1 dendrimer synthesis and applications: branching out into biology organic chemistry seminar april...
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Dendrimer Synthesis and Applications: Branching out into Biology
Organic Chemistry SeminarApril 28th, 2005
William PomerantzGellman Group
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Dendrimer- Greek roots:
dendra- tree, mer- segment
Dendritic: Nature’s Architecture
Tomalia, D., A.; Frechet, J. M. J. J. Polym. Sci., Part A: Polym. Chem. 2002, 40, 2719-2728http://inside.salve.edu/walsh/
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m
Neuronnm
Dendrimerm
Tree
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16 Å 67 Å
Insulin Cytochrome C Hemoglobin Prealbumin
Globular Dendrimers as Biomimetics
Maiti, P. K.; Tahir, C.; Wang, G.; Goddard, W. A. I. Macromolecules 2004, 37, 6236Tomalia, D., A.; Frechet, J. M. J. J. Polym. Sci., Part A: Polym. Chem. 2002, 40, 2719-2728
Molecular Modeling of Dendrimer Structure
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Dendrimer 101
Generation (GX) Defines the level of branching within the dendrimer shell. At high Generations dendrimers become spherical
G2G1
G0
Loading Reactive/diagnostic groups can be attached to to the surface of the dendrimers efficiently and with a predictable display
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G0
Core
Monomer
GX = generation
Generation Growth
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G1
GX = generation
Core
Monomer
Generation Growth
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G2
GX = generation
Core
Monomer
Generation Growth
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G3
GX = generation
Core
Monomer
Generation Growth
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Dendrimer 101
Generation (GX) Defines the level of branching within the dendrimer shell. At high Generations dendrimers become spherical
G2G1
G0
Loading Reactive/diagnostic groups can be attached to to the surface of the dendrimers efficiently and with a predictable display
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Dendrimer 101
Polydispersity Index (PDI) Indication of the distribution of molecular weights within a sample. PDI=1 is monodisperse
PDI= Mw/Mn
Time (Minutes)
Intensity
G4 G3 G2 G1
Size Exclusion Chromatography
de Brabander-van den Berg, E., M. M.; Meijer, E. W. Angew. Chem. Int. Ed. 1993, 32, 1308
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Synthesis Divergent
Convergent
Applications
Encapsulation
Gene Delivery
Cancer Therapy
Multivalency MRI
Conclusions and Outlook
Outline11
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Synthetic Considerations
Reagents
Reactions
High YieldingMinimal side reactionsPurify intermediates
Polydispersity (PDI)
Very Narrow PDI:PDI= Mw/Mn
CheapHigh ReactivityEasily Removable
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13Dendrimer CoreMultivalent Monomer
G0
G1
G3
G2
Divergent Dendrimer Synthesis13
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G0
G0.5
.. G1
PAMAM Dendrimer Synthesis (Divergent)
Very Narrow PDI:PDI= Mw/Mn
Tomalia, D., A. and co-workers. Macromolecules 1986, 19, 2466, Polym. J. (Tokyo) 1985, 17, 117
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N CO2Me
CO2Me
MeO2C
NH3
O
OMe
O
OMe
NH2H2N
NH2H2N
N
O
O
NH
HN
H2N
NH2
O
NH
H2N
N
O
O
NH
HN
N
N
O
NH
N
CO2Me
CO2Me
MeO2C
CO2Me
CO2Me
MeO2C
PAMAM
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Each reaction assumes 99.5% conversion
Polydispersity vs. Dendrimer Purity
Hummeln, J. C.; van Dongen, J. L. J.; Meijer, E. W. Chem. Eur. J. 1997, 3, 1489Kallos, G.; et al. J. Rapid Commun. Mass Spectrom 1991, 5, 383
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PAMAMDendrimer
NH
ON
CO2Me
CO2Me
NH
O HN
CO2Me
CO2Me
+
nn
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Polydispersity vs. Dendrimer Purity16
PAMAMDendrimer
Each reaction assumes 99.5% conversion
NH
ON
CO2Me
O NH
NH2NH
O
N NH
HN
O
O
nn
Hummeln, J. C.; van Dongen, J. L. J.; Meijer, E. W. Chem. Eur. J. 1997, 3, 1489Kallos, G.; et al. J. Rapid Commun. Mass Spectrom 1991, 5, 383
