CA2046124A1 - Cyclic peptides - Google Patents
Cyclic peptidesInfo
- Publication number
- CA2046124A1 CA2046124A1 CA002046124A CA2046124A CA2046124A1 CA 2046124 A1 CA2046124 A1 CA 2046124A1 CA 002046124 A CA002046124 A CA 002046124A CA 2046124 A CA2046124 A CA 2046124A CA 2046124 A1 CA2046124 A1 CA 2046124A1
- Authority
- CA
- Canada
- Prior art keywords
- group
- indicates
- cys
- acm
- val
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Landscapes
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE
A novel peptide represented by formula (I):
Cyclo-(Cys-Val-d-Phe-Pro)2 (I) wherein Cys indicates a cysteine group, Val indicates a valine group, d-Phe indicates a d-phenylalanine group, Pro indicates a proline group, and Cyclo indicates a cyclic form. This peptide is formed by way of active esterification or azidation of an intermediate:
Boc-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-OME
wherein Boc indicates an amino group protected by a t-butyloxycarbonyl group, Acm indicates an SH group protected by an acetamidomethyl group, and OMe indicates a methoxylated carboxyl group.
A novel peptide represented by formula (I):
Cyclo-(Cys-Val-d-Phe-Pro)2 (I) wherein Cys indicates a cysteine group, Val indicates a valine group, d-Phe indicates a d-phenylalanine group, Pro indicates a proline group, and Cyclo indicates a cyclic form. This peptide is formed by way of active esterification or azidation of an intermediate:
Boc-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-OME
wherein Boc indicates an amino group protected by a t-butyloxycarbonyl group, Acm indicates an SH group protected by an acetamidomethyl group, and OMe indicates a methoxylated carboxyl group.
Description
~046~24 NOVEL CYCLIC PEPTIDES
B~CKGROUND OF THE INVENTION
Field of the Invention This invention relates to a novel peptide, a method of making the same, and intermediates used for making the same, and metal cluster complexes formed with the same.
Description of the Prior Art Recently, the peculiarities of metal complexes have been attracted considerable attention in wide fields such as medicals and functional materials. For example, they are widely used as means for transferring metals in organisms. Also, in the field of functional materials, characteristics of complexed-metals are used to develop new materials and their enzyme-like functions are used to develop catalysts. Thus, the metal complexes contribute to various technical fields.
Further, peptides themselves are widely used as diagnostic and therapeutic agents. For example, tuberactinomycin and gramicidin S are strongly antibacterial and known as antibiotics.
Also, there are many peptides which exhibit physiological activities with respect to plants of the higher orders and insects. For example, destruxin and beauvelicin show activity with respect to insects, which is based on antibacterial effects against filamentous fungi.
6~
It has been known that metals exist in organisms while being held by peptides, namel~, as a cluster. E'or example, iron exists as an active center of an iron-sulfur protein having the structure of [Fe4S4(S-Cys)4]n-and contributing to electron transport. So far, variousstudies have been conducted with respect to materials similar to the clusters existing in organisms, i.e.
synthetic clusters. However, the conventional synthetic clusters exhibit oxidation-reduction potentials lower than those of the clusters in organisms and functions similar to those of the latter cannot be expected from the former.
SUMMARY OF THE INVENTION
It is thus an object of the present invention to provide a novel peptide whic~ can form clusters which are functionally close to those in organisms.
It is another object of the present invention to provide a method of making the novel peptide.
It is further object of the present invention to provide novel intermediates for making the novel peptide.
It is still another object of the present invention to provide a novel metal cluster complex.
In order to provide the novel metal cluster-complex which satisfies the forgoing demands, the inventor has conducted deligent studies and found it necessary to meet the following conditions:
l)As described in C. W. Carter, Jr., et al, Proc.
Natl. Acad. Sci. USA, 69, p.3526 (1972), the cluster which exists at the active center of the iron sulfur protein is covered with a peptide chain formed by hydrophobic amino acids and their hydrophobic environment stabilizes the cluster. Accordingly, a hydrophobic environment is necessary.
B~CKGROUND OF THE INVENTION
Field of the Invention This invention relates to a novel peptide, a method of making the same, and intermediates used for making the same, and metal cluster complexes formed with the same.
Description of the Prior Art Recently, the peculiarities of metal complexes have been attracted considerable attention in wide fields such as medicals and functional materials. For example, they are widely used as means for transferring metals in organisms. Also, in the field of functional materials, characteristics of complexed-metals are used to develop new materials and their enzyme-like functions are used to develop catalysts. Thus, the metal complexes contribute to various technical fields.
Further, peptides themselves are widely used as diagnostic and therapeutic agents. For example, tuberactinomycin and gramicidin S are strongly antibacterial and known as antibiotics.
Also, there are many peptides which exhibit physiological activities with respect to plants of the higher orders and insects. For example, destruxin and beauvelicin show activity with respect to insects, which is based on antibacterial effects against filamentous fungi.
6~
It has been known that metals exist in organisms while being held by peptides, namel~, as a cluster. E'or example, iron exists as an active center of an iron-sulfur protein having the structure of [Fe4S4(S-Cys)4]n-and contributing to electron transport. So far, variousstudies have been conducted with respect to materials similar to the clusters existing in organisms, i.e.
synthetic clusters. However, the conventional synthetic clusters exhibit oxidation-reduction potentials lower than those of the clusters in organisms and functions similar to those of the latter cannot be expected from the former.
SUMMARY OF THE INVENTION
It is thus an object of the present invention to provide a novel peptide whic~ can form clusters which are functionally close to those in organisms.
It is another object of the present invention to provide a method of making the novel peptide.
It is further object of the present invention to provide novel intermediates for making the novel peptide.
It is still another object of the present invention to provide a novel metal cluster complex.
In order to provide the novel metal cluster-complex which satisfies the forgoing demands, the inventor has conducted deligent studies and found it necessary to meet the following conditions:
l)As described in C. W. Carter, Jr., et al, Proc.
Natl. Acad. Sci. USA, 69, p.3526 (1972), the cluster which exists at the active center of the iron sulfur protein is covered with a peptide chain formed by hydrophobic amino acids and their hydrophobic environment stabilizes the cluster. Accordingly, a hydrophobic environment is necessary.
2) The whole sturcture of the cluster which coordinates the ligand or at least a local structure thereof near the metal is distorted.
3) Since it is difficult for materials other than peptides to synthesize a large ligand having four coordinating sulfur molecules within its molecule, a novel peptide is necessary.
4) When a peptide having amino and carboxyl groups at its ends is used as a ligand, there is a possibility that they may contribute to coordination. Accordingly, the peptide should not have polar groups such as amino group and carboxyl group at its end.
5) ~hen a peptide has a relatively large structure, it becomes flexible and may form complicated mixtures during complexing. In this case, the evaluation of its function becomes difficult. Accordingly, the peptide should have a certain hardness.
The inventor has found that a cyclic peptide is quite excellent in satisfying the foregoing conditions.
Gramicidin Sl which is an antibiotic, is a well-known cyclic peptide. However, since it does not have any coordinating amino acid slde chain, it cannot coordinate metals. However, the inventor has found that, while ~6~
maintaining its maln chain structure, four cysteines can be introduced thereinto to form a novel cyclic peptide which can coordinate metals and satisfy the above-mentioned conditions. As the result of diligent studies, the present invention has been achieved.
Namely, the present invention provides:
1) a novel cyclic peptide represented by ~ormula (I) Cyclo-(Cys-Val-d-Phe-Pro)2 (I) wherei.n Cys indicates a cysteine group, Val indicates a valine group, d-Phe indicates a d-phenylalanine group, Pro indicates a proline group, and Cyclo indicates a cyclic form, whose constitutional formula is represented by formula (I'):
0 CH2Ph C - NH - CH - C - N
HCCH2SH '1--HN C = O
O=C NH
HN CH3 C = O
O=C NH
HN 3HC -C = O
O=C NH
~ HSCH2CH
N -C -CH -NH -C
' O CHzPh S:) 2) methods of making the same;
3) novel intermediates used for making the same as follows:
i) Boc-Val-Cys(Acm)-OMe wherein Boc indicates an amino group protected by a t-butyloxycarbonyl group, Acm indicates an SH groupprotected by an acetamidomethyl group, and OMe indicates a methoxylated carboxyl group, which also apply to the following:
ii) H-Val-Cys(Acm)-OMe wherein H indicates a freed amino group, which also applies to the following:
iii) Boc-Cys(Acm)-Val-Cys(Acm)-OMe iv) Boc-Cys(Acm)-Val-Cys(Acm)-OH
v) Boc-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-OMe vi) Boc-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-OH
vii) Boc-Cys(Acm)-Val-CyslAcm)-d-Phe-Pro-ONSu wherein ONSu indicates an N-oxysuccinimido group, which also applies to the followin~:
viii) H-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-ONSu or hydrochlorates thereof ix) Cyclo-[Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro]2 x) Boc-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-NHNH2 wherein NHNH2 indicates a hydrazidated carboxyl group xi) Boc-[Cys(Acm)-Val-CyS(Acm)-d-phe-pro]2 xii) Boc-[Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro]2NHNH2 xiii) H-[Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro]2NHNH2 or hydrochlorates thereof xiv) Cyclo-[Cys(HgCl)-Val-Cys(HgCl)-d-Phe-Pro] 2 Z~ ~6 and 4) a metal or heavy metal cluster complex of the above-mentioned novel cyclic peptide (I) formed by a reaction of said novel cyclic peptide with a cluster of a metal or heavy metal selected from the group consisting of mercury, cadmium, zinc, molybdenum, cobalt, nickel, iron, titanium, palladium, manganese, silicon, and calcium.
The novel cyclic peptide (I) in accordance with the present invention exhibits an antibacterial activity and can be useful in the fields of medicines and agricultural chemicals.
Also, the cluster complexes obtained from the novel cyclic peptide (I) can exhibit high oxidation-reduction potentials which are closer to those of the clusters in organisms as compared with the conventional synthetic clusters. Accordingly, they can be useful as active agents in organisms in the fields of medicines and functional materials.
The novel cyclic peptide (I) in accordance with the present invention can be made by various methods.
Industrially, the following methods are advantageous.
1) Method (a) While protection and unprotection of polar groups of amino acids are repeated, a sequential elongation method is used to form a peptide in which cysteines are protected, which peptide is represented by the following formula:
Boc-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-OMe Then, its methoxy group is changed to an OH group.
Thereafter, a free acid and N-oxysuccinimide are used so that the peptide is subjected to actlve esterification.
An active esterification method is used to effect dimerization of thus modified peptide (cf. Figure 1).
2) Method (b) ~he above-mentioned peptide:
Boc-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-OMe is subjected to an azidation method, namely, is reacted with hydrazine monohydrate to yield hydrazide and then is reacted with isoamyl nitrite under a weakly acid condition so as to be azidated. The azidated peptide is reacted with a free amino acid of the other synthesizing block, namly, the peptide represented by the following formula: -H-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-OMe HCl (Cf. Figure 2.) In both cases, since the SH-group of Cys in the cyclic peptide is protected by Acm, an HgCl-adduct is formed and then HgCl2 is removed to form the novel cyclic peptide (I).
The cluster complex in accordance with the present invention can easily be formed when an organic solvent solution of the novel cyclic peptide (I) is mixed with an organic solvent solution of a metal or heavy metal cluster.
~ ~4~a~
BRIEF DESCRIPTION CF THE DRA~INGS
Figure 1 is a flow sheet showing a scheme for synthesizing the novel cyclic peptide in accordance with the present invention, Cyclo-~Cys-Val-Cys-d-Phe-Pro) 2 (I), by an active esterification;
Figure 2 is a flow sheet showing synthesizing steps of that peptide by an azidation method;
Figure 3 is a graph showing the results of measurement of molecular weight of an intermediate in accordance with the present invention, Cyclo-[Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro]2 (14), by FD-MS;
Figures 4A and 4B are charts showing 500MHz lH-NMR
spectral data of the above-mentioned intermediate;
Figure 5 is a chart showing the results of measurement of 1H-lHCOSY2D-N~R of the above-mentioned intermediate;
Figure 6 is a chart showing 500MHz lH-NMR spectral data of the novel cyclic peptide in accordance with the present invention;
Figure 7 is a chart showing an absorption spectrum of the four-iron cluster itself used in Example 3;
Figure 8 is a graph showing titration curves of the four-iron cluster and peptide in Example 3; and Figure 9 is a chart showing the results of measurment of ultraviolet-visible light absorption spectra of solutions E, F, and G in Experiment 1.
