WO1989001000A1

WO1989001000A1 - Novel polypeptides for the use of preparing films which are carrying hydrophobic groups

Info

Publication number: WO1989001000A1
Application number: PCT/FI1988/000120
Authority: WO
Inventors: Jorma Virtanen; Paavo Kinnunen
Original assignee: K & V Licencing Oy
Priority date: 1987-07-31
Filing date: 1988-07-22
Publication date: 1989-02-09
Also published as: FI873336A0

Abstract

The present invention concerns novel macromolecules, i.e. polypeptides having formula (I), wherein each of the groups Ri means independently the amino acid residue (II), (III), (IV), (V) and/or (VI), which over the carbon atom is bound to the frame, and Xi means a) an aliphatic long-chained hydrocarbon group which may contain double bonds, or b) an aliphatic long-chained hydrocarbon group which may contain double bonds, and which contains covalently bound a group Z which with an adjacent Z group may participate in the transfer of a photon, electron or proton, R' means hydrogen, lower alkyl, especially methyl or ethyl, benzyl or allyl, R'' means hydrogen, t-butyloxycarbonyl, benzyloxycarbonyl or fluorenylmethyloxycarbonyl, and the integer n indicates the number of repeating units, whereby the amino acids forming two consecutive units in the molecule are of opposite optical configuration, i.e. D and L, respectively. The compounds may be used for the manufacture of Langmuir-Blodgett films which, depending on the nature of the group Z, may be tailor-made to exhibit specific electrical properties, or in the case the side chains are saturated may be used as insulating films.

Description

NOVEL POLYPEPTIDES FOR THE USE OF PREPARING FILMS WHICH ARE CARRYING HYDROPHOBIC GROUPS

The present invention concerns novel macromolecules which are characterized by a molecular frame formed by similar ba¬ sic molecular units, viz. amino acid units, which carries hydrophobic groups in the form of substantially aliphatic hydrocarbon chains covalently bound to these units, which chains may contain groups imparting specific properties to the macromolecule, especially a fluorescent group, a redox group or an acid-base pair. Such macromolecules may be used for the preparation of films exhibiting, for example, speci¬ fic electrical or fluorescent properties, using Langmuir- Blodgett film-forming techniques, for use in a variety of applications which are based on the detection and/or measu¬ rement of changes in potential or fluorescence. Another field of use, especially of macromolecules containing redox groups, is as chemical catalysts.

The novel macromolecules correspond to the general formula

wherein each of the groups R¹ means independently the amino acid residue

0

II

-CH₂ -CH₂ -CH₂ -CH2 -NH -C -, 0 0 0

II II If

- CH2-O-C-, -CH₂-S-, -CH₂-C-0- and/or -CH₂-CH₂-C-0- which over the carbon atom is bound to the frame, and Xi means a) an aliphatic long-chained hydrocarbon group which may contain double bonds, or

b) an aliphatic long-chained hydrocarbon group which may contain double bonds, and which contains covalently bound a group Z which with an adjacent Z group may participate in the transfer of a photon, electron or proton,

R¹ means hydrogen, lower alkyl, especially methyl or ethyl, benzyl or allyl, R" means hydrogen, t-butyloxycarbonyl, ben¬ zyloxycarbonyl or fluorenylmethyloxycarbonyl, and the inte¬ ger n indicates the number of repeating units, whereby the amino acids forming two consecutive units

respectively, are of opposite optical configuration, i.e. D and L, respectively.

The long-chained aliphatic hydrocarbon group a) is prefe¬ rably of the formula -(CH )_m - CH3, wherein m is advantage¬ ously from about 3 to 24, preferably from 10 to 20, and it may contain up to 6 double bonds. The group mentioned under b) has preferably the formula -(CH^j_ς -Z -(CH₂)]_ -H, wherein J and 1 are independently integers from about 0 to 16, pro¬ vided that k + 1 about 24 , and it may contain up to 6 double bonds. Preferably k is from about 4 to 12.

The value of the integer n is not strictly critical but is for practical purposes prefrably 5 or greater, such as from 5 to 100, typically from 5 to 40.

