CA1271300A - Solid phase process for synthesizing peptides - Google Patents
Solid phase process for synthesizing peptidesInfo
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- CA1271300A CA1271300A CA000506495A CA506495A CA1271300A CA 1271300 A CA1271300 A CA 1271300A CA 000506495 A CA000506495 A CA 000506495A CA 506495 A CA506495 A CA 506495A CA 1271300 A CA1271300 A CA 1271300A
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Abstract
SOLID PHASE PROCESS FOR
SYNTHESIZING PEPTIDES
ABSTRACT OF THE DISCLOSURE
Thymosin .alpha.1 and other peptide amides are synthesized in the solid phase using methylbenzhydrylamine resin as the support and hydrogen bromide as the deprotecting and cleaving agents. The N-terminal 14 amino acid partial sequence of thymosin .alpha.1, Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-OH is synthesized on benzyl ester resin.
Using a mixture of hydrogen bromide, anisole and thioanisole as the cleavage and deprotection composition, the yields of the cleaved peptide and the selectivity of the deprotection is highly improved. For example, yields of thymosin .alpha.1 have been increased by about 90% as compared to the use of hydrogen bromide alone. By using hydrogen bromide rather than hydrogen fluoride, the synthesis can use conventional laboratory glassware and the synthesis can be easily scaled up to commercial production.
SYNTHESIZING PEPTIDES
ABSTRACT OF THE DISCLOSURE
Thymosin .alpha.1 and other peptide amides are synthesized in the solid phase using methylbenzhydrylamine resin as the support and hydrogen bromide as the deprotecting and cleaving agents. The N-terminal 14 amino acid partial sequence of thymosin .alpha.1, Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-OH is synthesized on benzyl ester resin.
Using a mixture of hydrogen bromide, anisole and thioanisole as the cleavage and deprotection composition, the yields of the cleaved peptide and the selectivity of the deprotection is highly improved. For example, yields of thymosin .alpha.1 have been increased by about 90% as compared to the use of hydrogen bromide alone. By using hydrogen bromide rather than hydrogen fluoride, the synthesis can use conventional laboratory glassware and the synthesis can be easily scaled up to commercial production.
Description
~ 7~
I SOLID PHASE PROCESS FOR
¦ SYNTHESIZING PEPTIDES
¦ BACKGROUND OF THE INVENTIO~
, _ . .. ..
I Field of Invention I _ . ___ _ ¦ This invention relates to an improved process for synthesis of peptides by the ~olid phase method. More particu-larly, the invention relates to the synthesis of peptide amides by solid phase peptide synthesis using a novel resin l support, me~hylbenzhydrylamine resin, and hydrogen bromide ¦ as the cleava&e and deprotecting agent for separating the resin-bound peptide from the resin support. In a particular aspect, the inventicn relates to the solid phase synthesis of thymosin al The invention also relates to an improved l process for synthesizing peptides, and particularly, thymosin l ~1 and the thymosin l-Nl_l4 fragment, by solid phase synthesis ¦ using an improved cleavage/deprotecting composi~ion, namely hydrogen bromide with a mixture of anisole and thioanisole.
Description of the Prior Art I
l The function of peptides in human health has received ¦ much recognition in recent years, see, e.g. "Peptides: A
Medical Rediscovery" by Joseph Alper, High Technology, Vol.
3, No. 9, September 1983, pages 60-63. Peptides have been ¦ shown to function, for example, as hormones for regulating ¦ growth, reproduction, and immunology, and as neurotransmitters.
¦ While ac~ual and positive biological results have been observed for several dozens of different peptides, still much work remains to be done to determine how the petides work in 1~71;~0 ¦ the body. For this purpose, lar~e quantities of the peptides are required. Also, in order for the peptides of known biological activity to be of practical significance in health care, they must be made readily available in large quantities and at relatively low C06t.
In addition to the "natural" peptides, i.e. those produced ln the body, "synthetic" peptides or analogs, which may be considered as derivatives of natural peptides, but having one or more chemical modifications in the molecular structure of the natural peptide, are also valuable for their modified chemical properties, such 85 resistance to enzyme degradation. Here again, the availability of relatively inexpensive means to rapidly and inexpensively produce synthetic peptides would be highly valuable.
Presently, various procedures for synthesizing peptides, i.e. for formation of a peptide linkage between amino acids, are known. Conventional procedures include both solution-phase (or liquid phase) methods and solid-phase methods. A general discussion of peptide synthesis can be found, for example, in Schroder E. Lubke: "The Peptides," Vol. l (1966), Academic Press, New York, U.S.A. and Neurath and Hill, "The Proteins,"
Vol. 2 (1976~, Academic Press, New York, U.S.A. Generally, these procedures involve the reaction between the free amino group of an amino acid or residue thereof having its carboxyl group, hydroxyl group or other reactive group(s) protected, and the ree primary carboxylic group of another amino acid or residue thereof having its amino group or other reactive group(s) protected to form a peptide bond. Each amino acid in the desired sequence can be added singly successively to another amino acid or residue thereof or separate peptide fragments with the desired amino acid sequence can be synthesize '~2 7~'3~3~
and then condens~ed to provide ~he desired peptide.
In the past ~en to twenty years the synthesis of peptides has been simpliXied by utilizing the solid phase synthesis method according to the general principles developed by Merrifield, [R.B. Merrifield, J. Am. Chem. Soc. 85, 2149-2154 (1963~; Stewart, et al, Solid Phase Peptide Synthesis, Freeman & Co., San Francisco, CA (1969); Barany, et 81, The Peptides, Analysis, Synthe&is and Biology, Vol. 2, pages 1-284, (1980)~.
Briefly in solid-phase peptide synthesis (SPPS), the carboxyl group of the first amino acid in a peptide is chemically bound to the surface o tiny, in~oluble beads, with the other reactive sites on the amino acid being temporarily blocked. The amino acid-bound resin beads are placed in a reaction vessel and unbound acid is washed away. After chemically unblocking the reactive amino group, the next amino acid in the desired sequence of the object peptide is added with a chemical coupling a8ent such that the two acids bind together, via a peptide bond. The synthesis is continued by repeating the foregoing process with successive amino acids in the sequence being added one at a time until the total peptide sequence is built up on the resin. Upon completion of the desired peptide sequence, the protected peptide is cleaved from the resin support, and all protecting groups are removed. The cleavage reaction and removsl of protecting groups may be accomplished simultaneously or sequentially.
SPPS has recently been adapted to automated and computerized instrumentation offering substantial improvements in speed, eficiency, and reliability. However, the use of such instrumentation has been substantially limited to production of peptides on a relatively small scale, on the ! ~ 7 ~ 3(~
order of 8 few mllligrams per production cycle. Also, the procedure inherently results in the production of unwanted by-products, unreacted acids, solvents, coupling or decoupling agents~ cleavage products and so on, making the subsequent 5 purification procedures troublesome.
Although the art iB aware of various different materials used as the insoluble beads, including glass, silica, synthetic resins, etc., the synthetic resins are most commonly used as the support material (insoluble beads). Conventional materials used as the resin include the styrene-divinyl benzene resin modified with a reactive group, such 8S chloro-methylated styrene-divinyl benzene resin and benzhydrylamine resin (BHA). BHA is particularly useul for synthesis of peptide amides, i.e. peptides of which the C-terminal amino acid has a carboxylamide (-CONH2) group, since the amide group can be formed directly. The benzhydrylaminopolystyrene-divinyl benzene resin support is described by P. Rivaille, et al, Helv. Chim. Acta., 54, 2772 (1971).
Hydrogen fluoride, HF, is commonly used as the cleavage agent, and also as the deprotecting agent for removing the various blocking group~.
Unfortunately, HF, whether in liquid, anhydrous, or gaseous form, is highly corrosive and toxic requiring special handling and special plastic materials. As a result, the scale-up of the SPPS procedure from the laboratory scale to com~,ercial scale production (e.g. on the order of one or more grams per production run) has proven extremely difficult It has been known to use the less corrosive and less toxic hydrogen bromide (HBr) in place of HF as the cleavage agent, alone, or together with anisole as a catalyst for cleaving the bound protec~ed peptide from the supporting ~ 3~3~
resin. However, HBr alone or with anisole does not function effectively as a cleavage agent with BHA resin, apparently due to ~he relatively high acid stability of BHA resin.
Accordingly, there is a great need to provide a method whereby peptides can be produced economically and simply using chemical substances which are non-corrosive to ordinary laboratory glasswares and of relatively low toxicity and which can produce the peptides on a commercial scale.
Objects of the Invention Therefore, it is an object of this invention to provide a safe, simple method for producing peptides by the solid phase peptide synthesis method which avoids the use of highly corroslve and toxic hydrogen fluoride.
It is another object to produce peptides according to the SPPS method which can be scaled up to commercial scale production, and which uses ordinary laboratory glassware, plastics, etc., for handling, storin~, and transporting all of the chemicals required for the process, including the cleavage and deprotec~ing agent.
Still another object of the invention is the production of peptides by the SPPS method in which the cleavage step results ;n less side reactions and produces fewer unwanted by-products, thereby greatly simplifyin~ the subsequent purification step(s), A further object of the invention is to provide a solid phase peptide synthesis method in which the yield of peptide at the cleavage step is improved over the conventiona processes using HF or HBr with anisole.
~ 1 5 ~.2 7 ~(3~
A specif~ic object of this invention is to provide an improved process for synthesizing thymosin 1 by solid phase peptide synthesis using HBr as the cleavage and de-protecting agent for synthesizin~ thymosin 1 and thymosin ~1-Nl_l4 fragment by solid phase peptide synthesis.
Summary of the Invention These and other objects of the invention which will become apparent from the following detailed description and spe~iic embodiments are provided by a method for synthesizi lg peptide amides having the amino acid sequence represented by the formula Yp-X-NH2, where Yp represents the N-terminal peptide fragment and X-NH2 represents the C-terminal smino acid having an amide group (-CONH2) in its molecular s~ructure in the solid phase wherein hydrogen bromide is used as a depro~ecting and cleaving agent and methylbenzhydrylamine resin is used as the solid support. In accordance with a preferred embodiment of the invention, the cleavage step is c~rried out in trifluoroacetic acid in the presence of anisole. In accordance with a more preferred embodiment of the invention, the cleavage step is carried out in ~he presence of a mixture of anisole and thioanisole whereby the yields of peptide are substantially improved and production of unwanted by-products substantially reduced.
