimal faisant appel a des resines echangeuses d'ions comme agents de piegeage - Google Patents

Procede de synthese peptidique a isolement minimal faisant appel a des resines echangeuses d'ions comme agents de piegeage

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Publication number
EP1180115A1
EP1180115A1 EP00936209A EP00936209A EP1180115A1 EP 1180115 A1 EP1180115 A1 EP 1180115A1 EP 00936209 A EP00936209 A EP 00936209A EP 00936209 A EP00936209 A EP 00936209A EP 1180115 A1 EP1180115 A1 EP 1180115A1
Authority
EP
European Patent Office
Prior art keywords
solution
amino acid
reaction
column
reactant
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00936209A
Other languages
German (de)
English (en)
Inventor
John C. Tolle
Jean-Christophe Califano
Madhup K. Dhaon
Howard A. Sachs
James K. Blodgett
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Abbott Laboratories
Original Assignee
Abbott Laboratories
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Filing date
Publication date
Application filed by Abbott Laboratories filed Critical Abbott Laboratories
Publication of EP1180115A1 publication Critical patent/EP1180115A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/02—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to synthetic chemical processes. More particularly, the present invention concerns a solution-phase process, particularly adapted to the production of commercial-scale quantities of polypeptides, which minimizes the requirement of isolation of intermediates.
  • the first stage of solid-phase peptide synthesis consists of the assembly of a peptide chain on a supporting insoluble polymer or resin by sequential reactions of protected amino acid derivatives.
  • the peptide chain is cleaved from the solid resin support with concurrent or subsequent cleavage of side-chain protecting groups to give the crude free peptide.
  • polypeptides are pieced together by classical solution chemistry which facilitates joining together individual amino acids, or di-, tri-. tetra- or oligopeptide fragments of the final polypeptide in which sites of unwanted reaction have been appropriately protected.
  • the smaller fragments are themselves similarly prepared by piecing together individual amino acids or smaller protected fragments, etc.
  • solution-phase method a fragment formed in a previous step by reaction of an unprotected N-terminal amino function can be reacted in a subsequent step at its unprotected C-terminal carboxyl function.
  • This possibility is not open to the solid-phase method since, as stated above, the growing peptide is "blocked " at C-terminus by attachment to the supporting resin.
  • Solution-phase peptide synthesis despite being free of the one-terminus-only synthesis limitation of solid-phase peptide synthesis, suffers from a shortcoming: the need to frequently isolate and purify the growing peptide. All reactions are carried out in solution, resulting in a mixture which contains the desired product as well as unwanted unreacted reagents and byproducts.
  • an improved method of peptide synthesis is provided which capitalizes on the principal advantages of both the solid-phase and solution-phase methods of peptide synthesis.
  • the method disclosed is capable of large-scale production of peptides in solution, is not subject to the one-terminus-only limitation of the solid-phase method.
  • the method of the present invention does not require the frequent isolation of intermediates in a lengthy synthetic sequence nor, necessarily, the removal of all contaminants from the reaction mixture prior to subsequent processing steps.
  • a process for the synthesis of a polypeptide having a pre-determined number and sequence of amino acid residues is provided.
  • the process comprises sequentially the steps of first exposing, in solution, a first substrate amino acid or peptide fragment of the desired polypeptide product, the first substrate amino acid or peptide fragment being protected at either its N- or C-terminus, to a stoichiometric excess of a second reactant amino acid or peptide fragment of the desired polypeptide. the second reactant amino acid or peptide fragment being protected at the other of its N- or C-terminus. to form a condensation product of the substrate and reactant. The resulting condensation product is protected at both its N- and C-termini.
  • reaction solution is contacted with an insoluble scavenger having a reactive functionality complementary to the unprotected N- or C-terminal functionality of the first amino acid or peptide fragment, to sequester the excess of the second reactant amino acid or peptide fragment.
  • the sequestered excess second reactant amino acid or peptide fragment is removed from the reaction solution, leaving the condensation product and reaction by-products in solution. Should it become necessary or desirable to decrease the volume of the reaction solution, which increases during the process of the present invention, the condensation product may be precipitated or crystallized, then place back in a solution of lesser volume.
  • This solution in the fourth step, is subjected to a reaction which removes the protecting group from either the N- or C- terminus of the condensation product of the first step.
  • the first through fourth steps are repeated as a cycle, with the deprotected condensation product of each previous fourth step becoming the substrate peptide fragment of each successive first step, until the desired peptide is produced.
  • the product is isolated and deprotected, if needed, of any terminal or side-chain protecting groups.
  • FIGURE 1 is a schematic representation of the process steps for preparing polypeptides according to the method of the present invention.
  • FIGURE 2 is a schematic representation of a commercial-scale semi-automatic process apparatus for preparing polypeptides in accordance with the process of the present invention.
  • a protected or blocked amino acid or peptide is one in which the reactive functionality of either or both the N-terminal amino group and/or the C-terminal carboxyl group have been blocked by reaction with a "blocking group " to prevent their reactivit ⁇ .
  • other functional groups of the amino acid or peptide such as side- chain amino groups in lysine or hydroxy groups in serine. threonine, or tyrosine; and carboxyl groups in aspartic or glutamic acid residues may be blocked by appropriate blocking groups to prevent unwanted reactions.
  • Blocking groups suitable for the protection of amine, hydroxy 1, and carboxyl functions are well known in the art. Blocking groups and methods for their attachment and cleavage are fully set out in "Protective Groups in Organic Synthesis," 2d Edition, by T. W. Greene, et al , John Wiley & Sons, Inc., New York 1991 .
  • the general process of the present invention is depicted in schematic block diagram.
  • the first amino acid or peptide fragment substrate suitably protected except for either its N-terminal amino group or its C-terminal carboxyl function is exposed, in a suitable solvent, to the reactant amino acid or peptide fragment.
  • the reactant may be a single amino acid or some peptide fragment of the end-product polypeptide. suitably protected at the other of its N-terminal amino group or C-terminal carboxyl group as well as at reactive side- chain functional groups.
  • the carboxyl terminus of the N-terminal protected amino acid or peptide fragment is activated by conversion to an active ester of the types well known in the art.
  • a preferred ester activating group for the process of the present invention is the N- hydroxysuccinimide ester group (-OSu).
  • the protecting group for the N-terminus of the reactant or substrate amino acid or peptide fragment is a group which is "orthogonal" to protecting groups which are employed in protecting side-chain amino, hydroxyl, and carboxyl groups in either the reactant or substrate.
