HIGH PERFORMANCE PEPTIDE SYNTHESIS

Abstract: In the method for the synthesis of a polypeptide chain on an insoluble solid support wherein an amino acid is introduced into a reactor packed with an insoluble solid support containing substituent groups reactive with said amino acid, coupling said amino acid to said support by condensation reaction with said reactive substituent groups, introducing a second similar or dissimilar amino acid into said reactor and coupling said second amino acid to said first amino acid and repeating the process until the desired polypeptide is obtained, the improvement wherein a pressurized flow system is used to obtain rapid reactions and quantitative yields of the desired products.

Description: - - HIGH PERFORMANCE PEPTIDE SYNTHESIS Technical Field This invention relates to the synthesis of peptides, polypeptides and proteins. More particularly, the inven¬ tion relates to an improvement in peptide, polypeptide and protein synthesis wherein the peptide, polypeptide or pro¬ tein is "grown" on an insoluble support or carrier by a series of stepwise coupling reactions. Background Art Classically, sequential polypeptides have been pre- pared by extremely laborious techniques wherein the inter¬ mediates have been isolated after the addition of each amino acid moiety. This has made the synthesis complicated and the preparation of long chain polypeptides nearly impossible because of low yields and/or racemization or other side reactions. In 1963, Merrifield (J. Amer. Chem. Soσ. , 85, 2149) and Letsinger and Kornet (J. Amer. Chem. Soc. , 85 , 2045) suggested the use of an insoluble polymer support for the growing peptide chain. This process permitted the separation of the growing peptide chain, prepared by classical synthetic methods, from reagents and by-products, without isolating the intermediate. In such a process the insoluble support is provided a reactive substituent group as by σhlorom thylation, carboxylation, hydroxymethylation, etc. and a protected amino acid coupled thereto either via the amino or carboxyl group. Then using a series of de- protection and coupling reactions the peptide is synthe¬ sized in a stepwise manner on the insoluble support. Although in principle the peptide may be assembled either from the amine or the carboxyl terminus, in practice the latter is preferred, mainly because of lower extents of racemization when Nα-urethane-protected, carboxyl activated amino acids are used for coupling reactions. After each deprotection step the insoluble support and its appended peptide chain are neutralized if necessary and washed - 2 - before the addition of the next amino acid residue. Finally, the polypeptide is removed from the solid sup¬ port by use of a suitable cleaving reagent, and any neces¬ sary deprotections completed. The final peptide is then subjected to extensive purification. Although the solid-phase method of peptide synthe¬ sis,originally developed by Merrifield, has revolution¬ ized the field of synthetic peptide chemistry, the nethod is complicated by many problems, especially when applied to large peptides ( > 10 amino acides) . For example, the reactive sites on the insoluble supports employed in the synthesis are located on and within the support at varying degrees or depths of accessibility. Because of shrinkage and swelling of the resin during a normal synthetic pro- cedure, the number of "inaccessible sites" may change throughout the procedure. This means that a reactive site which is inaccessible during one particular cycle may in practice become accessible during a subsequent cycle. Because of the heterogeneous nature of the reaction sites on the support and their variability with cycles or steps, reactions in the Merrifield synthetic procedure are often incomplete and unpredictable even though a substantial excess of the amino acid reactant is used. When very long polypeptide chains are synthesized this failure to obtain 100% reaction during every step of the synthesis gives rise to a large variety of "contaminant" polypeptides with "failure sequences" (i.e. sequences containing dele¬ tion of one or more amino acid residues) . Since these "contaminant" polypeptides are similar to the desired product they are difficult or impossible to separate and reduce the yield of desired peptide. Another drawback of the Merrifield method is that long reaction times are ordinarily required to cc * _--plete the coupling reaction. Depending on the amino acid reactant used, the addition of a single residue to the - 3 - sequence may take anywhere from 3 to 24 hours or more. - β Consequently, it is difficult to do more than 2 or 3 couplings during a 24 hour period. These long coupling times may also lead to many undesirable side reactions 5 which, after many coupling reactions, result in a marked decrease in yields and purity of the desired peptide. These problems