Patent Application: US-57586404-A

Abstract:
the present invention therefore presents modules from which helical constraints can be built by very flexible strategies . the peptide bonds involved partially compensate the hydrophobic nature of the disulfide bonds , which are also included into the constraint strategy . thus , the invention presents solutions , by means of which peptide bonds or closure of disulfide bridges can be used alternatively for closure of the constraint . this offers greater synthetic flexibility . moreover , the peptide bonds are more hydrophilic than disulfide bridges alone and offer the advantage of better solubility of the product in an aqueous surrounding . it is possible to attach solvation tags like glycosyl moieties , polyethylenglycol or other suitable extensions or appendices to the helical constraint structure . usually , such a hydrophilic helical constraint structure replaces two hydrophobic amino acid side chains and thus improves pharmacologic properties of the molecule .

Description:
in general , the invention provides structures which can be adapted to almost every synthetic problem during the synthesis of helically stabilized peptides . structurally , the bridges , which are constructed alongside the sequence of the peptide , comprise a flexible covalent backbone with at least one amide bond and one disulfide bond . closure of the bridge by the disulfide bond will e . g . be a good way of formation of the bridge . but if necessary , the bridge can be closed e . g . by on - resin closure of one peptide bond , while the disulfide bridge was already introduced as a ready to use building block . the skilled person knows other possible ways or is able to find other possible ways for performing the invention after reading and understanding the present description of the invention . in a preferred embodiment of the invention , the amide - bond containing building blocks which form the bridge structures are made by solid phase synthesis . the peptide chemist is familiar with these methods . thus one of the advantages of these building blocks is that they are synthetically easily available for peptide chemists . below , a series of six general formulas will present the whole range of the invention . the invention encompasses helical constrained peptides represented by formula ( 1 ) to ( 7 ). wherein x is hydrogen or any amino acid or any peptide , y is any amino acid sequence consisting of six amino acids , z is hydroxyl or any amino acid or any peptide , a , b , c and d are independently selected from the integers 1 to 3 , provided that a + b + c + d is any integer in the range from 5 to 9 ; at each independent position of w , w can be freely chosen from hydrogen , a hydroxyl -, carboxyl - or amino group , an alkyl moiety with at least one hydroxyl -, carboxyl - or amino group , a polyethyleneglycol moiety , or a naturally occurring or artificial sugar molecule , and the peptides can consist of natural and / or unnatural d - and / or l - amino acids . examples 1 to 4 demonstrate the application of this formula . wherein x is hydrogen or any amino acid or any peptide , y is any amino acid sequence consisting of six amino acids , z is hydroxyl or any amino acid or any peptide , a , b and d are independently selected from the integers 1 to 5 , provided that a + b + d is any integer in the range from 7 to 11 ; at each independent position of w , w can be freely chosen from hydrogen , a hydroxyl -, carboxyl - or amino group , an alkyl moiety with at least one hydroxyl -, carboxyl - or amino group , a polyethyleneglycol moiety , or a naturally occurring or artificial sugar molecule , and the peptides can consist of natural and / or unnatural d - and / or l - amino acids . example 5 illustrates this formula . wherein x is hydrogen or any amino acid or any peptide , y is any amino acid sequence consisting of six amino acids , z is hydroxyl or any amino acid or any peptide , a , b and d are independently selected from the integers 1 to 5 , provided that a + b + d is any integer in the range from 7 to 11 ; at each independent position of w , w can be freely chosen from hydrogen , a hydroxyl -, carboxyl - or amino group , an alkyl moiety with at least one hydroxyl -, carboxyl - or amino group , a polyethyleneglycol moiety , or a naturally occurring or artificial sugar molecule , and the peptides can consist of natural and / or unnatural d - and / or l - amino