PEPTIDOMIMETICS POSSESSING PHOTO-CONTROLLED BIOLOGICAL ACTIVITY

Abstract

The present invention relates to pharmaceutically and/or diagnostically active compounds, in particular peptide analogues (peptidomimetics), which can be reversibly controlled between an active and an inactive state by irradiation with light of different wavelengths. The present invention further relates to an intermediate compound usable in the manufacture of such pharmaceutically and/or diagnostically active compounds, as well as a manufacturing method thereof.

Claims

1-19. (canceled)

20. A photo-switchable molecular system comprising a scheme comprising formulae IVa and IVb ##STR00023## wherein R.sub.1 and R.sub.4 are independently selected from the group consisting of H, an alkyl group, alkenyl group, alkynyl group, alkoxy group, aryl group, heteroaryl group, cyano group, nitro group, phosphate group and sulfoxyl group; R.sub.2 and R.sub.3 are independently selected from the group consisting of an alkyl group, alkenyl group, alkynyl group, alkoxy group, aryl group, heteroaryl group, cyano group, nitro group, phosphate group and sulfoxyl group; X represents (CH.sub.xF.sub.y).sub.z, wherein x+y=2, x=0, 1 or 2, y=0, 1 or 2 and z=2 to 4; and Y.sub.1 and Y.sub.2 are independently selected from S, SO.sub.2, N, N-alkyl, or 0.

21. The photo-switchable molecular system according to claim 20, wherein R.sub.1 and R.sub.4 are H.

22. The photo-switchable molecular system according to claim 20, wherein R.sub.2 and R.sub.3 are methyl.

23. The photo-switchable molecular system according to claim 20, wherein X is CH.sub.2CH.sub.2CH.sub.2 or CF.sub.2CF.sub.2CF.sub.2.

24. The photo-switchable molecular system according to claim 20, wherein Y.sub.1 and Y.sub.2 are S.

25. The photo-switchable molecular system according to claim 20, wherein R.sub.1 and R.sub.4 are independently selected from the group consisting of H and a C.sub.1-C.sub.6 alkyl group, R.sub.2 and R.sub.3 are independently selected from the group consisting of a methyl group and an ethyl group and X is CH.sub.2CH.sub.2CH.sub.2 or CF.sub.2CF.sub.2CF.sub.2.

26. The photo-switchable molecular system according to claim 20, wherein each of R.sub.1 and R.sub.4 is H, each of R.sub.2 and R.sub.3 is a methyl group, X is CH.sub.2CH.sub.2CH.sub.2 or CF.sub.2CF.sub.2CF.sub.2, and each of Y.sub.1 and Y.sub.2 is S.

27. A method of photo-isomerizing an active compound, the method comprising: applying ultraviolet (UV) light to a compound of formula IVa to form a compound of formula IVb ##STR00024## wherein R.sub.1 and R.sub.4 are independently selected from the group consisting of H, an alkyl group, alkenyl group, alkynyl group, alkoxy group, aryl group, heteroaryl group, cyano group, nitro group, phosphate group and sulfoxyl group; R.sub.2 and R.sub.3 are independently selected from the group consisting of an alkyl group, alkenyl group, alkynyl group, alkoxy group, aryl group, heteroaryl group, cyano group, nitro group, phosphate group and sulfoxyl group; X represents (CH.sub.xF.sub.y).sub.z, wherein x+y=2, x=0, 1 or 2, y=0, 1 or 2 and z=2 to 4; and Y.sub.1 and Y.sub.2 are independently selected from S, SO.sub.2, N, N-alkyl, or 0.

28. The method according to claim 27 further comprising applying visible light to the compound of formula IVb to form the compound of formula IVa.

29. The method according to claim 27, wherein R.sub.1 and R.sub.4 are H.

30. The method according to claim 27, wherein R.sub.2 and R.sub.3 are methyl.

31. The method according to claim 27, wherein X is CH.sub.2CH.sub.2CH.sub.2 or CF.sub.2CF.sub.2CF.sub.2.

32. The method according to claim 27, wherein Y.sub.1 and Y.sub.2 are S.

33. The method according to claim 27, wherein R.sub.1 and R.sub.4 are independently selected from the group consisting of H and a C.sub.1-C.sub.6 alkyl group, R.sub.2 and R.sub.3 are independently selected from the group consisting of a methyl group and an ethyl group and X is CH.sub.2CH.sub.2CH.sub.2 or CF.sub.2CF.sub.2CF.sub.2.