  • the N-terminal protecting group on the reactant or substrate amino acid or peptide fragment should be one which is easily removed under conditions which do not remove the side-chain protecting groups or the C-terminal blocking group of the other of the reactant or substrate.
  • Two protecting groups are said to be “orthogonal " if chemical processes which are employed to remove one do not remove the other.
  • a preferred N-terminal protecting group for the reactant or substrate amino acid or peptide fragment is one which is easily cleaved under conditions of hydrogenolysis, or catalytic hydrogenation.
  • sulfur is known to "poison " or inactivate hydrogenation catalysts
  • the preferred embodiment of the process of the present invention is limited to the synthesis of peptides which do not contain side-chain sulfhydryl or thioether groups; i.e.. to the synthesis of non-cysteine- containing and non-methionine-containing peptides.
  • the side-chain amino, hydroxyl and carboxyl blocking groups are selected from blocking groups well known in the art which are not cleaved under hydrogenolysis conditions.
  • a preferred N-terminal amino protecting group for the reactant or substrate amino acids and peptide fragments utilized in the process of the present invention is the benzyloxycarbonyl group, sometimes termed the carbobenzoxy group, and denoted “Cbz” or simply "Z” in chemical shorthand.
  • the Z group is easily cleaved by hydrogenolysis under mild conditions from the N-terminal amino groups of protected amino acids or peptides while leaving unaffected the less reactive protecting groups which have been used to protect side-chain functional groups.
  • Preferred blocking groups for the C-terminus of the reactant or substrate amino acid or peptide fragment are simple ester groups such as the tert-b ty ⁇ ester group and the like.
  • the substrate (unprotected at either its N-terminus or C-terminus) is allowed to react with the reactant (unprotected at the other of its N- or C-terminus) until analysis of aliquot samples periodically taken from the reaction mixture indicate substantially complete reaction.
  • the reactant is employed in stoichiometric excess to the amount of substrate in order to drive the condensation reaction to completion.
  • an amount greater than 1.0, up to about 1.1 moles of reactant amino acid or peptide fragment is employed per mole of substrate amino acid or peptide fragment.
  • the amount of excess required in each particular coupling reaction will vary according to the chemical nature of the substrate and reactant to be coupled.
  • the reaction solution is contacted with a scavenger.
  • the scavenger is both insoluble in the solvent system employed, and possesses a functionality which is complementary to that of the unprotected teminus of the substrate amino acid or peptide fragment.
  • the substrate amino acid or peptide fragment is unprotected at its N-terminus and protected against reaction at its C-terminus.
  • the reactant amino acid or peptide fragment is protected at its N-terminus. preferably by a Z group, and is activated at its C-terminus.
  • the scavenger possesses an active amine functionality.
  • the preferred scavenger is an amine-functionalized resin, such as aminomethyl-functionalized resins known in the art, particularly commercially available aminomethyl-modified styrene- divinylbenzene copolymers.
  • the method of contacting the reaction solution with the scavenger resin may be by either adding resin to the reaction vessel, or vice versa, with circulation of the reaction solution through a column of the resin being the preferred method.
  • This method permits the repeated recirculation of the reaction solution through the resin column to ensure that the removal of excess reactant amino acid or peptide fragment is complete. Should it become necessary or desirable to decrease the reaction volume, which increases during each cycle of the process of the present invention, the condensation product may be precipitated or crystallized, then redissolved in a smaller volume of a suitable organic solvent.
  • the desired N-terminal or C-terminal blocking group of the condensation product of the first step of the process is removed. If, in a subsequent step, the reactant amino acid or peptide fragment is one in which the carboxyl terminus is unprotected, then the N-terminal protecting group of the condensation product is removed, and vice versa.
  • new segments either single amino acids or short peptide fragments, are added to the growing polypeptide by utilizing a C-terminally activated reactant and an N-terminally unprotected substrate. This is because of the preferred N-terminal Z protecting group and the preferred C-terminal OSu activating group. Hence, in this step of the preferred embodiment of the process, the N-terminal Z group is removed from the condensation product.
  • the preferred method for this deprotection step is catalytic hydrogenolysis in the presence of a palladium catalyst at pressures ranging between atmospheric pressure and about 30 to 50 psi (206.8 kPa to 344.7 kPa).
  • Preferred catalysts are those which can readily be removed from solution by filtration such as palladium supported on Deloxan® (organofunctional polysiloxane polymers manufactured by DEGUSSA AG. Weissfrauenstrasse 9. Frankfort am Main, Germany), and palladium supported on carbon, alumina (A1 0 3 ). or silica (Si0 2 ).
  • the hydrogenolysis leaves in the solution, once the catalyst has been removed by filtration, only the N-hydroxysuccinimide by-product of the initial coupling reaction, the N-terminally deprotected condensation product, and toluene which, together with carbon dioxide is the by-product of the Z-group hydrogenolysis. (It should be noted that the N-hydroxysuccinimide and toluene by-products interfere with neither the catalytic hydrogenation deprotection step, nor subsequent condensations.
  • isolation may be carried our following completion of a particular fourth or first step, respectively.
  • the deprotection coupling and scavenging steps are repeated using the deprotected condensation product of each cycle as the substrate material of each subsequent cycle until the desired polypeptide sequence is achieved.
  • the system 100 comprises a first reactor vessel 102 for carrying out amino acid/peptide fragment condensation or coupling reactions, a second reactor 106 for carrying out de-protecting reactions, a resin column
  • anahsis may be automated by means of a detector immersed in the reactor vessel content, sensitive to one or more of the reactant and substrate or a by-product of the coupling reaction. A signal from the sensor is sent to numeric processor 116 and used to determine the termination of the reaction step.
  • the mixture is stirred by means of stirrer 126 and the temperature is controlled to a level near ambient, generally less than about 30°C to about 35°C during the course of the reaction.
  • the reactor vessel 102 may be of a type well known in the process arts for controlling chemical reaction temperature.
  • vessel 102 may be equipped with an immersion cooling coil, cooling coils welded to the outside of the vessel itself, or may be of a double-jacketed type which permits the circulation of a coolant between the walls of the vessel.
  • the circulation of coolant is controlled by a conventional feed-back loop connected to numeric controller 116 which reacts to a temperature sensor, not shown, immersed in the contents of reactor vessel 102 and uses that temperature to control the circulation of reactor vessel coolant.
  • the solution recycled through the column and reactor vessel is periodically analyzed for completeness of removal of excess reactant.