DESCRIPTION OF THE PREFERRED EMBODIMENT
12~
The following examples wil] explain the present invention in detail.
Example 1 I) Making of Cyclo-[Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro~2 (14) by Active Esterification Method (Cf.
Figure 1) 1) Synthesis of Boc-Cys(Acm)-OH (1) H-Cys(Acm)-OH HCl (4.58g, 20mmol) was dissolved in 12ml of water.
To this solution, 4.20ml (20mmol) of triethylamine (abridged as "TEA" in the following) was added, while being stirred and cooled with ice. Then, the mixture was stirred at room temperature. Thereafter, dioxane solution (12ml) of 5.42g (20mmol) of 2-tertiary butyloxycarbonyloxyimino-2-phenylacetonitrile (abridged as "BOC-ON" in the following) was added dropwise thereto and the resulting mixture was stirred at room temperature for 3 hours. After the reaction, TEA was further added thereto to dissolve the insolubles. Then, the solvent was evaporated under a vacuum to leave a dried product.
It was dissolved in ethyl acetate and then was washed with water. The water layer was fùrther acidified and extracted with ethyl acetate. The extracted layer was dried with sodium sulfate anhydride. After the solvent was evaporated under a vacuum, the dried product was recrystallized in ether to yield the aimed white amorphous product (1).
Yield: 3.50g (59.9~) 2) Synthesis of H-Cys(Acm)-Me HCl (2) 26.75g (0.30mmol) of N-(hydroxymethyl)acetamide (abridged as "Acm" in the following) and 46.33g (0.27mmol) of H-Cys-OMe.HCl were dissloved in 70ml of S water. While the solution was cooled with ice, llml of 35% hydrochloric acid was added thereto. After the mixture was reacted at 0C for 2 hours, the gas in the reaction vessel was replaced with argon. Then the reaction vessel was left for five days. Vacuum evaporation was repeated with the addition of 20ml of ethanol anhydride to remove water from the mixture.
After 200ml of ethanol was added thereto and the insolubles were removed by filtration, the aimed product ~2) of a white crystalline powder form was recrystallized from ether.
Yield: 28.72g (43.8%) The physical properties of the product thus obtained were as follows:
lH-NMR(DMso-d6) ~DSS(Ppm)=l-88 (s, 9H, Acm CH3) 3.13 (d, 2H, Cys H~) 3.76 (s, 3H, OCH3) 3.82-4.56 (m, 3H, Cys Ha, Acm CH2) 8.20-9.20 (m, 2H, Acm HN, Cys HN) 3) Synthesis of Boc-Val-Cys(Acm)-OMe (3) 4.85g (20mmol) of the above-mentioned product (2) was dissolved in 50ml of N,N-dimethylformamide (abridged as "DMF" in the following). While the solution was cooled - 1.0-with ice and stirred, 2.19ml (20mmol) of N~
methylmorpholine (abridged as "NMM" in the following) was added thereto. After the mixture was stirred at room temperature for one hour, 4.35g (20mmol) of Boc-Val-OH, 2.97g (22mmol) of N-hydroxybenzotriazol (abridged as "HO~t" in the following), 4.13g (20mmol) of dicyclohexylcarbodiimide (abridged as "DCC" in the following) were added thereto in this order. Then the resulting mixture was sequentially stirred at -3C for 5.5 hours, at 0C for 12 hours, and at room temperature for 5.5 hours. After DMF was evaporated under a vacuum, 50ml of ethyl acetate was added to the resulting product.
Then the crystal d~eposited therefrom was separated by filtration, while the filtrate was sequentially washed with lM sodium hydrogencarbQnate aqueous solution, water, lM citric acid aqueous solution, and water. Thereafter, the washed filtrate was dried with sodium sulfate anhydride. The solvent was evaporated therefrom under a vacuum to leave a crude product. Then it was purified by a column chromatography to yield the aimed product (3) of a white powder form.
Yield: 4.52g (55.7~) The physical properties of the product thus obtained were as follows:
Elementary Analysis (~ by weight): Cl7H3lO6N3S
Empirical Value C:50.21, X:7.44, N:10.56 Calculated Value C:50.34, H:7.72, N:10.36 lH-NMR ( DMSO-d6 ) ~04~
(Ppm)-o-86 (m, 6H, Val HY) 1.40 (s, 9H, Boc CH3) 1.72-2.12 (m, 4H, Acm CH3, Val HP) 2.92 (m, 2H, Cys H~) 3.64 (m, 3H, OCH3) 3.82 (m, lH, Val H~) 4.00-4.62 (m, 4H, Cys Ha, Acm CH2) 6.60 (d, lH, Val HN) 8.00-8.68 (m,2H, Cys HN, Acm HN) 4) Synthesis of H-Val-Cys(Acm)-OCH3 (4) To 1.62g (4mmol) of the above-mentioned product (3), 30ml of 4M hydrochloric acid/dioxane was added. The resulting mixture was stirred at room temperature for 1.5 hours. Dioxane was evaporated therefrom under a vacuum to leave the aimed product (4~) of a white powder form.
5) Synthesis of Boc-Cys(Acm)-Val-Cys(Acm)-OMe (5) The above-mentioned product (4) was dissolved in 30ml of DMF. While the solution is cooled with ice and stirred, NMM (0.44g, 4mmol) was added thereto. Twenty minutes thereafter, Boc-Cys(Acm)-OH (1.17g, 4mmol), HOBt (0.59g, 4.4mmol), and DCC (0.83g, 4mmol) were added thereto in this order. Then the mixture was stirred at -3C for 5 hours, at 0C ~or 11 hours, and at room temperature for 5 hours. After DMF was evaporated under a vacuum, chloroform was added to the remaining product.
After the insolubles were separated by filtration, the filtrate was sequentially washed with lM sodium hydrogencarbonate aqueous solution, water, lM citric acid aqueous solution, and water. The extracted layer obtained after the washing was dried with sodium sulfate anhydride and the solvent was evaporated to leave a crude product. Then it was purified by a column chromatography to yield the aimed white amorphous product (5).
Yield: 1.67g (71.8%) The physical properties of the product thus obtained were as follows:
Elementary Analysis (% by weight): C23H4leN5S2 Empirical Value C:46.95, H:6.92, N:12.03 Calculated Value C:46.91, H:7.20, N:11.90 Remarks: Since 0.5mol of water of crystallization is considered to be contained therein, the value is that of C23H4lOgNsS2 0-5H2O-lH-NMR(DMso-d6) (PPm)=0-86 (m, 6H, Val Hr) 1.40 (s, 9H, Boc CH3) 1.60-2.20 (m, 7H, Acm CH3, Val H~) 2.46-3.00 (m, 4H, Cys H~) 3.64 (m, 3H, OCH3) 3.80-4.60 (m, 7H, Val Ha, Cys Ha, Acm CH2) 7.06, 7.54 (d, 2H, HN) 8.32-8.68 (t, 3H, Cys HN, Acm HN) 6) Synthesis of Boc-Cys(Acm)-Val-Cys(Acm)-OH (6) 1.47g (2.53mmol) of the above-mentioned product (5) was dissolved in 25ml of methanol. After lM sodium hydroxide aqueous solution was added thereto, the mixture was stirred at room temperature for 3 hours. Then, after 15ml of water was added thereto, methanol was evaporated from the mixture under a vacuum. After unreacted esters were removed by 20ml of ether, 30ml of lM citric acid aqueous solution was added to the water layer to weakly acidify the latter. Then, extraction was effected with ethyl acetate. The extracted material was dried with sodium sulfate anhydride and then the solvent was evaporated therefrom under a vacuum to leave the aimed white amorphous product (6).
Yield: 1.15g (80.2%) The physical properties of the product thus obtained were as follows:
[ a ]D=-25 . 7 (c 0.65, MeOH 23C) Elementary Analysis (% by weight): C22H39O8N5S2 Empirical Value -C:44.65, H:6.64, N:10.24 Calculated Value C:44.63, H:7.35, N:10.85 Remarks: The calculated value of C22H3gO8N5S2-0.5AcOEt-2H2o-lH-NMR(DMSO-d6) SDSS(Ppm)=0-88 (m, 6H, Val HY) 1.40 (s, 9H, Boc CH3) 1.60-2.20 (m, 7H, Acm CH3, Val H~) 2.56-3.00 (m, 4H, Cys H~) 3.80-4.60 (m, 7H, Val Ha, Cys Ha, Acm CH2) 7.08, 7.52 (d, 2H, HN) 8.16-8.68 (m, 4H, Cys HN, Acm HN) 12.2 (br, lH, OH) 7) Synthesis of Boc-d-Phe-OH (7) 9.90g (60mmol) of d-Phe-OH was added to 180ml of a dioxane-water (2:1) mixed solvent. Then, 60ml (60mmol) of lM sodium hydroxide aqueous solution was added thereto to dissolve d-Phe-OH. After 14.43g (66mmol) of di-(tertiary butyl)-dicarbonate ~abridged as "(BOC)2O" in the following] was added thereto, the mixture was stirred at room temperature for 3 hours. After dioxane was evaporated under a vacuum, 80ml of ethyl acetate was added to the remaining product which was being cooled with ice. After being acidified with 20ml of 5~
potassium hydrogensulfate aqueous solution, the mixture was separated. The resulting water layer was further extracted with ethyl acetate. The extracted layers were combined together and dried with sodium sulfate anhydride. The solvent was^evaporated therefrom under a vacuum to leave a crude product. Then, when it was recrystallized in ether-petroleum ether, the aimed product (7) of a white powder form was obtained.
Yield: 10.83g (68.1%) The physical properties of the product thus obtained were as follows:
H-NMR(CDCl~) S(Ppm)=l-4o (s, 9H, Boc CH3) 3.12 (d, 2H, Phe H~) 4.26-4.76 (m, lH, Phe Ha) 4.76-5.12 (m, lH, Phe OH) 6.88-7.56 (m, 5H, Phe C6H5) 8.72 (s, lH, Phe HN) ~6~2~
8) Synthesis of Boc-d-Phe-Pro-OMe (8) 3.31g (20mmol) of Pro-OMe.HCl was dissolved in 50ml of DMF. While the solution was cooled with ice and stirred, 2.19ml (20mmol) of NMM was added thereto. Ten minutes thereafter, 5.31g (20mmol) of the above-mentioned product (7), 2.97g (22mmol) of HOBt, 4.13g (20mmol) of DCC were added to the mixture in this order. The resulting mixture was sequentially stirred at -3C for 5 hours, at 0C for 10 hours, and at room temperature for 9 hours. Then, after DMF was evaporated from the mixture, 50ml of ethyl acetate was added thereto. After cooling, deposited crystals were separated by filtration. The filtrate was sequentially washed with lM sodium hydrogencarbonate aqueous solution, water, lM citric acid aqueous solution, and water. ~Then, the washed product was dried with sodium sulfate anhydride. Thereafter, the solvent was evaporated from the dried product to leave a crude product. Then, it was purified by a column chromatography to yield the aimed white amorphous product.
Yield: 6.73g (89.5%) The physical properties of the product thus obtained were as follows:
lH-NMR(DMSO-d6) ~DSS ( ppm)=1.32 (s, 9H, Boc CH~) 1.44-2.28 (m, 4H, Pro H~Y) 2.60-2.92 (m, 2H, Pro H~) 3.00-3.40 (m, 2H, Phe H~) 2046~4 3.60 (s, 3H, OCH3) 3.84-4.60 (m, 2H, Phe H~, Pro Ha) 6.60-7.60 (m, 6H, Phe C6H5, Phe HN) 9) Synthesis of H-d-Phe-Pro-OMe HCl (9) To 0.75g (2.Oml) of the above-mentioned product (8), 30ml of 4M hydrochloric acid/dioxane was added and the mixture was stirred at room temperature for 1.5 hours. Dioxane was repeatedly evaporated from the mixture under a vacuum, while several milliliters of ether was repeatedly added thereto. Thus, the aimed product (9) of a white powder form was quantitatively obtained.
The inventor has found that a cyclic peptide is quite excellent in satisfying the foregoing conditions.