34. The method according to claim 27, wherein each of R.sub.1 and R.sub.4 is H, each of R.sub.2 and R.sub.3 is a methyl group, X is CH.sub.2CH.sub.2CH.sub.2 or CF.sub.2CF.sub.2CF.sub.2, and each of Y.sub.1 and Y.sub.2 is S.

35. An intermediate compound represented by the general formula II or a salt thereof: ##STR00025## usable for the synthesis of a peptidomimetic compound represented by the general formula Ia: ##STR00026## wherein ZZ represents a protecting group; R.sub.1 and R.sub.4 are independently selected from the group consisting of H, an alkyl group, alkenyl group, alkynyl group, alkoxy group, aryl group, heteroaryl group, cyano group, nitro group, phosphate group and sulfoxyl group; R.sub.2 and R.sub.3 are independently selected from the group consisting of an alkyl group, alkenyl group, alkynyl group, alkoxy group, aryl group, heteroaryl group, cyano group, nitro group, phosphate group and sulfoxyl group; X represents (CH.sub.xF.sub.y).sub.z, wherein x+y=2, x=0, 1 or 2, y=0, 1 or 2 and z=2 to 4; Y.sub.1 and Y.sub.2 are independently selected from the group consisting of S, SO.sub.2, N, N-alkyl, or 0; P.sub.1 and P.sub.3 each independently represents a single amino acid residue or a peptide sequence of two or more amino acid residues; P.sub.2 is absent or represents a single amino acid residue or a peptide sequence of two or more amino acid residues; Q is C or N; R.sub.5 is selected from the group consisting of H, an alkyl group, heteroalkyl group, alkenyl group, heteroalkenyl group, alkynyl group or a heteroalkynyl group, and is bound to Q or may form a ring together with Q and N, or R.sub.5 is absent; R.sub.6 is selected from the group consisting of H, an alkyl group, heteroalkyl group, alkenyl group, heteroalkenyl group, alkynyl group, heteroalkynyl group, alkoxy group, aryl group, and heteroaryl group, or is absent; and R.sub.7 is selected from the group consisting of H, an amino acid side chain, an alkyl group, heteroalkyl group, alkenyl group, heteroalkenyl group, alkynyl group, heteroalkynyl group, alkoxy group, aryl group or a heteroaryl group; with the proviso that when P.sub.2 is absent, P.sub.1 and P.sub.3 are not bonded to each other; with the proviso that when Q is N, R.sub.5 is absent, and with the proviso that when R.sub.5 forms a ring together with Q and N, R.sub.6 is absent.

36. The intermediate compound according to claim 35, wherein ZZ is selected from the group consisting of t-butyloxycarbonyl (Boc) and fluorenylmethoxycarbonyl (Fmoc).

37. The intermediate compound according to claim 35, wherein R.sub.1 and R.sub.4 are independently selected from the group consisting of H and a C.sub.1-C.sub.6 alkyl group, R.sub.2 and R.sub.3 are independently selected from the group consisting of a methyl group and an ethyl group and X is CH.sub.2CH.sub.2CH.sub.2 or CF.sub.2CF.sub.2CF.sub.2.

38. The intermediate compound according to claim 35, wherein each of R.sub.1 and R.sub.4 is H, each of R.sub.2 and R.sub.3 is a methyl group, X is CH.sub.2CH.sub.2CH.sub.2 or CF.sub.2CF.sub.2CF.sub.2, and each of Y.sub.1 and Y.sub.2 is S.

39. The intermediate compound according to claim 35, wherein the peptidomimetic compound is represented by one of the following formulae GS-Sw (Lf), GS-Sw (FP) and GS-Sw (PV): ##STR00027##

40. A method of manufacturing the intermediate compound II-1 or a salt thereof defined in claim 35, represented by the general formula II, wherein Q is N, R.sub.6 and R.sub.7 is H, and R.sub.5 is absent ##STR00028## wherein ZZ represents a protecting group; R.sub.1 and R.sub.4 are independently selected from the group consisting of H, an alkyl group, alkenyl group, alkynyl group, alkoxy group, aryl group, heteroaryl group, cyano group, nitro group, phosphate group and sulfoxyl group; R.sub.2 and R.sub.3 are independently selected from the group consisting of an alkyl group, alkenyl group, alkynyl group, alkoxy group, aryl group, heteroaryl group, cyano group, nitro group, phosphate group and sulfoxyl group; X represents (CH.sub.xF.sub.y).sub.z, wherein x+y=2, x=0, 1 or 2, y=0, 1 or 2 and z=2 to 4; Y.sub.1 and Y.sub.2 are independently selected from the group consisting of S, SO.sub.2, N, N-alkyl, or 0; comprising the steps of: dissolving a dicarboxylic acid compound represented by the general formula III-1, a coupling reagent, a base and ZZ-hydrazine in a solvent; ##STR00029## wherein each of R.sub.1 to R.sub.4, X, Y.sub.1 and Y.sub.2 is as defined above; stirring the mixture for 30 minutes to 24 hours; and pouring the mixture into excess of water to obtain a compound of formula II-1 or a salt thereof as a precipitate.