  • this ma ⁇ be my means of physical removal of aliquot samples from the reactor vessel from time to time, or by a sensor immersed in the reactor vessel contents or fixed in-line in the piping connection between the reactor vessel 102 and resin column 104
  • the sensor is sensitive to excess reactant and is attached by feed-back control loop to numeric processor 116 and the signal from the recirculated product solution is used to control when the recirculation of the reaction solution through the resin column 104 may be stopped
  • the numeric controller closes appropriate valves and opens others to transfer, by means of pump 122, the eluate from resin column 102 to holding tank 114
  • a first wash solvent from first solvent tank 112 is then circulated through resin column 104 and back to first solvent tank 112 to flush an> remaining product from the column
  • This recirculating flush of column 104 may be for either a predetermined number of cycles or for a predetermined time
  • the first solvent wash solution containing any product flushed from resin column 104, is added to the contents of holding tank 114
  • a second wash or flush of column 104 may be carried out if desired by employing a second solvent, initially held in second solvent tank 110 As in the case of the wash of column 104 with the first solvent, the second solvent is recirculated between the column 104 and tank 110. either for a pre-determined number of cycles, or for a pre-determined time, as controlled by numeric processor unit 116 After complete flushing or washing of column 104, the second solvent solution, containing any additional product washed from column 104 is added to the contents of holding tank 114
  • N-methylpyrrohdone, and low molecular weight alcohols such as iso- propanol and the like, with cost and availability being concerns secondary to polarity and non- reactivity
  • Preferred solvents for the process of the present invention include dimethylformamide and /so-propanol
  • the holding tank 114 contains a solution comprising the coupled reaction product, together with the reaction solvent and the wash solvents, and (in the preferred embodiment of the process) toluene, tertiary amine salt (if the substrate or reactant amino acid or peptide fragment is employed in a salt form requiring neutralization prior to coupling), and N-hydroxysuccinimide, but substantially free of contaminating excess reactant and substrate.
  • the holding tank contents are next transferred by means of pump 124 to second reactor vessel 106 for removal of the Z-blocking group from the N-terminus of the coupled reaction product.
  • the preferred benzyloxycarbonyl (“Z " ) protecting group is easily removed hydrogenolysis under comparatively mild conditions.
  • second reactor 106 is shown as a hydrogenator fitted with gas inlet pipes 134, 136, and 138 and vent pipe 140.
  • Gas inlet pipes 134, 136, and 138 are connected to sources (not shown) of flush gases such as argon and nitrogen. and reaction gas, hydrogen.
  • the flow of gases into and out of second reactor 106 is controlled by valves 1, 2, 3, and 4 which, in turn are controlled by numeric processor 116.
  • the hydrogenator 116 is purged with flushing gases, initially flushed with hydrogen, and then charged at a higher pressure with hydrogen for the catalytic hydrogenolysis.
  • Process step 1 Reactant and substrate charged to reactor vessel 102 and allowed to react.
  • Process step 2 Reactor vessel 102 contents are recirculated through column 104.
  • Process step 3 Reactor vessel 102 contents are transferred to holding tank 114.
  • Process step 4 Cyclical first solvent flush of column 104 is carried out.
  • Process step 5 First solvent flush is transferred to holding tank 114.
  • Process step 6 Cyclical second solvent flush of column 104 is carried out.
  • Process step 7 Second solvent flush is transferred to holding tank 114.
  • Process step 8 Contents of holding tank 114 are transferred to second reactor vessel 106.
  • Process step 9 Second reactor vessel 106 is purged with first purge gas.
  • Process step 10 Second reactor vessel 106 is purged with second purge gas.
  • Process step 1 1 Second reactor vessel 106 is purged with hydrogen.
  • Process step 1 1 Second reactor vessel 106 is pressurized with hydrogen and the hydrogenolysis reaction is allowed to proceed.
  • Process step 12 Contents of second reactor vessel 106 are filtered to remove hydrogenation catalyst and, if construction of the desired peptide is incomplete, transferred to first reactor vessel 102 for addition of next amino acid or peptide fragment to the growing peptide chain.
  • Process step 13 If construction of the desired peptide is incomplete, repeat steps 1 - 12 as required.
  • Process step 14 If construction of the peptide is complete, the contents of holding tank 114 from step 7 are collected for further processing.
  • step l a, 2a, 3a and 4a for the first cycle of the process of the invention.
  • Step l a) Preparation of Benzyloxycarbonyl-Lvsyl(ter/-butyloxycarbonyl)-
  • Z-Lys(Boc)-OSu reagent from Step l a is then circulated repetitively through the resin column for about one hour in order to remove excess Z-Lys(Boc)-OSu Circulation is continued, if necessary, until analysis indicates complete removal of the excess reactant
  • the NMP reaction solution containing the blocked Z-Lys(Boc)-Lys(Boc)-OMe dipeptide product is set aside
  • the resin column is then washed by recirculating 15 mL ( 1 1 75 g) of z_.o-propanol through the column for about thirty minutes
  • the / ⁇ o-propanol solution, containing blocked dipeptide product washed from the column, is set aside
  • the column is next washed by recirculating 30 mL (30 99 g) of NMP through the column for about thirty minutes
  • the NMP solution, containing blocked dipeptide product washed from the column is set aside
  • the resin column is given a final wash
  • the hydrogen is then vented from the hydrogenator vessel, and the vessel is purged twice with nitrogen An aliquot sample of the reaction mixture is taken for analysis of the completeness of reaction If the analysis indicates incomplete reaction, the hydrogenator vessel is purged twice with hydrogen, recharged w ith hydrogen to a pressure of 35-45 psi (234 5-3 10 3 kPa). and the mixture is again stirred at a temperature of about 30°C for an additional hour The hydrogenator vessel is evacuated and then purged twice ith nitrogen, and an aliquot sample of the reaction mixture is again taken for analysis of the completeness of reaction The above steps of 14
  • the hvdrogen is vented from the hydrogenator vessel, the vessel is purged with nitrogen, and the vessel contents are filtered to remove the catalyst The hydrogenator vessel is rinsed with NMP. and the rinse solution is added to the reaction mixture filtrate
  • Step l b) Preparation of Benzylow carbonv 1-Leuc ⁇ l-Lvsyl(/er/ - but ⁇ loxycarbonyl)-Lvs ⁇ ne(/grt-butylox ⁇ carbonyl)-meth ⁇ l Ester Z-Leu-OSu (3 98 g, 1 1 mmol).
  • DIEA (1 29 g, 1 1 mmol) and the NMPA -propanol solution of the N-terminal deprotected dipeptide product of Step 4a ( 10 mmol.
