WO2001051505A1

WO2001051505A1 - Nα-2-(4-NITROPHENYLSULFONYL)ETHOXYCARBONYL-AMINO ACID FLUORIDES AND PROCESS FOR THE PREPARATION THEREOF

Info

Publication number: WO2001051505A1
Application number: PCT/KR1999/000810
Authority: WO
Inventors: Hack-Joo Kim; Weonu Chweh; Young-Cheol Kim
Original assignee: Hyundai Pharmaceutical Ind. Co., Ltd.
Priority date: 1999-12-24
Filing date: 1999-12-24
Publication date: 2001-07-19
Also published as: AU1894000A

Abstract

There are provided by the present invention new Nα-2-(4-nitrophenylsulfonyl)ethoxycarbonyl-amino acid fluorides as described in the general formula (I) wherein R1 represents hydrogen and R2 may represent hydrogen, isopropyl, 2-methylpropyl, tert-butoxymethyl, benzyl, 2-(tert-butoxycarbonyl)ethyl, 4-(tert-butoxycarbamido)butyl or 4-tert-butoxybenzyl, and process for the preparation thereof. By using the amino acid fluorides according to the present invention, a certain desired peptide may be easily synthesized without activation step.

Description

Nα-2-(4-NITROPHENYLSULFONYL)ETHOXYCARBONYL-AMINO ACID FLUORIDES AND PROCESS FOR THE PREPARATION THEREOF

TECHNICAL FIELD

The present invention provides a process for preparing a protected amino acid derivative for solid phase peptide synthesis, i.e. a Nα-protected amino acid fluoride obtained starting from a Nα-2-(4-nitrophenysulfonyl)ethoxycarbonyl-amino acid (Nα- Nsc-amino acid) by substituting a carboxyl group at the terminal of various Nα- protected amino acids with fluoride in solid phase peptide synthesis. The compound thus produced is a Nα-2-(4-nitrophenysulfonyl)ethoxycarbonyl-amino acid (Nα-Nsc- amino cid) fluoride having the following formula (I):

in which

R, represents hydrogen; and

R₂ represents hydrogen, isopropyl, 2-methypropyl, tert-butoxymethyl, benzyl, 2-(tert- butoxycarbonyl)ethyl, 4-(tert-butoxycarbamido)butyl or 4-tert-butoxybenzyl.

BACKGROUND ART

Solid phase peptide synthesis is widely employed for the preparation of biologically active peptides, which are used in medical and biological research and also as active substance in pharmacy, veterinary and diagnostics. The essence of solid phase peptide synthesis can be outlined as a stepwise elongation of a peptide chain by means of repeated cycles of chemical reactions, beginning from the first C-terminal amino acid attached to an insoluble carrier. During the course of the synthesis target products of all reactions remain bound to the carrier, whereas excessive reactants and side-products are removed by filtration and washing of the carrier.

In order to perform the solid phase synthesis of a peptide, the first amino acid (C-terminal of the target amino acid sequence) with the protected α-amino group is linked covalently to an insoluble polymeric carrier through the free carboxyl group by ester or amide bond formation. Then, Nα-protective group is selectively cleaved from thus obtained Nα-protected aminoacyl-polymer to form the aminoacyl-polymer with the free α-amino group. This polymer is further acylated with the next Nα-protected amino acid to obtain Nα-protected dipeptide-polymer. Such synthetic cycles, which consist of Nα-protection cleavage and of subsequent acylation of free amino group with following Nα-protected amino acid, are repeated until the assembly of target amino acid sequence is completed.

In practical solid phase synthesis, since a large molar excessive amount (2 to 10-fold) of acylating agent is usually employed to assure a complete conversion, all reactive groups in side chains of the amino acids, such as amino, carboxyl, hydroxyl, thiol, guanidino groups, should be blocked with appropriate protective groups. The protective groups for this purpose must be selected carefully to provide reliable and permanent protection of the side chains under conditions of peptidyl -polymer acylations and during the cleavage of temporary Nα-protection. On the other hand, these side- chain protective groups must provide the opportunity to deprotect the synthesized peptide in one or two stages quantitatively and without damage of its structure. In most cases, the peptidyl-polymer linkage also should be cleaved simultaneously. It is evident that the structure and the chemical properties of permanent protective groups for side-chains of amino acids are determined not only by the nature of reactive function to be protected but in a great extent by the structure and the chemical properties of the employed temporary Nα-protective group. Therefore, temporary Nα-protection is the key element of the whole strategy of solid phase peptide synthesis.

