MXPA01012082A - Minimal isolation peptide synthesis process using ion-exchange resins as scavenging agents. - Google Patents

Abstract

A process for the production of a polypeptide having a pre-determined number and sequence of amino acid residues, comprising the steps of first exposing a first substrate amino acid or peptide fragment to a stoichiometric excess of a second reactant amino acid or peptide fragment to form a condensation product; second, contacting the reaction solution from the first step with an insoluble scavenger to sequester the excess of the second reactant amino acid or peptide fragment; third, removing from the solution the sequestered excess second reactant amino acid or peptide fragment; fourth, subjecting the reaction solution to a reaction which removes the protecting group from either the N- or C-terminus of the condensation product of the first step; and fifth, if necessary, repeating the first through fourth steps. The method is capable of large-scale production of peptides in solution, is not subject to the one-terminus-only limitation of the solid-phase method, possesses the "cleanliness" of the solid-phase method and, like the solid-phase method, is capable of automation. Most importantly, however, 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 contaminating by-products from the reaction mixture prior to subsequent processing steps.

Description

SYNTHESIS PROCESS OF MINIMUM INSULATION PEPTIDES. USING ION EXCHANGE RESINS AS DEPURATING AGENTS This application is a continuation in part of the United States of America Patent Application Serial Number 09 / 322,762, filed May 26, 1999.

TECHNICAL FIELD The present invention relates to synthetic chemical processes. More particularly, the present invention has to do with a solution phase process, particularly adapted to the production of commercial scale quantities of polypeptides, which minimizes the requirement for isolation of intermediates.

BACKGROUND OF THE INVENTION Prior to the discovery of R. Bruce Merrifield, in 1963, of a solid-phase method for synthesizing polypeptides (RB Merrifield,. Am. Chem. Soc. 85: 2149-2154 (1963)), the processes for Preparation of peptides that contained more than a small number of amino acid residues, were difficult and time consuming. Since that time, the so-called Merrifield solid phase technique has been used for the production of a large number of peptides.

AAd ?? -?. ^ * * .l 4. ?? lt * > íl .. ~ -Al ~ »- *? =? .. -.1 .. J, F..FF, - -" ".-- *. ^. r? ^ i ^^. ^. - ,, * > "TW rfÜl The first stage of the synthesis of the solid phase peptide consists of the assembly of a chain of peptides in a polymer or insoluble support resin, by means of the sequential reactions of amino acid derivatives • 5 protected. In a subsequent step, the chain of peptides from the solid resin support is dissociated, with the concurrent or subsequent dissociation of side chain protecting groups, to give the crude free peptide. This technique has been adapted to be used in two 10 alternative methods. In the stationary solid phase variant originally used by Merrifield, the reagents and washing solvents are passed through a column of resin globules to which the growing peptide chain is attached, and on which this is supported.

This synthetic method is limited to the addition of new amino acid residues to the chain of growing peptides, only at the N-terminus, since the chain is typically fixed to the resin at its C-terminus. typical stationary, are added 20 individual amino acids at the N terminus of the growing peptide chain, until the desired polypeptide is obtained. In a less frequently used alternative, small fragments of peptides consisting of multiple amino acid residues are added to the growing peptide 25 fixed to the resin.

In an alternative solid phase peptide synthesis method, the resin pellets are slurried in a container, and exposed to successive wash solution or reagent solutions, with each being removed, • 5 typically through a filter in the bottom of the container, before the next one is added. The slurry method of solid phase peptide synthesis is limited in the same way to a peptide synthesis of only one term. Both variants described Previously, the solid phase peptide synthesis method is capable of being automated, and is well suited • for the preparation of milligram quantities to multiple grams of peptides. A number of automated phase peptide synthesizers are commercially available 15 solid, which employ a microcomputer to open and close the valves that control the sequence and duration of the flow of different reagents and washing solvents applied to a resin in which the peptide is supported. ^^ growth. When quantities of peptides are required that 20 exceed hundreds of grams, the synthetic solid phase method is generally inadequate, and the solution phase method is used. In the solution phase method, pieces of polypeptides are put together by solution chemistry25 classic, which facilitates the union of amino acids A & & amp; «< Ai, ^ - rfe # J &gBÉaHjßj ^ &. ^^^ * ~ A? &A ** iH H? A * - + ^? Individual proteins, or fragments of di-, tri-, tetra- or oligopeptides of the final polypeptide in which the undesired reaction sites have been appropriately protected. The smallest fragments are prepared by themselves • 5 similar way, by joining the pieces of individual amino acids or smaller protected fragments, and so on. By judiciously mapping that synthesis, it is possible to minimize the number of steps required for the production of a desired end product. Unlike 10 of the solid phase method, the solution phase method is free from the limitation of only one term in the • synthesis of a peptide. In the solution phase method, a fragment formed in a previous step can be reacted by the reaction of an amino function with terminal N 15 protected, in a subsequent step in its carboxyl function with unprotected C-terminus. This possibility is not open to the solid phase method since, as stated above, the growing peptide is "blocked" at the C-terminus by binding to the support resin. The synthesis of solution phase peptides, despite being free from the limitation of synthesis of only one term of solid phase peptide synthesis, suffers from one drawback: the need to isolate and frequently purify the growing peptide. All 25 reactions are carried out in solution, resulting in a mixture containing the desired product, as well as unwanted unreacted reagents and by-products. After many steps of synthesis, the solution would become loaded with appreciable amounts of these contaminants, which, if left in solution, would affect the subsequent steps of the synthesis or load the isolation and purification of the desired final product peptide. . As a consequence, it is prudent and often necessary to isolate intermediates as peptide synthesis proceeds. Each of these isolates adds to the cost and time of the synthesis, and decreases the overall yield of the desired peptide. With the advent of combinatorial chemistry techniques in recent years, methods have been made available to prepare small amounts of large libraries of compounds. Recently, D.L. Flynn, and collaborators, JL. Am. Chem. Soc. 119: 4874-4881 (1997) have described the use of resins to sequester excess reagents and byproducts of combinatorial chemistry steps. In the method described therein, the authors use resins called CMR / R, or complementary molecular recognition / reactivity, to purify the products of each parallel step in a combinatorial chemistry scheme to prepare libraries of compounds. The process involves the synthesis of members of the library by means of solution phase methods, followed by --- »B¡? 3tá ^^^ * ¿^^^^ y¡.

«P the removal of reagents, reagents, by-products and / or catalysts in excess of the solution phase, by incubating the reaction mixture through the CMR / R resins. 5 R.J. Booth, et al., J. Am. Chem. Soc. 119: 4882-4886 (1997) also describe the use of derivatives supported by polystyrene-divinylbenzene of tris- (2-aminoethyl) amine, to cool the reagents in excess of the raw reaction products, obtained from the 10 parallel synthesis of the solution phase of the amides, in the construction of combinatorial libraries. L.M. Gayo, and collaborators, Tetrahedron Letters. 38 (4): 513-516 (1997) describe the production of a combinatorial library of amides by means of first 15 react, in parallel, a number of amines with a slight excess of an acid chloride, to produce the desired amides. After the reaction, the mixture is treated with water to convert the acid chloride in excess to the corresponding carboxylic acid which, together with the by-product 20 of HCl from the formation of the amide and the hydrolysis of the acid chloride, is removed by means of a purification resin. S.D. Brown et al., J. Am. Chem. Soc. 118: 6331-6332 (1996) describe a "capture of 25 resin "for the combinatorial synthesis of compounds of tetra-substituted ethylene, using the reaction between Jbis (boryl) alkenes and alkyl halides. An aryl iodide fixed to the resin captures an intermediate product in the reaction, to produce a desired product. • 5 Although these techniques describe alternative ways to use functionalized resins to cool reactions, or to capture excess reagents, by-products or desired products in combinatorial chemistry schemes, none has provided an effective method 10 for preparing commercial scale quantities of therapeutically useful compounds, particularly peptides. The • Particular problems of large-scale production, characteristic of commercial synthesis, are of little concern in the construction of a library 15 combinatorial in the initial stages of drug discovery. There is therefore a need for improved methods for synthesizing commercial scale amounts of peptides that provide most of the advantages of the peptides. • 20 methods of both solid phase and solution phase, while minimizing the inconveniences of each.

SUMMARY OF THE INVENTION In accordance with the present invention, an improved method of peptide synthesis is provided, the ..tf? fji. t._l- which capitalizes the main advantages of the peptide synthesis methods of both solid phase and solution phase. The method described is capable of producing large-scale peptides in solution, is not subject to the limitation of only one term of the solid phase method, has the "cleanliness" of the solid phase method and, like the method of solid phase, is capable of automation. More importantly, the method of the present invention does not require the frequent isolation of intermediates in a prolonged synthetic sequence nor, neceily, the removal of all contaminants from the reaction mixture prior to the subsequent processing steps. In accordance with the main embodiment of the present invention, there is provided a process for the synthesis of a polypeptide having a number and sequence of previously determined amino acid residues. In its most general aspect, the process sequentially comprises the steps of first exposing, in solution, a first amino acid of the substrate or peptide fragment of the desired polypeptide product, the first amino acid of the substrate or peptide fragment being protected in, either its term N or C, with a stoichiometric excess of a second amino acid of the reagent or peptide fragment of the desired polypeptide, the second amino acid of the reagent or L? .AAMr.lrlf * - * - .íí.i. t? 1 peptide fragment being protected in the other of its N or C terms, to form a condensation product of the substrate and the reagent. The resulting condensation product is protected in both N and C terms. In the second step of the process, the reaction solution is contacted with an insoluble scavenger having a reactive functionality complementary to the functionality with non-protected N or C terminus. of the first amino acid or peptide fragment, to sequester the excess of the second 10 amino acid of the peptide reagent or fragment. In the third step, the second amino acid is removed • of excess reagent or peptide fragment sequestered from the reaction solution, leaving the condensation product and reaction byproducts in solution. If it came to It is necey or desirable to decrease the volume of the reaction solution, which is increased during the process of the present invention, the condensation product can be precipitated or crystallized, and then replaced in a • lower volume solution. This solution, in the fourth step, is subjected to a reaction that removes the protective group from either the N or C term of the condensation product of the first step. If, at this point, the desired polypeptide sequence is not yet achieved, the first to fourth steps are repeated as one cycle, with the condensation product 25 unprotected from each fourth previous step becoming the . »~ T .. *. * ~ F..A.tot» A,. «..-« &A ~ * J., "j | t, a ,,.,. ,.-_ ". ... ^ ... ^. Jh »-. . < ^ - A¿iH.

Peptide fragment from the substrate of each successive first step, until the desired peptide is produced. At the point at which the desired polypeptide sequence has been produced, the product is isolated and deprotected, if necessary, • 5 of any terminal or side chain protecting groups.

DESCRIPTION OF THE DRAWINGS OF THE DRAWINGS In the Drawings, which are part of the description of the present invention: Figure 1 is a schematic representation of the • steps of the process for preparing polypeptides according to the method of the present invention. Figure 2 is a schematic representation of a commercial-scale semiautomatic process apparatus for preparing polypeptides according to the process of the present invention.