  • a column of aminomethyl resin (2 g) is prepared as described above in Step 2a-3a, and the NMPAopropanol reaction mixture of the blocked t ⁇ peptide from Step l b is circulated repetitively through the resin column for about one hour periodically analyzing the column eluate for absence of excess Z-Leu-OSu
  • the NMP/zso-propanol reaction solution of the blocked t ⁇ peptide is set aside, and the column is washed by recirculating 15 mL ( 1 1 75 g) of z_.o-propanol through the column for about thirty minutes
  • the . ⁇ O-propanol solution, containing blocked t ⁇ peptide product washed from the column is set aside
  • the column is next washed by recirculating 30 mL (30 99 g) of NMP through the column for about thirty minutes
  • the NMP solution, containing blocked t ⁇ peptide product washed from the column is set aside
  • Step l c) Preparation of Benzyloxycarbonyl-Isoleucyl-Leucyl-Lvsyl(/er/- butyloxycarbonyl)-Lvsine(ter/-b ⁇ tyloxycarbonyl)-methyl Ester Z-Ile-OSu (3.98 g. 1 1 mmol) is mixed in a 1 -liter glass reactor vessel with the NMP/z ' -.o propanol solution of the N-terminal deprotected tripeptide product from Step 4b. The mixture is stirred at room temperature until all solids are dissolved.
  • the resulting mixture is allowed to react at room temperature for at least 2 hours, at which time an aliquot sample of the reaction mixture is analyzed for completeness of reaction. If the analysis indicates that the reaction is incomplete, the reaction mixture is stirred at room temperature for an additional hour and the reaction mixture is analyzed again. When analysis indicates the coupling reaction is complete, the reactor contents are held at room temperature with slow stirring.
  • a column of aminomethyl resin (2 g) is prepared as described above in Step 2a-3a, and the NMP/zso-propanol reaction mixture of the blocked tetrapeptide from Step l c is then circulated repetitively through the resin column for about one hour in order to remove excess Z- Ile-OSu. Circulation is continued, if necessary, until analysis indicates complete removal of the excess reactant. At this point, the NMP/.so-propanol reaction solution containing the blocked tetrapeptide product is set aside.
  • the resin column is then washed by recirculating 15 mL ( 1 1.75 g) of z ' _.o-propanol through the column for about thirty minutes.
  • the z ' .sopropanol solution, containing blocked tetrapeptide product washed from the column, is set aside.
  • the column is next washed by recirculating 30 mL (30.99 g) of NMP through the column for about thirty minutes.
  • NMP solution containing blocked tetrapeptide product washed from the column, is set aside.
  • the resin column is given a final wash by recirculating 15 mL ( 1 1 .75 g) of ..vo-propanol through the column for about thirty minutes.
  • the NMP/.so-propanol reaction solution of blocked tetrapeptide product, the NMP wash solution, and the two wo-propanol wash solutions are combined.
  • Step I d) Preparation of Benzyloxycarbonyl-Lvsine(ter.-butyloxycarbonyl)- lsoleucyl-Leucyl-Lvsyl(t- , rz-butyloxycarbonyl)-L ⁇ sine(tert- butyloxycarbonyP-methyl Ester Z-Lys(Boc)-OSu (5.25 g, 1 1 mmol) is mixed in a 1 -liter glass reactor vessel with the
  • NMP/._.o-propanol solution of the N-terminal deprotected tetrapeptide product from Step 4c The mixture is stirred at room temperature until all solids are dissolved. The resulting mixture is allowed to react at room temperature for at least 2 hours, at which time an aliquot sample of the reaction mixture is analyzed for completeness of reaction. If the analysis indicates that the reaction is incomplete, the reaction mixture is stirred at room temperature for an additional hour and the reaction mixture is analyzed again. When analysis indicates the coupling reaction is complete, the reactor contents are held at room temperature with slow stirring.
  • Steps 2d-3d Scavenging/Sequestration of Excess Reactant Z-Lvs(Boc)-OSu and Precipitation/Crystallization of Condensation Product
  • a column of aminomethyl resin (2 gm) is prepared as described above in Step 2a-3a. and the NMP/._>o-propanol reaction mixture of the blocked pentapeptide from Step I d is then circulated repetitively through the resin column for about one hour in order to remove excess Z-Lys(Boc)- OSu. Circulation is continued, if necessary, until analysis indicates complete removal of the excess reactant. At this point, the NMP/ .y-.-propanol reaction solution containing the blocked pentapeptide product is set aside.
  • the resin column is then washed by recirculating 15 mL ( 1 1.75 g) of ⁇ o-propanol through the column for about thirty minutes.
  • the column is next washed by recirculating 30 mL (30.99 g) of NMP through the column for about thirty minutes.
  • the NMP solution, containing blocked pentapeptide product washed from the column, is set aside.
  • the resin column is given a final wash by recirculating 15 mL ( 1 1 .75 g) of / ⁇ o-propanol through the column for about thirty minutes.
  • the NMPAo-propanol reaction solution of blocked pentapeptide product, the NMP wash solution, and the two wo-propanol ash solutions are combined.
  • Z-Val-OSu (3.30 g, 9.50 mmol) is mixed in a 1 -liter glass reactor vessel with the NMP solution of the N-terminal deprotected pentapeptide product from Step 4d. The mixture is stirred at room temperature until all solids are dissolved. The resulting mixture is allowed to react at room temperature for at least 2 hours, at which time an aliquot sample of the reaction mixture is analyzed for completeness of reaction. If the analysis indicates that the reaction is incomplete, the reaction mixture is stirred at room temperature for an additional hour and the reaction mixture is analyzed again. When analysis indicates the coupling reaction is complete, the reactor contents are held at room temperature with slow stirring.
  • Steps 2e-3e Scavenging/Sequestration of Excess Reactant Z-Val-OSu
  • a column of aminomethyl resin ( 1.60 g) is prepared as described above in Step 2a-3a, and the NMP reaction mixture of the blocked hexapeptide from Step l e is then circulated repetitively through the resin column for about one hour in order to remove excess Z-Val-OSu.