generally tend to limit the usefulness of the Merrifield synthesis to peptides with chainlengt-hs of about 10 to 15 amino acids (more or less, depending on 10 the amino acid sequence) . Scale-up of the conventional Merrifield method has also proved to be difficult and problematical. Disclosure of the Invention It is an object of the invention therefore to pro- 15 vide a method for the synthesis of polypeptides and pro¬ teins which overcomes the aforementioned problems asso¬ ciated with the Merrifield method or approach. More specifically, it is an object of the inven¬ tion to provide a method for the synthesis of polypep- 20 tides and proteins which offers extraordinarily rapid reaction rates compared to conventional solid-phase reactions. Yet another object of the invention is to provide an automatable method wherein long chain polypeptides 25 of greater than 20 amino acids can be prepared on a large scale, with little, if any, side reactions. A further object of the invention is to provide a method wherein coupling reactions are complete at every stage of the synthesis regardless of the sequence or 30 structure of the peptide so that the polypeptide or pro¬ tein product of desired chain length or size requires minimum purification or in many instances no purification after cleavage from the support. These objects are obtained by an improvement in 35 the method for the synthesis of polypeptide or protein - 4 - chains on an insoluble solid support wherein an amino acid is passed in a continuous flow through a reactor packed with an insoluble solid support containing substituent groups reactive with said amino acid and coupled to said support by condensation reaction with said reactive sub¬ stituent groups, a second similar or dissimilar amino acid is passed in a continuous flow through said reactor and coupled to said first amino acid and the process repeated until the desired polypeptide is obtained, which improvement comprises conducting the reaction in a pressurized flow reactor. By the term "pressurized" as used herein and iμ the appended claims is meant under a pressure of at 2 least ambient pressure plus 25 psi or 20.0003625 dyne/cm . Preferably the flow reactor is pressurized to at least 100 psi or 0.00145 dynecm 2, up to 1,000 psi or 0-0145 dyne/ Ccϋm2 although pressures of up to 10,000 psi or 0.145 dyne/cm 2 or more may be used. In general, the flow rate of the reactants and reagents employed will fall in the range of 4 to 50 ml per minute or more depending on the size of the reactor used. In a preferred aspect of the invention the first amino acid passed through the reactor packed with the insoluble support containing substituent groups reactive with said amino acid, is a protected amino acid. I-n accordance with this aspect of the invention a protected amino acid is coupled to said support by condensation reac¬ tion with said substituent groups, said coupled first amino acid is deprotected by passing a deprotecting agent through the flow reactor, a second similar or dissimilar protected amino acid is passed through said reactor and coupled to said first coupled amino acid, said coupled second amino acid is deprotected and the process repeated until the desired polypeptide is obtained, the improvement being that during said synthesis the reactants and reagents are passed through the reactor in - 5 - a continuous flow and the reaction pressure in said reac¬ tor is at the defined elevated pressures in order to ob¬ tain rapid reactions and quantitative yields of the de¬ sired products. It has been surprisingly found that conducting the steps of the synthesis in such a pressurized flow system ensures complete coupling reactions between the amino acids introduced and all of the available reactive sites on or within the insoluble support within a greatly reduced time period which is largely independent of the sequence of the polypeptide or protein. Similarly, it has been found that 100% deprotection is effected when this aspect of the in¬ vention is employed. In addition, any wash or neutrali¬ zation operations that may be employed are greatly facil- . itated. As a consequence, formation of by-product poly¬ peptides, that is, polypeptides of shorter chain lengths or polypeptides formed as a result of rearrangement of the activated amino acids or other side reactions is com¬ pletely eliminated. Quantitative yields of desired long- chain polypeptides and proteins are therefore made possible and the separation or purification problems that ordinarily plague conventional solid-phase synthesis in this regard are overcome. Also, the reaction rates achieved under the pressurized flow system of the invention compared to con- ventional solid-phase polypeptide coupling reactions are unexpectedly rapid. For example, coupling reactions which typically take, hours by the conventional procedures are completed in minutes. Such