Detailed Description of the Invention • As used throughout this specification and the appended claims, the terms "protected" or "blocked", as applied to amino acids and peptides, have the meanings commonly accepted in the art. That is, a protected or blocked amino acid or peptide is one in which the reactive functionality of either or both of the amino group with terminal N and / or the C-terminal carboxyl group, has been blocked by reaction with a "blocking group" to avoid its reactivity. Additionally, other functional groups of the amino acid or peptide may be blocked, such as side chain amino groups on lysine, or hydroxy groups on serine, threonine, or tyrosine; and carboxyl groups in residues of aspartic or glutamic acid, by means of appropriate blocking groups, to avoid unwanted reactions. Blocking groups suitable for the protection of the amine, hydroxyl, and carboxyl functions are well known in the art. Blocking groups and methods for their union and dissociation are fully established in "Protective Groups in Organic Synthesis", 2nd Edition, by T.W. Greene, and collaborators, John Wiley & Sons, Inc., New York, 1991. When referring to different blocking groups, shorthand designations of acronyms commonly used by chemists are used throughout this specification. The definitions of acronyms can be found, for example, in T.W. Green, and collaborators, in the aforementioned work, on pages xi-xvi. The abbreviation "OSu" refers to the fraction derived by the removal of the hydroxyl hydrogen atom of N-hydroxysuccinimide. The blocking group, alternatively known as "Cbz" or simply "Z" is the The benzyloxycarbonyl protecting group. The blocking group, alternatively known as "tBoc", or simply "Boc", is the tert-butyloxycarbonyl protecting group. Referring to Figure 1, the process is illustrated 5 of the present invention in schematic block diagram. In the first step of the process, the first amino acid of the substrate or appropriately protected peptide fragment is exposed, except for either its N-terminal amino group or its C-terminal carboxyl function, in a Suitable solvent, to the amino acid of the reagent or peptide fragment. The reagent can be a single amino acid or some • peptide fragment of the polypeptide of the final product, suitably protected in the other of its amino group with N-terminal or C-terminal carboxyl group, as well as in 15 reactive functional groups of side chain. The carboxyl terminus of the protected N-terminal 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 20 the N-hydroxysuccinimide ester group (-OSu). The protecting group for the N-terminus of the amino acid of the peptide or substrate reagent or fragment is a group that is "orthogonal" to the protecting groups that are used to protect the amino, hydroxyl, and carboxyl groups of 25 side chain in either the reagent or the substrate. For other 1 part, the protective group with N-terminus in the amino acid of the peptide or substrate reagent or fragment must be one that is easily removed under conditions that do not remove the side chain protecting groups, or the C-terminal blocker group of the other reagent or the substrate. It is said that two protective groups are "orthogonal" if the chemical processes that are used to remove one do not remove the other. A preferred protecting group with N-terminal preferred for the amino acid of the reagent or The peptide or substrate fragment is one that is easily dissociated under hydrogenolysis conditions, or ^ Catalytic hydrogenation. Since it is known that sulfur "poisons" or inactivates inactive hydrogenation catalysts, the preferred embodiment of the present process The invention is limited to the synthesis of peptides that do not contain sulfhydryl or side chain thioether groups; that is, to the synthesis of peptides that do not contain cysteine and that do not contain methionine. The amino blocking groups, • hydroxyl, and side chain carboxyl are selected from blocking groups well known in the art, which do not dissociate under hydrogenolysis conditions. A N-terminal amino protecting group, preferred for the amino acids and peptide fragments of the reagent or substrate that are used in the process of the present invention, is the benzyloxycarbonyl group, sometimes * ~ *, * F *. i i. called the carbobenzoxy group, and denoted "Cbz" or simply "Z" in chemical shorthand. The Z group is easily dissociated by hydrogenolysis under mild conditions of the N-terminal amino groups of the protected amino acids or peptides, while leaving the less reactive protecting groups that have been used to protect the side chain functional groups unaffected. Preferred blocking groups for the C-terminus of the amino acid of the peptide or substrate reagent or fragment are simple ester groups such as the tert-butyl ester group and the like. The substrate (unprotected in either its N term or its C term) is allowed to react the reagent (unprotected in the other of its N or C terms) until the analysis of the aliquot samples that are periodically taken from the reaction indicate the substantially complete reaction. In the process of the present invention, the reagent is employed in stoichiometric excess to the amount of the substrate, for the purpose of bringing the condensation reaction to completion. Preferably, an amount greater than 1.0, up to about 1.1 moles of amino acid of the peptide reagent or fragment, is used per mole of amino acid of the substrate or peptide fragment. The amount of excess that is required in each particular coupling reaction will vary according to the chemical nature of the substrate and the reagent to be coupled. Without However, it is within the experience of the process chemist to determine with a small-scale table reaction, the required molar ratio of the reagent to the substrate, before committing to the cost of a large-scale preparation. • 5 scale. When the initial reaction between the amino acid of the peptide substrate or fragment and the amino acid of the peptide reagent or fragment is essentially complete, the reaction solution is contacted with a scavenger. He The debugger is either insoluble in the solvent system used, or has a functionality that is complementary • that of the unprotected amino acid term of the substrate or peptide fragment. In the preferred embodiment of the process of the present invention, the fragment of The amino acid or peptide is deprotected at the N-terminus, and protected against the reaction at its C-terminus. Correspondingly, the amino acid of the peptide reagent or fragment is protected at its N-terminus, preferably by a Z-group, and is activated at your term C. In this In the preferred embodiment of the process, the debugger has an active amine functionality. The preferred scavenger is an amine functionalized resin, such as the aminomethyl functionalized resins known in the art, particularly the styrene-divinylbenzene copolymers 25 modified aminomethyl, commercially available.

The method of contacting the reaction solution with the scrubber resin can be either by adding the resin to the reaction vessel, or vice versa, with the circulation of the reaction solution through a column of the resin being the preferred method. This method allows repeated recirculation of the reaction solution through the resin column, to ensure complete removal of the amino acid from the excess reagent or peptide fragment. If it becomes necessary or desirable to decrease the volume of the reaction, which increases during each cycle of the process of the present invention, the condensation product can be precipitated or crystallized, and then re-dissolved in a smaller volume of a suitable organic solvent. In the next step of the process, the blocking group with N terminal or desired C terminal of the condensation product of the first step of the process is removed. If, in a subsequent step, the amino acid of the peptide reagent or fragment is one in which the carboxyl terminus is deprotected, then the protective group with N-terminal of the condensation product is removed, and vice versa. In the preferred embodiment of the process of this invention, new segments, either individual amino acids or short peptide peptide fragments, are added to the growing polypeptide, by using a reagent C terminally active, and an N-terminally deprotected substrate. This is due to the protective group Z with preferred terminal N, and to the activating group OSu with preferred terminal C. Therefore, in this step of the preferred embodiment of the process, the group Z with terminal N of the condensation product is removed. The preferred method for this deprotection step is catalytic hydrogenolysis in the presence of a palladium catalyst, at pressures ranging from atmospheric pressure to about 30 to 50 psi (206.8 kPa to 344.7 kPa). Preferred catalysts are those which can be easily removed from the 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 (Al203), or silica (Si02). The hydrogenolysis leaves in the solution, once the catalyst has been removed by filtration, only the by-product of N-hydroxysuccinimide from the initial coupling reaction, the N-terminally deprotected condensation product, and toluene which, together with the dioxide of carbon is the byproduct of the hydrogenolysis of the Z group. (It should be noted that the N-hydroxysuccinimide and toluene by-products do not interfere with the catalytic hydrogenation deprotection step, nor with I? A-tt? itFi.?...í. , - .Ji fc ». F -, tj t subsequent condensations. The absence of any need for its removal in multi-step synthesis is a particular advantage of the preferred process of the present invention). If, after removal of the amino acid from the excess peptide reagent or fragment, the polypeptide sequence of the desired final product has been completely constructed, the process is terminated, except for the isolation of the polypeptide and deprotection by conventional methods, if needed or if desired. The isolation is further simplified in the preferred embodiment of the process of the present invention, since the only remaining contaminants in the reaction solution are N-hydroxysuccinimide, tertiary amine salt (if the amino acid of the substrate or reagent or fragment is used) of peptide in salt form, requiring neutralization before coupling), and toluene, all of which are easily removed by simple precipitation or crystallization of the desired final product polypeptide. Those skilled in the art will realize that, when a polypeptide bearing, for example, a free N-terminus, is the desired final product, or when recrystallization alone can serve to remove the amino acid from the reagent or excess peptide fragment. , the insulation can be done after the completion of a room or first particular step, respectively. If, however, as is the more general case, the synthesis of the polypeptide of the desired final product is incomplete after removal of the amino acid from reagent 5 or excess peptide fragment, the deprotection and depuration coupling steps are repeated, using the unprotected condensation product of each cycle as the substrate material of each subsequent cycle until the desired polypeptide sequence is achieved. 10 Referring to Figure 2, a modality of a commercial scale processing system is shown, • semiautomatic, to be used to perform the process of the present invention. The system 100 comprises a first reactor vessel 102 for carrying out the condensation of the Amino acid / peptide fragment or coupling reactions, a second reactor 106 to perform the deprotection reactions, a resin column 104, a filter 108, a product containment tank 110, first and ^^ second solvent tanks 112 and 114, and a unit of Processor Control 116. Although stainless steel reaction vessels can be used, it is preferred that all reactor vessels and tanks are coated with glass. It should be noted that the layout of the process in Figure 2 is only schematic. The 25 representation of the filter as a plate press filter - 0 and - frame, and of pumps as centrifugal pumps, is merely for illustrative purposes. The selection of the actual equipment that will be used will be within the experience and knowledge of an expert chemical engineer.

• The process described above would be carried out in an apparatus such as the one shown in Figure 2, in a semi-automatic manner, essentially requiring only the manual loading of the reagents and solvents in different stages of the process. The operation of valves, pumps and 10 agitators is controlled by means of the digital processor control unit 116, which is programmed before each • personalized synthesis, based on the materials used. Table 1 below gives the steps of the process to help understand the flow of the process through the 15 appliance. The control valves are opened and closed, as appropriate, for a given step of the process. The first reaction step is performed in the reactor 102, until it is essentially complete as indicated by the analysis of the samples from the 20 reactor. The samples can be physically removed by the operator and analyzed or, in an alternative mode, the analysis can be automated by means of a detector submerged in the contents of the reactor vessel, sensitive to one or more of the reagent and substrate, or a 25 by-product of the coupling reaction. It sends a signal from the detector to the digital processor 116, and used to determine the completion of the reaction step. The mixture is stirred by means of stirrer 126, and the temperature is controlled at a level close to the ambient, • generally less than about 30 ° C to about 35 ° C, during the course of the reaction. The reactor vessel 102 can be of a type well known in the process techniques for controlling the chemical reaction temperature. For example, the container 102 may be equipped 10 with an immersion cooling coil, cooling coils welded to the outside of the vessel itself, or # can be of a double jacket type, allowing the circulation of a coolant between the walls of the container. In any of these modalities, the The circulation of the refrigerant is controlled by means of a conventional closed feedback path, connected to the digital controller 116, which reacts to a temperature sensor, is not shown, submerged in the contents of the reactor vessel 102, and uses that 20 temperature to control the circulation of the coolant of the reactor vessel. When analyzes of the contents of the reactor 102 indicate that the reaction is substantially complete, the contents are cycled through the column 25 of resin 104, to remove excess reagent. By "circulating in cycle" it is meant to recirculate, by means of the pump 120 and associated pipe and valves, the contents of the reactor vessel 102 through the resin column 104, and back into the reactor vessel 5 102 The excess reagent is taken and maintained by chemical reaction with the resin, allowing only the coupled product and the by-products of the reaction to return to the reactor vessel. The solution recycled through the column and the The reactor vessel is periodically analyzed to see the total removal of the excess reagent. How I know • described above, this can be by physically removing the aliquot samples from the reactor vessel from time to time, or by means of a detector 15 submerged in the contents of the reactor vessel, or fixed in line in the pipe connection between the reactor vessel 102 and the resin column 104. The detector is sensitive to the excess reagent, and is bonded by means of the ^^ Closed trajectory of feedback control to 20 digital processor 116, and the signal from the recirculated product solution is used to control when the recirculation of the reaction solution can be stopped through the resin column 104. When the analysis indicates that the reaction solution 25 is substantially free of excess reagent, the r i? ? ? The numerical controller closes the appropriate valves and opens others to transfer, by means of the pump 122, the eluate of the resin column 104 to the storage tank 114. A first wash solvent of the first solvent tank 112 is then circulated through the from the resin column 104 and back to the first solvent tank 112, to flush any remaining product from the column. The recirculating jet of column 114 may be for either a predetermined number of cycles, or for a pre-determined time, as controlled by digital processor 116. When the washing or jet cleaning of column 104 is complete with the first solvent, the first solvent wash solution, which contains any jetted product from the resin column 104, is added to the contents of the storage tank 114. After the jet cleaning of the resin column 104 with the first solvent , if desired, a second washing or jet cleaning of the column 104 can be carried out, by means of the use of a second solvent, initially contained in a second tank of solvents 110. As in the case of the washing of the column 104 with the first solvent, the second solvent is recirculated between column 104 and tank 110, either for a predetermined number of cycles, or for a predetermined time, as controlled by the digital processor unit 116. After llá.¿. ¿? ** ¿¿i.-t., ¿... - ». * A A, A i ti í. * the jet cleaning or washing of the column 104, the second solvent solution, which contains any additional washing product from the column 104, is added to the contents of the container tank 114. • 5 Although the embodiment of the apparatus shown in FIG.