In another aspect of the invention, which is broadly applicsble to the solid phase peptide synthesis for producing a peptide of a desired amino acid sequence by the steps o~
(a) temporarily chemically protecting the reactive amino group and any other reactive groups, other than the carboxylic acid group at the alpha-position, on the C-terminal ~ ~ 7 ~)( amino acid of thè peptide;
(b) chemically bonding the protected C-terminal amino acid Vi8 the carboxylic acid (-COOH) group thereof to a resin support;
(c) chemically deprotecting the reactive amino group of the resin-bound protected amino acid;
(d) chemically coupling via a peptide bond the next amino acid in the desired sequence by contacting the resin-bound amino acid from step (c) with said next amino ~cid having all of the reactive groups thereof, other than the carboxylic acid group at the alpha-position, chemically protected, in the presence of a coupling agent;
(e) chemically deprotecting the reactive amino group of .the coupled smino acid from step (d);
(f) continuing the synehesis by repeating steps (d) and (e) with successive amino acids in the desired sequence being added one at a time until the total desired sequence of the protected peptide i8 built up on the resin, and (g) cleaving the protected peptide from the resin support and deprotecting the protected sie-chain reactive groups the yield of the clesved and deprotected peptide is increased by contacting the resin supported-protected peptide in step (g) with a cleavage and deprotecting composition whic~ includes a mixture of hydrogen bromide, anisole and thioanisole.
In a specific embodiment, the present invention provides a method of synthesizing thymosin ~l in the solid phase wherein hydrogen bromide, rather than the highly corrosive hydrogen fluoride, i9 used as a deprotecting and cleaving agent and methylbenzhydrylamine resin is used as solid support.
In this specific embodiment also, by using a mixture of l~t7~C)~
¦ anisole and thioanisole in the deprotecting and cleaving ¦ mixture, the yield of thymosin 1 is substantially improved.
I Detailed Descrlption of the Invention I
¦ Although it has long been known to use hydrogen ¦ bromide as the cleavage agent for cleaving the peptide-resin ¦ bond in the solid phase protein synthesis method ~he use ¦ of HBr has been limited to certain resin supports, primarily ¦ chloromethylated polystyrene-divinylbenzene; HBr does not, ¦ however, effectively function as the cleavage or deprotecting ¦ agent with benzhydrylamine resin.
¦ The present invention is based on the surprising ¦ discovery tha~ by using me~hylbenzhydrylamine resin as the resin support the cleavage of the bound peptide from the support can be performed using hydrogen bromide as the cleaving agent with a high yield and high degree of specificity of cleavage and deprotecting, not possible with benzhydrylamine resin. Furthermore, the yield of peptide is substantially increased when the deprotec~ing and clesv~ge with HBr is carried out in the presence of an~sole and still further improvements are achieved according to the present invention by using a mixture of anisole and thioanisole.
Details of the invention will be described below in connection with the production of thymosin 1 It should be understood, however, that the methylbenzhydrylamine resin support can be used in the production of any peptide the ¦ C-terminal amino acid cf which includes an amide radical ¦ group, such as aspargine (Asn) ~-aminosuccinamic acid) ¦ NH2 CH COOH , and glutamine (Gln), NH2-CH-COOH , or CH2-CONH2 CH2CH2cONH2 .. . ~ 3~
generally, any ~ino acid in which the carboxylic acid group is modified to an amide group. An example oE a peptide falling in the latter group i9 the calcitonin peptide in which the N-terminal (position 32) amino acid is Pro~NH2, i.e. .. N ~ where .......... represents the residue of the pep~ide at positions 1-31 (see e.g. U.S. Patent 4,217,268 for a disclosure of the complete structure of calcitonin and its des-X2 derivatives, as well as the solid phase synthesis thereof using benzhydrylamine resin support and HF cleavage/
deprotection). In general, any peptide which can be produced by solid phsse synthesis on benzhydrylamine (BHA) resin support can be produced with the methylbenzhydrylamine (MBHA) resin support according to the present invention, In addition to thymosin ~i and calcitonin mentioned above~ representative of other known peptides which can be produced by the improved procedure of this invention include oxy~ocin, vasopressin, LH-RH antagonists (see e.g. U.S. Patent 4,431,635, U.S.
Patent 4,530,920), the glutamine ~Gln) termina~ed synthetic hormone-like peptides disclosed in U.S. Patent 4,389,342, etc. Also, ragments of peptides which have the -CONH2 groups can be synthesized by the solid phase synthesis method using MBHA resin support and HBr cleavage. For instance, mention can be made, for example, of the biologically active peptides which are fragments of the sauvagine peptide disclosed 2S in USP 4,474,765; fragment 521:Ala-Ser-Pro-Ser-Gln of the Fc region of immunoglobulin E tIgE) useful in blocking mammalian allergic reaction (disclosed in U.S. Patent 4,161,522);
pep~ide fragments o~ formulas (VI), (VII), (IX), (X), (XI), (XV), and (SVI) of U.S. Patent 4,497,801, etc.
~ ~ 7 ~tO
¦ Accordin~ly, it should be understood that the improved ¦ solid phase peptide synthesis of this invention is applicable ¦ to the production of any peptide having an amide ( CONH2) ¦ radical as part of the C-terminal amino acid whether in 5 ¦ the form of a "naturally" occurring amino acid, e.g. aspargine ¦ ~Asn) or glutamine (Gln) or as an amino-group substituted ¦ amino acid, the amino-group substituent replacing the hydroxyl ¦ group of a carboxylic acid radical. Thus, the peptides ¦ produ~ed by ~he improved SPPS method of this invention may ¦ be generally referred to as peptide amides and may be represente ¦ by the formula l Yp-X-NH2 ¦ where Yp r~presents the N-terminal peptide fra8ment and l X-NH2 represents the C-terminal amino acid having l an amide group (-CONH2).
¦ It ig understood that peptide sequences are written according to the generally accepted convention whereby the N-terminal ¦ amino acid is on the left snd the C-terminal amino acid ¦ is on the right. The N-~erminsl peptide fragment Yp will ¦ have at least two, preferably at least 3, amino acids bonded ¦ ~oge~her by peptide bonds, -CONH- There is no particular upper limit to the number of amino acids in the Yp peptide fragment. For instance, it is possible to use SPPS methodology l to synthesize peptides containing as many as 40 or more ¦ amino acids, and polypeptides containing, for example, up ¦ to about 50 or 60 or more amino acids ean be synthesized.
¦ In the C-terminal amino acid repre~ented by X-NH2, X may ¦ be any of the well known and reasonably accessible amino ¦ acids as de~cribed, for example, in generai textooks on ¦ peptide chemistry; for instance, see K.D. Kopple, "Peptides and Amino Acids," W.A. Ben~amin, Inc., New York, 1966, pp. 4-7. Furthermore, the optically active amino acids may be in either the D-, Lor DL- form.
The me~hylbenzhydrylamine resin used in the subject invention is prepared from commercially available polystyrene re~in beads ~1% divinyl benzene, 200-400 mesh U.S. Standard) by reacting the same with p-toluoyl chloride in the presence of a Lewis acid such as aluminum chloride in an inert solvent such as dichloroethane at a low temperature, preferably 0 to 5C to form a p-toluoyl resin, CH3~C6H4-C0-C6H4-resin.
This resin is reacted with a mixture of ammonium formate, formamide and formic acid at reflux temperature ~160-170C) for 24 hours to yield N-formyl methylbenzhydrylamine resin, CH3-C6H4-CHtNH-CO-H)-C6H4- resin. Upon hydrolysis in dilute hydrochloric acid, the desired methylbenzhydrylamine resin, i-e- CH3-C6~4-~H(NH2-HC1)-~6H4- resin, is formed.
According to a specific embodiment of the invention, thymosin 1 having the following sequence Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-~lu-Asn-OH
is produced by solid phase peptide synthesis using the so-produc d methylbenzhydrylamine resin.
The methylbenzhydrylamine resin so formed is neutralized and acylated with N-Boc-~-benzyl-L-aspartic acid in the presence of dicyclohexylcarbodiimide to give Boc-asparaginyl resin. The solid phase synthesis is then continued by the sequential incorporation of one amino acid unit in each cycle to form a peptide unit which is acetylated to give an acetylated octacosapep~ide resin of the structure:
7 ~ 3~
Ac-Ser(Bzl)-Asp(OBzl)-Ala-Ala-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl) Ile-Thr(Bzl)-Thr(Bzl)-Lys(ClZ)-Asp(OBzl)-Leu-Lys(ClZ)-Glu(OBzl)-Lys(ClZ)-Lys(ClZ)-Glu(OBZl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl) Asp(NH-CH(C6H4-CH3~-C6H4-resin)-OBzl.
The protected thymosin ~l-resin so obtained is suspended in trifluoroacetic acid and treated with dry hydrogen bromide ga~ at ordinary temperature (20-25C) for about one hour in order to cleave the polypeptide from the resin and at the same time remove all the protecting groups from the side chains of the amino acid residues. Although hydrogen bromide may be used alone it is preferred to include anisole and., most preferably, a mixture of anisole and thioanisole in the ~rifluoroacetic acid when treating with dry hydrogen bromide gas. The present inventor has found that when a mixture of HBr, TFA and anisole is used as the deprotecting and cleaving mixture, the yield of thymosin ~1 is increased by about SO% over the instance when a ~ixture of only TFA
and HBr is used, When a mixture of HBr, TFA, anisole and thioanisole is used, the yield is improved by about 90~/O. The volume ratio of anisole: thioanisole is preferably from about 20:80 to about 80:20, most preferably about 50:50 (i.e. about 1:1).