rapid rates of reaction result in the complete elimination of undesirable side reactions that occur because of the tendency of activated amino acids to decompose or rearrange during the long reaction times required for conventional synthetic procedures In a similar manner, a pressurized flow reactor may be used to increase rates and efficiencies of reactions in sequencing of polypeptides, proteins and nucleic acids. "BUREA - 6 - The use of a pressurized flow reactor in accordance with the teachings of the invention results in increased rates of reaction and also drives the reaction to completion, the both of which combine to result in rapid synthesis of pure materials in complicated, repetitive, sequential synthetic procedures. Modes for Carrying Out the Invention It should be understood that the term "polypeptides" as used in the specification and the appended claims is meant to include peptides and proteins. In the synthesis of the present invention, an insoluble solid support or matrix, advantageously in bead form such as any of the conventional solid-phase polymeric substrates conventionally employed for the synthesis of polypeptides can be utilized. Typical of such polymeric resins are crosslinked polystyrene resins, crosslinked polyacrylamides, glass beads, clays, celite, crosslinked dextran, and similar insoluble solid supports which either naturally contain reactive sites for coupling with the amino acid components or which can be provided with such reactive sites. Insoluble supports particularly preferred are derivatized, crosslinked polystyrene resins, such as chloromethylated or hydroxymethylated, crosslinked poly¬ styrene resins, benzhydrylamine resins and the like. The crosslinked polystyrenes are normally copolymers of styrene and a crosslinking agent preferably formed by way of a pearl or bead polymerization process using an aqueous suspension system. Preferred crosslinking agents for preparation of the crosslinked polystyrene resins include divinyl compounds such as para-divinylbenzene, meta- divinylbenzene, divinylcyclohexane, butadiene, and the like. Ordinarily, use of resin supports containing higher than normal levels of crosslinking (e.g. 2% rather than 1% crosslinked polystyrene) or even non-swellable macroporous resins is preferred, since swelling of the resin and consequent variability in the reactivity of - 7 - reactive sites is minimized. For this reason, the use of a single solvent system with relatively low concentrations (1-20% by volume) of reagents is generally preferred. The use of high pressures in the preferred aspect of this invention also minimizes swelling effects arising from the increase in the ratio of peptide support as the syn¬ thesis of the peptide proceeds, which can create problems, especially for larger peptides. Several preliminary operations are necessary before the synthesis of a peptide can be started. First, the supporting resin containing the C-terminal amino acid of the proposed peptide chain must be prepared. This can be accomplished in the same reactor and under the pressurized flow conditions of the present invention. For example, hydroxymethylated resins may be esterified in the flow reactor by esterification with a protected amino acid mixed or symmetrical anhydride, catalyzed by 4-dimethyl- aminopyridine. However, since the attachment of the first (i.e. C-terminal) protected amino acid residue involves the formation of an ester linkage and therefore involves a relatively longer reaction time than the subsequent amino acid coupling reactions, it is often preferred to complete the first attachment beforehand and store the C- terminal amino acid-substituted resin until needed. The insoluble solid support containing the protected C-terminal amino acid may be prepared for instance by es- terifying a suitably protected amino acid with the reactive site or substituent group on the insoluble support such as σhloromethy1ated or hydroxymethylated crosslinked poly- styrene resins. The esterification reaction is accomplished directly with the chloromethylated resin or via suitable activation of the protected amino acid in the case of the hydroxymethylated resin. There are a considerable number of protecting groups for terminal reactive amino -groups which have been employed in peptide synthesis. The pro¬ tecting groups of choice in prior art syntheses' have been - 8 - the benzyloxycarbonyl and especially the t-butyl-oxycar- bonyl groups but, because of the extremely strong acidic conditions required for their removal, more labile groups are preferred for high performance peptide synthesis. In order to minimize the formation of contaminants in the desired product careful choice of protecting groups and method of attachment of the first amino acid to the • resin support are recommended. For example, it is impor¬ tant that sidechain protecting groups, as well as the link- age