IE46470B1 - A process for synthesizing a peptide chain - Google Patents

Description

The present invention relates to a process for synthesizing a peptide chain.

The area of peptide synthesis has received considerable attention in recent years. A significant problem has existed in synthetically achieving a high molecular weight, pure polypeptide wherein the amino acid sequence of the peptide actually prepared corresponds to that sought. To approach realization of the synthesis with the desired purity has heretofore been quite laborious.

The synthetic preparation of a polypeptide is a multi-stage procedure whereby a desired product is constructed by sequential chemical reactions of a precursor and an added compound, with the precursor at any given stage being the chemically reacted, reactable precursor from the preceding stage. Thus, the procedure is reiterative.

In this process a first amino acid is reacted with a second to form a dipeptide, schematically represented by formula I Η 0 A I II I HN C CH \ z\/ \ CH N COH A' H 0 (I) The peptide so formed at this stage is separated from the unreacted acids and a third amino acid is then reacted with the dipeptide to form a tripeptide.

The procedure is reiterated until a polypeptide having the desired amino acid sequence, customarily termed the target peptide, is obtained. Sequence failure, whereby a portion of the elongated polypeptide chains have an improper amino acid sequence, can result from several causes. One can be the failure to remove residual free acid from the reaction mixture prior to reaction with a subsequent amino acid. The presence of such unreacted acid presents the possibility that a portion of the chains will be improperly elongated with the residual acid rather than with the acid desired at that stage of the sequence.

Yet a further and perhaps more significant cause of sequence failure is the incomplete reaction of all of the chains present with the amino acid added at each stage of the synthesis. In the preparation of low molecular weight polypeptides, the presence of chains containing different numbers of acids can be analytically ascertained and the desired peptide chains isolated. Conventional analytical techniques do not permit this to be done with respect to the higher molecular weight varieties, however, because the difference in molecular weight between properly and improperly synthesized chains is simply too small to be detectable.

A 6 4 7 0 - - 4 _ ./ The peptide synthesis procedure described above has been represented as involving the sequential reaction of amino acids with a polypeptide chain. Xn this respect, there are two approaches; one being growth of the peptide from the C-terminal end (the end of the chain with the 1.--C— OH group) and the other being growth from the N-terminal end (the end with the -NH^ group).- It is well recognized that during the synthesis either the alpha amino group (N-terminal route) or carboxyl group (C-terminal route) of the added acid must be blocked ' so that the added acid reacts with the polypeptide chain and reaction between the molecules of added acid cannot occur. It is necessary, therefore, that the blocked .group of the added acid, after reaction with the chain, be deblocked for the subsequent acid addition step. Failure to achieve deblocking at any stage of the synthesis can introduce a sequence failure. Moreover, deblocking must be accomplished in a fashion whereby the peptide being synthesized is not Adversely affected.

In order to achieve solubility, conventional methods of peptide synthesis customarily are carried out in a non-aqueous medium, particularly for high molecular weight peptides containing protected amino acid side chains. This has necessitated the use of harsh coupling reactions to effect peptide bond formation and the accompanying likelihood of chain disruption such as racemization or fragmentation. Moreover, especially with respect to the higher molecular weight polypeptides, non-aqueous reaction solutions may not permit the peptide to assume its - 5 naturally occurring configuration. In nature, of course, the peptides are manufactured in an aqueous environment.

According to the present invention there is provided a process for synthesizing a peptide chain having a predetermined sequence of amino acids, the process comprising reacting a precursor which comprises a first amino acid segment (as herein defined) of the sequence, the first amino acid segment having a free terminal carboxyl group or a free terminal amino group, with a second amino acid segment having a free Na-amino group and a blocked carboxyl group susceptible to enzymatic hydrolysis when the precursor has a free terminal carboxyl group, or a free carboxyl group and a blocked Naamino group susceptible to enzymatic hydrolysis when the precursor has a free terminal amino group, and deblocking the product peptide enzymatically in an aqueous medium.

As used herein the term segment or amino acid segment refers to an amino acid or a series of amino acids present in the desired peptide chain.

Peptide chain elongation in solution can be accomplished by reacting a precursor with the segment which follows in the desired sequence. The precursor can of course, comprise the initial amino acid of the chain or a chain of amino acids onto which additional amino acids are to be attached.

In polypeptide synthesis the added segment is an amino acid residue and the precursor is an elongatable peptide chain having either a free terminal amino or carboxyl group. Peptide bond formation and chain elongation thus is accomplished through either acylation of the amino group on the chain by the carboxyl moiety of the acid being added (N-terminal - 6 route) or acylation of the amino group of the added acid by the carboxyl group on the growing chain (Cterminal route).

In a preferred embodiment, the precursor is part of a larger complex which is water soluble and which contains a water soluble support attached to the precursor at the non-elongatable end thereof. That is, for C-terminal chain growth the chain is anchored to the support at the Na-group of the first amino acid residue of the sequence. For N-terminal growth the chain is anchored to the support through the carboxyl group of the first amino acid residue of the sequence. Chain elongation, therefore, is effected while the precursor is a part of the complex. So that the complex is stable in aqueous medium, attachment between the water-soluble support and precursor is preferably covalent and effected in a manner which permits subsequent release so that eventual recovery of synthesized, 'pure target fragment can be effected. If the growing chain is covalently complexed to the water soluble support aqueous solubility of the chain during the addition reaction is markedly enhanced, even when the chain is quite large.

Water soluble synthetic polymers are a class of substances which can be employed as supports. Representative examples of this class of substances are polyvinyl alcohol, polyvinylpyrrolidone, poly(acrylamide-acrylic acid) or polyethylenemaleic anhydride. Polyamides such as those amino acid polymers containing a glutamic acid or aspartic acid segment also are suitable substances for use as supports. Another useful substance which can be used as a support is polyethyleneglycol .

Water soluble polynucleotides also constitute a useful class of substances which can be employed as supports. Representative examples of useful polynu46470 - 7 cleotides, named as acids, include polyadenylic acid, polyuridylic acid, polythymidylic acid, polycytidylic acid and polyguanylic acid. Preferably the acids have at least ten repeating ribosephosphate moieties and are commercially available.

The manner of attachment of the first amino acid, or a short chain peptide, to the support should be such that the target peptide can be thereafter removed from the support under mild conditions. Therefore, in a preferred embodiment, an endopeptidase-specific spacer arm is included as a portion of the support and it is to this portion that the first protected amino acid segment of the target peptide is added.

The configuration of this spacer varies with respect to the synthetic route, that is C or N-terminal, to be employed. The use of an endopeptidase specific spacer arm has the advantage that only mild pH and temperature conditions are required for the removal of the target peptide from the support. While, for example, saponification can be used in an N-terminal route if the first amino acid of the peptide is attached directly to a polynucleotide support, saponification is harsh and can lead to racemization.

