EP0403600A1

EP0403600A1 - Deprotection of protected peptides

Info

Publication number: EP0403600A1
Application number: EP89910271A
Authority: EP
Inventors: Petrakis Konstantinos; Emil Thomas Kaiser
Original assignee: Rockefeller University
Current assignee: Rockefeller University
Priority date: 1988-08-18
Filing date: 1989-08-16
Publication date: 1990-12-27
Also published as: JPH02503681A; AU4208889A; JPH0647598B2; EP0403600A4; WO1990002138A1

Abstract

On a mis au point un procédé d'élimination de groupes protecteurs à partir de peptides et si désiré, de clivage du peptide à partir d'une résine porteuse, employant des résines d'acide fort en présence d'intercepteurs nucléophiles.A process has been developed for removing protective groups from peptides and, if desired, for cleaving the peptide from a carrier resin, employing strong acid resins in the presence of nucleophilic interceptors.

Description

DEPROTECTION OF PROTECTED PEPTIDES

FIELD OF THE INVENTION

1. This invention relates to improved procedures for deprotection of polypeptides employed in the synthesis of proteins and other biologically important polypeptides, including cleavage of polypeptides from a carrier resin used in solid phase synthesis.

BRIEF DESCRIPTION OF PRIOR ART

Chemical synthesis of peptides is carried out through condensation of the carboxy group of an amino acid ^'or peptide with the amino group of another amino acid or peptide forming a peptide bond. Peptides may be synthesized by condensing single amino acids with a growing peptide chain (stepwise synthesis), or by condensing preformed peptide segments (segment coupling synthesis). Since peptide bond formation involves specific carboxy and amino groups, all other functional groups on the molecule must carry protective groups. These protective groups must be such that they are easy to introduce, resist the

conditions for peptide bond formation, and are selectively removed from the completed peptide sequence. When the synthetic target contains amino acids with reactive side chain groups, e.g. amino, carboxy1, thio, hydroxyl, guanidino, etc. protection becomes a challenging problem. These groups must carry

protective groups which are stable during peptide bond formation and during the unmasking of amino or carboxyl groups which will serve as intermediates for further couplings. Removal of these and other protective groups is usually the ultimate step in a chemical synthesis of a polypeptide. Solution couplings have been the historical method for peptide synthesis. However, solution chemistry is not practical for stepwise synthesis of large peptides. Solid-phase synthesis (SPS), pioneered by R.B. Merrifield, offers a rapid and efficient method for synthesis of peptides up to about 40 amino acids. Its main advantages are (1) nearly quantitative coupling yields, and (2) rapid and simple purifications of peptide intermediates. In classical SPS, the peptide primary sequence is assembled on an insoluble support, usually a chloromethylated polystyrene resin, and the desired peptide is isolated after cleavage of the support and protective groups. The synthesis often starts with

attachment of the carboxy terminal amino acid carrying a t-butoxycarbonyl (Boc) amino protective group to the chloromethyl support through a benzyl ester bond. The resulting resin ester is treated with an acid to selectively remove Boc, neutralized, and coupled with the required second Boc amino acid to give a dipeptidyl resin intermediate. Suitable washings are used to purify the intermediate. Repetition of this acidolysis -neutralization-coupling cycle ultimately leads to the desired peptide-sequence. Because the synthesis and purification cycles are simple and rapid, they have been automated and peptide synthesizers are now commonly used (See for example, Barany, G. and Merrifield, R.B., The Peptides, Vol. 2, Academic Press Inc., New York, 1979, pp 1-284; Kemp-Vellaccio, Organic Chemistry, pp. 1030-1032 (1980)).

For synthesis of large peptides containing 50 to 150 amino acids, segment coupling is the method of choice. This method involves synthesis of suitably protected peptide segments each containing 10 to 15 amino acids. The segments are coupled, in solution, on a solid support, or through a combination of these techniques to give the target primary sequence in protected form. Cleavage of protective groups and, if necessary, separation from the resin furnishes the desired peptide.

An important contribution in this area is the solid-phase method of De Grado and Kaiser (W.F. DeGrado and Kaiser, E.T., J. Org. Chem 1982, 47, 3258). According to this method, the carboxy terminal amino acid of the desired segment, with its amino group protected with Boc, is attached to a p-nitro benzophenone oxime polysytrene resin through an ester bond involving the oxime moiety. The peptide segment is prepared through selective acidolysis-neutralization-coupling cycles, and is finally cleaved from the resin, in fully protected form, by a nucleophile such as N-hydroxypiperidine, or an amino acid. Segment couplings on the oxime resin and, when necessary, in solution furnish the

protected primary sequence of the desired peptide.