For the purpose of solid phase peptide synthesis, Nα-tert-butoxycarbonyl amino acids (Boc-amino acids) are well known and widely used (R. B. Merrifield, Biochemistry, 1964, N. 3, p. 1385). Tert-butoxycarbonyl (Boc) group can be removed by the action of acidic reagents of medium strength, such as, for example, trifluoroacetic acid and its solutions in chlorinated hydrocarbons, solutions of hydrogen chloride in organic solvents, boron trifluoride/diethyl ether complex and some other acids, to form isobutylene and carbon dioxide.

Together with temporary Να-Boc-protection, a protective group is employed for permanent blocking of side chains, which is stable during Να-Boc cleavage but can be cleaved by a stronger acidic reagent with the simultaneous fission of peptidyl- polymer bond. The known reagents, which can be used for this purpose include liquid hydrogen fluoride, trifuoromethanesulfonic acid and their mixtures with anisole, thioanisole or dimethylsulfide. The main drawback of the synthetic strategy with the use of temporary Να-Boc-protection is the application of an acid hydrolysis for the cleavage of both temporary and permanent protective groups, which cannot provide a complete stability of permanent protection. As the length of synthesized peptide grows, a permanent protective group experiences the repeated action of acidic reagents during a Boc cleavage step, which can result in a partial loss of these groups and the accumulation of by-products. Otherwise, the final treatment of peptidyl-polymer complex with a superacidic reagent may cause a partial destruction of the target peptide. Due to an extremely hazardous property of superacids, it should also be noticed that superacids must be handled with a special equipment and an appropriate safety measures.

To avoid the use of superacidic reagents for deprotection of the final peptides, it is regarded that several acid-sensitive groups recently proposed as a temporary Να- protective group are suitable to a permanent side-chain protection in the so-called tert- butyl type, which can be cleaved by acidic reagents of medium strength. As a typical example of such Nα-protective groups, l-(3,5-di-tert-butylphenyl)-l- methylethoxycarbonyl (t-Bumeoc) group has been disclosed in the literature (Collect. Czech. Chem. Commun., 1992, V. 57, p. 1707). Nα-tert-Bumeoc group can be cleaved by 1% trifluoroacetic acid dissolved in dichloromethane and used together with a permanent protective group in the form of t-butyl cleavable by neat trifluoroacetic acid or its concentrated solutions. In this case, although the use of superacids can be avoided, a general principle of differential acid hydrolysis still remains unchanged.

Another approach to accomplish the strategy of solid phase peptide synthesis has been made R. B. Merrifield in Science, 1986, V. 232, p. 341. This approach, called "orthogonality principle", is based on the assumption that temporary and permanent protective groups can be removed by totally distinct reagents according to totally different chemical mechanisms, so that temporary Nα-protection can be cleaved with an absolute selectivity to provide the complete preservation of permanent protection, and vice versa. At the present time, the "orthogonality principle" has been commonly accepted as a guideline for the development of efficient strategies of solid phase peptide synthesis.

As an example of implementation of the "orthogonality principle" the use of Nα-dithiasuccinylamino acid (Dts-amino acid) in solid phase synthesis has been described in Int. J. Peptide and Protein Res., 1987, V. 30. p. 740. Nα-dithiasuccinyl (Dts) protective group is quite resistant to acidic reagents of medium strength and is cleaved smoothly by thiol reagents in neutral media with the liberation of amino group and formation of carbon thiooxide. Application of Dts-amino acid in practical synthesis is still limited due to the lack of effective methods for their preparation.