between the resin and the growing peptide chain be completely stable to the various conditions used through¬ out the synthesis. However, these protecting groups and the linkage to the resin should be sufficiently labile so that final deprotection of the peptide and its cleavage from the support may be accomplished -under the mildest possible conditions without decomposition of sensitive amino acids or destruction of the peptide. Conventional solid-phase techniques use Nα-t-butyloxycarbonyl protec¬ tion in conjunction with benzyl sidechain protection and attachment to the resin. t-Butyloxycarbonyl group removal requires relatively strongly acidic conditions (e.g. 50% trifluoroacetic acid:dichloromethane) , while final depro¬ tection and cleavage from the resin requires even more strongly acidic conditions (e.g. anhydrous hydrogen fluoride or hydrogen bromide/acetic acid) , which can " lead to extensive decomposition of the final product. Further¬ more, the lack of complete selectivity during N -deprotec¬ tion can lead to partial cleavage of the growing peptide chain and consequent reduction in yield, as well as the possibility of contamination by truncated sequences. Termination by trifluoroacetylation of the amine terτainus during deprotection with trifluoroacetic acid has also been shown to be a serious side reaction in this synthetic scheme. - 9 - Thus, while t-butyloxycarbonyl and benzyloxycarbonyl protecting groups can.be used in the present invention, it is preferred that Nα-protecting groups which are removed under the mildest possible conditions be used. Illustrative of such groups are biphenylisopropyloxycarbonyl, adamanty- lisopropyloxycarbonyl and nitrophenylsulfenyl groups which are removable under mild, acidic conditions, or the 9-fluorenyImetHyIoxycHxbonyl or trifluoroacetyl groups which are cleaved under mild, basic conditions. For resin attachment, resins of the p-alkoxybenzyl alcohol type as [proposed by Wang, J. Amer. Chem. Soc. 95, 1328 (1973)], which are cleaved under relatively mild, acidic conditions (e.g. 50:50 trifluoroacetic acid: dichloromethane) are superior. A preferred combination for the invention utilizes Nα-fluorenylmethyloxycarbonyl in conjunction with t-butyl sidechain protection and the p-alkoxybenzyl alcohol resin. This results in complete selectivity, with Nα-deprotection under basic conditions and final cleavage and sidechain deprotection under acidic condi- tions. Use of this combination also reduces the danger of cleavage of the growing peptide chain from the resin during the synthesis and minimizes decomposition during final deprotection and cleavage of the product. Furthermore, coupling cycles are shortened substantially since no neutralization is required after the deprotection step. After the first C-terminal amino acid is coupled to the support, the resulting product is commonly analyzed using standard procedures such as spectrophotometric or quantitative amino acid analysis to determine the amino acid content for the purpose of calculating the amounts of subsequent amino acid reactants and deprotecting agents to be used in the synthesis. The thus-prepared C-terminal amino acid-containing, support is packed into a suitable continuous flow reactor. The reactor may take any desired shape or form so long " URE - 10 - as it is capable of withstanding the elevated pressures under which the synthesis of the present invention may be conducted. The preferred reactors, however, are column reactors having an inlet and an outlet so as to maximize the contact time between solvent or reactants and the growing peptide chains on the resin, thus maximizing the efficiency of the process. The remaining synthesis to form the desired poly¬ peptide sequence is carried out as follows. Before coupl- ing of the second amino acid residue can take place, the first residue already on the support must be deprotected. Deprotection of the first amino acid residue on the .resin as well as on each of the subsequently coupled amino acid residues can be carried out by pumping through the reactor an appropriate deprotecting agent. The deprotecting agents employed for this purpose are well known to those of ordi¬ nary skill in the peptide synthesis art and the particular deprotecting agent employed in any given instance will depend of course upon the protecting group on the amino acid/resin. If the protecting group is t-butyloxycarbonyl, trifluoroacetic acid, methanesulfonic acid or hydrochloric acid in a suitable solvent such as dioxane or dichloro- methane may be used. On the other hand, if the protecting group is biphenylisopropyloxycarbonyl, mild acidic solvoly- sis (for example with 1% trifluoroacetic acid in dich- loromethane) is the preferred method of deprotection. When the Nα-protecting group is 9-fluorenylmethyloxycarbonyl the preferred deprotecting agent is piperidine in dimethy- formamide (DMF) . The concentrations of the particular deprotecting agent in the solvent will vary depending again upon the particular protecting agent employed but will ordinarily range from about 5 to 50% by volume. A suffi¬ cient volume of deprotecting agent is pumped through the reactor over a period of time sufficient to effect complete removal of the protecting groups. Frequently, multiple -BURE - 11 - reactor volumes (for example 2-10 column volumes over a period of 5-30 minutes) of the solution containing the deprotecting agent are passed through the reactor to ensure complete removal of the protecting group. 