ugent contribution to polexgene (summary of wp 2) polexgene midterm meeting ghent 17-18/12/2007 pbm...
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UGent contribution to PolExGene(Summary of WP 2)
PolExGene Midterm MeetingGhent 17-18/12/2007
PBM-GGentent
UU
Polymer Chemistry & Biomaterials GroupPolymer Chemistry & Biomaterials Group
V.Vermeersch, S. Van Vlierberghe, P. Dubruel & E. Schacht
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
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’)
(CH2CH2N) (CH2CH2NH)
(CH2CH2NH2)
x y
Commercially available (Sigma)Branched – Mw 25.000
CH3
CCH2 *
C O
O
CH2
CH2
N
H3C CH3
*n
CH3
CCH2
C O
O
CH2
CH2
N
H3C CH3
AIBN
Toluene
pDMAEMASynthesis
Synthesis-partSynthesis-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
H2N CH COOH
(CH2)2
COOH
OH
H2SO4/H2O
H2N CH COOH
(CH2)2
C O
O
CH2
Protecting the carboxylic group of glutamic acid
Confirmation by 1H-NMR
• Step 2: BG-NCA formationStep 2: BG-NCA formation
V. Vermeersch – PolExGene Ghent - 18/12/2007 pag. 5
Synthesis of poly-L-glutamine derivative
H2N CH COOH
(CH2)2
C O
O
CH2
(CH2)2
C O
O
CH2
O
HN
O
O
O
CCl OCCl3
EtOAc
γ-benzylglutamate-N-carboxyanhydrid
Cyclisation: reaction with diphosgene (dangerous)
Cyclic molecule polymer obtained by ring-opening-polymerisation (ROP)
Product structure confirmed through 1H-NMR & IR
V. Vermeersch – PolExGene Ghent - 18/12/2007 pag. 6
Synthesis of poly-L-glutamine derivative
• Step 3: PolymerisationStep 3: Polymerisation
(CH2)2
O
HN
O
O
CH2Cl2/EtOAC (6/1)
C O
O
CH2
Bu3N
(CH2)2
C O
O
CH2
CHNH C* *
O
n
poly--benzylglutamate
Polymerisation:
Tertiary amine H-abstraction active species propagation
Product confirmed through 1H-NMR & IR; Mw determined by viscosimetry
V. Vermeersch – PolExGene Ghent - 18/12/2007 pag. 7
• Step 4: AminolysisStep 4: Aminolysis
Synthesis of poly-L-glutamine derivative
(CH2)2
C O
O
CH2
CHNH C* *
O
n(CH2)2
C O
NH
CHNH C* *
O
n
CH2
N
CH3 CH3
DMF, 2-HP
DMAEAAminolysis: removal of protective group + modification with tertiary amines.
Copolymerisation:only aminolysis is differentusing 2 reagents (DMAEA & TrAEA)instead of 1 (DMAEA)
Ratio of DMAEA/TrAEA added determines the copolymer ratio
DMF, 2-HP
DMAEA, TrAEA
(CH2)2
C O
NH
CHNH C*
O
x
CH2
N
H3C CH3
2
(CH2)2
C O
NH
CHNH C *
O
(CH2)2
NH2
y
Product structure confirmed through 1H-NMR & IR; Mw determined by GPC
pDMAEG
p(DMAEG-co-AEG)
Chemical analysis
V. Vermeersch – PolExGene Ghent - 18/12/2007 pag. 8
All products where analysed using 1H-NMR
5.0 4.5 4.0 3.5 3.0 2.5 2.0
CH2
C O
NH
CHNH C* *
O
n
CH2
N
CH3 CH3
CH2
CH2
a
b
d
c
e
f
a
bcd
e
f
5.0 4.5 4.0 3.5 3.0 2.5 2.0
CH2
C O
NH
CHNH C* *
O
n
CH2
N
CH3 CH3
CH2
CH2
a
b
d
c
e
f
a
bcd
e
f
3.5 3.4 3.3 3.2 3.1 3.0 2.9
CH2
N
H3C CH3
CH2
CH2
NH2
CH2
3.5 3.4 3.3 3.2 3.1 3.0 2.9
CH2
N
H3C CH3
CH2
CH2
NH2
CH2
pDMAEG p(DMAEG-co-AEG)
Ratio of integrals=
Copolymer ratio
Chemical analysis