The excess acids are then evaporated off at 40C
under partial vacuum and the anisole and thioanisole are washed off with ether. Crude thymosin ~1 is extracted from the residue with 1% ammonium acetate and desalted on a Sephadex G-10 column using 0.1 N acetic acid as the eluent. Thereafter, the thymosln ~1 may be purified by high pressure liquid chromatography (Clg reversed phase column, 5.7x30 cm).
lSephadex" is a trademark of Pharmacia AB, of Uppsala, Sweden . ~d 7~ t~
When sub~ected ~ analytical high pressure liquid chromatography the so obtained thymosin 1 behaves identically to reference thymosin ~1 prepared by the fragment condensation method.
Moreover, ~he presently synthesized thymosin ~1 has been found indistinguishsble from natural thymosin ~1 and gives satisfactory amino acid analysis.
It is noted that the combination of methylbenzhydrylamin , resin and hydrogen bromide must be used in order to obtain the desired results. If benzhydrylamine resin were used as the support resin, hydrogen fluoride must be used in the deprotecting and cleaving step. This repre~ents a signi-ficant difference between the method described in U.S. Patent 4,148,7~8 disclosing the SPPS method for producing thymosin ~1 and the present invention.
In the description above and following, the abbrevia tions have the following meaning according to standard customary nomenclature:
Boc, t-butyloxycarboxy; Bzl, benzyl; DCC, dicyclohexyl-csrbodiimide; Z, benzyloxycarbonyl; TFA, trifluoroacetic acid, and ClZ, 2-chlorobenzyloxycarbonyl.
While specific protectin~ groups have been employed in describing the preferred embodiment for synthesis of ~hymosin 1. it is within the skill of the art to utilize equivalen~ conventional protecting groups.
For example, -Ser(Rl) is u~ilized as the protected form o serine wherein Rl is a conventional protecting group for the hydroxyl group of the serine residues such as benzyl, acetyl, benzoyl, tert-butyl, trityl, 4-bromobenzyl,
I SOLID PHASE PROCESS FOR
¦ SYNTHESIZING PEPTIDES
¦ BACKGROUND OF THE INVENTIO~
, _ . .. ..
I Field of Invention I _ . ___ _ ¦ This invention relates to an improved process for synthesis of peptides by the ~olid phase method. More particu-larly, the invention relates to the synthesis of peptide amides by solid phase peptide synthesis using a novel resin l support, me~hylbenzhydrylamine resin, and hydrogen bromide ¦ as the cleava&e and deprotecting agent for separating the resin-bound peptide from the resin support. In a particular aspect, the inventicn relates to the solid phase synthesis of thymosin al The invention also relates to an improved l process for synthesizing peptides, and particularly, thymosin l ~1 and the thymosin l-Nl_l4 fragment, by solid phase synthesis ¦ using an improved cleavage/deprotecting composi~ion, namely hydrogen bromide with a mixture of anisole and thioanisole.
Description of the Prior Art I
l The function of peptides in human health has received ¦ much recognition in recent years, see, e.g. "Peptides: A
Medical Rediscovery" by Joseph Alper, High Technology, Vol.
3, No. 9, September 1983, pages 60-63. Peptides have been ¦ shown to function, for example, as hormones for regulating ¦ growth, reproduction, and immunology, and as neurotransmitters.
¦ While ac~ual and positive biological results have been observed for several dozens of different peptides, still much work remains to be done to determine how the petides work in 1~71;~0 ¦ the body. For this purpose, lar~e quantities of the peptides are required. Also, in order for the peptides of known biological activity to be of practical significance in health care, they must be made readily available in large quantities and at relatively low C06t.
In addition to the "natural" peptides, i.e. those produced ln the body, "synthetic" peptides or analogs, which may be considered as derivatives of natural peptides, but having one or more chemical modifications in the molecular structure of the natural peptide, are also valuable for their modified chemical properties, such 85 resistance to enzyme degradation. Here again, the availability of relatively inexpensive means to rapidly and inexpensively produce synthetic peptides would be highly valuable.
Presently, various procedures for synthesizing peptides, i.e. for formation of a peptide linkage between amino acids, are known. Conventional procedures include both solution-phase (or liquid phase) methods and solid-phase methods. A general discussion of peptide synthesis can be found, for example, in Schroder E. Lubke: "The Peptides," Vol. l (1966), Academic Press, New York, U.S.A. and Neurath and Hill, "The Proteins,"
Vol. 2 (1976~, Academic Press, New York, U.S.A. Generally, these procedures involve the reaction between the free amino group of an amino acid or residue thereof having its carboxyl group, hydroxyl group or other reactive group(s) protected, and the ree primary carboxylic group of another amino acid or residue thereof having its amino group or other reactive group(s) protected to form a peptide bond. Each amino acid in the desired sequence can be added singly successively to another amino acid or residue thereof or separate peptide fragments with the desired amino acid sequence can be synthesize '~2 7~'3~3~
and then condens~ed to provide ~he desired peptide.
In the past ~en to twenty years the synthesis of peptides has been simpliXied by utilizing the solid phase synthesis method according to the general principles developed by Merrifield, [R.B. Merrifield, J. Am. Chem. Soc. 85, 2149-2154 (1963~; Stewart, et al, Solid Phase Peptide Synthesis, Freeman & Co., San Francisco, CA (1969); Barany, et 81, The Peptides, Analysis, Synthe&is and Biology, Vol. 2, pages 1-284, (1980)~.
Briefly in solid-phase peptide synthesis (SPPS), the carboxyl group of the first amino acid in a peptide is chemically bound to the surface o tiny, in~oluble beads, with the other reactive sites on the amino acid being temporarily blocked. The amino acid-bound resin beads are placed in a reaction vessel and unbound acid is washed away. After chemically unblocking the reactive amino group, the next amino acid in the desired sequence of the object peptide is added with a chemical coupling a8ent such that the two acids bind together, via a peptide bond. The synthesis is continued by repeating the foregoing process with successive amino acids in the sequence being added one at a time until the total peptide sequence is built up on the resin. Upon completion of the desired peptide sequence, the protected peptide is cleaved from the resin support, and all protecting groups are removed. The cleavage reaction and removsl of protecting groups may be accomplished simultaneously or sequentially.
SPPS has recently been adapted to automated and computerized instrumentation offering substantial improvements in speed, eficiency, and reliability. However, the use of such instrumentation has been substantially limited to production of peptides on a relatively small scale, on the ! ~ 7 ~ 3(~
order of 8 few mllligrams per production cycle. Also, the procedure inherently results in the production of unwanted by-products, unreacted acids, solvents, coupling or decoupling agents~ cleavage products and so on, making the subsequent 5 purification procedures troublesome.
Although the art iB aware of various different materials used as the insoluble beads, including glass, silica, synthetic resins, etc., the synthetic resins are most commonly used as the support material (insoluble beads). Conventional materials used as the resin include the styrene-divinyl benzene resin modified with a reactive group, such 8S chloro-methylated styrene-divinyl benzene resin and benzhydrylamine resin (BHA). BHA is particularly useul for synthesis of peptide amides, i.e. peptides of which the C-terminal amino acid has a carboxylamide (-CONH2) group, since the amide group can be formed directly. The benzhydrylaminopolystyrene-divinyl benzene resin support is described by P. Rivaille, et al, Helv. Chim. Acta., 54, 2772 (1971).
Hydrogen fluoride, HF, is commonly used as the cleavage agent, and also as the deprotecting agent for removing the various blocking group~.
Unfortunately, HF, whether in liquid, anhydrous, or gaseous form, is highly corrosive and toxic requiring special handling and special plastic materials. As a result, the scale-up of the SPPS procedure from the laboratory scale to com~,ercial scale production (e.g. on the order of one or more grams per production run) has proven extremely difficult It has been known to use the less corrosive and less toxic hydrogen bromide (HBr) in place of HF as the cleavage agent, alone, or together with anisole as a catalyst for cleaving the bound protec~ed peptide from the supporting ~ 3~3~
resin. However, HBr alone or with anisole does not function effectively as a cleavage agent with BHA resin, apparently due to ~he relatively high acid stability of BHA resin.
Accordingly, there is a great need to provide a method whereby peptides can be produced economically and simply using chemical substances which are non-corrosive to ordinary laboratory glasswares and of relatively low toxicity and which can produce the peptides on a commercial scale.
Objects of the Invention Therefore, it is an object of this invention to provide a safe, simple method for producing peptides by the solid phase peptide synthesis method which avoids the use of highly corroslve and toxic hydrogen fluoride.
It is another object to produce peptides according to the SPPS method which can be scaled up to commercial scale production, and which uses ordinary laboratory glassware, plastics, etc., for handling, storin~, and transporting all of the chemicals required for the process, including the cleavage and deprotec~ing agent.
Still another object of the invention is the production of peptides by the SPPS method in which the cleavage step results ;n less side reactions and produces fewer unwanted by-products, thereby greatly simplifyin~ the subsequent purification step(s), A further object of the invention is to provide a solid phase peptide synthesis method in which the yield of peptide at the cleavage step is improved over the conventiona processes using HF or HBr with anisole.
~ 1 5 ~.2 7 ~(3~
A specif~ic object of this invention is to provide an improved process for synthesizing thymosin 1 by solid phase peptide synthesis using HBr as the cleavage and de-protecting agent for synthesizin~ thymosin 1 and thymosin ~1-Nl_l4 fragment by solid phase peptide synthesis.
Summary of the Invention These and other objects of the invention which will become apparent from the following detailed description and spe~iic embodiments are provided by a method for synthesizi lg peptide amides having the amino acid sequence represented by the formula Yp-X-NH2, where Yp represents the N-terminal peptide fragment and X-NH2 represents the C-terminal smino acid having an amide group (-CONH2) in its molecular s~ructure in the solid phase wherein hydrogen bromide is used as a depro~ecting and cleaving agent and methylbenzhydrylamine resin is used as the solid support. In accordance with a preferred embodiment of the invention, the cleavage step is c~rried out in trifluoroacetic acid in the presence of anisole. In accordance with a more preferred embodiment of the invention, the cleavage step is carried out in ~he presence of a mixture of anisole and thioanisole whereby the yields of peptide are substantially improved and production of unwanted by-products substantially reduced.
