of branched polymers for - uwaterloo.ca
TRANSCRIPT
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AMINO ACIDS FOR THE SYNTHESIS
OF BRANCHED POLYMERS FOR
CONTROLLED DRUG DELIVERYAPPLICATIONS
Greg Whitton, Mario GauthierDepartment of Chemistry
IPR Symposium May 15, 2007IP
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OUTLINE
Project ObjectivesBackgroundSynthesisResultsFuture WorkAcknowledgements
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PROJECT OBJECTIVES
Synthesize biocompatible dendritic (arborescent) polymers with a narrow molecular weight distribution (MWD)Suitable for microencapsulation
Intravenous drug deliveryDemonstrate the feasibility of the synthetic scheme proposed
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BACKGROUND
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Linear diblock copolymer micelles(Mössmer et al. Macromolecules 2000, 33, 4791)
Surfactant micelles(Ohno et al. Macromolecules 2002, 35, 8989)
Used for microencapsulationBoth assemblies have critical micelle concentrations (unstable at C < CMC)Molecules in micelle interchange with free molecules (dynamic structure)
MICELLES
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ARBORESCENT MICELLESBranched structure obtained from grafting onto scheme
Static structure (stable, no CMC)Biocompatible? Amino acids
Linear G0 G1
G21) Functionalization
2) Grafting*
Hydrophilic segment
Hydrophobic segment
1) Functionalization
2) Grafting
*
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SYNTHESIS
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SYNTHETIC SCHEMERing opening polymerization
Linear poly(γ-benzyl L-glutamate) (core side chains)Linear poly(γ-tert-butyl L-glutamate) (shell side chains)
Partial deprotection of benzyl esterSubstrate with randomly distributed grafting sites
GraftingComb-branched polymer (Generation zero, G0)Higher generations G1, G2 …..etc.
CharacterizationSize Exclusion Chromatrography1H NMR Spectroscopy
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RING OPENING POLYMERIZATIONN-carboxyanhydride of amino acidsInitiated by a primary amine (n-hexylamine)Susceptible to side reactions
N
O
H
OO
O
OR
N N O R
O H
O
NH2
On-C6H13
H
O RODMF / 25oC
nn-C6H13 NH2
-CO2
R = benzyl or tert-butyl ‘living’ primary amine end groupCH 2
C CH3
CH3 CH3
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SIDE REACTIONS IN ROPDestroy ‘living’ primary amineBroaden MWDE.g. end group cyclization
Specific to poly(γ-benzyl L-glutamate)
n N NO H
O
NHO
n-C6H13H
OO
n
+OHN N O
O H
O
NH2
On-C6H13
H
OO
γ-lactam ring
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SOLUTION TO SIDE REACTIONS
Several methods in literatureTo preserve ‘living’ character (primary amine) of chainTo maintain a narrow MWD
E.g. lower reaction temperaturePropagation and side reactions have different activation energies (Ea)
Side reaction Ea > Propagation Ea
Lowering T decreases side reactions more rapidly
Arrhenius k = Ae-Ea/RTIPR 20
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MORE SOLUTIONS
Hydrochloride salt initiatorDecreases reactivity of primary amine chain ends in reactionLonger reaction times
High vacuum techniquesRemoves water and other contaminants from reactionAzeotropic drying of monomerTime-consuming procedure
Difunctional initiatorSide reactions not prevented, but ‘living’ primary amine introduced as protected chain end
OO
NH
OO
NH2
CH3
H-Lys(Boc)-OMe
n-C6H13 NH3+
Cl-n-C6H13 NH2 + ClH
DormantReactive
