Presentation is loading. Please wait.

Presentation is loading. Please wait.

UGent contribution to PolExGene (Summary of WP 2) PolExGene Midterm Meeting Ghent 17-18/12/2007 PBM G ent - G entU Polymer Chemistry & Biomaterials Group.

Similar presentations


Presentation on theme: "UGent contribution to PolExGene (Summary of WP 2) PolExGene Midterm Meeting Ghent 17-18/12/2007 PBM G ent - G entU Polymer Chemistry & Biomaterials Group."— Presentation transcript:

1 UGent contribution to PolExGene (Summary of WP 2) PolExGene Midterm Meeting Ghent 17-18/12/2007 PBM G ent - G entU Polymer Chemistry & Biomaterials Group V.Vermeersch, S. Van Vlierberghe, P. Dubruel & E. Schacht

2 Workpackage Contents V. Vermeersch – PolExGene Ghent - 18/12/2007 pag. 2 Workpackage 2 Development of CPP-containing polymers Objective to develop and characterise cell penetrating peptide (CPP) cationic polymers and CPP-derivatives polymer conjugates with varying substitution degrees of CPP a range of immobilisation strategies will be evaluated. Reference polymers: Polyethyleneimine (PEI) & PDMAEMA (V04) Specific poly-α-aminoacids: polyglutamine derived homo- & copolymers polyarginine & polylysine homo- & copolymers

3 Selection of reference polymers V. Vermeersch – PolExGene Ghent - 18/12/2007 pag. 3 Polyethyleneimine (PEI) Widely used & extensively studied polymer with excellent transfection results (but high cytotoxicity ) Poly(dimethylaminoethyl)methacrylate (PDMAEMA – V04) Proven efficiency in previous Ph.D work (‘In house knowledge’) Commercially available (Sigma) Branched – Mw pDMAEMA Synthesis

4 Synthesis-part Total synthesis consists of 4 reaction-steps Step 1: γ -benzylglutamate V. Vermeersch – PolExGene Ghent - 18/12/2007 pag. 4 Synthesis of poly-L-glutamine derivative Protecting the carboxylic group of glutamic acid Confirmation by 1 H-NMR

5 Step 2: BG-NCA formation Step 2: BG-NCA formation V. Vermeersch – PolExGene Ghent - 18/12/2007 pag. 5 Synthesis of poly-L-glutamine derivative γ -benzylglutamate- N-carboxyanhydrid Cyclisation: reaction with diphosgene (dangerous) Cyclic moleculepolymer obtained by ring-opening-polymerisation (ROP) Product structure confirmed through 1 H-NMR & IR

6 V. Vermeersch – PolExGene Ghent - 18/12/2007 pag. 6 Synthesis of poly-L-glutamine derivative Step 3: Polymerisation Step 3: Polymerisation poly-  -benzylglutamate Polymerisation: Tertiary amine H-abstraction active species propagation Product confirmed through 1 H-NMR & IR; M w determined by viscosimetry

7 V. Vermeersch – PolExGene Ghent - 18/12/2007 pag. 7 Step 4: Aminolysis Step 4: Aminolysis Synthesis of poly-L-glutamine derivative Aminolysis: removal of protective group + modification with tertiary amines. Copolymerisation: only aminolysis is different using 2 reagents (DMAEA & TrAEA) instead of 1 (DMAEA) Ratio of DMAEA/TrAEA added determines the copolymer ratio Product structure confirmed through 1H-NMR & IR; M w determined by GPC pDMAEG p(DMAEG-co-AEG)

8 Chemical analysis V. Vermeersch – PolExGene Ghent - 18/12/2007 pag. 8 All products where analysed using 1 H-NMR pDMAEG p(DMAEG-co-AEG) Ratio of integrals = Copolymer ratio

9 Chemical analysis V. Vermeersch – PolExGene Ghent - 18/12/2007 pag. 9 IR analysis is an excellent tool to visualise conversion control polymerisation aminolysis Anhydride peak Ester peak Amide peak BG-NCA pBGpDMAEG

10 Synthesis of poly-L-arginine derivatives V. Vermeersch – PolExGene Ghent - 18/12/2007 pag. 10 A different poly-  -aminoacid with biocompatible properties for gene delivery is poly-L-arginine Synthesis Polymerisation of R-NCA Guanidylation of polyornithine Mimicking the structure using poly-agmatine Grafting onto poly-L-lysine Up to now polymerisation & grafting method were tested

11 Synthesis of poly-L-arginine derivatives V. Vermeersch – PolExGene Ghent - 18/12/2007 pag. 11 Formation of R-NCA: N-carboxybenzyl-L-arginine + PBr 3 Polymerisation is reached by using an amine initiator (R-NH 2, R 3 N) Side reactions are inevitable guanidium group protected by a H + (abstraction by initiator possible) Product is dialysed against Milli-Q – Analysed by 1 H-NMR & IR M w determined by GPC (low DPup to 7000 Da with low yield)

12 Synthesis of poly-L-arginine derivatives V. Vermeersch – PolExGene Ghent - 18/12/2007 pag. 12 R-NCA grafting of PLL was therefore also tested Guanidinium group/chain Lysine chain PLL + KOtBu initiator solution High Mw PLL ( Da) Up to Da PLArg grafts

13 Fluorescent Marker Coupling V. Vermeersch – PolExGene Ghent - 18/12/2007 pag. 13 For visualising & in-vitro tracking of polyplexes Oregon Green 488 Fluorescent marker Polymer Mol% coupled V07a1,02 V07b6,1 V07c7,3 V085,2 PEI+OREG2,7 Characterised by UV (  = 488 nm)

14 GMBS Covalent linking to Ac-Cys-Penetratine Intermediate: GMBS coupling Peptide Coupling V. Vermeersch – PolExGene Ghent - 18/12/2007 pag. 14 CPP-coupled p(DMAEG-co-AEG) V09 = V05 + CPP (2,5 mol%)

15 WP2 related products (sent-out) PolExGene codeBase polymerMwMw PolydispersityAnalysis pEI V04pDMAEMA ,86NMR V01pDMAEG ,24NMR, IR V03pDMAEG ,36NMR, IR V05pDMAEG-AEG ,51NMR, IR V06pDMAEG-AEG ,43NMR, IR PolExGene codePolymer compositionMol% coupledAnalysis V07aV05 + Oreg. Green1,02 UV ( = 488) V07bV05 + Oreg. Green6,1 UV ( = 488) V07cV05 + Oreg. Green7,3 UV ( = 488) V08aV06 + Oreg. Green5,2 UV ( = 488) PEI+OREGPEI + Oreg. Green2,7 UV ( = 488) V09V05 + Penetratine5,2 UV ( = 280) V. Vermeersch – PolExGene Ghent - 18/12/2007 pag. 15

16 V. Vermeersch – PolExGene Ghent – 18/12/2007 pag. 16 Future Synthesis Other coupling reactions planned: V05 & V06 Penetratine coupling to V06 Coupling of other CPP Varying the CPP density Continued synthesis of arginine-based polymers Coupling to polymers via cysteine-function Further exploring the efficiency of various initiators PLL with different M w as backbone for pLL-pArg copolymers Alternative strategy using polyornithine will be explored Startup synthesis of polyagmatine agmatine ornithine


Download ppt "UGent contribution to PolExGene (Summary of WP 2) PolExGene Midterm Meeting Ghent 17-18/12/2007 PBM G ent - G entU Polymer Chemistry & Biomaterials Group."

Similar presentations


Ads by Google