In another aspect of the invention, which is broadly applicsble to the solid phase peptide synthesis for producing a peptide of a desired amino acid sequence by the steps o~
(a) temporarily chemically protecting the reactive amino group and any other reactive groups, other than the carboxylic acid group at the alpha-position, on the C-terminal ~ ~ 7 ~)( amino acid of thè peptide;
(b) chemically bonding the protected C-terminal amino acid Vi8 the carboxylic acid (-COOH) group thereof to a resin support;
(c) chemically deprotecting the reactive amino group of the resin-bound protected amino acid;
(d) chemically coupling via a peptide bond the next amino acid in the desired sequence by contacting the resin-bound amino acid from step (c) with said next amino ~cid having all of the reactive groups thereof, other than the carboxylic acid group at the alpha-position, chemically protected, in the presence of a coupling agent;
(e) chemically deprotecting the reactive amino group of .the coupled smino acid from step (d);
(f) continuing the synehesis by repeating steps (d) and (e) with successive amino acids in the desired sequence being added one at a time until the total desired sequence of the protected peptide i8 built up on the resin, and (g) cleaving the protected peptide from the resin support and deprotecting the protected sie-chain reactive groups the yield of the clesved and deprotected peptide is increased by contacting the resin supported-protected peptide in step (g) with a cleavage and deprotecting composition whic~ includes a mixture of hydrogen bromide, anisole and thioanisole.
In a specific embodiment, the present invention provides a method of synthesizing thymosin ~l in the solid phase wherein hydrogen bromide, rather than the highly corrosive hydrogen fluoride, i9 used as a deprotecting and cleaving agent and methylbenzhydrylamine resin is used as solid support.
In this specific embodiment also, by using a mixture of l~t7~C)~
¦ anisole and thioanisole in the deprotecting and cleaving ¦ mixture, the yield of thymosin 1 is substantially improved.
I Detailed Descrlption of the Invention I
¦ Although it has long been known to use hydrogen ¦ bromide as the cleavage agent for cleaving the peptide-resin ¦ bond in the solid phase protein synthesis method ~he use ¦ of HBr has been limited to certain resin supports, primarily ¦ chloromethylated polystyrene-divinylbenzene; HBr does not, ¦ however, effectively function as the cleavage or deprotecting ¦ agent with benzhydrylamine resin.
¦ The present invention is based on the surprising ¦ discovery tha~ by using me~hylbenzhydrylamine resin as the resin support the cleavage of the bound peptide from the support can be performed using hydrogen bromide as the cleaving agent with a high yield and high degree of specificity of cleavage and deprotecting, not possible with benzhydrylamine resin. Furthermore, the yield of peptide is substantially increased when the deprotec~ing and clesv~ge with HBr is carried out in the presence of an~sole and still further improvements are achieved according to the present invention by using a mixture of anisole and thioanisole.
Details of the invention will be described below in connection with the production of thymosin 1 It should be understood, however, that the methylbenzhydrylamine resin support can be used in the production of any peptide the ¦ C-terminal amino acid cf which includes an amide radical ¦ group, such as aspargine (Asn) ~-aminosuccinamic acid) ¦ NH2 CH COOH , and glutamine (Gln), NH2-CH-COOH , or CH2-CONH2 CH2CH2cONH2 .. . ~ 3~
generally, any ~ino acid in which the carboxylic acid group is modified to an amide group. An example oE a peptide falling in the latter group i9 the calcitonin peptide in which the N-terminal (position 32) amino acid is Pro~NH2, i.e. .. N ~ where .......... represents the residue of the pep~ide at positions 1-31 (see e.g. U.S. Patent 4,217,268 for a disclosure of the complete structure of calcitonin and its des-X2 derivatives, as well as the solid phase synthesis thereof using benzhydrylamine resin support and HF cleavage/
deprotection). In general, any peptide which can be produced by solid phsse synthesis on benzhydrylamine (BHA) resin support can be produced with the methylbenzhydrylamine (MBHA) resin support according to the present invention, In addition to thymosin ~i and calcitonin mentioned above~ representative of other known peptides which can be produced by the improved procedure of this invention include oxy~ocin, vasopressin, LH-RH antagonists (see e.g. U.S. Patent 4,431,635, U.S.
Patent 4,530,920), the glutamine ~Gln) termina~ed synthetic hormone-like peptides disclosed in U.S. Patent 4,389,342, etc. Also, ragments of peptides which have the -CONH2 groups can be synthesized by the solid phase synthesis method using MBHA resin support and HBr cleavage. For instance, mention can be made, for example, of the biologically active peptides which are fragments of the sauvagine peptide disclosed 2S in USP 4,474,765; fragment 521:Ala-Ser-Pro-Ser-Gln of the Fc region of immunoglobulin E tIgE) useful in blocking mammalian allergic reaction (disclosed in U.S. Patent 4,161,522);
pep~ide fragments o~ formulas (VI), (VII), (IX), (X), (XI), (XV), and (SVI) of U.S. Patent 4,497,801, etc.
~ ~ 7 ~tO
¦ Accordin~ly, it should be understood that the improved ¦ solid phase peptide synthesis of this invention is applicable ¦ to the production of any peptide having an amide ( CONH2) ¦ radical as part of the C-terminal amino acid whether in 5 ¦ the form of a "naturally" occurring amino acid, e.g. aspargine ¦ ~Asn) or glutamine (Gln) or as an amino-group substituted ¦ amino acid, the amino-group substituent replacing the hydroxyl ¦ group of a carboxylic acid radical. Thus, the peptides ¦ produ~ed by ~he improved SPPS method of this invention may ¦ be generally referred to as peptide amides and may be represente ¦ by the formula l Yp-X-NH2 ¦ where Yp r~presents the N-terminal peptide fra8ment and l X-NH2 represents the C-terminal amino acid having l an amide group (-CONH2).
¦ It ig understood that peptide sequences are written according to the generally accepted convention whereby the N-terminal ¦ amino acid is on the left snd the C-terminal amino acid ¦ is on the right. The N-~erminsl peptide fragment Yp will ¦ have at least two, preferably at least 3, amino acids bonded ¦ ~oge~her by peptide bonds, -CONH- There is no particular upper limit to the number of amino acids in the Yp peptide fragment. For instance, it is possible to use SPPS methodology l to synthesize peptides containing as many as 40 or more ¦ amino acids, and polypeptides containing, for example, up ¦ to about 50 or 60 or more amino acids ean be synthesized.
¦ In the C-terminal amino acid repre~ented by X-NH2, X may ¦ be any of the well known and reasonably accessible amino ¦ acids as de~cribed, for example, in generai textooks on ¦ peptide chemistry; for instance, see K.D. Kopple, "Peptides and Amino Acids," W.A. Ben~amin, Inc., New York, 1966, pp. 4-7. Furthermore, the optically active amino acids may be in either the D-, Lor DL- form.
The me~hylbenzhydrylamine resin used in the subject invention is prepared from commercially available polystyrene re~in beads ~1% divinyl benzene, 200-400 mesh U.S. Standard) by reacting the same with p-toluoyl chloride in the presence of a Lewis acid such as aluminum chloride in an inert solvent such as dichloroethane at a low temperature, preferably 0 to 5C to form a p-toluoyl resin, CH3~C6H4-C0-C6H4-resin.
This resin is reacted with a mixture of ammonium formate, formamide and formic acid at reflux temperature ~160-170C) for 24 hours to yield N-formyl methylbenzhydrylamine resin, CH3-C6H4-CHtNH-CO-H)-C6H4- resin. Upon hydrolysis in dilute hydrochloric acid, the desired methylbenzhydrylamine resin, i-e- CH3-C6~4-~H(NH2-HC1)-~6H4- resin, is formed.
According to a specific embodiment of the invention, thymosin 1 having the following sequence Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-~lu-Asn-OH
is produced by solid phase peptide synthesis using the so-produc d methylbenzhydrylamine resin.
The methylbenzhydrylamine resin so formed is neutralized and acylated with N-Boc-~-benzyl-L-aspartic acid in the presence of dicyclohexylcarbodiimide to give Boc-asparaginyl resin. The solid phase synthesis is then continued by the sequential incorporation of one amino acid unit in each cycle to form a peptide unit which is acetylated to give an acetylated octacosapep~ide resin of the structure:
7 ~ 3~
Ac-Ser(Bzl)-Asp(OBzl)-Ala-Ala-Val-Asp(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl) Ile-Thr(Bzl)-Thr(Bzl)-Lys(ClZ)-Asp(OBzl)-Leu-Lys(ClZ)-Glu(OBzl)-Lys(ClZ)-Lys(ClZ)-Glu(OBZl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl) Asp(NH-CH(C6H4-CH3~-C6H4-resin)-OBzl.
The protected thymosin ~l-resin so obtained is suspended in trifluoroacetic acid and treated with dry hydrogen bromide ga~ at ordinary temperature (20-25C) for about one hour in order to cleave the polypeptide from the resin and at the same time remove all the protecting groups from the side chains of the amino acid residues. Although hydrogen bromide may be used alone it is preferred to include anisole and., most preferably, a mixture of anisole and thioanisole in the ~rifluoroacetic acid when treating with dry hydrogen bromide gas. The present inventor has found that when a mixture of HBr, TFA and anisole is used as the deprotecting and cleaving mixture, the yield of thymosin ~1 is increased by about SO% over the instance when a ~ixture of only TFA
and HBr is used, When a mixture of HBr, TFA, anisole and thioanisole is used, the yield is improved by about 90~/O. The volume ratio of anisole: thioanisole is preferably from about 20:80 to about 80:20, most preferably about 50:50 (i.e. about 1:1).