As stated above, chemical methods of peptide synthesis involve an ultimate deprotection step which may include removal of the peptide from an SPS resin. In most cases, deprotection is carried out by a strong acid. Hydrogen fluoride [ Lenard, J. et al, Journal of the American Chemical Society, 89:181-182 (1967)], sulfonic acids [Yajima, H. et al, Chem, Pharm, Bull. 22:1087-1094 (1974)], and other superacids operating under S_n1 cleaving conditions, and. hydrogen bromide [Merrifield, R.B., Biochemistry, Vol. 3, 1385-1390 (1964)] have been used. Many side reactions are associated with these acidolyses. Thus, nucleophilic side-chain groups such as those of tyrosine, methionine, cysteine and tryptophan can undergo alkylation by benzyl, t-butyl, and other carbocations generated during acidolysis (Martinez, J. et al, Synthesis, 1981, 333-335), and the side chain of aspartic or glutamic acid residues can be dehydrated becoming acylating agents [Feinberg, R.S. and Merrifield R.B., J. Am. Chem. Soc., 97;3485-3494 (1975)]. Examples of side-reactions involving specific amino acid residues include the following:

(1 ) Tryptophan undergoes alkylation during the acidolytic steps of SPS and in the final acidolysis aimed at producing the synthetic target (Chino, N. et al, Peptide Chemistry 1977,

Protein Research Foundation, pp. 27-32, Osaka, Japan). Thus, removal of Boc from peptides by trifluoroacetic acid (TFA) causes up to 30% t-butylation of tryptophan residues. Although

nucleophilic scavengers supress this reaction, it is best controlled by N -formylation of the tryptophan. However, Nⁱ- formyl tryptophan is stable to strong acids, including HF and sulfonic acids. Deprotection of the tryptophan is usually accomplished after acidolytic deprotection of other groups on the peptide by treatment with aqueous base or other nucleophiles, thus requiring an additional step with the potential for further side reactions.

(2) Alkylation and oxidation of methionine sulfur is frequently encountered during SPS [Hoffman, K. Journal of the American Chemical Society 87:631 (1965)]. The problem can be simplified by using methionine sulfoxide instead of methionine, and reducing the residue at the end of the synthesis. This additional step, however, is occasionally slow and accompanied by side reactions [Houghton, R.A. Analytical Biochemistry 98:36 (1979)].

(3) In the case of tyrosine, designing a side-chain protective group has been a challenge. The benzyl group was found to be unsuitable because during superacid deprotection significant amounts of 3-benzyl tyrosine formed. This side reaction could be controlled to varying degrees by blocking the phenolic OH with 2,6-dichlorobenzyl, 2-bromobenzoxy carbonyl, or cyclohexyl groups.

Over the past 20 years there have been many attempts to overcome problems associated with peptide deprotection. An important contribution was the discovery that nucleophiles accelerate acidolysis and act as scavengers to limit side reactions. Brady [Journal of Organic Chemistry 42:143 (1977)] observed that dimethyl sulfide (DMS) in trifluoroacetic acid (TFA) enhanced the rate of cleavage of the benzoxycarbonyl (Z) group. Kiso et al [Chem. Pharm. Bull. 28:673 (1980)] and Yajima et al [Chem. Pharm. Bull. 28:1214-1218 (1980)] found that Z and other benzyl groups are cleaved exceptionally fast by thioanisole in TFA. Finally, Tarn and co-workers (U.S. Patent 4,507,230) have described improved HF/DMS cleavages of peptides which operate by an S 2 mechanism, and Yajima et al (J. Chem. Soc. Chem. Common. 1987, pp 274-275) have reported deprotections by trimethylsilyl triflate/thioanisole in TFA, which also appear to operate by an S_n2 mechanism. Modifications of the Tam and Yajima methods using ethane dithiol as an additive also deprotect Nⁱ-formyl tryptophan residues.

Despite the recent improvements, super acid-catalyzed deprotections of peptides remain highly problematic. The acids required are extremely corrosive and toxic, and are difficult to handle, especially in large scale operations. HF, in particular, attacks ordinary laboratory and industrial containers and

reaction vessels, and therefore requires special handling. It is listed as a transportation hazzard.

BRIEF DESCRIPTION OF THE INVENTION

It has now been discovered that protected peptides, which are synthesized by solution or solid-phase methods, are

deprotected rapidly, efficiently and under mild conditions by strong acidic resins in an acidic solvent containing nucleophilic scavengers. Cleavage takes place at ice bath or ambient

temperatures, i.e. 0º to 45ºC. Reaction time is not critical and depends upon several factors apparent to those skilled in the art, such as temperature, number of protective groups to be removed, selected resin, composition of reaction mixture, etc.