Thus, considering attachment in more detail, for synthesis via the C-terminal route, the ribose of the 3' end of a polynucleotide, for example, is oxidized to a dialdehyde which is subsequently coupled to an endopeptidase specific spacer arm by reductive alkylation. This involves formation of an amine, dialdehyde adduct followed by reduction of that adduct in aqueous solution with, for example, sodium borohydride. Therefore, the arm has, on one end, a primary amino group reactable with the oxidized ribose moiety of the nucleotide. The other end thereof contains a carboxyl group which, after having acylated a Na-amino group of an added acid, can be hydrolyzed by the action of an 0 - 8 endopeptidase. Polypeptides themselves containing a carboxyl terminated arginine or lysine residue constitute a useful class of such spacers, especially where the N-terminal residue, or other residues, are from hydrophobic acids requiring no protection such as glycine, alanine, or valine. The dipeptide, glycylL-arginine, is a useful spacer arm which can be coupled, by reductive alkylation, to a polynucleotide according to the method of Royer, et al., 64 478 (1975). The dipeptide spacer, attached via a tertiary amine linkage to the nucleotide, thus has a free carboxyl group available for addition of a first carboxyl protected amino acid or polyacid segment for the preparation of a target peptide by the C-terminal route.

When the use of polyethyleneglycol or polyvinyl alcohol including an endopeptidase-specific spacer arm is required as the polymeric support, the alcohol of the polyethyleneglycol or polyvinyl alcohol is converted to an alkoxide derivative, for example by reaction with potassium tert-butoxide, and the alkoxide derivative is reacted with ethyl bromoacetate to provide the oarboxymethyl derivative. This derivative is hydrolyzed to give the free acid which is coupled to the endopeptidase specific spacer arm using a water soluble carbodiimide activator.

Polyvinylpyrrolidone, poly (acrylamide-acrylic acid) and polyethylenemaleic anhydride are subjected to basic hydrolysis to form derivatives having free carboxyl groups. The polyamides are selected from those having a free carboxyl group. These substances also are coupled with the spacer arm as stated above.

After preparation, removal of the target peptide from its complex with the support can be effected by use of a highly specific endopeptidase, for example, trypsin, which cleaves only those peptide bonds whose carbonyl is that of arginine (or lysine). The enzyme - 9 for this cleavage can be used either bound to a support or free in solution at mild alkaline pH.

For N-terminal peptide synthesis, preparation of the polynucleotide support again requires oxidation of the ribose moiety, with a spacer arm being attached thereto through a secondary amine leakage via reductive alkylation. However, in this case, the end of the spacer arm disposed for covalent coupling to the first amino acid to be added must contain a free amino group so that N-terminal chain growth can occur. Thus, the spacer arm contains a primary amine group in both terminal positions, one to react with the nucleotide and the other to complex with the first acid residue of the sequence. Moreover, in order to also have the required endopeptidase activity necessary for eventual removal of the target peptide from the support, the amine group used for covalent coupling to the first acid residue of the desired sequence is provided by the alpha-amino group of arginine or lysine or derivatives thereof.

One convenient manner of preparing the support for N-terminal chain growth is to first reductively couple a short chain diamine to the aldehyde containing nucleotide, Royer et al., supra. Thereafter Na-amino protected arginine or lysine is attached through the acid carboxyl group to the available amine of the diamine, for example by using a water soluble carbodiimide activator, and the protecting group removed, for example by using the enzyme L-pyrrolidonecarboxylpeptidase, to yield the desired support having a free amino group available for peptide synthesis by the N-terminal route.

When use of a vinyl polymer or polyamide support is desired, derivatives containing a free carboxyl group are prepared as above. The carboxyl derivative is coupled with a diamine, such as ethylene46470 - 10 diamine, using a carbodiimide. The Na-amino protected arginine or lysine spacer arm is attached to the diamine as indicated above for polynucleotide supports.

Subsequent recovery of the target peptide from the support can be effected by the two stage use of an arginine or lysine specific endopeptidase as previously discussed followed by an arginine or lysine specific exopeptidase, for example carboxypeptidase B. The first enzyme releases the arginine or lysine terminat10 ing target peptide from the remainder of the support while the second removes the C-terminal arginine or lysine residue and simultaneously liberates the target peptide. As stated above, both enzymes may be used bound or in solution at mild alkaline pH.

If lysine or arginine is to be present in the target peptide, the side chains of these amino acid segments must be protected. If such side chains are not protected, the target peptide itself would be fragmented by the endopeptidase used for separation from the support. Typical protecting groups are trifluoroacetyl for the epsilon amino group of lysine and nitro group protection for the guanidinium side chain of arginine. Deprotection of these residues can be accomplished by routine procedures well known for this purpose.

In order to prevent the added amino acid from reacting with itself during chain elongation, the primary alpha amino group or, as the case may be, the carboxyl group thereof, as well as other reactive groups except for the intended reactive moiety, must be appropriately blocked or protected. As hereinafter discussed, the blocking or protecting group for an alpha amino group or carboxyl group is one which can be enzymatically removed. Hereinafter, the symbol, a, refers to the term alpha. 6 4 7 0 -lilt is preferred, particularly with respect to peptide synthesis by the C-terminal route, to use, nonactivated amino acid ester derivatives containing a free Na-amino group to effect reaction with the precursor. Compounds within this class include those prepared from single amino acids as well as other compounds, for example, those containing one or more peptide bonds prepared from the same or different amino acids. These amino acid derivatives containing an ester blocked carboxyl group can be represented as follows: A 0 NH2-CH— (-ϋ— N — I -)n-(i-0-R J Η H A (II) wherein n is zero or an integer; A is an amino acid side chain which can be different in each repeating unit when n is greater than zero; and R is a blocking group which prevents the derivative from acylating a molecule containing a free amino group. Preferably, R is a short chain, straight or branched alkyl group, having less than 10 carbon atoms, which as hereinafter discussed can be removed enzymatically. Other preferred ester groups are the benzyl and ο-, m-, or p-nitrobenzyl groups.

The derivatives represented by formula II above are prepared by known esterification techniques such as the acid-catalyzed reaction of an amino acid with an alcohol. Using these derivatives, reactions with a precursor containing a free carboxyl group can be accomplished at near zero temperature in water at acid - 12 pH utilizing a water soluble carbbdiimide as a coupling reagent.

When an N-terminal route is selected, again the conventional means of coupling an Na-blocked acid to the free amino precursor involving use of a water soluble carbodiimide is an attractive and practical approach.

As will be discussed, the Na-blocking group is enzymatically removable.

Another coupling means is the use of active Unblocked acid derivatives to effect reaction with the precursor. Active amino acid esters are one example of such derivatives. As is recognized (Bodanszky and Klausner, The Chemistry of Polypeptides, ed.

Katsoyannis, p. 21, Plenum, 1973), these active esters spontaneously form peptide bonds in solution at room temperature with minimal adverse racemization.