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Convergent Synthesis
Dendrimer Core Multivalent MonomerG3Hawker, C. J.; Frechet, J. M. J. J. Am. Chem. Soc 1990, 112, 7638-7647
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Dendrimer Wedge Synthesis (Convergent)
Frechet and co-workers; J. Am. Chem. Soc. 1990, 112, 7638 J. Control. Release 2000, 65, 121
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K2CO3, 18-Crown-6
G0 G1
G0
G2
BnO Br
HO
HO OHBnO
HO
O
OBn
O
BnO
O
OBn
O
HO
O O
OBn
O
BnO
O
1. CBr4, PPh3
2. K2CO3, 18-Crown-6
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G2 Dendrimer Synthesis (Convergent)19
G2
CBr4, PPh3
G2-Dendrimer
G2-Br
K2CO3, 18-Crown-6
BnO
O
OBn
O
Br
O O
OBn
O
BnO
O
R OH
OH
OH
HO
Frechet and co-workers; J. Am. Chem. Soc. 1990, 112, 7638 J. Control. Release 2000, 65, 121
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G2-Dendrimer
R = Bn
R = H
R = (CH2CH2O)16CH3
Pd/C, H2
K2CO3, CH3(OCH2CH2)16OMs
G2 Dendrimer Synthesis (Convergent)
RO
O
OR
O
O
OO
OR
O
RO
O
OR
O
OR
O
O
O
O
OR
OOR
O
RO
O
RO
O
O
O
O
RO
ORO
O
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Frechet and co-workers; J. Am. Chem. Soc. 1990, 112, 7638 J. Control. Release 2000, 65, 121
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Rapid synthesisCheap reagentsExponential growthLarge dendrimers attainable
Fewer simultaneous reactionsStandard purification Intermediates characterizableDifferentiationMonodisperse
Convergent
Slower growth processMid-sized dendrimers
Multiple side reactions (intra/inter) Large excess of reagentsLow polydispersity
DivergentAdvantages:
Disadvantages:
Synthetic Comparison21
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Synthesis Divergent
Convergent
Applications
Encapsulation
Gene Delivery
Cancer Therapy
Multivalency MRI
Conclusions and Outlook
Outline22
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Guests =
Molecular Encapsulation
Surface
Interbranch
Core
Jansen, J. F. G. A.; de Brabander-van den Berg, E., M. M.; Meijer, E. W. Science 1994, 266, 1226
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Space-Filling Model
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Guests =
Molecular Encapsulation
Surface
Interbranch
Core
Jansen, J. F. G. A.; de Brabander-van den Berg, E., M. M.; Meijer, E. W. Science 1994, 266, 1226
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Space-Filling Model
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Molecular Encapsulation-“Dendritic-Box”
Jansen, J. F. G. A.; et al. Science 1994, 266, 1226de Brabander-van den Berg, et al. Angew. Chem. Int. Ed. 1993, 32, 1308
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Raney-Co, H2
PPI Dendrimer
NH2 CN
AcOH
NH2
N
N
H2N
NH2NH2
NH2
BOX
Lid
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Molecular Encapsulation-“Dendritic-Box”
Jansen, J. F. G. A.; et al. Science 1994, 266, 1226de Brabander-van den Berg, et al. Angew. Chem. Int. Ed. 1993, 32, 1308
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PPI Dendrimer
Raney-Co, H2
NH2 CN
AcOH
NH2
N
N
H2N
NH2NH2
NH2
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1) Probe + Coupling
1) Coupling 2) Probe
1) Probe + Coupling 2) 12M HCl
“Phe-Box” Probe Encapsulation
Jansen, J. F. G. A.; et al. Science 1994, 266, 1226
N
O
OH
O
ESR Probe
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NH2
64
N
O
O
O
O
NHBoc
NH
O
NHBoc
Probe
Probe
64
Ph
PhCH2Cl2, Et3N
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Unimolecular Micelle Containers
“Frechet” Polyaryl- ether dendrimer
Liu, M.; Kono, K.; Frechet, J. M. J. J. Control. Release 2000, 65, 121
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[Pyrene]water
10-6 M
[Dendrimer]
G3
G2
G18.0 x 10-7M
H2O
=
G1 G2 G3
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Drug Encapsulation and Release
Liu, M.; Kono, K.; Frechet, J. M. J. J. Control. Release 2000, 65, 121
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Indomethacin
Drug + G3
Free Drug
Time (hr)
% Release
pH =737oC
=N
CH2COOHO
O
Cl
Water bath
Dialysis membrane