The compounds according to the invention are thus macromo¬ lecules of peptide type exhibiting a well-defined structure. They differ from regular polymers, the structure and also the molecular weight of which may not be correspondingly exactly tailored. It is prior known (e.g. Journal of Polymer Science, Vol. 15, 1977, p. 1499) to make polypeptides containing hydro- phobic groups, the basic peptide units of which, however, are of a single stereoisomeric form, usually the L-form. Such peptides have the tendency to form Λ-helical structures whereby the molecules will form a hydrophobic "shell" resul¬ ting in quite unsatisfactory LB-properties. The mutual dis¬ tances between the groups contained in the side chains in¬ crease in such a structure and it is not possible to predict which groups will be in contact with each other. When using such polypeptides in the films it will consequently not be possible to obtain specific properties which depend on the interaction between such groups.

Contrary to the known polypeptides, the gist of the present invention lies in the fact that - as is evident from the formula I - in the novel polypeptide structure all the the hydrophobic groups R¹-X¹ are directed to the same side of the plane defined by the chain • • *NH-CO-NH-CO •••• ^{Sucn a} structure is formed when one uses alternating optical confi¬ guration —D-L-D-L-... of the single amino acids used for making the macromolecule. In such a macromolecule the side chains will be parallel and located at a mutual distance of o , . about 3,4 A which is optimal with respect to the interaction of the molecules. By means of the described arrangement it is thus possible to avoid disturbing torsional movements of the molecules and the structure provides for the manufacture of LB-films of very good quality.

By choosing suitably the groups X¹ and Z as well as their mutual positions in the molecule, the properties of the macromolecule, especially the electrical properties, may be tailored to any specific application or use. Such properties will, subseqent to activation of the macromolecule, for example by using light, external voltage, or heat, manifest themselves and are detected as a change in fluorescence or electric potential. When preparing films according to the invention, the macro¬ molecules orient themselves on the liquid surface used for making the film in a symmetrical and a parallel fashion so that the hydrophilic groups are directed into the liquid and the hydrophobic groups in a direction outwards from the sur¬ face plane. This symmetrical arrangement is facilitated by the formation of hydrogen bonds between a keto group and an amide nitrogen of adjacent molecules in the films, in the following manner:

Due to the hydrogen bonds, the compounds form relatively stiff films. The deposition properties of the films re¬ main, however, good.

The group X¹ means as an aliphatic hydrocarbon group a) a saturated or unsaturated alkyl group. A saturated alkyl group is electrically inert, preventing the transfer of electrons, wherefore a film made of a macromolecule^" contai¬ ning only saturated side chains may be used, for example, as an insulating layer.

On the other hand, by incorporating into such an aliphatic hydrocarbon group double bonds, a tunnelling effect may be obtained in a film or aggregate of the said type. The transfer of electrons may thus be directed (channelled) in a desired manner by introducing into suitable positions of the side chains double bonds mediating the transfer of electrons.

As saturated groups X^x useful for the purpose may be menti¬ oned:

tridecyl - (CH₂ ⁾ι₂ ~^CH3 pentadecyl - <CH )i4 -CH3 heptadecyl - (CH )ιg -CH3

As unsaturated groups χⁱ useful for the purpose the heptadec-8-en-l-yl group may be mentioned

H H / t - (CH2.7 -C = C-(CH₂)₇ -CH3

H H tπcos-14-en-l-yl: -(CH₂)₁₃ -C = C-(CH₂)₇ -CH₃

nonadeca-4,7,10,13-tetraen-l-yl

H H -(CH₂)3 -(C = C- CH₂)₄ -(CH₂)₃ -CH₃.

The second group b) is formed by the saturated or unsatu¬ rated hydrocarbon groups containing a fluorescent group Z and/or a group Z capable of electron/proton transfer.

Energy transfer following absorption of a photon may take place either as exciton transfer, when the photon is transferred between similar groups, or as a so called Fδrster-transfer between different groups. Typical fluo¬ rescent groups participating in exciton transfer are the polycyclic aromatic groups, for example the following mono- or divalent residues derived from anthracene, pyrene or perylene, i.a.

Fδrster-energy transfer may take place between groups ex¬ hibiting mutually compatible spectra, whereby the emission spectrum of the donor group at least partly should be com¬ patible or coincide with the absorption spectrum of the acceptor group.

Typical groups capable of Fδrster-energy transfer are the afore mentioned polycyclic aromatic groups, especially py- renyl as a photon donor an as its acceptor amino-aryl groups substituted with nitro, nitrile and/or lower alkoxy groups, such as 1,4,6-trinitro-anilino-, 2-methoxy-4- nitro- or -5-nitro-anilino groups.