Gramicidin Sl which is an antibiotic, is a well-known cyclic peptide. However, since it does not have any coordinating amino acid slde chain, it cannot coordinate metals. However, the inventor has found that, while ~6~
maintaining its maln chain structure, four cysteines can be introduced thereinto to form a novel cyclic peptide which can coordinate metals and satisfy the above-mentioned conditions. As the result of diligent studies, the present invention has been achieved.
Namely, the present invention provides:
1) a novel cyclic peptide represented by ~ormula (I) Cyclo-(Cys-Val-d-Phe-Pro)2 (I) wherei.n Cys indicates a cysteine group, Val indicates a valine group, d-Phe indicates a d-phenylalanine group, Pro indicates a proline group, and Cyclo indicates a cyclic form, whose constitutional formula is represented by formula (I'):
0 CH2Ph C - NH - CH - C - N
HCCH2SH '1--HN C = O
O=C NH
HN CH3 C = O
O=C NH
HN 3HC -C = O
O=C NH
~ HSCH2CH
N -C -CH -NH -C
' O CHzPh S:) 2) methods of making the same;
3) novel intermediates used for making the same as follows:
i) Boc-Val-Cys(Acm)-OMe wherein Boc indicates an amino group protected by a t-butyloxycarbonyl group, Acm indicates an SH groupprotected by an acetamidomethyl group, and OMe indicates a methoxylated carboxyl group, which also apply to the following:
ii) H-Val-Cys(Acm)-OMe wherein H indicates a freed amino group, which also applies to the following:
iii) Boc-Cys(Acm)-Val-Cys(Acm)-OMe iv) Boc-Cys(Acm)-Val-Cys(Acm)-OH
v) Boc-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-OMe vi) Boc-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-OH
vii) Boc-Cys(Acm)-Val-CyslAcm)-d-Phe-Pro-ONSu wherein ONSu indicates an N-oxysuccinimido group, which also applies to the followin~:
viii) H-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-ONSu or hydrochlorates thereof ix) Cyclo-[Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro]2 x) Boc-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-NHNH2 wherein NHNH2 indicates a hydrazidated carboxyl group xi) Boc-[Cys(Acm)-Val-CyS(Acm)-d-phe-pro]2 xii) Boc-[Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro]2NHNH2 xiii) H-[Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro]2NHNH2 or hydrochlorates thereof xiv) Cyclo-[Cys(HgCl)-Val-Cys(HgCl)-d-Phe-Pro] 2 Z~ ~6 and 4) a metal or heavy metal cluster complex of the above-mentioned novel cyclic peptide (I) formed by a reaction of said novel cyclic peptide with a cluster of a metal or heavy metal selected from the group consisting of mercury, cadmium, zinc, molybdenum, cobalt, nickel, iron, titanium, palladium, manganese, silicon, and calcium.
The novel cyclic peptide (I) in accordance with the present invention exhibits an antibacterial activity and can be useful in the fields of medicines and agricultural chemicals.
Also, the cluster complexes obtained from the novel cyclic peptide (I) can exhibit high oxidation-reduction potentials which are closer to those of the clusters in organisms as compared with the conventional synthetic clusters. Accordingly, they can be useful as active agents in organisms in the fields of medicines and functional materials.
The novel cyclic peptide (I) in accordance with the present invention can be made by various methods.
Industrially, the following methods are advantageous.
1) Method (a) While protection and unprotection of polar groups of amino acids are repeated, a sequential elongation method is used to form a peptide in which cysteines are protected, which peptide is represented by the following formula:
Boc-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-OMe Then, its methoxy group is changed to an OH group.
Thereafter, a free acid and N-oxysuccinimide are used so that the peptide is subjected to actlve esterification.
An active esterification method is used to effect dimerization of thus modified peptide (cf. Figure 1).
2) Method (b) ~he above-mentioned peptide:
Boc-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-OMe is subjected to an azidation method, namely, is reacted with hydrazine monohydrate to yield hydrazide and then is reacted with isoamyl nitrite under a weakly acid condition so as to be azidated. The azidated peptide is reacted with a free amino acid of the other synthesizing block, namly, the peptide represented by the following formula: -H-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-OMe HCl (Cf. Figure 2.) In both cases, since the SH-group of Cys in the cyclic peptide is protected by Acm, an HgCl-adduct is formed and then HgCl2 is removed to form the novel cyclic peptide (I).
The cluster complex in accordance with the present invention can easily be formed when an organic solvent solution of the novel cyclic peptide (I) is mixed with an organic solvent solution of a metal or heavy metal cluster.
~ ~4~a~
BRIEF DESCRIPTION CF THE DRA~INGS
Figure 1 is a flow sheet showing a scheme for synthesizing the novel cyclic peptide in accordance with the present invention, Cyclo-~Cys-Val-Cys-d-Phe-Pro) 2 (I), by an active esterification;
Figure 2 is a flow sheet showing synthesizing steps of that peptide by an azidation method;
Figure 3 is a graph showing the results of measurement of molecular weight of an intermediate in accordance with the present invention, Cyclo-[Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro]2 (14), by FD-MS;
Figures 4A and 4B are charts showing 500MHz lH-NMR
spectral data of the above-mentioned intermediate;
Figure 5 is a chart showing the results of measurement of 1H-lHCOSY2D-N~R of the above-mentioned intermediate;
Figure 6 is a chart showing 500MHz lH-NMR spectral data of the novel cyclic peptide in accordance with the present invention;
Figure 7 is a chart showing an absorption spectrum of the four-iron cluster itself used in Example 3;
Figure 8 is a graph showing titration curves of the four-iron cluster and peptide in Example 3; and Figure 9 is a chart showing the results of measurment of ultraviolet-visible light absorption spectra of solutions E, F, and G in Experiment 1.
DESCRIPTION OF THE PREFERRED EMBODIMENT
12~
The following examples wil] explain the present invention in detail.
Example 1 I) Making of Cyclo-[Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro~2 (14) by Active Esterification Method (Cf.
Figure 1) 1) Synthesis of Boc-Cys(Acm)-OH (1) H-Cys(Acm)-OH HCl (4.58g, 20mmol) was dissolved in 12ml of water.
To this solution, 4.20ml (20mmol) of triethylamine (abridged as "TEA" in the following) was added, while being stirred and cooled with ice. Then, the mixture was stirred at room temperature. Thereafter, dioxane solution (12ml) of 5.42g (20mmol) of 2-tertiary butyloxycarbonyloxyimino-2-phenylacetonitrile (abridged as "BOC-ON" in the following) was added dropwise thereto and the resulting mixture was stirred at room temperature for 3 hours. After the reaction, TEA was further added thereto to dissolve the insolubles. Then, the solvent was evaporated under a vacuum to leave a dried product.
It was dissolved in ethyl acetate and then was washed with water. The water layer was fùrther acidified and extracted with ethyl acetate. The extracted layer was dried with sodium sulfate anhydride. After the solvent was evaporated under a vacuum, the dried product was recrystallized in ether to yield the aimed white amorphous product (1).
Yield: 3.50g (59.9~) 2) Synthesis of H-Cys(Acm)-Me HCl (2) 26.75g (0.30mmol) of N-(hydroxymethyl)acetamide (abridged as "Acm" in the following) and 46.33g (0.27mmol) of H-Cys-OMe.HCl were dissloved in 70ml of S water. While the solution was cooled with ice, llml of 35% hydrochloric acid was added thereto. After the mixture was reacted at 0C for 2 hours, the gas in the reaction vessel was replaced with argon. Then the reaction vessel was left for five days. Vacuum evaporation was repeated with the addition of 20ml of ethanol anhydride to remove water from the mixture.
After 200ml of ethanol was added thereto and the insolubles were removed by filtration, the aimed product ~2) of a white crystalline powder form was recrystallized from ether.
Yield: 28.72g (43.8%) The physical properties of the product thus obtained were as follows:
lH-NMR(DMso-d6) ~DSS(Ppm)=l-88 (s, 9H, Acm CH3) 3.13 (d, 2H, Cys H~) 3.76 (s, 3H, OCH3) 3.82-4.56 (m, 3H, Cys Ha, Acm CH2) 8.20-9.20 (m, 2H, Acm HN, Cys HN) 3) Synthesis of Boc-Val-Cys(Acm)-OMe (3) 4.85g (20mmol) of the above-mentioned product (2) was dissolved in 50ml of N,N-dimethylformamide (abridged as "DMF" in the following). While the solution was cooled - 1.0-with ice and stirred, 2.19ml (20mmol) of N~
methylmorpholine (abridged as "NMM" in the following) was added thereto. After the mixture was stirred at room temperature for one hour, 4.35g (20mmol) of Boc-Val-OH, 2.97g (22mmol) of N-hydroxybenzotriazol (abridged as "HO~t" in the following), 4.13g (20mmol) of dicyclohexylcarbodiimide (abridged as "DCC" in the following) were added thereto in this order. Then the resulting mixture was sequentially stirred at -3C for 5.5 hours, at 0C for 12 hours, and at room temperature for 5.5 hours. After DMF was evaporated under a vacuum, 50ml of ethyl acetate was added to the resulting product.
Then the crystal d~eposited therefrom was separated by filtration, while the filtrate was sequentially washed with lM sodium hydrogencarbQnate aqueous solution, water, lM citric acid aqueous solution, and water. Thereafter, the washed filtrate was dried with sodium sulfate anhydride. The solvent was evaporated therefrom under a vacuum to leave a crude product. Then it was purified by a column chromatography to yield the aimed product (3) of a white powder form.
Yield: 4.52g (55.7~) The physical properties of the product thus obtained were as follows:
Elementary Analysis (~ by weight): Cl7H3lO6N3S
Empirical Value C:50.21, X:7.44, N:10.56 Calculated Value C:50.34, H:7.72, N:10.36 lH-NMR ( DMSO-d6 ) ~04~
(Ppm)-o-86 (m, 6H, Val HY) 1.40 (s, 9H, Boc CH3) 1.72-2.12 (m, 4H, Acm CH3, Val HP) 2.92 (m, 2H, Cys H~) 3.64 (m, 3H, OCH3) 3.82 (m, lH, Val H~) 4.00-4.62 (m, 4H, Cys Ha, Acm CH2) 6.60 (d, lH, Val HN) 8.00-8.68 (m,2H, Cys HN, Acm HN) 4) Synthesis of H-Val-Cys(Acm)-OCH3 (4) To 1.62g (4mmol) of the above-mentioned product (3), 30ml of 4M hydrochloric acid/dioxane was added. The resulting mixture was stirred at room temperature for 1.5 hours. Dioxane was evaporated therefrom under a vacuum to leave the aimed product (4~) of a white powder form.
5) Synthesis of Boc-Cys(Acm)-Val-Cys(Acm)-OMe (5) The above-mentioned product (4) was dissolved in 30ml of DMF. While the solution is cooled with ice and stirred, NMM (0.44g, 4mmol) was added thereto. Twenty minutes thereafter, Boc-Cys(Acm)-OH (1.17g, 4mmol), HOBt (0.59g, 4.4mmol), and DCC (0.83g, 4mmol) were added thereto in this order. Then the mixture was stirred at -3C for 5 hours, at 0C ~or 11 hours, and at room temperature for 5 hours. After DMF was evaporated under a vacuum, chloroform was added to the remaining product.
After the insolubles were separated by filtration, the filtrate was sequentially washed with lM sodium hydrogencarbonate aqueous solution, water, lM citric acid aqueous solution, and water. The extracted layer obtained after the washing was dried with sodium sulfate anhydride and the solvent was evaporated to leave a crude product. Then it was purified by a column chromatography to yield the aimed white amorphous product (5).