The most widely known and employed strategy of solid phase synthesis according to the "orthogonality principle" is based on the use of Nα-9- fluorenylmethoxycarbonylamino acids (Fmoc-amino acids), as described by C. D. Chang and J. Meienhofer in Int. J. Peptide and Protein Res., 1975, N. 11, p. 246. Να- 9-fluorenylmethoxycarbonyl (Fmoc) group is resistant to acidic reagents and is cleaved according to the β-elimination mechanism by organic bases in aprotic solvents, for example, by morpholine diethylamine, piperazine, or piperidine in dimethylformamide (DMF) or dichloromethane, amino group being liberated and dibenzofulvene being formed together with CO₂. In solid phase synthesis the cleavage of Fmoc group is preferably performed by the treatment of Να-protected peptidyl-polymer with 20 to 50% piperidine solution in DMF during 10 to 30 minutes. Said conditions can allow to use a permanent acid-sensitive protection of t-butyl type together with temporary Να- Fmoc-protection, thus providing the "othogonality" of the synthetic strategy.

Να-Fmoc-amino acids have been widely used in automatic and semi-automatic synthesizer of all types, as well as in manual solid phase peptide synthesis. However, due to an extreme base sensitivity and instability in neutral aprotic solvents of Να- Fmoc-protective group the acylation conditions and further the purity of used solvents should be carefully controlled. Special care should be taken when Να-Fmoc-amino acids are used for the synthesis of peptides exceeding 30 residues in length. In addition, a relatively high production cost prevents the use of Fmoc-derivatives in a large scale peptide preparation.

For such reasons, it has still been required to develop the novel Να-protected amino acid derivatives useful for effective strategy of solid phase peptide synthesis. To satisfy such requirement Να-2-(4-nitrophenylsulfonyl)ethoxycarbonyl-amino acids (Nα-Nsc-amino acids) have been developed and WO 06/25394 relating to their preparation and their application to solid phase peptide synthesis has been registered as a patent in US, Europe, Japan, Australia and Korea, of which the right is owned by Hyundai Pharmaceutical Co., Ltd. as the applicant of the present application. The study relating to them has been continuously and extensively conducted, and as a result, WO 98/17638 relating to Nα-Nsc-amino acids, which were not included in WO 96/25394. is in progress of patent examination procedure in several countries in the world.

In the above patents and numerous references, it has been identified that Nsc- amino acids used as the novel starting material in the present invention have a superior stability and efficiency in comparison to the prior Fmoc-amino acids, and therefore, in applying to the actual synthetic procedures can increase the stability and efficiency and provide a good yield to secure the economic advantage.

Numerous studies have continuously been made by other researchers to secure the superiority in solid phase peptide synthesis. According to this, an effort to make up for the problems involved in the synthesis of peptides using Fmoc-amino acids as mentioned above and to obtain the substances providing an equivalent effect and efficacy has been made and thus, the study about Fmoc-amino acid fluorides in order to solve the problem of impurities produced during activation step and a long process time in case of the synthesis using Fmoc-amino acids has been disclosed in several reports (Carpino, et al., Ace. Chem. Res. 1996, V29, -268-274). However, such study could never be commercialized due to the problem of stability of the materials. As one alternative, which can overcome the problem that in spite of the advantage of synthesis Fmoc-amino acid fluorides could not be commercialized due to their poor stability and storability, the present invention has been made.

Nα-Nsc-amino acid fluorides used in the present invention are obtained from fluorination of Nα-Nsc-amino acids and using the same the method for obtaining the desired peptides by a direct coupling of amino acids without any activation step, which is regarded as their unique advantage corresponding to Nα-Nsc-amino acids in solid phase peptide synthesis, has been explored.

DETAILED DESCRIPTION OF INVENTION The synthesis of numerous peptides using Nα-Nsc-amino acids has been successfully progressed under general condensing conditions. Such synthetic method is disclosed, of course, in the above-mentioned patents together with its specific examples and further, the matters related to them are reported in numerous papers as the product of study. The matters of such study takes advantages of the stability and relatively increased hydrophilic property of Nα-Nsc-amino acids to improve the major problem of the prior synthetic methods, which may be caused during the procedure for obtaining the desired synthesized product by cleaving Nα-protected amino acid halides with water. Thus, since Nα-Nsc-amino acids can be readily synthesized, and are cheap and economically useful, they are necessarily required for application to a peptide synthesis using a solid support.