Figure 2 illustrates only two solvent wash tanks, 110 and 112, those skilled in the chemical processing art will understand that a third tank and subsequent tanks for solvents can be added 10 additional, as required. However, for most purposes, two tanks will be sufficient, with the • judicious choice of washing solvents for each particular reaction step. Polar solvent can be used as the washing solvent (s), with the limitation that the The solvent must not be reactive with the C-terminal activating groups, the functional groups, or the protecting groups on either the substrate or the reagent. In addition, these solvents must be used to solubilize effectively ^^ both the substrate and the reagent. The right solvents 20 include dimethyl sulfoxide, dimethylformamide (DMF), N-methylpyrrolidone, and low molecular weight alcohols such as iso-propanol and the like, with cost and availability being concerns secondary to polarity and non-reactivity. The preferred solvents for The process of the present invention includes dimethylformamide and iso-propanol. In this step of the process, the holding tank 114 contains a solution comprising the coupled reaction product, together with the reaction solvent and the solvents * 5 washing, and (in the preferred embodiment of the process) toluene, tertiary amine salt (if the amino acid of the substrate or reagent or peptide fragment is used in a salt form, requiring neutralization before coupling), and N-hydroxysuccinimide, but substantially 10 free from contaminating the reagent and excess substrate. The content of the container tank is then transferred, by means of the pump 124, to the second vessel of the reactor 106 for the removal of the blocking group Z from the N terminus of the coupled reaction product. As described above, the The preferred benzyloxycarbonyl ("Z") protecting group is easily removed by hydrogenolysis under comparatively mild conditions. In Figure 2, the second reactor 106 is shown as a hydrogenator adapted with the gas inlet pipes 134, 136, and 138 and the fan pipe 140. The gas inlet pipes 134, 136, and 138 are connected to the gas inlet pipes 134, 136, and 138. the sources (not shown) of jet cleaning gases such as argon and nitrogen, and the reaction gas, hydrogen. The flow of gases in and out of the second reactor 106 is controlled by means of valves 1, 2, 3, and 25 which, in turn, are controlled by the processor number 116. The hydrogenator 116 is purged with the jet cleaning gases, initially jet cleaned with hydrogen, and then charged at a higher pressure with hydrogen for the catalytic hydrogenolysis. When the reaction is complete, the appropriate valves are closed and opened to circulate the content of the hydrogenator 106, through the filter 108, to remove and collect the hydrogenation catalyst. From the filter 108 the reaction mixture is returned to the first reactor vessel 102 for subsequent reaction steps, or to the container tank 114 of the final product.

Table 1 Step 1 of the process: The reagent and the substrate are charged to the reaction vessel 102, and allowed to react. Step 2 of the process: The content of the reactor vessel 102 is recirculated through the column 104. Step 3 of the process: The contents of the reactor vessel 102 are transferred to the container tank 114. Step 4 of the process: The cleaning is carried out to a cyclic jet of column 104 with the first solvent. Step 5 of the process: The first jet solvent is transferred to the l. ai. íf¿..¿A- £ ¿. ^^^^? ^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ container tank 114. Step 6 of the process: The cyclic jet cleaning of column 104 is carried out with the second solvent. Step 7 of the process: The second jet solvent is transferred to the holding tank 114. Step 8 of the process: The contents of the holding tank 114 are transferred to the second vessel of the reactor 106. Step 9 of the process: The second reactor vessel is purged 106 with the first purge gas. Step 10 of the process: The second reactor vessel 106 is purged with the second purge gas. Step 11 of the process: The second reactor vessel 106 is purged with hydrogen. Step 11 of the process: The second reactor vessel 106 is pressurized with hydrogen, and the hydrogenolysis reaction is allowed to proceed. Step 12 of the process: The contents of the second reactor vessel 106 are filtered, to remove the hydrogenation catalyst and, if the desired peptide construct is incomplete, it is transferred to the first reactor vessel 102 for the addition of the following amino acid fragment or peptide to the growing peptide chain.

Step 13 of the process: If the desired peptide construct is incomplete, repeat steps 1-12, as required. Step 14 of the process: If the peptide construction is complete, • 5 the contents of the container tank 114 of step 7 are collected for further processing.

The following examples are presented to enable an expert in the art to better understand the process of • present invention. However, these examples will not be read as limiting the scope of the invention, as defined by the appended claims. The numbers of 15 individual passages are designated in accordance with Figure 1. The numbering for the cyclically repeated steps follows the format: step 1, 2a, 3a and 4a for the first cycle of the process of the invention; step Ib, 2b, 3b and 4b for the second cycle; etc. EXAMPLE 1 Preparation of Ester-Benzyloxycarbonyl-Lysyl (Tert-Butyloxycarbonyl) -Alanyl-Phenylalanyl-Vallylsilyl (ert-butyloxycarbonyl) -Isoleuyl-eucyl-isyl (t-butyloxycarbonyl) -25 isine bu iloxycarbonyl) -me ilo PaSQ la) Preparation of Benzyloxycarbonyl-Lysyl Ester (fcert-butyloxycarbonyl) - Lisin (fcert-butyloxycarbonyl) -methyl Lys (Boc) -OMe are mixed. HCl (2.96 grams, 10 mmol), • 5 and Z-Lys (Boc) -OSu (5.25 grams, 11 mmol) with 40 milliliters (41.32 grams) of N-Methyl-2-pyrrolidinone (NMP), in a 50 milliliter glass reactor, and shake the mix at room temperature, until the solids dissolve. Diisopropylethylamine (DIEA) (1.24 grams, 10.50 mmol) is slowly added to the contents of the reactor, for a period of about fifteen to thirty minutes. It allows the • Resulting mixture react at room temperature for approximately two hours. After this period of time, an aliquot of the reaction mixture is taken to analyze if the reaction is complete. 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 re-analyzed. When this analysis indicates that the coupling reaction 20 is complete, the contents of the reactor are maintained at room temperature with slow stirring.

Steps 2a-3a) Purification / Sequestration of Z-Lys Reagent (Boc) -QSu in Excess 25 Aminomethyl resin (2 grams) is mixed with 30 milliliters (30.99 grams) of NMP in a second reactor vessel. The resulting mixture is stirred at room temperature until a homogeneous slurry is obtained. The resin / NMP slurry is loaded onto the glass column, the resin slurry is allowed to settle within a packed bed, and any excess NMP is drained from the column. The reaction mixture of NMP containing the product Z-Lys (Boc) -Lys (Boc) -10 OMe and the reagent Z-Lys (Boc) -OSu in excess of Step 1 is then repetitively circulated through the resin column, for about one • hour, with the purpose of removing the Z-Lys (Boc) -OSu in excess. Circulation continues, if necessary, until the analysis indicates the complete removal of the reagent in 15 excess At this point, the NMP reaction solution containing the dipeptide Z-Lys (Boc) -Lys (Boc) -MOe blocked product is reserved. The resin column is then washed by recirculating 15 milliliters (11.75 grams) of iso-propanol to • through the column, for approximately thirty 20 minutes. The solution of iso-propanol, which contains the blocked dipeptide product, which was washed from the column, is reserved. The column is then washed by recirculating 30 milliliters (30.99 grams) of NMP through the column, for about thirty minutes. The NMP solution, containing the blocked dipeptide product, which was washed from the column, is reserved. A final wash is given to the resin column by recirculating 15 milliliters (11.75 grams) of iso-propanol through the column, for about thirty minutes. The NMP reaction solution of the blocked dipeptide, the NMP wash solution, and the two wash solutions of iso-propanol are combined.

Step 4a) Deprotection of the N Terminal of the Ester Product from Paso la. Benzyloxycarbonyl-Lysyl Ester (tert-butyloxycarbonyl) -Lisin (fcerfc-bu-yloxycarbonyl) -methyl Palladium-Deloxan® (0.60 grams) and para-toluenesulfonic acid (pTSA) (1.90 grams, 10 mmol) are placed in a 250 milliliter hydrogenator vessel. The hydrogenator vessel is flooded with argon, and the combined solutions of NMP / iso-propanol from Steps 2a-3a are charged to the hydrogenator vessel. The container is sealed and evacuated to a pressure of 20-25 inches of mercury (67.7-84.6 kPa) and purged three times with hydrogen. Then hydrogen is charged to the hydrogenator vessel at a pressure of 35-45 psi (234.5- 310.3 kPa), and the mixture is stirred at about 30 ° C, for about 2 hours. Then the hydrogen in the container is vented hydrogenator, and the vessel is purged twice with nitrogen. An aliquot sample of the reaction mixture is taken to analyze if the reaction is complete. If the analysis indicates that the reaction is incomplete, the 9 5 Hydrogenator vessel is purged twice with hydrogen, refilled with hydrogen at a pressure of 35-45 psi (234.5-310.3 kPa), and the mixture is stirred again at a temperature of about 30 ° C, for an additional hour . The hydrogenator vessel is evacuated and then purged two 10 times with nitrogen, and an aliquot of the reaction mixture is taken again to analyze if the reaction is • complete. The above steps of hydrogenation and analysis of the reaction mixture are repeated until the analysis indicates the substantial completion of the reaction of 15 hydrogenolysis. After the completion of the hydrogenolysis reaction, hydrogen is vented from the hydrogenating vessel, the vessel is purged with nitrogen, and the contents of the vessel are filtered to remove the • 20 catalyst. The hydrogenating vessel is rinsed with NMP, and the rinsing solution is added to the filtrate of the reaction mixture.

Step Ib) Preparation of Benzyloxycarbonyl-25-leucyl-Lisyl ester (fcupfc-butyloxycarbonyl) -Lisin (tert-butyloxycarbonyl) -methyl Z-Leu-OSu (3.98 grams, 11 mmol), DIEA (1.29 grams, 11 mmol) and the solution of NMP / i so-propanol • 5 of the deprotected dipeptide product in the N-terminal of the Step 4a (10 mmol, assuming a complete reaction), in a 1 liter glass reactor, and stir the mixture at room temperature, until all the solids are dissolved. The mixture is then stirred and allowed to 10 react at room temperature for approximately two hours. At the end of this time, a sample is taken • aliquot of the reaction mixture to analyze if the reaction is complete. If the analysis indicates that the reaction is incomplete, stir the reaction mixture to the 15 room temperature for an additional hour, and the reaction mixture is re-analyzed. When the analysis indicates that the coupling reaction is complete, the contents of the reactor are maintained at room temperature with slow stirring. Steps 2b-3b Purification / Sequestration of Excess Z-Leu-OSu Reagent An aminomethyl resin column (2 grams) is prepared as described above in Step 2a-3a, and the reaction mixture is repeatedly circulated. of NMP I i so-propanol of the blocked tripeptide of Step Ib, through the resin column for approximately one hour, periodically analyzing the eluate of the column to see the absence of Z-Leu-OSu in excess. When the assay indicates a substantially pure product, the blocked tripeptide NMP / iso-propanol reaction solution is reserved, and the column is washed by recirculating 15 milliliters (11.75 grams) of iso-propanol through the column, for about thirty minutes. The solution of iso-propanol, which contains the blocked tripeptide product, which was washed from the column, is reserved. The column is then washed by recirculating 30 milliliters (30.99 grams) of NMP through the column, for about thirty minutes. The NMP solution, which contains the blocked tripeptide product, which was washed from the column, is reserved. The reaction solution of NMP / iso-propanol of the blocked tripeptide product, and the washing solutions of iso-propanol and NMP are combined.

Step 4b) Deprotection of Terminal N of the Product of Step Ib. Benzyloxycarbonyl-Leucyl-Lysyl ester (tert-u-yloxy carbonyl) -Lisin (fcßrfc-bu-yloxycarbonyl) -methyl Using the same process as that of Step 4a, but Í-? After the addition of pTSA, the benzyloxycarbonyl protecting group in the tripeptide product Z-Leu-Lys (Boc) -Lys (Boc) -OMe of step Ib is removed by the addition of pTSA by hydrogenolysis.

