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1
Dendrimer Synthesis and Applications: Branching out into Biology
Organic Chemistry SeminarApril 28th, 2005
William PomerantzGellman Group
2
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/
2
m
Neuronnm
Dendrimerm
Tree
3
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
4
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
5
G0
Core
Monomer
GX = generation
Generation Growth
6
G1
GX = generation
Core
Monomer
Generation Growth
7
G2
GX = generation
Core
Monomer
Generation Growth
8
G3
GX = generation
Core
Monomer
Generation Growth
9
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
10
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
11
Synthesis Divergent
Convergent
Applications
Encapsulation
Gene Delivery
Cancer Therapy
Multivalency MRI
Conclusions and Outlook
Outline11
12
Synthetic Considerations
Reagents
Reactions
High YieldingMinimal side reactionsPurify intermediates
Polydispersity (PDI)
Very Narrow PDI:PDI= Mw/Mn
CheapHigh ReactivityEasily Removable
12
13Dendrimer CoreMultivalent Monomer
G0
G1
G3
G2
Divergent Dendrimer Synthesis13
14
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
14
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
15
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
15
PAMAMDendrimer
NH
ON
CO2Me
CO2Me
NH
O HN
CO2Me
CO2Me
+
nn
16
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
17
Convergent Synthesis
Dendrimer Core Multivalent MonomerG3Hawker, C. J.; Frechet, J. M. J. J. Am. Chem. Soc 1990, 112, 7638-7647
18
Dendrimer Wedge Synthesis (Convergent)
Frechet and co-workers; J. Am. Chem. Soc. 1990, 112, 7638 J. Control. Release 2000, 65, 121
18
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
19
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
20
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
20
Frechet and co-workers; J. Am. Chem. Soc. 1990, 112, 7638 J. Control. Release 2000, 65, 121
21
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
Synthesis Divergent
Convergent
Applications
Encapsulation
Gene Delivery
Cancer Therapy
Multivalency MRI
Conclusions and Outlook
Outline22
23
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
23
Space-Filling Model
24
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
24
Space-Filling Model
25
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
25
Raney-Co, H2
PPI Dendrimer
NH2 CN
AcOH
NH2
N
N
H2N
NH2NH2
NH2
BOX
Lid
26
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
26
PPI Dendrimer
Raney-Co, H2
NH2 CN
AcOH
NH2
N
N
H2N
NH2NH2
NH2
27
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
27
NH2
64
N
O
O
O
O
NHBoc
NH
O
NHBoc
Probe
Probe
64
Ph
PhCH2Cl2, Et3N
28
Unimolecular Micelle Containers
“Frechet” Polyaryl- ether dendrimer
Liu, M.; Kono, K.; Frechet, J. M. J. J. Control. Release 2000, 65, 121
28
[Pyrene]water
10-6 M
[Dendrimer]
G3
G2
G18.0 x 10-7M
H2O
=
G1 G2 G3
29
Drug Encapsulation and Release
Liu, M.; Kono, K.; Frechet, J. M. J. J. Control. Release 2000, 65, 121
29
Indomethacin
Drug + G3
Free Drug
Time (hr)
% Release
pH =737oC
=N
CH2COOHO
O
Cl
Water bath
Dialysis membrane
30
Synthesis Divergent
Convergent
Applications
Encapsulation
Gene Delivery
Cancer Therapy
Multivalency MRI
Conclusions and Outlook
Outline30
31
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
31
32
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
32
Polycation
1.
2. 3.
DNA
endosome
Nucleus
DNA
Nucleus
endosomeDNA
H+
DNA
DNA
Bind DNA
Cellular Uptake
Endosomal Release Transfection
33
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
33
+/- Charge ratio
NH
C
O
NH2
H OH115
PAMAM
G2-G10NH2
x
34
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
34
+/- Charge ratio
NH
C
O
NH2
H OH115
PAMAM
G2-G10NH2
x
PAMAMNH2
x-y
Defect
H2O
Superfect
35
Transfection/Cytotoxicity Comparison
Gebhart, C. L.; Kabanov, A. V. J. Control. Release 2001, 73, 401
35
Defect PAMAM
CellViability
PPIDendrimer
PEI 22KLinear
Luciferase(ng/mg)
PEI/PluronicCopolymer
PEI 25KBranched
PEI 50KLinear
NH
HN
NHn
PEI
36
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
36
Defect PAMAM
PhePAMAM
37
Synthesis Divergent
Convergent
Applications
Encapsulation
Gene Delivery
Cancer Therapy
Multivalency MRI
Conclusions and Outlook
Outline37
38
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
38
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
39
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
39
40
Dendrimer Design Features
Frechet, J. M. J. and co-workers. Bioconjugate Chem. 2002, 13, 443, Macromolecules 1998, 31, 4061, Bioconjugate Chem. 2002, 13, 453
40
= Drug
High MW Dendrimer
High MW Dendrimer
Modular Approach
41
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
41
O
O
O
O
ORO
RO
ORO
RO
=
=
OH
O O
42
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
42
=
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
43
Dendrimer Cytotoxicity
De Jesus, O. L. P.; Ihre, H. R.; Frechet, J. M. J.; Szoka, F. C. J. Bioconjugate Chem. 2002, 13, 453
43
Half-Lives of I-III all < 10 Min.
% Cell Viability
[Dendrimer] (mg/mL)
12 kDa
4 kDa
24 kDa
44
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
45
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
46
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
47
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
48
Synthesis Divergent
Convergent
Applications
Encapsulation
Gene Delivery
Cancer Therapy
Multivalency MRI
Conclusions and Outlook
Outline48
49
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
50
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
51
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
52
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
53
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
54
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
55
65 Å betweenbinding sites
Clustering vs. Multivalency55
monovalent glycocluster multivalent
Woller, E. K.; Cloninger, M. J. Org. Lett. 2002, 4, 7
56
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
57
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
58
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
59
Synthesis Divergent
Convergent
Applications
Encapsulation
Gene Delivery
Cancer Therapy
Multivalency MRI
Conclusions and Outlook
Outline59
60
Gd3+
[Xe] 4f7
MRI Contrast Agents
Caravan, P.; Ellison, J. J.; McMurray, T. J.; Lauffer, R. B. Chem. Rev. 1999, 99, 2293
60
61
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
62
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
63
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
64
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
65
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.
65
66
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.
66
67
67Acknowledgements
Sam GellmanGellman Group
Practice Talk Attendees
Chris ParadiseKatie AlfareKevin “HP” SchultzAdam Garske
Charlie FryJennifer Moran
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