Yield: 1.67g (71.8%) The physical properties of the product thus obtained were as follows:
Elementary Analysis (% by weight): C23H4leN5S2 Empirical Value C:46.95, H:6.92, N:12.03 Calculated Value C:46.91, H:7.20, N:11.90 Remarks: Since 0.5mol of water of crystallization is considered to be contained therein, the value is that of C23H4lOgNsS2 0-5H2O-lH-NMR(DMso-d6) (PPm)=0-86 (m, 6H, Val Hr) 1.40 (s, 9H, Boc CH3) 1.60-2.20 (m, 7H, Acm CH3, Val H~) 2.46-3.00 (m, 4H, Cys H~) 3.64 (m, 3H, OCH3) 3.80-4.60 (m, 7H, Val Ha, Cys Ha, Acm CH2) 7.06, 7.54 (d, 2H, HN) 8.32-8.68 (t, 3H, Cys HN, Acm HN) 6) Synthesis of Boc-Cys(Acm)-Val-Cys(Acm)-OH (6) 1.47g (2.53mmol) of the above-mentioned product (5) was dissolved in 25ml of methanol. After lM sodium hydroxide aqueous solution was added thereto, the mixture was stirred at room temperature for 3 hours. Then, after 15ml of water was added thereto, methanol was evaporated from the mixture under a vacuum. After unreacted esters were removed by 20ml of ether, 30ml of lM citric acid aqueous solution was added to the water layer to weakly acidify the latter. Then, extraction was effected with ethyl acetate. The extracted material was dried with sodium sulfate anhydride and then the solvent was evaporated therefrom under a vacuum to leave the aimed white amorphous product (6).
Yield: 1.15g (80.2%) The physical properties of the product thus obtained were as follows:
[ a ]D=-25 . 7 (c 0.65, MeOH 23C) Elementary Analysis (% by weight): C22H39O8N5S2 Empirical Value -C:44.65, H:6.64, N:10.24 Calculated Value C:44.63, H:7.35, N:10.85 Remarks: The calculated value of C22H3gO8N5S2-0.5AcOEt-2H2o-lH-NMR(DMSO-d6) SDSS(Ppm)=0-88 (m, 6H, Val HY) 1.40 (s, 9H, Boc CH3) 1.60-2.20 (m, 7H, Acm CH3, Val H~) 2.56-3.00 (m, 4H, Cys H~) 3.80-4.60 (m, 7H, Val Ha, Cys Ha, Acm CH2) 7.08, 7.52 (d, 2H, HN) 8.16-8.68 (m, 4H, Cys HN, Acm HN) 12.2 (br, lH, OH) 7) Synthesis of Boc-d-Phe-OH (7) 9.90g (60mmol) of d-Phe-OH was added to 180ml of a dioxane-water (2:1) mixed solvent. Then, 60ml (60mmol) of lM sodium hydroxide aqueous solution was added thereto to dissolve d-Phe-OH. After 14.43g (66mmol) of di-(tertiary butyl)-dicarbonate ~abridged as "(BOC)2O" in the following] was added thereto, the mixture was stirred at room temperature for 3 hours. After dioxane was evaporated under a vacuum, 80ml of ethyl acetate was added to the remaining product which was being cooled with ice. After being acidified with 20ml of 5~
potassium hydrogensulfate aqueous solution, the mixture was separated. The resulting water layer was further extracted with ethyl acetate. The extracted layers were combined together and dried with sodium sulfate anhydride. The solvent was^evaporated therefrom under a vacuum to leave a crude product. Then, when it was recrystallized in ether-petroleum ether, the aimed product (7) of a white powder form was obtained.
Yield: 10.83g (68.1%) The physical properties of the product thus obtained were as follows:
H-NMR(CDCl~) S(Ppm)=l-4o (s, 9H, Boc CH3) 3.12 (d, 2H, Phe H~) 4.26-4.76 (m, lH, Phe Ha) 4.76-5.12 (m, lH, Phe OH) 6.88-7.56 (m, 5H, Phe C6H5) 8.72 (s, lH, Phe HN) ~6~2~
8) Synthesis of Boc-d-Phe-Pro-OMe (8) 3.31g (20mmol) of Pro-OMe.HCl was dissolved in 50ml of DMF. While the solution was cooled with ice and stirred, 2.19ml (20mmol) of NMM was added thereto. Ten minutes thereafter, 5.31g (20mmol) of the above-mentioned product (7), 2.97g (22mmol) of HOBt, 4.13g (20mmol) of DCC were added to the mixture in this order. The resulting mixture was sequentially stirred at -3C for 5 hours, at 0C for 10 hours, and at room temperature for 9 hours. Then, after DMF was evaporated from the mixture, 50ml of ethyl acetate was added thereto. After cooling, deposited crystals were separated by filtration. The filtrate was sequentially washed with lM sodium hydrogencarbonate aqueous solution, water, lM citric acid aqueous solution, and water. ~Then, the washed product was dried with sodium sulfate anhydride. Thereafter, the solvent was evaporated from the dried product to leave a crude product. Then, it was purified by a column chromatography to yield the aimed white amorphous product.
Yield: 6.73g (89.5%) The physical properties of the product thus obtained were as follows:
lH-NMR(DMSO-d6) ~DSS ( ppm)=1.32 (s, 9H, Boc CH~) 1.44-2.28 (m, 4H, Pro H~Y) 2.60-2.92 (m, 2H, Pro H~) 3.00-3.40 (m, 2H, Phe H~) 2046~4 3.60 (s, 3H, OCH3) 3.84-4.60 (m, 2H, Phe H~, Pro Ha) 6.60-7.60 (m, 6H, Phe C6H5, Phe HN) 9) Synthesis of H-d-Phe-Pro-OMe HCl (9) To 0.75g (2.Oml) of the above-mentioned product (8), 30ml of 4M hydrochloric acid/dioxane was added and the mixture was stirred at room temperature for 1.5 hours. Dioxane was repeatedly evaporated from the mixture under a vacuum, while several milliliters of ether was repeatedly added thereto. Thus, the aimed product (9) of a white powder form was quantitatively obtained.
10) Synthesis of Boc-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-OMe (10) 0.67g (2.Ommol) of the above-mentioned product (9) was dissolved in 30ml of DMF. While the solution was cooled with ice and stirred, 220 ~1 of NMM was added thereto. Ten minutes thereafter, a DMF solution of 1.13g (2.0mmol) of the above-mentioned product (6), 0.30g (2.2mmol) of HOBt, 0.41g (2.Ommol) of DCC were added to the mixture in this order. The resulting mixture was sequentially stirred at -3C for 5 hours, at 0C for 12.5 hours, and at room temperature for 7 hours. Thenr after DMF was evaporated under a vacuum and the residue was dissolved in chloroform, the impurities were separated by filtration. The filtrate was sequentially washed with lM
sodium hydrogencarbonate aqueous solution, water, lM
citric acid aqueous solution, and water. Then, the ;~0461~4 organic layer was dried with sodium sulfate anhydride.
Thereafter, the solvent was evaporated from the dried product to leave a crude product. Then, it was purified by a column chromatography to yield the aimed yellowish white amorphous product.
Yield: 0.86g (52.3%) The physical properties of the product thus obtained were as follows:
Elementary Analysis (~ by weight): C37H57OloN7S2 Empirical Value C:54.16, H:6.88, N:11.34 Calculated Value C:53.92, H:6.99, N:11.90 lH-NMR ( DMso-d6 ) (pPm)=o-88 (m, 6H, Val Hr) 1.40 (s, 9H, Boc CH3) 1.48-2.24 ~m, llH, Acm CH3, Val H~, ~ro H~, 9r 2.60-3.00 (m, 6H, Pro H~, Cys H~) 3.00-3.40 (m, 2H, Phe H~) 3.60 (s, 3H, OCH3) 3.80-5.00 (m, 9H, Val Ha, Cys Ha, Phe Ha, Pro Ha, Acm CH2) 6.80-7.40 (m, 5H, Phe C6H5) 7.40-8.60 (m, 6H, Cys HN, Acm HN, Val HN, Phe HN) 11) Synthesis of Boc-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-OH (11) :;
21~4~12a~
l.OOg (1.2mmol) of the above-mentioned product (10) was dissolved in lOml of methanol. After 1.44ml (1.44mmol) of lM sodium hydroxide aqueous solution was added thereto, the solution was stirred at room temperature for 5 hours. lOml of water was added to the resulting reaction solution and methanol was evaporated therefrom under a vacuum. The remaining aqueous solution was separated with addition of ether. While being cooled with ice and stirred, the water layer was weakly acidified with addition of 5ml of lM citric acid aqeous solution and then subjected to extraction with ethyl acetate. The extracted layer was dried with sodium sulfate anhydride. Then, the solvent was evaporated under a vacuum to leave the aimed yellowish white amorphous product (11).
Yield: 0.85g (86.5~) -The physical properties of the product thus obtained were as follows:
Elementary Analysis (% by weight): C36H55OloN7S2 Empirical Value C:53.71, H:6.86, N:11.02 Calculated Value C:53.48, H:7.08, N:10.91 Remarks: The calculated value as C36HssOloN7S2 AcOEt lH--NMR(DMSO--d6) ~Dss(PPm)=0-86 (m, 6EI, Val HY) 1.44 (s, 9H, Boc CH3) 1.52-2.28 (m, llH, Acm CH3, Val H~, Pro H~
Z046~24 2.60-3.80 ~m, 8H, Pro H~, Cys H~, Phe H~) 3.80-5.00 (m, 9H, Val HQ, Cys Ha, Phe Ha, Pro H~) 6.80-7.36 ~m, 5H, Phe C6H5) 7.~0-8.60 (m, 6H, Cys HN, Acm H
Val HN, Phe HN) 12) Synthesis of Boc-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-ONSu (12) 0.74g (0.9mmol) of the above-mentioned product (11) was dissolved in 10ml of DMF. While the solution was cooled with ice, 0.21g (1.8mmol) of HONSu and 0.359 (1.8mmol) of 1-ethyl-3-(3-dimethylaminopropyl)-1~ carbodiimide hydrochlorate (abridged as "EDC.HCl" in the following) were added thereto in this order. The resulting mixture was stirred at 0C for 12 hours. After DMF was evaporated under a vacuum from the mixture, the residue was dissolved in 20ml of chloroform. Then, the solution was washed with lM sodium hydrogencarbonate and water. The organic layer obtained thereby was dried with sodium sulfate anhydride. Thereafter, the solvent was evaporated under a vacuum to leave a crude product.
Then, it was purified by a column chromatography to yield the aimed reddish white amorphous product (12).
Yield: 0.61g (73.6%) The physical properties of the product thus obtained were as follows:
Z 1:)4~
Elementary Analysis (% by weight): C40H58Ol2N8S2 Empirical Value C:50.61, H:6.24, N:10.96 Calculated Value C:50.30, H:6.11, N:11.59 Remarks: The calculated value as C40H58Ol2N8S2 0.5CHCl3 lH-NMR(DMSO-d6) ss(PP~n)=0-86 (m, 6H, Val Hr) 1.40 (s, 9H, Boc CH3) 1.52-2.20 (m, llH, Acm CH3, Val Ha, Pro H~r) 2.60 (s, 4H, ONSu) 2.60-3.80 (m, 6H, Pro Hr, Cys Ha, Phe Ha) 3.80-5.00 (m, 9H, Val Ha, Cys Ha, Phe Ha, Pro Ha ~ Acm CH2) 6.80-7.36 (m, 5H, Phe C6H5) 7.40-8.60 (m, 6H, Cys HN, Acm H~, Val HN, Phe HN) 13) Synthesis of H-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-ONSu HCl (13) To l.llg (1.22mmol) of the above-mentioned product (12), 20ml of 4M hydrochloric acid/dioxane solution was added. After the mixture was stirred at room temperature for 1 hour, dioxane was repeatedly evaporated under a vacuum with addition of several milliliters of ether.
After this evaporation process was repeated for several 261~6~4 times, the aimed product of a pale reddish purple powder form was quantitatively obtained.
Yield: 1.079 14) Synthesis of Cyclo~[Cys(Acm~-Val-Cys(Acm)-d-Phe-Pro]2 (14) 560mg (0.62mmol) of the above-mentioned product (13) was dissolved in lOml of DMF. While being vigorously stirred at room temperature, the mixture was added dropwise to 200ml of pyridine for 1.5 hours. After the mixture was further stirred at room temperature ~or 28.5 hours, pyridine was repeatedly evaporated therefrom under a vacuum, while the residue was repeatedly dissolved in chloroform, to leave a crude brown amorphous product. The crude yield was 1.069.
0.53g of the crude produt was dissolved in a mixed solvent of methanol-water. After being purified by a column filled with an ion-exchange resin, the solution was repeatedly purified by a column chromatography to yield the aimed yellowish white amorphous product.
Yield: 20.2mg (3.8%) Figure 3 is a graph showing the results of measurement of molecular weight of this product by FD-MS.
Figures 4A and 4B are charts showing 500MHz lH-NMR
spectral data thereof. Figure 5 is a chart showing the results of measurement of 1H-lHCOSY2D-NMR thereof.