The excess acids are then evaporated off at 40C
under partial vacuum and the anisole and thioanisole are washed off with ether. Crude thymosin ~1 is extracted from the residue with 1% ammonium acetate and desalted on a Sephadex G-10 column using 0.1 N acetic acid as the eluent. Thereafter, the thymosln ~1 may be purified by high pressure liquid chromatography (Clg reversed phase column, 5.7x30 cm).
lSephadex" is a trademark of Pharmacia AB, of Uppsala, Sweden . ~d 7~ t~
When sub~ected ~ analytical high pressure liquid chromatography the so obtained thymosin 1 behaves identically to reference thymosin ~1 prepared by the fragment condensation method.
Moreover, ~he presently synthesized thymosin ~1 has been found indistinguishsble from natural thymosin ~1 and gives satisfactory amino acid analysis.
It is noted that the combination of methylbenzhydrylamin , resin and hydrogen bromide must be used in order to obtain the desired results. If benzhydrylamine resin were used as the support resin, hydrogen fluoride must be used in the deprotecting and cleaving step. This repre~ents a signi-ficant difference between the method described in U.S. Patent 4,148,7~8 disclosing the SPPS method for producing thymosin ~1 and the present invention.
In the description above and following, the abbrevia tions have the following meaning according to standard customary nomenclature:
Boc, t-butyloxycarboxy; Bzl, benzyl; DCC, dicyclohexyl-csrbodiimide; Z, benzyloxycarbonyl; TFA, trifluoroacetic acid, and ClZ, 2-chlorobenzyloxycarbonyl.
While specific protectin~ groups have been employed in describing the preferred embodiment for synthesis of ~hymosin 1. it is within the skill of the art to utilize equivalen~ conventional protecting groups.
For example, -Ser(Rl) is u~ilized as the protected form o serine wherein Rl is a conventional protecting group for the hydroxyl group of the serine residues such as benzyl, acetyl, benzoyl, tert-butyl, trityl, 4-bromobenzyl,
2,6-dichlorobenzyl and benzyloxycarbonyl, -Asp-(OR2) is utilized as the protected form of aspartic acid wh~rein R2 i8 ~ conventional protecting group for carboxyl groups 1 ~ 7 1~()0 selected from esters such as aryl esters, particularly phenyl or phenyl substituted with lower alkyl, halo, nitro, thio or substitut~d thio, i.e., methylthio, aralkyl esters such as benzyl or benzyl substituted with methoxy, halo or nitro, lower alkyl esters such as methyl, ethyl, tert-butyl and tert~amyl, substituted lower alkyl esters such as 2-haloethyl dimethylaminoethyl and cyanomethyl, benzhydryl esters and phenacyl esters; -Thr(Rl) - is utilized as the protected form of threonine wherein Rl i8 as defined above; -Glu(OR2~-is utilized as the protected form of glutamic acid wherein R2 is as defined above; -Lys(R3) i5 utilized as the protected form of lysine wherein R3 is a conventionsl ~-amino protecting group selected from benzyloxycarbonyl which may be optionally substituted in the aromatic ring such as by 2-chloro, 4-chloro, 2-bromo, 4-bromo, 2,4-dichloro, 4-nitro, 4-methoxy, 3,5-dimethox ~, 4-methyl, 2,4,6-trimethyl, 4-phenylazo, 4-~4-methoxyphenylszo), 2-(N,N-dimethylcarbonamido), 4-dihydroxyboryl, and 2-nitro-4,5-;
dimethoxy, other urethane type protecting groups, such as 4-toluene~ulfonylethyloxycarbonyl, 9-fluorenylmeth910xycarbonyl and related base cleavable groups, cyclopentyloxycarbonyl and related nitrogen containing urethane groups; acyl groups such as formyl, trifluoroacetyl, phthaloyl, benzenesulfonyl, acetoacetyl, chloroacetyl, 2-nitrobenzoyl, 4-toluene-sulfonyl;
and -Asp-OR2 is utilized as the protected form of asparagine wherein R2 is as defined above.
Although the above description has been given with particular emphasis for the solid phase peptide synthesis of thymosin ~l, with its specified amino acid sequence and specified protected side chain groups~ the practitioner will readily recognize that certain general conditions will ~L~"7~ 3~) .~
preferably be selected regardless of the particular peptide being synthe~ized. For example, it i8 well recognized that the ~-amino group of esch amino acid employed in the peptlde synthesis mu~ be protected during the coupling reaction to prevent side reactions involving the reactive ~-amino function. Similarly, for those amino acids containing reactive side-chain functional groups (e.g. sulfhydryl, ~-amino, hydroxyl, carboxyl), such functional groups need also be protected both during the initial coupling of the amino acid contsining the side-chain group and during the coupling of subsequent amino acid~. Suitable protecting groups are known in the art lSee for example9 Protective Groups in nic Chemistry, M. McOmie, Editor, Plenum Press, N.Y., 1973.]
In selecting a particular ptoecting group, the following conditions must be observed: an ~-amino protecting group must: (1) be stable and render the -amino function inert under the conditions employed in the coupling reaction, and (2) must be readily removable after the coupling reaction under conditions that will not remove the side chain protecting groups or alter the structure of the peptide fragment.
A side chain protec~ing group must: (l) be stable and render the side chain functional group inert under the condition employed in the coupling reaction, (2) be stable under the condi~ions employed in removing the -amino protecting group and ~3) be readily removable upon completion of the desired amino acld sequence under reaction conditions that will not alter the structure of the peptide chain, or racemization of any of the chiral centers contained therein. Suitable protecting groups for the ~-amino function are t-butyloxy-~ 7~
carbonyl (Boc), ~enzyloxycarbonyl (Cbz), biphenylisopropyl-oxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, l,l-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl, 2 - cyano-t-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl and the like, especially t-butyloxycarbonyl (Boc).
A~ examples of carboxyl-protecting groups, there may be mentioned such ester-forming groups as those capable of giving alkyl ester (e.g. methyl, ethyl, propyl, butyl, t-butyl, etc., esters), benzyl ester, p-nitrobenzyl ester, p-methoxybenzyl e~ter, p-chlorobenzyl ester, benzhydryl e~ter, etc. and hydrazide-forming groups such as those capable of giving carbobenzoxy hydrazide, t-butyloxy-carbonyl hydrazide, trityl hydrazide, etc.
As group~ for protecting the guanidino group of arginine, there may be mentioned nitro, tosyl, p-methoxybenzene-sulfonyl, carbobenzoxy, isobornyloxycarbonyl, admantyloxycarbony L, etc. The guanidino group may also be protected in the form of 8 salt with an acid (e.g. benzenesulfonic acid, toluene-sulfonic acid, hydrochloric acid, sulfuric acid, etc.).
The hydroxyl group of threonine may be protected, for example, by way of known esterification or etherifica~ion.
As examples of groups suitable for said esterification, there may be mentioned lower alkanoyl groups (e.g. acetyl), aroyl groups (e.g. benzoyl), and groups derived from carbonic acid, such as benzyloxycarbonyl, ethyloxycarbonyl, etc.
As groups suitable for said etherification, there may be mentioned benzyl, tetrahydropyranyl, t-butyl, etc. The hydroxyl ~roup of threonine, however, need not necessarily be protected.
Methionine may be protected in the form of a sulfoxide.
Other preferred side chain protective groups for particular amino acids include; for tyrosine: benzyl, o-bromobenzyloxy-~L~71~3()0 carbonyl 9 2,6-dichlorobenzyl, isopropyl, cyclohexyl, cyclopentyl and acetyl; for histidine: benzyl, p-toluenesulfonyl and 2,4-dinitrophenyl; $or tryptophan: formyl.
According to the present invention, the production of ~he peptide amides is carried out using methylbenzhydrylamine ~polystyrene-divinyl benzene) resin as the solid support.
However, for the production of other types of peptides using the novel HBr, anisole, thioanisole cleavage/deprotecting composition of this invention, other conventional solid suppor~s to which the C-terminal amino acid i8 attached can be used. Suitable solid supports useful for the solid phase peptide synthesis are those materials which are iner~
to the reagentc and reaction conditions of the stepwise condensation-deprotection reactions, as well as being insoluble in the media used. Suitable solid supports are chloromethyl-polystyrene-divinylbenzene polymer, hydroxymethyl-polystyrene-divinylbenzene polymer functionalized, ~rosslinked poly-N-acrylylpyrrolidine resins, and the like, especially chloromethyl polystyrene-1% divinylbenzene polymer.
The N~-Boc-amino acid or similarly protected C-terminal amino acid is at~ached to the methyl benzhydrylamine resin by means of an N,N'-dicyclohexylcarbodiimide (DCC)/l-hydroxy-benzotriazole (HBT) mediated coupling for from sbout 2 to about 24 hours, preferably about 12 hours at a temperature of between about 10 and 50C., preferably 25C., in a solvent such as dichloromethane or DMF, preferably dichloromethane.
The attachment to the chloromethyl polystyrenedivinylbenzene type of resin is made by means of the reaction of the Nl-protected amino acid, especially the Boc-amino acid, as its cesium, tetramethylammonium, triethylammonium, 4,5-diaza-bicyclo-[S.4.0]undec-5-ene, or similar salt in ethanol, ... 3L~71~ 3 acetonitrile, N,N-dimethylformamide (DMF), and the like, especially the cesium salt in DMF, with the chloromethyl resin at an ~leva~ed temperature, for example, between about 40 and 60C., preferably about $0C for from about 12 to S 48 hours, preferably about 24 hours. The removal of the Nl-protecting groups may be performed in the presence of, for example, a solution of trifluoroacetic acid in methylene chloride, hydrogen chloride in dioxane, hydrogen chloride in acetic acid, or other strong acid solution, preferably 50% trifluoroacetic acid in dichloromethane at about ambient temperature. The coupling of successive protected amino acids can be carried out in an automatic polypeptide synthesizer as well known in the art. Each protected amino acid i5 preferably introduced in approximately 2.5 or more molar exces~ and the coupling may be carried out in dichloromethane, dichloromethane/DMF mixtures, DMF and the like, especially in methylene chloride at about ambient temperature. Other solven~s which are known to be useful for the purpose of peptide-forming condensation reactionJ for example, dimethyl-sulfoxide, pyridine, chloroform, dioxane, te~rahydrofuran, ethyl acetate, N-methylpyrrolidone, etc., as well as suitable mixtures thereof may also be used.