5 After the deprotecting step, the resin is washed with a suitable solvent, normally the solvent in which the deprotecting agent was dissolved, in order to remove ex¬ cess deprotecting agent. If the deprotecting agent is an acid- the peptide on the resin must be neutralized by wash- 10 ing with an appropriate base such as triethylamine in a solvent such as diσhloromethane. Any excess triethylamine and triethylammoniu chloride, or trifluoroacetate, formed may be removed by repeated washings with a suitable solvent such as dichloro ethane or dimethylformamide. The free 1.5 α-amino group, thus prepared, is now ready for coupling with the next protected amino acid. The next, Nα-protected amino acid is first activated, that is, converted into a reactive form, for instance, by converting the amino acid into an active ester or anhydride 0 or by activation with dicychlohexylcarbodiimide, carbonyldi- imidazole or other activating agents. In the high perfor¬ mance peptide synthesis method of the present invention the most preferred activated amino acid derivatives are the mixed anhydride, symmetrical anhydride or active ester 5 derivatives (such as the p-nitrophenyl esters catalyzed by 1-hydroxybenzotriazole) . These activated amino acids are preferred because of the short coupling times, quanti¬ tative yields and minimization or complete elimination of side reactions they provide. A solution of the activated 0 protected second amino is then passed into and through the reactor packed with the support now containing an unprotected C-terminal amino acid. In general, an excess of the activated, protected amino acid per equivalent of the first amino acid on the resin is employed although 5 the excess required may be limited by recycling solutions - 12 - through the column. Again the common practice is to pass more than one reactor volume of the activated, protected amino acid through the reactor to ensure complete reaction. It should be understood that any of the conventional methods of activating amino acids for the purpose of coupling with another amino acid may be applied to this method. These procedures should be well-known to anyone skilled in the art of peptide chemistry. After the coupling of the second protected amino acid to the first amino acid, the attached protected amino acid is then deprotected, neutralized (if necessary) and washed as described above before coupling of the next amino acid derivative is effected. This procedure is repeated until the desired sequence of amino acids has been assembled on the insoluble support. While the above described methods progress from the carboxyl terminus end toward the amine terminus end of the peptide, it should be understood that the reverse direction of synthesis, that is, from amine terminus to carboxyl terminus can be employed. For instance, a prepared or selected insoluble support having sites such as alkyloxy- carbonyl or aryloxycarbonyl chloride groups, activated carboxyl groups, etc. , reactive with the amine group of an amino acid can be used. In practice synthesis of a peptide from its carboxyl terminus is generally preferred, mainly because of lower extents of racemization when Nα-urethane-protected, carboxyl activated amino acids are used for coupling reactions. Resins of the p-alkoxybenzyl alcohol type are preferred because of the relatively mild cleavage conditions (for example 50% trifluoroacetic acid: dichloromethane) which are required. These mild cleavage conditions minimize or eliminate completely the destruc¬ tion of sensitive amino acids during the cleavage process. ' U E4 - 13 - The entire series of coupling reactions in the above- described methods, from the second amino acid, (and if desired the first amino acid) to the last is conducted in a pressurized reactor, preferably under a reactor pressure of at least 40 psi or 0.00058 dyne/cm up to 2 1,000 psi or 0.0145 dyne/cm , although pressures of up 2 to 10,000 psi or 0.145 dyne/cm or more may be used. Reactor pressures of this level can be generated by use of commercial pressurizing equipment and methods. For example, any of the commercially available recipro¬ cating pumps capable of generating the required pres¬ sures and flow rates can be used and the reactants, rea¬ gents and wash solvents pumped directly