The superior utility of Nα-Nsc-amino acids are under commercialization so as to be readily used by many persons and further, an effort to design techniques for solid phase peptide synthesis comparable or superior to them are in progress. As a result of such a series of study, some cases showing the result of synthesis in a high efficiency but in a lower stability in comparison to Nα-Nsc-amino acids have been reported. In conventional peptide synthesis, the reaction was conducted by addition of Nα-Nsc- amino acids and activating agents. However, due to the presence of activating agents the products obtained after completion of the reaction may contain many impurities, which are required to separate by washing and removing. Such separation step is, of course, not difficult to practice, but requires the consumption of time and lowers the relative yield of the desired product.

The present invention provides a momentum for synthesis of peptides, which were technically difficult to practice, by adding fluorides to Nα-Nsc-amino acids through an easy fluorination to obtain Nα-Nsc-amino acid fluorides for increasing the utility of Nα-Nsc-amino acids, and then directly applying Nα-Nsc-amino acid fluorides thus obtained to the actual synthesis of peptides. In case of the synthesis using Nα- Nsc-amino acids to which fluorides are added, the synthetic reaction can be readily proceeded without addition of any activating agent and the reaction products are obtained in a high purity. Contrary to this, the reaction product obtained from the synthetic reaction practiced by simply adding activating agents to Nα-Nsc-amino acids contains some impurities remained therein and therefore, are produced in a lower yield. Of course, in case that the synthesis is practiced by adding activating agents, the reaction is more stably proceeded in comparison to the synthesis only using Nα-Nsc- amino acid fluorides so that the peptide can be prepared in a large scale. However, due to one additional step for activation the synthesis may be regarded as being somewhat disadvantageous in view of the time required for synthesis.

According to the present invention, peptides having amino acids chain which were very difficult to synthesize according to the peptide synthesis using the well- known prior urethane-protected amino acid halides, particularly, Fmoc-amino acids or Fmoc-amino acid-halides, can be readily synthesized by applying Nα-Nsc-amino acid halides, to show an increase in convenience and efficiency of peptide synthesis.

First, the method for synthesis of Nα-Nsc-amino acid fluorides can be represented by the following reaction scheme:

Scheme 1 fluorinating agent

organic solvent (ID (I)

In the above reaction scheme, R, represents hydrogen and R₂ represents hydrogen, isopropyl, 2-methylpropyl, tert-butoxymethyl, benzyl, 2-(tert- butoxycarbonyl)ethyl, 4-(tert-butoxycarbamido)butyl or 4-tert-butoxybenzyl. In order to synthesize the desired product according to the above reaction scheme, the relevant Nsc-amino acid is used as the starting material to which a fluorinating agent, for example, cyanuric fluoride (3.0 eq.) and pyridine (1.0 eq.), in an organic solvent, for example, dichloromethane (DCM) or tetrahydrofuran (THF) solvent is added to proceed the reaction. This fluorination reaction is completed within about 30 minutes at room temperature.

As the solvent, dichloromethane (DCM) can be generally used except for Nsc-

Gly-OH, Nsc-Phe-OH and Nsc-Leu-OH, which have a slight solubility in dichloromethane (DCM) solvent. In addition, in order to avoid the aggregation of reactants during the reaction it is useful to dilute the solvent concentration from 0.04 M to 0.02M.

The completion of this reaction can be confirmed first by a thin layer chromatography (TLC) and then by a high performance liquid chromatography (HPLC) in absolute methanol solvent after conversion into a methyl ester. The yield of the product is about 70% to 95% and the purity is about 90% or more.

Nα-Nsc-amino acid fluoride derivatives prepared by fluorination of Nsc-amino acids exhibit some differences in a nuclear magnetic resonance (NMR). Thus, the product containing fluoride group can be very readily analyzed by discovering the peak around 1840 cm^"' according to an infrared (IR) spectroscopy.