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Synthesis Divergent
Convergent
Applications
Encapsulation
Gene Delivery
Cancer Therapy
Multivalency MRI
Conclusions and Outlook
Outline30
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www.comet.itrcindia.org
Gene Delivery
DNA transported to nucleusvia viral or synthetic molecules
CellGene Product
http://www.ornl.gov/sci/techresources/Human_Genome/medicine/genetherapy.shtml
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Key Processes for Gene Delivery
Boussif, O.; et al. Proc. Natl. Acad. Sci. USA 1995, 92, 7297. Behr, J. P. Acc. Chem. Res. 1993, 26, 274
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Polycation
1.
2. 3.
DNA
endosome
Nucleus
DNA
Nucleus
endosomeDNA
H+
DNA
DNA
Bind DNA
Cellular Uptake
Endosomal Release Transfection
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Gn
pLys115
Dendrimer Transfection Efficiency
Haenzler, J.; Szoka, F. C. J. Bioconjugate Chem. 1993, 4, 372Tang, M. X.; Redemann, C. T.; Szoka, F. C. J. Bioconjugate Chem. 1996, 7, 703
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+/- Charge ratio
NH
C
O
NH2
H OH115
PAMAM
G2-G10NH2
x
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Gn
pLys115
Dendrimer Transfection Efficiency
Haenzler, J.; Szoka, F. C. J. Bioconjugate Chem. 1993, 4, 372Tang, M. X.; Redemann, C. T.; Szoka, F. C. J. Bioconjugate Chem. 1996, 7, 703
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+/- Charge ratio
NH
C
O
NH2
H OH115
PAMAM
G2-G10NH2
x
PAMAMNH2
x-y
Defect
H2O
Superfect
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Transfection/Cytotoxicity Comparison
Gebhart, C. L.; Kabanov, A. V. J. Control. Release 2001, 73, 401
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Defect PAMAM
CellViability
PPIDendrimer
PEI 22KLinear
Luciferase(ng/mg)
PEI/PluronicCopolymer
PEI 25KBranched
PEI 50KLinear
NH
HN
NHn
PEI
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Increasing Transfection vs. Toxicity
Phe(64)-G4 Lipofectamine Superfect
Kono, K.; et al. Bioconjugate Chem. 2005, 16, 208, Malik, N.; et al. J. Control. Release 2000, 65, 133
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Defect PAMAM
PhePAMAM
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Synthesis Divergent
Convergent
Applications
Encapsulation
Gene Delivery
Cancer Therapy
Multivalency MRI
Conclusions and Outlook
Outline37
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CH2
CH3
GlyPheLeuGly
x y
CH3
CONH
OH
CH2
(Macromolecular) Drug Biocompatibility
Drug Criteria:
• Water Soluble
• Low Cytotoxicity
• Biodistribution tissue/cell specificity
• Bioavailability half-life in body, degradable
• Reproducible Pharmacokinetics
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DrugD
rug
DrugD
rug
Drug
HPMA Co-polymer
Duncan, and co-workers S. Hum Exp Toxicol 1998, 17, 93, Eur. J. Cancer 1995, 5, 766
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Enhanced Permeability and Retention (EPR) Passive Targeting of Tumor Cells
Duncan, R. Nat Rev Drug Discov 2003, 2, 347-360, Matsumara, Y.; et al. Cancer Res. 1986, 6, 6387
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Dendrimer Design Features
Frechet, J. M. J. and co-workers. Bioconjugate Chem. 2002, 13, 443, Macromolecules 1998, 31, 4061, Bioconjugate Chem. 2002, 13, 453
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= Drug
High MW Dendrimer
High MW Dendrimer
Modular Approach
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G4 Dendrimer 12 kDa
G4 Dendrimer 4 kDa
G2 Dendrimer 24 kDa
Core
I
II
III
Evaluating Dendrimer Size for EPR
De Jesus, O. L. P.; Ihre, H. R.; Frechet, J. M. J.; Szoka, F. C. J. Bioconjugate Chem. 2002, 13, 453
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O
O
O
O
ORO
RO
ORO
RO
=
=
OH
O O
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Conjugation of Doxorubicin (DOX)
De Jesus, O. L. P.; Ihre, H. R.; Frechet, J. M. J.; Szoka, F. C. J. Bioconjugate Chem. 2002, 13, 453
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=
DOX
1. TFA/MeOH
2. H+, DOX
O
O
O OH
OH
O
O
OHNH2
OH
OOH
O
O
OH
HO
Cl O
ONO2
80%O
O
O
O
O
NH
O
1.