acids . example 6 illustrates this formula . wherein x is hydrogen or any amino acid or any peptide or any compound represented by formula ( 1 ) to ( 2 ), y is any amino acid sequence consisting of six amino acids , z is hydroxyl or any amino acid or any peptide or any compound represented by formula ( 1 ) to ( 6 ), a , b , c and d are independently selected from the integers 1 to 3 , provided that a + b + c + d is any integer in the range from 5 to 9 and the peptides can consist of natural and / or unnatural d - and / or l - amino acids ; at each independent position of w , w can be freely chosen from hydrogen , a hydroxyl -, carboxyl - or amino group , an alkyl moiety with at least one hydroxyl -, carboxyl - or amino group , a polyethyleneglycol moiety , or a naturally occurring or artificial sugar molecule , and the peptides can consist of natural and / or unnatural d - and / or l - amino acids . example 7 illustrates the application of this formula . wherein x is hydrogen or any amino acid or any peptide or any compound represented by formula ( 1 ) to ( 6 ), y is any amino acid sequence consisting of six amino acids , z is hydroxyl or any amino acid or any peptide or any compound represented by formula ( 1 ) to ( 6 ), a , b and d are independently selected from the integers 1 to 5 , provided that a + b + d is any integer in the range from 7 to 11 and the peptides can consist of natural and / or unnatural d - and / or l - amino acids ; at each independent position of w , w can be freely chosen from hydrogen , a hydroxyl -, carboxyl - or amino group , an alkyl moiety with at least one hydroxyl -, carboxyl - or amino group , a polyethyleneglycol moiety , or a naturally occurring or artificial sugar molecule , and the peptides can consist of natural and / or unnatural d - and / or l - amino acids . example 8 illustrates this type of formula . wherein x is hydrogen or any amino acid or any peptide or any compound represented by formula ( 1 ) to ( 6 ), y is any amino acid sequence consisting of six amino acids , z is hydroxyl or any amino acid or any peptide or any compound represented by formula ( 1 ) to ( 6 ), a , b and d are independently selected from the integers 1 to 5 , provided that a + b + d is any integer in the range from 7 to 11 and the peptides can consist of natural and / or unnatural d - and / or l - amino acids ; at each independent position of w , w can be freely chosen from hydrogen , a hydroxyl -, carboxyl - or amino group , an alkyl moiety with at least one hydroxyl -, carboxyl - or amino group , a polyethyleneglycol moiety , or a naturally occurring or artificial sugar molecule , and the peptides can consist of natural and / or unnatural d - and / or l - amino acids . examples 9 and 10 illustrate the application of this formula . amino acids described in this invention can be of the naturally occuring l stereoisomer form as well as the enantiomeric d form . the one - letter code refers to the accepted standard polypeptide nomenclature , but can mean alternatively a d - or l - amino acid : by way of a non - limiting example a constraint building block was prepared as follows . cysteamine ( 10 mmol ) was dissolved in 20 ml trifluoroacetic acid . the solution was stirred at room temperature and a solution of acetamidomethanol ( 12 mmol ) was added dropwise over a period of 30 minutes . the mixture was stirred for additional 120 minutes and the volatile parts removed in vacuo . the residue was dissolved in 80 ml water and the ph adjusted to 9 . the product was then extracted with chloroform / isopropanol ( 3 / 1 ) and the solvents removed in vacuo . the crude product was then dissolved in a minimum of dcm and this solution added to a mixture of boc - β - ala ( 10 mmol ), cl — hobt ( 10 mmol ), diea ( 10 mmol ) and dic ( 20 mmol ) in a minimum of dcm . after 12 hours the solution was washed with satured sodiumhydrogencarbonate , sodiumhydrogensulfate and sodiumchloride , dried over sodiumsulfate and the solvent removed in vacuo . the residue was dissolved in 10 ml trifluoroacetic acid and stirred for 60 minutes . trifluoroacetic acid was then removed by coevaporation with dcm and the residue dissolved in dcm . the solution was neutralized by addition of diea and poured into a mixture of fmoc - glu - otbu ( 10 mmol ), cl — hobt ( 10 mmol ), diea ( 10 mmol ) and dic ( 20 mmol ). after 12 hours the solution was washed with satured sodiumhydrogencarbonate , sodiumhydrogensulfate and sodiumchloride , dried over sodiumsulfate and the solvent removed in vacuo . the residue was dissolved in 10 ml trifluoroacetic acid and stirred for 60 minutes . trifluoroacetic acid then was removed by coevaporation with dcm and the residue dissolved in dcm . the crude constraint building block was purified by hplc on a kromasil c - 18 column , eluted with acetonitrile - water gradient containing 0 . 