For groups capable of electron transfer, i.e. as redox groups, as donors the afore mentioned polycyclic aromatic groups, typically pyrene, as well as lower alkoxy, hydroxy and/or amino and/or loweralkyl amino substituted aryl groups, such as 1,4-phenylene groups or naphtyl groups come into question.

Groups exhibiting electron acceptor properties are such groups which are derived for example from benzo-, naphto- and anthraquinones and which may be further substituted, typically with cyano and/or halogen, such as the following groups

Other suitable groups include

Typical proton donors are the aromatic groups, such as phe¬ nyl, naphtyl and anthranyl groups containing hydroxy groups, e.g. 2-hydroxy-l-naphtyl or 4-hydroxy-l-naphtyl, 2,5-dihydroxy-l-4-phenylene, 9,10-dihydroxy-2,6-anthrylene as well as groups of tetrathio- and tetraselenofulvalenyl type of the formulas

Proton acceptors are typically aromatic groups containing a weakly basic amino group, wherein the nitrogen atom may form part of a cyclic system, anilino groups, whereby the benzene ring may be substituted by lower alkoxy and/or nitro, e.g. 4-phenylaminophenyl and 2-methoxy-4-nitro- anilino, and carbazolyl, for example 4-carbazolyl.

The macromolecules according to the invention may be pre¬ pared using conventional techniques, for example by in¬ creasing the molecular chain one amino acid unit at a ti¬ me.

Thus the polypeptides (I) are prepared from amino acids corresponding to the group

using methods known from peptide chemistry. For example, when the amino acid to be coupled is serine or cysteine the Ct-amino group is first protected with conventional protec- ting groups known from peptide chemistry, such as with t- butyloxycarbonyl or fluorenylmethyloxycarbonyl groups (t-BOC and FMOC protection, respectively), whereafter the group X is attached by reacting a carboxylic acid or its functional derivative corresponding to X, such as an acid chloride or anhydride, with the hydroxy group in serine and the mercapto group in cysteine, to form an ester and sulfide bond respec¬ tively. When the amino acid to be coupled is lysine, the group X may be attached in a known manner prior to protecti¬ on of the° -amino group in the manner described, for example by reacting the acid or its functional derivate correspon¬ ding to X with the second amino group in a conventional man¬ ner to form an amide bond. Thereafter theo -amino group is protected for the synthesis. The group X may be coupled over en ester bond to an amino acid or peptide containing a se¬ cond carboxylic acid group, such as aspartic and glutamic acid. - For the manufacture of the polypeptides, preferably a peptide synthetisator is used.

Longer polypeptide chains may be prepared by connecting the carboxylic end of one polypeptide to the amino acid end of the other.

In case the side chains are the same and the molecular weight need not be exactly specified it is simplest to first manufacture a dipeptide containing the D- and L- forms of the same amino acid. This is polymerised in a normal manner. Instead of the group X there may in the amino acid be a protecting group, which only after polyme¬ risation is replaced with the group X.

As mentioned above, the new macromolecules according to the invention are well suitable e.g. for the formation of monomolecular films using Langmuir-Blodgett techniques whereby specific electrical properties may be imparted to the films for various applications wherein use is made of fluorescence changes or changes in the electrical potenti¬ al as a consequence of transfer of an electron or a pro¬ ton, such as in various electronic, electric, electroche¬ mical or photochemical applications, such as in micro cir¬ cuits, photodetectors, sensors, microphones, micro lasers, semiconductor lasers etc. - A second field of use of the macromolecules are as chemical catalysts, either as such or coated onto a suitable support material.

