| A polypeptide generally refers to a compound of ?-amino acid monomers linked by peptide bonds.The therapeutic peptides have many advantages such as clear biological function,specific action,low toxicity and have been widely used in clinical for stimulation of hematopoiesis,anti-tumor and immunomodulatory effects etc.In order to overcome their poor stability and short half-life,chemical modification with polymers such as polyethylene glycol(PEG)is considered to be a simple and effective strategy.Considering that the existing chemically modified polypeptide drugs still have problems such as poor selectivity of the reaction site,low modification yield,and easy loss of activity,we attempted to use enzyme catalysis for site-directed modification of peptide drugs with PEG or hyaluronic acid(HA).The tool enzyme is a microbial transglutaminase(mTGase)derived from Streptomcyes mobaraensis and can catalyze the formation of a new amide bond between the y-carboxamide group of glutamine(Gln)and the primary amine group of lysine(Lys).Accordingly,this present study included three active peptides,thymopentin(TP5;amino acid sequence RKDVY),antitumor polypeptide PCK3145(amino acid sequence EWQTDNCETCTCYGT)and designed anti-angiogenic active peptide ES2-TH(amino acid sequence IVRRADRAAVPGGCGKRK),each of which contained Gln or Lys residues.The end-functionalized PEG or HA derivatives were used as modifiers for site-specific modification catalyzed by mTGase.The pharmacokinetic profiles and in vitro activities of the resulting PEG/HA-polypeptide conjugates were determined and compared with the unmodified polypeptides.The aim of this study was to explore highly efficient and site-specific modification of peptide drugs with PEG/HA by using mTGase catalysis.The research content and main achievements of this thesis are as follows:1.Enzymatic synthesis and characterization of PEG-polypeptide conjugates(1)Synthesis and characterization of PEG-ZQGThe carboxyl group of the benzyloxycarbonyl-L-glutamine-glycine(ZQG),a dipeptides substrate of mTGase,was activated by EDC/NHS,then reacted with an amino group of tert-butylamine polyethylene glycolamine(BOC-PEG-NH2)to give PEG-ZQG,and the reaction yield was 75.3%.The UV-visible scan spectra between 200 nm and 800 nm showed that both PEG-ZQG and ZQG have characteristic absorption peaks at 255 nm.The 1H NMR spectrum of PEG-ZQG showed a characteristic signal peak of ZQG benzene ring at ?=7.47-7.17 ppm,so the PEG-ZQG containing a single Gin residue was successfully synthesized.(2)Preparation and characterization of PEG-TP5TP5 containing a single Lys residue was attempted to covalently attach to the Gln residue of PEG-ZQG under the catalysis of mTGase.The reaction solution was purified by Superdex 30 gel column to obtain the new product(named as PEG-TP5),and the reaction yield was 78.5%.HPLC analysis showed that the purity of PEG-TP5 was 96.6%.Similar to TP5,the UV-visible scan of PEG-TP5 showed characteristic absorption peaks at 220 nm and 275 nm.Its 1H NMR spectrum showed the characteristic signal peaks of the benzene ring of the TP5 tyrosine residue at ?= 7.02 ppm and ?=6.73 ppm.The characteristic signal peak of two methyl groups of TP5 proline residue was found at ? = 6.73 ppm.The signal peak of lysine residue side chain C4-H in PEG-TP5 shifted to the lower field(? = 3.18 ppm)compared to the TP5(?=2.53 ppm),indicating that PEG-ZQG was conjugated with TP5 lysine side chain amino group.The integral ratio of the benzene ring signal peak of TP5 to the tert-butyl signal peak of BOC-PEG-NH2(? = 1.32 ppm)in the conjugate is 0.41:1.Each TP5 molecule contains 4 aromatic hydrogens,and each BOC-PEG-NH2 molecule contains 9 t-butyl hydrogens.Thus,the ratio of TP5 to PEG molecules in PEG-TP5 was calculated as 1:1.2.Enzymatic synthesis and characterization of HA-peptide(1)Preparation and characterization of 6-O-sulfated HAHA was first converted to tetrabutylammonium salt by ion exchange method,and then reacted with different concentrations of sulfur trioxide pyridine complex to obtain sulfated HA(SHA)with different sulfation.Elemental analysis showed that the molar ratios of sulfur to nitrogen of SHA-1,SHA-2,SHA-3 and SHA-4 were 0.0500,0.9333,1.453 and 2.888,respectively.The 1H NMR spectrum of SHA-2 showed that only the signal peak of C6-H at ?