The active esters can be prepared by reacting the acid moiety of a Na-protected amino acid with an 2o alcohol having substituents which make it readily displaceable by an attacking amino group on the precursor chain. The preparatory reaction can be accomplished in an organic solvent in the presence of a carbodiimide. Aliphatic alcohols containing one or more electron withdrawing groups, phenol (and thiophenol) derivatives and hydroxylamine derivatives are useful alcohols. Particular examples of useful active esters are those containing the following displaceable leaving groups: cyanomethyl, carboethoxy30 methyl, propargyl, N-hydroxysuccinimide, N-hydroxylphthalimide, p-nitrophenyl, 2,4,5-trichlorophenyl, as well as others given in the foregoing reference.

While the active esters are preferred amino acid derivatives prepared with other readily displaceable groups on the carboxyl moiety are also useful. These 6 4 7 0 - 13 groups include, for example, those such as azido, imidazole, halo, acyl and phosphoryl.

With enzymatically deblockable Na-groups, the active amino acid derivatives, and especially the esters, constitute a useful class of compounds for peptide synthesis using the N-terminal route. Both the acylation reaction with the chain and the deblocking procedure for subsequent elongation can be accomplished in solution under very mild conditions, thus minimizing any adverse effects on the polymer being synthesized. Also, as will be hereinafter discussed, the use of the active esters can obviate blocking other side chains on certain amino acids which ordinarily need appropriate protection.

Xn essential aspects, the compounds constituting the above class of active esters are those which contain an amino acid derivative having an activated terminal carboxyl group and an N-blocking group susceptible to removal by a corresponding and specific enzyme. In one embodiment, these compounds can be represented as follows: A 0 A 0 il I Bgz-NH -— C (-C N C —) -C—X Η Η H (III) wherein B^z is an enzymatically removable Na-blocking group; X is a group readily displaceable by an amino group; and n and A are as identified with respect to formula II. The L-pyrrolidonecarboxyl (pyroglutamyl) group is a useful Na-acyl blocking group. Kurath and Thomas, Helv. Chim. Acta, 56, 1658 (1973) and Doolittle, Methods in Enzymol, 19, 558 (1970) illustrate the manner in which L-pyrrolidonecarboxylic acid can be 7 0 - 14 used to prepare the Na-L-pyrrolidonecarboxyl derivatives of amino acids.

To obtain high conversion, the elongation reaction can be accomplished with a large excess of the added sequencing segment, amounting to at least a 2:1 equivalent ratio, and preferably at least 5:1. However, the solution after reaction may nevertheless contain unreacted transformable precursor. In a reiterative procedure, the presence of such unreacted precursor can produce a sequence failure. While blocking of the unreacted precursor could be accomplished prior to the next reaction, blocking does not alter the molecular structure of the precursor and the possibility of sequence failure continues to exist if deblocking were to occur subsequently.

There, in a preferred embodiment pruning is effected. Pruning is selectively and chemically removing unreacted precursor from reacted compound after the formation of the latter, thereby leaving a properly reacted compound which is free of material containing the base molecular structure of the unreacted precursor. Pruning is important in achieving ultimate separation and recovery of a pure target peptide.

With particular respect to the synthesis of polypeptides, which are elongated through acylation of a terminal amino on the precursor using Na-blocked, free carboxyl segments of through acylation of a free Na-amino group on a C-terminal carboxyl blocked segment by a free carboxyl group on the precursor, pruning can be effected by enzymatically hydrolyzing those precursor chains which failed to elongate and thereby degrading such chains. The unreacted chains, of course, still contain either a free amino group in the case of N-terminal growth, or a carboxyl group in the C-terminal case, and thus can be enzymatically 6 4 70 - 15 attacked using an appropriate enzyme. On the other hand, those chains which did properly elongate will have their terminal reactive group (either amino or carboxyl) protected by a blocking group and will not undergo hydrolysis.

A preferred method of enzymatic pruning is to pass the reaction solution through a column which contains a water insoluble support material having immobilized on its surface an enzyme which selectively hydrolyzes substances either from the N-terminus or C-terminus.

An aminopeptidase, such as aminopeptidase M or leucine aminopeptidase, is suitable for hydrolysis at the Nterminus (Royer and Andrews, 1973, J. Biol. Chem., 248, 1807). The hydrolysis is carried out at a temperature of from 0° to 50°C. and at a pH of 6.5 to 7.5. For hydrolysis directed at the C-terminus, a carboxypeptidase such as carboxypeptidase A, B, C or Y is useful. These Y and C enzymes, at pH of from 4 to 6, have been demonstrated as having non-specific, Cterminal exopeptidase activity. Hayashi et al., J.

Biol Chem. 248, 2296 (1973) and Kuhn et al., Biochemistry, 13, 3871 (1974). A temperature from 0 to 60°C. is employed. All of these enzymes are specific for L-amino acid residues and, as will be hereinafter discussed, unreacted precursor will only be present in the L-isomer form.

An alternative method of pruning involves scavenging the unreacted precursor from the reaction solution, such as by attaching it to a water insoluble support, and thereafter separating the solution from the support. With particular respect to a precursor having a free amino terminus, a manner of accomplishing this is to immobilize onto a support an electrophilic reagent which has specific covalent reactivity for the free terminal amino group of the unreacted precursor and, thereafter, pass the reaction solution into /^6470 - 16 intimate contact with the support in order to bond the unreacted precursor thereto. A suitable electrophilic reagent is the mixed disulfide formed by reaction of a tir'd derivative and merdaptosuccinio anhydride For C-terminal scavenging, a support containing free primary amino groups can be used in conjunction with water soluble carbodiimides.

Subsequent to pruning of unreacted precursor, the properly elongated chains are separated and recovered from excess unreacted amino acid. In a preferred embodiment this separation is effected while the elongated chains are reversibly coupled to an insoluble support. The term reversibly coupled as used herein refers to coupling by means of a non15 covalent and non-ionic association between two substances which have a specific affinity for each other in an aqueous medium, which affinity can be dissipated without chemical reaction. Reversible coupling thus permits attachment to and release from the support without the use of harsh conditions which might adversely effect the transformed compound.

Reversible coupling of the elongated chains to an insoluble support can be achieved if the precursor is part of a larger, water soluble complex which contains a polynucleotide support attached to the precursor through the non-elongatable end thereof.

The insoluble support is conveniently contained in a column and has covalently affixed to its surface a polynucleotide adsorbent which as specific affinity for the polynucleotide support complexed to the reacted precursor. As the solution containing the complex is passed through the column, the elongated precursor is reversibly coupled to the insoluble support by affinitive interaction between the support and the adsorbent. Separation of the elongated chains, in complexed form, from chemically unrelated substances, - 17 such as the unreacted amino acid reactant which does not contain the covalently bonded support, is thereby effected. The coupling can be simply reversed by heat, the institution of a competing association, or a change of pH. In order to achieve reversible coupleing, the polynucleotide selected as the water-soluble support should have a base which is complementary, as to spatial arrangement and affinitive interaction, with the base of the polynucleotide adsorbent. Examples of useful complementary base pairs are adenine with either uracil or thymine and cytosine with guanine.