Special advantages of the deprotection method include exceptionally easy isolation of product and acidic resin, which allows the artisan to regenerate and re-use the resin for further deprotections. At the end of the reaction, the deprotected peptide remains bound on the acidic resin and is isolated by treatment of the resin with a mild base followed by a filtration. The neutralized resin also isolated through this filtration, can be regenerated by treatment with a strong acid. This is in sharp contrast to other deprotection procedures, such as those

mentioned above, which involve separation of deprotected peptides from complex product mixtures and make re-use of the acid used for the deprotection difficult.

DETAILED DESCRIPTION OF THE INVENTION

This invention will be best understood by consideration of the Figure, which shows a simplified version of what is presently believed to be the mechanism of the reaction. In the Figure, the benzyl protective group of a serine-containing peptide (1) is cleaved by a sulfonic acid resin ( 2 ) in TFA containing DMS (3 ) .

The first equation shows an equilibrium between reactants 1, 2 and 3 and their resin-bound form 4. The degree to which the equilibrium is shifted to the bound state depends on the acidity of the resin. In the case of Nafion resins (see below), which have an estimated pKa ≥ -12 (Olah, G. Synthesis, 1986, 513), and other strong acid resins the shift to 4 is significant. Salt bridges and/or hydrogen bonds bind 1 and 3 to the resin. One form of 4, a structure such as 5, in which the protected serine oxygen forms a strong hydrogen-bond or salt bridge with a resin SO₃H group, undergoes cleavage through an S_n2-type displacement by DMS producing resin-bound product 6 . The by-product 7 can also form a salt with the resin. Through a similar mechanism, other protected residues of peptide 1 may also be cleaved. The desired product 8 is isolated by filtration of the resin-bound peptide, treatment of the resin with dilute aqueous ammonia to liberate the deprotected peptide, and purification of the crude product in the ammonia solution by standard techniques. The resin, isolated as the ammonium salt 9, can be regenerated by treatment with acid and re-used.

It will be apparent to those skilled in the art that this procedure can be readily automated in the same manner as in SPS.

A unique feature and special advantage of the deprotection mechanism shown in the Figure is the reversible immobilization of protected peptide 1, of peptide intermediates, of product 8 and of by-product 7 on the insoluble resin 2. This immobilization limits exposure of peptide to strong acid and reactive alkylating agents (such as 7 ) , both of which can be destructive. By contrast, during homogeneous deprotections (by HF or sulfonic acids) peptide forms are exposed to strong acid and alkylating by-products continuously, throughout the course of the reaction. Therefore, peptide deprotections by the method of this invention utilizing a strong acid resin are superior to homogeneous superacid-promoted cleavages

The reaction of this invention may take place in an acid solvent such as a low molecular weight carboxylic acids or its fluorinated or chlorinated derivatives. Choice of reaction solvent will be influenced by the solubility properties of the protected peptide. Trifluoroacetic acid is especially preferred because it dissolves a wide range of peptides. Its considerable acidity also accelerates deprotection rates. It is especially useful when the peptide is to be cleaved from an SPS resin as well as deprotected. However, other acids with pKa values of from about 4 to -4 can also be employed.

Neutral solvents may be employed in association with the acid solvents to form mixed solvents. Suitable neutral solvents include low molecular weight halogenated alcohols and

hydrocarbons containing up to about four carbon atoms such as trifluoroethanol, hexafluoroisopropanol, ethylene dichloride, chloroform, dichloroethane, etc. Trifluoroethanol is a preferred neutral solvent because it dissolves many peptides and is relatively non-toxic. Deprotection rates in either acid or mixed solvents are accelerated by addition of acids of pKa from about -8 to -13. These additives are especially useful when the peptide, in addition to being deprotected, is also to be cleaved from an SPS carrier resin. Example of such acids include sulfonic acids (methane sulfonic, trifluoromethane sulfonic, etc.), hydrogen halides, and Lewis acids such as aluminum trichloride, trimethyl silyl halides, trimethylsilyl triflate etc. The preferred amounts of strong acid are from 1 to 10 equivalents per

equivalent of protected amino acid residue to be cleaved. At the end of the deprotection reaction the strong acid additive should be neutralized with a mild base such as pyridine to avoid complications in product isolation.

Cleavage rates may also be accelerated by addition of suitable salts of strong acids. These are protonated by the acid resin of the reaction mixture and act as additional cleavaging agents. Preferred salts are alkali and alkaline earth metal salts which are derived from acids with pK values of from about -8 to -13, and are soluble in the reaction mixture. Examples include lithium trifluoromethane sulfonate, lithium fluoride, sodium bromide, and lithium or potasium iodide. The amount of salt employed is from about 0.1 to 5 equivalents of salt for every equivalent of acid group on the resin.