  • the benzyloxycarbonyl protecting group on the Z-Val-Lys(Boc)-I le-Leu-Lys(Boc)-Lys(Boc)-OMe hexapeptide product of Step 1 e is removed b ⁇ hydrogenolysis
  • Step I f) Preparation of Benzyloxycarbonyl-Phenylalanyl-Valyl-Lvs ⁇ ne(ter.- butyloxycarbonyl)-Isoleucyl-Leucyl-Lvsyl(.er.-butyloxycarbonyl)-
  • Steps 2f-3f Scavenging/Sequestration of Excess Reactant Z-Phe-OSu
  • a column of l resin ( 1 60 g) is prepared as described abo ⁇ e in Step 2a-3a. and the NMP/iso-propanol reaction mixture of the blocked heptapeptide from Step I f is then circulated repetitively through the resin column for about one hour in order to remo ⁇ e excess Z-Phe-OSu Circulation is continued, if necessary , until analysis indicates complete removal of the excess reactant At this point the NMP/. so-propanol reaction solution containing the blocked heptapeptide product is set aside.
  • the resin column is then washed by recirculating 15 mL ( 1 1.75 g) of .so-propanol through the column for about thirty minutes.
  • the column is next washed by recirculating 30 mL (30.99 g) of NMP through the column for about thirty minutes.
  • the NMP solution, containing blocked heptapeptide product washed from the column, is set aside.
  • the resin column is given a final wash by recirculating 15 mL ( 1 1 .75 g) of .-.o-propanol through the column for about thirty minutes.
  • the NMPAo-propanol reaction solution of blocked heptapeptide. the NMP wash solution, and the two / ' so-propanol wash solutions are combined.
  • OMe heptapeptide product of Step I f is removed by hydrogenolysis.
  • NMP//->o-propanol solution of the N-terminal deprotected heptapeptide product from Step 4f The mixture is stirred at room temperature until all solids are dissolved. The resulting mixture is allowed to react at room temperature for at least 2 hours, at which time an aliquot sample of the reaction mixture is analyzed for completeness of reaction. If the analysis indicates that the reaction is incomplete, the reaction mixture is stirred at room temperature for an additional hour and the reaction mixture is analyzed again. When this analysis indicates the coupling reaction is complete, the reactor contents are held at room temperature with slow stirring.
  • a column of aminomethyl resin ( 1.60 g) is prepared as described above in Step 2a-3a. and the NMP// ' io-propanol reaction mixture of the blocked octapeptide from Step l g is then circulated repetitively through the resin column for about one hour in order to remove excess Z-Ala-OSu.
  • Step 4g N-Terminal Deprotection of the Benzyloxycarbonyl-Alanv l-Phenylalanyl-Valyl- Lvsyl(tgrt-butyloxycarbonyl)-Isoleucyl-Leuc ⁇ l-Lvsyl(tgr/- butyloxycarbonyl)-Lys ⁇ ne(tgrt-butyloxycarbon ⁇ l )-meth ⁇ l Ester Product of
  • Step l Using the same process as that of Step 4a. but without the addition of pTSA, the benzyloxycarbonyl protecting group on the Z-Ala-Phe-Val-Lys(Boc)-I le-Leu-Lys(Boc)-Lys(Boc)- OMe octapeptide product of Step l g is removed by hydrogenolysis
  • Step l h Preparation of Benzylox ⁇ carbonyl-Lvsyl(/g/ .-butyloxycarbonyl-Alanyl-
  • Steps 2h-3h A column of aminomethyl resin ( 1 60 g) is prepared as described above in Step 2a-3a. and the MP/zso-propanol reaction mixture of the blocked nonapeptide from Step l h is then circulated repetitively through the resin column for about one hour in order to remove excess Z-Lys(Boc)- OSu Circulation is continued, if necessary, until analysis indicates complete removal of the excess reactant At this point, the NMPAo-propanol reaction solution containing the blocked nonapeptide product is set aside The resin column is then washed by recirculating 15 mL ( 1 1 75 g) of ._.o-propano] through the column for about thirty minutes The zso-propanol solution 21
  • the column is next washed by recirculating 30 mL (30.99 g) of NMP through the column for about thirty minutes.
  • the NMP solution, containing blocked nonapeptide product washed from the column is set aside.
  • the resin column is given a final wash by recirculating 15 mL ( 1 1.75 g) of z ' _.o-propanol through the column for about thirty minutes.
  • the NMP/z ' _.o-propanol reaction solution of blocked nonapeptide, the NMP wash solution, and the two /-.o-propanol wash solutions are combined.
  • Ala-Phe-Val-Lys(Boc)-Ile-Leu-Lys(Boc)-Lys(Boc)-OMe is 13.28 g (94% of theoretical yield from
  • Gly-OMe.HCl (6.25 g, 50 mmol) and Z-Phe-OSu (21 .80 g, 55 mmol) are mixed with 60 mL (56.52 g) of dimethylformamide (DMF) in a 250 mL glass reactor, and the mixture is stirred at room temperature until the solids are dissolved.
  • Diisopropylethylamine (DIEA) (6.49 g, 55 mmol) is added slowly to the reactor contents over a period of from about fifteen to thirty minutes. The resulting mixture is allowed to react at room temperature for about two hours.
  • Steps 2a-3a) Scavenging/Sequestration of Excess Reactant Z-Phe-OSu Aminomethyl resin (8 30 g) is mixed with 50 mL (47 10 g) of DMF in a second reactor vessel The resulting mixture is stirred at room temperature until a homogeneous slurry is obtained The resin/DMF slurry is charged to a glass column, the resin slurry is allowed to settle into a packed bed, and any excess DMF is drained from the column The DMF reaction mixture containing the Z-Phe-Gly-OMe product and excess Z-Phe-OSu reagent from Step l a is then circulated repetitively through the resin column for about one hour in order to remove excess Z-Phe-OSu Circulation is continued if necessary, until analysis indicates complete removal of the excess reactant At this point, the DMF reaction solution containing the blocked Z-Phe-Gl> -OMe dipeptide product is set aside The resin column is then washed by recirculating 20
  • the vessel is sealed and evacuated to a pressure of 20-25 inches of mercury (67 7 - 84 6 kPa) and purged three times with hydrogen Hydrogen is then charged to the hydrogenator vessel to a pressure of 35-45 psi (234 5-3 10 3 kPa) and the mixture is stirred at about 30°C for about 2 hours
  • the hy drogen is then vented from the hydrogenator vessel, and the vessel is purged twice with nitrogen An aliquot sample of the reaction mixture is taken for analysis of the completeness of reaction If the analysis indicates incomplete reaction, the hydrogenator vessel is purged twice with hydrogen, recharged with hydrogen to a pressure of 35-45 psi (234 5-3 10 3 kPa).
