WO1996017872A1 - Intermediate for producing surfactant peptide - Google Patents

Abstract

A peptide derivative having the sequence as specified herein below: Xaa-Pro-Val-Xbb-Xcc-Lys-Arg, (wherein Xaa is absent or represents Cys or Ser; Xbb represents His or Asn; and Xcc represents Leu or Ile). It can readily and efficiently provide a peptide highly soluble in methanol, etc., and the obtained surfactant peptide can readily be compounded with a lipid mixture and hence is suitable for preparing a lung surfactant. As the surfactant has a good suspensibility and a potent surface activity, it is useful as a remedy for respiratory distress syndrome.

Description

 Description Intermediate for the production of surfactant surfactants

 The present invention relates to peptide derivatives. More specifically, the present invention relates to a peptide derivative which is an intermediate for producing a synthetic peptide useful as an active ingredient for treating respiratory distress syndrome. Background art

 Respiratory distress syndrome is a disease in which the surface activity of the alveolar surface is reduced due to lack of pulmonary surfactant and the alveoli collapse, resulting in severe respiratory distress. Is high. It is known that pulmonary surfactant preparations are effective for respiratory distress syndrome in newborns. In recent years, as apoproteins specific to mammalian lung surfactant, lipophilic surfactant topoprotein A and surfactant topoprotein D, and hydrophobic surfactant topoprotein B (hereinafter referred to as “SP-B”). And surfactant Apoprotein C (hereinafter referred to as “SP-c”) [Reviews on the structure and function of apoproteins (Toyoaki Akino, Yoshio Kuroki, Respiration and Circulation, Vol. 38, No. 1) No. 8, pp. 722, 1990; edited by Hiroki Yasuda et al., Biosurfactants, Chapter 2 Surfactant biochemistry — Surfactants and apoproteins, pp. 131, 1990, Inc. (Science Forum)].

Human lung-derived SP-C [SEQ ID NO: 1] is an apoprotein with 35 amino acids, a rich N-terminal amino acid, Val-rich, and extremely strong hydrophobicity. In addition, the lungs of pest [SEQ ID NO: 2], pig [SEQ ID NO: 3], rat, etc. The isolated SP-C also consists of 34 to 35 amino acids, and the amino acid sequence at the N-terminal varies depending on the animal species, but has very high homology to humans.

 The present inventors have described below a hydrophilic peptide portion having a specific sequence which is a partial structure of SP-C on the N-terminal side, and a hydrophobic peptide portion mainly composed of Leu and / or Nle on the C-terminal side. Synthetic peptides containing amino acid sequences can be easily isolated and purified, can be manufactured in large quantities, can be formic acid, trifluoroacetic acid (TFA), trifluoroethanol, Dissolves well in dimethyl sulfoxide (DMSO), form-of-cloth, mixed form-of-form-methanol, methanol, ethylenechlorohydrin or tetrahydrofuran. High, even if the lung surfactant prepared from the mixture of the synthetic peptide and the lipid is produced by a conventional freeze-drying method without a suspending agent and at -20 ° C or lower. Turbidity He knew that it had good and strong surface activity, and filed a patent application (PCT / JP94 / 02057).

 Xaa-Pro- Val-Xbb-Xcc-Lys- Arg- W

(Xaa is absent or represents Cys or Ser, Xbb represents His or Asn, Xcc represents Leu or lie, and W represents a hydrophobic peptide moiety.)

 The present invention is used as an intermediate for the production of the above-mentioned synthetic peptide (hereinafter referred to as “surfactant peptide”) which exhibits a strong surface activity by being combined with a lipid mixture. It is a peptide derivative in which the N-terminal and functional side chain of a hydrophilic peptide represented by the following specific sequence are protected (hereinafter, referred to as “the peptide derivative of the present invention”).

Xaa-Pro-Val-Xbb-Xcc-Lys-Arg (Xaa is absent or represents Cys or Ser, Xbb represents His or Asn, and Xcc represents Leu or lie.)

