SYNTHESIS OF PEPTIDE ANALOGS

Technical Field

The present invention relates to the synthesis of pep- tide analogs. More particularly, the invention relates to a novel polymeric disc, wafer or other similarly shaped res¬ in and a method for its use in solid phase peptide synthesis ("SPPS") .

The present invention permits the rapid production of peptide analogs, i.e., numerous peptides differing from one another by only a single amino acid or small number or a- mino acids. The synthesis of analogs according to the pres¬ ent invention can take place at a rapid rate while assuring that the reagents necessary to synthesize the a ^' nalogs under- go quantitatively complete reactions so as to minimize unde¬ sirable side-reaction products which could result in the production of "deletion peptides" or "deletion sequences."

Statement of Exploitation of Invention in Industry

The invention addresses and satisfies the industrial need for a rapid and quantitatively efficient method of syn¬ thesizing long-chain peptides. Within recent years, new hor¬ mones and relating, factors, inhibitors, growth factors, tox¬ ins, and ion carriers and antibiotics have been discovered. This and related activity has created an increased need for the chemical synthesis of peptides and small proteins. It is important to be able to make the parent compound tm con¬ firm its structure and in some cases to provide the peptide in larger quantities than can be conveniently obtained from natural sources.

Synthetic peptide analogs are essential for structure- function studies designed to investigate the mechanism of

action and to produce inhibitors or superagonists of improv¬ ed selectivity and duration of action. The synthesis of im- munogenic peptides has great potential for the development of vaccines and can play an important role in the detection 5 and isolation of new gene products. The present invention greatly simplifies and increases the efficiency of the task of preparing synthetic peptides.

Background Art

Solid phase peptide synthesis was introduced by Dr. R.

10 Bruce Merrifield in 1963 when Dr. Merrifield attached a grow¬ ing peptide chain to a solid support. Merrifield, R. B. (1963) J. Am. Chem. Soc. 85, 2149-2154. The procedures e- nunciated by Dr. Merrifield for SPPS were as follows: An a- mino acid corresponding to the C-terminal of the target pep-

15 tide is covalently attached to an insoluble polymeric sup¬ port (the "resin"-)- The next amino acid, with a protected alpha-amino acid, is activated and reacted with the resin- bound amino acid to yield an amino-protected dipeptide on the resin. Excess reactants and co-products are removed by fil-

20 tration and washing- The a ino-protecting group is removed and chain extension is continued with the third and subse¬ quent protected amino acids. After the target protected pep¬ tide chain has been built up in this stepwise fashion, all side chain groups are removed and the anchoring bond between

25 the peptide and the resin is cleaved by suitable chemical means thereby releasing the crude peptide product into solu¬ tion. The desired peptide then undergoes an extensive puri¬ fication procedure and is then characterized. Kent, S. & Clark-Lewis, I. "Modern Methods for the Chemical Synthesis of

30 Biologically Active Peptides," Division of Biology 147-75,

California Institute of Technology, Pasadena, California 91125 U.S.A.; Houghten, R. A., Chang, W. C. & Li, C. H. (1980), Int. J. Pept. Protein Res., 16 311-320; Houghten, R._ A., Ostresh, J. M. & Klipstein, F. A. (1984), Eur . J. Biochem. ,

35 145, 157-162; Stewart, J. M. & Young, J. D., Solid Pha^se Pep-

tide Synthesis, Pierce Chemical Company (2d ed. 1984). See, also, Geysen, H. M., Meloen, R. H. & Barteling, S. J. (1984) Proc. Natl. Acad. Sci . USA, 81, 3998-4002; Matthes, H. W. D., Zenke, W. M. , Grundstrom, T., Staub, A., Wintzerith, M. & Chambon P. , (1984) The EMBO Journal, 3_, 801-805.

The resin employed in standard SPPS is known as the "Merrifield resin" and is a polystyrene bead of 100 - 200 mi¬ crons in size. The resin typically contains 0.5 - 2.0% di- vinylbenzene cross-linkage and contains 0.2 to 0.8 mmole of p_-chloromethyl groups per gram resin. The number of p_-chloro methyl groups determines the number of individual chains per gram and their ultimate size. The size of the bead allows for a rapid penetration of reagents in SPPS. The percent¬ age of cross-linkage determines the extent to which the res- in shrinks and swells during solvent changes. A large shrink-and-swell effect is preferred.

Dr. Merrifield had adopted known techniques of peptide chemistry, which were being used by others in solution phase synthesis, for solid phase peptide synthesis. In doing so, Dr. Merrifield eliminated the intensive purification procedur required between each chemical step; the solid-phase proce¬ dure only required filtration and rinsing of the solid sup¬ port with fresh solvent. Solid phase synthesis permitted chemists to add 5 - 6 amino acids per day rather than one or two amino acids per week.