The reaction temperature for the condensation/coupling reaction may be selected from the range known to be useful for the purpose of peptide-forming condensation reactions.
Thus, it may normally be within the range of about -40C
to about 60C, and preferably, abou~ -20C to about 0C.
The coupling agent is normally DCC in dichloromethane but may be N,N'-di-iso-propylcarbodiimide or other carbodiimide ether alone or in the presence of HBT, N-hydroxysuccinimide, ethyl 2-hydroxyimino-2 cyanoacetate, other N-hydroxyimides or oximes. Alternatively, protected amino acid active esters ~` ~ ~7~
(e.g. p-nitrophenyl, pentafluorophenyl and the like) or symmetrical anhydrides may be used.
The coupling, deprotection/cleavage reactions and preparation of derivatives of the polypeptides are suitably carried out at temperatures between about -10 and ~50C, most preferably about 20-25C. The exact temperature for any particular reaction will, of course, be dependent upon the substrates, reagents, solven~s and so forth, all being well within ~he skill of the practitioner. The fully deprotecte polypeptide may then be purified by a sequence of chromatographi steps employing any or all of the following types: ion exchange on a weakly basic resin in the acetate form; gel permeation chromatography, e.g. on Sephadex G-25; hydrophobic adsorption chromatography on underivatized polystyrene-divinyl-benzene (for example, Amberlite XAD~; silica gel adsorption chromatography; lon exchange chromatography, e.g. on Sephadex G-25, or countercurrent distribution; high performance liquid chromatography (HPLC), especially reverse phase HPLC on octyl- or octadecylsilyl-silica bonded phase column packing.
In another specific embodiment of the present invention, the N-terminal 14 amino partial sequence of thymosin ~1.
Ac-Ser-Asp--Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-OH
(thymosin ~l-Nl_l4) is synthesized on benzyl ester resin.
The protected thymosin al-Nl_l4-resin is deprotected and cleaved by treatment with hydrogen bromide, trifluoroacetic acid, anisole and thioanisole in a manner similar to that described above for thymosin ~1 Purification of the crude product by preparative high pressure liquid chromatography yields a pure thymosin ~l-Nl_l4 compound which is found to be identical to a reference compound synthesized previously by the fragment condensation method in solution.
2"Amberlite XAD" is a trademark of Rohm and Haas Company of Philadelphia, Pennsylvania, U.S.A.
This inv~ention is further illustrated by the following non-limiting examples.
Example 1 Methylbenzhydrylamine Resin 50.9 gm of polystyrene resin beads (copolystyrene-1%-divinyl benzene, 200-400 mesh beads) was suspended in 500 ml of dichloroethane. The mixture was cooled in an ice-bath with gentle mechanical s~irring until the temperature went below 5C.
15.5 g of p-toluoyl chloride and 13.3 g of aluminum chloride were mixed in 250 ml of dichloroethane in a dropping funnel. The solution was added dropwise to the cooled, ~tirred suspen~ion of the resin bead~ over a period of about 40 min., care being taken not to allow the reaction to warm up above 5C. The stirring was continued for 4 hours at room temperature when the resin was washed sequentially with lsopropanol, isopropanol-water (1:1 mixture), isopropanol and dried to give 53.8 g of p-toluoyl resin. This resin was then mixed with 168 g of ammonium formate, 201 ml of formamide, 134 ml of formic acid and 350 ml of nitrobenzene.
The mixture was gradually heated up to 165-170C under reflux and maintained for 1 day during which time about 115 ml of the aqueous phase was collected into a Dean-Stark trap.
The resin was washed and dried as above to yield 55.5 g of N-formyl me~hylbenzhydrylamine resin. The product was hydrolyzed in a mixture of 300 ml each of 12 N HCl and isopropan 1 under reflux for 3 hr. Washing and drying the resulting resin provided 54.9 g of the hydrochloride form of methylbenz-hydrylamine resin. It showed a very strong positive reaction to ninhydrin reagent and incorporated 0.4 mmol of Boc-Ala-OH
when neutralized and coupled with Boc-Ala-OH in the presence of dicyclohexylcarbodiimide.
~ ~'7~
Ac-Ser-Asp-Ala Ala-Val-Asp.Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-V~l-Vfll-Glu-Glu-~Ala-Glu-Asn-OH
(Thym~sin ~1) 5.02 gm of methylbenzhydrylamine resin obtained in Example 1 was placed in a peptide synthesis flask equipped with a sintered glass filter at the bottom and a mechanical stirrer at the top. The resin wa~ washed with 20 volumes of 10% triethylamine in methylene chloride and stirred with a fresh portion of the same solution for 10 min. The neutralize methylbenzhydrylamine resin was then reacte~ with Boc-L-aspartic acid a-benzyl ester (1.95 g, 6 mmol) and dicyclohexylcarbodiimid (1.24 g, 6 mmol) for 2 hours to form Boc-~-benzyl-L-aspartic 8cyl methylbenzhydrylamine resin. The resin turned from ninhydrin positive tc ninhydrin negative after this reaction.
The solid phase peptide synthesis was then continued by performing the following steps wherein in each cycle one amino acid was incorporsted sequentially into the growing peptide chain on the resin:
(1) prcwash with 40% trifluoroacetic acid in CH2C12;
(2) stir for 28 minutes with 40% trifluoroacetic acid;
dimethoxy, other urethane type protecting groups, such as 4-toluene~ulfonylethyloxycarbonyl, 9-fluorenylmeth910xycarbonyl and related base cleavable groups, cyclopentyloxycarbonyl and related nitrogen containing urethane groups; acyl groups such as formyl, trifluoroacetyl, phthaloyl, benzenesulfonyl, acetoacetyl, chloroacetyl, 2-nitrobenzoyl, 4-toluene-sulfonyl;
and -Asp-OR2 is utilized as the protected form of asparagine wherein R2 is as defined above.
Although the above description has been given with particular emphasis for the solid phase peptide synthesis of thymosin ~l, with its specified amino acid sequence and specified protected side chain groups~ the practitioner will readily recognize that certain general conditions will ~L~"7~ 3~) .~
preferably be selected regardless of the particular peptide being synthe~ized. For example, it i8 well recognized that the ~-amino group of esch amino acid employed in the peptlde synthesis mu~ be protected during the coupling reaction to prevent side reactions involving the reactive ~-amino function. Similarly, for those amino acids containing reactive side-chain functional groups (e.g. sulfhydryl, ~-amino, hydroxyl, carboxyl), such functional groups need also be protected both during the initial coupling of the amino acid contsining the side-chain group and during the coupling of subsequent amino acid~. Suitable protecting groups are known in the art lSee for example9 Protective Groups in nic Chemistry, M. McOmie, Editor, Plenum Press, N.Y., 1973.]
In selecting a particular ptoecting group, the following conditions must be observed: an ~-amino protecting group must: (1) be stable and render the -amino function inert under the conditions employed in the coupling reaction, and (2) must be readily removable after the coupling reaction under conditions that will not remove the side chain protecting groups or alter the structure of the peptide fragment.
A side chain protec~ing group must: (l) be stable and render the side chain functional group inert under the condition employed in the coupling reaction, (2) be stable under the condi~ions employed in removing the -amino protecting group and ~3) be readily removable upon completion of the desired amino acld sequence under reaction conditions that will not alter the structure of the peptide chain, or racemization of any of the chiral centers contained therein. Suitable protecting groups for the ~-amino function are t-butyloxy-~ 7~
carbonyl (Boc), ~enzyloxycarbonyl (Cbz), biphenylisopropyl-oxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, l,l-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl, 2 - cyano-t-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl and the like, especially t-butyloxycarbonyl (Boc).
A~ examples of carboxyl-protecting groups, there may be mentioned such ester-forming groups as those capable of giving alkyl ester (e.g. methyl, ethyl, propyl, butyl, t-butyl, etc., esters), benzyl ester, p-nitrobenzyl ester, p-methoxybenzyl e~ter, p-chlorobenzyl ester, benzhydryl e~ter, etc. and hydrazide-forming groups such as those capable of giving carbobenzoxy hydrazide, t-butyloxy-carbonyl hydrazide, trityl hydrazide, etc.
As group~ for protecting the guanidino group of arginine, there may be mentioned nitro, tosyl, p-methoxybenzene-sulfonyl, carbobenzoxy, isobornyloxycarbonyl, admantyloxycarbony L, etc. The guanidino group may also be protected in the form of 8 salt with an acid (e.g. benzenesulfonic acid, toluene-sulfonic acid, hydrochloric acid, sulfuric acid, etc.).
The hydroxyl group of threonine may be protected, for example, by way of known esterification or etherifica~ion.
As examples of groups suitable for said esterification, there may be mentioned lower alkanoyl groups (e.g. acetyl), aroyl groups (e.g. benzoyl), and groups derived from carbonic acid, such as benzyloxycarbonyl, ethyloxycarbonyl, etc.
As groups suitable for said etherification, there may be mentioned benzyl, tetrahydropyranyl, t-butyl, etc. The hydroxyl ~roup of threonine, however, need not necessarily be protected.
Methionine may be protected in the form of a sulfoxide.
Other preferred side chain protective groups for particular amino acids include; for tyrosine: benzyl, o-bromobenzyloxy-~L~71~3()0 carbonyl 9 2,6-dichlorobenzyl, isopropyl, cyclohexyl, cyclopentyl and acetyl; for histidine: benzyl, p-toluenesulfonyl and 2,4-dinitrophenyl; $or tryptophan: formyl.
According to the present invention, the production of ~he peptide amides is carried out using methylbenzhydrylamine ~polystyrene-divinyl benzene) resin as the solid support.