into and through the reactor. Alternatively, the reactants, reagents and wash solvents may be pumped through the reactor by means of pressurization with an inert gas such as nitrogen and the pressure in the reactor regulated by controlling the volume of inert gas released to transfer the reactants, reagents and wash materials into and through the reactor. The scale of the peptide syntheses in the pres¬ surized flow reactor may vary widely, being limited solely by the capacity of the pressurized pun-ping means utilized. For example, reactors in the range 5-100 ml in volume may be used for the production of up to tens of grams of pep¬ tides, although larger reactors (up to 1000 ml or more) may be used if necessary. The completed peptide sequence can be removed from the insoluble support by any of the standard methods as, for instance, by cleavage with anhydrous hydrogen fluoride, transesterification, acidolysis, aminolysis, etc. This cleavage is most conveniently accomplished by extruding the peptide-support from the reactor and treating it with the cleavage reagent at atmospheric pressure. However, the cleavage may also be carried out in the reactor and - 14 - under the elevated reactor pressures of the invention pro¬ vided the reactor is resistant to the cleavage reagent. After cleavage, the resulting peptide is found to be remarkably homogeneous and to require no or minimal purification. Because of the very low contamination of by-products overall yields are found to be surprisingly high and whatever purification is necessary can be carried out with relative ease. Such purifications, if required, are preferably carried out by partition chromatography, ion exchange chromatography, gel permeation chromatography, countercurrent distribution and the like. Illustrations of peptides, polypeptides and pro¬ teins which can be obtained by the method of the inven¬ tion are enkephalins, angiotensin, oxytocin, vasopressin, leuteinizing hormone releasing hormone, somatostatin, gastrin, insulin, glucagon, ribonuclease, endorphins, etc. The following examples are included to further illustrate the present invention. In all of the examples, the nitrogen-pres-suri ed system described in Example 2 of United States Patent No. 4,192,798, incorporated herein by reference, was employed. Example -- : Synthesis of L-Leucyl-L-alanyl-glycyl-L-valine using 9-Fluorenylmethyloxycarbonyl (Fmoc)- protected Amino Acid Mixed Anhydrides p-Benzyloxybenzyl alcohol polystyrene resin (2% crosslinked) was esterified with Fmoc-L-valine by the method of Wang -[J. Amer. Chem. Soc. 95_, 1328 (1973)] to give a substitution level of 0.6 moles/gram as determined by the spectrophotometric method of Meienhofer et al. [Int. J. Pept. Prot. Res. 13_, 35 (1979)]. The resin (1.2 g) was swollen in dry DMF, packed into a stainless steel column reactor (1.0 x 5 cm) and treated with 10% (w.v) peperi- dine in D-MF a t a flow-rate of 5 ml/minute for 10 minutes. The column reactor was washed to neutral pH with DI-F at a flow-rate of approximately 20 ml/minute over a 5 minute - - 15 - period before treating with a solution of Fmoc-glycine mixed anhydride [prepared by treating a solution of Fmoc- glycine (3.5 mmoles) and N-methylmorpholine (3.5 mmoles) in DMF (40 ml) with isobutyl chloroformate (3.2 moles)] • 5 over a 10 minute period. The cleavage, washing and coupling cycles were repeated using equivalent amounts of Fmoc-L- alanine and Boc-L-leucine mixed anhydrides. The synthesis was conducted under 50 psi nitrogen pressure. After comple¬ tion of the synthesis the Boσ-tetrapeptide resin was 10 transferred from the column reactor to a sintered funnel where it was washed copiously with DMF, methanol and dich- loromethane and then suspended in 75% trifluoroacetic acid in dichloromethane (25 ml) at room temperature. After stirring for 30 minutes, the resin was filtered and washed 15 with methanol. The combined filtrates were evaporated and the residue washed with ether to give a quantitative yield (0.34 g) of the tetrapeptide. Analysis of the pro¬ duct by thin-layer and high performance liquid chromato¬ graphy showed the product to be homogeneous ( 99.9% pure) 20 with no detectable contamination by "failure sequences" or "wrong-opening" products. Example 2: Synthesis of L-Leucyl-L-alanyl-glycyl-L-valine * using Fmoc-protected Amino Acid Symmetrical Anhydrides 25 The synthesis described under Example 1 was repeated using ' 1 gram of Fmoc-valine resin (substitution = 0.52 mmoles/gram) and preformed symmetrical anhydrides (2.3 equivalents) for activation. The symmetrical anhydrides were prepared by treating the appropriate Fmoc-amino acid 30 (2.4 mmoles) with dicyclohexylcarbodiimide (1.2 mmoles) in » DMF (12 ml) . The solution was filtered to remove dicy- clohexylurea prior to use. Couplings were allowed to pro¬ ceed for 10 minutes. Deprotection, cleavage and isola¬ tion of the