It should be appreciated that polynucleotides of the copolymer type also can be used, especially when they are of the block form containing alternating and repeating segments of complementary base pairs.

In this instance, of course, the same polynucleotide can be used as both the water-soluble support and adsorbent.

When the elongation reaction is carried out using a precursor which contains a polyethylene glycol, vinyl polymer of polyamide support separation of the unreacted added amine acid segment is carried out by conventional methods.

Deblocking of the elongated complex is effected enzymatically before it is used as a further precursor in subsequent stages. It is, of course, necessary that the blocking group on the elongated complex be selectively degradable by enzymatic action.

Turning first to that aspect of the present invention wherein chain elongation is accomplished through the C-terminal end of a growing chain by reaction with an amino acid segment of Formula II, the blocking group on the acid segment is preferably a short chain alkyl group or benzyl group coupled to the acid through an ester linkage. One reason for - 18 this is that deblocking after reaction with the precursor can be accomplished enzymatically using an esterase, thereby hydrolyzing off the ester group to yield the free C-terminal carboxyl group for subsequ5 ent elongation of the chain. A carboxypeptidase such as carboxypeptidase Y is useful for this purpose so long as the pH is maintained in the range of pH of from 8 to 9, preferably at pH 8.5. At a pH of 8.5 this enzyme exhibits optimum esterase activity to the exclusion of peptidase activity, while, as previously discussed, at a lower pH it is exclusively an exopeptidase. The hydrolysis reaction is carried out at a temperature of from 0° to 60°C.

A further significant advantage accompanying the use of this particular enzyme for deblocking is that hydrolysis is only effected with respect to esters of L-amino acids. Thus, those chains containing blocked esters of D-amino acids are not hydrolyzed by the enzyme and are not available for subsequent growth.

As a result, a high degree of optical purity with respect to the target peptide can be achieved.

When growth from the N-terminus is desired, the L-pyrrolidonecarboxy group is a useful blocking agent for the α-amino group on the added acid. The elon25 gated complex containing this blocking group then is exposed to an enzyme, such as L-pyrrolidonecarboxylpeptidase, which has the necessary specificity at a temperature of from 0° to 60°C. and a pH of 7 to 8.

This enzyme is effective in deblocking only derivatives of L-amino acids. Thus, any D-isomer terminating chains remain blocked and are effectively no longer available for subsequent growth.

In either of the foregoing cases, intimate contact should be achieved between the blocking group on the chains and the enzyme in order to effect substantially complete deblocking of the L-terminated 6 4 7 0 - 19 chains. Accordingly, it is preferred that contact be achieved while the elongated precursor is dissolved in an aqueous medium. Moreover, in order to easily separate the deblocked compound and the enzyme and to minimize enzyme loss, the enzyme preferably is immobilized on a water insoluble support. Therefore, a preferred manner of accomplishimg the deblocking is to pass the aqueous solution of the blocked elongated complex through a column which contains an insoluble support having the enzyme immobilized thereon. As should be apparent, with respect to C-terminal synthesis, a column containing carboxypeptidase Y immobilized on a water insoluble support may be used both for pruning unreacted precursor and for deblocking the terminal carboxyl group of the blocked elongated complex merely by adjusting the pH to achieve the desired exopeptidase or esterase activity respectively.

Turning now to the combined use of the above features in a preferred multistage polypeptide synthesis, the initial step is the reaction of a first amino acid derivative with a water soluble support to form a water soluble covalent complex containing the first amino acid residue of the interded sequence. The added acid contains an enzymatically removable Na-amino or C-carboxy protecting group depending on the route selected. Unreacted amino acid derivative is removed from the reaction solution containing the initial complex by passing the solution through a column containing a water insoluble support which has immobilized on its surface an adsorbent which can affinitively interact with the water-soluble support. Preferably, the water-soluble support and adsorbent are both polynucleotides such as polyuridylic acid and polyadenylic acid respectively, and the solution is maintained at about 4°C. The insoluble support then is washed several times with 7.5 pH phosphate buffer at this 6 4 7 0 - 20 temperature. Thereafter, the complex is eluted from the insoluble support as an aqueous solution free of the added acid derivative by simple drawing buffer through the column at an elevated temperature, pre5 ferably from 4O°-6O°C.

The solution so obtained then is passed through another column in order to remove the blocking group on the terminal acid segment of the complex. Accordingly, this column contains an insoluble support having immobilized on its surface an enzyme having specificity for the protecting group. Then the solution is introduced back into a clean reaction vessel and, since the blocking group has been removed, chain elongation can be effected with the second amino acid of the intended sequence. As with the first acid, the second acid is derived so as to be appropriately Na or C-blocked.

The foregoing procedure is then reiterated to successively add the desired acids to the complex containing the growing polypeptide chain until a short polypeptide, for example hexapeptide, has been prepared. It will be noted that, up to this point, pruning of chains which failed to react with added acid has not been employed.

As will become apparent, there is no particular advantage to be derived from including this step in the early stages of the synthesis, although it can be used without any adverse consequences if desired.

At this point, the solution recovered after separation of unreacted acid from the short chain peptide is enzymatically treated to release the elongated chains from the water soluble support. The solution is passed back over the insoluble support containing the immobilized adsorbent to remove the separated water soluble support and the terminal amino or carboxyl blocked short chain target peptide then is 6 4 7 0 - 21 isolated from the solution. Since the occurrence of sequence failure in any of the foregoing steps results in the presence of chains having less than, for example, six amino acid residues, separation and isolation of the desired short chain peptide easily can be accomplished by conventional techniques, such as ion exchange or gel filtration chromatography.

The preparation of long chain polypeptides can be effected by using a short chain polypeptide as a precursor. The short chain polypeptide precursor may be prepared by the present process as illustrated above or synthetically prepared by other methods. In addition, naturally occurring short chain polypeptides may be used as the precursor. In any event, the pure short chain polypeptide is attached to the biospeoific soluble support enzymatic deblocking is effected, and the short chain peptide complex then is used as the precursor for chain elongation with the next amino acid derivative. It is at this point that the above described pruning of unreacted chains preferably is initiated.

To this end, the reaction solution, which contains the complex of the elongated polypeptide, unreacted excess blocked acid, and unreacted complex of the short chain polypeptide precursor, is passed through another column containing an insoluble support having an alphaamino group or terminal carboxyl-specific exopeptidase immobilized on its surface.

On passing through this column, the unreacted precursor chains,which contain an unblocked terminal amino or carboxyl group, are enzymatically degraded and, therefore, pruned from the desired product chains. Thereafter, this step is incorporated into the above described reiterative sequencing procedure as the chain is elongated with additional amino acid derivatives.

Finally, after the desired target polypeptide has been prepared, the polypeptide chains are released - 22 from the biospecific water soluble support ahd the target peptide separated and purified. It will be appreciated that, due to the incorporation of the enzymatic degradation step for each sequence after the preparation of the short chain polypeptide precursor, the final reaction solution contains very few and, preferably, no polypeptide chains which differ from the target peptide by less than the number of amino acid residues in the precursor. Thus, conventional separation techniques can be used.