  • the hydrogenator vessel is evacuated and then purged tw ice w ith nitrogen, and an aliquot sample of the reaction mixture is again taken for analysis of the completeness of reaction
  • the abov e steps of hydrogenation and reaction mixture analysis are repeated until the analysis indicates substantial completion of the hydrogenolysis reaction
  • the hydrogen is vented from the hydrogenator vessel, the vessel is purged with nitrogen, and the vessel contents are filtered to remove the catalyst.
  • the hydrogenator vessel is rinsed with DMF. and the rinse solution is added to the reaction mixture filtrate.
  • Step l b) Preparation of Benzyloxycarbonyl-Lvsyl(/gr/-but ⁇ loxycarbonyl)-
  • Steps 2b-3b) Scavenging/Sequestration of Excess Reactant Z-Lvs(Boc)-OSu A column of aminomethyl resin (8.30 g) is prepared as described above in step 2a-3a, and the DMF/zso-propanol reaction mixture from Step l b is circulated repetitively through the resin column for about one hour, periodically analyzing the column eluate for absence of excess Z- Lys(Boc)-OSu. When the analysis indicates substantially pure product, the DMF/zso-propanol reaction solution of the blocked tripeptide product is set aside, and the column is washed by recirculating 20 mL ( 15.66 g) of zso-propanol through the column for about thirty minutes.
  • the zso-propanol solution, containing blocked tripeptide product washed from the column, is set aside.
  • the column is next washed by recirculating 40 mL (37.68 g) of DMF through the column for about thirty minutes.
  • the DMF solution, containing blocked tripeptide product washed from the column is set aside.
  • the resin column is given a final wash by recirculating 20 mL ( 15.66 g) of zso-propanol through the column for about thirty minutes.
  • the DMF/zso-propanol reaction solution of blocked tripeptide product and the zso-propanol and DMF wash solutions are combined.
  • Step 4b N-Terminal Deprotection of the Benzyloxycarbom l-Lvsyl(fgrz- butyloxycarbonvD-Phenylalanyl-Glvcine-methyl Ester Product of Step l b Using the same process as that of Step 4a. but without the addition of pTSA, the benzyloxycarbonyl protecting group on the N-terminus of the Z-Lys(Boc)-Phe-Gly-OMe tripeptide product of Step l b is removed by hydrogenolysis.
  • Step l c) Preparation of Benzyloxycarbonyl-Lvsyl(/g/-t-butyloxycarbonyl)-
  • Steps 2c-3c) Scavenging/Sequestration of Excess Reactant Z-Lvs(Boc)-OSu A column of aminomethyl resin (8.30 g) is prepared as described above in Step 2a-3a, and the DMF/zso-propanol reaction mixture from Step l c is circulated repetitively through the resin column for about one hour, periodically analyzing the column eluate for absence of excess Z-
  • Lys(Boc)-OSu When the analysis indicates substantially pure product, the DMF/zso-propanol solution of reaction product is set aside, and the column is washed by recirculating 20 mL ( 15.66 g) of zso-propanol through the column for about thirty minutes. The zso-propanol solution, containing blocked tetrapeptide product washed from the column, is set aside. The column is next washed by recirculating 40 mL (37.68 g) of DMF through the column for about thirty minutes.
  • the DMF solution, containing blocked tetrapeptide product washed from the column, is set aside.
  • the resin column is given a final wash by recirculating 20 mL ( 15.66 g) of zso-propanol through the column for about thirty minutes.
  • the DMF/zso-propanol reaction solution of blocked tetrapeptide product and the zso-propanol and DMF wash solutions are combined.
  • Step I d) Preparation of Benzyloxycarbon ⁇ 1-Alanyl-
  • a column of aminomethyl resin (3 45 g) is prepared as described above in Step 2a-3a, and the NMP reaction mixture of the blocked hexapeptide from Step 1 e is then circulated repetitively through the resin column for about one hour in order to remove excess Z-Ly s(Boc)-OSu Circulation is continued, if necessary, until analysis indicates complete removal of the excess reactant At this point, the NMP reaction solution containing the blocked hexapeptide product is set aside The resin column is then washed by recirculating 20 mL ( 15 66 g) of zso-propanol through the column for about thirty minutes The zso-propanol solution, containing blocked hexapeptide product washed from the column, is set aside The column is next washed by recirculating 40 mL (41 32 g) of NMP through the column for about thirty minutes The NMP solution, containing blocked hexapeptide product washed from the column, is set aside The resin column is given a
  • Step I f Preparation of Benzyloxycarbonyl-Lvsyl(.grz-but ⁇ lo ⁇ carbonv
  • Steps 2f-3f Scavenging/Sequestration of Excess Reactant Z-Lys(Boc)-OSu
  • a column of aminomethyl resin (3 45 g) is prepared as described above in Step 2a-3a. and the NMP/zso-propanol reaction mixture from Step I f is circulated repetitiv ely through the resin column for about one hour, periodically analyzing the column eluate for absence of excess Z-
  • Steps 2g-3g Scavenging/Sequestration of Excess Reactant Z-Leu-OSu and Precipitation/Crystallization of the Condensation Product
  • a column of ammomethyl resin (3 45 g) is prepared as described above in Step 2a-3a, and the NMP/zso-propanol reaction mixture from Step l g is circulated repetitively through the resin column for about one hour, periodically analyzing the column eluate for absence of excess Z-Leu- OSu When the analysis indicates substantially pure product, the NMP/zso-propanol solution of reaction product is set aside, and the column is washed by recirculating 40 mL (3 1 32 g) of zso- propanol through the column for about thirty minutes The zso-propanol solution, containing blocked octapeptide product washed from the column, is set aside The column is next washed by recirculating 80 mL (81 64 g) of NMP through the column for about thirty minutes The NMP solution, containing blocked octapeptide product washed from the column, is set aside The resin column is given a final wash by recirculating 40 mL (31
  • Step l h) Preparation of Benzyloxycarbonyl-Phenylalanyl-Leuc 1-LvsvKtgr/- butyloxycarbonyl)-Lvsyl(/gr/-butylox ⁇ carbony l)- Alanyl-LvsvKtgr/- butyloxycarbonyl)-Lysyl(tert-butyloxycarbonyl)-Phenylalanyl-Glvcine- methyl Ester
  • Z-Phe-OSu (5.70 g. 14.38 mmol) is mixed in a 1 -liter glass reactor vessel with the NMP solution of the N-terminal deprotected octapeptide product from Step 4g. The mixture is stirred at room temperature until all solids are dissolved. The resulting mixture is allowed to react at room temperature for at least 2 hours, at which time an aliquot sample of the reaction mixture is analyzed for completeness of reaction. If the analysis indicates that the reaction is incomplete, the mixture is stirred at room temperature for an additional hour and the reaction mixture is again analyzed. When this analysis indicates the coupling reaction is complete, the reactor contents are held at room temperature with slow stirring.