While the solid phase technique had revolutionized bio- medical research in industry and academia, this procedure has remained essentially unchanged since its inception in the early 1960's. With the explosive pace at which biotechnical research has been advancing in the industrialized nations of the world, substantially more peptides, particularly an¬ alogs of greater complexity are needed in industry and re¬ search than ever before.

The ever increasing demand for analog peptides has ^r

been approached in several ways, but no approach, thus far, has proven completely satisfactory. One highly expensive and labor intensive method has been to use a series of reac¬ tion vessels, e.g., Stewart, J. M. & Young, J. D., Solid Phase Peptide Synthesis, Pierce Chemical Company, pp. 125- 130 (2d ed. 1984), rather than use a single reaction vessel.

Thereafter, the "pin" method was developed which re¬ sulted in the synthesis of peptides on the surface of a dowel rod. The concept was to employ may rods along a plate with each rod entering a different reaction well. The draw¬ backs inherent in the pin method are multi-fold- First, the formed peptide remains on the dowel during biological test¬ ing; there is no guarantee that the conformation of the bound peptide duplicates the conformation in solution. Secondly, and more importantly, each analog in actuality represented a separate synthesis. Accordingly, if one dowel were to show a superior biological response, there would be no means of determining whether the reactions involved in the synthesis of a particular analog was superior or whether an analog syn- thesized was biologically superior.

Subsequent to the pin method, the "tea bag" method was developed where a resin was placed in individual packets similar in design to ordinary tea bags. See, Houghten, R. A., (1985) Proc. Natl . Acad. Sci . USA 82, 5131-5135. The concept of the tea bag method was that many tea bags could be placed into the same reaction vessel so that many pep¬ tides could be synthesized together. When the point of dif¬ ference or deviation was reached in the formation of parti¬ cular peptides, i.e., the point where an analog would differ from a similar peptide by a single or small number of amino acids, each tea bag would be separated by hand and reacted separately for the differing amino acids. Following the necessary separate reactions, the tea bags would all be re¬ turned to the same reaction vessel for the continued forma- tion of those portions of the analogs which would be common to several peptide-s, thereby minimizing experimental error. Theoretically and initially, the tea bag method appeared to

be ideal. In practice, however, the tea bag mesh would necessarily be prohibitively small. The flow of reagents to the resin would be inhibited. Consequently, many reactions would fail to go to completion thereby resulting in the syn- thesis of peptides having deletion sequences. The result¬ ing truncated peptides could not, without possibly great dif¬ ficulty, be separated from analog peptides having the prop¬ er sequence.

Disclosure of Invention

In accordance with the present invention, provided is a novel polymeric disc, wafer or similarly shaped resin for carrying out the synthesis of peptide analogs via the solid phase peptide synthesis techniques generally known and de¬ scribed above. The polymeric disc of the present invention may be made out of those resins presently used in bead form in SPPS, such as, for example, benzhydryla ine resins, e.g., p_-methyl-benzhydrylamine (MBHA) resin, Boc-aminoacyl-4-(oxy- methyl)-phenylacetamidomethyl (Pam) resin, polyamide- resins and chloromethyl resin materials (the "classical Merrifield" resin), among others. If the disc is made of cross-linked polystyrene, preferably there would be a 2 - 5% cross-link¬ age .

The inventive polymeric disc, which it will be under¬ stood as including all suitably shaped and sized resins, not merely that which may be thought of as a circular disc, should be sufficiently thin so as to allow for the rapid penetration of reagents to insure that the required reactions may run to completion. The disc of the invention should, preferably, have a thickness of 200 - 400 um. Aside from this parameter, i.e., the thickness of the disc, the pre¬ cise shape of the disc, it should be emphasized, may be any shape having any suitable length or width whatsoever depend¬ ing upon the requirements of the user.

As part of the present invention, a process for the- synthesis of peptide analogs, usi-ng the polymeric resin

disc of the invention is further disclosed.

Brief Description of the Drawing

In the Drawing:

The FIGURE outlines the experimental process of solid phase peptide synthesis.

Best Mode for Carrying Out the Invention

The concept of solid phase peptide synthesis and, as will be explained, as it relates to the present invention may be best understood by reference to the experimental pro- cedure outlined in the FIGURE, wherein X is a reactive group such as a p_-chloromethyl group; L is a labile protecting group; and S is a stable side-chain blocking group to pre¬ vent side chain reactions during the peptide synthesis.