Step 5) Preparation of Benzyloxycarbonyl-Isoleucyl-Leucyl-Lysyl Ester (fc-rfc-butyloxycarbonyl) -Lisin (fcert-butyloxycarbonyl) -methyl Z-Ile-OSu (3.98 grams, 11 mmol) is mixed in 10 ml. 1 liter glass reactor vessel with the NMP / iso-propanol solution of the deprotected tripeptide product in the • terminal N of 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 of the reaction mixture is analyzed to see if the reaction is complete. 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 the analysis indicates that the coupling reaction is complete, the contents of the reactor are maintained at room temperature with slow stirring. 25 Steps 2c-3c) Purification / Sequestration of Reagent Z- to ?? u? tllLáM, ti I SUI-OSu Excess column aminomethyl resin (2 g) is prepared as described above in Step 2 to 3, and then is repetitively circulating the reaction mixture of NMP / iso-propanol of the tetrapeptide blocked from Step 1, through the resin column, for about one hour, in order to remove excess Z-Ile-OSu. Circulation continues, if necessary, until the analysis indicates complete removal of the excess reagent. At this point, the reaction solution of NMP / iso-propanol containing the blocked tetrapeptide product is reserved. The resin column is washed by recirculating 15 milliliters (11.75 grams) of isopropanol through the column, for about thirty minutes. The solution of iso-propanol, which contains the blocked tetrapeptide product, which was washed from the column, is reserved. The column is then washed by recirculating 30 milliliters (30.99 grams) of NMP through the column, for about thirty minutes. The NMP solution is reserved, which contains the blocked tetrapeptide product, which was washed from the column. A final wash is given to the resin column by recirculating 15 milliliters (11.75 grams) of iso-propanol through the column, for about thirty minutes. The NMP / iso-propanol reaction solution of the product is combined blocked tetrapeptide, the NMP wash solution, and the two wash solutions of iso-propanol.

Step 4c) Deprotection of the Terminal N of the Product of the Passage. Benzyl Oxi-arbonyl-Isoleucyl-Leucyl-Lisyl (tßrt-butyloxycarbonyl) -Lisinffciferf-butyloxycarbonyl) -methyl ester Using the same process as that of Step 4a, but without the addition of pTSA, the benzyloxycarbonyl protecting group is removed by hydrogenolysis. the product • tetrapeptide Z-Ile-Leu-Lys (Boc) -Lys (Boc) -OMe del Paso le.

Step Id) Preparation of Benzyloxycarbonyl-15-bonyl-lysine (tert-butyloxycarbonyl) -Isoleucyl-leucyl-lysyl (fcert-butyloxycarbonyl) -Lisinf tert-butyloxycarbonyl) -methyl ^ ^ Z-Lys (Boc) - OSu (5.25 grams, 11 mmol) in F 20 a 1 liter glass reactor vessel with the NMP / iso-propanol solution of the deprotected tetrapeptide product in the N-terminal of 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 - «* * & .á * i. < It is analyzed an aliquot sample of the reaction mixture to see if the reaction is complete. If the analysis indicates that the reaction is incomplete, the reaction mixture is stirred at room temperature for an additional hour 5, and the reaction mixture is retested. When the analysis indicates that the coupling reaction is complete, the contents of the reactor are maintained at room temperature with slow stirring. 10 Steps 2d-3d) Purification / Sequestration of Reagent Z-Lys (Boc) -OSu in Excess and Precipita¬ • tion / Crystallization of the Condensation Product A column of aminomethyl resin is prepared (2) 15 grams) as described above in Step 2a-3a, and then the NMP / iso-propanol reaction mixture of the blocked pentapeptide of Step Id, is passed repetitively, through the resin column, during ^^ approximately one hour, with the purpose of removing the Z-F 20 Lys (Boc) -OSu in excess. The circulation continues, if necessary, until the analysis indicates the complete removal of the excess reagent. At this point, the reaction solution of NMP / iso-propanol containing the blocked pentapeptide product is reserved. The resin column 25 is then washed by recirculating 15 milliliters (11.75 grams) of iso- propanol through the column, for approximately thirty minutes. The solution of iso-propanol, which contains the blocked pentapeptide product, which was washed from the column, is reserved. The column is then washed by recirculating 30 milliliters (30.99 grams) of NMP through the column, for about thirty minutes. The NMP solution, which contains the blocked pentapeptide product, which was washed from the column, is reserved. A final wash is given to the resin column by recirculating 15 milliliters (11.75 grams) of iso-propanol through the column, for about thirty minutes. The NMP / iso-propanol reaction solution of the blocked pentapeptide product, the NMP wash solution, and the two wash solutions of iso-propanol are combined. To the solution of NMP / iso-propanol (± 350 milliliters) of the blocked pentapeptide is added pre-cooled water (150 milliliters), causing precipitation / crystallization of the pentapeptide product Z-Lys (Boc) -Ile-Leu-Lys (Boc) - Lys (Boc) -OMe. The mixture is stirred for 24 hours, after which time the pentapeptide is isolated by filtration. The resulting solid is re-slurried with water (200 milliliters) for 4 hours, and refiltered, then dried under vacuum. The dry solid is recrystallized from MeOH / EtOAc (40/45 milliliters) at 70 ° C, then stirred for 2 hours at room temperature. environment in the mother liquor. The recrystallized solid is isolated by filtration, dried twice with a volume of EtOAc (15 milliliters) and dried under vacuum. Concentrate the MeOH / EtOAc filtrate to dryness, with solidification 5 concomitant Z-Lys (Boc) -Ile-Leu-Lys (Boc) -Lys (Boc) -OMe additional. The resulting solid is recrystallized from MeOH / EtOAc (10/10 milliliters), filtered and rinsed with two volumes of EtOAc (8 milliliters), then dried under vacuum. The obtained solid (0.48 grams) is combined with the Z-10 Lys (Boc) -Ile-Leu-Lys (Boc) -Lys (Boc) -OMe (8.80 grams) of the first recrystallization, to give a total of 9.28 grams ^ B (78 percent of theoretical yield).

Step 4d) Deprotection of Terminal N of the Product of Step 3d. Benzyloxycarbonyl-Lisyl (tert-butyloxycarbonyl) -Isoleucyl-eucyl-isyl (tert-butyloxycarbonyl) -Lisin (tert-buoyloxycarbonyl) -methyl ester 20 Mix Z-Lys (Boc) -Ile-Leu -Lys (Boc) -Lys (Boc) -OMe (9.28 grams, 0.86 mmol) with 60 milliliters of NMP in a 1 liter glass reactor. Then, using the same process as that of Step 4a, but without the addition of pTSA, the protective group is removed by hydrogenolysis 25 benzyloxycarbonyl in the N-terminus of the pentapeptide product (from 3d Step). Step) Preparation of Benzyloxycarbonyl-Valyl-Lisin Ester (tert-butyloxycarbonyl) -Isoleucyl-Leucyl-Lisyl (tert-butyloxy -carbonyl) -Lisi (fcerfc-butyloxy-carbonyl) -methyl Z mixture -Val-OSu (3.30 grams, 9.50 mmol) in a 1 liter glass reactor vessel with the NMP solution of the deprotected pentapeptide product at the N-terminus of 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 to see if the reaction is complete. 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 re-analyzed. When the analysis indicates that the coupling reaction is complete, the contents of the reactor are maintained at room temperature with slow stirring.

Steps 2e-3e) Depuration / Sequestration of Z-Val-OSu Reagent in Excess An aminomethyl resin column is prepared (1.60 grams) as described above in Step 2a-3a, and then the NMP reaction mixture of the blocked hexapeptide from Step 1 is repeatedly circulated through the resin column, for about one 5 hours, for the purpose to remove the Z-Val-OSu in excess. Circulation continues, if necessary, until the analysis indicates complete removal of the excess reagent. At this point, the NMP reaction solution containing the blocked hexapeptide product is reserved. Afterwards, the 10 resin column by recirculating 15 milliliters (11.75 grams) of iso-propanol through the column, for about thirty minutes. The solution of iso-propanol, which contains the blocked hexapeptide product, which was washed from the column, is reserved. Later 15 washes the column by recirculating 30 milliliters (30.99 grams) of NMP through the column for approximately thirty minutes. The NMP solution, which contains the blocked hexapeptide product, is reserved. • washed from the column. A final wash is given to the resin column by recirculating 15 milliliters (11.75 grams) of iso-propanol through the column, for about thirty minutes. The NMP reaction solution of the blocked hexapeptide, the NMP wash solution, and the two isopropanol wash solutions are combined.

Step 4e) Check out Terminal N of the Step Product. Benzyloxycarbonyl-Valyl-Lisyl Ester (tert-bu- • 5-tyloxycarbonyl) -Isoleucyl-Leucyl-Lysyl (fcert-butyloxycarbonyl) -Lisin (tert-butyloxycarbonyl) -methyl Using the same process as that of Step 4a, but without the addition of pTSA, the benzyloxycarbonyl protecting group in the hexapeptide product Z-Val-Lys (Boc) -Ile-Leu-Lys (Boc) -Lys (Boc) -OMe from the benzyloxycarbonyl group is removed by hydrogenolysis.

• Step him.

Step lf) Preparation of Benzyloxycarbonyl Ester-lalanyl-Valyl-Lisin (t &r-butyloxycarbonyl) -Isoleucil-Leuc 1 -Lisyl (tert-butyloxycarbonyl) -L sin (fcert-butyloxycarbonyl) -methyl Z-Phe-OSu mixture (3.77 grams, 9.50 mmol) in a 1 liter glass reactor vessel with the NMP / iso-propanol solution of the deprotected hexapeptide product in the N-terminal of Step 4e. 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 to see if the reaction is complete. 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 re-analyzed. When the analysis indicates that the coupling reaction is complete, the contents of the reactor are maintained at room temperature with slow stirring.

Steps 2f-3f) Depuration / Sequestration of Z-Phe-OSu Reagent in Excess An aminomethyl resin column (1.60 grams) is prepared as described above in Step 2a-3a, and then the reaction mixture is repetitively circulated of NMP / iso-propanol from the heptapeptide blocked from Step lf, through the resin column, for about one hour, in order to remove excess Z-Phe-OSu. Circulation continues, if necessary, until the analysis indicates complete removal of the excess reagent. At this point, the reaction solution of NMP / iso-propanol containing the blocked heptapeptide product is reserved. The resin column is then washed by recirculating 15 milliliters (11.75 grams) of isopropanol through the column for about thirty minutes. The iso-propanol solution is reserved, which i- i you contains the blocked heptapeptide product, which was washed from the column. The column is then washed by recirculating 30 milliliters (30.99 grams) of NMP through the column, for about thirty minutes. The NMP solution, containing the blocked heptapeptide product, which was washed from the column, is reserved. A final wash is given to the resin column, by recirculating 15 milliliters (11.75 grams) of iso-propanol through the column, for about thirty minutes. The reaction solution of NMP / iso-propanol from the blocked heptapeptide, the washing solution of NMP, and the two washing solutions of iso-propanol are combined.

Step 4f) Protection of Terminal N of the Product of Step lf. Benzyloxycarbonyl-Phenylalanyl-Vallyl-Lisyl Ester (fcert-butyloxycarbonyl) -Ioleoyl-Leucyl-Tissil (Terfc-butyloxycarbonyl) -Lisin (tert-butyloxycarbonyl) -methyl Using the same process as that of Step 4a, but without the addition of the pTSA, the benzyloxycarbonyl protecting group in the hexapeptide product Z-Phe-Val-Lys (Boc) -Ile-Leu-Lys (Boc) -Lys (Boc) -OMe from Step lf is removed by hydrogenolysis.

Step ls) Preparation of Benzyloxycarbonyl-Alanyl-Phenylalanyl-Valyl Ester (fcßrt-butyloxycarbonyl) -Isoleucil- • 5 Leucyl-Lysyl (fcupfc-butyloxycarbonyl) - Lys (t-rfc-butyloxycarbonyl) -methyl Z-megcla- Ala-OSu (3.04 grams, 9.50 mmol) in a ^ 1 liter glass reactor vessel with the solution of NMP / iso-propanol of the heptapeptide product deprotected in the 10 terminal N of Step 4f. The mixture is stirred at room temperature until all solids are dissolved. Allowed • that the resulting mixture reacts at room temperature for at least 2 hours, at which time an aliquot of the reaction mixture is analyzed to see if the 15 reaction is complete. 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 re-analyzed. When the analysis indicates that the coupling reaction is complete, the 20 contents of the reactor at room temperature with slow stirring.