Furthermore, it will be appreciated that the foregoing, generally described reiterative procedure, is useful with respect to both the C-terminus and Nterminus routes to peptide synthesis. The principal differences between the two routes reside in the manner in which the growing chain is attached to the soluble support and in the selection of blocking groups and enzymes. Also, there can be a difference in the manner in which activation for chain elongation is accomplished, for example the use of active esters for N-terminal growth versus carbodiimide mediated coupling. The latter, which is useful with respect to both C- and Nterminal growth, is preferred. Most preferred is the C-terminal approach to chain elongation.

In light of the foregoing discussion, it will be appreciated that the difficulties associated with a sequence failure at any stage can be substantially eliminated so long as (1) a pure transformable precursor is used for reaction at some intermediate stage of the procedure and (2) degradation of unreacted precursor is utilized in all stages subsequent thereto. The minimun point at which the use of a pure polypeptide precursor is necessary depends on the ultimate molecular weight of the polypeptide to be fashioned.

The molecular weight of the pure polypeptide precursor can be considered as the minimum molecular weight 64 70 - 23 difference which will exist in the final polymer solution between the desired polypeptide and incomplete polymer chains. Therefore, as the desired polypeptide increases in molecular weight, the minimum difference between the weight of the pure and incomplete chains becomes smaller for any given pure polypeptide precursor, and the difficulty in eventual purification increases.

Accordingly, while a pure hexapeptide precursor is suitable for the ultimate preparation of a polypeptide containing twenty to about thirty-five amino acid residues, it is to be understood that correspondingly larger pure polypeptide precursors should be used initially for the preparation of higher molecular weight, pure polymers.

Thus, it is apparent that the illustrated procedure itself can be used to prepare pure, high molecular weight precursors for subsequent use in the preparation of even higher molecular weight compounds. For example, a polypeptide containing 35 acid residues prepared as described above can itself be used as the pure precursor for the preparation of a polymer containing upwards of 100 amino acid residues.

The process of the present invention may be employed in the synthesis of such peptides as glucagon, enkephalin, ACTH, calcitonin, vasopressin, pentagastrin and endorphin.

As indicated previously, reactive groups other than the alpha amino or carboxyl group on some amino acids must be blocked or protected during chain elongation. As is well recognized, these groups are the epsilon amino group of lysine, the imidazole group of histidine, the phenolic hydroxyl group of tyrosine, the carboxyl groups of glutamic and aspartic acids, the thiol group of cysteine, and the guanidinium group of arginine. Typically, removal of these blocking groups 6 4 7 0 - 24 is the final step of the complete synthesis procedure The following table sets forth examples of recognized protecting groups; see also, Solid Phase Peptide Synthesis, Stewart and Young, Freeman and Co., 1969, pp. 13-23, for blocking groups as well as the conditions under which they can be removed.

TABLE Acid_Blocking Group Removal Lysine acetamido or trifluoroacetyl Histidine Tyrosine Cysteine Arginine ethoxyformyl acetyl acetamidomethyl (Veber et al., 1972, J. Am Chem. Soc., 94, 5456) nitro pH 9, 1.2M hydrazine or 1M piperidine at 0°C pH 9, 1.2M hydrazine pH 9, 1.2M hydrazine mercuric acetate and then H2S (to be removed last) catalytic hydrogenolysis A particular aspect of the present invention resides in the fact that only certain minimal protection of side groups is necessary. This fact, in combination with the use of a high molecular weight bio-specific support in a preferred embodiment, is advantageous in achieving water solubility of the growing polypeptide.

Thus, when the above described active esters of amino acids are utilized for chain elongation (Nterminal growth), only the epsilon amino group of lysine and the thiol group of cysteine need be blocked. This results in advantages accompanying initial amino acid preparation as well as avoiding the necessity for eventual deblocking and potential adverse ZO effect on the prepared poly4 6 4 7 0 - 25 peptide. For C-terminal growth, the carboxylate side groups of glutamic and aspartic acid need to be protected. Also, in view of the procedure discussed above for separating· the peptide from the handle, in both routes the ε-amino group of lysine and the guanidinium group of arginine also require protection where either of these acids are included in both the handle and the target peptide.

It has been indicated that a water insoluble support for the immobilized adsorbent and enzymes is employed. While a variety of known water insoluble materials either organic or inorganic, can be used so long as they do not adversely affect the growing polymer chain or complex thereof, the insoluble support preferably is rigid and dimensionally stable in changing solution so that it can tolerate various reaction conditions. Porous glass beads constitute an expecially preferred class of rigid supports. As hereinafter illustrated, these beads can be suitably derivatized and activated so as to effect immobilization of enzymes and adsorbents thereof. Pierce Chemical Company of Rockford, Illinois, is a commercial source of such beads which are manufactured by Corning Works. A oarticularly useful support is Glycophase G porous glass beads. These beads are the 2,3-dihydroxypropyloxypropyltrimethoxysilane derivative of porous glass.

Also, it is to be understood that the aqueous solutions referred to herein can contain organic solvents or other ingredients which do not adversely affect the desirable features of the described procedures. Use of organic solvents such as dimethylformamide or methanol, in fact, are considered to be desirable with respect to the preparation of high molecular weight polypeptides in order to assure solubility. However, the type thereof and amount Λ6470 - 26 should be selected so as not to interfere with reversible coupling in the separation step.

The following examples illustrate the manner in. which the present invention can be accomplished: Example 1 A. Preparation of Immobilized Enzymes Five columns are prepared each containing a particular enzyme immobilized on 10 grams of Glycophase G porous glass beads (74-126 micron, pore diameter about 550 angstroms) obtained from Pierce Chemical Company or, with respect to carboxypeptidase Y, cross linked Sepharose (Registered Trade Mark) from Pharmacia., Depending on the enzyme used, the glass is activated to facilitate enzyme attachment by one of three approaches. Approach (a) involves adding 10 grams of the beads to a beaker containing I water. Ten grams of cyanogen bromide is then added and, while maintaining the solution temperature at 20°C., the pH of the solution is held constant at 11 by the continuous addition of cold 6 N sodium hydroxide. Activation is considered complete, usually after about 15 minutes, when the uptake of the sodium hydroxide ends, as indicated by a change in pH. At that time, the beads are filtered from the solution and washed with a solution of 0.IM sodium bicarbonate at a pH of 9.5. Approach (b) involves reacting 10 g. of the beads with 0.5 g. paranitrobenzyl bromide in 50 ml. of dioxane for 24 hours at room temperature, followed by heating at 100°C. in a 10% by weight aqueous solution of dithionite. The arylamine derivatived Glycophase G beads are washed with distilled water and activated by diazotization in 30 ml of 0.5 N HCL at 0°C. with an excess of NaN02 followed by washing the activated beads with 3 liters of 3% by weight sulfamic acid 20 1. of distilled water. - 27 Approach (c) involves reaction with NalO^, Royer et al., supra. The columns and method of enzyme attachment (200 mg. of the following table. enzyme used) is given in Column Enzyme Activation Approach Method of Attachment 1 L-pyrrolidonecar- boxylpeptidase (a) 6 hours at pH 8, 4°C. in 0.1M 2pyrrolidone aqueous medium containing bicarbonate buffer. 2 Aminopeptidase M (b) At pH 7.6 in tris containing MnC^tl mM), Royer et al., J.Bio.Chem.,248(5) , 1807 (1973) 3 Carboxypeptidase Y Conversion of Sepha rose to hexamethylene diamine derivative by reductive alkylation followed by enzyme coupling with water soluble carbodiimide at pH 4.75, 12 hours, 04°C. 4 Trypsin (c) Reductive alkylat- ion 5 Carboxypeptidase B (c) Reductive alkylation 6 4 7 0 - 28 B. Preparation of Immobilized Polynucleotide Adsorbent The arylamine derivative of Glycophase G porous glass beads are prepared and activated by approach (b), supra. The beads are reacted at 0°C. for 3 hours with 100 mg. of commercial polyadenylic acid (Poly A) (MW above 1000) in buffer at pH 8. After washing, the beads contain about 1¾ by weight, of the acid.