V. Vermeersch – PolExGene Ghent - 18/12/2007 pag. 9
IR analysis is an excellent tool to visualise conversion control
polymerisation
aminolysis
Anhydride peakEster peak Amide peak
BG-NCA pBG pDMAEG
Synthesis of poly-L-arginine derivatives
V. Vermeersch – PolExGene Ghent - 18/12/2007 pag. 10
A different poly--aminoacid with biocompatible properties forgene 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
NH
NH2H2N
HN
O
O
O
Br
(CH2)3
CHNH C* *
O
n
NH
H2N NH2
Br
O NH
O
O
OH
NH
NH2HN
THF
PBr3 initiator
Synthesis of poly-L-arginine derivatives
V. Vermeersch – PolExGene Ghent - 18/12/2007 pag. 11
Formation of R-NCA: N-carboxybenzyl-L-arginine + PBr3
Polymerisation is reached by using an amine initiator (R-NH2, R3N)
Side reactions are inevitable guanidium group protected by a H+ (abstraction by initiator possible)
Product is dialysed against Milli-Q – Analysed by 1H-NMR & IR
Mw determined by GPC (low DP up to 7000 Da with low yield)
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/chainLysine chain
PLL + KOtBu initiator solutionHigh Mw PLL (117000 Da)
Up to 35000 Da PLArg grafts
Fluorescent Marker Coupling
V. Vermeersch – PolExGene Ghent - 18/12/2007 pag. 13
For visualising & in-vitro tracking of polyplexes
(CH2)2
C O
HN
CHNH C*
O
n
CH2
N
H3C CH3
2
(CH2)2
C O
HN
CHNH C
O
CH2
NH2
2
m-x(CH2)2
C O
HN
CHNH C *
O
CH2
NH
2
OO
F
OH
F
C O
HOOC
x(CH2CH2N) (CH2CH2NH)
(CH2CH2NH2)
x y(CH2CH2N) (CH2CH2NH)
(CH2CH3NH)
x y
z z
100-a a
OO
F
OH
F
C O
HOOCOregon Green 488
Fluorescentmarker
PolymerMol%
coupled
V07a 1,02
V07b 6,1
V07c 7,3
V08 5,2
PEI+OREG 2,7
Characterised by UV (= 488 nm)
(CH2)2
C O
HN
CHNH C*
O
n
CH2
N
H3C CH3
2
(CH2)2
C O
HN
CHNH C *
O
CH2
NH
2
m
C O
(CH2)3
NOO
GMBS
Covalent linking to Ac-Cys-Penetratine
Intermediate: GMBS coupling
Peptide Coupling
V. Vermeersch – PolExGene Ghent - 18/12/2007 pag. 14
(CH2)2
C O
NH
CHNH C*
O
m
CH2
N
H3C CH3
2
(CH2)2
C O
NH
CHNH C
O
CH2
NH2
2
n-x
C O
(CH2)3
NOO
(CH2)2
C O
NH
CHNH C *
O
CH2
NH
2
x
SCH2
Penetratine (CPP)
Ac
CPP-coupled p(DMAEG-co-AEG)
V09 = V05 + CPP (2,5 mol%)
WP2 related products (sent-out)
PolExGene code Base polymer Mw Polydispersity Analysis
pEI pEI 25.000 - -
V04 pDMAEMA 177.000 1,86 NMR
V01 pDMAEG 79.900 1,24 NMR, IR
V03 pDMAEG 242.000 1,36 NMR, IR
V05 pDMAEG-AEG 91.600 1,51 NMR, IR
V06 pDMAEG-AEG 242.000 1,43 NMR, IR
PolExGene code Polymer composition Mol% coupled Analysis
V07a V05 + Oreg. Green 1,02 UV ( = 488)
V07b V05 + Oreg. Green 6,1 UV ( = 488)
V07c V05 + Oreg. Green 7,3 UV ( = 488)
V08a V06 + Oreg. Green 5,2 UV ( = 488)
PEI+OREG PEI + Oreg. Green 2,7 UV ( = 488)
V09 V05 + Penetratine 5,2 UV ( = 280)
V. Vermeersch – PolExGene Ghent - 18/12/2007 pag. 15
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 Mw as backbone for pLL-pArg copolymers
• Alternative strategy using polyornithine will be explored
• Startup synthesis of polyagmatine
H2N COOH
NH2H2N
HN
NH2
NH2
agmatine
ornithine