41. The method according to claim 40 further comprising dissolving the precipitate in an organic solvent and washing the solution with aqueous sodium bicarbonate and hydrogen chloride solutions.

42. The method according to claim 40, wherein at least one of the following conditions are present: the solvent is selected from the group consisting of dimethylformamide, dimethylsulfoxide, hexamethylphosphotriamide; the protecting group is selected from the group consisting of t-butyloxycarbonyl (Boc) and fluorenylmethoxycarbonyl (Fmoc); and the coupling reagent is selected from the group consisting of carbodiimides, (N,N,N,N-Tetramethyl-O-(benzotriazol-1-yl (TBTU), 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) and (Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBop); and the base is selected from the group consisting of trimethylamine and diisopropylamine.

Description

THE FIGURES SHOW

[0090] FIG. 1 shows the cyclic antibiotic Gramicidin S (GS), and three photo-sensitive peptidomimetics derived from it. The GS analogues are shown in their open forms.

[0091] FIG. 2 shows kinetics of the photo-conversion of GS-Sw(FP) from its closed to its open form in a water-acetonitrile mixture, 3:1, at 25 C., 100 ug/ml concentration.

[0092] FIG. 3 shows analytical RP-HPLC chromatograms for GS-Sw(FP) acquired during the course of illumination of the peptidomimetic dissolved in a water-acetonitrile mixture, 3:1, 100 ug/ml by UV and visible light. The two neighboring peaks correspond to the two diastereomers of the closed form of the peptidomimetic (indicated).

[0093] FIG. 4 shows UV/VIS absorbance spectra of the peptidomimetics, GS-Sw(FP) in the open (dotted line) and closed (solid line). The signal at 400 nm is an instrumental artifact.

[0094] FIG. 5 shows an antimicrobial effect of the peptidomimetic GS-SwFP in the open form on Staphylococcus xylosus growth. The compound was applied to the bacterial lawn at different concentrations and then irradiated by visible light. Some geometrical shapes were cut out from a paper covering the entire Petri dish, such that the photo-switchable compound was converted into the open form only in those small areas exposed to the light. (A) 6 ug/ml of the closed form were converted into the open form by about 60% (as calculated from the curve on FIG. 2 and described in example 3); (B) 6 ug/ml, converted to 80%; (C) 8 ug/ml, converted to 60%; (D) 8 ug/ml, converted to 80%; Where the photo-switchable GS analogue is successfully activated, it exhibits a pronounced antimicrobial activity as seen from the transparent areas, where no bacterial growth in observed upon incubation at 37 C. for 18 hours.

[0095] FIG. 6 shows intermediate compound II-2 (compound 4), and particularly preferred examples thereof (compounds 4a, 4 b, 4c). Compound 4a contains a residue of L-proline and is an example of the building block with the amino acid fragment that contains a cyclic aliphatic ring; compound 4b contains a residue of N-methyl glycine and is an example of the photoswitching building block with N-substituted amino acid fragment; compound 4c contains a residue of glycine (amino acid side chain=H) and is the closest example to the building block where the NH-fragment is substituted by CH.sub.2.

[0096] FIG. 7 shows the .sup.1H NMR spectrum of compound 4a.

[0097] FIG. 8 shows the .sup.1H NMR spectrum of compound 14.

[0098] FIG. 9 shows the .sup.1H NMR spectrum of compound 7.

[0099] FIG. 10 shows the .sup.1H NMR spectrum of compound 8.

DETAILED DESCRIPTION

[0100] The peptidomimetic compounds of the present invention are chemically and thermally stable and reversibly transform between their biologically active and inactive (or less active) forms in high conversion efficiency through irradiation of light having suitable wavelengths. Furthermore, the peptidomimetic compounds of the present invention are biocompatible and resistant to photo-destruction and proteases. Consequently, the pharmaceutical and/or diagnostic activity of the peptidomimetics of the present invention may be effectively switched on and off, which renders the peptidomimetic compounds of the present invention particularly advantageous in the specific treatment of localized disorders in a patient. By only activating the pharmaceutical and/or diagnostic properties of the peptidomimetic at the desired site of action (and deactivating outside that area), side-effects are reduced and the therapeutic index is significantly increased. Particularly, the peptidomimetic compounds of the present invention may be readily employed in a variety of established applications, including photodynamic therapy.