The Langmuir-Blodgett film-forming technique in short is based on letting a film-forming surface active com¬ pound orient itself at the interface between two diffe¬ rent phases, for example the interface between a liquid, such as water, glycerol etc, and a gas,^" such as air, argon etc, the hydrophilic part of the molecule orient¬ ing itself towards the- liquid and the hydrophobic part, i.e. the lipophilic part, orienting itself in a direc¬ tion away from the liquid. A surface active compound dissolved in a suitable organic solvent or solvent mix¬ ture, for example in chloroform or cyclohexane, is spread onto the surface of a liquid contained in a trough. From the film spread as a monomolecular layer, for examp¬ le onto a water surface, the solvent evaporates rapidly. By means of a boom which rests on top of the trough and which is in contact with the liquid surface, the avai¬ lable surface area of the liquid-gas-interface is re¬ stricted, the total area of the monomolecular surface film thus decreasing or increasing. By means of the boom it is thus possible to regulate the surface tension of the film, which is inversely proportional to the surfa¬ ce pressure of the film, and which is determined by mea¬ suring the force exerted on a sensor in the film by means of a sensitive balance. When a support forming the substrate is transported vertically through the interface, preferably at constant speed, the film is deposited as a mo¬ nomolecular layer onto the substrate, the lipophilic part towards the substrate when the support is transported through the interface in a direction from air to water, and the hydrophilic part towards the substrate when the support is transported through the film in the opposite direction. The thickness of the individual film layer is substantially dependant on the organic compound used for the preparation of the film and especially on the length of the aliphatic chains contained therein, and it is usually of the order of 20 to 30 A. - The films may also be deposited using so-called horizontal deposition, whereby the support is lifted through the interface from the water phase horizontally. This mode of deposition is practical when the film to be deposited is very stiff.

By transporting the support several times through the interface, substantially any number of film layers may be deposited onto the substrate.

In the appended Figure is shown a compression isotherm, which curve is a characteristic of the film-forming proper¬ ties of a compound, and which in this case has been obtained for a LB-film made from the compound of the Example 1. The isotherm indicates that the film is somewhat stiff due to the fromation of hydrogen bonds. The transfer properties of the film were very good.

When such a film was deposited at 30 dyne/cm onto a quartz glass slide its absorbance was measured to be 0.14 at 345 nm. The absorbance of multilayer films increases linearly with the number of film layers. The absorbance measured cor¬ responds to the expected density of the pyrene groups in the compound.

The following Examples illustrate the invention. In the Examples the following abbreviations are used: M = myris- toyl; PD = pyrene decanoyl, D-Lys = D-lysine; L-Lys = L- lysine, BOC = t-butyloxycarbonyl; Me = methyl; Asp = aspartic acid; TU = tetrathiofulvalenyl undecanoic acid; PH pyrene hexanol.

Example 1

Preparation of

BOC-(PD-L-Lys)-(M-D-Lys)-(M-L-Lys)-(M-D-Lys)-(PD-L-Lys)-Me

a. Coupling of BOC-Pyrenyldecanoyl-L-lysine to a resin

1 g of hydroxymethyl resin (Merrifield) and 0.75 g PD-L- Lys were mixed in 25 ml of dichloromethane containing 12.2 mg of dimethylaminopyridine and 1 ml of 1M dicyclohexyl- carbodi-imide-dimethylformamide solution. It was filtered the following day, washed with dichlorometane and dried at 30°C.

b. Peptide synthesis

The resin used in the step a was transferred to a peptide synthetisator wherein the coupling of the amino acid deri¬ vatives was every time carried out using the following scheme:

1. Washing three times with dichloromethane (duration 3x30 sec. )

2. Addition of 15 ml of trifluoroacetic acid solution (5 in)

3. Addition of 15 ml of trifluoroacetic acid solution (15 min. )

4. Washing five times with dichloromethane (5x30 sec.)

5. Neutralization with triethyl amine (3x 1 min.)

6. Washing five times with dichloromethane (5x30 )

7. Addition of the next amino acid derivative

8. Addition of the dicyclocarbodi-imide solution c. Releasing the peptide from the resin

100 mg of resin was added to a 10 ml bottle. 4 ml of methanol, 4 ml of toluene and 1 ml of triethylamine were added. The mixture was heated over night at 50°C. After filtering and evaporation of the solution 18 mg of the title product was obtained, m.p. 198-200°C.

Example 2

Removing the protecting groups

The product according to the Example 1 was dissolved in 2 ml of dichloromethane while heating on a bath at 40°C. Dry hydrochloric acid gas was fed for 0.5 hours, whereby thin layer indicated that the starting material had disappeared and that a new product had formed, which was coloured by ninhydrine. The solution was evaporated to deryness. The residue was dissolved in 2 ml of dichloromethane/methanol 1:1 and 0.1 ml of 0.1 M aqueous sodium hydroxide solution was added. The mixtue was evaporated to dryness and the re¬ sidue purified in a LH-20 Sephadex-column of a volume of 5 ml and which was eluted with a 2:l-mixture of dichloromethane/methanol. Pure pentapeptide (PD-L-Lys )- (M-D-Lys)-(M-L-Lys)-(M-D-Lys )-(PD-L-Lys) was obtained in an amount of 5.6 mg.