EXAMPLE 2 (N-terminal approach) A. Preparation of active esters of Na-L-Pyrrolidonecarboxyl-L-amino acids Samples of the following L-pyrrolidonecarboxyl blocked amino acids are obtained in substantially pure form by the method of Kurath and Thomas, supra: L-Alanine Glycine; S-ACM-L-Cysteine; L-Phenylalanine; ε-TFA-L-Lysine; L-Arginine L-Leucine; L-Serine; L-Tyrosine; L-Valine; wherein ACM stands for acetamidomethyl and TFA stands for trifluoroacetyl. Thus, 0.1 moles of a commercially available protected pyrolidone carboxylic acid (Z-PC-OH) and 0.11 moles of N-hydroxysuccinimide (NHS) are cooled to 0°C in 100 ml. 1,2-dimethoxyethane. Dicyclohexyl carbodiimide (0.11 moles) is added. The reaction mixture is stirred for 2 hours at 0-40°C. and then overnight at room temperature. The dicyclohexyl urea is removed by filtration and washed with additional 1,2-dimethoxyethane. The filtrates are reduced at 40°C. in vacuo. The N-protected pyrrolidonecarboxyl N-hydroxysuccinimide (Z-PC-NHS) ester 6 4 7 0 - 29 is crystallized from 2-propanol. The Z-PC-NHS (6.0 moles) is dissolved in 20 ml. dioxane. A solution (10 ml.) containing NaCO3'H2O (7.6 mmoles) and the desired amino acid (7.6 mmoles) is added and the mixture stirred for 4 hours at room temperature. The volume is reduced to 4 ml. After neutralization with IN HCl, a precipitate results which is washed with two, ml.-portions of water. The material (Z-PC-Acid) is recrystailized from 2-propanol, Z-PC-Acid (about 1 g.), Pd-black (1 g.) and 200 ml. of 50% by weight aqueous methanol are placed in a 3-necked flask. Hydrogen gas is passed through the suspension for 3 hours at room temperature. After removal of methanol and water, the L-pyrrolidonecarboxyl blocked amino acid is crystallized from methanol diethyl ester.

Having obtained the L-pyrrolidonecarboxyl blocked amino acid by the above method, the N-hydroxysuccinimide active ester thereof is prepared by adding one equivalent of each blocked acid to separate reaction vessels containing ethyl acetate, one equivalent of N-hydroxysuccinimide and 1.1 equivalents of dicyclohexylcarbodiimide. Esterification of the free carboxyl group is effected by stirring for 4 hours at room temperature. The reaction solutions are then removed from the vessels. Precipitated dicylohexyl urea is filtered off and the active esters purified by recrystallization from ethyl acetate-petroleum ether.

B. Preparation of Polynucleotide-First Acid Complex Polyuridylic acid (Poly U)(10 mg.) is oxidized with 0.01 M NalO^ for 18 hours. The resulting aldehyde containing product is reductively alkylated with hexamethylene diamine according to Royer, et al., Biochem. Biophys. Research Commun., 64, 478 (1975).

The solution at 4°C., is drawn through the column prepared in Example IB containing immobilized Poly A 6 4 7 0 - 30 which selectively adsorbs the diamine derivatized Poly U. After washing the column and raising its temperature to 55°C., the diamine derivatived Poly ϋ is eluted therefrom in phosphate buffer (pH 7.5). PC-L5 Arg-OH then is bound to the diamine derivatived Poly U using water-soluble 1 - ethyl - 3 - (3 - dimethylaminopropyl) - carbodiimide at pH 4.75 and the PC blocking group then is removed by passing the solution through column 1 prepared as in Example IA. The pro10 duct formed is Poly U-L-Arg.

Subsequently, a large excess of the activated ester of valine prepared as in Example 2A is added to the solution and the solution is stirred for two hours. The product formed is Poly U-L-Arg-L-Val.

C. The Procedure Two-hundred millilitres of the solution from 2B above containing the support - L-Arg-L-Val complex, at 4°C., is passed by suction at 5 ml/minute through the column prepared in Example IB containing the immobili20 zed polyadenylic acid beads. The column is then washed at 4°C. with 1 liter portions of water at 15 ml/ min. to remove unreacted acid. Thereafter the temperat ure of the column is raised to 55°C. and a 200 ml. portion of phosphate buffer (pH 7.5) at this temperat ure is drawn through the column at 5 ml/min. to elute the support-Arg-Val complex.

The solution, at pH 7.5 and 25°C., containing the complex is then passed, at 5 ml/min., through packed column 1 containing the immobilized L-pyrroli30 donecarboxylpeptidase to remove the L-pyrrolidonecarboxyl blocking group, thus providing 200 ml. of solution which contains the complex of support-Arg-Val with a free alpha-amino group.

This solution is then added back into a clean reaction vessel and the foregoing procedure repeated 64 7ο - 31 with the sequential addition of samples containing the activated and blocked lysine, phenylalanine, cysteine, and alanine.

After preparation of the hexapeptide support5 Arg-Val-Lys-Phe-Cys-Ala-PC solution free of unreacted alanine derivative and before removal of the blocking group, the solution is passed through column 4, containing immobilized trypsin, to release the chains from the support. Separation of the pure, blocked hexapeptide is then effected by passing the solution over the column containing immobilized polyadenylic acid to remove the support followed by gel filtration chromatography.

Subsequently the carboxyl group of the hexapeptide is reactivated by esterification and recomplexed to the support as in 2A and B above, deblocked (column 1) and reacted with the sample of glycine prepared as in 2A. Passage of the resulting solution, at 5 ml/min, is then effected, at 35°C., through the column containing immobilized aminopeptidase M (column 2) to degrade any of the hexapeptide chains which failed to react with glycine. This step is thereafter incorporated into the synethesis procedure for the addition of the leucine, tyrosine and serine.