However, for the production of other types of peptides using the novel HBr, anisole, thioanisole cleavage/deprotecting composition of this invention, other conventional solid suppor~s to which the C-terminal amino acid i8 attached can be used. Suitable solid supports useful for the solid phase peptide synthesis are those materials which are iner~
to the reagentc and reaction conditions of the stepwise condensation-deprotection reactions, as well as being insoluble in the media used. Suitable solid supports are chloromethyl-polystyrene-divinylbenzene polymer, hydroxymethyl-polystyrene-divinylbenzene polymer functionalized, ~rosslinked poly-N-acrylylpyrrolidine resins, and the like, especially chloromethyl polystyrene-1% divinylbenzene polymer.
The N~-Boc-amino acid or similarly protected C-terminal amino acid is at~ached to the methyl benzhydrylamine resin by means of an N,N'-dicyclohexylcarbodiimide (DCC)/l-hydroxy-benzotriazole (HBT) mediated coupling for from sbout 2 to about 24 hours, preferably about 12 hours at a temperature of between about 10 and 50C., preferably 25C., in a solvent such as dichloromethane or DMF, preferably dichloromethane.
The attachment to the chloromethyl polystyrenedivinylbenzene type of resin is made by means of the reaction of the Nl-protected amino acid, especially the Boc-amino acid, as its cesium, tetramethylammonium, triethylammonium, 4,5-diaza-bicyclo-[S.4.0]undec-5-ene, or similar salt in ethanol, ... 3L~71~ 3 acetonitrile, N,N-dimethylformamide (DMF), and the like, especially the cesium salt in DMF, with the chloromethyl resin at an ~leva~ed temperature, for example, between about 40 and 60C., preferably about $0C for from about 12 to S 48 hours, preferably about 24 hours. The removal of the Nl-protecting groups may be performed in the presence of, for example, a solution of trifluoroacetic acid in methylene chloride, hydrogen chloride in dioxane, hydrogen chloride in acetic acid, or other strong acid solution, preferably 50% trifluoroacetic acid in dichloromethane at about ambient temperature. The coupling of successive protected amino acids can be carried out in an automatic polypeptide synthesizer as well known in the art. Each protected amino acid i5 preferably introduced in approximately 2.5 or more molar exces~ and the coupling may be carried out in dichloromethane, dichloromethane/DMF mixtures, DMF and the like, especially in methylene chloride at about ambient temperature. Other solven~s which are known to be useful for the purpose of peptide-forming condensation reactionJ for example, dimethyl-sulfoxide, pyridine, chloroform, dioxane, te~rahydrofuran, ethyl acetate, N-methylpyrrolidone, etc., as well as suitable mixtures thereof may also be used.
The reaction temperature for the condensation/coupling reaction may be selected from the range known to be useful for the purpose of peptide-forming condensation reactions.
Thus, it may normally be within the range of about -40C
to about 60C, and preferably, abou~ -20C to about 0C.
The coupling agent is normally DCC in dichloromethane but may be N,N'-di-iso-propylcarbodiimide or other carbodiimide ether alone or in the presence of HBT, N-hydroxysuccinimide, ethyl 2-hydroxyimino-2 cyanoacetate, other N-hydroxyimides or oximes. Alternatively, protected amino acid active esters ~` ~ ~7~
(e.g. p-nitrophenyl, pentafluorophenyl and the like) or symmetrical anhydrides may be used.
The coupling, deprotection/cleavage reactions and preparation of derivatives of the polypeptides are suitably carried out at temperatures between about -10 and ~50C, most preferably about 20-25C. The exact temperature for any particular reaction will, of course, be dependent upon the substrates, reagents, solven~s and so forth, all being well within ~he skill of the practitioner. The fully deprotecte polypeptide may then be purified by a sequence of chromatographi steps employing any or all of the following types: ion exchange on a weakly basic resin in the acetate form; gel permeation chromatography, e.g. on Sephadex G-25; hydrophobic adsorption chromatography on underivatized polystyrene-divinyl-benzene (for example, Amberlite XAD~; silica gel adsorption chromatography; lon exchange chromatography, e.g. on Sephadex G-25, or countercurrent distribution; high performance liquid chromatography (HPLC), especially reverse phase HPLC on octyl- or octadecylsilyl-silica bonded phase column packing.
In another specific embodiment of the present invention, the N-terminal 14 amino partial sequence of thymosin ~1.
Ac-Ser-Asp--Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-OH
(thymosin ~l-Nl_l4) is synthesized on benzyl ester resin.
The protected thymosin al-Nl_l4-resin is deprotected and cleaved by treatment with hydrogen bromide, trifluoroacetic acid, anisole and thioanisole in a manner similar to that described above for thymosin ~1 Purification of the crude product by preparative high pressure liquid chromatography yields a pure thymosin ~l-Nl_l4 compound which is found to be identical to a reference compound synthesized previously by the fragment condensation method in solution.
2"Amberlite XAD" is a trademark of Rohm and Haas Company of Philadelphia, Pennsylvania, U.S.A.
This inv~ention is further illustrated by the following non-limiting examples.
Example 1 Methylbenzhydrylamine Resin 50.9 gm of polystyrene resin beads (copolystyrene-1%-divinyl benzene, 200-400 mesh beads) was suspended in 500 ml of dichloroethane. The mixture was cooled in an ice-bath with gentle mechanical s~irring until the temperature went below 5C.
15.5 g of p-toluoyl chloride and 13.3 g of aluminum chloride were mixed in 250 ml of dichloroethane in a dropping funnel. The solution was added dropwise to the cooled, ~tirred suspen~ion of the resin bead~ over a period of about 40 min., care being taken not to allow the reaction to warm up above 5C. The stirring was continued for 4 hours at room temperature when the resin was washed sequentially with lsopropanol, isopropanol-water (1:1 mixture), isopropanol and dried to give 53.8 g of p-toluoyl resin. This resin was then mixed with 168 g of ammonium formate, 201 ml of formamide, 134 ml of formic acid and 350 ml of nitrobenzene.
The mixture was gradually heated up to 165-170C under reflux and maintained for 1 day during which time about 115 ml of the aqueous phase was collected into a Dean-Stark trap.
The resin was washed and dried as above to yield 55.5 g of N-formyl me~hylbenzhydrylamine resin. The product was hydrolyzed in a mixture of 300 ml each of 12 N HCl and isopropan 1 under reflux for 3 hr. Washing and drying the resulting resin provided 54.9 g of the hydrochloride form of methylbenz-hydrylamine resin. It showed a very strong positive reaction to ninhydrin reagent and incorporated 0.4 mmol of Boc-Ala-OH
when neutralized and coupled with Boc-Ala-OH in the presence of dicyclohexylcarbodiimide.
~ ~'7~
Ac-Ser-Asp-Ala Ala-Val-Asp.Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-V~l-Vfll-Glu-Glu-~Ala-Glu-Asn-OH
(Thym~sin ~1) 5.02 gm of methylbenzhydrylamine resin obtained in Example 1 was placed in a peptide synthesis flask equipped with a sintered glass filter at the bottom and a mechanical stirrer at the top. The resin wa~ washed with 20 volumes of 10% triethylamine in methylene chloride and stirred with a fresh portion of the same solution for 10 min. The neutralize methylbenzhydrylamine resin was then reacte~ with Boc-L-aspartic acid a-benzyl ester (1.95 g, 6 mmol) and dicyclohexylcarbodiimid (1.24 g, 6 mmol) for 2 hours to form Boc-~-benzyl-L-aspartic 8cyl methylbenzhydrylamine resin. The resin turned from ninhydrin positive tc ninhydrin negative after this reaction.
The solid phase peptide synthesis was then continued by performing the following steps wherein in each cycle one amino acid was incorporsted sequentially into the growing peptide chain on the resin:
(1) prcwash with 40% trifluoroacetic acid in CH2C12;
(2) stir for 28 minutes with 40% trifluoroacetic acid;
(3) three washings with CH2C12;
(4) prewash with 10% triethylamine in CH2C12;
(5) stir for 5 minutes with 10% ~riethylamine;
(6) wash three times with CH2C12;
(7) stir for 120 minutes with 6 mmol each of Boc-Glu(OBzl)-OH and DCC;
(8) wash once with CH2C12;
(9) wash three times with 50% i-propanol in CH2C12;
(10) wash three times with CH2C12;
. 1;~
~11) test~for ninhydrin color reaction; if positive, repeat eteps 7-11; if negative, go to the next synthetic cycle.
The syntheti~ cycle was repeated using the following amino acids sequentially and one a t a time in step 7 of each cycle: Boc-Ala-OH, Boc-Glu(OBzlj-OH, Boc-Glu(OBzl)-OH, Boc-Val-OH, ~oc-Val-OH, Boc-Glu(OBzl)-OH, Boc-Lys~ClZ)-OH, Boc-Lys(ClZ)-OH, Boc-Glu(OBzl)-OH, Boc-Lys(ClZ)-OH, Boc-Leu-OH, Boc-Leu-OH, Boc-Asp(OBzl)-OH, Boc-Lys(ClZ)-OH, Boc-Thr(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Ile-OH, Boc-Glu(O~zl) OH, Boc-Ser(Bzl)-OH, Boc-Ser(Bzl~-OH, Boc-Thr(Bzl)-OH, Boc-Asp(OBzl)-OH, Boc-Yal-OH, Boc-Ala-OH, Boc-Ala-OH, Boc-Asp(OBzl)-OH, Boc-Ser(Bzl)-OH, and CH3COOH. The protec~ed thymosin l-resin so obtained, Ac-Ser(Bzl)-Asp(OBzl)-Ala-Ala-Val-Asp(OBzl)-Thr(Bzl) Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-Ile-Thr(Bzl)-Thr(Bzl)-Lys(ClZ)-Asp(OBzl)-Leu-Lys(ClZ)-Glu(OBzl)-Lys(ClZ)-Lys(ClZ)-Glu(OBzl)-Val-Val-Glu-(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asp(HN-CH(C6H4-CH3)-C6H4-resin)-OBæl, weighed 1608 gm. Part (1.01 gm) of this material was mixed with 2 ml each of anisole and thioanisole and 16 ml trifluoroacetic acid. A gentle stream of dry HBr gaæ was bubbled through the magnetically stirred suspension for 60 minutes at room temperature. The excess acids were then removed by evaporation at 40C in a rotary evaporator and the residue washed with ether several times to remove the excess anisole and thioanisole. The peptide was extracted into 1% a~monium scetate (2 x 25 ml) and the solution desalted on a Sephadex G-10 column (2.6 x 9S cm) using 0.1 M acetic acid as eluant, monitored at 230 nm. The material present in the major peak was lyophil~zed to give 0.33 g of crude product which on purification on preparative high pressure liquid chromatography (Clg reverse phase column, eluted with 10-11.25% CH3CN in pH 5.0 potassium phosphate buffer) ~.~ 7~ 3~
provided 0.10 g of pure thymosin ~ has the amino acid composition o: A~p, 4.00; Thr, 3.12; Ser, 2.64; Glu, 6.00;
Ala, 2.92; Val, 1.92; Ile, 1.04; Leu, 0.96; Lys, 3.98 (hydrolysi in 6 N HCl, 110C, 24 hr). The product migrated identically in analytical HPLC with the reference compound prepared by the fragmetlt condensa~ion method in ~olution.