The deprotection reaction of this invention, with or without cleavage from a carrier resin, takes place in the presence of nucleσphiles. Nucleophiles are molecules which are particularly reactive towards positively-polarized molecular centers. Typical nucleophiles include molecules which contain heteroatoms with unshared pairs of electrons such as sulfur and oxygen. For purposes of describing and claiming this invention they will be referred to as nucleophilic scavengers. A particularly useful nucleophilic scavenger is the dimethyl sulfide shown in the

Figure.

Phenols and aromatic ethers are typical oxygen containing nucleophilic scavengers, sometimes referred to as carbonium ion scavengers, which are particularly useful in limiting alkylation side-reactions during deprotection. These scavengers are

especially useful when the peptide contains tyrosine or other aromatic amino acids subject to alkylation. The scavengers are particularly effective in preventing e.g. benzylation by benzyl carbocations generated during acidolysis of benzyl-protected peptides.

The aromatic group of the phenol or aromatic ether is usually phenyl and may be further substituted with other groups such as hydroxyl, mercapto, halogen or alkyl groups containing from 1 to 10 carbon atoms. Typical examples of preferred

scavengers of this type are the cresols, phenol and anisole.

Other equivalent nucleophiles will be readily apparent to those skilled in the art. A wide variety of nucleophilic scavengers which function as such because of the presence of unshared electrons on a sulfur atom are available for use in this invention. Typically, they are alkyl and aromatic thiols and thioethers, including S-trialkylsilyl derivatives thereof in which the alkyl groups of the trialkysilyl moiety may be the same or different and contain up to four carbon atoms. The alkyl group of an alkyl thiol or alkyl thioether will preferably contain up to three carbon atoms, but may contain up to ten carbon atoms. The aromatic group of an aromatic thiol or thioether is normally a phenyl group. Both the alkyl and the aromatic moiety may be further substituted with hydroxyl, mercapto, halogen or alkyl groups containing from 1 to 10 carbon atoms. Nucleophilic scavengers of the sulfur class which may be mentioned by way of example include thiophenol, the thiocresols, hydroxythiophenols, ethane dithiol, thioanisole, dimethyl sulfides, methylthiotrimethyl silane and

phenylthiotrimethyl silane. Dimethyl sulfide, thioanisole and phenylthiotrimethyl silane are especially preferred because they are readily available, strongly nucleophilic and volatile.

It will be noted that some nucleophilic scavengers such as hydroxythiophenols are of both the sulfur and oxygen class.

Scavengers from both classes can be used alone, but, as will be seen from the examples, it is common to utilize members from both classes in the same reaction mixture.

If the peptide contains Nⁱ-formyl residues, it is useful to include a dithiol such as an alkyl dithiol containing up to 10 carbon atoms, which may be in a straight or branched chain, in the reaction mixture. Ethane dithiol or its S-trimethylsilyated derivatives silyl group are especially preferred. They assist in deformylation by rapid production of thio orthoformates [Tarn, J.P. et al J. Am. Chem. Soc. 108, 5244 (1986)].

These same reagents also assist in protecting the sulfur of cysteine and methionine by preventing its oxidation or alkylation by carbocations;

Resins capable of deprotecting peptides according to the method of this invention include polystyrene polymers containing sulfonic and phosphonic acid groups (some are

commercially available under the trade name Dowex) and various fluorinated sulfonic acid resins of a wide range of structures such as those sold under the trade name Nafion. In general, acidic polymer resin formulations with pK values of from about -4 to -13 may be used for protective group removal. It will be apparent to the skilled artisan that the acidity of the resin used will determine the type and rate of protective group removal to a significant extent. Lewis acidic derivatives of the resins described above, such as their trialkylsilyl sulfonate

derivatives, are also useful for carrying out deprotections.

Normally a large molar excess of resin will be employed to insure as complete a reaction as possible. The Nafion resins are perfluorinated polymers having sulfonic acid groups in the amount of about 0.01 to 5

milliequiv./gram. Typically, they are prepared by polymerizing sulfonated perfluorinated vinyl compounds, or by copolymerizing sulfonated perfluorinated vinyl ethers with perfluoroethylene and/or perfluoroolefins. They are described, for example in United States Patents 4,041,847; 4,052,474 and 4,052,475.

For claim purposes, these perfluorinated polymers, their trialkylsilyl sulfonate derivatives, and their equivalents will be referred to as sulfonated perfluorinated resins. Preferred sulfonated perfluorinated resins for carrying out the procedure of this invention are pertrimethylsilylated Nafion (Murata, S. and Noyori, R. Tetrahedron Letters, 21, 1980, 767-768) and Nafion-H polymers.