NH2NHBoc, DMAP2.84%
NHBoc
, Pyr
HN NHBoc
O
O
O
O
O
NH
O
NHN
N
R
R
OH
OH
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Dendrimer Cytotoxicity
De Jesus, O. L. P.; Ihre, H. R.; Frechet, J. M. J.; Szoka, F. C. J. Bioconjugate Chem. 2002, 13, 453
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Half-Lives of I-III all < 10 Min.
% Cell Viability
[Dendrimer] (mg/mL)
12 kDa
4 kDa
24 kDa
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Drug Release Studies
De Jesus, O. L. P.; Ihre, H. R.; Frechet, J. M. J.; Szoka, F. C. J. Bioconjugate Chem. 2002, 13, 453
44
=
DOX
O
O
O OH
OH
O
O
OHNH2
OH
OOH
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Gilles, E. R.; Frechet, J. M. J. J. Am. Chem. Soc. 2002, 124, 14137
G1-10 kDaG1-20 kDa
G2-5 kDaG2-10 kDaG2-20 kDa
G3-5 kDaG3-10 kDaG3-20 kDa
45Higher MW “Bow-Tie” Dendrimers
O
O
O
O
O
O
OPhPh
, DMAP
H2, Pd/C
1.
2.
O
O
O
O O
O
O
OO
OO
O
O
O
O
O
O
O
O
O
O
O
HO
HO
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Higher MW “Bow-Tie” Dendrimers
Gilles, E. R.; Frechet, J. M. J. J. Am. Chem. Soc. 2002, 124, 14137
G1-10 kDaG1-20 kDa
G2-5 kDaG2-10 kDaG2-20 kDa
G3-5 kDaG3-10 kDaG3-20 kDa
46
O
O
O
O
O
O
OPhPh
, DMAP
H2, Pd/C
1.
2.
O
O
O
O O
O
O
OO
OO
O
O
O
O
O
O
O
O
O
O
O
HO
HO
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Biodistribution and Bioavailibility In Vivo
Gilles, E. R.; Frechet, J. M. J. J. Am. Chem. Soc. 2002, 124, 14137
47
G1-10 kDa 8 +/- 1G2-10 kDa 26 +/- 6G3-10 kDa 40 +/- 4G3-20 kDa 50 +/- 10
Plasma Half-Life (hrs)
% Dose/g tissue
G3-10 kDa
G3-20 kDa
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Synthesis Divergent
Convergent
Applications
Encapsulation
Gene Delivery
Cancer Therapy
Multivalency MRI
Conclusions and Outlook
Outline48
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Multivalent Glycoreceptors
**Binding sites shallow, Ka Monomer ~ 10-3 M
Bertozzi, C. R.; Kiessling, L. L. Science 2001, 291, 2357. Lundquist, J. J.; Toone, E. J. Chem. Rev. 2002, 102, 555. Lee, Y. C.; Lee, R. T. Acc. Chem. Res. 1995, 28, 321
49
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PAMAM
Dendrimers for Multivalent Display
Size control can affect mechanism of binding
Dendrimer amenable to modular design
50
= Sugar
Lundquist, J. J.; Toone, E. J. Chem. Rev. 2002, 102, 555. Kanai, M.; et al. J. Am. Chem. Soc. 1997, 119, 9931.
