UGent contribution to PolExGene (Summary of WP 2) PolExGene Midterm Meeting Ghent 17-18/12/2007 PBM...

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


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


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



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



• 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


• Step 4: AminolysisStep 4: Aminolysis


(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


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


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


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



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)



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


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


(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. 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

UGent contribution to PolExGene (Summary of WP 2) PolExGene Midterm Meeting Ghent 17-18/12/2007 PBM...

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