1 % v / v trifluoroacetic acid and lyophylized . another example of the synthesis of a building block is the following procedure : a solution of n - α - fmoc - l - glutamic acid γ - allyl ester ( 20 mmol , 8 . 19 g ) and diisopropylethylamine ( diea ) in a minimum of dry dichloromethane ( dcm ) is added to 10 g of 2 - chlorotrityl resin ( 200 - 400 mesh , capacity 1 . 4 mmol / g ). the mixture is shaken for 60 minutes . after removing of the reaction solution by filtration the resin is washed 3 times with each 80 ml of dcm / methanol / diea ( 80 / 15 / 5 ). 80 ml of dcm / meoh / diea ( 80 / 15 / 5 ) are added to the resin which is shaken for 10 minutes and the solvent mixture is filtered off . this procedure is repeated once . the resin is washed 6 times with 200 ml dcm each . in order to determine the loading of the resin with the amino acid the wet resin is weighed and an aliquot of about 100 mg is taken and dried at the air . the fmoc group is removed by dcm / piperidine and the amount of the piperidine / dibenzofulvene adduct determined by uv absorption at 330 nm . typically , the loading is about 0 . 64 mmol / g dried resin . the resin is washed 3 times with 200 ml dcm each under argon . 200 ml dry dcm is added , argon is passed through the mixture for 15 minutes , 115 mmol phenyl silane ( 12 . 5 g ) and 1 ml diea is added and argon is passed another 30 seconds through the mixture . 4 . 33 mmol pd ( pph 3 ) 4 ( 5 g ) ( tetrakis ( triphenylphosphine ) palladium ( 0 )) is added . after 3 hours the resin is washed 5 times with 200 ml dcm each , 5 times with 200 ml dmf each , once again washed 5 times with 200 ml dcm each , and 5 times with 200 ml dmf each . a solution of 19 . 5 mmol β - alanine allylester hydrochlorid ( 3 . 23 g ), 25 mmol 6 - chloro - 1 - hydroxybenztriazole ( cl — hobt ) ( 4 . 24 g ), 75 mmol diea ( 12 . 39 ml ) and 25 mmol pybop ( 13 g ) ( benzotriazole - 1 - yl - oxy - tris - pyrrolidino - phosphonium hexafluorophosphate ) in 150 ml dmf is added to the deprocted resin . the mixture is shaken at room temperature ovemight . the resin is washed 6 - times with 100 ml dmf each . the resin is washed 3 times with 200 ml dcm each under argon . 200 ml dry dcm is added , argon is passed through the mixture for 15 minutes , 115 mmol phenyl silane ( 12 . 5 g ) and 1 ml diea is added and argon is passed another 30 seconds through the mixture . 4 . 33 mmol pd ( pph 3 ) 4 ( 5 g ) ( tetrakis ( triphenylphosphine ) palladium ( 0 )) is added . after 3 hours the resin is washed 5 times with 200 ml dcm each , 5 times with 200 ml dmf each , once again washed 5 times with 200 ml dcm each , and 5 times with 200 ml dmf each . a solution of 25 mmol cl — hobt ( 4 . 24 g ), 25 mmol pybop ( 13 g ) and 25 mmol diea ( 4 . 13 ml ) is added to the deprotected resin in 75 ml dmf . after 1 minute a solution of 25 mmol s - trityl - cysteamin - hydrochlorid ( 8 . 89 g ) and 50 mmol diea ( 8 . 26 ml ) in 75 ml dmf is added . the mixture is shaken overnight at room temperature . afterwards the resin is washed 4 times with 100 ml dmf each and 3 times 100 ml dcm each . ( a ) the resin is shaken for 2 hours with 200 ml 2 , 2 , 2 - trifluoroethanol / dcm ( 50 / 50 ). the volatile compounds are removed in vaclio . dcm is added and removed in vacuo 3 times . yield of crude product : 5 . 3 g ( 66 %) ( b ) the resin is shaken for 4 . 5 hours with 200 ml 2 , 2 , 2 - trifluoroethanol / dcm ( 90 / 10 ). the volatile compounds are removed in vacuo . dcm is added and removed in vacuo 3 times . yield of crude product : 0 . 5 g ( 6 %) ( c ) the resin is shaken for 3 days with 200 ml 2 , 2 , 2 - trifluoroethanol / dcm ( 50 / 50 ). the volatile compounds are removed in vacuo . dcm is added and removed in vacuo 3 times . yield of crude product : 0 . 73 g ( 9 %) total yield : 6 . 53 g ( 8 . 80 mmol , 82 % according to initial resin loading ) 1 h - nmr ( 400 mhz , dmso - d 6 ), δ [ ppm ]: 7 . 93 ( t , 1h , j = 5 . 6 hz ), 7 . 89 ( d , 2h , j = 7 . 4 hz ), 7 . 82 ( t , 1h , j = 5 . 6 hz ), 7 . 72 ( d , 2h , j = 7 . 