The storage and preservation are important to fluoride derivatives. Nsc-amino acid fluorides can be stored and preserved under nitrogen atmosphere or in a refrigerator for about one month without decomposition. It is regarded that such storage stability is far superior to that of the fluoride derivatives of other Nα-protected amino acids, particularly Fmoc-amino acid-fluorides. In addition, in view of the stability in solvent, it can be identified that Nsc-amino acid fluorides can be retained in DMF for 24 hours or more. Table 1. Definition of R, and R₂ in the compounds of formula (I)

The utility of Nα-Nsc-amino acid fluorides thus prepared can be proved as follows. First, in actual synthesis Nα-Nsc-amino acid fluorides can readily accomplish the synthesis of peptides, which are difficult to synthesize by means of Fmoc-protected amino acid fluorides. Examples of such peptides include Leu-enkephalin, A-VI-5, β- amyloid peptide (16-20), etc. It could be observed that peptides thus synthesized exhibit more increased activity.

The protocol of such reactions is as follows. All of the coupling reactions were carried out in the presence of DMF and the deprotection was carried out in 1 % DBU-20% piperidine/DMF solvent. This is the standard protocol, in which the condensation reaction is conducted for 40 minutes and the deprotection is conducted for 14 minutes (10 minutes/3 minutes/1 minute). PEG-PS resin was used as the solid phase support and the synthetic reactions were conducted following the manual for a synthesizer (Pioneer model). Finally, a suitable scavenger was added in the presence of trifluoroacetic acid (TFA) and then, the reaction mixture was stirred for about 2 hours to terminate the cleavage reaction.

Specific examples of the synthesis accomplished through the above reactions can be shown in the following.

EXAMPLE 1

Synthesis of Nsc-Val-F

374 mg (1 mmol) of Nsc-Nal-OH was dissolved in 5 ml of dichloromethane (DCM), and 266 μl (3 mmol) of cyanuric fluoride and 81 μl (1 mmol) of thoroughly dried pyridine were added thereto. The reaction mixture was then stirred under nitrogen atmosphere for 30 minutes.

Water-soluble white precipitate was filtered off and the filtrate was immersed in saline-cooled ice water and then filtered. The residue was absorbed into dried anhydrous magnesium sulfate (MgSO₄) g to remove any retained moisture and then filtered. The filtrate was evaporated and dried. The organic layer was recrystallized from dichloromethane (DCM)-hexane solution to obtain the white solid (Yield: 82%). The physical properties of Nsc-amino acid fluorides thus synthesized are summarized in the following Table 2.

Table 2. Physical properties of Nsc-amino acid fluorides

Note)

a measuring apparatus: BUCHI 531 model

b JASCO DIP 1000 model / 25°C

c JASCO FT/IR 300E model / KBr-Pellet

d After conversion into methyl ester by treatment with anhydrous MeOH, identification by HPLC

EXAMPLE 2

Synthesis of Nsc- lv-F

332.3 mg (1 mmol) of Nsc-Gly-OH was added to 50 ml of dry dichloromethane (DCM) and then stirred in suspension. 266 μl of cyanuric fluoride and thoroughly dried pyridine were added and the reaction mixture was then stirred for 3 hours under nitrogen. The reaction was terminated by addition of ice to the reaction solution and the organic layer was extracted with dichloromethane (DCM), dried over anhydrous magnesium sulfate (MgSO₄), filtered and then concentrated. The concentrate was treated with dichloromethane (DCM)-diethyl ether to obtain the white solid (Yield: 79.2%).

EXAMPLE 3

Synthesis of Nsc-Phe-F

422.4 mg (1 mmol) of Nsc-Phe-OH was added to 50 ml of dry dichloromethane (DCM) and then stirred in suspension. 266 μl of cyanuric fluoride and 81 μl of thoroughly dried pyridine were added and the reaction mixture was then stirred for 6 hours under nitrogen. The reaction solution was concentrated and then, ethyl acetate and ice were added to the residue. The organic layer was extracted with ethyl acetate, dried over anhydrous magnesium sulfate (MgSO₄) and then concentrated. The concentrate was treated with dichloromethane (DCM) to obtain the white solid (Yield: 84.9%).