In FD-MS, the molecular weight of the aimed product (M.W. 1383.76) is indicated as a peak of 1383.
While Cyclo-[Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro]2 has 8 2~:)461~4 amide protons and 4 NH protons of the Acm groups, only 4 and 2 of them are respectively observed. It indicates that the product is a symmetrical cyclic peptide.
II) Method of Making Cyclo-(Cys-Val-Cys-d-Phe-Pro)2 From Cyclo-[Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro]2 (14) 15) Synthesis of Cyclo-[Cys(HgCl)-Val-Cys(HgCl)-d-Phe-Pro]2 (15) 55.8g (4.0 x 10-5mol) of the above mentioned product (14) was dissolved in 2ml of dimethyl sulfoxide (abridged as "DMSO" in the following). After 87.2mg (3.2 x 10-4mol) of mercury chloride was added thereto, the solution was stirred at room temperature for 13 hours.
When 20ml of water was added thereto, a white precipitate was formed. It was then cooled with an ice bath and separated by filtration. The resulting solid was dried under a vacuum to yield the aimed product of a white powder form (15).
Yield: 60mg (70%) 16) Synthesis of Cyclo-[Cys(HgCl)-Val-Cys(HgCl)-d-Phe-Pro]2 (15) [Alternative Method To 15)]
28.5mg (2.1 x 10-5mol) of the above-mentioned product (14) was dissolved in lml of DMSO. 44.7mg (1.7 x 10-4mol) of mercury chloride was added to the solution and the mixture was stirred at room temperature for 21 hours.
DMSO was evaporated therefrom under a vacuum and then 20ml of dichloromethane was added thereto to form a white L2~
precipitate. The precipitate was washed with 50ml of dichloromethane. After dichloromethane was washed with water, the remaining product was dried with sodium sulfate anhydride and then the solvent was evaporated therefrom under a vacuum to leave the aimed product in a pale yellow oil form. The yield thereof was 30.Omg (71%). Chloroform was used to recover the compound from the drying agent and the water layer. The recovered compound was assembled with the white precipitate. The total yield was 55.8mg.
sodium hydrogencarbonate aqueous solution, water, lM
citric acid aqueous solution, and water. Then, the ;~0461~4 organic layer was dried with sodium sulfate anhydride.
Thereafter, the solvent was evaporated from the dried product to leave a crude product. Then, it was purified by a column chromatography to yield the aimed yellowish white amorphous product.
Yield: 0.86g (52.3%) The physical properties of the product thus obtained were as follows:
Elementary Analysis (~ by weight): C37H57OloN7S2 Empirical Value C:54.16, H:6.88, N:11.34 Calculated Value C:53.92, H:6.99, N:11.90 lH-NMR ( DMso-d6 ) (pPm)=o-88 (m, 6H, Val Hr) 1.40 (s, 9H, Boc CH3) 1.48-2.24 ~m, llH, Acm CH3, Val H~, ~ro H~, 9r 2.60-3.00 (m, 6H, Pro H~, Cys H~) 3.00-3.40 (m, 2H, Phe H~) 3.60 (s, 3H, OCH3) 3.80-5.00 (m, 9H, Val Ha, Cys Ha, Phe Ha, Pro Ha, Acm CH2) 6.80-7.40 (m, 5H, Phe C6H5) 7.40-8.60 (m, 6H, Cys HN, Acm HN, Val HN, Phe HN) 11) Synthesis of Boc-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-OH (11) :;
21~4~12a~
l.OOg (1.2mmol) of the above-mentioned product (10) was dissolved in lOml of methanol. After 1.44ml (1.44mmol) of lM sodium hydroxide aqueous solution was added thereto, the solution was stirred at room temperature for 5 hours. lOml of water was added to the resulting reaction solution and methanol was evaporated therefrom under a vacuum. The remaining aqueous solution was separated with addition of ether. While being cooled with ice and stirred, the water layer was weakly acidified with addition of 5ml of lM citric acid aqeous solution and then subjected to extraction with ethyl acetate. The extracted layer was dried with sodium sulfate anhydride. Then, the solvent was evaporated under a vacuum to leave the aimed yellowish white amorphous product (11).
Yield: 0.85g (86.5~) -The physical properties of the product thus obtained were as follows:
Elementary Analysis (% by weight): C36H55OloN7S2 Empirical Value C:53.71, H:6.86, N:11.02 Calculated Value C:53.48, H:7.08, N:10.91 Remarks: The calculated value as C36HssOloN7S2 AcOEt lH--NMR(DMSO--d6) ~Dss(PPm)=0-86 (m, 6EI, Val HY) 1.44 (s, 9H, Boc CH3) 1.52-2.28 (m, llH, Acm CH3, Val H~, Pro H~
Z046~24 2.60-3.80 ~m, 8H, Pro H~, Cys H~, Phe H~) 3.80-5.00 (m, 9H, Val HQ, Cys Ha, Phe Ha, Pro H~) 6.80-7.36 ~m, 5H, Phe C6H5) 7.~0-8.60 (m, 6H, Cys HN, Acm H
Val HN, Phe HN) 12) Synthesis of Boc-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-ONSu (12) 0.74g (0.9mmol) of the above-mentioned product (11) was dissolved in 10ml of DMF. While the solution was cooled with ice, 0.21g (1.8mmol) of HONSu and 0.359 (1.8mmol) of 1-ethyl-3-(3-dimethylaminopropyl)-1~ carbodiimide hydrochlorate (abridged as "EDC.HCl" in the following) were added thereto in this order. The resulting mixture was stirred at 0C for 12 hours. After DMF was evaporated under a vacuum from the mixture, the residue was dissolved in 20ml of chloroform. Then, the solution was washed with lM sodium hydrogencarbonate and water. The organic layer obtained thereby was dried with sodium sulfate anhydride. Thereafter, the solvent was evaporated under a vacuum to leave a crude product.
Then, it was purified by a column chromatography to yield the aimed reddish white amorphous product (12).
Yield: 0.61g (73.6%) The physical properties of the product thus obtained were as follows:
Z 1:)4~
Elementary Analysis (% by weight): C40H58Ol2N8S2 Empirical Value C:50.61, H:6.24, N:10.96 Calculated Value C:50.30, H:6.11, N:11.59 Remarks: The calculated value as C40H58Ol2N8S2 0.5CHCl3 lH-NMR(DMSO-d6) ss(PP~n)=0-86 (m, 6H, Val Hr) 1.40 (s, 9H, Boc CH3) 1.52-2.20 (m, llH, Acm CH3, Val Ha, Pro H~r) 2.60 (s, 4H, ONSu) 2.60-3.80 (m, 6H, Pro Hr, Cys Ha, Phe Ha) 3.80-5.00 (m, 9H, Val Ha, Cys Ha, Phe Ha, Pro Ha ~ Acm CH2) 6.80-7.36 (m, 5H, Phe C6H5) 7.40-8.60 (m, 6H, Cys HN, Acm H~, Val HN, Phe HN) 13) Synthesis of H-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-ONSu HCl (13) To l.llg (1.22mmol) of the above-mentioned product (12), 20ml of 4M hydrochloric acid/dioxane solution was added. After the mixture was stirred at room temperature for 1 hour, dioxane was repeatedly evaporated under a vacuum with addition of several milliliters of ether.
After this evaporation process was repeated for several 261~6~4 times, the aimed product of a pale reddish purple powder form was quantitatively obtained.
Yield: 1.079 14) Synthesis of Cyclo~[Cys(Acm~-Val-Cys(Acm)-d-Phe-Pro]2 (14) 560mg (0.62mmol) of the above-mentioned product (13) was dissolved in lOml of DMF. While being vigorously stirred at room temperature, the mixture was added dropwise to 200ml of pyridine for 1.5 hours. After the mixture was further stirred at room temperature ~or 28.5 hours, pyridine was repeatedly evaporated therefrom under a vacuum, while the residue was repeatedly dissolved in chloroform, to leave a crude brown amorphous product. The crude yield was 1.069.
0.53g of the crude produt was dissolved in a mixed solvent of methanol-water. After being purified by a column filled with an ion-exchange resin, the solution was repeatedly purified by a column chromatography to yield the aimed yellowish white amorphous product.
Yield: 20.2mg (3.8%) Figure 3 is a graph showing the results of measurement of molecular weight of this product by FD-MS.
Figures 4A and 4B are charts showing 500MHz lH-NMR
spectral data thereof. Figure 5 is a chart showing the results of measurement of 1H-lHCOSY2D-NMR thereof.
In FD-MS, the molecular weight of the aimed product (M.W. 1383.76) is indicated as a peak of 1383.
While Cyclo-[Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro]2 has 8 2~:)461~4 amide protons and 4 NH protons of the Acm groups, only 4 and 2 of them are respectively observed. It indicates that the product is a symmetrical cyclic peptide.
II) Method of Making Cyclo-(Cys-Val-Cys-d-Phe-Pro)2 From Cyclo-[Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro]2 (14) 15) Synthesis of Cyclo-[Cys(HgCl)-Val-Cys(HgCl)-d-Phe-Pro]2 (15) 55.8g (4.0 x 10-5mol) of the above mentioned product (14) was dissolved in 2ml of dimethyl sulfoxide (abridged as "DMSO" in the following). After 87.2mg (3.2 x 10-4mol) of mercury chloride was added thereto, the solution was stirred at room temperature for 13 hours.
When 20ml of water was added thereto, a white precipitate was formed. It was then cooled with an ice bath and separated by filtration. The resulting solid was dried under a vacuum to yield the aimed product of a white powder form (15).
Yield: 60mg (70%) 16) Synthesis of Cyclo-[Cys(HgCl)-Val-Cys(HgCl)-d-Phe-Pro]2 (15) [Alternative Method To 15)]
28.5mg (2.1 x 10-5mol) of the above-mentioned product (14) was dissolved in lml of DMSO. 44.7mg (1.7 x 10-4mol) of mercury chloride was added to the solution and the mixture was stirred at room temperature for 21 hours.
DMSO was evaporated therefrom under a vacuum and then 20ml of dichloromethane was added thereto to form a white L2~
precipitate. The precipitate was washed with 50ml of dichloromethane. After dichloromethane was washed with water, the remaining product was dried with sodium sulfate anhydride and then the solvent was evaporated therefrom under a vacuum to leave the aimed product in a pale yellow oil form. The yield thereof was 30.Omg (71%). Chloroform was used to recover the compound from the drying agent and the water layer. The recovered compound was assembled with the white precipitate. The total yield was 55.8mg.
17) Synthesis of Cyclo-(Cys-Val-Cys-d-Phe-Pro)2 (I) In an argon atmosphere, not more than lOml of deaerated methanol was added to 0.06g of the above-mentioned product (15). The latter was not dissolved but dispersed in the former. To~~the resulting white turbid solution, hydrogen sulfate was introduced for 15 minutes.
After the solution was deaerated, the precipitate of mercury sulfide was separated by filtration. Then, methanol was evaporated therefrom under a vacuum to leave the aimed product (I) of a white powder form. The yield was 7.2mg (22~).
Figure 6 is a chart showing 500MHz lH-NMR spectral data of the product.
After the solution was deaerated, the precipitate of mercury sulfide was separated by filtration. Then, methanol was evaporated therefrom under a vacuum to leave the aimed product (I) of a white powder form. The yield was 7.2mg (22~).
Figure 6 is a chart showing 500MHz lH-NMR spectral data of the product.
18) Synthesis of Cyclo-(Cys-Val-Cys-d-Phe-Pro)2 (I) [Alternative Method To 17)]
The synthesis was conducted in the same way as 17) except that distilled DMSO was used as the reaction 2al~6~2:4 solvent. The aimed product (I) was obtained with the same yield.
Example 1 I) Making of Cyclo[Cys(Acm)-Val-Cys(Acm)-d-Phe-Prol2 (14) by Azidation Method (Cf. Figure 2) 1) Synthesis of Boc-Cys(Acm)-Val(Cys)-d-Phe-Pro-NHNH2 (16) 209.7mg (0.254mmol) of the product (10) of Example 1 was dissolved in 5.2ml of methanol. 0.62ml (12.8mmol) of hydrazine monohydrate was added thereto and the mixture was stirred at room temperature for 31.5 hours.
Then, methanol was evaporated under a reduced pressure.