4 hz ), 7 . 66 ( d , 1h , j = 7 . 9 hz ), 7 . 41 ( t , 2h , j - 7 . 4 hz ), 7 . 36 - 7 . 20 ( m , 17h ), 4 . 30 - 4 . 15 ( m , 3h ), 3 . 95 - 3 . 89 ( m , 1h ), 3 . 23 - 3 . 14 ( m , 2h ), 2 . 99 - 2 . 92 ( m , 2h ), 2 . 24 - 2 . 10 ( m , 6h ), 2 . 00 - 1 . 90 ( m , 1h ), 1 . 80 - 1 . 68 ( m , 1h ), lc - esi - ms : m / z =[ m + h ]: 743 ( 95 %), [ m + na ]: 765 ( 100 %). the sequences given in the examples below harbour target specific sequences , which can be used in the way described below , but might be modified without loss of desired action by means of single or multiple amino acid exchange operations . a substitution mutation of this sort can be made to change an amino acid in the resulting peptide in a non - conservative manner ( i . e . by changing an amino acid belonging to a grouping of amino acids having a particular charge or size or other characterisitics to a grouping of amino acids with other grouping parameters ) or in a conservative manner ( i . e . by changing amino acids within one grouping of amino acids ). such a conservative change generally leads to less change in the structure and function of the resulting protein . a non - conservative change is more likely to alter the structure , activity or function of the resulting protein , although — if done at the right place — might be without deleterious effect on the target - interactions . the present invention should be considered to include sequences containing conservative and non - conservative changes , which do not significantly alter the activity or binding characteristics of the resulting modified peptide as compared to the original sequence . the following is one example of various groupings of amino acids : amino acids with charged polar r groups ( negatively charged at ph 6 ): particularly preferred conservative substitutions are : lys for arg and vice versa ; glu for asp and vice versa ; ser for thr and vice versa ; gin for asn and vice versa . moreover , the invention includes modifications of the given binding regions of the peptide by amino acids , which transfer specific desired properties to the peptide . such improvements include n - and / ot c - terminal modifications , which protect the peptides against exopeptidas cleavage . preferred solutions of this problem include the use non - natural amino acids in terminal positions , especially preferred is the use of d - amino acids . non - conservative exchanges inside the peptide sequence might be used to transfer better water solubility to non - binding but hydrophobic regions of the peptide . chelating amino acids in n - or c - terminal postions can be use to enable the peptide to bind to metal - activated surfaces in order to assist purification and refolding during the production process . the bridge in example 1 connects the side chains of glutamine ( glutamic acid respectively ) and cysteine via beta - alanine and 2 - aminoethanthiol . this compound represents an antagonist for the interleukin - 2 receptor . the last step of the synthesis of the cyclic helical constraint bridge is normally the formation of a disulfide bridge : it has to be pointed out that this constraint bridge is custom - designed by molecular modelling . the bridge which connects the sidechains of amino acid i and i + 7 has appropriate size and orientation to stabilize the helix without strain . furthermore , the bridge is stabilized by an aspartate side chain in position i + 3 which acts as a supporting pillar . the hydrogen bond from one of the amide nh group to the aspartate side chain stabilizes the constraint and faciliates the synthesis of the bridge , because the correct conformation which leads to the formation of the disulfide bond is also stabilized . the three - dimensional molecular model in fig1 demonstrates the stabilizing effect of the hydrogen bond to the aspartate side chain at position i + 3 . thereby the invention provides a new way of stabilizing helical constraints by hydrogen bonds by amide structures in the bridge , combined with a disulfide bridge which is easy to form . another aspect in this invention is the stabilisation of the bridge from i to i + 7 by a hydrogen bond from a glutamine side chain in position i + 4 . in this case , the supporting pillar is the hydrogen bond donor and the constraint bridge is the hydrogen bond acceptor . this is in contrast to the previous structure , where the supporting pillar was the hydrogen bond acceptor