G2-PAMAM
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Concanavalin A
MW (monomer) = 26.5 kDa
Possesing one saccharide and 1 metal binding site
Binds -D-mannose and -D-glucose
Mandal, D. K.; Kishore, N.; Brewer, C. F. Biochemistry 1994, 33, 1149Derewenda, Z.; et al. EMBO J. 1989, 8, 2189
tetramer 65 Å between binding sites
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G1-G4 PAMAM Glycodendrimers
Page, D.; Roy, R. Bioorg. Med. Chem. Lett. 1996, 4, 1949. Page, D.; Roy, R. Bioconjugate Chem. 1997, 8, 714
52
p-NO2--D-Mannose
NHn
SNH
OPAMAMG1G2G3G4
n =4,8,16,32
O OH
HHO
H
H
HHO
HOH2C
H
O O OH
HHO
H
H
HHOHOH2C
H
SCN
2. MeOH/1M NaOMe
O O OH
HHO
H
H
HHOHOH2C
H
O2N
NH2 nn =4,8,16,32
PAMAMG1G2G3G4
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Concanavalin A
Binding Enhancement
Page, D.; Roy, R. Bioconjugate Chem. 1997, 8, 714. Images of Concanavalin A and Peal Lectin from: Derewenda, Z.; et al. EMBO J. 1989, 8, 2189, http://spec.ch.man.ac.uk/99stuf/interests_98.html
53
pNO2Ph--D-Mannose 105 1 G1(4-mer) 12.4 2.1 G2(8-mer) 4.1 3.2 G3(16-mer) 3.1 2.1 G4(32-mer) 2.3 1.4
Compound IC50(M) Rel. Potency
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Loading of G1-G6 PAMAMs
Woller, E. K.; Cloninger, M. J. Org. Lett. 2002, 4, 7
54
DMF, 8 hr rt1.
2.NaOMe/MeOH
Theo. No. Amines No. Sugar Avg. % Gen. (MALDI) (MALDI) Loading
1 8(1430) 8(5280) 1002 16(3260) 16(10960) 100: : : :6 256(50800) 173(133500) 67
NH2n
PAMAMG1-G6
n =8,16,30,54,92,173
OO
O OH
H
HO
H
H
HHO
HOH2C
HSCN
n
OO
O OH
H
HO
H
H
HHO
HOH2C
HHN
S
HNPAMAM
G1-G6
n =8,16,30,54,92,173
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65 Å betweenbinding sites
Clustering vs. Multivalency55
monovalent glycocluster multivalent
Woller, E. K.; Cloninger, M. J. Org. Lett. 2002, 4, 7
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Loading Effects on Binding
Woller, E. K.; et al. J. Am. Chem. Soc. 2003, 125, 8820
56
Precipitation Assay >2 sugars available/ Con. A
Con. A:Dendrimer ratioincreases with generation
Binding Assay
Area/Sugar
Rel. Act./Mannose
G3
G4
G5
G6
y
OO
O OH
H
HO
H
H
HHO
HOH2C
HHN
S
HN
PAMAMG3-G6
x
OHO
HN
S
HN
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CD Docetaxel Glycodendrimer
Dendrimers: A Modular Approach
Benito, J. M.; Gomez-Garcia, M.; Mellet, C. O.; Baussanne, I.; Defaye, J.; Fernandez, J. M. G. J. Am. Chem. Soc. 2004, 126, 10355
57
YFD = Your Favorite Drug
YFDCell
Recognition OO
O OH
H
HO
H
H
HHO
HOH2C
HHN
S
HN
n
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Binding of Con A: A Model Study
Benito, J. M.; et al. J. Am. Chem. Soc. 2004, 126, 10355
58
Compound Valency IC50(M) Rel Inhibition
CD-1-Mannose 1 1360 1CD-6-Mannose 6 10 22.7CD-6-Mannose + Docetax. 6 6 38
DocetaxelCD-6-Mannose
S
NH O
O
O
OHO
HOOH OH
O
HO OH OH
OH
O
OH
OH
OH
OH
O
O
O O
OH
OH
OH
HO
O OH
OH
HOHO
O
OHHOHO
HO
NH
S
NH
HNHN
OHN
5S
HN-CD
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Synthesis Divergent
Convergent
Applications
Encapsulation
Gene Delivery
Cancer Therapy
Multivalency MRI