product as described under Example I gave 0.242 35 g of the tetrapeptide (99%) yield) which was homogeneous - 16 - ( 99% pure) by chromatographic analysis. Example 3: Synthesis of L-Leucyl-L-alanyl-glycyl-L-valine using Fmoc-protected Amino Acid p-Nitrophenyl Esters The synthesis described under Example 2 was repeated using p-nitrophenyl esters catalyzed by 1-hydroxybenzo- triazole for activation. Couplings were allowed to pro¬ ceed for 15 minutes using solutions of the appropriate Fmoc-amino acid p-nitrophenyl esters (1.2 mmoles, 2.3 equivalents) and 1-hydroxybenzotriazole (1.2 mmoles) in DMF (12 ml) . Deprotection and cleavage of the product as described under Example ϊ gave 0.24 g of the product (98% yield) which contained only traces (<2%) of contami¬ nant peptides on chromatographic analysis. Example 4: Synthesis of L-Leucyl-L-alanyl-glycyl-L- valine using Biphenylisopropyloxycarbonyl (Bpoc) - protected Amino Acid Mixed Anhydrides p-Benzyloxybenzyl alcohol polystyrene resin (2% crosslinked) was esterified with Bpoc-L-valine by the method of Wang, to give a substitution level, as deter¬ mined by nitrogen analysis, of 0.49 mmoles/gram. This resin (1.2 g) was swollen in dichloromethane and packed into a stainless steel column reactor (1.0 x 5 cm). Cleavage of the Bpoc-group was effected by passing a 1% solution of trifluoroacetic acid in dichloromethane through the column for 10 minutes at a flow-rate of 5 ml/minute. The column was subsequently washed with dichloromethane (10 ml/min. , 5 minutes) , neutralized with 5% diisopropy- lethylamine in dichloromethane (10 ml/min., 5 minutes) and finally washed to neutrality with dichloromethane (10 ml/min. , 5 minutes) . Bpoc-glycine mixed anhydride (0.03 M, 80 ml) (generated as described under Example 1) was passed through the column over a 15 minute period. The resin was washed with dichloromethane (10 ml/min., 10 minutes) before repeating the total cycle using Bpoc- - 17 - alanine and Boc-leucine mixed anhydrides. The entire synthesis was carried out under 200 psi or 0.0029 dyne/ 2 cm nitrogen pressure. The tetrapeptide was isolated as described under Example 1 (0.273 g, 99% yield) and shown to be homogeneous (> 9% pure) by chromatographic analysis. Example 5: Synthesis of L-Prolyl-L-prolyl-L-valine using Fmoc-protected Amino Acid Mixed Anhydrides Fmoc-L-valine p-benzyloxybenzyl ester polystyrene resin (1 g, subst- = 0.5 mmoles/gram) was swollen in dich¬ loromethane and packed into a column reactor (1.0 x 5 cm) and coupled with Fmoc-proline and then with Boc-proline mixed anhydrides (8 equivalents of each) by the general procedure described under Example 1. The synthesis was carried out in dichloromethane as solvent, except for Fmoc- cleavage cycles which utilized 10% piperidine in DMF. The tripeptide was cleaved and deprotected as described in Example 1 and shown to contain 2% isobutyldxycarbonyl-L- prolyl-L-valine (the "wrong-opening" product) by reverse phase high performance liquid chromatography. This was a surprising result in that proline-to-proline coupl¬ ings by the mixed anhydride method in solution synthe¬ ses of the prior art are known to be problematical and give substantial amounts (up to 10%) of the "wrong-opening" product [see for example, Katsoyannis and Schwartz in "Methods of Ξnzymology" £7, 555 (1977)]. Example 6: Synthesis of Methionine -Enkephalin (L-Tyro- sylglycyl-glycyl-L-phenylalanyl-L-methionine) using Fmoc-Amino Acid Mixed Anhydrides Fmoc-L-methionine p-benzyloxybenzyl ester poly- styrene resin (1.1 g, subst. = 0.59 mmoles/gram) was swollen in dimethylacetamide and packed into a stainless steel column (1.0 x 5 cm) . After deprotection as des¬ cribed under Example 1, the mixed anhydrides (3.6 mmoles, 5.5 equivalents) of Fmoc-L-phenylalanine, Fmoc-glycine ' E - 18 - ( twice) and finally Boc-O-t-butyl-L-tyrosine were coupled as 0. 1M solutions in dimethylacetamide for 10 minutes per coupling. The entire synthesis was conducted under 2 1000 psi or 0.0145 dyne/cm nitrogen pressure. The pep- 5 tide-resin was removed from the reactor and cleaved by suspending in thioanisole:dimethylsulfide:trifluoroacetic acid (2:8^20) at room temperature under nitrogen for 1 hour. The resin was filterecϊ7 ~" washed with methanol, the filtrate evaporated and the residue washed with ether 10 to give methionine -enkephalin in quantitative yield (0.495 g) . Analysis by reverse phase high performance liquid chromatography showed the product to be > 95% pure, with only trace contamination by peptide impurities. - Example 7: Synthesis of Delta-Sleep Inducing Peptide -•-* (L-Tryptσphanyl-L-alanyl-glycyl-glycyl-L- aspartyl-L-alanyl-L-seryl-glycyl-L-σlutamic acid) using Fmoc-protected Amino Acid ixed Anhydrides Fmoc-α-t-butylglutamic acid p-benzyloxybenzyl 20 ester polystyrene resin (8 g, substitution = 0.20 mmoles/ gram) was swollen in DMF and packed into a stainless steel