Steps 2s-3s) Depuration / Sequestration of Z-Ala-OSu Reagent in Excess 25 A column of aminomethyl resin (1.60 grams) is prepared as described above in Step 2a-3a, and then the mixture is repetitively circulated. NMP / iso-propanol reaction of the blocked octapeptide from Step lg, through the resin column, for about an hour, for the purpose of removing excess Z-Ala-OSu. Circulation continues, if necessary, until the analysis indicates complete removal of the excess reagent. At this point, the reaction solution of NMP / iso-propanol containing the blocked octapeptide product is reserved. The resin column is then washed by recirculating 15 milliliters (11.75 grams) of iso¬ • propanol through the column, for approximately thirty minutes. The solution of iso-propanol, which contains the blocked octapeptide product, which was washed from the column, is reserved. The column is then washed by recirculating 30 milliliters (30.99 grams) of NMP through the column, for about thirty minutes. The NMP solution, which contains the octapeptide product, is reserved • blocked, which was washed from the column. A final wash is given to the resin column by recirculating 15 milliliters (11.75 grams) of iso-propanol through the column, for about thirty minutes. The reaction solution of NMP / iso-propanol of the blocked octapeptide, the washing solution of NMP, and the two wash solutions of iso-propanol are combined.

N¿A.jtutAfat * M t > . .. JuLLit, Step 4s) Deprotection of the Terminal N of the Step Product la. Benzyloxycarbonyl-Alanyl-Phenylalanyl-5-Valyl-Lisyl (tert-butyloxycarbonyl) -Isoleucyl-Leucyl-Lysyl (tert-butyloxycarbonyl) -Lisin (fcert-butyloxycarbonyl) -methyl ester Using the same process as that of Step 4a , but without the addition of pTSA, the benzyloxycarbonyl protecting group in the product is removed by hydrogenolysis • octapeptide Z-Ala-Phe-Val-Lys (Boc) -Ile-Leu-Lys (Boc) -Lys (Boc) -MOe from Step lg. 15 Step lh) Preparation of Benzyloxycarbonyl-Lysyl Ester (tert-butyloxycarbonyl) - Alanyl-Phenylalanyl-Vallyl-Lisin (fcefc-butyloxycarbonyl) - Isoleucyl-Leucyl-l-Lisyl (tert-butyloxycarbonyl) -Lisin 20 (tert-butyloxycarbonyl) ) -methyl Z-Lys (Boc) -OSu (4.54 grams, 9.50 mmol) is mixed in a 1 liter glass reactor vessel with the NMP / iso-propanol solution of the octaprotein product deprotected at the N-terminal of Step 4g . The mixture is stirred 25 at room temperature until all the solid 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 to see if the reaction is complete. 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 re-analyzed. When the analysis indicates that the coupling reaction is complete, the contents of the reactor are maintained at room temperature with slow stirring.

Steps 2h-3h) Purification / Sequestration of Z- T.ys Reagent (Boc) -OSn in Excess An aminomethyl resin column (1.60 grams) is prepared as described above in Step 2a-3a, and then circulated repetitively the NMP / iso-propanol reaction mixture of the nonapeptide blocked from the Step lh, through the resin column, for about an hour, in order to remove the Z-Lys (Boc) -OSu in excess. Circulation continues, if necessary, until the analysis indicates complete removal of the excess reagent. At this point, the reaction solution of NMP / iso-propanol containing the blocked nonapeptide product is reserved. The resin column is then washed by recirculating 15 milliliters (11.75 grams) of iso- > ii-.iai, .fataJ-. ?, M4., .. -.t aaa.j.) LÜliinf - »- • - *» «- r ... j ",,., ^ ...... .A, _ ^ AJLj, i.l propanol through the column, for approximately thirty minutes. The solution of iso-propanol, which contains the blocked nonapeptide product, which was washed from the column, is reserved. The column is then washed by recirculating 5 milliliters (30.99 grams) of NMP through the column, for about thirty minutes. The NMP solution is reserved, which contains the blocked nonapeptide product, which was washed from the column. A final wash is given to the resin column by means of recirculating. 10 milliliters (11.75 grams) of iso-propanol through the column, for about thirty minutes. It combines • the reaction solution of NMP / iso-propanol of the blocked nonapeptide, the washing solution of NMP, and the two washing solutions of iso-propanol. 15 Step 5) Isolation of Ester from Benzyloxycarbonyl -Lisyl (fcerfc-butyloxycarbonyl) -Alanyl-Phenylalanyl-Valil-Lisin ^ (fcert-butyloxycarbonyl) -Iaoleucyl- 20 Leuci -Lisyl (fcupfc-butyloxycarbonyl) - Lisin (fcert-butyloxycarbonyl) -methyl To the solution of NMP / iso-propanol of the blocked nonapeptide from Step 2h-3h water previously cooled is added (300 milliliters), causing precipitation / crystallization 25 of the nonapeptide product Z-Lys (Boc) -Ala-Phe-Val-Lys (Boc) -Ile- Íri-AA ^.,. *. A .. ^ t¿ ^^ ^. ^^ < * Leu-Lys (Boc) -Lys (Boc) -OMe. The mixture is stirred for 24 hours, after which time the nonapeptide is isolated by filtration. The resulting solid is re-slurried with water (250 milliliters) for 4 hours, and refiltered, then dried under vacuum. The yield of Z-Lys (Boc) -Ala-Phe-Val-Lys (Boc) -Ile-Leu-Lys (Boc) -Lys (Boc) -OMe is 13.28 grams (94 percent of the theoretical yield from Z-Lys (Boc) -Ile-Leu-Lys (Boc) -Lys (Boc) - OMe, 82 percent overall yield). Example 2 Preparation of Ester d = Benzyloxycarbonyl-Lysyl (f-butyloxycarbonyl) -Fenylalanyl-Leuci-1-Lysyl (tert-butyloxycarbonyl) -Lisyl (fcert-butyloxycarbonyl) -Alanyl-Lysyl (fcert-butyl-15-oxycarbonyl) -Lisi] (fcerfc-butyloxycarbonyl) -Fenylalanyl-Glycine-methyl Step a) Preparation of Benzyloxycarbonyl-Phenylalanyl-Glycine-methyl Ester 20 Gly-OMe.HCl (6.25 grams, 50 mmol) and Z-Phe-OSu (21.80 grams, 55 mmol) are mixed with 60 milliliters (56.52 g). grams) of dimethylformamide (DMF) in a 250 milliliter glass reactor, and the mixture is stirred at room temperature until the solids dissolve. Diisopropylethylamine (DIEA) is added slowly (6.49 grams, 55 mmoles) to the contents of the reactor, for a period of about fifteen to thirty minutes. The resulting mixture is allowed to react at room temperature for about two hours. After this period of time, an aliquot sample of the reaction mixture is taken to analyze if the reaction is complete. 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 re-analyzed. When this analysis indicates that the coupling reaction is complete, the contents of the reactor are maintained at room temperature with slow stirring.

Steps 2a-3a) Purification / Sequestration of Reagent Z- P e-QSu in Excess Aminomethyl resin (8.30 grams) is mixed with 50 milliliters (47.10 grams) 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 loaded onto a glass column, the resin slurry is allowed to settle within a packed bed, and any excess DMF is drained from the column. The reaction mixture of DMF containing the product Z-Phe-Gly-OMe and the At the same time, the reagent Z-Phe-OSu in excess of the Step la, through the resin column, for about an hour, for the purpose of Remove the Z-Phe-OSu in excess. Circulation continues, if necessary, until the analysis indicates complete removal of the excess reagent. At this point, the reaction solution of DMF containing the blocked dipeptide Z-Phe-Gly-OMe product is reserved. The resin column is then washed by recirculating 20 milliliters - (15.66 grams) of iso-propanol through the column, for about thirty minutes. The solution of iso-propanol, which contains the blocked dipeptide product, which was washed from the column, is reserved. The column is then washed by recirculating 40 milliliters (37.68 grams) of DMF through the column, for about thirty minutes. The DMF solution, which contains the blocked dipeptide product, which was washed from the column, is reserved. A final wash is given to the resin column by recirculating 20 milliliters (15.66 grams) of iso-propanol through the column, for about thirty minutes. The reaction solution of DMF of the blocked dipeptide, the washing solution of DMF, and the two washing solutions of iso-propanol are combined.

Step 4a) Deprotection of the Terminal N of the Step Product la. Ester de Ben- ¿Aal'tafc - ¿íaÍb¿ aíííH £% Cycloxycarbonyl-Phenylalanyl-Gl icinomile Palladium-Deloxan® (0.90 grams) and para-toluenesulfonic acid (pTSA) (10 grams, 52.05 mmol) are placed in a 5 1 liter hydrogenator container. The hydrogenator vessel is flooded with argon, and the combined DMF / iso-propanol solutions from Step 2a-3a are charged to the hydrogenator vessel. The container is sealed and evacuated to a pressure of 20-25 inches of mercury (67.7-84.6 kPa) and 10 purge three times with hydrogen. Then hydrogen is charged to the hydrogenator vessel at a pressure of 35-45 psi (234.5- 310.3 kPa), and the mixture is stirred at about 30 ° C, for about 2 hours. Then the hydrogen in the container is vented 15 hydrogenator, and the vessel is purged twice with nitrogen. An aliquot sample of the reaction mixture is taken to analyze if the reaction is complete. If the analysis indicates that the reaction is incomplete, the hydrogenator vessel is purged twice with hydrogen, 20 refill with hydrogen at a pressure of 35-45 psi (234.5-310.3 kPa), and the mixture is stirred again 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 is taken again 25 of the reaction mixture to analyze if the reaction is complete The above steps of hydrogenation and analysis of the reaction mixture are repeated until the analysis indicates substantial completion of the hydrogenolysis reaction. After the completion of the hydrogenolysis reaction, hydrogen is vented from the hydrogenating vessel, the vessel is purged with nitrogen, and the contents of the vessel are filtered to remove the catalyst. The hydrogenating vessel is rinsed with DMF, and the rinsing solution is added to the filtrate of the reaction mixture.

Step Ib) Preparation of Benzyloxycarbonyl-Lysyl (tert-butyloxycarbonyl) -phenylalanyl-glycine-methyl Ester Z-Lys (Boc) -OSu (26.20 grams, 55 mmol), DIEA (6.49 grams, 55 mmol) are placed and the DMF / iso-propanol solution of the deprotected dipeptide product at the N-terminal of Step 4a (50 mmol, assuming a complete reaction), in a 1 liter glass reactor, and the mixture is stirred at room temperature, until that all the solids dissolve. The mixture is then stirred and allowed to react at room temperature for about two hours. At the end of this time, an aliquot sample of the reaction mixture is taken to analyze - ^? IM if the reaction is complete. 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 re-analyzed. When this analysis • 5 indicate that the coupling reaction is complete, the contents of the reactor are maintained at room temperature with slow stirring.

Steps 2b-3b Purification / Sequestration of Z-10 Lys (Boc) Reagent -OSu in Excess An aminomethyl resin column is prepared (8.30 • grams) as described above in Step 2a-3a, and the reaction mixture of DMF / iso-propanol from Step Ib is repeatedly circulated through the 15 resin for approximately one hour, periodically analyzing the eluate of the column to see the absence of Z-Lys (Boc) -OSu in excess. When the analysis indicates a substantially pure product, the solution of ^^ reaction of DMF / iso-propanol of the tripeptide product 20 blocked, and the column is washed by recirculating 20 milliliters (15.66 grams) of iso-propanol through the column, for about thirty minutes. The solution of iso-propanol, which contains the blocked tripeptide product, which was washed from the column, is reserved. Later 25 wash the column by recirculating 40 milliliters (37.68) grams) of DMF through the column, for approximately thirty minutes. The DMF solution, which contains the blocked tripeptide product, which was washed from the column, is reserved. The column is given a final wash by recirculating 20 milliliters (15.66 grams) of isopropanol, through the column, for approximately thirty minutes. The reaction solution of DMF / iso-propanol of the blocked tripeptide product, and the washing solutions of iso-propanol and DMF are combined. 10 Step 4b) Deprotection of Terminal N of the • Product of Step Ib. Benzyloxycarbonyl-Lysyl Ester (tert-butyloxycarbonyl) -Fenylalanyl-Glycine-methyl Using the same process as that of Step 4a, but without the addition of pTSA, the benzyloxycarbonyl protecting group is removed by hydrogenolysis at the N-terminus of the product tripeptide Z-Lys (Boc) -Phe-Gly-OMe from Step Ib.