Example 3 Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-OH
(Thymosin l-Nl-14) 0 200 gm of Boc-Lys~ClZ)-OCH2-C6H~-resin was placed in a peptide synthesis flask and the solid phase synthesis carried out as described in Example 2, wi~h 3 mmol each of DCC and the following amino acids in step 7 in Example 2 of each cycle: Boc-Thr(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Ile-OH, Boc-Glu(OBzl)-OH, Boc-Ser(Bzl)-OH, Boc-Ser(Bzl)-OH, Boc-Thr(Bzl) O
Boc-Asp(OBzl)-OH, Boc-Val-OH, Boc-Al 8- OH, Boc-Ala-OH, Boc-Asp(OBzl)-OH, Boc-Ser(Bzl)-OH and CH3COOH. The protected thymosin l-Nl_l4-resin, Ac-Ser(Bzl)-Asp(OBzl)-Ala-Ala-Val-Asp-(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-lle-Thr(Bzl)-Thr(Bzl _ Lys(ClZ)-OCH2-C6H4-resin, weighed 3.28 gm. Part of the so obtained material ~1.0 gm) was mixed with 2 ml each of anisole and thioanisole and 16 ml TFA. Dry HBr gas was gently bubbled through the mixture under magnetic stirring for 60 minutes at room temperature. Evaporation of the acids and extraction of the peptide in a manner similar to that described above in Example 2 afforded 0.32 gm of crude product which on purification by preparative HPLC
(polystyrene PRP-l column, with 20% CH3CN in 0.05% TFA as eluant) gsve 0.21 gm of the desired thymosin 1-Nl_l4.
Its amino acid composition is ~sp, 2.00; Thr, 3.17; Ser, .
r,~, 2.75; Glu, 0.99; Ala, 2.00; Val, 0.97; Ile, 0,94; Lys, 1.07 (hydrolysis in 6 N HCl, 110C, 24 hr). It migrated identically with the reference compound prep~red by the fragment condensatio method on analytical HPLC.
. 1;~
~11) test~for ninhydrin color reaction; if positive, repeat eteps 7-11; if negative, go to the next synthetic cycle.
The syntheti~ cycle was repeated using the following amino acids sequentially and one a t a time in step 7 of each cycle: Boc-Ala-OH, Boc-Glu(OBzlj-OH, Boc-Glu(OBzl)-OH, Boc-Val-OH, ~oc-Val-OH, Boc-Glu(OBzl)-OH, Boc-Lys~ClZ)-OH, Boc-Lys(ClZ)-OH, Boc-Glu(OBzl)-OH, Boc-Lys(ClZ)-OH, Boc-Leu-OH, Boc-Leu-OH, Boc-Asp(OBzl)-OH, Boc-Lys(ClZ)-OH, Boc-Thr(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Ile-OH, Boc-Glu(O~zl) OH, Boc-Ser(Bzl)-OH, Boc-Ser(Bzl~-OH, Boc-Thr(Bzl)-OH, Boc-Asp(OBzl)-OH, Boc-Yal-OH, Boc-Ala-OH, Boc-Ala-OH, Boc-Asp(OBzl)-OH, Boc-Ser(Bzl)-OH, and CH3COOH. The protec~ed thymosin l-resin so obtained, Ac-Ser(Bzl)-Asp(OBzl)-Ala-Ala-Val-Asp(OBzl)-Thr(Bzl) Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-Ile-Thr(Bzl)-Thr(Bzl)-Lys(ClZ)-Asp(OBzl)-Leu-Lys(ClZ)-Glu(OBzl)-Lys(ClZ)-Lys(ClZ)-Glu(OBzl)-Val-Val-Glu-(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asp(HN-CH(C6H4-CH3)-C6H4-resin)-OBæl, weighed 1608 gm. Part (1.01 gm) of this material was mixed with 2 ml each of anisole and thioanisole and 16 ml trifluoroacetic acid. A gentle stream of dry HBr gaæ was bubbled through the magnetically stirred suspension for 60 minutes at room temperature. The excess acids were then removed by evaporation at 40C in a rotary evaporator and the residue washed with ether several times to remove the excess anisole and thioanisole. The peptide was extracted into 1% a~monium scetate (2 x 25 ml) and the solution desalted on a Sephadex G-10 column (2.6 x 9S cm) using 0.1 M acetic acid as eluant, monitored at 230 nm. The material present in the major peak was lyophil~zed to give 0.33 g of crude product which on purification on preparative high pressure liquid chromatography (Clg reverse phase column, eluted with 10-11.25% CH3CN in pH 5.0 potassium phosphate buffer) ~.~ 7~ 3~
provided 0.10 g of pure thymosin ~ has the amino acid composition o: A~p, 4.00; Thr, 3.12; Ser, 2.64; Glu, 6.00;
Ala, 2.92; Val, 1.92; Ile, 1.04; Leu, 0.96; Lys, 3.98 (hydrolysi in 6 N HCl, 110C, 24 hr). The product migrated identically in analytical HPLC with the reference compound prepared by the fragmetlt condensa~ion method in ~olution.
Example 3 Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-OH
(Thymosin l-Nl-14) 0 200 gm of Boc-Lys~ClZ)-OCH2-C6H~-resin was placed in a peptide synthesis flask and the solid phase synthesis carried out as described in Example 2, wi~h 3 mmol each of DCC and the following amino acids in step 7 in Example 2 of each cycle: Boc-Thr(Bzl)-OH, Boc-Thr(Bzl)-OH, Boc-Ile-OH, Boc-Glu(OBzl)-OH, Boc-Ser(Bzl)-OH, Boc-Ser(Bzl)-OH, Boc-Thr(Bzl) O
Boc-Asp(OBzl)-OH, Boc-Val-OH, Boc-Al 8- OH, Boc-Ala-OH, Boc-Asp(OBzl)-OH, Boc-Ser(Bzl)-OH and CH3COOH. The protected thymosin l-Nl_l4-resin, Ac-Ser(Bzl)-Asp(OBzl)-Ala-Ala-Val-Asp-(OBzl)-Thr(Bzl)-Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-lle-Thr(Bzl)-Thr(Bzl _ Lys(ClZ)-OCH2-C6H4-resin, weighed 3.28 gm. Part of the so obtained material ~1.0 gm) was mixed with 2 ml each of anisole and thioanisole and 16 ml TFA. Dry HBr gas was gently bubbled through the mixture under magnetic stirring for 60 minutes at room temperature. Evaporation of the acids and extraction of the peptide in a manner similar to that described above in Example 2 afforded 0.32 gm of crude product which on purification by preparative HPLC
(polystyrene PRP-l column, with 20% CH3CN in 0.05% TFA as eluant) gsve 0.21 gm of the desired thymosin 1-Nl_l4.
Its amino acid composition is ~sp, 2.00; Thr, 3.17; Ser, .
r,~, 2.75; Glu, 0.99; Ala, 2.00; Val, 0.97; Ile, 0,94; Lys, 1.07 (hydrolysis in 6 N HCl, 110C, 24 hr). It migrated identically with the reference compound prep~red by the fragment condensatio method on analytical HPLC.
Claims (2)
1. In a solid phase synthesis of the thymosin ?1, polypeptide having the sequence:
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH by forming the polypeptide having the above sequence anchored to a resin, said polypeptide optionally having one or more protected functional groups; and cleaving the polypeptide from the resin and deprotecting protected functional groups of the polypeptide the improvement comprising using methylbenzhydrylamine resin and cleaving and deprotecting the polypeptide by contacting the polypeptide with hydrogen bromide, anisole, thioanisole and trifluoroacetic acid and wherein the anisole and thioanisole are present in a volume ratio of about 4:1 to about 1:4 with respect to each other.
Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH by forming the polypeptide having the above sequence anchored to a resin, said polypeptide optionally having one or more protected functional groups; and cleaving the polypeptide from the resin and deprotecting protected functional groups of the polypeptide the improvement comprising using methylbenzhydrylamine resin and cleaving and deprotecting the polypeptide by contacting the polypeptide with hydrogen bromide, anisole, thioanisole and trifluoroacetic acid and wherein the anisole and thioanisole are present in a volume ratio of about 4:1 to about 1:4 with respect to each other.
2. The process of claim 1 wherein the volume ratio o anisole to thioanisole is about 1:1.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US722,218 | 1976-09-10 | ||
| US72221885A | 1985-04-11 | 1985-04-11 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1271300A true CA1271300A (en) | 1990-07-03 |
Family
ID=24900940
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000506495A Expired CA1271300A (en) | 1985-04-11 | 1986-04-11 | Solid phase process for synthesizing peptides |
Country Status (2)
| Country | Link |
|---|---|
| CA (1) | CA1271300A (en) |
| IL (1) | IL78479A0 (en) |
-
1986
- 1986-04-11 CA CA000506495A patent/CA1271300A/en not_active Expired
- 1986-04-11 IL IL78479A patent/IL78479A0/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| IL78479A0 (en) | 1986-08-31 |
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