  • a column of aminomethyl resin (2.40 g) is prepared as described above in Step 2a-3a, and the NMP reaction mixture of the blocked nonapeptide from Step l h is then circulated repetitively through the resin column for about one hour in order to remove excess Z-Phe-OSu. Circulation is continued, if necessary, until analysis indicates complete removal of the excess reactant. At this point, the NMP reaction solution containing the blocked nonapeptide product is set aside. The resin column is then washed by recirculating 20 L ( 15.66 g) of zso-propanol through the column for about thirty minutes. The zso-propanol solution, containing blocked nonapeptide product washed from the column, is set aside.
  • the column is next washed by recirculating 40 mL (41.32 g) of NMP through the column for about thirty minutes.
  • the NMP solution, containing blocked nonapeptide product washed from the column, is set aside.
  • the resin column is given a final wash by recirculating 20 mL ( 15.66 g) of zso-propanol through the column for about thirty minutes.
  • the NMP reaction solution of blocked nonapeptide, the NMP wash solution, and the two zso-propanol wash solutions are combined.
  • Step 4a Using the same process as that of Step 4a, but without the addition of pTSA, the benzyloxycarbonyl protecting group on the N-terminus of the Z-Phe-Leu-Lys(Boc)-Lys(Boc)-Ala- Lys(Boc)-Lys(Boc)-Phe-Gly-OMe nonapeptide product of Step l h is removed by hydrogenolysis.
  • Step l i) Preparation of Benzyloxycarbonyl-Lvsyl(/grt-butyloxycarbonyl)-
  • Lys(Boc)-Lys(Boc)-Ala-Lys(Boc)-Lys(Boc)-Phe-Gly-OMe decapeptide product The decapeptide is isolated by filtration, rinsed with 1 liter of methanol, and dried under vacuum.
  • the yield of Z- Lys(Boc)-Phe-Leu-Lys(Boc)-Lys(Boc)-Ala-Lys(Boc)-Lys(Boc)-Phe-Gly-OMe is 19.02 g (79% of theoretical yield from Z-Leu-Lys(Boc)-Lys(Boc)-Ala-Lys(Boc)-Lys(Boc)-Phe-Gly-OMe, 41 % overall yield).
  • Step 1 Preparation of 9-Fluorenylmethoxycarbonyl-pg/ ⁇ -Alanyl-Leucyl-Alanyl-Leucine-/grt-butyl Ester Step 1 ) Preparation of Benzyloxycarbonyl-Alanyl-Leucine- /grz-butyl Ester Leu-OtBu HCl ( 184 g, 0.82 mol), and Z-Ala-OSu (290 g. 0.91 mol) are mixed with 1 .9 liters ( 1790 g) of dimethylformamide in a 5-liter glass reactor, and the mixture is stirred at room temperature until the solids are dissolved.
  • Diisopropylethylamine ( 1 15 g (0.97 mol) is added slowly to the reactor contents over a period of from about fifteen to thirty minutes. The resulting mixture is allowed to react at room temperature for about two hours. Aliquot samples of the reactor contents are then taken for analysis of the completeness of the coupling reaction. When these analyses indicate the reaction is complete, the reactor contents are held at room temperature with slow stirring.
  • Aminomethyl resin 164 g
  • 1 .6 liters 1550 g
  • dimethylformamide dimethylformamide
  • the resulting mixture is stirred at room temperature until a homogeneous slurry is obtained.
  • the resin/DMF slurry is charged to a glass column, the resin slurry is allowed to settle into a packed bed, and any excess DMF is drained from the column.
  • the reaction mixture containing the Z-Ala-Leu-OtBu dipeptide product and excess Z-Ala- OSu reagent is then circulated repetitively through the resin column for about one hour.
  • the DMF solution containing the Z-Ala-Leu-O-Z-Bu dipeptide product is set aside, and an aliquot sample is removed for verification of the completeness of the scavenging reaction. Circulation is continued, if necessary, until analysis indicates complete removal of the excess reactant.
  • the resin column is then washed by recirculating 785 mL (615 g) of zso-propanol through the column for about thirty minutes.
  • O-t-Bu dipeptide product washed from the column is set aside.
  • the column is next washed by recirculating 1.56 liters ( 1470 g) of dimethyl-formamide through the column for about thirty minutes.
  • the DMF solution, containing the Z-Ala-Leu-O-t-Bu dipeptide product washed from the column is set aside.
  • the resin column is given a final wash by recirculating 785 mL (615 g) of zso-propanol through the column for about thirty minutes.
  • the original reaction solution in DMF, the DMF wash solution, and the two zso-propanol wash solutions are combined.
  • Step 4) - N-Terminal Deprotection of the Benzyloxycarbonyl-Alanyl-Leucine-tert- butyl Ester Product of Step 1 )
  • Palladium-Deloxan® (16 g) is placed in a 2-gallon (7.57 L) hydrogenator vessel.
  • the hydrogenator vessel is flooded with argon, and the combined DMF/zso-propanol solutions from steps (2) and (3) are charged to the hydrogenator.
  • the vessel is sealed and evacuated to a pressure of 20-25 inches of mercury (67.7 - 84.6 kPa) and purged three times with hydrogen. Hydrogen is then charged to the hydrogenator vessel to a pressure of 35-45 psi (234.5-3 10.3 kPa) and the mixture is stirred at about 30°C for about 2 hours.
  • the hydrogenator vessel is then vented to vacuum, repressurized w ith hydrogen to a pressure of 35-45 psi (234.5-3 10.3 kPa). and hydrogenation is continued for about one hour at 30°C .
  • the vessel is the purged and evacuated twice with nitrogen, and an aliquot sample of the reaction mixture is taken for analysis of the completeness of reaction. If the analysis indicates WO 00/71569 PCT/USOO/l 4152
  • the hydrogenator vessel is purged twice with hydrogen, recharged with hydrogen to a pressure of 35-45 psi (234.5-310.3 kPa) and the mixture is again stirred at a temperature of about 30°C for an additional hour.
  • the hydrogenator vessel is evacuated and then purged twice with nitrogen, and an aliquot sample of the reaction mixture is again taken for analysis of the completeness of reaction. The above steps of hydrogenation and reaction mixture analysis are repeated until the analysis indicates substantial completion of the hydrogenolysis reaction.