As shown in the Figure, the deprotected peptide is released from the selected acid resin employed by treatment with dilute aqueous ammonia. This is the currently preferred reagent since it is readily available, volatile and easy to use. However, other alkaline reagents such as carbonates, phosphates, alkyl amines, pyridine, and other aromatic amines may also be used. As indicated above, this invention is applicable to the removal of a wide variety of protective groups. They include nitro, p-toluenesulfonyl, benzyl, 2,6-dichlorobenzyl, 2- chlorobenzyl, 2-bromobenzyl, carbobenzoxy, substituted

carbobenzoxy, methyl benzyl, methoxy benzyl, benzhydryl, formyl and t-butoxycarbonyl. In general all protective groups commonly used in peptide synthesis, including SPS, which can be cleaved by acids, are removed by the procedure of this invention. As would be expected some protecting groups are removed easier than others. In general, the more stable protecting groups will require the use of more strongly acidic resins.

The peptides which serve as substrates for the deprotection procedure of this invention can be obtained by any of a variety of procedures, as indicated above. When the peptide is

synthesized by SPS, deprotection and cleavage may be accomplished as follows. The peptide may first be detached from the support and then completely deprotected using the method of the

invention. For example, if the peptide is attached to the support through an ester bond, separation from the resin support may be accomplished by hydrogenolysis (Anwer, M.K. and Spatola, A. J. Org. Chem. 1983, 48, 3503-3507). Certain peptidyl

benzhydryl amine supports may also be cleaved by

hydrogt.n/palladium catalyst systems (Colombo, R. J. Chem. Soc, Chem. Cυmmum, 1981, 1012-1013). Alternatively, a peptidyl resin, especially one in which the peptide is anchored through an ester bond, may be cleaved and the resulting peptide completely

deprotected by using the method of this invention. In this case, the reaction is conducted in an acidic solvent such as TFA or, preferably, TFA containing a stronger acid additive as described above. A preferred method of synthesis of peptides for deprotection by the methods of the invention is segment synthesis-coupling, using the oxime resin SPS as discussed by Kaiser (J. Am. Chem. Soc. 1985, 107, 7087-7092). As stated above the usual reactants involved in the

deprotection of peptides according to the procedure of the invention are the selected sulfur-containing nucleophilic scavenger, scavengers limiting specific amino acid side- reactions, and strong acid resin. These reactants are normally employed in a molar excess compared to the amino acid protective group or groups to be removed to insure as complete a reaction as possible. Typically it will be a large excess. As shown in the Examples, it may be as high as 500 to 1000%. Neither reaction temperature nor time is critical. The reaction is conveniently carried out at a temperature between 0º-45ºC. during a period between 1-20 hours.

It has been observed that the benzyl group used to protect serine can be removed by the acid resin alone, without the need of nucleophilic scavengers. It is therefore apparent that when the peptide to be deprotected does not contain amino acids prone to side reactions, nucleophilic scavengers will not be necessary for efficient deprotection to occur. In fact, even with

sensitive amino acids, nucleophilic scavengers may be

unnecessary. This is because, as stated above, throughout the course of the deprotection, peptide molecules (i.e. protected peptide, intermediates, and peptide product) and the carbocations generated are bound on the acid resin, which minimizes side reactions. The following examples illustrate the process of the invention. Examples 1 through 11 describe the deprotection of individual amino acids, and illustrate how the process would be applied to peptides. Example 12 illustrates the process applied to the deprotection of a nonapeptide with three different protective groups. Example 13 outlines the deprotection and concurrent cleavage of a heptadecamer attached to an SPS support through an ester bond and containing nine protective groups, and

Example 15 describes the deprotection of an octamer with seven protective groups.

Example 1

Conversion of t-Butoxycarbonyl Aspartic Acid

β-Benzyl Ester (1) to Aspartic Acid

To a solution of dimethyl sulfide (25.5^1, 0.35 mmole) and m-cresol (21.6 μl, 0.20 mmole) in trifluoroacetic acid (1.5 ml) was added 1 (17.0 mg, 0.05 mmole) and Nafion-H (0.06 g, 0.55 mmole). The resulting mixture was stirred vigorously under nitrogen for 3 hours, and filtered to give the resin-bound product. The resin was washed with methylene chloride (6x5 ml) and with ether (2×5 ml), stirred with 1.0 M ammonium hydroxide (4 ml) for 1 hour at 0 C, filtered off, and washed with 1M ammonium hydroxide (5x2 ml). The pooled aqueous filtrate and washings were subjected to quantitative amino acid analysis and found to contain 0.05 mmole of aspartic acid. Example 2

Conversion of t-Butoxycarbonyl -O- Benzyl Serine (2) to Serine

Serine 2 (15 mg, 0.05 mmole) was deprotected by a mixture of Nafion-H (0.69 g, 0.63 mmole), dimethyl sulfide (25.5 μl, 0.35 mmole), m-cresol (21.6 μl, 0.2 mmole), and trifluoroacetic acid