[0101] In addition, the intermediate compound of the present invention easily allows to readily prepare a large variety of peptidomimetic compounds, e.g. using natural peptides as templates. Synthesis of such peptidomimetics is simple and can be achieved by using standard methods such as convergent synthesis, parallel synthesis, automated solid-phase synthesis, etc.

[0102] In the following, the present invention is further illustrated by the following examples, but is not limited thereby.

Example 1

Synthesis of the Photo-Switchable Building Block (1 b)

[0103] ##STR00017##

[0104] The starting dicarboxylic acid 2 used for the synthesis of Ib was obtained as described in the literature [S. Gronowitz, K. Stenhamar, L. Svensson, Heterocycles 1981, 15, 947; T. B. Norsten, N. R. Branda, J. Am. Chem. Soc. 2001, 123, 1784].

[0105] Compound 2 (5 g, 14.3 mmol) was dissolved in dimethylformamide (25 ml). N,N-diisopropylcarbodiimide (DIC, 1.76 g, 14 mmol) and subsequently N,N-diisopropylethylamine (DIPEA, 3.7 g, 28.6 mmol) were added to the solution. Fmoc-hydrazine (Fmoc-NHNH.sub.2; 3.56 g; 14 mmol) was added immediately. After stirring the reaction mixture overnight it was poured into water (100 ml). The precipitate was filtered, dissolved in dichloromethane (200 ml) and washed twice with 0.5 M aq solution of sodium bicarbonate (100 ml), then with 0.5 M aq solution of hydrochloric acid (100 ml) in order to remove the unreacted dicarboxylic acid.

[0106] The organic phase was dried with magnesium sulfate. Evaporation of dichloromethane under reduced pressure gave the crude material which contained, along with the desirable 1 b, also the by-product 3. The by-product did not interfere with the solid-phase peptide synthesis, so the obtained material was used without additional purification. The analytically pure 1 b can be obtained using RP-HPLC (acetonitrile/water mixture as the eluent).

[0107] .sup.1H-NMR (500 MHz, DMSO-d.sub.6), 5=1.90 (s, 3H, CH.sub.3), 1.94 (s, 3H, CH.sub.3), 1.95-2.05 (m, 2H), 2.79 (t, J=7.8 Hz, 4H), 4.2-4.4 (system CH.sub.2CH, two rotamers 4:1), 7.17-7.91 (m, aromatic protons, 101), 9.00-9.36 (rotamers 1:4, 11), 10.22-10.46 (rotamers 4:1, 11).

Example 2

Synthesis and Isolation of GS Analogues (General Procedure)

[0108] Synthesis of GS analogues: cyclo(.sup.DFPVO-1 b-PVOL), cyclo(.sup.DFPVOL-1 b-VOL) and cyclo(.sup.DFPVOL.sup.DF-1 b-OL) (GS-Sw(LF), GS-Sw(FP), GS-Sw(PV)).

[0109] The known peptide antibiotic Gramicidin S (GS) was used as a template. This cyclic decapeptide is known to exist in an antiparallel 3-sheet with the strands fixed by two -turns ([PVOL.sup.DFPVOL.sup.DF].sub.cyclo, with O=ornithine, and .sup.DF=D-phenylalanine). Four hydrogen bonds stabilize the overall amphipatic conformation of the molecule (cf. FIG. 1). GS is strongly membrane-active against Gram-positive bacteria, but has some undesirable hemolytic side-effects on red blood cells, and it is significantly protease-resistant. The unpolar diarylethene photo-switchable fragment in the open-form is well suited to replace the unpolar dipeptide units in one of the -turns, either L.sup.DF, .sup.DFP, or PV, thereby giving the respective peptidomimetics GS-Sw(LF), GS-Sw(FP), and GS-Sw(PV) (also cf. FIG. 1).

