monomeric peptide was synthesized as peptide amide by an automated synthesizer ( multisyntech , witten ; germany ) on a rink amide mbha resin ( nova biochem ) using 9 - fluorenytmethoxycarbonyl ( fmoc ) chemistry and o -( benzottriazol - 1 - yl )- n , n , n ′, n ′- tetramethyluronium hexafluorophosphate / 1 , 3 - diisopropylethylamine activation . branched peptide molecules ( maps ) were synthesized on fmoc 4 - lys 2 - lys - βala wang resin . side chain protecting groups were trityl for gln tert - butoxycarbonyl for lys , 2 , 4 , 6 , 7 - pentamethyldihydrobenzofuran - 5 - sullonyl for arg , and tert - butyl ether for ser . peptides were then cleaved from the resin and deprotected with trifluoroacetic acid containing water and triisopropylsilane ( 95 / 2 . 5 ,/ 2 . 5 ). crude peptides were purified by reversed - phase chromatography on a vydac c18 column . tfacetate - acetate counter ion exchange is performed on a ion - exchange resin ( ag1 - x8 acetate , biorad ) and the peptides are lyophilized and isolated as peptide ch 3 coo —. identity and purity of final products was confirmed by ettan ™ maldi - tof mass spectrometry ( ms ) ( amersham biosciences ). the structure of a tetra - branched m33 peptide as described above is reported in fig1 . minimum inhibitory concentration ( mic ) was determined by a standard microdilution assay as recommended by the national committee for clinical laboratory standards ( nccls ) using cation - supplemented mueller - hinton ( mh ) broth ( oxoid ltd . basingstoke , uk ) and a bacterial inoculum of 5 × 10 4 cfu per well , in a final volume of 100 μl . results were recorded by visual inspection after 24 h of incubation at 37 ° c . peptide mics were determined against strains of several bacterial species , including gram - negative and gram - positive pathogens ( table 1 and 2 ). activity of tetra - branched d - m33 was retained against mdr strains with various resistance mechanisms ( such as extended - spectrum beta - lactamases and carbapenemases ), including mdr p . aeruginosa strains from cf patients . the antimicrobial activity of tetra - branched d - m33 appeared slightly better with respect to tetra - branched l - m33 for mdr strains of pseudomonas and one strain of klebsiella and sensitively better respect to tetra - branched l - m33 for the two strains of staphyloccoccus aureus reported in table 1 . minimal inhibitory concentration of tetra - branched d - m33 was consequently tested against a panel of gram - positive bacteria and fungi for which tetra - branched l - m33 was not active at concentrations below 24 μm ( table 2 ). surprisingly , tetra - branched d - m33 is active against all microorganisms tested , with the better efficacy ( 0 . 35 μm ) for the strains of s . epidermidis and one strain of s . capitis . mic of 0 . 35 μm was never reported for tetra - branched l - m33 against gram - negative bacteria . haemolysis of fresh human erythrocytes was determined using the method of parpart , summarized as follows . a calibration curve was constructed by suspending fresh human erythrocytes in phosphate buffer ( ph 7 . 4 , 110 mm sodium phosphate ) with various concentrations of nacl and incubated for 30 min at room temperature . samples were centrifuged at 500 × g for 5 minutes , and haemoglobin release was monitored by measuring the absorbance of supernatants at 540 nm . the absorbance obtained with 0 . 1 % nacl corresponded to 100 % lysis and that with 1 % nacl , to 0 % lysis . peptides dissolved in pbs were added to human erythrocyte solution at several concentrations . the resulting suspension was incubated separately at 37 ° c . for 2 h and 24 h . release of hemoglobin was monitored by measuring the absorbance of the suspernatant at 540 nm after centrifuging and haemolysis percentage was calculated using the calibration curve . a very important feature is that , contrary to most antimicrobial peptides described so far , tetra - branched d - m33 shows a negligible haemolysis grade ( fig2 ), suggesting their possible use also through systemic administrations . 8 μl of a 1 - mg / ml solution of peptides was incubated at 37 ° c . with 20 μl human serum ( fig3 ) or plasma . samples withdrawn at time 0 ( fig3 a ) and after 24 h ( fig3 b ) were precipitated with 200 μl methanol , centrifuged for 1 min at 10 000 × g and diluted with 800 μl 0 . 1 % tfa in water . these solutions were analyzed by hplc using a c18 - column controls for peptide retention time in the crude mixture were obtained by adding the same concentration of tetra - branched peptides to supernatants of plasma or serum treated with methanol and centrifuged as above , running the mixture immediately . ms analysis of the supernatant of crude solutions was performed on an ettan maldi tof mass spectrometer . the results are reported in fig3 . tetra - branched d - m33 peptide was analysed for its antibacterial activity in mice infected with lethal amounts of the gram - negative bacterium e . coli or the gram - positive bacterium s . aureus . the smallest number of bacteria causing 100 % lethal infection ( ld100 ) after intra - peritoneal ( ip ) injection was 1 . 5 × 10 9 with e . coli and 5 × 10 6 with s aureus . bacterial ld100 killed mice in 15 - 24 hours . balb - c mice were infected with the ld100 of bacteria and treated 1 hour later with the peptide by ip administration . following infection with bacteria , d - m33 protected 100 % of animals from signs of sepsis and death ( seven - day survival ) when administered in a single dose at a concentration of 10 mg / kg for e . coli ( fig4 ) and 25 mg / kg for s . aureus ( fig5 ). d - m33 did not produce apparent toxicity signs in animals treated ip with a peptide dose of 100 mg / kg ( not shown ), 10 time the dose reported in fig4 and 4 times the dose of fig5 . d - m33 proved more stable to bacterial proteases than l - m33 . fig6 shows the incubation of both peptides with the s . aureus protease aureolysin , which appears particularly active against l - m33 and inactive within 24 hours against d - m33 . falciani c , et al ., 2007 , chem biol drug des , 69 : 216 - 21 hancock r e and sahl h g , 2006 , nat biotecnol 24 : 1551 - 7 stasko n a and schoenfisch m h , 2006 , j am chem soc , 28 : 8265 - 71 wenzel r p and . edmond m b , 2000 , n engl j med 343 : 1961 - 3
the instant invention refers to an antibacterial peptide with all amino acids in d - configuration , possessing strong antimicrobial activity against gram - negative and gram - positive bacteria and candida strains . the peptide can be in linear form multimerised on a skeleton of polyacrylamide , of dextrane units or on a skeleton of ethylene glycol units . the peptide is resistant proteolysis especially when synthesized in the tetra - branched form where identical peptide sequences are linked together by an appropriate molecular scaffold .