(1.5 ml), according to the procedure of Example 1. Production of serine was quantitative, as shown by quantitative amino-acid analysis. Example 3

Conversion of N -Butoxycarbonyl-O-2,6- dichlorobenzyl Tyrosine (3) to Tyrosine

Tyrosine 3 (22 mg, 0.05 mmole) was deprotected by a mixture of Nafion-H (0.61g, 0.55 mmole), dimethylsulfide

(25.5 μl, 0.35 mmole), m-cresol (21.6 μl , 0.20 mmole), and trifluoracetic acid (1.5 ml), according to the procedure of Example 1. Conversion to tyrosine was complete, as shown by quantitative amino acid analysis. Example 4

Conversion of N^α-t-Butoxycarbonyl-N^α-2- chlorobenzoxycarbonyl Lysine (4) to Lysine

Lysine 4 (21 mg, 0.05 mmole) was cleaved by a mixture of Nafion-H (0.63 g. 0.57 mmole), dimethyl sulfide (25.5 μl , 0.35 mmole), m-cresol (21.6 μl , 0.20 mmole), and trifluoracetic acid (1.5 ml), according to the procedure of Example 1. Conversion to lysine was complete, as shown by quantitative amino acid analysis.

Example 5 Conversion ofN^α -t-Butoxycarbonyl-N^im-t-Benzoxymethyl Histidine (5) to Histidine

Histidine 5 (19.0 m, 0.05 mmole) was cleaved by a mixture of Nafion-H (0.64 g, 0.58 mmole), dimethyl sulfide (25.5 μl, 0.35 mmole), m-cresol (21.6 μl , 0.20 mmole), and trifluoracetic acid {1.5 ml), according to the procedure of Example 1. Conversion to histidine was complete, as shown by quantitative amino acid analysis..

Example 6 Conversion of N^α-t-Butoxycarbonyl-Nⁱⁿ- Formyl Tryptophan (6) to L-Tryptophan

Tryptophan derivative 6 (17 mg, 0.05 mmole) was cleaved by a mixture of Nafion-H (797 mg, 0.72 mmole), dimethylsulfide (25.5 μl , 0.35 mmole), m-cresol (21.6/ιl, 0.20 mmole), ethanedithiol (30 ul, 0.29 mmole), and trifluoroacetic acid (1.5 ml) according to the procedure of Example 1. Conversion to tryptophan was complete, as shown by quantitative amino acid analysis.

Example 7

Conversion of N^α-t-Butoxycarbonyl-N^g-mesitylene

sulfonyl-L-Argine (7) to Arginine

Arginine derivative 7 (27.8 mg, 0.05 mmole) is cleaved by a mixture of Nafion-H (0.60 g. 0.55 mmole), dimethylsulfide (25.5 μl , 0.35 mmole), m-cresol (21.6μ1, 0.20 mmole), and

trifluoroacetic acid (1.5 ml) according to the procedure of Example 1.

Example 8

Conversion of N^α-t-Butoxycarbonyl-N^g-Tosyl- L-Arginine (8) to Arginine

A mixture of the arginine derivative J3 (11 mg, 0.025 mmole) pertrimethylsilyl Nafion (Nafion-TMS; See Murata, S. Tetrahedron Letters, 1980, 21, 767 for preparation), (400 mg, 0.32 mmole), phenylthiotrimethylsilane (20,111, 0.1 mmole), m-cresol (11μ1, 0.1 mmole), and trifluoroacetic acid (1 ml) was stirred under nitrogen for 3 hours. The reaction mixture was filtered to give the resin-bound product. The resin was washed with methylene chloride (6×5 ml) and with ether (2×5 ml), stirred with a mixture of 1.0 M ammonium hydroxide (4ml) and 1 M ammonium fluoride

(1 ml) for 1 hour at 0ºC, filtered off, and washed with 1.0M ammonium hydroxide (10×2 ml). The pooled aqueous filtrate and washings were concentrated to a volume of about 1 ml and washed with ether (4×1 ml). Amino acid analysis of the aqueous solution showed that cleavage of 8 to arginine was quantitative.