Referring to the FIGURE, a synthetic polymer, such as the polymeric disc of the present invention, would bear re¬ active groups, X. The amino acid which will form the C-ter- minal residue of the peptide to be synthesized is converted to a derivative in which its amino acid group is protected by a labile protecting group, L. Any standard protecting group, such as, for example, the Boc group, may be used in conjunction with the present invention. The foregoing deri¬ vative of the C-terminal amino acid is coupled to the reac¬ tive polymer. At this point, the repetitive cyclic part of SPPS begins. A reagent is applied to the protected a ino- acyl polymer to remove the labile blocking group, L, from the amino acid residue. The reagent employed must not, in any way, harm the link of the C-terminal residue to the polymer. Moreover, if the amino acidi.attach.ed to the poly¬ mer (and all amino acids in the peptide to be synthesized) contains a side-chain reactive functional group, that func¬ tional group must be blocked by a stable blocking group, S, which will remain completely intact throughout the synthesis, but which can be removed finally to yield the free peptide. Following removal of the labile protecting group, the next

amino acid is coupled to the aminoacyl polymer by use of a suitable coupling reaction. Again, the alpha-amino acid must be protected with the labile group.

This cycle of deprotection and coupling is then repeat- ed with each amino acid which is to be incorporated into the peptide chain. For the deprotection reaction, standard acidolysis methods, such as, a 25% solution of trifluoroace- tic acid in dichloromethane may be used. Dicyclohexyl-car- bodii ide (DDC) may be employed as the coupling agent, as well as other suitable coupling agents for use with the present invention, a different type of reagent, e.g., anhy¬ drous liquid hydrogen fluoride, is applied to cleave the pep¬ tide from the polymer and allow it to be dissolved. The blocking groups, which have protected side-chain functional groups, must also be removed, and are usually chosen so that they can be removed simultaneously with the cleavage of the peptide from the resin. The peptide can also be cleaved from the resin by ammonia and amines to yield peptide amides.

The present invention concerns the polymer support to be employed in the foregoing SPPS framework. The support must be insoluble and have satisfactory means of attaching the first amino acid to it. The polymeric disc of the pres¬ ent invention, i.e., the polymer support, may be made out of those resin materials presently used for SPPS when such is carried out with fine bead resins via conventional means. The polymeric disc, which may have any desired shape suit¬ able for the user (e.g., any suitable length or width) should preferably have a thickness of 200 - 400 μ . The resin of the present invention may be made out of, for example, a benzhydrylamine, e.g., p_-methylbenzhydrylamine , Boc-amino- acyl-4-(oxymethyl)-phenylacetamidomethyl„.(Pam) , polyamide, p_-hydroxymethyl resin, "Wang resins (i.e., hydrazide resin, ether resin and p_-alkoxybenzyl alcohol resin) and cross-link¬ ed polystyrene, among other materials. Wang, S. S. (1973) J. Amer . Chem. Soc. 93 , 1328-1333. If cross-linked polysty¬ rene is to be the material of the resin, the composition of the resin should be at least 1% divinylbenzene; a resin with

substantially less than 1% divinylbenzene would be too frag¬ ile to be of any use to the chemist.

Additionally, the inventive resin, to be effective, need not rely upon permeation, but may effectively act via a sur- face reaction. Thus, a hybrid-type resin is possible. Such a hybrid resin may have a strong, inert support, or backing, made of, for example, plastics or nylons (e.g., Nylon-66), or other materials.

The present invention further includes a method for use of the novel polymeric disc. In the synthesis of analogs, discs would be individually tagged. Peptide synthesis upon the severally tagged discs would take place within one reac¬ tion vessel in accordance with known principles of SPPS. When the point of deviation in the peptides is reached, i.e., where the amino acid or acids which are to differ from one peptide analog to another in the. synthesis process is reach¬ ed, the discs of the invention can be separated by hand or other procedure, e.g., tongs, reacted separately in differ¬ ent reaction vessels and then, subsequently, again placed in the same reaction vessel to continue or complete the syn¬ thesis of the analog chain with those amino acids generally common to the peptide analogs. Unlike prior methods, such as the tea bag method, where the bag mesh is too dense to allow for proper permeation, the present invention permits the necessary reactions to run to completion.

Finally, a hybrid-type resin, having an inert support as described above, which, relies upon a surface reaction, can also be conveniently transferred between reaction ves¬ sels with conventional tongs.

The invention will now be more fully described by ref¬ erence to the following Example. It should, however, be un¬ derstood tϊiat the following Example is_ for purposes of illus¬ tration only and not meant for the purpose of defining the limits or scope of the invention.