and the constraint bridge was the hydrogen bond donor . the respective three - dimensional model can be seen in fig2 . the constraint bridge from amino acid i to i + 7 has appropriate size and orientation to stabilize the helix without strain . this bridge is stabilized by two custom - designed supporting pillars from two opposite sides represented by the sidechains of two standard amino acids . an aspartate side chain at position i + 3 acts as a hydrogen - bond acceptor which connects to an amide nh group of the constraint bridge . synchronously the constraint bridge is stabilized at the opposite side by a lysine side chain at position i + 4 which acts as a hydrogen bond donor for a carbonyl group of the constraint bridge . the three - dimensional molecular models in fig3 a and 3b demonstrate the stabilizing effect of the two supporting pillars from two opposite sides of the constraint bridge . the bridge in example 4 connects the side chains of glutamine ( glutamic acid respectively ) and cysteine via glycine and 3 - aminopropan - 1 - thiol . this compound represents an antagonist for the interleukin - 2 receptor . the bridge in example 5 connects the side chains of glutamine ( glutamic acid respectively ) and homocysteine via glycine and 2 - aminoethanthiol . this compound represents an antagonist for the interleukin - 2 receptor . the bridge in example 6 connects the side chains of asparagine ( aspartic acid respectively ) and cysteine via beta - alanine and 3 - aminopropan - 1 - thiol . this compound represents an antagonist for the interleukin - 2 receptor . the bridge in example 7 connects the side chains of glutamine ( glutamic acid respectively ) and homocysteine via 5 - aminopentari - 1 - thiol . the bridge backbone ist substituted with a sidechain containing two hydroxyl groups to improve the solubility of the compound . the bridge in example 8 connects the side chains of lysine and homocysteine via 3 - thiopropionic acid . this compound represents an antagonist for the interleukin - 2 receptor . the bridge in example 9 connects the side chains of cysteine and glutamine ( glutamic acid respectively ) via beta - alanine and 2 - aminoethanthiol . this compound represents an antagonist for the interleukin - 4 receptor . the bridge in example 10 connects the side chains of cysteine and glutamine ( glutamic acid respectively ) via omega - aminohexanthiol which is glycosylated to improve the pharmacokinetic properties of the compound . this compound represents an antagonist for the interleukin - 4 receptor . the bridges in example 11 and example 12 connect the side chains of homocysteine and lysine via 4 - thiobutyric acid . this compounds represent binding molecules for the erythropoietin receptor . circular dichroism can be used to determine whether a peptide is helical or not . in a cd spectrum , a zero point at 200 nm and a minimum in “ w ” form between 200 and 250 nm are indications for a helical structure . both criteria are independent of peptide concentration in solution . to find out how the helicity of the peptide in this example is influenced by trifluorethanol ( tfe ), the peptide with unclosed constraint was solved in tfe / h 2 o 1 : 9 , 2 : 8 , 3 : 7 , 4 : 6 , and 5 : 5 . regarding fig4 it can be noticed that zero points move to larger wave lengths and bands at ≈ 222 nm become more pronounced with increasing tfe concentration . from these facts it can be concluded that the amount of helical structure rises with increasing tfe concentration . the peptide with closed helical constraint was solved in tfe / h 2 o 1 : 9 and 5 : 5 , respectively . in comparison to the spectra of peptides without helical constraint , zero points can be found at larger wave lengths and the band at 222 nm is stronger . therefore it can be concluded that the helical constraint bridge attached to the peptide leads to a larger amount of helical structure . helical content is a significant factor for biological effectivity of this peptide . fig5 describes a nk - 92 proliferation assay of the helical constrained il - 2r binding peptide described above ( pep15cd , right ) in comparison with the corresponding native unconstrained peptide ( pep15c , left ). the activity of the constrained helical peptide shows that the constraint bridge is effective and fixes the bioactive conformation of the peptide . braisted , a ., k . j . judice , et al . 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