column reactor (1.5 x 15 cm) . After deprotection (10 minutes) as described under Example 1 the resin was treated sequentially with the mixed anhydrides (16 mmoles/ 25 loo ml; 10 minutes) of Fmoc-glycine, Fmoc-O-t-butyl-L- serine, Fmoc-L-alanine, Fmoc-S-t-butyl-L-aspartic acid, Fmoc-glycine (twice), Fmoc-L-alanine, and finally 3oc-L- tryptophan. The synthesis was carried out under 1000 psi 2 or 0.0145 dyne/cm nitrogen pressure. The peptide-resin 30 was removed from the reactor and cleaved and deprotected by suspending in trifluoroacetic acid:ethanedithiol: * dichloromethane (90:10:20) for 1.75 hours under nitrogen at room temperature. After filtration and evaporation of the solvent the residue was washed with ether to give 35 delta-sleep inducing peptide (1.70 g, 100%) . Analysis by " E - 19 - reverse phase high performance liquid chromatography showed the peptide to be>95% pure, with only trace contamination by peptide impurities. " BU

Claims: - 20 - It is Claimed: 1. In the continuous flow method for the synthe¬ sis of a polypeptide chain on an insoluble solid support wherein a protected amino acid is passed in a continuous flow through a flow reactor packed with an insoluble solid support containing substituent groups reactive with said protected amino acid, and coupled to said support by conden¬ sation reaction with said reactive substituent groups, a second similar or dissimilar protected amino acid is passed through said reactor in a continuous flow and coupled to said first amino acid and the process repeated until the desired polypeptide is obtained, the improvement comprising conducting the reaction in said flow reactor under pressure. 2. The improvement of Claim 1 wherein the reaction 2 pressure is at least about 40 psi or 0.00058 dyne/cm . 3. The improvement of Claim 1 wherein the reactive substituent groups on said insoluble support are protected amino acids. 4. The improvement of Claim 3 wherein the protected amino acids are Nα-fluorenylmethyloxycarbonyl amino acids. 5. The improvement of Claim 3 wherein the protected amino acids are N -biphenylisopropyloxycarbonyl amino acids. 6. The improvement of Claim 3 wherein the protected amino acids are Nα-adamantylisopropyloxycarbonyl amino acids. 7. The improvement of Claim 1 wherein said support is a crosslinked polystyrene. 8. The improvement of Claim 1 wherein said support is a crosslinked polyacrylamide. 9. The improvement of Claim 1 wherein the protec¬ ted amino acids are employed as their mixed anhydrides. 10. The improvement of Claim 1 wherein the protec¬ ted amino acids are employed as their symmetrical anhydrides. BU - 21 - 11. The improvement of Claim 1 wherein the protec¬ ted amino acids are employed as their active esters. 12. In the method for synthesis of a polypeptide * chain on an insoluble solid support wherein a first protected amino acid is passed through a reactor packed with an in- 5 soluble solid support containing substituent groups reac¬ tive with said protected amino acid and coupled to said support by condensation reaction with said substituent groups; said amino acid is deprotected by passing through said reactor a deprotecting agent, a second similar or 10 dissimilar protected amino acid is passed through said reac¬ tor and coupled to said first coupled amino acid, said coup¬ led second amino acid is deprotected by passing through said reactor a deprotecting agent and repeating the process until ., ' the desired peptide is obtained, the improvement wherein 5 the reactants and reagents are passed through the reactor in a continuous flow under pressure. 13. The improvement of Claim 12 wherein the reac- tion pressure is at least 40 psi or 0.00058 dyne/cm 2. 14. The improvement of Claim 13 wherein the reac- 0 tive substituent groups on said insoluble support are pro¬ tected amino acids. 15. The improvement of Claim 14 wherein the pro¬ tected amino acids are Nα-fluorenylmethyloxycarbonyl amino acids. 5 16. The improvement of Claim 14 wherein the pro¬ tected amino acids are Nα-biphenylisopropyloxycarbonyl amino acids. 17. The improvement of Claim 14 wherein the pro¬ tected amino acids are Nα-adamantylisopropyloxycarbonyl 0 amino acids. 18. The improvement of Claim 14 wherein the in¬ soluble support is a crosslinked polystyrene. 19. The improvement of Claim 14 wherein the insolu¬ ble support is a crosslinked polyacrylamide. - 22 - 20. The improvement of Claim 12 wherein the pro¬ tected amino acids are employed as their mixed anhydrides 21. The improvement of Claim 12 wherein the pro¬ tected amino acids are employed as their symmetrical anhydrides. 22. The improvement of Claim 12 wherein the pro¬ tected amino acids are employed as their active esters. - U " RE

Inventors: FULLER WILLIAM DEAN, GOODMAN MURRAY, VERLANDER MICHAEL S
Applicants: BIORESEARCH INC
Application year: 1982
Main classification: C07C103
IPC codes: C07K1, C40B40, C40B60