[0110] Standard Fmoc-based solid-phase synthesis and commercially available reagents were used for the synthesis of all the GS analogues. .sup.DPhenylalanine pre-loaded chlorotrityl resin with loading of 0.73 mmol/g (200 mg, 1 equiv) was used to synthesize the linear precursors. Coupling of the amino acid was performed using the following molar ratios of the reagents: Fmoc-amino acid (4 equiv), HOBt (4 equiv), HBTU (3.9 equiv), DIPEA (8 equiv). Incorporation of the diarylethene building block was performed by coupling with 1 b (in the form of the crude mixture as obtained in example 1 above; the amount was taken to provide 1.5 equiv of 1 b, the photo-switchable fragment in the open form), HOBt (1.5 equiv), HBTU (1.45 equiv.), DIPEA (3 equiv). The coupling time in all cases was 1 hour. N-Fmoc-deprotection was carried out by treating the resin with 20% piperidine in DMF for mm. After completing the synthesis, the resin was washed with dichloromethane and dried under vacuum for 24 h. The linear precursors were cleaved from the resin by a mixture of hexafluoroisopropanol and dichloromethane (1:3) (maintaining the side chain protection of ornithine residues). The volatile products from the filtered solution were blown off by argon flow. After dissolving the residue in an acetonitrile-water (1:1) mixture and subsequent lyophilization, the crude linear precursors were obtained and used for the cyclization without further purification. The conversion of the linear precursors into the targeted cyclic peptidomimetics was done in dichloromethane (1 L, the precursor did not dissolve completely) by addition solution of PyBOP (3 equiv) and HOBt (3 equiv) in dimethylformamide (1 ml) followed by DIPEA (6 equiv) to the suspension of the corresponding precursor. The reaction mixture was stirred for 8 h and additional amounts (the same as above) of the reagents (PyBOP, HOBt, DIPEA) were added. After 16 h, the solvent was evaporated under reduced pressure and the residue was lyophilized. The deprotection cocktail (trifluoroacetic acid, triisopropylsilane and water, 92.5:2.5:5 by volume, 10 ml) was added to the residue. After 15 min, the volatiles were blown off by argon flow and the residue was lyophilized.

[0111] The crude cyclic peptidomimetics were purified using RP-HPLC in two steps: first on a preparative C18 column (VYDAC, 22250 mm) with a linear A:B gradient of 8% B/min and 17 ml/min flow rate, followed by the second step on a C18 semipreparative column (VYDAC, 10250 mm) with a linear A:B gradient of 4% B/min and a 6 ml/min flow rate, where A is a mixture of 10% acetonitrile and 90% of the 5 mM HCl; B is a mixture of 90% acetonitrile and 10% of the 5 mM HCl. The purity of the peptidomimetics was checked on the analytical C18 column (VYDAC, 4.6250 mm) with a linear A:B gradient of 1% B/min and a 1.5 ml/min flow rate. The identity of each peptidomimetic was confirmed by MALDI-TOF mass spectrometry; m/z=1225.4 [GS-Sw(LF)], 1241.5 [GS-Sw(FP)], 1289.5 [GS-Sw(PV)].

Example 3

Characterization of Photochromic Properties of the GS Analogues

[0112] Each of the GS analogues was tested for photo-conversion efficiency from the more flexible state of the diarylethene unit (open form) to the rigid state (closed form) upon irradiation by UV light. Solutions of each peptidomimetic, GS-Sw(LF), GS-Sw(FP), and GS-Sw(PV), were prepared with a concentration of 100 ug/ml (in a water-acetonitrile mixture, 3:1). Then the extent of conversion from the open state to the closed state upon irradiation by UV light was determined using RP-HPLC (analytical C18 column, linear A:B gradient of 4% B/min, 1.5 ml/min flow rate) after 0, 5, 25, 50 and 75 mM of light exposition. A standard short-wavelength UV lamp (SPECTROLINEXX-15F/F) was used, and the solutions were placed in 10 cm distance from the lamp at 25 C.

[0113] The transformation proceeds up to 35-80%, depending on the conditions (see FIGS. 2 and 3 for results on GS-Sw(FP)). The extent of conversion could be considerably enhanced by the addition of chaotropic agents to the solutions, when the GS analogues were switched to the closed in 1 M aq solution of urea (see below).

[0114] The reverse photo-conversion of the peptidomimetic GS-Sw(FP) from the closed to the open form by visible light was also tested. A solution of peptide in the pink-colored closed-form (in a water-acetonitrile mixture, 3:1, 100 ug/ml) was used. The conversion of the peptidomimetic from the closed-form to the open-form was determined by RP-HPLC (analytical C18 column, linear A:B gradient of 4% B/min, 1.5 ml/min flow rate) after 0.25, 1.5, 5.5, 7.5 mm of irradiation by visible light. A bright halogen lamp (250 Watts) was used, and the solutions were placed in 10 cm distance from the light source. The obtained data fitted well to the exponential equation y=1exp(t/T), where y is a conversion of closed form into the open form, t is the time of the illumination and r is the half-conversion time. In order to achieve 60% transformation, the time of illumination should be 7.5 min, while 80% conversion is achieved in 12.5 min, etc. The conversion from closed to open could be achieved to 100%.