• Step 20) Preparation of Benzyloxycarbonyl-T.isyl Ester (fcert-butyloxycarbonyl) - Lysyl (fcerfc-butyloxycarbonyl) -Fenylalanyl-Glycine-methyl Z-Lys (Boc) -OSu (26.20 grams, 55 mmol) are placed ), 25 and the solution of DMF / iso-propanol of the tripeptide product unprotected from Step 4b, in a 1 liter glass reactor, and the mixture is stirred at room temperature, until all solids are dissolved. The mixture is then stirred and allowed to react at room temperature during • 5 approximately two hours. At the end of this time, an aliquot sample of the reaction mixture is taken to analyze if the reaction is complete. 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 retested. When this analysis indicates that the coupling reaction is complete, the • Reactor content at room temperature with slow stirring. 15 Steps 2c c Debug / Sequester Z-Lys Reagent (Boc) -OSu in Excess An aminomethyl resin column (8.30 grams) is prepared as described above in Step 2a-3a, and the mixture is repeatedly circulated of reaction DMF / iso-propanol from the Step, through the resin column for about one hour, periodically analyzing the eluate of the column to see the absence of Z-Lys (Boc) -OSu in excess. When the analysis indicates a substantially pure product, the solution of Reaction of DMF / iso-propanol of the reaction product, and wash the column by recirculating 20 milliliters (15.66 grams) of iso-propanol through the column, for approximately thirty minutes. The solution of iso-propanol, which contains the blocked tetrapeptide product F 5, which was washed from the column, is reserved. The column is then washed by recirculating 40 milliliters (37.68 grams) of DMF through the column, for about thirty minutes. The DMF solution, which contains the blocked tetrapeptide product, which was washed from the 10 column. The column is given a final wash by recirculating 20 milliliters (15.66 grams) of iso-propanol, a • through the column, for approximately thirty minutes. The DMF / isopropanol reaction solution of the blocked tetrapeptide product is combined and the 15 washing solutions of iso-propanol and DMF.

Step 4c) Deprotection of the N Terminal of the Step Product. Benzyloxycarbonyl-Lysyl Ester (tert-butyloxy- • 20-carbonyl) -Lisyl (fcert-butyloxycarbon-1) -Fenylalanyl-Glycidyl-ethyl Using the same process as that of Step 4a, but without the addition of pTSA, removes by benzolysis the benzyloxycarbonyl protecting group at the N-terminus of the 25 tetrapeptide product Z-Lys (Boc) -Lys (Boc) -Phe-Gly-OMe lJk *,! J, AÚ *, * «. * i * L..A-. i ^ J * ~.

Step him. Step Id) Preparation of Benzyloxycarbonyl Ester-Alanyl-Lysyl (fcerfc-butyloxycarbonyl) -Lisyl (terfc-bn oxycarbonyl) -5-Phenylalanyl-Glycine-methyl Z-Ala-OSu (16.00 grams, 50 mmol ), and the DMF / iso-propanol solution of the deprotected tetrapeptide product from Step 4c, in a 1 liter glass reactor, and the mixture is stirred at room temperature, until 10 Dissolve all solids. The mixture is then stirred and allowed to react at room temperature for about two hours. At the end of this time, an aliquot sample of the reaction mixture is taken to analyze if the reaction is complete. If the analysis indicates that the The reaction is incomplete, the mixture is stirred at room temperature for an additional hour, and the reaction mixture is re-analyzed. When this analysis indicates that the coupling reaction is complete, the ^^ content of the reactor at room temperature with stirring F 20 slow. (Note: Because in this particular coupling reaction no theoretical excess of Z-Ala-OSu was used, Step 2d was not required, and was not conducted). 25 Step 3d) Precipitation / Crystallization of the Condensation Product To the solution of DMF / iso-propanol (± 500 milliliters) of the blocked pentapeptide of Step Id, pre-cooled water (250 milliliters) is added, causing the 5 precipitation / crystallization of the product pentapeptide Z-Ala-Lys (Boc) -Lys (Boc) -Phe-Gly-OMe. The pentapeptide is isolated by filtration, dried with DMF / water (500/250 milliliters), then with 2 liters of water. The resulting solid is redissolved in DMF (700 milliliters), 10 precipitates / crystallizes by the addition of water (350 milliliters), is collected by filtration and dried under • empty. The yield of Z-Ala-Lys (Boc) -Lys (Boc) -Phe-Gly-OMe is 28.26 grams (62 percent of theoretical yield). 15 Step 4d) Deprotection of the N Terminal of the Step 3d Product. Benzyloxycarbonyl-Alanyl-Lysyl Ester (fcerfc-butoyloxycarbonyl) -Lisyl (tert-butyloxycarbonyl) -Fenylalanyl-Glyc-methyl- ^^ 20 Mix Z-Ala-Lys (Boc) -Lys (Boc) -Phe- Gly-OMe (16.90 grams, 18.84 mmol) with 160 milliliters of NMP in a 1 liter glass reactor. Then, using the same process as that of Step 4a, but without the addition of the pTSA, the benzyloxycarbonyl protecting group 25 is removed by hydrogenolysis at the N-terminus of the pentapeptide product (from Step 3d).

Step) Preparation of Benzyloxycarbonyl Ester (fcfc-butyloxycarbonyl) - Alanyl-Lysyl (fcupfc-bu-yloxycarbonyl) - Lysyl (fcupfc-butyloxycarbonyl) -phenyl-5-alanyl-glycine-methyl Z-Lys ( Boc) -OSu (9.89 grams, 20.72 mmol) and DIEA (1.22 grams, 10.36 mmol), in a 1 liter glass reactor vessel, with the NMP solution of the pentapeptide product deprotected at the N-terminal of the Step 10 4d. The mixture is stirred at room temperature, until all the solids are dissolved. It allows the mixture • Resulting reaction at room temperature for at least two hours, at which time an aliquot sample of the reaction mixture is analyzed, to see if the reaction 15 is complete. 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 retested. When this analysis indicates that the coupling reaction is complete, the content of the • 20 reactor at room temperature with slow stirring.

Steps 2e-3e) Depuration / Sequestration of Z-Lys (Boc) -OSu Reagent in Excess An aminomethyl resin column (3.45 25 grams) is prepared as described above in Step 2a-3a, and the NMP reaction mixture of the blocked hexapeptide from Step 1 is then repeated repetitively through the resin column for about one hour for the purpose of removing the Z-Lys (Boc) -OSu in • 5 excess. Circulation continues, if necessary, until the analysis indicates complete removal of the excess reagent. At this point, the NMP reaction solution containing the blocked hexapeptide product is reserved. The resin column is then washed by means of recirculating. 10 milliliters (15.66 grams) of iso-propanol through the column, for about thirty minutes. It reserves • the iso-propanol solution, which contains the blocked hexapeptide product, which was washed from the column. The column is then washed by recirculating 40 milliliters (41.32). 15 grams) of NMP through the column, for approximately thirty minutes. The NMP solution, containing the blocked hexapeptide product, which was washed from the column, is reserved. A final wash is given to the resin column by recirculating 20 milliliters (15.66 grams) ^^ 20 of iso-propanol through the column, for about thirty minutes. The NMP reaction solution of the blocked hexapeptide, the NMP wash solution, and the two isopropanol wash solutions are combined. 25 Step 4e) Deprotection of Terminal N of the Step Product Je. Benzyl ester oxycarbonyl-Lysyl (terfc-butyloxycarbonyl) alanyl-Ti sil (oxycarbonyl fcerfc-buty) -Lisil (terfc-butyloxycarbonyl) phenylalanyl-glycine-methyl Acetate Using the same process as that of Step 4a, but without the addition of pTSA, it is removed by hydrogenolysis benzyloxycarbonyl protecting group at the N-hexapeptide product Z-Lys (Boc) -Ala-Lys (Boc) -Lys (Boc) -Phe-Gly-OMe Step le.

Step lf) Preparation of ester-Lysyl Bonil benzyloxycarbonyl (fcert-butyloxycarbonyl) - Lysyl (tert-butyloxycarbonyl) -Alanil- Lysyl (terfc-butyloxycarbonyl) -Lisil (fcerfc-butyloxycarbonyl) glycine methyl -phenylalanyl- are placed Z- Lys (Boc) -OSu (9.89 g, 20.72 mmol), and NMP solution / isopropanol hexapeptide deprotected product of Step 4a, in a glass reactor of 1 liter, and the mixture is stirred at room temperature, until all solids dissolve. The resulting mixture is allowed to react at room temperature for approximately two hours, at which time a t. *; ** 3 »í« shows aliquot of the reaction mixture to analyze if the reaction is complete. If the analysis indicates that the reaction is incomplete, the mixture is stirred at room temperature for an additional hour, and the mixture is retested • Reaction When this analysis indicates that the coupling reaction is complete, the contents of the reactor are maintained at room temperature with slow stirring.

Steps 2f-3f) Depuration / Sequestration of Z-10 Lys (Boc) Reagent -OSu in Excess An aminomethyl resin column is prepared (3.45 • grams) as described above in Step 2a-3a, and the reaction mixture is repetitively circulated.

NMP / iso-propanol from Step lf, through the column 15 resin, for approximately one hour, periodically analyzing the eluate of the column to see the absence of Z-Lys (Boc) -OSu in excess. When the analysis indicates that the product is substantially pure, the NMP / iso-propanol solution of the reaction product is reserved, and the ^^ 20 resin column by recirculating 40 milliliters (31.32 grams) of iso-propanol through the column, for about thirty minutes. The solution of iso-propanol, which contains the blocked heptapeptide product, which was washed from the column, is reserved. Later 25 washes the column by recirculating 80 milliliters (82.64 grams) of NMP through the column, for about thirty minutes. The NMP solution, containing the blocked heptapeptide product, which was washed from the column, is reserved. A final wash is given to the resin column by recirculating 40 milliliters (31.32 grams) of iso-propanol through the column, for about thirty minutes. The reaction solution of NMP / iso-propanol from the blocked heptapeptide product, and the washing solutions of iso-propanol and NMP are combined. 10 Step 4f) Deprotection of Terminal N of the • Product of Step lf. Ben- ester ciloxicarbonil-Lysyl (terfc-butyloxycarbonyl) -Lisil (tert-butiloxicarbo- 15 nil) alanyl-LisiKfcert-butiloxicarho- nil) -Lisil (fcert-butyloxycarbonyl) -Fe- nilalanil-methyl-glycine Using the same process that that of Step 4a, but without the addition of pTSA, is removed by hydrogenolysis ^^ 20 the benzyloxycarbonyl protecting group at the N-terminus of the heptapeptide product Z-Lys (Boc) -Lys (Boc) -Ala-Lys (Boc) -Lys (Boc) -Phe-Gly-OMe from Step lf.