 There are two methods for chemical production of peptides: the stepwise elongation method and the fragment condensation method. The fragment condensation method is easier to purify the target compound and is more suitable for large-scale synthesis than the sequential elongation method. It is a manufacturing method and has the feature that loss due to accidental failure can be prevented. The peptide derivative of the present invention is a peptide derivative capable of easily producing surfactant peptide by condensing with a previously prepared hydrophobic peptide moiety.

The protecting group for the N-terminus and the functional side chain of the peptide derivative of the present invention is not particularly limited as long as it is a protecting group used in usual peptide synthesis. The protecting groups of 9-fluorenylmethyloxycarbonyl (Fmoc), 2-chlorobenzyloxycarbonyl (2-CLZ) or t-butyloxycarbonyl (Boc) are the protective groups of Lys. Boc, carbobenzoxy (Z) or tosyl (Tos) groups as bases, Trt, Fmoc, Boc, Dnp, Bom, Bzl or Tos groups as His protecting groups, and Arg protecting groups as Mtr, Pmc, Mts or Tos groups. That is, specific examples of the peptide derivative of the present invention include Fmoc-Pro-Val-His (, rt) -Leu-Lys (, Boc) -Argi _> tr), Fmoc-Pro-Val-Asn-Leu-Lys ( (Boc) -Arg (Mtr), Fmoc-Pro-Val-Asn-Ile-Lys (Boc) -Arg (Mtr) or Fmoc-Cys (Acm)-Pro-Val-His (Trt)-Leu-Ly s (Boc )-Arg (Mtr).

Surfactant peptide produced from the peptide derivative of the present invention and a hydrophobic peptide portion is mixed with choline phosphoglyceride, acidic phospholipid and fatty acid as a lipid mixture. Thus, a pulmonary surfactant useful as a therapeutic agent for respiratory distress syndrome can be produced. BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, the present invention will be described in more detail with reference to Examples.

 In the following examples, the molecular weight of the synthesized peptides was measured by the fast atom bombardment method (FABMS). The mass spectrometer used was JMS-S102A (produced by Honden Electronics Co., Ltd.), and the ion source used was a cesium gun (lOKeV).

 [Example 1] Fmoc-Pro-Vat His (Trt) -Leu-Lys (Boc) -Arg (Mtr) Solid phase synthesis of the title peptide derivative A using a peptide synthesizer system 9050 (Millipore) did.

 As the first resin, N-α-9-fluorenylmethyoxycarbonyl mono-ω- 4-methoxy-2,3,6, -trimethylbenzenesulfonyl-arginine (Fmoc-Arg (Mtr)) N-α-91-fluorenylmethyloxycarbonyl combined with methoxy-41-alkoxybenzyl alcohol-resin (Sasrin resin, trade name of Bachem, Inc.) ω— 4-Methoxy-2. 3, 6, — Trimethylbenzenesulfonyl arginine 0-resin (Fmoc—Arg (Mtr) —〇—resin) (0.20 mmol) and a peptide synthesizer system According to the synthesis protocol of Model 9050, amino acid was sequentially extended in the N-terminal direction on the resin while monitoring, to synthesize a peptide 0-resin in which the N-terminal and the functional group were completely protected.

Thereafter, the completely protected peptide resin was washed five times with methanol and dried under reduced pressure. A TFA-dichloromethane (1:99, V / V) solution was added to the dried peptide-0-resin (330 mg) while stirring under ice cooling, and the mixture was stirred under ice cooling for 30 minutes. After stirring for 90 minutes at room temperature, the peptide was cut out of the resin with the protecting group attached, and a glass filter (G3) And filtered. The filtrate was concentrated under reduced pressure to about 5 ml by an evaporator, and ethyl ether was added to precipitate the peptide. The peptide precipitate was collected by filtration with a glass filter (G3), washed five times with getyl ether, and dried under reduced pressure to obtain 180 mg of peptide derivative A.