[0115] Stock solutions were prepared for all seven HPLC-purified compounds (wild type GS, and both open and closed forms for each of the three peptidomimetics), with a concentration of 1 mg/ml as verified by analytical RP-HPLC. To prepare the stock solutions of GS and its analogues in open form, the corresponding compounds were weighed and dissolved in 50% ethanol to obtain the desired 1 mg/ml concentration. To prepare the stock solutions of the GS analogues in the closed form, the following procedure was used:

[0116] The compounds were dissolved at a concentration of 100 ug/ml in 1 M aq urea and exposed to UV light for 25 min as described above. The open and closed forms were separated using RP-HPLC (preparative C18 column, linear A:B gradient of 8% B/min, 17 ml/min flow rate) and lyophilized. The corresponding retention times are listed in Table 1. The lyophilized fractions corresponding to the closed form of the peptidomimetics were dissolved in a small amount of 50% ethanol, and the concentrations were determined by analytical RP-HPLC. All these manipulations were done in the dark.

TABLE-US-00001 TABLE 1 Retention times at which GS and its analogues were eluted from the analytical HPLC C18 column (VYDAC, 4.6 250 mm) with a linear A:B gradient of 1% B/min and a 1.5 ml/min flow rate[[)]]. GS- GS- GS- GS- GS- GS- Sw(LF), Sw(FP), Sw(PV), Sw(LF), Sw(FP), Sw(PV), GS open open open closed closed closed RT [min] 44.9 34.9 40.1 41.5 24.2 26.4 31.6

[0117] The two isolated forms of the peptidomimetics have different absorbance spectra, showing the characteristics features of compounds bearing the diarylethene chromophores [M. Irie. Photochromism of diarylethene single molecules and single crystals. Photochem. Photobiol. Sci. 2010, 9, 1535-1542]. The UV/VIS absorbance spectra for one of the peptidomimetics, GS-Sw(FP) in the closed and open states are shown in FIG. 4.

Example 4

Photo-Switching the Antimicrobial Activity

[0118] The antimicrobial activities of GS and its analogues were measured using broth microdilution assay using a standard protocol [Daniel Amsterdam (1996). Susceptibility testing of antimicrobials in liquid media. In: Antibiotics in laboratory medicine, Loman, V., ed., 4th ed. Williams and Wilkins, Baltimore, Md., pp. 52-111]. The peptidomimetic compounds were tested against bacteria strains Escherichia coli DSM 1103, Staphylococcus aureus DSM 1104, Staphylococcus epidermidis DSM 1708, and Staphylococcus xylosus DSM 20267. GS analogues in the closed form were prepared in advance by RP-HPLC and stored protecting them from light. The corresponding minimal inhibitory concentrations (MIC) are listed in Table 2, where a small MIC value indicates a high antimicrobial activity, and vice versa. All photo-switchable GS analogues are thus seen to have a good antimicrobial activity in the open form, while they are much less active when the photo-switch is in the rigid closed state.

TABLE-US-00002 TABLE 2 Antimicrobial activities of GS and its photo-switchable analogues. Values of minimal inhibitory concentration (MIC) are given in mg/ml. GS- GS- GS- GS- GS- GS- Sw(LF), Sw(FP), Sw(PV), Sw(LF), Sw(FP), Sw(PV), GS open open open closed closed closed E. coli 8 >128 128 >128 64 >128 >128 S. aureus 2 8 4 4 128 32 16 S. epidermidis 2 16 8 4 128 64 32 S. xylosus 1 8 8 4 128 32 32

[0119] As seen in Table 2, it is possible to define therapeutically important concentration ranges in which the peptidomimetics in the open form suppress bacterial growth, while being inactive in the closed form. One further experiment aimed at finding these optimal conditions for treatment with the peptidomimetic GS-SwFP is illustrated in FIG. 5.

Example 5

Photo-Switching the Hemolytic Activity

[0120] Another biological activity of GS, GS-Sw(LF), GS-Sw(FP), and GS-Sw(PV), which is important for practical (in vivo) applications, is the hemolytic activity, and this can also be reversibly activated and deactivated by light. It should be noted that the hemolytic activity is the major side-effect of many antimicrobial peptides when applied systemically, which hinders their application as drugs.