EXAMPLE 4

Synthesis of Nsc-Leu-F

388.4 mg (1 mmol) of Nsc-Leu-OH was added to 50 ml of dry dichloromethane (DCM) and then stirred in suspension. 266 μl of cyanuric fluoride and 81 μl of thoroughly dried pyridine were added and the reaction mixture was then stirred for 6 hours under nitrogen. The reaction solution was concentrated and then, ethyl acetate and ice were added to the residue. The organic layer was extracted with ethyl acetate, dried over anhydrous magnesium sulfate (MgSO₄) and then concentrated. The concentrate was treated with diethyl ether and petroleum ether to obtain the white solid (Yield: 70%). EXAMPLE 5

Synthesis of Nsc-TyrftBuVF

422.4 mg (1 mmol) of Nsc-Tyr(tBu)-OH was added to 50 ml of dry dichloromethane (DCM) and then stirred in solution. 266 μl of cyanuric fluoride and

81 μl of thoroughly dried pyridine were added and the reaction mixture was then stirred for 6 hours under nitrogen. The reaction was terminated by addition of ice to the reaction solution and the organic layer was extracted with dichloromethane (DCM). The extract was dried over anhydrous magnesium sulfate (MgSO₄), concentrated and then treated with diethyl ether to obtain the white solid (Yield: 95.2%).

EXAMPLE 6

Synthesis of Nsc-Lys(Boc F

503.5 mg (1 mmol) of Nsc-Lys(Boc)-OH was dissolved in 5 ml of dry dichloromethane (DCM). 266 μl of cyanuric fluoride and 81 μl of pyridine were added thereto and the reaction mixture was then stirred for 30 minutes under nitrogen atmosphere. The white solid produced during the reaction was filtered off and ice water was introduced into the filtrate to separate only the dichloromethane (DCM) layer, which was then dried over magnesium sulfate (MgSO₄) dried with dichloromethane (DCM), filtered and then evaporated. The residue was recrystallized from dichloromethane (DCM)-hexane solution to obtain the white solid (78%).

EXAMPLE 7

Synthesis of Nsc-Glu(OtBu)-F

460.5 mg (1 mmol) of Nsc-Glu(OtBu)-OH was dissolved in 5 ml of dry dichloromethane (DCM). 266 μl of cyanuric fluoride and 81 μl of pyridine were added thereto and the reaction mixture was then stirred for 30 minutes under nitrogen atmosphere. The white solid produced during the reaction was filtered off and ice water was introduced into the filtrate to separate only the dichloromethane (DCM) layer, which was then dried over magnesium sulfate (MgSO₄) dried with dichloromethane (DCM), filtered and then evaporated. The residue was recrystallized from dichloromethane (DCM)-hexane solution to obtain the white solid (81%).

EXAMPLE 8

Synthesis of Nsc-Ser(tBuVF

418.4 mg (1 mmol) of Nsc-Ser(tBu)-OH was dissolved in 5 ml of dry dichloromethane (DCM). 266 μl of cyanuric fluoride and 81 μl of pyridine were added thereto and the reaction mixture was then stirred for 30 minutes under nitrogen atmosphere. The white solid produced during the reaction was filtered off and ice water was introduced into the filtrate to separate only the dichloromethane (DCM) layer, which was then dried over magnesium sulfate (MgSO₄) dried with dichloromethane (DCM), filtered and then evaporated. The residue was recrystallized from dichloromethane (DCM)-hexane solution to obtain the white solid (84%).

EXAMPLE 9

Synthesis of Leu-enkephalin using Nsc-amino acid fluorides

Sequence : Tyr-Gly-Gly-Phe-Leu

Resin : Fmoc-Leu-Peg-PS (0.2 mmol/g) used 100 mg

Coupling condition : 2 excess amino acid fluoride without base Reaction media : DMF (reaction volume 1 ml)

Deprotection : 1% DBU-20%) piperidine in DMF (5 min/1 min/1 min)

TFA-cleavage : 95% TFA / 2.5% water / 2.5% phenol

Fmoc-Leu-Peg-PS resin was introduced into a Ramps-kit reaction vessel, activated with 2 ml of dichloromethane (DCM) and then thoroughly washed with DMF. 1 ml of deprotection solvent was added and then Ramps-kit reaction vessel was well shaken for 5 minites. The reaction solution was then filtered and then 1 ml of deprotection solvent was added again. The reaction solution was shaken again for one minute and then filtered. 1 ml of deprotection solvent was added again and then, the reaction solution was shaken again for one minute and then filtered. The respective filtrates were separately preserved in order to observe the deprotection process.