The residue was subjected to evaporation under a vacuum with addition of distilled water. Then, the evaporation under a vacuum was sequentially repeated with addition of methanol and ether. Thereby, the aimed product of a yellow powder form (16) was obtained.
Yield: 197.2mg (94.0%) Hydrazide Test; positive 2) Synthesis of Boc-[Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro]2 (17) 197.2mg (0.24mmol) of the above-mentioned product (16) was dissolved in 1.22ml of DME' and then cooled to -50C. To this solution, 180 ~1 (0.72mmol) of 4M
hydrochloric acid/dioxane was added. Thereafter, 34 ~1 (0.24mmol) of isoamyl nitrite was added thereto and the mixture was stirred for 1.5 hours. Further, 8 ~1 of isoamyl nitrite was added thereto and the mixture was -2~-20~6~4 stirred for 0.5 hour. The reaction solution was cooled to -70C. To the cooled solution, 101 ~1 (0.72mmol) of TEA, 1.22ml of a DMF solution of 187.3mg (0.25mmol) of H-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-OMe.HCl cooled to -70C, and 26 ~1 (0.24 mmol) of NMM were added in this order. About 30 minutes thereafter, the mixture was transferred to an ice bath of 0C. While 9 ~1 of NMM was added thereto and the pH is adjusted to 7, the mixture was stirred. Then, DMF was evaporated therefrom under a vacuum. The evaporation was sequentially repeated with addition of methanol and ether. After 3ml of chloroform was added thereto, the remaining product was sequentially washed with water, lM sodium hydrogencarbonate aqueous solution, water, lM citric acid aqueous solution, and water. After the organic layer was dried with sodium sulfate anhydride, the solvent was evaporated under a vacuum to leave 251.6mg of a crude product. The crude yield was 69.lmg.
The crude product was purified by a column chromatography to yield the aimed product (17) in a pale brown amorphous form.
Yield: 168.Omg (46.1~) 3) Synthesis of Boc-[Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro]2NHNH2 (18) 10.3mg (6.79 x 10-6mol) of the above-mentioned product (17) was dissolved in 140 ~1 of methanol. 16.5 ~1 (3.40 x 10-4 mol) of hydrazine monohydrate was added thereto at room temperature and the mixture was stirred Z046~24 at the same temperature for 41 hours. Then, methanol was evaporated under a vacuum. The residue was subjected to evaporation under a reduced pressure with addition of 100 ~1 of methanol and 500 microliters of water. Further, the vacuum evaporation was effected with addition of ether to leave the aimed product of a white powder form (18).
Yield: 10.4mg (100%) 4) Synthesis of H-[Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro]2-NHNH2 HC1 (19) 10.4mg (6.86 x 10-6mol) of the above-mentioned product (18) was dissolved in 0.3ml of methanol. O.lml of 4M hydrochloric acid/dioxane was added dropwise thereto and the mixture was stirred at room temperature for 1.5 hours. Then, the solvent was evaporated therefrom under a reduced pressure and the remaining product was washed with ether for several times.
Thereafter, the washed product was dried under a vacuum to quantitatively yield the aimed product (19) of a yellowish white powder. The yield was 10.2mg.
5) Synthesis of Cyclo-[Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro]2 (14) 10.2mg (6.86 x 10-6mol) of the above-mentioned product (19) was dissolved in 200 micoliters of DMF and cooled to -30C. Then, 6 ~1 (3.5 equivalent weights) of 4M hydrochloric acid/dioxane solution was added thereto.
10 minutes thereafter, 10 ~1 (7.06 x 10-6mol) of isoamyl nitrite which had been diluted to 1/10 by DMF was added Z~)~6~
to the mixture. 1.5 hours thereafter, 2 microliters of diluted isoamyl nitrite was added thereto and the mixture was stirred for 6.5 hours. The reaction solution was cooled to -60C. Then, DMF cooled to -60C was added thereto. Thereafter, 6 ~1 of NMM (8 equivalent weights~
was added thereto and the p~ was adjusted to 7.30 minutes thereafter, the mixture was stirred at -20C for 44 hours. Then, the solvent was evaporated therefrom under a vacuum and the remaining product was sequentially washed with methanol and ether. Thereafter, the solvent was evaporated thereform under a vacuum to leave the aimed product of a yellowish white powder form. The crude yield was 14.1mg. It was then purified by a column chromatography to yield the aimed product (14).
II) Method of Making Cyclo-(Cys-Val-Cys-d-Phe-Pro~2 From Cyclo-[Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro]2 (14) The method same as that shown in Example 1 II) was used to obtain the aimed product (I).
Example 3 Synthesis of Cyclic Peptide-4-Iron Cluster Complex by Reaction Between Cyclo-(Cys-Val-Cys-d-Phe-Pro)2 (I) And (NEt4)2[Fe4S4-(S-t-Bu)4] (abridged as "4-iron cluster" in the following) The cyclic peptide (I) was dissolved in DMSO to form 0.8mM solution. Then, the 4-iron cluster was dissolved in DMSO to form 0.66mM solution. To Xml of the solution of (I), (2-X)ml of the 4-iron cluster solution was added. After the mixture was shaken for 10 minutes, the ultraviolet-visible light absorption spectrum thereof was measured and the absorption maximums at 305nm and 419nm of each spectrum were plotted. Figure 7 shows the absorption spectrum of the 4-iron cluster alone. Figure 3 shows their titration curves.
In view of the titration curves of Figure 8, it was found that the 4-iron cluster and the cyclic peptide were reacted at a ratio of about 1:1.2 to 1.3.
~ccordingly, the generation of the aimed complex was confirmed.
Experiment 1 Core Extraction From Cyclic Peptide-4-Iron Cluster Using Thiophenol 2mg of the cyclic peptide was dissolved in 1.2ml of DMSO (solution A, whose concentration corresponds to 1.52 x 10-3M). Then, 0.2ml of solution A was sampled and 0~31ml of DMSO was added thereto to form a solution (solution B, whose-concentration corresponds to 5.96 x 10-4M).
The synthesis was conducted in the same way as 17) except that distilled DMSO was used as the reaction 2al~6~2:4 solvent. The aimed product (I) was obtained with the same yield.
Example 1 I) Making of Cyclo[Cys(Acm)-Val-Cys(Acm)-d-Phe-Prol2 (14) by Azidation Method (Cf. Figure 2) 1) Synthesis of Boc-Cys(Acm)-Val(Cys)-d-Phe-Pro-NHNH2 (16) 209.7mg (0.254mmol) of the product (10) of Example 1 was dissolved in 5.2ml of methanol. 0.62ml (12.8mmol) of hydrazine monohydrate was added thereto and the mixture was stirred at room temperature for 31.5 hours.
Then, methanol was evaporated under a reduced pressure.
The residue was subjected to evaporation under a vacuum with addition of distilled water. Then, the evaporation under a vacuum was sequentially repeated with addition of methanol and ether. Thereby, the aimed product of a yellow powder form (16) was obtained.
Yield: 197.2mg (94.0%) Hydrazide Test; positive 2) Synthesis of Boc-[Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro]2 (17) 197.2mg (0.24mmol) of the above-mentioned product (16) was dissolved in 1.22ml of DME' and then cooled to -50C. To this solution, 180 ~1 (0.72mmol) of 4M
hydrochloric acid/dioxane was added. Thereafter, 34 ~1 (0.24mmol) of isoamyl nitrite was added thereto and the mixture was stirred for 1.5 hours. Further, 8 ~1 of isoamyl nitrite was added thereto and the mixture was -2~-20~6~4 stirred for 0.5 hour. The reaction solution was cooled to -70C. To the cooled solution, 101 ~1 (0.72mmol) of TEA, 1.22ml of a DMF solution of 187.3mg (0.25mmol) of H-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-OMe.HCl cooled to -70C, and 26 ~1 (0.24 mmol) of NMM were added in this order. About 30 minutes thereafter, the mixture was transferred to an ice bath of 0C. While 9 ~1 of NMM was added thereto and the pH is adjusted to 7, the mixture was stirred. Then, DMF was evaporated therefrom under a vacuum. The evaporation was sequentially repeated with addition of methanol and ether. After 3ml of chloroform was added thereto, the remaining product was sequentially washed with water, lM sodium hydrogencarbonate aqueous solution, water, lM citric acid aqueous solution, and water. After the organic layer was dried with sodium sulfate anhydride, the solvent was evaporated under a vacuum to leave 251.6mg of a crude product. The crude yield was 69.lmg.
The crude product was purified by a column chromatography to yield the aimed product (17) in a pale brown amorphous form.
Yield: 168.Omg (46.1~) 3) Synthesis of Boc-[Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro]2NHNH2 (18) 10.3mg (6.79 x 10-6mol) of the above-mentioned product (17) was dissolved in 140 ~1 of methanol. 16.5 ~1 (3.40 x 10-4 mol) of hydrazine monohydrate was added thereto at room temperature and the mixture was stirred Z046~24 at the same temperature for 41 hours. Then, methanol was evaporated under a vacuum. The residue was subjected to evaporation under a reduced pressure with addition of 100 ~1 of methanol and 500 microliters of water. Further, the vacuum evaporation was effected with addition of ether to leave the aimed product of a white powder form (18).
Yield: 10.4mg (100%) 4) Synthesis of H-[Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro]2-NHNH2 HC1 (19) 10.4mg (6.86 x 10-6mol) of the above-mentioned product (18) was dissolved in 0.3ml of methanol. O.lml of 4M hydrochloric acid/dioxane was added dropwise thereto and the mixture was stirred at room temperature for 1.5 hours. Then, the solvent was evaporated therefrom under a reduced pressure and the remaining product was washed with ether for several times.
Thereafter, the washed product was dried under a vacuum to quantitatively yield the aimed product (19) of a yellowish white powder. The yield was 10.2mg.
5) Synthesis of Cyclo-[Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro]2 (14) 10.2mg (6.86 x 10-6mol) of the above-mentioned product (19) was dissolved in 200 micoliters of DMF and cooled to -30C. Then, 6 ~1 (3.5 equivalent weights) of 4M hydrochloric acid/dioxane solution was added thereto.
10 minutes thereafter, 10 ~1 (7.06 x 10-6mol) of isoamyl nitrite which had been diluted to 1/10 by DMF was added Z~)~6~
to the mixture. 1.5 hours thereafter, 2 microliters of diluted isoamyl nitrite was added thereto and the mixture was stirred for 6.5 hours. The reaction solution was cooled to -60C. Then, DMF cooled to -60C was added thereto. Thereafter, 6 ~1 of NMM (8 equivalent weights~
was added thereto and the p~ was adjusted to 7.30 minutes thereafter, the mixture was stirred at -20C for 44 hours. Then, the solvent was evaporated therefrom under a vacuum and the remaining product was sequentially washed with methanol and ether. Thereafter, the solvent was evaporated thereform under a vacuum to leave the aimed product of a yellowish white powder form. The crude yield was 14.1mg. It was then purified by a column chromatography to yield the aimed product (14).
II) Method of Making Cyclo-(Cys-Val-Cys-d-Phe-Pro~2 From Cyclo-[Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro]2 (14) The method same as that shown in Example 1 II) was used to obtain the aimed product (I).
Example 3 Synthesis of Cyclic Peptide-4-Iron Cluster Complex by Reaction Between Cyclo-(Cys-Val-Cys-d-Phe-Pro)2 (I) And (NEt4)2[Fe4S4-(S-t-Bu)4] (abridged as "4-iron cluster" in the following) The cyclic peptide (I) was dissolved in DMSO to form 0.8mM solution. Then, the 4-iron cluster was dissolved in DMSO to form 0.66mM solution. To Xml of the solution of (I), (2-X)ml of the 4-iron cluster solution was added. After the mixture was shaken for 10 minutes, the ultraviolet-visible light absorption spectrum thereof was measured and the absorption maximums at 305nm and 419nm of each spectrum were plotted. Figure 7 shows the absorption spectrum of the 4-iron cluster alone. Figure 3 shows their titration curves.
In view of the titration curves of Figure 8, it was found that the 4-iron cluster and the cyclic peptide were reacted at a ratio of about 1:1.2 to 1.3.
~ccordingly, the generation of the aimed complex was confirmed.
Experiment 1 Core Extraction From Cyclic Peptide-4-Iron Cluster Using Thiophenol 2mg of the cyclic peptide was dissolved in 1.2ml of DMSO (solution A, whose concentration corresponds to 1.52 x 10-3M). Then, 0.2ml of solution A was sampled and 0~31ml of DMSO was added thereto to form a solution (solution B, whose-concentration corresponds to 5.96 x 10-4M).