= 3.4 ppm shifted toward the lower field,while the signal peaks of C2-H,C3-H and C4-H did not.Therefore,SHA-2 is a 6-O-sulfated HA derivative.(2)Preparation and characterization of HA-HMDBecause there was no free amino group in the HA.a reductive amination reaction is used to attach 1,6-hexamethylene diamine(HMD)to the reducing end of HA.The 1H NMR spectrum of HA-HMD showed three signal peaks were observed at ?=1.35 ppm.?=1.60 ppm and ?= 2.90 ppm with similar integral values,which were attributed to?-H,?-H and ?-H of HMD,respectively,confirming the structure of HA-HMD as expected.(3)Preparation and characterization of HA-ZQGHA-ZQG was prepared by covalently linking the terminal amino group of HA-HMD with the carboxyl group of ZQG by a similar method to synthetic PEG-ZQG,and the reaction yield was 86.9%.The 1H NMR spectrum of HA-ZQG had a characteristic signal peak of benzene ring of ZQG at ?=7.45-7.25 ppm,indicating that the synthesis of HA-ZQG containing Gln was successful.(4)Preparation and characterization of HA-TP5The TG5 and HA-ZQC were modified by catalyzed with mTGase to yield the new product(HA-TP5).The reaction yield was 82.7%and the purity of HA-TP5 was 95.1%.The 1H NMR spectrum of HA-TP5 showed the characteristic absorption peaks of the benzene ring of TP5 tyrosine residues at ?=7.02 ppm and ?= 6.73 ppm.The ?-H signal peak of the TP5 amino acid appeared at ?=3.78 ppm and a signal peak of methyl group in TP5 proline appeared at ?= 0.99 ppm.So TP5 was covalently modified by HA.According to the 1H NMR spectrum,the ratio of the anomeric hydrogen signal peak of HA to the peak value of the amino acid a-H signal peak was 1:0.29.Each TP5 molecule contained 5 ?-H and each HA molecule contained an average of 18 anomeric hydrogens,therefore an average of one TP5 was attached to one HA.(5)Preparation and characterization of HA-PC K3145Modification of a single Gin residue of PCK3145 with a primary amino group-containing HA-HMD under mTGase catalysis gave a new product(HA-PCK3145),with a yield of 81.7%and a purity of 88.3%.The 1H NMR spectrum of HA-PCK3145 showed that a characteristic absorption peak of benzene ring in PCK3145 tyrosine and tryptophan at ? = 8.32 ppm,and that the signal peak of each amino acid a-H in PCK3145 appeared at ?=3.88-3.77 ppm.A signal peak present at the ? = 2.51 ppm was attributed to the glutamic acid residue,glutamine residue.This indicated that the Gin residue of PCK3145 was modified by HA.According to the integral calculation,the ratio of the anomeric hydrogen to the amino acid a-H of HA was 1:0.8,which was calculated according to the average of 15 a-H in each PCK3145 molecule and 18 anomeric hydrogen per HA molecule.So one PCK3145 polypeptide was ligated to one HA.(6)Preparation and characterization of HA-ES2-THThe Lys of ES2-TH was modified with HA-HMD to obtain a new product(HA-ES2-TH),with a reaction yield of 85.0%and a purity of 92.1%.The 1H NMR spectrum of HA-ES2-TH showed that the small peaks at ? = 3.76-3.94 ppm were characteristic signal peaks of each amino acid ?-H of ES2-TH.,A peak at ? = 2.56 ppm was the methylene group directly attached to the N or S atom on lysine,arginine or cysteine.The peak appearing at ? = 1.21 ppm was the signal peak of the methyl group in isoleucine and valine.The results indicated that ES2-TH was successfully modified by HA.The integral ratio of the anomeric hydrogen of HA to the ?-H signal peak of amino acid was 1:1.24,which was calculated according to the average of 18 ?-H in each ES2-TH molecule and 18 anomeric hydrogen per HA molecule.So one ES2-TH polypeptide was ligated to one HA.(7)Preparation and characterization of SHA-ES2-THFinally,ES-TH was modified with SHA-HMD to obtain SHA-ES2-TH,with a reaction yield of 80.0%and a purity of 96.4%.Similar to the HA-ES2-TH spectrum,the 1H NMR spectrum of SHA-ES2-TH showed that the small peaks at ?= 3.76-3.94 ppm were the characteristic signal peaks of the respective amino acids ?-H of ES2-TH,and that a peak at ? = 2.56 ppm was a signal peak for the methylene group directly attached to the N or S atom on lysine,arginine or cysteine,and that the peak appearing at ?=1.21 ppm was the signal peak of the methyl group in isoleucine and valine.The integral ratio of the anomeric hydrogen of SHA to the a-H signal peak of amino acid was 1:1.18,which was calculated according to the average of 18 ?