  • the hydrogenator vessel Upon completion of the hydrogenolysis reaction, the hydrogenator vessel is vented to vacuum, purged with nitrogen, and the vessel contents are filtered to remove the catalyst. The hydrogenator vessel is rinsed with dimethylformamide. and the rinse solution is added to the reaction mixture filtrate.
  • Step 1 ') Preparation of Benzyloxycarbonyl-Leucyl-Alanyl-Leucine- /grt-butyl Ester Z-Leu-OSu (328 g, 0.90 mol) and the solution of the deprotected dipeptide product of step 4) (0.82 mol, assuming complete reaction) are placed in a 12-liter glass reactor, and the mixture is stirred at room temperature until all solids are dissolved. The mixture is then stirred at room temperature for about two hours. At the end of this time, an aliquot sample of the reaction mixture is taken for analysis of the completeness of the coupling reaction. If the analysis indicates that the reaction is incomplete, the mixture is stirred at room temperature for an additional hour and the reaction mixture is again analyzed. When these analyses indicate the coupling reaction is complete, the reactor contents are held at room temperature with slow stirring.
  • Steps 2'-3 ' Scavenging/Sequestration of Excess Reactant Z-Leu-OSu
  • a column of aminomethyl resin (164 g) is prepared as described above in steps 2) and 3) and the reaction mixture from step 1 ') is circulated repetitively through the resin column for about one hour, periodically analyzing the column eluate for absence of excess Z-Leu-OSu.
  • the solution of Z-Leu-Ala-Leu-O-t-Bu is set aside, and the column is washed by recirculating 785 mL (615 g) of zso-propanol through the column for about thirty minutes.
  • the column is next washed by recirculating 1.56 liters ( 1470 g) of dimethylformamide through the column for about thirty minutes.
  • the DMF solution, containing of Z-Leu-Ala-Leu-O-t-Bu tripeptide product washed from the column is set aside.
  • the resin column is given a final wash by circulating 785 mL (615 g) of zso-propanol through the column for about thirty minutes.
  • the original solution of of Z-Leu-Ala- Leu-O-t-Bu tripeptide product, the two zso-propanol wash solutions, and the DMF wash solution are combined.
  • Step 4 ' N-Terminal Deprotection of the Benzyloxycarbonyl-Leucyl-Alanyl- Leucine - tgrt-butyl Ester Product of Step 1 ')
  • step 4 the benzyloxycarbonyl protecting group on the N-terminus of the Z-Leu-Ala-Leu-O-t-Bu tripeptide product of step 1 ') is removed by hydrogenolysis.
  • Step 1 Preparation of 9-Fluorenylmethoxycarbonyl-og/o-Alanyl-Leucyl-Alanyl-
  • Fmoc- 'gto-alanine-N-hydroxysuccinimide ester (Fmoc- ⁇ -Ala-OSu, 336 g, 0.82 mol) is mixed in a 50-liter glass reactor vessel with the DMF/zso-propanol solution of the N-terminal deprotected tripeptide product from step 4'). The mixture is stirred at room temperature until all solids are dissolved. The resulting mixture is stirred at room temperature for at least 2 hours, at which time an aliquot sample of the reaction mixture is analyzed for completeness of the coupling reaction. If the analysis indicates that the reaction is incomplete, the mixture is stirred at room temperature for an additional hour after which the reaction mixture is again analyzed.
  • pre-cooled distilled water is added to the 50-liter reactor vessel, maintaining the temperature below 25°C, causing the precipitation/crystallization of the Fmoc- ⁇ -Ala-Leu-Ala-Leu-O-t-Bu tetrapeptide product.
  • the quantity of distilled water added is calculated using the equation:
  • the resulting slurry of tetrapeptide is mixed well in the 50-liter vessel at room temperature, after which time dimethylformamide/water(55:45, v/v) is added to aid in the flowability of the slurry.

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Abstract

L'invention concerne un procédé de production d'un polypeptide dont le nombre et les séquences de résidus d'acides aminés sont prédéterminés. Ce procédé consiste à exposer un premier fragment peptidique ou d'acide aminé sous forme de substrat à un excès stoechiométrique d'un second fragment peptidique ou d'acide aminé réactif de manière à obtenir un produit de condensation ; à mettre la solution réactionnelle en contact avec un agent de piégeage insoluble pour piéger l'excès de second fragment peptidique ou d'acide aminé réactif ; à éliminer de la solution le second fragment piégé ; à soumettre la solution réactionnelle à une réaction qui élimine le groupe de protection à partir de la terminaison N ou C du produit de condensation obtenu à la première étape ; si nécessaire, à répéter les étapes précédentes. Ainsi, on assure de hauts rendement de production peptidique sans limitation (une seule terminaison) imposée par la synthèse en phase solide. Le procédé de cette invention est aussi « propre » que la synthèse en phase solide et peut en outre être automatisé. Ce procédé présente encore un avantage considérable, il n'est pas nécessaire d'isoler des intermédiaires dans une séquence synthétique longue ni d'éliminer tous les sous-produits contaminants du mélange réactionnel avant d'exécuter des étapes de traitement ultérieures.
EP00936209A 1999-05-26 2000-05-23 Procede de synthese peptidique a isolement minimal faisant appel a des resines echangeuses d'ions comme agents de piegeage Withdrawn EP1180115A1 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US528899 1983-09-02
US32276299A 1999-05-26 1999-05-26
US322762 1999-05-26
US52889900A 2000-03-20 2000-03-20
PCT/US2000/014152 WO2000071569A1 (fr) 1999-05-26 2000-05-23 Procede de synthese peptidique a isolement minimal faisant appel a des resines echangeuses d'ions comme agents de piegeage

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AU2002300066B2 (en) * 2001-07-19 2004-06-03 Merck Sharp & Dohme B.V. Process for Rapid Solution Synthesis of Peptides
TWI247012B (en) 2001-07-19 2006-01-11 Akzo Nobel Nv Process for rapid solution synthesis of peptides
TWI243826B (en) 2001-07-19 2005-11-21 Akzo Nobel Nv Process for the preparation of peptides
ES2729197T3 (es) 2006-03-01 2019-10-30 Kaneka Corp Método de producción de péptidos
WO2016140232A1 (fr) * 2015-03-04 2016-09-09 Jitsubo株式会社 Procédé de synthèse peptidique
CA3126705A1 (fr) 2019-02-01 2020-08-06 Gap Peptides Llc Strategie de synthese pour groupe de protection gap
SE2050293A1 (en) * 2020-03-17 2021-09-18 Peptisystems Ab Peptide synthesis and system thereof

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