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GRAFTING
Substrate coupling with side chainsSubstrate activated with carbodiimide/HOBt
Standard peptide chemistry
Stoichiometry varied to maximize grafting yieldProcess repeated for higher generations
G0 polymer
NN N
O H
On-C6H13
H
OO
CO2H
H
O
NHO
RO
R O
CH3N
N N
O H
On-C6H13
HOR
O H
O
NHO
RO
O NH
O
CH3
DIC/HOBtxy
y
x
DIC = 1,3-DiisopropylcarbodiimideHOBt = 1-hydroxybenzotriazole
DMF
NH2
+
Substrate
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DEPROTECTION OFPoly(L-glutamate esters)
1) Acidolysis of poly(γ-benzyl L-glutamate) homopolymer
Random acidolysis assumedStoichiometry varied to obtain desired deprotection level
SUBSTRATE
N N O R
O H
O
NH
On-C6H13
H
OO
O
CH3
nPartial Hydrolysis
xyHBr/TFA
NN N
O H
On-C6H13
H
OO
CO2H
H
O
NHO
RO
O
CH3
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NN N
O H
On-C 6H13
H
OO
H
O
NHO
RO
O
O
O
CH3
CH3
CH3 CH3
xy
TFAN
N N
O H
On-C 6H13
H
OO
CO 2H
H
O
NHO
RO
O
CH3x
y
DEPROTECTION OFPoly(L-glutamate esters)
2) Acidolysis of benzyl- and tert-butyl-glutamate copolymer
Random copolymerization assumedStoichiometry varied for desired tert-butyl ester content
SUBSTRATEIPR 20
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Last cycle: poly(γ-tert-butyl L-glutamate) chainsSelective cleavage of tert-butyl groups yields
hydrophilic shell
Result: Water-soluble dendritic micelles
SHELL ADDITION
Poly(benzyl L-glutamate)
TFA
Poly(tert-butyl L-glutamate) Poly(L-glutamic acid)
Selective DeprotectionIP
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Characterization
Size Exclusion Chromatography (SEC)Apparent molecular weights (Mn, Mw)PDI for linear and graft polymersGrafting yield
1H NMR SpectroscopyNumber-average degree of polymerization (DPn)
Absolute Mn for side chainsDeprotection level of substrate
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RESULTS
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RING OPENING POLYMERIZATION
a Polymerization temperature = 00C; b H-Lys(Boc)-OMe initiator; c High vacuum technique; *number in name refers to run number
4,70020201.144,0004 dPoly(Bz-Glu)-35a
3,60015101.213,6002 hPoly(Bz-Glu)-37
8,00035401.245,20010 dPoly(Bz-Glu)-36ac
12,20054501.217,2005 dPoly(Bz-Glu)-30c
4,70020201.284,30024 hPoly(Bz-Glu)-26b
5,40023201.274,0005 dPoly(Bz-Glu)-25a
MnH NMR
1H NMR DPnTarget DPPDIMn
app
(SEC)Reaction
TimeSample Name*
PDI values ranging from 1.14-1.28, satisfactoryChain functionality (-NH2) level will determine which
method is bestIP
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Tail on low molecular weight side of peakPossible side reactions, slow initiation, or SEC column adsorption
PDI = Mw/Mn = 1.24
Solvent N,N-dimethylacetamide (1g/L LiCl) at ~800C, flow rate 1 mL/min (DRI = differential refractometer index detector signal intensity)
SEC Analysis Poly(Bz-Glu)-36
900
1400
1900
2400
2900
16 17 18 19 20 21 22 23 24 25 26Elution Volume (mL)
DR
I
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1H NMR ANALYSIS
Terminal -CH3Area= 0.0288
ppm1H NMR Spectrum of Poly(Bz-Glu)-33
-CH2-Area= 0.4026
2 DPn = 0.4026 DPn = 21.03 0.0288
Backbone
-CH-
CH 3 N H
O
N
O
H
O
H 2 C
O
N H 2
O
On
Mn ~ 5,000
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DEPROTECTION OFPoly(L-glutamate esters)
Good correlation between 1H NMR analysis and –CO2H titration resultsTarget acidolysis level of ~30% by both methods
Sample number or letter refers to homopolymer or copolymer used for reaction. Reaction time 3 h.