19.4mg of the 4-iron cluster was dissolved in 16.06ml of DMSO (solution C, whose concentration corresponds to 1.25 x 10-3M). lml of solution C was sampled and 1.53ml of DMSO was added thereto to form a solution (solution D, whose conentration corresponds to 4.9 x 10-4M).
0.51ml of solution D was sampled and 0.51ml of DMSO was added thereto to form solution E. Solution D
was added to solution B to form solution F. Further, 3 ;2~41~ 4 ~1 of thiophenol which had been diluted to 1/40 by DMSO
was added to solution F (1.2 equivalent weights per the cyclic peptide) for core extraction (solution G).
I~ltraviolet-visible light absorption was measured for each of solution E (containing the 4-iron cluster alone), solution F (containing the cyclic peptide-4-iron cluster complex), and solution G (in which the 4-iron complex was core-extracted from the cyclic peptide-4-iron complex by thiophenol). Their spectra are shown in Figure 9. In Figure 9, spectra (a), (b), and (c) correspond to solutions E, F, and G, respectively.
The maximum of 457nm in spectrum (c) is specific to [Fe4S4(Sph)4]2-. Accordingly, it was found that the 4-iron cluster maintained its structure in the cyclic peptide which was a ligand. ~
Experiment 2 Measurement of Oxidation-Reduction Potential of Cyclic Peptide-4-Iron Cluster Complex by Derivative Pulse Polarography lml of the 4-iron cluster solution C was added to the cyclic peptide solution A of Experiment 1 and the mixture was concentrated to about lml to form colution H.
34mg of tetra-n-butylammonium perchlorate (n-Bu4NClO4) was added thereto as an electrolyte. After the base line of a blank (DMSO lml + n-~u4NClO4 34mg) was measured, solution H was subjected to a derivative pulse polarography, by which the oxidation-reduction potential was measured. As results, it was found that, although 2~
the 4-iron cluster by itself exhibited a low oxidation-reduction potential, it had an oxidation-reduction potential higher than that of the 4-iron cluster alone, in fact, the highest oxidation-reduction potential in the peptide-4-iron clusters which had been synthesized so far, when the cyclic peptide in accordance with the present invention coordinated therewith.
Table 1 shows the oxidation-reduction potential of the peptide-4-iron cluster complex in accordance with the present invention. For comparison, the oxidation-reduction potentials of the 4-iron cluster itself and conventionally synthesized peptide-4-iron clusters are also shown therein.
Z~4~
Table 1 Measurement of Oxidation-Ruduction Potentials Oxidation-Reduction Potential El/2V (vs. Ag~/Ag) _ Comparative Example 1 [Fe4S4(S-t-Bu4)4]Z~ -1.37 (in DMF) Comparative Example 2 [Fe4S4(SPh)4]2- -0.99 (in DMF) Comparative Example 3 [Fe4S4(Boc-(Gly-Cys-Gly)4-NH2] 2- -O . 86 (in DMSO) Comparative Example 4 [Fe4S4(z-Cys-Gly-Aly-Ala-OMe)2]2- -0.79 (CH2C12, 233K) Cyclic Peptide-4-Iron Cluster Complex of Experiment 2 -0.70 (in DMSO) _ _
0.51ml of solution D was sampled and 0.51ml of DMSO was added thereto to form solution E. Solution D
was added to solution B to form solution F. Further, 3 ;2~41~ 4 ~1 of thiophenol which had been diluted to 1/40 by DMSO
was added to solution F (1.2 equivalent weights per the cyclic peptide) for core extraction (solution G).
I~ltraviolet-visible light absorption was measured for each of solution E (containing the 4-iron cluster alone), solution F (containing the cyclic peptide-4-iron cluster complex), and solution G (in which the 4-iron complex was core-extracted from the cyclic peptide-4-iron complex by thiophenol). Their spectra are shown in Figure 9. In Figure 9, spectra (a), (b), and (c) correspond to solutions E, F, and G, respectively.
The maximum of 457nm in spectrum (c) is specific to [Fe4S4(Sph)4]2-. Accordingly, it was found that the 4-iron cluster maintained its structure in the cyclic peptide which was a ligand. ~
Experiment 2 Measurement of Oxidation-Reduction Potential of Cyclic Peptide-4-Iron Cluster Complex by Derivative Pulse Polarography lml of the 4-iron cluster solution C was added to the cyclic peptide solution A of Experiment 1 and the mixture was concentrated to about lml to form colution H.
34mg of tetra-n-butylammonium perchlorate (n-Bu4NClO4) was added thereto as an electrolyte. After the base line of a blank (DMSO lml + n-~u4NClO4 34mg) was measured, solution H was subjected to a derivative pulse polarography, by which the oxidation-reduction potential was measured. As results, it was found that, although 2~
the 4-iron cluster by itself exhibited a low oxidation-reduction potential, it had an oxidation-reduction potential higher than that of the 4-iron cluster alone, in fact, the highest oxidation-reduction potential in the peptide-4-iron clusters which had been synthesized so far, when the cyclic peptide in accordance with the present invention coordinated therewith.
Table 1 shows the oxidation-reduction potential of the peptide-4-iron cluster complex in accordance with the present invention. For comparison, the oxidation-reduction potentials of the 4-iron cluster itself and conventionally synthesized peptide-4-iron clusters are also shown therein.
Z~4~
Table 1 Measurement of Oxidation-Ruduction Potentials Oxidation-Reduction Potential El/2V (vs. Ag~/Ag) _ Comparative Example 1 [Fe4S4(S-t-Bu4)4]Z~ -1.37 (in DMF) Comparative Example 2 [Fe4S4(SPh)4]2- -0.99 (in DMF) Comparative Example 3 [Fe4S4(Boc-(Gly-Cys-Gly)4-NH2] 2- -O . 86 (in DMSO) Comparative Example 4 [Fe4S4(z-Cys-Gly-Aly-Ala-OMe)2]2- -0.79 (CH2C12, 233K) Cyclic Peptide-4-Iron Cluster Complex of Experiment 2 -0.70 (in DMSO) _ _
Claims (20)
1. A novel peptide represented by formula (I):
(I) wherein Cys indicates a cysteine group, Val indicates a valine group, d-Phe indicates a d-phenylalanine group, Pro indicates a proline group, and Cyclo indicates a cyclic form.
(I) wherein Cys indicates a cysteine group, Val indicates a valine group, d-Phe indicates a d-phenylalanine group, Pro indicates a proline group, and Cyclo indicates a cyclic form.
2. A method of making the cyclic peptide defined in claim 1, said method comprising the steps of:
repeatedly protecting and unprotecting a polar group of a constitutive amino acid to form, by a sequential elongation method, an intermediate peptide, in which cysteines are protected, represented by the following formula:
wherein Boc indicates an amino group protected by a t-butyloxycarbonyl group, Cys indicates a cysteine group, Val indicates a valine group, d-Phe indicates a d-phenylalanine group, Pro indicates a proline group, Acm indicates an SH group protected by an acetamidomethyl group, and OMe indicates a methoxylated carboxyl group;
and then dimerizing said intermediate peptide by an active esterification to form said cyclic peptide.
repeatedly protecting and unprotecting a polar group of a constitutive amino acid to form, by a sequential elongation method, an intermediate peptide, in which cysteines are protected, represented by the following formula:
wherein Boc indicates an amino group protected by a t-butyloxycarbonyl group, Cys indicates a cysteine group, Val indicates a valine group, d-Phe indicates a d-phenylalanine group, Pro indicates a proline group, Acm indicates an SH group protected by an acetamidomethyl group, and OMe indicates a methoxylated carboxyl group;
and then dimerizing said intermediate peptide by an active esterification to form said cyclic peptide.
3. A method as defined in claim 2, which comprises the steps of forming intermediates represented by the following formulas:
wherein Boc indicates an amino group protected by a t-butyloxycarbonyl group, Cys indicates a cysteine group, Val indicates a valine group, d-Phe indicates a d-phenylalanine group, Pro indicates a proline group, Acm indicates an SH group protected by an acetamidomethyl group, OMe indicates a methoxylated carboxyl group, H
indicates a freed amino group, ONSu indicates an N-oxysuccinimido group, and Cyclo indicates a cyclic form.
wherein Boc indicates an amino group protected by a t-butyloxycarbonyl group, Cys indicates a cysteine group, Val indicates a valine group, d-Phe indicates a d-phenylalanine group, Pro indicates a proline group, Acm indicates an SH group protected by an acetamidomethyl group, OMe indicates a methoxylated carboxyl group, H
indicates a freed amino group, ONSu indicates an N-oxysuccinimido group, and Cyclo indicates a cyclic form.
4. A method of making the cyclic peptide defined in claim 1, said method comprising the steps of:
repeatedly protecting and unprotecting a polar group of a constitutive amino acid to form, by a sequential elongation method, an intermediate peptide, in which cysteines are protected, represented by the following formula:
wherein Boc indicates an amino group protected by a t-butyloxycarbonyl group, Cys indicates a cysteine group, Val indicates a valine group, d-Phe indicates a d-phenylalanine group, Pro indicates a proline group, Acm indicates an SH group protected by an acetamidomethyl group, and OMe indicates a methoxylated carboxyl group;
azidating said intermediate peptide;
reacting the azidated peptide with a peptide represented by the following formula:
to form said cyclic peptide.
repeatedly protecting and unprotecting a polar group of a constitutive amino acid to form, by a sequential elongation method, an intermediate peptide, in which cysteines are protected, represented by the following formula:
wherein Boc indicates an amino group protected by a t-butyloxycarbonyl group, Cys indicates a cysteine group, Val indicates a valine group, d-Phe indicates a d-phenylalanine group, Pro indicates a proline group, Acm indicates an SH group protected by an acetamidomethyl group, and OMe indicates a methoxylated carboxyl group;
azidating said intermediate peptide;
reacting the azidated peptide with a peptide represented by the following formula:
to form said cyclic peptide.
5. A method as defined in claim 4, which comprises the steps of forming intermediates represented by the following formulas:
wherein Boc indicates an amino group protected by a t-butyloxycarbonyl group, Cys indicates a cysteine group, Val indicates a valine group, d-Phe indicates a d-phenylalanine group, Pro indicates a proline group, Acm indicates an SH group protected by an acetamidomethyl group, NHNH2 indicates a hydrazidated carboxyl group, and H indicates a freed amino group.
wherein Boc indicates an amino group protected by a t-butyloxycarbonyl group, Cys indicates a cysteine group, Val indicates a valine group, d-Phe indicates a d-phenylalanine group, Pro indicates a proline group, Acm indicates an SH group protected by an acetamidomethyl group, NHNH2 indicates a hydrazidated carboxyl group, and H indicates a freed amino group.
6. A novel peptide represented by the following formula:
wherein Boc indicates an amino group protected by a t-butyloxycarbonyl group, Val indicates a valine group, Cys indicates a cysteine group, Acm indicates an SH group protected by an acetamidomethyl group, and OMe indicates a methoxylated carboxyl group.
wherein Boc indicates an amino group protected by a t-butyloxycarbonyl group, Val indicates a valine group, Cys indicates a cysteine group, Acm indicates an SH group protected by an acetamidomethyl group, and OMe indicates a methoxylated carboxyl group.
7. A novel peptide represented by the following formula:
H-Val-Cys(Acm)-OMe wherein H indicates a freed amino group, Val indicates a valine group, Cys indicates a cysteine group, Acm indicates an SH group protected by an acetamidomethyl group, and OMe indicates a methoxylated carboxyl group.
H-Val-Cys(Acm)-OMe wherein H indicates a freed amino group, Val indicates a valine group, Cys indicates a cysteine group, Acm indicates an SH group protected by an acetamidomethyl group, and OMe indicates a methoxylated carboxyl group.
8. A novel peptide represented by the following formula:
Boc-Cys(Acm)-Val-Cys(Acm)-OMe wherein Boc indicates an amino group protected by a t-butyloxycarbonyl group, Cys indicates a cysteine group, Val indicates a valine group, Acm indicates an SH group protected by an acetamidomethyl group, and OMe indicates a methoxylated carboxyl group.