Example 9

Conversion of N^g-nitro

Arginine (9) to Arginine

Nitroarginine 9 (5.5 mg, 0.025 mmole) was cleaved by a mixture of Nafion-TMS (400 mg, 0.32 mmole), phenylthiotrimethylsilane (20 /αl, 0.1 mmole), m-cresol (11 μl , 0.1 mmole), and trifluoracetic acid (1ml), according to the procedure of Example 8. Conversion to arginine was quantitative, as shown by

quantitative amino acid analysis. Example 1 0

Conversion of N^α-t-Butoxycarbonyl-S-p- Methylbenzyl Cysteine (10) to Cysteine

Cysteine 10) (8 mg, 0.025 mmole) was cleaved by a mixture of Nafion-TMS (400 mg, 0.32 mmole), phenylthiotrimethylsilane (20 μl, 0.1 mmole), m-cresol (11 /μl, 0.1 mmole), and trifluoroacetic acid (1 ml), according to the procedure of Example 8. Conversion to cysteine was quantitative, as shown by quantitative amino-acid analysis. Example 11

Conversion of N^α-t-Butoxycarbonyl-O-Benzyl-Serine (2) in

Trifluoroethanol to Serine

Serine 2 (30 mg, 0.10 mmole) is deprotected by a mixture of Nafion-H (1.39 g, 1.26 mmole), dimethyl sulfide (51 μl , 0.70 mmole), m-cresol (43.2/11, 0.4 mmole), trifluoromethane sulfonic acid (42 mg, 0.30 mmole), and trifluoroethanol (2 ml) using a modified version of the procedure of Example 1. The modification involves the addition of 0.3 mmole of pyridine to the reaction mixture when the reaction is judged complete.

Example 1 2

Deprotection of Boc-Val-Glu(Bzl)-Ile-Tyr (Cl₂Bzl)-

Pro-Val-Ala-Ala-Leu-OH (11) Furnishing

H-Val-Glu-Ile-Tyr-Pro-Val-Ala-Ala-Leu-OH (12) To the protected nonapeptide 11 , (14.4 mg) prepared on an oxime resin using the method of DeGrado and Kaiser, in

trifluoroacetic acid (2 ml) containing dimethylsulfide (26 μl , 0.35 mmole) and m-cresol (22 μl, 0.2 mmole) was added Nafion-H (0.68 g, 0.62 mmole) and the resulting mixture was stirred under nitrogen for 4.5 hours. The reaction mixture was filtered to give the resin-bound product. The resin was washed with

methylene chloride (5×5 ml) and with ether (3×5 ml), stirred with 1.0M ammonium hydroxide (4 ml) for 1 hour at 0 C, filtered off, and washed with 1.0 M ammonium hydroxide (6×2 ml). The pooled aqueous filtrate and washings were washed with ether (3×15 ml) and lyophilized to give a white solid. The crude product was suspended in ether, filtered, and washed with ether (5×5 ml) and with ethyl acetate (3×2 ml). It was further purified by

preparative reverse-phase high performance liquid chromatography to give deprotected peptide 12, 10.1 mg, which gave satisfactory amino acid analysis and was chromatographically indistinguishable from an authentic sample of 12 prepared by catalytic

hydrogenolysis of 11 using palladium on carbon and ammonium formate in methanol (Anwer, M.K. and Spatola, A.F. Synthesis, 1950, 929).

Example 13

Deprotection of H-Tyr (Br-Z)-Gly-Gly-Phe-Leu-Lys(Cl-Z)- Lys (Cl-Z)Val-Lys-(Cl-Z)-Pro-Lys(Cl-Z)-Val-Lys(Cl-Z)-Val-Lys(Cl-Z)Ser(Bzl-Ser(Bzl)-O-CH₂-Resin (13) Producing H-Tyr-Gly-Gly-Phe-Leu-Lys-Lys-Val-Lys-Pro-Lys-Val-Lys-Val-Lys-Ser-Ser-OH (14).

This example illustrates cleavage of a peptide from an SPS resin and deprotection taking place in the same reaction mixture.

Heptadecamer 13 is synthesized on the chloromethyl support of Merrifield, using the solid-phase method. Initial loading of the resin is 0.33 mmole of Ser (Bzl)/g of resin. For cleavage, a mixture of 13 (23 mg) Nafion-TMS (1.0 g, 0.8 mmole),

trifluoromethanesulfonic acid (750 mg, 0.5 mmole) ,

phenylthiotrimethylsilane (740 μl), m-cresol (200μl), and TFA (5 ml) is shaken for 3 hours. The reaction mixture is neutralized with pyridine (395 mg, 0.5 mmole); the Nafion resin is carefully filtered off, washed with methylene chloride (5x1 ml) and with ether (3×1 ml), and shaken with a mixture of 1M ammonium

hydroxide (5 ml) and 1 M ammonium fluoride (3 ml) at 0°C for 1 hour. The, resin is filtered off and washed with 1.0M ammonium hydroxide (10×1 ml). The pooled filtrate and washings are concentrated to a volume of about 3 ml and the peptide product contained in this solution isolated by preparative reverse-phase high performance liquid chromatography. It is identified as heptadecamer 21 on tne basis of its amino acid analysis and mass spectrometry.