Conclusions and Outlook
Outline59
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Gd3+
[Xe] 4f7
MRI Contrast Agents
Caravan, P.; Ellison, J. J.; McMurray, T. J.; Lauffer, R. B. Chem. Rev. 1999, 99, 2293
60
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Signal Enhancement with Contrast Agents61
Gd
Gd
T1
Fast relaxation of nuclear spins can be enhanced by dipole-dipole interaction with paramagnetic ions
Relaxation is further enhanced by slowing down the rotational correlation time of the paramagnetic ion
Gd3+
Morgan, L. O.; Bloembergen, N. J. Chem. Phys. 1961, 34, 842. Wiener, E. C. et al. J. Am. Chem. Soc. 1996, 118, 7774
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Rotational Correlation Times
Free Chelate: 1.5 X 10-10
G2-TU DTPA: 9.3 X 10-10
G6-TU DTPA: 2.5 X 10-9
Rotational Correlation Time Effects
Wiener, E. C. et al. J. Am. Chem. Soc. 1996, 118, 7774, Kobayashi, H.; et al. J. Magn. Reson. Imaging 2004, 20, 512
62
PAMAM-TU-DTPA
G2-G6 HN
S
NH
N
COO-
HN-OOC
COO-
N COO-
-OOC
n
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Dendrimer-Chelate Half-life
Kobayashi, H.; Sato, N.; Hiraga, A.; Sage, T.; Nakamoto, Y.; Ueda, H.; Konishi, J.; Togashi, K.; Brechbiel, M. W. Magn. Res. Med. 2001, 45, 454
63
NN
N
O
O
NH
O
HN
OO O
O
O
Gd
OHH
C6H4NCS
Blood Half-Life (min.)G6-[Gd]: 13 +/- 3G5-[Gd]: 4.9 +/- 1.1G4-[Gd]: 2.5 +/- 0.9G3-[Gd]: 1.1 +/- 0.4Gd-DTPA: 0.4 +/- 0.2
Gd-DTPA
PAMAM
G=3-6
Gd
x = 32, 64 ,96,192
X
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G6-Gd G5-Gd G4-Gd G3-Gd Gd
Contrast Imaging in Mice
Microvasculature, able to be imaged with larger dendrimer generations 9 min. after injection
Kobayashi, H.; Sato, N.; Hiraga, A.; Sage, T.; Nakamoto, Y.; Ueda, H.; Konishi, J.; Togashi, K.; Brechbiel, M. W. Magn. Res. Med. 2001, 45, 454
64
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Summary
• Dendrimers’ 3-D structure allows for interesting host-guest properties as well as attachment of reactive/diagnostic groups.
• Low/monodisperse dendrimers predictably displaying a high degree of functionality, make for an interesting complement to conventional polymers.
• Care must be taken for analysis of biocompatibility when developing new materials for biological applications.
• Commercial availability of dendrimers such as PAMAM has encouraged many scientists to enter into an interdisciplinary field.
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Drug
A Dendrimer Perspective
• Dendrimer modularity, can enable attachment of multiple reporters, recognition groups, and cargo.
• Commercial dendrimer products are on the market (Gadomer-17: Sheering AG, Superfect: Qiagen) or in clinical trials. With increasing studies on biocompatibility this number should grow.
• However, the frontier still remains wide open and is limited only by one’s imagination.
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67Acknowledgements
Sam GellmanGellman Group
Practice Talk Attendees
Chris ParadiseKatie AlfareKevin “HP” SchultzAdam Garske
Charlie FryJennifer Moran