After completion of the deprotection, resin was washed three times with DMF, three times with dichloromethane (DCM), three times with MeOH and then three times with DMF. 17 mg (0.04 mmol) of Nsc-Phe-F was dissolved in 1 ml of DMF, introduced into the reaction vessel and then well shaken for 20 minutes. Then, the reaction mixture was washed three times with DMF, three times with dichloromethane (DCM), three times with MeOH and then three times with DMF. The deprotection step (De-Nsc) and condensation step were repeated two times in case of Nsc-Gly-F and one time in case of Nsc-Tyr(tBu)-F. After Nsc was finally removed, the reaction solution was well washed and then dried. 5 ml of TFA-cleavage solution was added to well dried resin and then, the mixture was allowed to stand for 2 hours, concentrated to remove THF and then treated with excess diethyl ether to obtain the white powder after filtration (Yield: 75%).

EXAMPLE 10 Synthesis of A-VI-5 peptide using Nsc-amino acid fluorides

Sequence : H-Val-Glu-Ser-Ser-Lys-OH

Resin : Fmoc-Lys(Boc)-PEG-PS

Loading : 0.18 mmol/g

Amount : 200 mg

Excess reagent : 5 times

Base : 1 equivalent DIEA

Coupling time : 20 minutes

Deblocking time : 10 minutes ( 1 % DBU / 20% piperidine / DMF)

Amino acid fluorides as used

Nsc-Val-F : 338 mg

Nsc-Ser(tBu)-F : 756 mg

Nsc-Glu(otBu)-F : 415 mg

After completion of the synthesis, the final cleavage reaction was conducted using TFA and the reaction solution was filtyered. To the resulting solution was added cold anhydrous ether to obtain the white solid (72%).

EXAMPLE 11

Synthesis of β-amyloid peptide using Nsc-amino acid fluorides Sequence : H-Lys-Leu-Nal-Phe-Phe-OH

Resin : Fmoc-Phe-PEG-PS

Loading : 0.21 mmol/g

Amount : 100 mg

Excess reagent : 5 times

Base : No base

Coupling time : 40 minutes

Deblocking time : 15 minutes (1% DBU / 20% piperidine / DMF)

Amino acid fluorides as used

Nsc-Nal-F : 395 mg

Νsc-Leu-F : 357 mg

Νsc-Lys(Boc)-F : 531 mg

After completion of the synthesis, the final cleavage reaction was conducted using TFA and the reaction solution was filtyered. To the resulting solution was added cold anhydrous ether to obtain the white solid (75%).

Claims

1. A Nα-2-(4-nitrophenylsulfonyl)ethoxycarbonyl-amino acid fluoride derivative represented by the following formula (I):

in which

R, represents hydrogen; and

2. A process for preparing Nα-2-(4-nitrophenylsulfonyl)ethoxycarbonyl-amino acid fluoride derivatives represented by the following formula (I), which comprises fluorinating a Nsc-amino acid of formula (II) in an organic solvent:

Nα -Nsc-NR]-CHR₂-COOH Nα -Nsc-NRι-CHR₂-COF

(II) (I)

wherein R, and R₂ are defined as in claim 1.

3. The process as defined in claim 2, characterized in that the organic solvent is dichloromethane or tetrahydrofuran and an agent for fluorination is cyanuric fluoride and pyridine.

4. A process for synthesis of peptides in a solid phase by using a Nα-2-(4- nitrophenylsulfonyl)ethoxycarbonyl-amino acid fluoride derivative represented by the following formula (I) without activation step:

wherein R, and R, are defined as in claim 1.