Step ls) Preparation of Benzyloxycarbonyl Ester-25-bonyl-Leucyl-Lysyl (fcerfc-butyloxycarbonate) bonil) -Lisyl (erfc-butyloxycarbonyl) - Alanyl-Lisyl (fcerfc-butyloxycarbonyl) - Lysyl (tert-butyloxycarbonyl) -phenyl-alanyl-glycine-methyl • 5 Z-Leu-OSu (7.50 grams, 20.72 mmoles) are placed , and the NMP / iso-propanol solution of the deprotected heptapeptide product from Step 4f, in a 1 liter glass reactor, and the mixture is stirred at room temperature, until all solids are dissolved. The mixture is then stirred and allowed to react at room temperature for about two hours. At the end of this time, it is taken • an aliquot sample of the reaction mixture to analyze if the reaction is complete. 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 retested. When this analysis indicates that the coupling reaction is complete, the contents of the reactor are maintained at room temperature with slow stirring. • 20 Steps 2g-3g) Purification / Sequestration of Z-Leu-OSu Reagent in Excess and Precipitation / Crystallization of the Condensation Product 25 An aminomethyl resin column is prepared (3.45 grams) as described above in Step 2a-3a, and the NMP / iso-propanol reaction mixture of Step Ig is repeatedly circulated through the resin column, for about one hour, analyzing • periodically eluate the column to see the absence of Z-Leu-OSu in excess. When the analysis indicates that the product is substantially pure, the NMP / iso-propanol solution of the reaction product is reserved, and the column is washed by recirculating 40 milliliters (31.32 grams) of iso-propanol through the column, for approximately thirty minutes. The solution of • iso-propanol, which contains the blocked octapeptide product, which was washed from the column. The column is then washed by recirculating 80 milliliters (81.64 grams) of NMP through the column, for about thirty minutes. The NMP solution is reserved, which contains the blocked octapeptide product, which was washed from the column. A final wash is given to the resin column by recirculating 40 milliliters (31.32 grams) of iso-propanol through the column, for about thirty minutes. The NMP / isopropanol reaction solution of the blocked octapeptide product and the washing solutions of iso-propanol and NMP are combined. To the solution of NMP / iso-propanol (± 500 milliliters) 25 of the blocked octapeptide is added water previously cooled (200 milliliters), causing the precipitation / crystallization of the octapeptide product Z-Leu-Lys (Boc) -Lys (Boc) -Ala-Lys (Boc) -Lys (Boc) -Phe-Gly-OMe. The octapeptide is isolated by filtration, is washed with NMP / water (500/200 • 5 milliliters), and 2 liters of water, then dried under vacuum. The yield of Z-Leu-Lys (Boc) -Lys (Boc) -Ala-Lys (Boc) -Lys (Boc) -Phe-Gly-OMe is 22.75 grams (82 percent of the theoretical yield of Z-Ala- Lys (Boc) -Lys (Boc) -Phe-Gly-OMe). 10 Step 4s) Deprotection of Terminal N of the Product of Step 3s. Ben- • Cryloxycarbonyl-Leucyl-Lisyl Ester (fcert-butyloxycarbonyl) -Lisyl (fcupfc-butyloxycarbonyl) -Alanyl-Lisyl (fcerfc-butyl-15-oxycarbonyl) -Lisyl (tert-butyloxycarbonyl) -Fenylalanyl-Glyc-methyl mixed Z-Leu-Lys (Boc) -Lys (Boc) -Ala-Lys (Boc) -Lys (Boc) -Phe-Gly-OMe (19.18 grams, 13.08 mmol) with 200 milliliters of NMP in a glass reactor 1 liter. ^^ 20 Then, using the same process as that of Step 4a, but without the addition of the pTSA, the benzyloxycarbonyl protecting group is removed by hydrogenolysis at the N-terminus of the blocked octapeptide product (from Step 3g). 25 Step 1) Preparation of Benzyloxy Ester bonyl-phenylalanyl-leucyl-lysi 1 (fcupf-butyloxycarboni 1) -lisi 1 (fcupfc-buty-oxycarbonyl) -lanyl-lysyl (fcupfc-butyl-oxycarbonyl) -lysyl (ert-butyloxycarbonyl) -phenylalanyl-glycine-methyl Z-Phe-OSu (5.70 grams, 14.38 mmol) is mixed in a 1 liter glass reactor vessel, with the solution NMP of the octaprotein product deprotected at the N-terminus of Step 4g. The mixture is stirred at room temperature, until all the solids are dissolved. The resulting mixture is allowed to react at room temperature for at least two hours, at which time an aliquot sample of the reaction mixture is analyzed to see if the reaction is complete. 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 retested. When this analysis indicates that the coupling reaction is complete, the contents of the reactor are maintained at room temperature with slow stirring.

Steps 2h-3h) Purification / Sequestration of Z-Phe-OSu Reagent in Excess An aminomethyl resin column (2.40 grams) is prepared as described above in Step 2a-3a, and then the mixture is repeatedly repeated.

NMP reaction of the blocked nonapeptide from Step 1, through the resin column, for about one hour, in order to remove the excess Z-Phe-OSu. Circulation continues, if necessary, until the analysis • 5 indicate the complete removal of the excess reagent. At this point the NMP reaction solution, which contains the blocked nonapeptide product, is reserved. The resin column is then washed by recirculating 20 milliliters (15.66 grams) of iso-propanol through the column, during 10 approximately thirty minutes. The iso-propanol solution, which contains the blocked nonapeptide product, is reserved, • who washed from the column. The column is then washed by recirculating 40 milliliters (41.32 grams) of NMP, through the column, for about thirty 15 minutes. The NMP solution, containing the blocked nonapeptide product, which was washed from the column, is removed. A final wash is given to the resin column by recirculating 20 milliliters (15.66 grams) of iso-propanol to ^^ through the column, for about thirty ^^ 20 minutes. The NMP reaction solution of the blocked nonapeptide, the NMP washing solution, and the washing solutions of iso-propanol and NMP are combined.

Step 4h) Deprotection of Terminal N of the Product of Step lh. Ester de Ben- Cycloxycarbonyl-phenylalanyl-leucyl-lysyl (fcupfc-butyloxycarbon 1) -Li si 1 (fcupfc-b-tyloxycarbonyl) -lanyl-lysyl (fc-rfc-butyloxycarbonyl) -lysyl (ert-butyloxycarbonyl) -phenylalanyl-glycine-methyl Using the same process than that of Step 4a, but without the addition of pTSA, the benzyloxycarbonyl protecting group is removed by hydrogenolysis at the N-terminus of the nonapeptide product Z-Phe-Leu-Lys (Boc) -Lys (Boc) -Ala-Lys (Boc) -Lys (Boc) -Phe-Gly-OMe from Step lh.

Step li) Preparation of Benzyloxycarbonyl-lysyl (tert-butyloxycarbonyl) -phenylalanyl-leucyl-lysyl (tert-butyloxycarbonyl) -lysyl (tert-butyloxycarbonyl) -lanyl-lysyl (tert-butyloxycarbonyl) ) -Lisyl (fcfc-butyloxycarbonyl) -phenylalanyl-glycine-methyl Z-Lys (Boc) -OSu (6.55 grams, 13.73 mmoles), and the NMP / iso-propanol solution of the deprotected nonapeptide product from Step 4h, are placed in a 1 liter glass reactor, and the mixture is stirred at room temperature, until all the solids are dissolved. The resulting mixture is allowed to react at room temperature during t #? -? < > -fa * «M '» t > "- > '*'« r ^ < f, & ,. ,,,, .. t, «* jfe» ti > iiaMÍ * M »Mt < aMMt ^^,«? *** - JL && amp; A. approximately two hours, at which time an aliquot sample of the reaction mixture is taken to analyze if the reaction is complete. 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 re-analyzed. When this analysis indicates that the coupling reaction is complete, the contents of the reactor are maintained at room temperature with slow stirring. (Note: Clearance / sequestration of the Z-Lys (Boc) -OSu reagent in excess, as for the final sequence of Step 2i-3i, was not conducted before isolation of the final condensation product in Example 2).

Step 5) Isolation of Ester of Benzyloxy-15 carbonyl-Lysyl (fcerfc-butyloxycarbonyl) -Fenilalanyl-Leucyl-Lysyl (fcerfc- butyl oxycarbonyl) -Lisyl (fcerfc-butyl-oxycarbonyl) -Alanyl-Lysyl (fcerfc-butyl- oxycarbonyl) -Lisyl (tert-butyloxycarbonyl-20-phenylalanyl-glycine-methyl) To the solution of NMP / iso-propanol (± 260 milliliters) of the blocked decapeptide from Step li, precooled water (150 milliliters) is added, causing the precipitation / crystallization of the product decapeptide Z-25 Lys (Boc) -Phe-Leu-Lys (Boc) -Lys (Boc) -Ala-Lys (Boc) -Lys (Boc) -Phe- HÜill | t j-fai'i * ^ - - "*" - - - - * - * - »-.-i- * W * + t" > "• * - *" - F - "- - * - *** • '• "Gly-OMe The nonapeptide is isolated by filtration, dried 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 grams (79 per • 5 percent theoretical yield from Z-Leu-Lys (Boc) - Lys (Boc) -Ala-Lys (Boc) -Lys (Boc) -Phe-Gly-OMe, 41 percent overall yield).

Example 2 Preparation of Ester = 9-Fluorenylmethoxycarbonyl-Jefca-Alanyl-Leucyl-Alanyl-Leucin-tert-butyl • PaSO 1) Preparation of Benzyloxycarbonyl-Alanyl-Leucin-fcert-butyl Ester 15 Leu-OtBuDHCl are mixed (184 grams, 0.82 moles), and Z-Ala-OSu (290 grams, 0.91 moles), with 1.9 liters (1790 grams) of dimethylformamide in a 5-liter glass reactor, and the mixture is stirred at room temperature, until the solids dissolve. It is added slowly 20 diisopropylethylamine (115 grams, 0.97 moles) to the contents of the reactor, for a period of about fifteen to thirty minutes. The resulting mixture is allowed to react at room temperature for about two hours. Aliquots of the content are then taken 25 of the reactor to analyze whether the coupling reaction is complete When these analyzes indicate that the reaction is complete, the contents of the reactor are maintained at room temperature with slow stirring. • 5 Steps 2-3) Depuration / Sequestration of Reagent (Z-Ala-OSu in Excess) Aminomethyl resin (164 grams) is mixed with 1.6 liters (1550 grams) of dimethylformamide in a second reactor vessel. The resulting mixture is stirred to the 10 room temperature until a homogeneous slurry is obtained. The resin / DMF slurry is loaded onto a column of • glass, the resin slurry is allowed to settle within a packed bed, and any excess DMF is drained from the column. Thereafter, the reaction mixture containing the dipeptide product Z-Ala-Leu-OtBu and the excess Z-Ala-OSu reagent is circulated repetitively through the resin column for approximately one hour. At the end of this ^^ time, the DMF solution containing the product is set aside ^^ 20 dipeptide Z-Ala-Leu-O-t-Bu, and an aliquot is removed to verify if the depuration reaction is complete. Circulation continues, if necessary, until the analysis indicates complete removal of the excess reagent. Then the resin column is washed by means of recirculating 785 25 milliliters (615 grams) of iso-propanol through the column, for approximately thirty minutes. The solution of iso-propanol, containing the dipeptide product Z-Ala-Leu-O-t-Bu, which was washed from the column, is reserved. The column is then washed by recirculating 1.56 liters (1470 grams) of dimethylformamide through the column for about thirty minutes. The DMF solution, containing the dipeptide product Z-Ala-Leu-0-t-Bu, which was washed from the column, is reserved. A final wash is given to the resin column by recirculating 785 10 milliliters (615 grams) of iso-propanol through the column, for about thirty minutes. It combines • the original reaction solution in DMF, the DMF wash solution, and the two isopropanol wash solutions. 15 Step 4) Deprotection of the N Terminal of the Ester Product from Step 1). Benzyloxycarbonyl-Alanyl-Leucin-tert-Butyl Ester • 20 Palladium-Deloxan® (16 grams) is placed in a 2-gallon (7.57-liter) hydrogenator vessel. The hydrogenator vessel is flooded with argon, and the combined solutions of DMF / isopropanol from Steps (2) and (3) are charged to the hydrogenator vessel. The container is sealed and 25 evacuates at 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 at a pressure of 35-45 psi (234.5-310.3 kPa), and the mixture is stirred at about 30 ° C, for about 2 hours. 5 The hydrogenating vessel is then vented under vacuum, re-pressurized with hydrogen at a pressure of 35-45 (234.5-310.3 kPa), and hydrogenation is continued for about one hour at 30 ° C. The vessel is purged and evacuated twice with nitrogen, and a sample is taken 10 aliquot of the reaction mixture to analyze if the reaction is complete. If the analysis indicates that the reaction is incomplete, the hydrogenator vessel is purged twice with hydrogen, recharged with hydrogen at a pressure of 35-45 psi (234.5-310.3 kPa), and agitated again. 15 mix 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 of the reaction mixture is taken again to analyze if the reaction is complete. The previous steps of Hydrogenation and analysis of the reaction mixture are repeated until the analysis indicates substantial completion of the hydrogenolysis reaction. After completion of the hydrogenolysis reaction, the hydrogenator vessel is vented to the Vacuum, purge with nitrogen, and filter the contents of the vessel to remove the catalyst. The hydrogenating vessel is rinsed with dimethylformamide, and the rinsing solution is added to the filtrate of the reaction mixture. aso i ') Preparation of Benzyloxycarbonyl-Leucyl-Alanyl-Leucin-fcerf-butyl Ester Z-Leu-OSu (328 grams, 0.90 mole), and the solution of the unprotected dipeptide product from step 4) (0.82 moles) are placed. , assuming the complete reaction), in a 12 liter glass reactor, and the mixture is stirred at room temperature, until all the solids are dissolved. The mixture is then stirred at room temperature for approximately two hours. At the end of this time, an aliquot sample of the reaction mixture is taken to analyze if the coupling reaction is complete. 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 retested. When these analyzes indicate that the reaction is complete, the contents of the reactor are maintained at room temperature with slow stirring.