Example

The following procedure is suggested for the synthesis of the following three analogs of enkephalin:

(1) Tyr-Gly-Gly-Phe-Leu (Natural) (2) Tyr-DAla-Gly-Phe-Leu [D-Ala ] Enkephalin

4 (3) Tyr-Gly-Gly-DPhe-Leu [D-Phe ] Enkephalin

Step 1 : Place three 4" x 4" polymeric discs of the present invention into a flat glass reaction tank.

Step 2: Rinse the discs as follows: 2 x 100 ml of dichloromethane for 5 minutes each; and 2 x 100 ml of dimethylformamide for 5 minutes each.

Step 3: Add 1:1 molar ratio of Boc-amino acid cesium salt [(Boc-Leu-0 -) Cs+2 ] to the discs containing jD-chloromethyl groups overnight in a water bath at 50 degrees C.

Step 4: Rinse the discs of the invention in the following solvents ;

2 x 100 ml dimethylformamide for 2 minutes each 2 x 100 ml dimethylformamide :H_0 (1:1) for 2 minutes each 2 x 100 ml dimethylformamide for 2 minutes each

2 x 100 ml methanol for 2 minutes each

3 x 100 ml dichloromethane for 2 minutes each

Step 5: Deprotect with 100 ml of 45% trifluoroacetic acid in dichloromethane for 20 - 30 minutes.

Step 6: Rinse off trifluoroacetic acid with 6 x 100 ml washes of dichloromethane, 2 minutes each.

Step 7: Neutralize discs with 2 x 100 ml washes of 10% tri- ethylamine in dichloromethane, 2 minutes each.

Step 8: Rinse off triethylamine with 6 x 1,00 ml washes of

dichloromethane, 2 minutes each.

Step 9: Remove disc #3 and place into a second tank or re¬ action vessel.

Step 10: Add 3 molar excess of Boc-Phe-OH to discs ?I and #2 (6 moles total) in dichloromethane; and

Add 3 molar excess of Boc-DPhe-OH to disc #3 (3 moles total) in dichloromethane.

Step 11: Add 6 moles dicyclohexylcarbodiimide in dichloro¬ methane to discs £1 and 2; and Add 3 moles to disc #3.

Step 12: React for 1 hour, rinse with 6 x 100 ml dichloro¬ methane, 2 minutes each.

Step 13: Repeat Steps 7, 8, 10, 11 and 12.

Step 14: Deprotect by placing all three discs together in 45% trifluoroacetic acid/dichloromethane for 30 minutes.

Step 15: Rinse 6 x 100 ml dichloromethane.

Step 16: Neutralize as in Step 7.

Step 17: Rinse 6 x 100 ml dichloromethane.

Step 18: Add 9 moles total of Boc-Gly-OH and 9 moles of di- cyclohexylcarbodiimide in dichloromethane.

Step 19: React 1 hour and rinse with 6 x 100 ml dichloro¬ methane.

Step 20 : Neutralize and rinse.

Step 21: Repeat Steps 18 and 19.

Ste ^" 22: Deblock (as in Step 14) and rinse (as in Step 15)

Step 23: Neutralize and rinse.

Step 24: Remove disc #2 and place into a second tank (re¬ action vessel) .

Step 25: Add 6 moles of Boc-Gly-OH and 6 moles dicyclohex- ylcarbodiimide in dichloromethane to discs #1 and #3; and

Add 3 moles of Boc-DAla-OH and 3 moles of dicyclo- hexylcarbodiimide to disc #2.

Step 26 : React 1 hour and rinse discs.

Step 27: Neutralize and rinse.

Step 28: Repeat Steps 25 and 26.

Step 29: Place all three discs together, deprotect and rinse.

Step 30: Neutralize the discs and rinse.

Step 31 : React 9 moles of Boc-Tyr-OH and dicyclohexyl- carbodiimide in dichloromethane for 1 hour.

Step 32: Rinse, neutralize, rinse an repeat Step 31.

Step 33: Rinse, deprotect and rinse.

Step 34: Rinse 2 x 100 ml methanol (2 minutes each) .

Step 35: Dry, preferably, under vacuum.

Step 36: Cleave each disc using 90% hydrogen fluoride and 10% anisole for 1 hour at 0 degrees C, then evaporate hydrogen fluoride with vacuum at 0 degrees C.

Step 37: Extract anisole residue with 3 x 50 ml diethyl ether . _* -

Step 38: Extract anisole residue using 1%, 10% and 50% AcOH.

Step 39: Lyophilize peptide.

While only several embodiments of the present inven¬ tion have been shown and described, it will be obvious to those skilled in the art that many modifications may be made thereunto without departing from the spirit and scope of the invention.