In the same manner the hexapeptide (TU-L-Lys )-(Tϋ-D-Lys )- (TU-L-Lys)-(TU-D-Lys)-(TU-L-Lys)-(Tϋ-D-Lys) was prepared which has especially advantageous electricity conducting properties. Example 3

(PH-L- sp)-(PH-D-Asp)_n

6.4 g of /3-benzylester of N-t-BOC-L-aspartic acid was dis¬ solved in a mixture containing 100 ml of dichloromethane and 20 ml of dimethylformamide. 2.3 g of dicyclohexylcarbodi- imide in 40 ml of dichloromethane was added. The mixture was filtered after i hour and to the filtrate 2.3 g of /3-benzylester of D-aspartic acid and 1 g of triethylamine was added dissolved in 40 ml of dimethylformamide. The di¬ chloromethane was evaporated and the dimethylformamide solu¬ tion was poured into a 500 ml LH-20 Sephadex column which was eluted with a 3:l-mixture of dichloromethane/ethanol. The pure fractions were combined and evaporated. 3.8 g of the protected dipeptide was obtained. This was dissolved in 200 ml of dichloromethane at 40°C in a bath. Into the solu¬ tion dry hydrochloric acid gas was introduced during hour. The residue was evaporated to dryness and dissolved in 100 ml of a 3:l-mixture of dichloromethane/dimethylformamide. 1.8 g of dicyclohexylcarbodi-imide in 30 ml of dichloromet¬ hane was added. After filtering the dichloromethane was evaporated. 50 ml of ethanol and 0.5 g of Pd/C was added. Hydrogenation was carried out in a hydrogenation vessel at normal pressure for 2 hours. After filtering the ethanol was carefully evaporated from the filtrate and finally kept for several hours in a high vacuum. From the dimethylformamide solution one tenth was removed and to this was added 0.3 g of pyrenylhexanol, 0.23 g of dicyclohexylcarbodi-imide and 20 mg of dimethylaminopyridine in 5 ml of dichloromethane. Mixing was carried out for 6 hours in a 40°C bath. After filtering the filtrate was poured into a 100 ml LH-60 Sepha¬ dex column and eluted with a 3:l-mixture of dichloromethane/ethanol. 0.42 g of pure polypeptide (PH-L-Asp)-(PH-D-Asp)_{n was} obtained. Instead of pyrenehexanol other alcohols may be used. The corresponding polypeptide was made also using trimethyltet- raselenafulvalenyl-undecanol.

Claims

Cla ims

1. Compounds of the formula

wherein each of the groups R¹ means independently the amino acid residue

0 H -CH₂ -CH₂ -CH₂ -CH₂ -NH -C -,

0 0 0

- CH₂-0-C-, -CH₂-S-, -CH₂-C-0- and/or -CH₂-CH₂-C-0- which over the carbon atom is bound to the frame, and X¹ means

a) an aliphatic long-chained hydrocarbon group which may contain double bonds, or

R' means hydrogen, lower alkyl, especially methyl or ethyl, benzyl or allyl, R" means hydrogen, t-butyloxycarbonyl, ben¬ zyloxycarbonyl or fluorenylmethyloxycarbonyl, and the inte¬ ger n indicates the number of repeating units, whereby the amino acids forming two consecutive units

respectively are of opposite optical configuration, i.e. D and L, respectively.

2. Compounds according to the claim 1, c h a r a c t e r ¬ i z e d in that X¹ in the meaning of the group a) has the formula -(CH₂)_m - CH3, wherein m is from about 3 to 24, pre¬ ferably from 10 to 20, and optionally contains up to 6 double bonds.

3. Compounds according to the Claim 1 or 2, c h a r a c ¬ t e r i z e d in that X in the meaning of the group b) has the formula -(CH₂)_]ζ -Z -(CH₂)^ -H, wherein k and 1 are inde¬ pendently integers from about 0 to 16, provided that k + 1 <_ about 24, and optionally contains up to 6 double bonds, and

Z has the meaning in the claim 1.

4. Compounds according to any of the preceeding claims, c h a r a c t e r i z e d in that n is from 5 to 100, pre¬ ferably from 5 to 40.

5. Compounds according to the claim 1, c h a r a c t e r ¬ i z e d that they are used for the manufacture of Langmuir-Blodgett films.