30310.25:11.5Poly(Glu-OH)-343430
3331
0.30:0.700.30:0.70
0.51.0
Poly(Glu-OH)-BPoly(Glu-OH)-C
31340.25:11.0Poly(Glu-OH)-19
25270.2:10.25Poly(Glu-OH)-18-346490.35:10.25Poly(Glu-OH)-18-2
Titration % Cleavage
1H NMR % Cleavage
Mole Ratio of HBr or tert-butyl to Benzyl Units
Reaction Size (g)
Sample Name
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SELECTIVE DEPROTECTION
--C CH3
CH3 CH3
--C CH3
CH3 CH3
Essentially 100% of tert-butyl ester groups cleavedRemaining peaks from hexylamine initiator hydrocarbon chain (-CH2-)No benzyl ester groups cleaved
1H NMR Scan for Poly(Glu-OH)-B (Copolymer)
Before addition of TFA
After addition of TFA
ppm
C CH3
CH3 CH3
CH3(CH2)5NH2
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SEC Analysis PG-graft-PG-22
0
200
400
600
800
1000
1200
1400
19 20 21 22 23 24 25 26 27Elution Volume (mL)
D R
I
AREA 1
AREA 2
Graft Polymer
Linear Chains
Grafting Yield = AREA 1 x 100% AREA 1 + AREA 2 = 83%
Baseline resolution not achievedLarger MW difference needed between graft and linear polymers
Unreacted
GRAFTING REACTION
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69336221PG-g-PG-28 59336254PG-g-PG-2670335023PG-g-PG-2383315523PG-g-PG-2256315533PG-g-PG-2143335050PG-g-PG-1968335058PG-g-PG-18
Graft Yield% Graft SitesDPnSUBDPn
SIDESample Name
Molar ratios NH2 : CO2H : DIC : HOBt : TEA1 : 2 : 10 : 10 : 20
Grafting yield ranged from 43-83%
83% Grafting yield obtained when using side chains obtained at 00C and copolymer substrate
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FUTURE WORKDetermine ‘living’ chain content of samples by Non-aqueous Capillary Electrophoresis (NACE)
Confirm random acidolysis and copolymerization by 15N NMR spectroscopy
Systematic variations in side chain DPn, coupling site density to optimize grafting yield
Synthesize G1, G2, …etc. polymers
Last grafting cycle with tert-butyl esterIP
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ACKNOWLEDGEMENTS
Dr. DuhamelMembers of the Gauthier and Duhamel Groups
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1H NMR Spectrum of Monomer
5.21
1.00
2.22
1.10
2.28
1.19
N
O
H
OO
O
O
1.15
A
AA’
A’
B
BF
C
F
E
E
D
D
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Overall Synthetic Scheme
OH
O NH2
O
OR
N
O
H
OO
O
O
R
OCCl3Cl3CO
O
NN O
R
O H
O
NH2
On-C6H13
H
O RO
NN N
O H
On-C6H13
H
OO
CO2H
H
O
NH2
OR
O
R
NN N
O H
On-C6H13
HOR
O H
O
NH2
OR
O
O NH
R = Benzyl or tert-Bu
n-C6H13NH2
DMF / 25oC
n
Partial Hydrolysis
xyy
x
(A) + DIC
DIC = 1,3 Diisopropylcarbodiimide
1 2
3
4
-Explain each step briefly so audience can get an idea of what is going on
-mention more details on synthesis later
(A)
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ROP MECHANISM
N
O
H
OO
O
O
R
N+ NO H
On-C 6H13
OR
OH
HO-
n-C6H13NH2
DMF / 25oC H+ transfer
N N O
O H
On-C 6H13
H
OR
O
H- CO2
NN
H
O H
n-C 6H13H
OR
O
N N O R
O H
O
NH2
On-C 6H13
H
O RO
n
n-C 6H13 NH2
B
N
O
H
OO
O
O
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Activated Monomer Mechanism
Result: No initiator unit in chain, ill-defined polypeptides
n-C 6 H 13 N H 2
N
O