After the serine has been affixed to the growing chain and the complex separated from the reaction mixture, the solution containing the complex is passed through column 4 to remove the handle and treated with 1M piperidine at 0°C. for one hour to remove the trifluoroacetyl blocking group on the lysine residue. The resulting solution is then passed at 5 ml/min. through the packed column of immobilized pyrrolidonecarboxylpeptidase (column 1) to remove the terminal pyrrolidonecarboxyl group on the serine acid residue and the solution then treated at pH 4 with mercuric acetate and stirred for one hour at 25°C. to remove the acetamidomethyl protecting group on the cysteine residue. The 6 4 70 - 32 resulting thio compound is generated by bubbling H2S through the solution. Finally, the pure decapeptide HO - Arg - Val -Lys - Phe - Cys - Ala - Cly - Leu Tyr - Ser - NH2 so prepared is separted and isolated from residual Piily U and other ingredients as illustrated above, j EXAMPLE 3 A further column of packed porous glass beads is prepared. Glycophase G beads are used and initially are reacted at 40°C. in dimethylformamide with tosyl chloride (10% by weight of beads) and one equivalent, to the chloride, of triethylamine. Thereafter the glass is filtered, washed and reacted with an excess of thioacetic to displace tosyl on the bead surface. The beads then are separated from the reaction medium and added to a vessel containing water in order to hydrolyze the acid and yield the thiol derivative thereof. Subsequently, the beads are reacted in water at 25°C, with mercaptosuccinic anhydride in order to obtain beads with the mixed disulfide immobilized on their surface. The mixed disulfide is an electrophylic reagent which has specific covalent reactivity for primary terminal amino groups.

Example 1 is then again repeated except that the column containing the immobilized disulfide replaces column 2 containing the immobilized aminopeptidase M. Rather than degrading unreacted chains, passage through this column serves to scavenge such chains by the covalent reaction between the disulfide and the free terminal amino group on unreacted chains. Therefore, the reaction solution which is recovered from the column has the unreacted polypeptide chain removed therefrom.

After each scavenging operation an aqueous solution (pH 8) of sodium borohydride (0.5M) is passed through the column at 25°C. in order to remove the - 33 covalently bonded chains therefrom. The column is then reactivated by treatment with mercaptosuccinic anhydride to restore the disulfide bridge.

EXAMPLE 4 (C-Terminal Approach) A. Preparation of Ethyl Ester Protected Amino Acids The amino acids set forth in Example 2A are esterified by reaction with ethanol, under reflux, in the presence of a catalytic amount of anhydrous HCl.

The reaction volume is concentrated and upon cooling the amino acid esters crystallize as hydrochlorides. These protected acids also are commercially available.

B. Preparation of Support-First Amino Acid Complex Poly 0 (10 mg) is trated with NalO^ (0.01 M) for 18 hours. The resulting dialdehyde is coupled to the spacer arm dipeptide H-Gly-Arg-OH by reductive alkylation according to Royer et al., Biochem, and Biophys. Research Commun., 64, 478 (1975) .

The ethyl ester of valine, as prepared in 5A is attached via reaction in the presence of a watersoluble carbodiimide at pH 4.75, O-4°C., 12 hours.

C. The Procedure The basic reiterative procedure is the same as with respect to the N-terminal approach except that deblocking and pruning of unreacted chains is accomplished using column 3 containing immobilized carboxypeptidase Y. For pruning, the pH is 5.5 in a phosphate buffer. For deblocking the pH of the solution, prior to passage through the column, is adjusted to 8.5 by addition of NaOH. In both instances a column and solution temperature of 35°C. is used. For separation and recovery of the peptide from the handle, successive 470 - 34 passage through column 4 and the column of Example IB is employed with the respective solutions being at a pH of 8 (using Tris) and using a solution and column temperatures of 35°C.

EXAMPLE 5 Synthesis of H-Leu-Phe-Leu-OH A. Preparation of the Handle-/l’EG-CH2CO-Gly-Arg(NO2)_7 ! Polyethylene glycol (PEG)(14 g., Mol. Wt. 60007000) and potassium tertbutoxide (10 g.) were dissolved in t-butanol (150 ml.) by warming to 40°C.

Ethyl bromoacetate (5 ml.) was added over a period of 10 minutes. After an additional 2 hours of stirring at 40°C., the solvent was removed by a rotary evaporator.

The residue was dissolved in 100 ml, of 2 N NaOH and kept at room temperature for two hours. The pH of the mixture was then adjusted to 2.0. The PEG was extracted twice into 200 ml. of CHCl^. The organic extract was washed with water and then dried over Na2S0^. Evaporation of the solvent yielded 12 g. of carboxymethyl-PEG.

Glycine was added to the carboxymethyl-PEG with a water-soluble carbodiimide as follows. H-Gly-OEtHCl (1.4 g.) and carboxymethyl-PEG (3.4 g.) were dissolved in 25 ml. of ^0. The pH was adjusted to 6.0 with triethylamine. Ethyl dimethylaminopropyl carbodiimide 'HCl (EDC, 2 g.) was added and pH was maintained at 6.0 with a pH-Stat. After 3 hours at room temperature, the reaction mixture was acidified to pH 2.0 and the product was extracted into CHCl^. The organic layer was washed with 1 N HCl and water. After drying over NajSO^ the solvent was evaporated under reduced pressure. PEG-CH2CO-Gly-OEt(3.2 g.) was dissolved in 20 ml. of water and the pH adjusted to 10.5. The saponification was followed at this pH using a pH-stat, The titrant was 0.1 N NaOH. About 4 ml. of base was consumed over ί 6 4 7 0 - 35 the reaction period of one hour. Acidification and extraction with CHCl^ gave 3.3 g. of product. Amino acid analysis showed 100 mmoles of glycine/mg. polymer.

H-Arg(N02)-OMe'HCl was reacted in a manner analogous to the coupling of glycine ethyl ester. PEGGly-Arg(N02)-OMe (3.30 g.) was treated at pH 8.5 with 25 units of CPY immobilized on CL-Sepharose (R.T.M.) (Liberatore, et al., (1976) FEBS Letters 68, 45). The pH was maintained with 0.1 N NaOH for 5 hours at room temperature. The bound enzyme was removed by filtration.

B. Preparation of PEG“CH2CO-Gly-Arg(N02)Leu-Phe-LeuOH H-Leu-OEtH-Phe-OEt, and H-Leu-OEt were added successively as described above. The rate of deprotection improved dramatically as the chain length increased. The amino acid analysis for the final peptide was in agreement with theory.