 This crude peptide derivative A was added to TFA-dichloromethane (1:99, V

/ V) solution to prepare a 10 mg / ml sample solution, which is purified by HPLC using an Asahipak GS-510 (021.5 x 500 mm) column (trademark, manufactured by Asahi Kasei Corporation), The peptide derivative A was collected. As the eluent, a TFA-dichloromethane (1 '· 99, V / V) solution was used and eluted at a flow rate of 8. lmlZmin for 120 minutes. The presence of the peptide in the eluate was monitored by 245 nm (spectrophotometer; JASCO Corporation Model 870-UV) and a differential refractometer (Shimadzu Corporation Model RID-6A).

 FABMS (M + H-); 1527.0 (calculated molecular weight; 1525.8) [Example 2] Fmoc-Cys (Acm)-Pro-Val-His (Trt)-Leu-Lys

(Boc)-Arg (Mtr)

 The title peptide derivative B was synthesized and purified by a peptide synthesizer system 9050 (manufactured by Millipore) in the same manner as in Example 1.

 FABMS (M + H-); 1700.0 (calculated molecular weight; 1698.8) IR (KBr, cm '): 332.5.1, 1675-0, 17333.9

_{Ή - NMR (CDC1 3, ppm} ): 0. 76, 0. 85 (gem - CH 3 1. 38 1.95, 2.06 (one _{CH 3), 1. 63 (_} CH -), 2 55-3.23 (one CH ₂ —), 2.69 (aromatic-CH _a ), 3.00 (—〇CH ₃ ), .78 (one COCH ₃ ), 7.05-7.60 (aromatic -Η)

 [Reference Example 1] Synthesis of surfactant protein C (solid phase method)

樹脂 -α-Fluoronylmethyloxycarbonyl Luleucine-0-resin (Fmoc-Nle-0-resin) (0.20 mmol Z0.5 g) was used. After swelling the resin with DMF for 20 minutes, the resin was washed four times with DMF. A 20% piperidine-DMF solution was added and shaken to perform deprotection. This operation was repeated three times to completely perform the deprotection. The resin was then washed 9 times with DMF to remove excess piperidine in the resin. At this time, the residue of piperidine was confirmed with pH test paper.

 Then, add DMF (6 ml), Fmoc-Nle (0.5 mmol), N-hydroquinquinzotriazole (0.5 mmol) and N.N 'diisopropylpropyl posiimide (0.5 mmol) and shake for 90 minutes. Then, a condensation reaction was performed. The resin was then washed four times with DMF to remove excess reagent. Confirmation of this condensation reaction was performed by Kaiser test by the ninhydrin method.

 Thus, according to the synthesis plan, the amino acid was sequentially extended in the N-terminal direction on the resin, and H-Nle- (Nle-Nle-0-resin) was synthesized by a multipeptide solid-phase synthesis system.

Next, after adding DMF to the synthesized H-Nle- (Nle) ₁₄ -Nle-0-resin, the peptide derivative A of Example 1 was added to N-hydroxyquinobenzotriazole and N, N'-diisopropylcarpoide. The mixture was added and shaken for 8 hours, and the condensation reaction was performed twice. The confirmation of the condensation reaction was carried out by a force test using the ninhydrin method.

Then, to the obtained peptide-0-resin, a 20% solution of pyridin-DMF was added to deprotect the F-moc protecting group at the N-terminal. It was washed six times with methanol and dried under reduced pressure. While stirring the dried peptide-0-resin (100 mg) under ice cooling, m-cresol (0.2 ml), 1,2-ethanedithiol (0.5 ml), thioanisole (1 2 ml), TFA (7.5 ml) and trimethylsilyl bromide (4 ml), and the mixture was stirred under ice-cooling for 120 minutes to obtain a functional side chain. The peptide was cut out from the resin together with the deprotection, and filtered through a glass filter (G3). The filtrate was concentrated under reduced pressure to about 5 ml by an evaporator, and getyl ether was added to precipitate the peptide. The peptide precipitate was taken out using a glass filter (G3), washed with getyl ether five times, dried under reduced pressure, and further purified by HPLC to obtain a surfactant peptide of [SEQ ID NO: 4]. C was prepared.