[0121] To test the hemolytic activities of GS and its analogues, conserved human blood samples were obtained from Karlsruhe municipal hospital and washed four times in Tris buffer, pH 7.6, at 4.degree. C. Aliquots of the blood cells were incubated with different concentrations of the peptide/peptidomimetics for 30 mm at 37 C. and subsequently centrifuged. The absorption of the supernatant at 540 nm gives the extent of hemolysis, relative to 0% as taken from the peptide-free control and 100% after treatment with Triton X-100 (not to interfere with this analysis, the samples with GS analogues in the closed form were back-converted to their open forms by 30 min exposure to the visible light). The HC.sub.50 values, where 50% of the erythrocytes were lysed, were determined from the concentration dependent curves and are listed in Table 3. Small HC.sub.50 values indicate a high hemolytic activity, and vice versa. All GS analogues in the closed state were much less hemolytic than in the open state, just as it was seen for their antimicrobial activities. This proves by several independent assays that the biological activities of the photo-switchable GS analogues could be controlled by light.

TABLE-US-00003 TABLE 3 GS- GS- GS- GS- GS- GS- Sw(LF), Sw(FP), Sw(PV), Sw(LF), Sw(FP), Sw(PV), GS open open open closed closed closed H.sub.50, g/ml 12 47 >>128 6.5 72 6 58

Example 6

Synthesis of the Photo-Switchable Building Block 4a

[0122] Synthesis of 6.

[0123] The 15 g of compound 5 (0.0456 mol) was dissolved in 250 ml of dried tetrahydrofuran under inert atmosphere of argon gas. The solution was cooled to 78 C. by cooling bath with dry ice. To the cooled solution were added 20 ml (0.051 mol) of the 2.5 M butyllithium solution in hexane. The reaction mixture was let go to the temperature 10 C. and then again was cooled to 78 C. 4 g (0.0548 mol) of the dimethylformamide was added to the solution at 78 C. The solution was slowly (during half an hour) heated to 0 C. and stirred at that temperature for one more hour. Then solution was poured into 200 ml of water and 55 ml (0.055 mol) of 1 M hydrochloric acid was added. The product 6 was extracted by 200 ml of diethyl ether. Separated organic phase was dried by anhydrous magnesium sulfate and volatile solvents were removed under reduced pressure. Obtained 15 g of crude material without purification was used in next synthetic step.

[0124] Synthesis of 7.

[0125] The 15 g of crude compound 6 (approximately 0.0456 mol) was dissolved in 250 ml of toluene; 7.1 g (0.0684 mol) of 2,2-dimethyl-1,3-propanediol and 0.1 g of p-toluenesulfonic acid were added. Then the solution was refluxed with Dean-Stark apparatus until all the water, which has been formed in the reaction process, was removed (0.82 g). Toluene was removed under reduced pressure. Pure product 7 was obtained after column chromatography on silica gel using n-hexane/ethyl acetate 10:1 as eluent. The yield was 14.2 g (76% of theoretical) in two steps from 5 to 7.

[0126] Synthesis of 9.

[0127] The 3 g (0.00733 mol) of compound 7 was dissolved in 50 ml of dried tetrahydrofuran under inert atmosphere of argon gas. The solution was cooled to 78 C. by cooling bath with dry ice. To the cooled solution were added 3.52 ml (0.0088 mol) of the 2.5 M butyllithium solution in hexane. The reaction mixture was let go to the temperature 10 C. and then again was cooled to 78 C. 2.27 g (0.0088 mol) of the compound 8 was added to the solution at 78 C. Compound 8 was synthesized analogously to the protocol published by Z. H. Zhou et al, Heteroatom Chemistry, 2003, 7, 603-606. DOI: 10.1002/hc.10195. The solution was slowly (during half an hour) heated to 0 C. and stirred at that temperature for one more hour. Then solution was poured into 100 ml of water and 9 ml (0.009 mol) of 1 M hydrochloric acid was added. The product 9 was extracted by 100 ml of diethyl ether. Separated organic phase was dried by anhydrous magnesium sulfate and volatile solvents were removed under reduced pressure. Pure product 9 was obtained after column chromatography on silica gel using as the eluent n-hexane/ethyl acetate 5:1. The yield was 3.1 g (73% of theoretical).

[0128] Synthesis of 10.

[0129] The 3.1 g (0.0064 mol) of compound 9 was dissolved in 40 ml of ethanol. Then 7.42 g (0.032 mol) of freshly prepared argentum oxide and 0.5 g (0.0128 mol) sodium hydroxide were added and actively stirred for two hours. 20 ml (0.02 mol) of 1 M hydrochloric acid and 40 ml of ethanol were added. Formed precipitate was filtered on a paper filter and the solution with the product 10 was extracted twice by 100 ml of diethyl ether. Separated organic phases were combined and dried by anhydrous magnesium sulfate and volatile solvents were removed under reduced pressure. The yield was 3.2 g (100% of theoretical) of pure compound 10.