H
OO
O
O
R
O OO
O
O
R
N-
n-C 6 H 13 N H 3+
+N
O
H
OO
O
O
R
N
O OO
O
O
R
O
N H 2
O
O
R
-C O 2
Undergo normal polymerization
Repeat and create another active monomer
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Water-Induced PolymerizationWater not as nucleophilic as n-hexylamineAppears in solvent, glassware, atmosphere
No initiator unit in chain
N
O
H
OO
O
O
R
DMF / 25oC
OHN O
R
O H
O
NH2
O
O RO
nOH2-CO2
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CH3
O
O CH2 Ph
H Br
CH3
O
O+
CH2 PhH
Br-
CH3 C
O
OH
CH2 PhBr
CH3
O
O C
CH3
CH3
CH3
O CF3
O
H
CH3
O
O+
C
CH3
CH3
CH3H
O-F3C
O
CH3 C
O
OH
C+
CH3
CH3
CH3
++
+
Deprotection Mechanism
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NN N
O H
On-C 6H13
H
OO
H
O
N
OR
O
RH
O
OOH
NN N
O H
On-C 6H13
HOR
O H
O
NH2
OR
O
O NH
xy
yx
NN N
O H
On-C 6H13
H
OO
H
O
N
O RO
RH
O
OO
N
NH
xy
N C N (DIC)
Substrate (backbone)
NN
NOH
(HOBt)
NN N
O H
On-C 6H13
H
OO
H
O
N
OR
O
RH
O
OO
NN
N
xy
NH2
Side chains
+ +
NN
NOH
N NH H
ODIU
H+ transfer
Grafting Mechanism
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Grafting Side ReactionN C N
P CO2HP
OO
C NNH
PO
O
C NNH
PO
OH
PO
O
O
P+ N
H
O
NH
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RESULTS
4,90021201.1414,0003 dPoly(Bz-Glu)-33b
6,50028201.1515,4002 hPoly(Bz-Glu)-32e
6,60029201.1216,4002 hPoly(Bz-Glu)-31e
12,20054501.1721,2005 dPoly(Bz-Glu)-30e
501.1219,4004 dPoly(Bz-Glu)-30e
5,60024201.1511,20010 dPoly(Bz-Glu)-29c
4,90021201.1610,5006 dPoly(Bz-Glu)-29c
201.2011,70024 hPoly(Bz-Glu)-27d
4,70020201.1912,50024 hPoly(Bz-Glu)-26d
5,40023201.1411,5005 dPoly(Bz-Glu)-25b
4,20018201.128,1402 hPoly(Bz-Glu)-24d
5,10022201.1511,30024 hPoly(Bz-Glu)-23b
5,10022201.1911,2004 hPoly(Bz-Glu)-22
3,50014201.139,53013 dPoly(Bz-Glu)-21a
7,60033201.149,2205 dPoly(Bz-Glu)-20a
7,20031201.117,9603 dPoly(Bz-Glu)-20a
11,30050601.1816,1002 hPoly(Bz-Glu)-19
5,80025201.1811,5002 hPoly(Bz-Glu)-18
MnH NMR
1H NMR DPTarget DPPDIMn
appReaction
TimeSample Name*
Table 1: Polymerization of Glu-NCA Under Different Reaction Conditions
a hydrochloride salt initiator, b temperature decrease to 00C, c temperature decrease to -150C, d H-Lys(Boc)-OMe initiator e High vacuum technique.*number in sample name refers to run number
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15N NMR COPOLYMER
One peak suggests similar environment
Similar reactivities for copolymerization
Might need better resolution to confirmIP
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n
O
O
NH
O
NH
O
CH3 N
H
O
R
NH2R
O
t-Bu
O
O
NH
O
O
NH2
CH3
t-Bu
N
O
H
OO
R
O
CH3
O CH3
O
N
n
O
O
NH
O
NH
O
CH3 N
H
O
R
NHR
O
t-Bu
O
CH3
n
TFA
O
O
NH2
NH
O
CH3 N
H
O
R
NHR
O
CH3
2)-CO2
1)-t-Bu
Lysine initiation/deprotection
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N-CARBOXYANHYDRIDE(MONOMER) SYNTHESIS
92% product yield on 10 g reaction scaleProduct purity confirmation by 1H NMR
HO
O NH2
O
OR
N
O
H
OO
O
O
R
OCCl3Cl3CO
O
Reflux / EtAc+ + 2/3 CHCl31/3
γ-alkyl-L-glutamate
R=Benzyl or tert-Butyl
Triphosgene Monomer
CH 2
C CH3
CH3 CH3
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