FABMS (M + H ⁺ ); 256.2 (calculated molecular weight; 255.3). [Reference Example 2] Synthesis of surfactant peptide D (liquid phase method)

 H-Leu- (Leu), prepared in the same manner as in Reference Example 1. -Leu-0-resin is cut out of the resin and purified to H-Leu- (Leu) ,. -Leu-OH was obtained.

FABMS (M + H ⁺ ); 137.0 (calculated molecular weight: 137.8.8) Dissolve 2.75 g (0.2 mmol) of this peptide in 600 ml of methylene chloride under ice-cooling, and add 3 ml of concentrated sulfuric acid. Then, 300 ml of isobutylene was blown in and left at room temperature for 65 hours. After completion of the reaction, the reaction solution is neutralized with an aqueous solution of sodium hydrogen carbonate, the organic layer is washed with water, dried over sodium sulfate, and the solvent is concentrated under reduced pressure to give a protected C-terminal carboxyl group (H—Leu— (Leu), „-Leu-OBt) 2.08 g (90% yield).

FABMS (M + Η '); 143.1 (calculated molecular weight; 1431.9) Then, 2.4 g (0.15 mol) of peptide derivative B was dissolved in 500 ml of dimethylformamide (DMF) under ice-cooling. Then, N, N'-diisop pillcarbosimid 0.3 g (0.15 mmol), N-hydroxysuccinimide 0.17 g (0.15 mmol) and H-Leu- (Leu) ,. 2.15 g (0.15 mmol) of Leu-OBt was added, and the mixture was reacted at -20 ° C for 1 hour and at room temperature for 10 hours. After the completion of the reaction, the precipitated dicyclourea was removed by filtration, and water was added to the filtrate. The product was extracted with a black hole form, washed sequentially with a 10% aqueous sodium hydrogen carbonate solution, a 0.1N aqueous hydrochloric acid solution and distilled water, and the solvent was distilled off under reduced pressure. Then, after recrystallization from ethanol / water (4: 1), the crystals were dried under reduced pressure to obtain 3.58 g of a condensate (yield: 80%).

 FABMS (Μ + Η '); 3115.2 (Calculated molecular weight; 3113.9) The protecting group of each functional group of this condensate peptide is 1% (V / V) indole and TFA containing 0.1% (V / V) ethanediol, and the Acm group was prepared according to the method of Fujii et al. (J. Chemical Soc. Chem. Commun., No. 5, 283 (1989) Deprotection was performed according to)) to obtain crude surfactant peptide D of “SEQ ID NO: 5”. Purification was performed in the same manner as in Example 2 to prepare pure surfactant peptide D.

 FABMS (M + H); 2211.3 (Calculated molecular weight: 2209.9) [Amino acid composition analysis]

 The surfactant peptides obtained in Reference Examples 1 and 2 were treated with 12N hydrochloric acid-TFA containing 5% (v / v) phenol [2: 1 (V / V)] at 150 ° C under vacuum. After acid hydrolysis for 2, 4, 6, 12, 24, 48 and 72 hours to remove the acid, the hydrolysis products were analyzed by Shimadzu Amino Acid Automatic Analysis System (LC-9A). In the hydrolysis for 1 to 72 hours, the amino acid value showing higher recovery was adopted, and the amino acid composition value was calculated. The value almost coincided with the calculated value.

 (Reference Example 3)

1,2-dino-noremitoyl glycerol (3) -phosphocholin (21 Omg), 1-noreno-tomi-one 2-oleoyl-1 sn-glycero (3) -phospho-1L-serine (90.Omg) ) And palmitic acid (33.Omg) were dissolved in a chloroform solution (4: 1 (VZV)) (100 ml), and surfactant peptide C of SEQ ID NO: 4 (11 Omg) was dissolved in TFA (0. 5 ml). These solutions were mixed and evaporated to dryness under reduced pressure. The resulting residue The suspension was suspended in a water-ethanol mixture [9: 1 (V / V)] (110 ml) at 45 ° C for 25 minutes. This suspension was frozen at 55 ° C and dried at 100-120 Hg vacuum for 28 hours to give 348.7 mg of a white powder of lung surfactant.