Example 1 4

Conversion of O-Benzyl Serine (15) to

Serine Without Nucleophilic Scavengers

Serine 1 5 (10 mg., 0.05 mmole) is deprotected by a mixture of Nafion (550 mg, 0.5 mmol) and TFA (1 ml) following the procedure of Example 1. Production of serine was quantitative, as shown by quantitative amino acid analysis.

Example 1 5

Deprotection of Boc-Asp(Bzl)-Arg(NO₂)-Val-Arq(NO₂)- Lys(Cl-Z)-Lys(Cl-Z)-Ser(Bzl)-Gly-OH (16) Furnishing H-Asp-Arg- Val-Arq-Lys-Lys-Ser-Gly-OH (17) A mixture of octamer 1 6 (15 mg, 0.014 mmole), Nafion-TMS (1.3g, 1.04 mmole), phenylthiotrimethyl silane (70 μl, 0.38 mmole), m-cresol (40 μl, 0.37 mmole), and TFA (2.5ml) is stirred under nitrogen for 4.5 hours. The reaction mixture is filtered to give the resin-bound product. The resin is washed with CH₂Cl₂ (10×5ml) and with ether (5×5ml), dried by suction, and further washed with a mixture of 1.0M ammonium hydroxide (15 ml) and 1.0M NH.F (15ml) and with 1.0M ammonium hydroxide (20 ml). The pooled aqueous washings are lyophilized to give the crude deprotected peptide, which is purified by washing with ether and with ethyl acetate and by semi-preparative reverse phase HPLC. The product is characterized as 17 through its amino acid analysis and mass spectrum.

Claims

WHAT IS CLAIMED IS:

1. A method of removing protecting groups from a peptide which comprises reacting the peptide with a strong acid resin having a pKa value of from about -4 to -13 or with a Lewis acid derivative of such a resin in an acid solvent having a pKa value of from about 4 to -4.

2. A method of removing protecting groups from a peptide which comprises reacting the peptide with a strong acid resin having a pKa value of from about -4 to -13 or with a Lewis acid derivative of such a resin in an acid solvent having a pKa value of from about 4 to -4 in the presence of a nucleophilic

scavenger.

3. A method as in claim 2 wherein the resin is a sulfonated perfluorinated resin.

4. A method as in claim 2 wherein the resin is a sulfonated perfluorinated resin and the acid is trifluoroacetic acid.

5. A method as in claim 2 additionally employing a neutral solvent which is a low molecular weight halogenated alcohol or hydrocarbon containing up to about four carbon atoms.

6. A method as in claim 2, 3, 4 or 5 wherein the

nucleophilic scavenger is an alkyl or aromatic thiol, thioether, or a trialkylsilyl derivative of said thiol, a phenol or an aromatic ether.

7. A method as in claim 2, 3, 4 or 5 wherein the nucleophilic scavenger is selected from the group consisting of dimethyl sulfide, thioansole, phenylthiotrimethyl silane, phenol, anisole and cresols.

8. A method as in claim 2 conducted in the presence of an acid additive having a pKa value of from about -8 to -13 whereby the peptide is simultaneously cleaved from a carrier resin.

9. A method as in claim 8 wherein the resin is a sulfonated perfluorinated resin.

10. A method as in claim 8 wherein the resin is a sulfonated perfluorinated resin, the acid is trifluoroacetic acid and the acid additive is trifluoromethanesulfonic acid.

11. A method as in claim 8, 9 or 10 wherein the nucleophilic scavenger is an alkyl or aromatic thiol, theoether, a

trialkylsilyl derivative of said thiol, a phenol or an aromatic ether.

12. A method as in claim 8, 9 or 10 wherein the nucleophilic scavenger is selected from the group consisting of dimethyl sulfide, thioansole, phenylthiotrimethyl silane, phenol, anisole and cresols.

13. A method as in claim 2 conducted in the presence of a salt of an acid, said acid having a pKa value of from about -8 to -13 whereby the peptide is simultaneously cleaved from a carrier resin.

14. A method as in claim 13 wherein the resin is a

sulfonated perfluorinated resin.

15. A method as in claim 13 wherein the resin is a sulfonated perfluorinated resin, the acid is trifluoroacetic acid and the acid additive is trifluoromethanesulfonic acid.

16. A method as in claim 13, 14 or 15 wherein the

nucleophilic scavenger is an alkyl or aromatic thiol, thioether, or a trialkylsilyl derivative thereof or a thioether of said thiol, a phenol or an aromatic ether.

17. A method as in claim 13, 14 or 15 wherein the

nucleophilic scavenger is selected from the group consisting of dimethyl sulfide, thioansole, phenylthiotrimethyl silane, phenol, anisole and cresols.