-H in each ES2-TH molecule and 18 anomeric hydrogen per SHA molecule.Thus one ES2-TH polypeptide was ligated to one SHA.3.Polypeptide conjugate pharmacokinetic studyBALB/c mice were used as animal models.After a single administration of the tail vein,the serum drug concentrations at different time points were determined to evaluate the pharmacokinetic profile of the different polypeptide conjugates.The pharmacokinetic behavior of TP5,PEG-TP5 and HA-TP5 in mice was consistent with the two-compartment model.According to the blood concentration-time curve,their distribution half-life t1/2?was 0.5032 min.4.257mn and 4.01 min.respectively,and their elimination half-life ti/2p was 29.45 min,173.9 min and 224.5 min,respectively.Therefore,the half-life of TP5 was significantly prolonged after modification with PEG or HA.The area under the curve(AUC)of TP5,PEG-TP5 and HA-TP5 was 193.5?g·mL-min,924.2?g·mL-1 min and 2382?g·mL-1 min,respectively.Thus the bioavailability of PEG/HA-TP5 was significantly higher than that of TP5.Compared with PCK3145,the t1/2? of HA-PCK3145 in mice was extended from 22.37 min to 57.58 min,and ti/2? was extended from 219.5 min to 3032 min,indicating that the half-life of HA-modified PCK3145 was significantly prolonged.Compared with unmodified PCK3145,the AUC0-of HA-PCK3145 was extended from 1688?g·mL-1.min to 6717?g ·mL-1,min,indicating that the modified conjugate has higher bioavailability.Compared with ES2-TH,the t1/2? of HA-ES2-TH in mice was extended from 17.46 min to 34.99 min,and t1/2? was extended from 243.3 min to 1703 min,indicating that the half-life of HA-modified ES2-TH was significantly prolonged.Compared with unmodified ES2-TH.the AUC0-z of HA-ES2-TH was extended from 510.5?g·mL-1·min to 2958?g·mL-1 min,indicating that the modified conjugate has higher bioavailability.4.Characterization of in vitro activity of polypeptide conjugates(1)In vitro lymphocyte proliferative activity of PEG-TP5 and HA-TP5The effects of TP5 and PEG/HA-TP5 on the proliferation of mouse spleen lymphocytes in vitro were determined by MTT assay.The results showed that TP5,PEG-TP5 and HA-TP5 with concentrations of 18.4?pmol·L-1,36.8?mol·L-1,73.5pmol·L-1 and 147.1?mol·L-1,respectively,had significant stimulation in vitrolymphocyte proliferation activity(P<0.05),and that the lymphocyte proliferation rate of PEG-TP5 and HA-TP5 was not significantly different from that of TP5 at the same concentration of(P>0.05).Therefore,TP5 modified with PEG or HA modification catalyzed by mTGase had little effect on its in vitro immunological activity.(2)In vitro anti-tumor cell proliferation activity of HA-PCK3145The proliferation of HA-PCK3145 against human breast cancer cells was evaluated by MTT assay.The results showed that PCK3145 and HA-PCK3145 with concentrations of 14.26?mol·L-1,28.52?mol·L-1,57.04?mol·L-1 and 114.09?mol·L-1,respectively,had extremely significant inhibition effects on human breast cancer cells proliferation in vitro(P<0.01),and that the anti-tumor cell proliferation of HA-PCK3145 was comparable to that of PCK3145 at the same concentration(P>0.05).Therefore,PCK3145 catalyzed by mTGase for HA modification has little effect on its anti-tumor activity in vitro.(3)In vitro anti-vascular endothelial cell proliferation activity of HA-ES2-THThe proliferation of HA-ES2-TH against vascular endothelial cells was evaluated by MTT assay.The results showed that ES2,ES2-TH and HA-ES2-TH had extremely significant anti-proliferation effect on the vascular endothelial cells(P<0.01)in vitro when the concentrations were 26.18?mol·L-1,52.36?mol·L-1 and 104.71?mol·L-1,respectively.The HA-ES2-TH had the equivalent anti-tumor cell proliferation activity to ES2-TH and ES2 at the same concentrations(P>0.05).Therefore,ES2 in tandem with homing peptide and further modified with HA by mTGase catalysis had little effect on anti-angiogenic activity in vitro.In conclusion,the site-directed modification of TP5,PCK3145 and ES2-TH with PEG/HA catalyzed by mTGase enzyme can obtain a series of highly pure polypeptide conjugates.Compared with the unmodified polypeptide,the in vivo half-life of each polypeptide conjugate was significantly prolonged,the bioavailability was significantly improved,and the biological activity in vitro was not changed much.Therefore,site-directed enzymatic modification with PEG/HA is a new strategy for the development of promising peptide drugs. |
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