 No residual ethanol was found in this powder, and the content of each component with respect to the total weight of the surfactant was 1,0.2-dipalmitoylglycerol (3) -phosphocholin was 60.2% (W / W), 1—Palmi Toileu 2—Age Reoyl-sn—Glycerol (3) —Phospho-L-Serine 25.8% (WZW), Palmitic Acid 9.5% (W / W), Surfactant The peptide C was 3.2% (W / W) and water 1.3% (W / W). The following table shows the results obtained by measuring the surface activity and the alveolar cavity volume maintenance effect of this lung surfactant by the method described in W093 21225. Surface activity of pulmonary surfactant and maintenance of alveolar cavity volume

Industrial applicability

According to the peptide derivative of the present invention as described above, Highly efficient peptides can be produced easily and in large quantities. The pulmonary surfactant prepared from the surfactant peptide and the lipid mixture has a good suspension property and a strong surface activity, and thus is useful as a therapeutic agent for respiratory distress syndrome.

1

 11 SEQUENCE LISTING SEQ ID NO: 1

 Array length: 35

Sequence type: amino acid

 Topology: linear

Sequence type: peptide

Array

 Phe Gly lie Pro Cys Cys Pro Val His Leu Lys Arg Leu Leu lie Val 1 5 10 15

Val Val Val Val Val Leu He Val Val Val lie Val Gly Ala Leu Leu

 20 25 30

 Met Gly Leu

 35

SEQ ID NO: 2

Array length: 34

Sequence type: amino acid

 Topology: linear

Sequence type: peptide

Array

 Leu lie Pro Cys Cys Pro Val Asn lie Lys Arg Leu Leu lie Val Val

1 5 10 15

Val Val Val Val Leu Leu Val Val Val lie Val Gly Ala Leu Leu Met

 20 25 30

Gly Leu SEQ ID NO: 3

Array length: 35

Sequence type: amino acid

 Topology: linear

Sequence type: peptide

Array

 Leu Arg lie Pro Cys Cys Pro Val Asn Leu Lys Arg Leu Leu Val Val

1 5 10 15

Val Val Val Val Val Leu Val Val Val Val He Val Gly Ala Leu Leu

 20 25 30

 Met Gly Leu

 35

SEQ ID NO: 4

Array Length: 22

Sequence type: amino acid

Tona: Rossi '-: Linear

Array type ^ Petit

Array

 Pro Val His Leu Lys Arg Nle Nle Nle Nle Nle Nle Nle Nle Nle Nle 1 5 10 15

Nle Nle Nle Nle Nle Nle

 20

SEQ ID NO: 5

Array length: 19

Sequence type: amino acid

Topology: linear Sequence type: peptide

Array

 Cys Pro Val His Leu Lys Arg Leu Leu Leu Leu Leu Leu Leu Leu Leu 1 5 10 15 Leu Leu Leu

Claims

The scope of the claims 1. A peptide derivative represented by the following specific sequence in which the N-terminus and functional side chain of the peptide are protected.  Xaa-Pro-Val-Xbb-Xcc-Lys-Arg (Xaa is absent or represents Cys or Ser, Xbb represents His or Asn, Xcc represents Leu or lie.)  2. Sequence power, Fmoc-Pro-Val-His (Trt)-Leu-Lys (Boc) -Arg (Mtr), Fmoc-Pro-Val-Asn-Leu-Lys (Boc) -Arg (Mtr), Fmoc- Claim 1 which is Pro-Val-Asn-He-Lys (Boc)-Arg (Mtr) or Fmoc-Cys (Acm)-Pro-Val-His (Trt)-Leu-Lys (Boc)-Arg (Mtr). The peptide derivative of the above.