Steps 2 '-3') Purification / Sequestration of Reagent Z- '** Leu-OSu in Excess An aminomethyl resin column (164 grams) is prepared as described above in steps 2) and 3) and the • 5 reaction mixture from Step 1 '), through the resin column for about one hour, periodically analyzing the eluate of the column to see the absence of excess Z-Leu-OSu. When the analysis indicates a substantially pure tripeptide product, the 10 solution of Z-Leu-Ala-Leu-O-t-Bu, and the column is washed by recirculating 785 milliliters (615 grams) of isopropanol through the column, for about thirty minutes. The solution of iso-propanol, which contains the tripeptide product Z-Leu-Ala-Leu-O- -Bu, is reserved. 15 washed from the column. The column is then washed by recirculating 1.56 liters (1470 grams) of dimethylformamide through the column, for about thirty minutes. The DMF solution, containing the tripeptide product Z-Leu-Ala-Leu-O- -Bu, which was washed from the • 20 column. The resin column is given a final wash by recirculating 785 milliliters (615 grams) of isopropanol through the column, for about thirty minutes. The original solution of the tripeptide product Z-Leu-Ala-Leu-O- -Bu is combined, the two solutions 25 washing of iso-propanol, and the washing solution of DMF. aso 4 ') Deprotection of Terminal N of the Product of Step 1'). Benzyloxycarbonyl-Leucyl-Alanyl-Leucine-butyl-butyl ester Using the same process as that in Step 4), the benzyloxycarbonyl protecting group is removed by hydrogenolysis at the N-terminus of the tripeptide product Z-Leu-Ala-Leu- O-t-Bu of step 1 '). 10 Step 1 '') Preparation of Ester of 9-Fluorenyl- • methoxycarbonyl-beta-Alani-Leucyl-Alanyl-Leucin-tert-Butyl Fmoc-Jeta-alanin-N-15 hydroxysuccinimide ester (Fmoc-β-Ala) is mixed -OSu, 336 grams, 0.82 moles) in a 50-liter glass reactor vessel with the DMF / iso-propanol solution of the deprotected tripeptide product in the N-terminal of Step 4 '). The mixture is stirred at room temperature until all are dissolved F ^^ 20 the solids. 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 to see if the coupling reaction is complete. 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 re-analyzed. When these tests indicate that the reaction is complete, distilled water previously cooled to the • 50 liter reactor vessel, maintaining the temperature below 25 ° C, causing the precipitation / crystallization of the tetrapeptide product Fmoc- ß-Ala-Leu-Ala-Leu-O-t-Bu. The amount of distilled water added is calculated using the equation: 10 Kilograms of Distilled Water = [(Volume of the • Solution) / O .55] - [Volume of the Solution].

The resulting tetrapeptide slurry mixes well 15 in the 50 liter vessel at room temperature, after which dimethylformamide / water (55:45, volume / volume) is added to aid in the flowability of the slurry. The mixing is continued for about two hours at room temperature, after which time ^^ 20 isolates the tetrapeptide product by filtration, with the collection of the mother liquor in an appropriate recipient. An additional 4 kilograms of dimethylformamide / water (55:45 volume / volume) is added to the 50 liter reactor vessel, to wipe the residual tetrapeptide product, the 25 which is isolated by filtration, with the collection of ,. * '"" filtered in an appropriate receiver. Then approximately 4 kilograms of distilled water are added to the 50 liter glass reactor to wipe the remaining tetrapeptide product, which is likewise isolated by filtration, • with the collection of the filtrate in an appropriate receiver. The sample of the distilled water filtrate is analyzed to see the presence of dimethylformamide; distilled water is used to wash the filter cake of the combined tetrapeptide, until no dimethylformamide is detected in the filtrate. At this point, the filter cake is washed with about 4 kilograms of acetonitrile, with the filtrate being washed in an appropriate receiver. The cake of the wet acetonitrile filter is aspirated to dryness, for about one hour, and then 15 transferred to a glass drying tray and dried under vacuum and nitrogen flow at 30 ° C. The yield of Fmoc-ß-Ala-Leu-Ala-Leu-O- -Bu is 30.5 grams (60 percent theoretical yield). • twenty 25 i LA.A.?t?'* '** - - - .i. fcÍ .;

Claims (15)

1. A process for the synthesis of a polypeptide that has a number and sequence of amino acid residues • 5 previously determined, comprising, sequentially, the steps of: a) exposing, in solution, a first a first amino acid of the peptide substrate or fragment of the polypeptide, the first amino acid of the substrate or fragment of 10 peptide being protected in either its N or C terminus, with a stoichiometric excess of a second amino acid of the reagent • or peptide fragment of the polypeptide, the second amino acid of the reagent or peptide fragment being protected in the other of its N or C terms, to form a product of 15 condensation of the substrate and the reagent, the condensation product being protected in both terms N and C; b) contact the solution of step a) with an insoluble purifier that has a reactive functionality complementary to the functionality with terminal N or C not ^ Protected from the first amino acid of the substrate or peptide fragment, to sequester the excess of the second amino acid from the peptide reagent or fragment; c) remove the second amino acid from the reagent or fragment of excess sequestered peptide, leaving the 25 condensation product and to the byproducts of the reaction in solution; or driving the precipitation or subsequent crystallization of the condensation product, and then re-dissolving the condensation product, if the continuation of the synthesis is necessary or desired; • 5 d) removing the protective group from either the N or C term of the condensation product of step a); e) if the desired polypeptide sequence is not yet achieved, repeat steps a) to d), with the condensation product unprotected from each step d) previous 10 becoming the peptide fragment of the substrate of each step a) in succession; and • f) isolating and deprotecting, if necessary, the polypeptide product. final, once the desired peptide sequence is achieved. 15

2. The process, in accordance with the Claim 1, wherein the protecting group that was removed in step d) is removed from the term N.

3. The process, according to claim 1, wherein the protecting group that was removed ^ W 20 in step d) is removed from the term C.

4. The process, according to claim 1, wherein the insoluble scavenger employed in step b) is selected from the amine-functionalized resins and carboxyl. 25

5. The process, in accordance with the Claim 2, wherein the insoluble scavenger employed in step b) is an amine functionalized resin.

6. The process, according to claim 3, wherein the insoluble scavenger employed in step b) is a carboxyl-functionalized resin.

7. The process, according to Claim 5, wherein said resin is a polystyrene-divinylbenzene resin functionalized on aminomethyl.

8. A process for the synthesis of a polypeptide having a number and sequence of previously determined amino acid residues, comprising the steps of: a) exposing an amino acid solution of the substrate or fragment of said polypeptide, the substrate having a protecting group terminal C, which can not be removed by hydrogenolysis, with a stoichiometric excess of an amino acid of the reagent or a fragment of the polypeptide, the reagent having a N-terminal protectant group, which can be removed by hydrogenolysis, to form a product of condensation; b) contacting the solution of step a) with an amine-functionalized resin, to sequester the excess amino acid from the reagent or peptide fragment; c) remove the excess sequestered amino acid from the reagent or peptide fragment from the solution, leaving to the condensation product and to the by-products of the reaction in solution; or to conduct the precipitation or subsequent crystallization of the condensation product, and then to re-dissolve the condensation product, if the continuation of the synthesis is necessary or desired.; d) subjecting the solution of step c) to hydrogenolysis conditions, to remove the protective group from the N-terminus of said condensation product; e) repeating steps a) to d), using as the peptide fragment of the substrate for subsequent steps a) the unprotected condensation product of subsequent steps d) until the desired polypeptide has been produced.

9. The process, according to Claim 8, wherein the amine-functionalized resin is a resin modified by aminomethyl.

10. The process, according to claim 8, wherein the C-terminus of the reagent amino acid or a peptide fragment is activated, for reaction with amino functional groups.

11. The process according to claim 10, wherein the C-terminus of the amino acid of the reagent or a peptide fragment is activated by esterification with N-hydroxysuccinimide.

12. The process, in accordance with the r * Claim 8, wherein the N-terminal protective group of the reagent amino acid or the peptide fragment is benzyloxycarbonyl.

13. A process for the synthesis of a polypeptide 5 having a number and sequence of previously determined amino acid residues, comprising the steps of: a) exposing a solution of an amino acid of the substrate or fragment of said polypeptide, the substrate having a protective group of terminal C, with an excess Stoichiometric of an amino acid of the reagent or a fragment of the polypeptide, the reagent having an N-terminal benzyloxycarbonyl protecting group, and being activated by esterification with N-hydroxysuccinimide, to form a condensation product, until it is substantially 15 completes the reaction between the substrate and the reagent; b) contacting the solution of step a) with an amine-functionalized resin, to sequester and to remove from the solution the excess of the amino acid of the reagent or peptide fragment, leaving the condensation product and 20 to the byproducts of the reaction in solution, or to conduct the precipitation or subsequent crystallization of the condensation product, and then to re-dissolve the condensation product, if the continuation of the synthesis is necessary or desired; 25 c) submit the solution from step b) to conditions ti -? -? il? k? -.t & í.Xiit? í.,,. ... .iaatMtn .. ve *? ± u * ua? iák? x? r * t < a > . »Afeae tj-AjUi- * * of catalytic hydrogenation, to remove the benzyloxycarbonyl protecting group from the N term of the condensation product; d) removing, by filtration, the hydrogenation catalyst of the solution; e) repeating steps a) to d) until the desired polypeptide has been produced; and f) isolating and deprotecting, if desired or desired, the product polypeptide.

14. A process according to claim 13, wherein the synthesized polypeptide is Benzyloxycarbonyl-Lysyl Ester (tert-butyloxycarbonyl) -Alanyl-Phenylalanyl-Vallyl-Lysyl (tert-butyloxycarbonyl) -Isoleucyl-Leucyl-Lysyl (tert-butyloxycarbonyl) -Lisin (ert-butyloxycarbo¬ 15 nil) -methyl.

15. A process according to claim 13, wherein the synthesized polypeptide is 9-Fluorenylmethoxycarbonyl-Jeta-Alanyl-Leucyl-Alanyl-Leucin-tert-butyl Ester. twenty 25 SUMMARY A process for the production of a polypeptide having a number and sequence of amino acid residues previously determined, comprising the steps of first exposing a first amino acid of the substrate or peptide fragment to a stoichiometric excess of a second amino acid of the reagent or fragment of peptide to form a condensation product; Second, put 10 contact the reaction solution of the first step with an insoluble debugger to sequester the excess of the second • amino acid of the reagent or peptide fragment; third, remove the second amino acid from the reagent or fragment of excess sequestered peptide from the solution; in Fifthly, subjecting the reaction solution to a reaction that removes the protecting group from either the N or C terminus of the condensation product of the first step; and fifth, if necessary, repeat the steps first to fourth. The method is capable of producing peptides in large scale solution, is not subject to the limitation of only one term of the solid phase method, has the "cleanliness" of the solid phase method and, like the method of solid phase, is capable of automation. More important, however, the method of the present invention does not require the 25 frequent isolation of intermediaries in a sequence sfcc • *** » long synthetic or, necessarily, the removal of all polluting by-products from the reaction mixture, before the subsequent processing steps. * ^ - > '-í *.; *: