Patent Application: US-201615551116-A

Abstract:
the invention relates to cyclic peptides of 3 - 9 amino acids comprising 2 - 7 aliphatic and 0 - 2 polar amino acids that are capable of self - assembling , wherein said aliphatic amino acids are arranged in decreasing hydrophobicity from n - to c - terminus and at least a portion of the cyclic peptide has to have its amino acids in alternating d - and l - configuration , as well as their use in hydrogels as well as co - gels or co - hydrogels . the hydrogels of the invention may be used in nanomedicine or drug delivery , cell culture or alternatively in electronic devices .

Description:
we have previously described ultrashort peptide sequences ( 3 - 7 residues ) which have an innate tendency to self - assemble into helical fibers that ultimately result in hydrogel formation , see e . g . wo 2011 / 123061 , us 2014 / 0093473 a1 , wo 2014 / 104981 a1 of the inventors , and hauser et al . ( 2011 ), mishra et al . ( 2011 ). the microarchitecture of these nanofibrous hydrogels resemble extracellular matrix , opening avenues for widespread applications as biomimetic scaffolds for tissue engineering and three - dimensional cell culture . furthermore , the ultrashort peptide hydrogels demonstrate remarkable mechanical stiffness , thermostability , biocompatibility , in vitro and in vivo stability . in particular , the stability of these hydrogels offer attractive advantages to applications such as developing injectable therapies for degenerative disc disease and other tissue engineering applications requiring the construct to provide structural support over long durations . however , in developing these hydrogels for shorter - term applications , such as injectable matrices for drug and gene delivery , it is desirable to precisely control the drug release rate . however , when a co - hydrogel , containing a bioactive compound and the peptide was formulated , only a burst release could be observed , a sustained release was never achieved . this application describes a novel class of self - assembling aliphatic cyclic peptides . inspired by the structure of previously mentioned class of ultrashort self - assembling peptides , the cyclic peptides represent a head to tail macrocylized form of these peptides . however , to achieve self - assembly of cyclic peptide , the peptide contains alternate l - and d - amino acids ( with regards to the absolute configuration , fig1 ). in comparison to this , the parent peptides only contain one amino acid stereo isomer ( all l or all d ). although the self - assembling properties of cyclic peptides are well known ( mandal et al ., 2014 ; montenegro et al ., 2013 ; li et al ., 2012 ), most of the reported systems do not form hydrogels . hydrogel formation could this far only be achieved using rigid structures . recently several groups independently report on the hydrogel formation using functionalized cyclic dipeptides . however , a cyclic dipeptide not only represents the smallest possible cyclic peptide , but is often better described as a diketopiperazine unit , and can thus be not considered as a macrocyclic peptide . gelation of diketopiperazine is achieved through additional functionalization of the amino acid side chain and cannot be seen as an intrinsic molecular behavior ( manchineella and govindaraju , 2012 ; hoshizawa et al ., 2013 ; kleinsmann and nachtsheim , 2013 ). to the best of our knowledge no macrocyclic peptide which can self - assemble to form hydrogels is reported this far . in this disclosure we describe the synthesis of macrocyclic peptides which can self - assemble in water to form hydrogels made of nano - tubular fibres . these peptides are made entirely of aliphatic α - amino acids , and self - assembly is only achieved through non - covalent interaction . all fmoc protected amino acids , o -( b enzotriazol - 1 - yl )- n , n , n ′, n ′- tetramethyluronium tetrafluoroborate ( tbtu ), benzotriazol - 1 - yl - oxytripyrrolidinophosphonium hexafluorophosphate ( pybop ) were purchased from gl biochem ( shanghai ) ltd . dimethylformamide ( dmf ) ( analytical grade ) was purchased from fisher scientific uk . acetic anhydride ( ac 2 o ) and dimethyl sulfoxide ( dmso ) was purchased from sigma aldrich . n , n - diisopropylethylamine ( dipea ), dichloromethane ( dcm ), trifluoroacetic acid ( tfa ) and tis ( triisopropylsilane ) were purchased from alfa aesar , a johnson matthey company . piperidine was purchased from merck schuchardt ohg . diethyl ether ( et 2 o ) was purchased from tedia company inc . all chemicals were used as received . all peptide based compounds were purified on an agilent 1260 infinity preparative hplc system equipped with a phenomenex lunar c18 column ( 150 × 21 . 2 mm 5 μm ). the hplc was coupled over an active splitter to a sq - ms for mass triggered fraction collection . milliq water and hplc grade acetonitrile , both containing 0 . 1 % formic acid , were used as eluents . 1 h and 13 c nmr spectra were recorded on a bruker av - 400 ( 400 mhz ) instrument and all signals were referenced to the solvent residual peak . h - livags - oh was synthesized on wang resin ( gl biochem ) using spps following standard peptide synthesis protocols ( kirin et al ., 2007 ). the de - protection of fmoc was achieved by treating the resin with piperidine in dmf . the supernatant was filtered off and the resin washed with dmf . coupling of the appropriate fmoc - protected amino acid to the resin was done by treating the resin with a combined solution of the amino acid ( 3 equivalent ), tbtu ( 3 equivalent ) and dipea ( 3 equivalent ) in dmf . the filtering - cum - washing , de - protection , and coupling cycle was then repeated until all the amino acids of the peptide were linked . the fmoc deprotected peptide was cleaved from the resin using a mixture of tfa / h20 / tis ( 95 : 2 . 5 : 2 . 5 ). after precipitation with et 2 o the solid was collected by centrifugation washed with et 2 o and dried . cyclization was carried out in solution at a concentration of 0 . 5 mg / ml in dmf using a threefold access of tbtu and dipea . the cyclization reaction was followed by hplc - ms and if required , more coupling reagent was added to achieve full cyclization . afterwards the solvent was removed , and the product was purified by hplc - ms . see fig5 to 7 for nmr and esi - ms spectra . clk6 was synthesized using standard solid phase cyclization reactions procedure ( abbour and baudy - floc ′ h , 2013 ). in short : fmoc - lys - oallyl ( 1 . 05 mmol ) was coupled to 2 - chlorotrityl resin ( 2 . 1 g ) in dmf / ch2cl2 ( 1 : 3 ). for this purpose , ctc resin was washed once with ch 2 cl 2 , afterwards , fmoc - lys - oallyl , dissolved in dmf and ch 2 cl 2 was added followed by 5 equivalents of dipea . after 5 min an additional equivalent of dipea was added . the reaction was allowed to proceed for 30 min . afterwards , the resin was quenched with meoh to avoid side reactions . the following peptide was synthesized as described above . after fmoc - d - leu - oh was added , the allyl group was removed using pd ( pph4 ) 4 ( 0 . 1 mmol ) and 10 equivalents of phsih 3 . the reaction was allowed to proceed in ch2cl2 in an open vessel for 1 hours . hplc - ms confirmed full deprodection . afterwards , the resin was washed 5 times with dmf followed by fmoc deprodection . final cyclization was carried out in dmf on the resin using pybop ( 4 equiv . ), hoat ( 4 equiv .) and dipea ( 4 equiv .) as coupling reagent . small amounts of resin were cleaved to follow the reaction by hplc - ms . once complete cyclization was achieved , the peptide was cleaved from the resin as described above . after purification by hplc - ms the pure product was obtained by lyophilization . see fig8 to 10 for nmr and esi - ms spectra . hydrogel samples were shock frozen and kept at − 80 ° c . frozen samples were then freeze - dried . lyophilized samples were fixed onto a sample holder using a carbon conductive tape and sputtered with platinum from both the top and the sides in a jeol jfc - 1600 high resolution sputter coater . the coating current was 20 ma and the process lasted for 50 sec . the surface of interest was then examined with a jeol jsm - 7400f field emission scanning electron microscopy ( fesem ) system using an accelerating voltage of 2 kv . as discussed above , we have previously reported a new class of aliphatic amphiphilic ultrashort peptides which have an innate tendency to self - assemble in water to form biomimetic , nanofibrous hydrogels with very high mechanical strength and are extremely stable in vitro and in vivo . in this patent application , we explore the possibility of conduction a head to tail macro cyclization reaction to obtain cyclic peptides . to achive this goal , the previously reported peptides sequences , which have been proven to form hydrogels , can be cyclized . however , to facilitate self - assembly of cyclic peptides a peptide containing alternate absolute stereo configurations of the amino acids have to be synthesized ( fig1 ). two parent peptide sequences were chosen to conduct a proof of concept study : firstly , ac - livags - oh [ seq id no . 21 ] was used , since it can be cyclized in solution as an unprotected peptide . for this purpose h 2 n - livags - oh was synthesized by standard fmoc - solid phase peptide synthesis ( see above for details ), whereby leucine and valine was used in d - absolute configuration . it has to be noted , that glycine does not have a stereocenter and thus no l or d stereoisomer exists . cyclization of h 2 n - livags - oh was performed in solution using standard reaction conditions yielding clivags (= cls 6 ). see fig2 a . since solution was cyclization resulted in low yield , the cyclic analog of ac - livagk - nh 2 [ seq id no . 16 ] was synthesized entirely on the solid phase . for this purpose , an orthogonal synthetic approach was used , whereby fmoc - lys - oallyl was the starting amino acid . after the entire fmoc protected peptide was synthesized , the allyl protection group can be removed without cleaving the peptide from the resin . this allows that the final cyclization reaction is carried on solid phase and the cyclic peptide , clivagk (= clk 6 ) can be cleaved from the resin and purified by hplc - ms ( see above ). see fig2 b . in order to determine the minimum gelation concentration in water , the cyclic peptides were attempted to be dissolved in milliq water . as cls6 displayed a low solubility in water , the minimum gelation concentration could not be determined . however , to prove , that cls6 is able to self - assemble in water , cls6 was dissolved in hexafluoroisopropanol ( hfip ) and dropped slowly into water . a gelatinous “ precipitate ” is formed when cls6 is dropped into water proving the ability of the cyclic peptide to self - assemble in water . the low solubility of cls6 in water can be attributed to the absence of a charged amino acid residue . in order to introduce a charged amino acid residue clk6 was synthesized and its ability to form hydrogels was investigated . for this purpose clk6 was dissolved at a concentration of 10 mg / ml in water . however , full solubility was only achieved , when the peptide solution was heated at 60 ° c . for about 2 h . after standing at room temperature , an opaque sol gel was formed . in contrast , when a 5 mg / ml solution of clk6 was prepared in the same way , a clear hydrogel was formed overnight ( see fig3 ). further reduction of the peptide concentration only resulted in an increase in viscosity , but no hydrogel formation could be observed . our previous studies on the parent peptide ac - livagk - nh2 have shown stimuli responsive behaviour to salt , which allows to reduce the minimum gelation concentration by 50 %. to test , whether clk6 displays stimuli response to salt concentration , a 5 mg / ml 1 × pbs solution was prepared . for this purpose , clk6 was dissolved in 9 parts of water and afterwards 1 part of 10 × pbs solution was added . after vortexing , only peptide aggregation , resulting in precipitation of clk6 was observed ( fig3 ). morphological characterization of the cls 6 hydrogel scaffolds was done by field emission scanning electron microscopy ( fesem ) and representative images are shown in fig4 . a fibrillization of cls 6 is clearly visible in both images , confirming the ability of the compound to self - assemble in water . we report here the synthesis of two cyclic peptides which are derived from a class of ultrashort aliphatic peptides . the cyclic peptides were synthesized though a head to tail cyclization reaction , either in solution or on solid support . although one example , cls6 displays limited water solubility , the compounds still displays self - assembling properties , when a solution of cls6 dissolved in hfip is added drop wise to water . to increase the water solubility clk6 was synthesized , whereby the lysine residue bares a positive charge , which should increase the water solubility . upon solubilizing clk6 in water at 60 ° c . a hydrogel is formed after about 2 h standing at room temperature an opaque sol gel was formed . in contrast , when a 5 mg / ml solution of clk6 was prepared in the same way , a clear hydrogel was formed overnight . further reduction of the peptide concentration only resulted in an increase in viscosity , but no hydrogel formation could be observed . fesem studies of cls6 confirmed a fibre structure of the hydrogels proving its ability to self - assemble in water . this new material can be used for drug delivery , nano printing , as nano template , for nano wires and as additive in other peptide based hydrogels . it is to be understood that the described embodiment ( s ) have been provided only by way of exemplification of this invention , and that further modifications and improvements thereto , as would be apparent to persons skilled in the relevant art , are deemed to fall within the broad scope and ambit of the present invention described herein . s . abbour and m . baudy - 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