[0130] Synthesis of 4a.

[0131] The 3.2 g (0.0064 mol) of compound 10 was dissolved in 20 ml of dichloromethane. 2 ml of trifluoroacetic acid was added to the solution and solution was incubated for 2 hours at room temperature. Then volatile solvents were removed under reduced pressure. Then obtained yellow oil was dissolved in 50 ml of water/acetone 1:1. 1,075 g (0.0128 mol) of sodium bicarbonate and 3.3 g (0.0128 mol) of fluorenylmethoxycarbonyl chloride were added. Solution was kept under stirring for 4 hours at room temperature. Then slowly 12.8 ml (0.0128) of 1 M hydrochloric acid were added and product 4a was extracted twice by 100 ml of diethyl ether. Separated organic phases were combined and dried by anhydrous magnesium sulfate and volatile solvents were removed under reduced pressure. Pure compound 4a was obtained after column chromatography on silica gel with eluent n-hexane/ethyl acetate 5:1. The yield was 4 g (91% of theoretical).

##STR00018## ##STR00019##

Example 7

Synthesis of Compound 4b

[0132] Compound 4b was synthesized using the same protocols as in the case of 4a preparation.

##STR00020##

Example 8

Synthesis of Compound 4c

[0133] Synthesis of 14.

[0134] The 3 g of compound 7 (0.0073 mol) was dissolved in 75 ml of dried tetrahydrofuran under inert atmosphere of argon. The solution was cooled to 78 C. by cooling bath with dry ice. To the cooled solution were added 3.52 ml (0.0088) of the 2.5 M butyllithium solution in hexane. The reaction mixture was let go to the temperature 10 C. and then again was cooled to 78 C. 1.08 g (0.0088 mol) of the ethyl chloroacetate was added to the solution at 78 C. The solution as slowly (during half an hour) heated to 0 C. and stirred at that temperature for one more hour. Then solution was poured into 200 ml of water and 55 ml (0.055 mol) of 1M hydrochloric acid was added. The product 6 was extracted by 200 ml of diethyl ether. Separated organic phase was dried by anhydrous magnesium sulfate and volatile solvents were removed under reduced pressure. Pure compound 14 was obtained after column chromatography on silica gel with eluent n-hexane/ethyl acetate 4:1. The yield was 1.8 g (54% of theoretical).

[0135] Synthesis of 15.

[0136] Converting compound 14 to 15 was done using the same protocol as for converting compound 9 to 10 with 100% yield.

[0137] Synthesis of 16.

[0138] 1.8 g (0.00472 mol) of compound 15 was dissolved in 20 ml water/ethanol 1:1. 0.5 g (0.0076 mol) of sodium azide was added. The reaction mixture was stirred for 24 hours at 40 C. Then 50 ml of water were added and the product 16 was extracted by 100 ml of diethyl ether. Organic phase was dried by anhydrous magnesium sulfate and volatile solvents were removed under reduced pressure. Obtained 1.81 g of crude material was used in the next synthetic step without purification.

[0139] Synthesis of 4c.

[0140] 1.81 g (0.00470 mol) of compound 16 was dissolved in 20 ml of methanol in a 500 ml volume glass. 100 mg of palladium, 10% on carbon, were added. The air from the glass was pumped off and hydrogen gas was pumped in.

[0141] Afterwards the glass was connected to the balloon with hydrogen gas and solution was kept under stirring for 4 hours at room temperature. Then glass was connected to vacuum in order to remove the hydrogen gas and the solution was filtered. Methanol was removed yielding yellow oil. Then obtained yellow oil was dissolved in 50 ml of water/acetone 1:1. 0.79 g (0.0094 mol) of sodium bicarbonate and 2.4 g (0.0094 mol) of fluorenylmethoxycarbonyl chloride were added. Solution was actively stirred for 4 hours at room temperature. Then slowly 9.4 ml (0.0094) of 1 M hydrochloric acid were added and product 4a was extracted twice by 100 ml of diethyl ether. Separated organic phases were combined and dried by anhydrous magnesium sulfate and volatile solvents were removed under reduced pressure. Pure compound 4b was obtained after column chromatography on silica gel with eluent n-hexane/ethyl acetate 5:1. The yield was 2.74 g (90% of theoretical).

##STR00021## ##STR00022##