C. Release of .the Peptide The final product (1 g.) was dissolved in 20 ml. of MeOH/cyclohexene (1:1 V/V). Freshly prepared palladium black (250 mg.) was added and the reaction mixture was stirred with refluxing for 1 hour. The catalyst was filtered and the product was dried in vacuo (0.9 g. yield). The product was dissolved in 0.1 M N-ethyImorpholineacetate buffer at pH 8.0 and bound trypsin (1 g., prepared acc. to Royer et al., Methods in Enzymol, (1977) 47, 40) was added. The suspension was tumbled for 6 hours. The release of the peptide was followed by TLC using authentic H-Leu-Phe-Leu-OH as a standard. The yield of product was 80% based on the number of initiation sites available on the PEG support.

The procedures illustrated in the foregoing Examples are easy to accomplish, readily susceptible to automation, and applicable to the aqueous, synthetic 6 4 7 0 - 36 preparation of polypeptides from amino acids containing primary amino groups in the alpha position. As is well recognized, this includes all of the amino acids except proline and any analogs thereof. With respact to proline, the acylable amino group is a secondary amine and is not amenable to blocking and deblocking with the L-pyrrolidonecarboxyl group.

Accordingly, when the polypeptide being fashioned is to contain the proline residue and when an N-terminal route is used, it is necessary to block this acid by conventional techniques, such as with the carbobenzoxy (Z) group, and thereafter accomplish deblocking with, for example, HBr-glacial acetic acid. Therefore, in synthesizing a proline-containing polypeptide from the N-terminal, the above-described procedure is interrupted after the blocked-proline is added to the chain.

The chain can then be released from the support, isolated and the blocking group removed. The chain then is reattached to the handle and the procedure described herein reiterated for the addition of subsequent acids onto the chain.

The enzymatic deblocking described herein is generally applicable both as concerns polymer synthesis and the other multi-stage molecular transformation procedures involving sequential chemical reactions, so long as the procedure requires a removable blocking group.

Thus, with respect to conventional solid phase peptide preparation wherein chain elongation is effected with the chain covalently affixed to an insoluble support, it will be appreciated that appropriate enzymatic solutions can be successively passed over the support to remove unreacted chains and to deblock at each stage of the reiterative procedure. As to conventional solution methods in organic solvents, the use of immobilized enzymes as illustrated herein can, for 6 4 7 0 - 37 example, be conveniently employed by simply isolating the peptide chain population after each sequential acid reaction and dissolving it in an aqueous medium.

The above described process is advantageous in that the deblocking of a peptide is easy to accomplish and does not result in destruction of the chain being fashioned. In addition, the deblocking is accomplished in a manner which ensures the optical purity of the peptide being formed.

Furthermore, reiterative elongation of a growing peptide chain can be rapidly accomplished in an aqueous medium without disruption of the polymer chain. Additionally, elaborate protection of amino acid side chains which customarily decreases the aqueous solubility of the polymer being prepared is eliminated.

The process enables a substantially pure polypeptide of predictable amino acid sequence to be prepared, is susceptible to automation, and can readily be used to prepare pure, high molecular weight target peptides.

The described process is also advantageous in that in a preferred embodiment the isolation of an elongated polypeptide from its reaction environment so that the proper sequence can result on further reaction is easy to accomplish and involves a minimum expenditure of time and minimum peptide loss.

In another preferred embodiment, the difficulties attendant on the reiterative preparation of polymers which result from sequence failure due to incomplete reaction are minimised by efficiently removing failed sequences from the growing chain population so that recovery of a desired pure polypeptide can be accomplished more conveniently.

Claims (23)

1. CLAIMS:1. A process for synthesizing a peptide chain having a predetermined sequence amino .aids, the process comprising reacting a precursor which comprises a first amino acid segment (as herein defined) of the sequence, the first amino acid segment having a free terminal carboxyl group or a free terminal amino group, with a second amino acid segment having a free N a -amino group and a blocked carboxyl group susceptible to enzymatic hydrolysis when the precursor has a free terminal carboxyl group, or a free carboxyl group and a blocked N a -amino group susceptible to enzymatic hydrolysis when the precursor has a free terminal amino group, and deblocking the product peptide enzymatically in an aqueous medium.

2. A process according to claim 1, wherein the precursor comprises a first amino acid segment attaches to a water-soluble support.

3. A process according to claim 2, wherein the water-soluble support comprises a polynucleotide,

4. A process according to claim 2, wherein the water-soluble support comprises a polyethylene glycol.

5. A process according to any one of claims 1 to 4 wherein the precursor is reacted with the second amino acid segment in an aqueous medium.

6. A process according to any one of claims 1 to 5, wherein the second amino acid segment contains a blocked carboxyl group and the blocking group is an ester group.

7. A process according to claim 6, wherein the ester group is an alkyl ester group of less than 10 4 6 4 70 - 39 carbon atoms.

8. A process according to claim 6 or claim 7, wherein the carboxyl group is deblocked enzymatically using an esterase.

9. A process according to claim 8, wherein the carboxyl group is deblocked using carboxypeptidase Y at a pH of from 8 to 9 and at a temperature of from 0 to 60°C.

10. A process according to claim 9, wherein the pH is 8.5.

11. A process according to any one of claims 1 to 3, wherein the second amino acid segment contains a blocked amino group and the blocking group is the Lpyrrolidonecarboxyl group.

12. A process according to claim 11, wherein the amino group is deblocked using L-pyrrolidonecarboxylpeptidase at a pH of from 7 to 8 at a temperature of from 0 to 60°C.

13. A process according to any one of the preceding claims, wherein any unreacted precursor is removed enzymatically.

14. A process according to claim 13, wherein the precursor contains a free terminal amino group and the unreacted precursor is removed using aminopeptidase M at a temperature of from 0 to 50°C. and at a pH of from 6.5 to 7.5.

15. A process according to any one of claims 1 to 11, wherein any unreacted precursor is removed by scavenging. - 40

16. A process according to claim 2 or claim 3, wherein the first amino acid segment is attached to the water-soluble support by a linkage susceptible to enzymatic cleavage. 5

17. A process according to claim 16, wherein the peptide chain is attached to the water-soluble support by way of an arginine or lysine residue.

18. A process according to claim 16 or claim 17, wherein the deblocked product peptide chain is 10 released from the water-soluble support enzymatically.

19. A process according to claim 18, wherein the product peptide chain is released from the watersoluble support with trypsin.

20. A process according to claim 2 or claim 3, 15 wherein any unreacted second amino acid segment is removed before the product peptide is deblocked by reversibly coupling (as hereinbefore defined) the water-soluble support of the precursor to an appropriate adsorbent immobilized on an insoluble support, 20 separating the unreacted second amino acid segment, and releasing the water-soluble support from the adsorbent.

21. A process according to claim 20, wherein the water-soluble support is polyuridylic acid and the adsorbent is polyadenylic acid. 25

22. A process according to claim 20 or claim 21, wherein the water-soluble support is released from the adsorbent by heating.

23. A process according to any one of the preceding claims wherein the step of reacting a precursor 30 with an amino acid segment is repeated until the desired number of amino acid segments are added to the - 41 precursor.