Boc-Cys(Acm)-Val-Cys(Acm)-OMe wherein Boc indicates an amino group protected by a t-butyloxycarbonyl group, Cys indicates a cysteine group, Val indicates a valine group, Acm indicates an SH group protected by an acetamidomethyl group, and OMe indicates a methoxylated carboxyl group.
9. A novel peptide represented by the following formula:
Boc-Cys(Acm)-Val-Cys(Acm)-OH
wherein Boc indicates an amino group protected by a t-butyloxycarbonyl group, Cys indicates a cysteine group, Val indicates a valine group, and Acm indicates an SH
group protected by an acetamidomethyl group.
Boc-Cys(Acm)-Val-Cys(Acm)-OH
wherein Boc indicates an amino group protected by a t-butyloxycarbonyl group, Cys indicates a cysteine group, Val indicates a valine group, and Acm indicates an SH
group protected by an acetamidomethyl group.
10. A novel peptide represented by the following formula:
Boc-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-OMe wherein Boc indicates an amino group protected by a t-butyloxycarbonyl group, Cys indicates a cysteine group, Val indicates a valine group, d-Phe indicates a d-phenylalanine group, Pro indicates a proline group, Acm indicates an SH group protected by an acetamidomethyl group, and OMe indicates a methoxylated carboxyl group.
Boc-Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro-OMe wherein Boc indicates an amino group protected by a t-butyloxycarbonyl group, Cys indicates a cysteine group, Val indicates a valine group, d-Phe indicates a d-phenylalanine group, Pro indicates a proline group, Acm indicates an SH group protected by an acetamidomethyl group, and OMe indicates a methoxylated carboxyl group.
11. A novel peptide represented by the following formula:
wherein Boc indicates an amino group protected by a t-butyloxycarbonyl group, Cys indicates a cysteine group, Val indicates a valine group, d-Phe indicates a d-phenylalanine group, Pro indicates a proline group, and Acm indicates an SH group protected by an acetamidomethyl group.
wherein Boc indicates an amino group protected by a t-butyloxycarbonyl group, Cys indicates a cysteine group, Val indicates a valine group, d-Phe indicates a d-phenylalanine group, Pro indicates a proline group, and Acm indicates an SH group protected by an acetamidomethyl group.
12. A novel peptide represented by the following formula:
wherein Boc indicates an amino group protected by a t-butyloxycarbonyl group, Cys indicates a cysteine group, Val indicates a valine group, d-Phe indicates a d-phenylalanine group, Pro indicates a proline group, Acm indicates an SH group protected by an acetamidomethyl group, and ONSu indicates an N-oxysuccinimido group.
wherein Boc indicates an amino group protected by a t-butyloxycarbonyl group, Cys indicates a cysteine group, Val indicates a valine group, d-Phe indicates a d-phenylalanine group, Pro indicates a proline group, Acm indicates an SH group protected by an acetamidomethyl group, and ONSu indicates an N-oxysuccinimido group.
13. A novel peptide represented by the following formula:
wherein H indicates a freed amino group, Cys indicates a cysteine group, Val indicates a valine group, d-Phe indicates a d-phenylalanine group, Pro indicates a proline group, Acm indicates an SH group protected by an acetamidomethyl group, and ONSu indicates an N-oxysuccinimido group; or a hydrochlorate of said peptide.
wherein H indicates a freed amino group, Cys indicates a cysteine group, Val indicates a valine group, d-Phe indicates a d-phenylalanine group, Pro indicates a proline group, Acm indicates an SH group protected by an acetamidomethyl group, and ONSu indicates an N-oxysuccinimido group; or a hydrochlorate of said peptide.
14. A novel peptide represented by the following formula:
wherein Cys indicates a cysteine group, Val indicates a valine group, d-Phe indicates a d-phenylalanine group, Pro indicates a proline group, Acm indicates an SH group protected by an acetamidomethyl group, and Cyclo indicates a cyclic form.
wherein Cys indicates a cysteine group, Val indicates a valine group, d-Phe indicates a d-phenylalanine group, Pro indicates a proline group, Acm indicates an SH group protected by an acetamidomethyl group, and Cyclo indicates a cyclic form.
15. A novel peptide represented by the following formula:
wherein Boc indicates an amino group protected by a t-butyloxycarbonyl group, Cys indicates a cysteine group, Val indicates a valine group, d-Phe indicates a d-phenylalanine group, Pro indicates a proline group, Acm indicates an SH group protected by an acetamidomethyl group, and NHNH2 indicates a hydrazidated carboxyl group.
wherein Boc indicates an amino group protected by a t-butyloxycarbonyl group, Cys indicates a cysteine group, Val indicates a valine group, d-Phe indicates a d-phenylalanine group, Pro indicates a proline group, Acm indicates an SH group protected by an acetamidomethyl group, and NHNH2 indicates a hydrazidated carboxyl group.
16. A novel peptide represented by the following formula:
wherein Boc indicates an amino group protected by a t-butyloxycarbonyl group, Cys indicates a cysteine group, Val indicates a valine group, d-Phe indicates a d-phenylalanine group, Pro indicates a proline group, and Acm indicates an SH group protected by an acetamidomethyl group.
wherein Boc indicates an amino group protected by a t-butyloxycarbonyl group, Cys indicates a cysteine group, Val indicates a valine group, d-Phe indicates a d-phenylalanine group, Pro indicates a proline group, and Acm indicates an SH group protected by an acetamidomethyl group.
17. A novel peptide represented by the following formula:
Boc-[Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro]2NHNH2 wherein Boc indicates an amino group protected by a t-butyloxycarbonyl group, Cys indicates a cysteine group, Val indicates a valine group, d-Phe indicates a d-phenylalanine group, Pro indicates a proline group, Acm indicates an SH group protected by an acetamidomethyl group, and NHNH2 indicates a hydrazidated carboxyl group.
Boc-[Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro]2NHNH2 wherein Boc indicates an amino group protected by a t-butyloxycarbonyl group, Cys indicates a cysteine group, Val indicates a valine group, d-Phe indicates a d-phenylalanine group, Pro indicates a proline group, Acm indicates an SH group protected by an acetamidomethyl group, and NHNH2 indicates a hydrazidated carboxyl group.
18. A novel peptide represented by the following formula:
H-[Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro]2NHNH2 wherein H indicates a freed amino group, Cys indicates a cysteine group, Val indicates a valine group, d-Phe indicates a d-phenylalanine group, Pro indicates a proline group, Acm indicates an SH group protected by an acetamidomethyl group, and NHNH2 indicates a hydrazidated carboxyl group; or a hydrochlorate of said peptide.
H-[Cys(Acm)-Val-Cys(Acm)-d-Phe-Pro]2NHNH2 wherein H indicates a freed amino group, Cys indicates a cysteine group, Val indicates a valine group, d-Phe indicates a d-phenylalanine group, Pro indicates a proline group, Acm indicates an SH group protected by an acetamidomethyl group, and NHNH2 indicates a hydrazidated carboxyl group; or a hydrochlorate of said peptide.
19. A novel peptide represented by the following formula:
Cyclo-[Cys(HgCl)-Val-Cys(HgCl)-d-Phe-Pro]2 wherein Cys indicates a cysteine group, Val indicates a valine group, d-Phe indicates a d-phenylalanine group, Pro indicates a proline group, and Cyclo indicates a cyclic form.
Cyclo-[Cys(HgCl)-Val-Cys(HgCl)-d-Phe-Pro]2 wherein Cys indicates a cysteine group, Val indicates a valine group, d-Phe indicates a d-phenylalanine group, Pro indicates a proline group, and Cyclo indicates a cyclic form.
20. A novel metal or heavy metal cluster complex of the novel cyclic peptide in accordance with claim 1 formed by a reaction of said novel cyclic peptide with a cluster of a metal or heavy metal selected from the group consisting of mercury, cadmium, zinc, molybdenum, cobalt, nickel, iron, titanium, palladium, manganese, silicon, and calcium.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1088562A JPH02268197A (en) | 1989-04-07 | 1989-04-07 | Novel peptide and intermediate for production thereof |
| CA002046124A CA2046124A1 (en) | 1989-04-07 | 1991-07-03 | Cyclic peptides |
| CA002046484A CA2046484A1 (en) | 1989-04-07 | 1991-07-08 | Peptides |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1088562A JPH02268197A (en) | 1989-04-07 | 1989-04-07 | Novel peptide and intermediate for production thereof |
| CA002046124A CA2046124A1 (en) | 1989-04-07 | 1991-07-03 | Cyclic peptides |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2046124A1 true CA2046124A1 (en) | 1993-01-04 |
Family
ID=25674682
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002046124A Abandoned CA2046124A1 (en) | 1989-04-07 | 1991-07-03 | Cyclic peptides |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2046124A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115521362A (en) * | 2022-09-30 | 2022-12-27 | 哈尔滨工业大学（深圳） | Preparation method of pseudodestruxinB |
-
1991
- 1991-07-03 CA CA002046124A patent/CA2046124A1/en not_active Abandoned
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115521362A (en) * | 2022-09-30 | 2022-12-27 | 哈尔滨工业大学（深圳） | Preparation method of pseudodestruxinB |
| CN115521362B (en) * | 2022-09-30 | 2024-05-28 | 哈尔滨工业大学（深圳） | Preparation method of Pseudodestruxin B |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US5446128A (en) | Alpha-helix mimetics and methods relating thereto | |
| Gruff et al. | Trigonal-planar [M (SR) 3] 1-complexes of cadmium and mercury. Structural similarities between mercury-cysteine and cadmium-cysteine coordination centers | |
| Barišić et al. | Helically chiral ferrocene peptides containing 1′‐aminoferrocene‐1‐carboxylic acid subunits as turn inducers | |
| EP0345171B1 (en) | Metalloporphyrine derivatives, their therapeutical use and their use in the preparation of hybrid molecules | |
| Zouikri et al. | Azaproline as a β‐turn‐inducer residue opposed to proline | |
| Xu et al. | 1, 1′-Ferrocenoyl–oligoprolines. A synthetic, structural and electrochemical study | |
| Brown et al. | Characterization of a crystalline synthetic analog of copper (II)-bleomycin | |
| Ohno et al. | Influence of the distal para substituent through NH---S hydrogen bonds on the positive shift of the reduction potentials of [Fe4S4 (Z-cys-Gly-NHC6H4-pX) 4] 2-(X= H, OMe, F, Cl, CN) complexes | |
| CA2046124A1 (en) | Cyclic peptides | |
| Loiseau et al. | High yield synthesis of tentoxin, a cyclic tetrapeptide | |
| CA1218650A (en) | Process for the synthesis of pyrido-imidazo- rifamycins | |
| Matsueda et al. | Peptide synthesis by oxidation-reduction condensation. Ii. The use of disulfide as an oxidant | |
| US3875136A (en) | Process for the manufacture of peptides containing cystine | |
| EP0500170A2 (en) | Cyclic hexapeptides having antibiotic activity | |
| Goel et al. | The synthesis and structural characterization of N-para-ferrocenyl benzoyl dipeptide esters: The X-ray crystal structure of N-{para-(ferrocenyl) benzoyl}-l-alanine-glycine ethyl ester | |
| GUPTA et al. | Conformation of dehydropeptides: synthesis and solution structure of model peptides containing (E) and (Z) dehydroaminobutyric acid residues | |
| US4459225A (en) | Peptide amides and process for their manufacture | |
| Lawrance et al. | Tetraazacycloalkanes With Pendant Carboxylates. Copper (II)-Directed Syntheses and Crystal Structure of (Ethyl 1, 5, 9, 13-tetraazabicyclo [11.2. 2] heptadecane-7-carboxylate) copper (II) Perchlorate | |
| JPH04346999A (en) | Chain peptide and its production | |
| JPH04346998A (en) | Chain peptide and its production | |
| Hiratani et al. | N, N'-Di (8-quinolyl) glutaramide Exhibiting Highly Selective and Efficient Uphill Transport of Cu (II) through Liquid Membranes. | |
| US6060586A (en) | Ureins derived from alpha, omega-diamono acids and process for their preparation | |
| US2638467A (en) | Process for the manufacture of organic nitrogen-containing compounds | |
| CA2028954A1 (en) | Process for preparing monohydrate 7-(d-2amino-2-phenylacetamido)-3-cefem-4-carboxylic acid and intermediate formed in the process | |
| CA2379851A1 (en) | Pseudomycin n-acyl side-chain analogs |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued |