Core-clickable PEG-branch-azide bivalent-bottle-brush polymers by ROMP: Grafting-through and clicking-to

Article abstract of DOI:10.1021/ja108441d

The combination of highly efficient polymerizations with modular "click" coupling reactions has enabled the synthesis of a wide variety of novel nanoscopic structures. Here we demonstrate the facile synthesis of a new class of clickable, branched nanostru

Full text of DOI:10.1021/ja108441d

Published on Web 12/13/2010
Core-Clickable PEG-Branch-Azide Bivalent-Bottle-Brush
Polymers by ROMP: Grafting-Through and Clicking-To
Jeremiah A. Johnson,† Ying Y. Lu,† Alan O. Burts,† Yeon-Hee Lim,‡ M. G. Finn,‡
Jeffrey T. Koberstein,§ Nicholas J. Turro,| David A. Tirrell,*,† and
Robert H. Grubbs*,†
DiVision of Chemistry and Chemical Engineering, California Institute of Technology, 1200 E.
California BlVd., Pasadena, California 91125; Department of Chemistry and The Skaggs
Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road,
La Jolla, California 92037; Department of Chemical Engineering, Columbia UniVersity, 500
West 120th Street, New York, New York, 10027; and Department of Chemistry, Columbia
UniVersity, 3000 Broadway, New York, New York 10027
Received September 18, 2010; E-mail: rhg@caltech.edu; tirrell@caltech.edu
Abstract: The combination of highly efficient polymerizations with modular “click” coupling reactions has
enabled the synthesis of a wide variety of novel nanoscopic structures. Here we demonstrate the facile
synthesis of a new class of clickable, branched nanostructures, polyethylene glycol (PEG)-branch-azide
bivalent-brush polymers, facilitated by “graft-through” ring-opening metathesis polymerization of a branched
norbornene-PEG-chloride macromonomer followed by halide-azide exchange. The resulting bivalent-brush
polymers possess azide groups at the core near a polynorbornene backbone with PEG chains extended
into solution; the structure resembles a unimolecular micelle. We demonstrate copper-catalyzed azide-alkyne
cycloaddition (CuAAC) “click-to” coupling of a photocleavable doxorubicin (DOX)-alkyne derivative to the
azide core. The CuAAC coupling was quantitative across a wide range of nanoscopic sizes (∼6-∼50
nm); UV photolysis of the resulting DOX-loaded materials yielded free DOX that was therapeutically effective
against human cancer cells.
Introduction
mers, bottle-brush polymers, and hyperbranched polymers
generally display longer in vivo retention times compared to
The explosion of nanoscience is fuelled by problems that
require nanoscale solutions and by the inherent challenges of
nanoscale synthesis.1-3 In drug delivery, for example, plasma
clearance time of a polymer-drug conjugate is closely linked
to its nanoscopic size; materials smaller than 10 nm are excreted
rapidly whereas larger materials often display significantly
longer retention times.3-9 Nanoscopic architecture also plays
an important role in determining in vivo retention time.10-12
Branched polymeric structures such as dendrimers, star poly-
their linear polymer analogues.11,13 A wealth of research
attention has focused on synthetic approaches to branched,
stimuli-responsive, nanoscopic materials that possess orthogonal
functionality for incorporation of therapeutic, imaging, and
targeting molecules.14-25
(11) Fox, M. E.; Szoka, F. C.; Frechet, J. M. J. Acc. Chem. Res. 2009,
42, 1141–1151.
(12) Chen, B.; Jerger, K.; Frechet, J. M. J.; Szoka, F. C. J. Controlled
Release 2009, 140, 203–209.
† Division of Chemistry and Chemical Engineering, California Institute
of Technology.
(13) Grayson, S. M.; Godbey, W. T. J. Drug Targeting 2008, 16, 329–
356.
‡ Department of Chemistry and The Skaggs Institute for Chemical
Biology, The Scripps Research Institute.
(14) Duncan, R. Nat. ReV. Drug DiscoVery 2003, 2, 347–360.
(15) Bartlett, D. W.; Su, H.; Hildebrandt, I. J.; Weber, W. A.; Davis, M. E.
Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 15549–15554.
(16) Wu, P.; Malkoch, M.; Hunt, J. N.; Vestberg, R.; Kaltgrad, E.; Finn,
M. G.; Fokin, V. V.; Sharpless, K. B.; Hawker, C. J. Chem. Commun.
2005, 5775–5777.
§ Department of Chemical Engineering, Columbia University.
| Department of Chemistry, Columbia University.
(1) Whitesides, G. M. Small 2005, 1, 172–179.
(2) Steigerwald, M. L.; Brus, L. E. Acc. Chem. Res. 1990, 23, 183–188.
(3) Peer, D.; Karp, J. M.; Hong, S.; Farokhzad, O. C.; Margalit, R.;
Langer, R. Nat. Nanotechnol. 2007, 2, 751–760.
(17) Ihre, H. R.; Padilla De Jesus, O. L.; Szoka, F. C., Jr.; Frechet, J. M. J.
Bioconj. Chem. 2002, 13, 443–452.
(4) Matsumura, Y.; Maeda, H. Cancer Res. 1986, 46, 6387–6392.
(5) Gref, R.; Minamitake, Y.; Peracchia, M. T.; Trubetskoy, V.; Torchilin,
V.; Langer, R. Science 1994, 263, 1600–1603.
(18) Breitenkamp, K.; Emrick, T. J. Am. Chem. Soc. 2003, 125, 12070–
12071.
(19) Becker, M. L.; Remsen, E. E.; Pan, D.; Wooley, K. L. Bioconj. Chem.
2004, 15, 699–709.
(6) Gref, R.; Domb, A.; Quellec, P.; Blunk, T.; Mueller, R. H.; Verbavatz,
J. M.; Langer, R. AdV. Drug DeliVery ReV. 1995, 16, 215–233.
(7) Edwards, D. A.; Hanes, J.; Caponetti, G.; Hrkach, J.; Ben-Jebria,
A.; Eskew, M. L.; Mintzes, J.; Deaver, D.; Lotan, N.; Langer, R.
Science 1997, 276, 1868–1871.
(20) Such, G. K.; Tjipto, E.; Postma, A.; Johnston, A. P. R.; Caruso, F.
Nano Lett. 2007, 7, 1706–1710.
(21) Cengelli, F.; Grzyb, J. A.; Montoro, A.; Hofmann, H.; Hanessian,
S.; Juillerat-Jeanneret, L. ChemMedChem 2009, 4, 988–997.
(22) Tong, G. J.; Hsiao, S. C.; Carrico, Z. M.; Francis, M. B. J. Am. Chem.
Soc. 2009, 131, 11174–11178.
(8) Langer, R. Nature (London) 1998, 392, 5–10.
(9) Mammen, M.; Chio, S.-K.; Whitesides, G. M. Angew. Chem., Int.
Ed. 1998, 37, 2755–2794.
(23) Lee, S.-M.; Chen, H.; O’Halloran, T. V.; Nguyen, S. T. J. Am. Chem.
Soc. 2009, 131, 9311–9320.
(10) Qiu, L. Y.; Bae, Y. H. Pharm. Res. 2006, 23, 1–30.
9
10.1021/ja108441d  2011 American Chemical Society
J. AM. CHEM. SOC. 2011, 133, 559–566 559

A R T I C L E S
Johnson et al.
The most widely studied branched macromolecules for drug
delivery are dendrimers; their nanoscale, monodisperse, mul-
tivalent structures provide functional handles for elaboration with
bioactive groups of interest.26-28 One factor that limits wide-
spread application of dendrimers is synthetic difficulty. Though
recent synthetic advances are impressive,16,29-32 it remains a
challenge to prepare functionally diverse dendrimers of variable
sizes. To overcome this challenge, researchers have appended
linear polymers to dendrimers33-36 or prepared linear polymers
with dendritic fragments attached to their side chains. The latter
“dendronized linear polymers” have attracted attention as
nanoscopic building blocks; their cylindrical shapes can provide
important advantages in nanoscale fabrication when compared
to spherical dendrimers.37-43 Similar nanoscale structures can
be formed from bottle-brush-polymers that carry long chains
grafted at high density to linear polymer backbones.44-63 Bottle-
brush polymers are prepared by graft-to, graft-from, or graft-
through methodologies. The graft-to strategy requires an efficient
coupling reaction to attach a functional molecule to every
monomer unit of a linear polymer; “click”64 reactions such as
the copper-catalyzed azide-alkyne cycloaddition65,66 (CuAAC)
and thiol-ene coupling have proven useful in this regard.43,46,67-70
Graft-from and graft-through methodologies require highly
efficient polymerization reactions capable of initiation and
propagation in sterically demanding environments.48,51-53,71
Recent developments in efficient, controlled polymerization
coupled with new click reactions provide materials chemists
with the tools to generate novel functional nanoscopic
materials.72-77
Ring-opening metathesis polymerization (ROMP) of strained
alkene- (e.g., norbornene)-terminated macromonomers (MM)
initiated by ruthenium N-heterocyclic carbene complexes (e.g.,
1, Figure 1) has recently proven useful for graft-through
synthesis of functional bottle-brush polymers;50,78-83 fast initia-
tion leads to low polydispersities (PDIs) while release of ring
(24) Lee, J.-S.; Green, J. J.; Love, K. T.; Sunshine, J.; Langer, R.;
Anderson, D. G. Nano Lett. 2009, 9, 2402–2406.
(25) Shen, Y.; Jin, E.; Zhang, B.; Murphy, C. J.; Sui, M.; Zhao, J.; Wang,
J.; Tang, J.; Fan, M.; Van Kirk, E.; Murdoch, W. J. J. Am. Chem.
Soc. 2010, 132, 4259–4265.
(52) Neugebauer, D.; Sumerlin, B. S.; Matyjaszewski, K.; Goodhart, B.;
Sheiko, S. S. Polymer 2004, 45, 8173–8179.
(26) Helms, B.; Meijer, E. W. Science 2006, 313, 929–930.
(27) Tomalia, D. A.; Baker, H.; Dewald, J.; Hall, M.; Kallos, G.; Martin,
S.; Roeck, J.; Ryder, J.; Smith, P. Polym. J. 1985, 17, 117–132.
(28) Bosman, A. W.; Janssen, H. M.; Meijer, E. W. Chem. ReV. 1999,
99, 1665–1688.
(53) Sumerlin, B. S.; Neugebauer, D.; Matyjaszewski, K. Macromolecules
2005, 38, 702–708.
(54) Gao, H.; Matyjaszewski, K. J. Am. Chem. Soc. 2007, 129, 6633–
6639.
(55) Hadjichristidis, N.; Pitsikalis, M.; Iatrou, H.; Pispas, S. Macromol.
Rapid Commun. 2003, 24, 979–1013.
(56) Tsukahara, Y.; Mizuno, K.; Segawa, A.; Yamashita, Y. Macromol-
ecules 1989, 22, 1546–1552.
(57) Tsukahara, Y.; Tsutsumi, K.; Yamashita, Y.; Shimada, S. Macro-
molecules 1990, 23, 5201–5208.
(58) Dziezok, P.; Sheiko, S. S.; Fischer, K.; Schmidt, M.; Moller, M.
Angew. Chem., Int. Ed. 1998, 36, 2812–2815.
(59) Neiser, M. W.; Okuda, J.; Schmidt, M. Macromolecules 2003, 36,
5437–5439.
(60) Neiser, M. W.; Muth, S.; Kolb, U.; Harris, J. R.; Okuda, J.; Schmidt,
M. Angew. Chem., Int. Ed. 2004, 43, 3192–3195.
(61) Yuan, Y.-Y.; Du, Q.; Wang, Y.-C.; Wang, J. Macromolecules 2010,
43, 1739–1746.
(62) Li, C.; Ge, Z.; Fang, J.; Liu, S. Macromolecules 2009, 42, 2916–
2924.
(29) Montanez, M. I.; Campos, L. M.; Antoni, P.; Hed, Y.; Walter, M. V.;
Krull, B. T.; Khan, A.; Hult, A.; Hawker, C. J.; Malkoch, M.
Macromolecules 2010, 43, 6004–6013.
(30) Antoni, P.; Robb, M. J.; Campos, L.; Montanez, M.; Hult, A.;
Malmstrom, E.; Malkoch, M.; Hawker, C. J. Macromolecules 2010,
43, 6625–6631.
(31) Killops, K. L.; Campos, L. M.; Hawker, C. J. J. Am. Chem. Soc.
2008, 130, 5062–5064.
(32) Wu, P.; Feldman, A. K.; Nugent, A. K.; Hawker, C. J.; Scheel, A.;
Voit, B.; Pyun, J.; Frechet, J. M. J.; Sharpless, K. B.; Fokin, V. V.
Angew. Chem., Int. Ed. 2004, 43, 3928–3932.
(33) Lee, C. C.; Gillies, E. R.; Fox, M. E.; Guillaudeu, S. J.; Frechet,
J. M. J.; Dy, E. E.; Szoka, F. C. Proc. Natl. Acad. Sci. U.S.A. 2006,
103, 16649–16654.
(34) Gillies, E. R.; Dy, E.; Frechet, J. M. J.; Szoka, F. C. Mol. Pharm.
2005, 2, 129–138.
(63) Li, A.; Lu, Z.; Zhou, Q.; Qiu, F.; Yang, Y. J. Polym. Sci., Part A:
Polym. Chem. 2006, 44, 3942–3946.
(35) Gillies, E. R.; Frechet, J. M. J. J. Org. Chem. 2004, 69, 46–53.
(36) Gillies, E. R.; Frechet, J. M. J. J. Am. Chem. Soc. 2002, 124, 14137–
14146.
(64) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed.
2001, 40, 2004–2021.
(37) Grayson, S. M.; Frechet, J. M. J. Macromolecules 2001, 34, 6542–
6544.
(65) Tornoe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67,
3057–3064.
(38) Boydston, A. J.; Holcombe, T. W.; Unruh, D. A.; Frechet, J. M. J.;
Grubbs, R. H. J. Am. Chem. Soc. 2009, 131, 5388–5389.
(39) Zhang, A.; Zhang, B.; Waechtersbach, E.; Schmidt, M.; Schlueter,
A. D. Chem.sEur. J. 2003, 9, 6083–6092.
(66) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B.
Angew. Chem., Int. Ed. 2002, 41, 2596–2599.
(67) Sumerlin, B. S.; Tsarevsky, N. V.; Louche, G.; Lee, R. Y.;
Matyjaszewski, K. Macromolecules 2005, 38, 7540–7545.
(68) Gao, H.; Matyjaszewski, K. J. Am. Chem. Soc. 2007, 129, 6633–
6639.
(40) Lee, C. C.; Grayson, S. M.; Frechet, J. M. J. J. Polym. Sci., Part A:
Polym. Chem. 2004, 42, 3563–3578.
(41) Yoshida, M.; Fresco, Z. M.; Ohnishi, S.; Frechet, J. M. J. Macro-
molecules 2005, 38, 334–344.
(69) Tsarevsky, N. V.; Bernaerts, K. V.; Dufour, B.; Du Prez, F. E.;
Matyjaszewski, K. Macromolecules 2004, 37, 9308–9313.
(70) Campos, L. M.; Killops, K. L.; Sakai, R.; Paulusse, J. M. J.; Damiron,
D.; Drockenmuller, E.; Messmore, B. W.; Hawker, C. J. Macromol-
ecules 2008, 41, 7063–7070.
(42) Canilho, N.; Kaseemi, E.; Mezzenga, R.; Schlueter, A. D. J. Am.
Chem. Soc. 2006, 128, 13998–13999.
(43) Helms, B.; Mynar, J. L.; Hawker, C. J.; Frechet, J. M. J. J. Am.
Chem. Soc. 2004, 126, 15020–15021.
(71) Boerner, H. G.; Beers, K.; Matyjaszewski, K.; Sheiko, S. S.; Moeller,
M. Macromolecules 2001, 34, 4375–4383.
(44) Zhang, M.; Mueller, A. H. E. J. Polym. Sci., Part A: Polym. Chem.
2005, 43, 3461–3481.
(72) Golas, P. L.; Matyjaszewski, K. Chem. Soc. ReV. 2010, 39, 1338–
1354.
(45) Jiang, X.; Lok, M. C.; Hennink, W. E. Bioconj. Chem. 2007, 18,
2077–2084.
(73) Iha, R. K.; Wooley, K. L.; Nystrom, A. M.; Burke, D. J.; Kade, M. J.;
Hawker, C. J. Chem. ReV. 2009, 109, 5620–5686.
(74) Hawker, C. J.; Wooley, K. L. Science 2005, 309, 1200–1205.
(75) Johnson, J. A.; Finn, M. G.; Koberstein, J. T.; Turro, N. J. Macromol.
Rapid Commun. 2008, 29, 1052–1072.
(46) Tsarevsky, N. V.; Bencherif, S. A.; Matyjaszewski, K. Macromol-
ecules 2007, 40, 4439–4445.
(47) Lutz, J.-F.; Boerner, H. G.; Weichenhan, K. Macromolecules 2006,
39, 6376–6383.
(48) Allen, M. J.; Wangkanont, K.; Raines, R. T.; Kiessling, L. L.
Macromolecules 2009, 42, 4023–4027.
(76) Binder, W. H.; Sachsenhofer, R. Macromol. Rapid Commun. 2007,
28, 15–54.
(49) Cheng, G.; Boeker, A.; Zhang, M.; Krausch, G.; Mueller, A. H. E.
Macromolecules 2001, 34, 6883–6888.
(77) Hawker, C. J.; Fokin, V. V.; Finn, M. G.; Sharpless, K. B. Aust.
J. Chem. 2007, 60, 381–383.
(50) Lu, H.; Wang, J.; Lin, Y.; Cheng, J. J. Am. Chem. Soc. 2009, 131,
13582–13583.
(78) Xia, Y.; Kornfield, J. A.; Grubbs, R. H. Macromolecules 2009, 42,
3761–3766.
(51) Morandi, G.; Pascual, S.; Montembault, V.; Legoupy, S.; Delorme,
N.; Fontaine, L. Macromolecules 2009, 42, 6927–6931.
(79) Xia, Y.; Olsen, B. D.; Kornfield, J. A.; Grubbs, R. H. J. Am. Chem.
Soc. 2009, 131, 18525–18532.
9
560 J. AM. CHEM. SOC. VOL. 133, NO. 3, 2011

PEG-Branch-Azide Bivalent-Bottle-Brush Polymers by ROMP
A R T I C L E S
Figure 1. Schematic depiction of the “graft-through and click-to” approach described in this work. A: We start with a bivalent macromonomer (2) and
perform “graft-through” ROMP with catalyst 1 followed by in situ chloride-azide exchange. B: The resulting azido-bivalent-brush polymer is then functionalized
with an alkyne by click chemistry in a “click-to” step. If the alkyne partner is a drug molecule linked via a cleavable linker, then the resulting brush polymer
can release the drug in response to an external stimulus. In this work, we demonstrate controlled release of the anticancer agent doxorubicin (DOX) in
response to 365 nm UV light.
strain provides the driving force for high conversion in these
systems. Although catalyst 1 is incompatible with azides and
alkynes, its functional group tolerance enables use of monomers
bearing azide and alkyne precursors that are easily converted
to the requisite click groups after polymerization. Binder and
co-workers used ROMP combined with CuAAC to append
various small molecules to clickable linear polymers.84,85 We
have used ROMP in conjunction with CuAAC to generate cyclic
polymers by end-to-end coupling.86 To our knowledge, there
are no examples of water-soluble brush polymers prepared by
graft-through ROMP that are subsequently functionalized via
click reactions. Such a strategy combines the benefits of ROMP
for graft-through brush polymer synthesis with the modularity
and efficiency of CuAAC for clicking-to; branched nanoscopic
polymers with easily addressable functional groups can be
rapidly prepared over a wide range of sizes simply by altering
the ratio of catalyst to MM.
clickable polyethylene glycol (PEG)-azide branched polymer
material: a bivalent-bottle-brush polymer (Figure 1). Graft-
through ROMP of a PEG-chloride macromonomer (Figure 1,
Scheme 1, 6) gave bivalent-brush polymers of varying nano-
scopic sizes. These materials resemble first-generation den-
dronized polymers with branch points located near the linear
polymer backbone (Figure 1); attached on one side of the branch
point is a linear polymer and on the other side a small molecule
(e.g., a drug). This design incorporates a water-soluble PEG
domain that extends into solvent and a hydrophobic alkyl
chloride near the core. We hypothesize that PEG attachment to
one branch of these bivalent-brush polymers will confer the
biocompatibility widely observed for PEGylated systems.6,87
Core-functionalized nanomaterials have been elaborated with
functional small molecules by click chemistry;88-92 in this work,
we convert the core alkyl chlorides to azides by treatment with
sodium azide. Then, we utilize CuAAC to couple doxorubicin
(DOX), to the core via a photocleavable nitrobenzyloxylcarbonyl
(NBOC) linker. The latter enables controlled release of free
DOX in response to long-wavelength (∼365 nm) ultraviolet
In this report, we demonstrate the sequential combination of
graft-through- and click-to synthetic strategies to generate a
(80) Li, Z.; Ma, J.; Cheng, C.; Zhang, K.; Wooley, K. L. Macromolecules
2010, 43, 1182–1184.
(87) Otsuka, H.; Nagasaki, Y.; Kataoka, K. AdV. Drug DeliVery ReV. 2003,
55, 403–419.
(81) Li, Z.; Zhang, K.; Ma, J.; Cheng, C.; Wooley, K. L. J. Polym. Sci.,
Part A: Polym. Chem. 2009, 47, 5557–5563.
(88) O’Reilly, R. K.; Joralemon, M. J.; Hawker, C. J.; Wooley, K. L.
New J. Chem. 2007, 31, 718–724.
(82) Conrad, R. M.; Grubbs, R. H. Angew. Chem., Int. Ed. 2009, 48, 8328–
8330.
(83) Le, D.; Montembault, V.; Soutif, J. C.; Rutnakornpituk, M.; Fontaine,
L. Macromolecules 2010, 43, 5611–5617.
(89) O’Reilly, R. K.; Joralemon, M. J.; Hawker, C. J.; Wooley, K. L.
Chem.sEur. J. 2006, 12, 6776–6786.
(90) O’Reilly, R. K.; Joralemon, M. J.; Wooley, K. L.; Hawker, C. J.
Chem. Mater. 2005, 17, 5976–5988.
(84) Binder, W. H.; Kluger, C. Macromolecules 2004, 37, 9321–9330.
(85) Binder, W. H.; Kluger, C.; Josipovic, M.; Straif, C. J.; Friedbacher,
G. Macromolecules 2006, 39, 8092–8101.
(91) Joralemon, M. J.; O’Reilly, R. K.; Hawker, C. J.; Wooley, K. L.
J. Am. Chem. Soc. 2005, 127, 16892–16899.
(86) Clark, P. G.; Guidry, E. N.; Chan, W. Y.; Steinmetz, W. E.; Grubbs,
R. H. J. Am. Chem. Soc. 2010, 132, 3405–3412.
(92) Prasuhn, D. E., Jr.; Singh, P.; Strable, E.; Brown, S.; Manchester,
M.; Finn, M. G. J. Am. Chem. Soc. 2008, 130, 1328–1334.
9
J. AM. CHEM. SOC. VOL. 133, NO. 3, 2011 561

A R T I C L E S
Johnson et al.
Scheme 1. Synthesis of PEG-Norbornene-Chloride MM 6
Scheme 2. Synthesis of Azido-Bivalent-Brush Polymers (nN3) by
ROMP and Chloride-Azide Exchange
(UV) light; we demonstrate that released DOX is therapeutically
active against human cancer cells in culture.
Results and Discussion
Synthesis of MM 6. Clickable bivalent PEG brush polymers
can be generated from an MM containing a strained norbornene
derivative for ROMP, a PEG chain, and a functional group
suitable for CuAAC (Figure 1, Scheme 1). Since neither azides
nor alkynes are compatible with catalyst 1, we designed MM 6
to carry an alkyl chloride for facile conversion to an azide in a
post-ROMP modification step. We incorporated a 6-carbon
spacer between the strained norbornene moiety and the tertiary
amide branch point to reduce steric hindrance between the
growing polymer chain end and the branched side-chains. This
spacer may also facilitate subsequent side chain modification
reactions, such as chloride to azide conversion and CuAAC.
MM 6 was obtained in five steps from exo-N-(6-hydroxyhexyl)-
5-norbornene-2,3-dicarboximide (norb-C6OH, Scheme 1). The
synthesis commenced with Swern oxidation of norb-C6OH to
give aldehyde 2. Reductive amination using 2 and 3-chloro-
propylamine hydrochloride gave secondary amine 3, which was
readily converted to acid 4 by treatment with succinic anhydride.
Reaction of 4 with N-hydroxysuccinimide (NHS) in the presence
of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochlo-
ride (EDC) and catalytic 4-(dimethylamino)pyridine (DMAP)
gave NHS ester 5. We have prepared 5 on a multigram scale
and expect it to be useful for the preparation of a variety of
multifunctional ROMP monomers. In this work, commercially
available 3 kDa PEG-NH2 was treated with a slight excess of 5
to give MM 6. Excess 5 was used to ensure complete conversion
of every PEG-amine to amide 6; the only polymeric species in
the final reaction mixture was 6 and it was easily purified via
repeated precipitation in diethyl ether. The 1H NMR, 13C NMR,
and MALDI spectra for MM 6 are provided in the Supporting
Information.
Table 1. Characterization of nN3 Bivalent Brush Polymers by GPC
(room temp., 0.2 M LiBr in DMF, 1 mL/min) and DLS (aqueous)
n (theo.)a
DPb
Mn (GPC, kDa)
PDI
Rhc/nm
10
30
50
70
10
32
53
70
80
101
295
575
34.1
109
180
238
272
343
1,000
1,960
1.11
1.04
1.07
1.14
1.14
1.06
1.11
1.27
3.1 (0.3)
6.3 (0.8)
6.9 (0.9)
7.3 (0.3)
7.5 (0.8)
11 (1)
80
100
300
400
15 (1)
25 (2)
a Ratio of MM to catalyst (i.e., theoretical DP). This number is used
to identify nN3 and nDOX samples throughout the text. b DP observed
derived from Mn(GPC)/Mn(MM). c Hydrodynamic radii measured by
dynamic light scattering (DLS). DLS correlation functions were fit using
the CONTIN algorithm.
of variable sizes rapidly and efficiently; each of the polymers
described here was prepared by adding an aliquot of catalyst 1
to a vial containing MM 6, allowing 60 min for ROMP to
proceed, quenching the polymerization by addition of ethyl vinyl
ether, solvent exchange, and finally treatment with sodium azide
(Scheme 2). The first several steps were performed within a
few hours, whereas the final step, attaching azides to the polymer
side chains, was left for 48 h to ensure high conversion.
The polymer characterization data in Table 1 demonstrate
that ROMP of 6 is well-controlled up to the highest DP tested;
a small amount of catalyst deactivation and/or experimental error
may explain the larger-than-expected DP for the 400N3 samples.
In general, polymers with low polydispersities and molecular
weights defined by the ratio of 6:1 were obtained. The GPC
traces for all samples (Figure 2A) show monomodal molecular
weight distributions and very high (>95%) MM conversions.
All of the polymers were highly soluble (>100 mg/mL) in water;
Graft-Through ROMP of 6 and Halide-Azide Exchange to
Generate nN3 Bivalent-Brush Polymers. We pursued a one-pot
synthetic strategy to rapidly generate a library of azide-functional
brush polymers (nN3) with variable number-average degrees of
polymerization (DP) and hydrodynamic radii. A strength of this
approach is the ease of generating branched functional materials
9
562 J. AM. CHEM. SOC. VOL. 133, NO. 3, 2011

PEG-Branch-Azide Bivalent-Bottle-Brush Polymers by ROMP
A R T I C L E S
Figure 2. A: Representative GPC traces for nN3 bivalent-brush polymer samples show monomodal molecular weight distributions, low polydispersities,
and high conversions. B: Hydrodynamic diameter histograms for selected nN3 polymers obtained from DLS measurements using the CONTIN fitting algorithm.
The particle sizes increase with increasing DP and are varied over a wide range by adjusting the ratio of MM to catalyst (6:1) before graft-through ROMP.
an external stimulus leaving the brush polymer scaffold intact.
In this work, we designed 9 for controlled release of DOX in
response to UV light after click coupling to the nN3 polymer
scaffold (Scheme 3). Photoinitiated release from macromolecules
provides a means for site-specific, controlled drug delivery and
could potentially complement traditional phototherapeutic
methods.93-103 Though placement of azide groups near the core
of these bivalent-brush polymers should allow PEG to extend
into solution and increase the biocompatibility of these materials,
a buried location may make them difficult to functionalize with
subsequent reactions. Anticipating this challenge was one reason
we chose two highly efficient reactions, halide-azide exchange
and CuAAC, as the modification reactions; as described above,
Figure 3. FTIR spectra of nN3 bivalent-brush polymers normalized to the
strong PEG ether (C-O-C) antisymmetric stretch absorbance. The relative
amount of azide is approximately the same for each n value which suggests
CuAAC has proven useful for quantitative attachment of small
molecules to the side chains of ROMP polymers84,85 and we
that chloride-azide exchange is not sterically limited with increased DP.
expected that it would provide the best opportunity for high-
yield coupling of sterically demanding alkyne reagents to
bivalent-branch polymers. There are a variety of CuAAC
conditions throughout the literature and the catalyst/ligand/
solvent system depends on the application;104-107 perhaps the
most impressive applications of CuAAC are in the field of
aqueous DLS measurements (Table 1, Figure 2B) confirm that
their nanoscopic dimensions increased with DP and were tunable
over a broad range (radii from 3-25 nm) of relevant sizes for
drug delivery applications. Furthermore, DLS (Figure 2B) shows
that the particle size distributions are monomodal.
(93) Dolmans, D. E. J. G. J.; Fukumura, D.; Jain, R. K. Nat. ReV. Cancer
2003, 3, 380–387.
Graft-through ROMP ensures that every side chain unit
possesses both a PEG chain and an alkyl chloride; the weight
percentage of chloride is independent of DP. It follows that if
every chloride is converted to an azide then the weight
percentage of azide is also independent of DP. One may expect
side chain modification reactions with bivalent-brush polymers
to display DP-dependent conversion; perhaps increased DP
would lead to lower reaction yields because of increased steric
hindrance. We have found that for both chloride-azide exchange
and CuAAC reactions there is no apparent difference in reaction
yield across the DP values studied here. Figure 3 shows the
FTIR spectra for a subset of nN3 polymers normalized to the
PEG ether (C-O-C antisymmetric stretch) absorbance at
∼1100 cm-1. The intensities of the characteristic azide anti-
symmetric stretch (∼2100 cm-1) are the same for each DP,
which suggests that the azide:PEG ratio is the same for each
sample and that the chloride-azide exchange reaction occurred
with equal efficiency for each DP value.
(94) Bethea, D.; Fullmer, B.; Syed, S.; Seltzer, G.; Tiano, J.; Rischko,
C.; Gillespie, L.; Brown, D.; Gasparro, F. P. J. Dermatol. Sci. 1999,
19, 78–88.
(95) Mal, N. K.; Fujiwara, M.; Tanaka, Y. Nature 2003, 421, 350–353.
(96) Skwarczynski, M.; Noguchi, M.; Hirota, S.; Sohma, Y.; Kimura, T.;
Hayashi, Y.; Kiso, Y. Bioorg. Med. Chem. Lett. 2006, 16, 4492–
4496.
(97) McCoy, C. P.; Rooney, C.; Edwards, C. R.; Jones, D. S.; Gorman,
S. P. J. Am. Chem. Soc. 2007, 129, 9572–9573.
(98) Jiang, M. Y.; Dolphin, D. J. Am. Chem. Soc. 2008, 130, 4236–4237.
(99) Agasti, S. S.; Chompoosor, A.; You, C.-C.; Ghosh, P.; Kim, C. K.;
Rotello, V. M. J. Am. Chem. Soc. 2009, 131, 5728–5729.
(100) Lee, H.-i.; Wu, W.; Oh, J. K.; Mueller, L.; Sherwood, G.; Peteanu,
L.; Kowalewski, T.; Matyjaszewski, K. Angew. Chem., Int. Ed. 2007,
46, 2453–2457.
(101) Pastine, S. J.; Okawa, D.; Zettl, A.; Frechet, J. M. J. J. Am. Chem.
Soc. 2009, 131, 13586–13587.
(102) Kim, H.-C.; Hartner, S.; Behe, M.; Behr Thomas, M.; Hampp Norbert,
A. J. Biomed. Opt. 2006, 11, 34024.
(103) Haertner, S.; Kim, H.-C.; Hampp, N. J. Polym. Sci., Part A: Polym.
Chem. 2007, 45, 2443–2452.
(104) Lewis, W. G.; Magallon, F. G.; Fokin, V. V.; Finn, M. G. J. Am.
Chem. Soc. 2004, 126, 9152–9153.
Click Chemistry Using DOX-NBOC-Alkyne 9. The ultimate
application of these nN3 bivalent-brush polymers is defined by
how they are decorated with functional groups. For drug delivery
applications, one can imagine clicking a drug-alkyne derivative
attached via a cleavable linker group (Figure 1). The resulting
polymer would undergo controlled drug release in response to
(105) Rodionov, V. O.; Fokin, V. V.; Finn, M. G. Angew. Chem., Int. Ed.
2005, 44, 2210–2215.
(106) Rodionov, V. O.; Presolski, S. I.; Diaz, D. D.; Fokin, V. V.; Finn,
M. G. J. Am. Chem. Soc. 2007, 129, 12705–12712.
(107) Rodionov, V. O.; Presolski, S. I.; Gardinier, S.; Lim, Y.-H.; Finn,
M. G. J. Am. Chem. Soc. 2007, 129, 12696–12704.
9
J. AM. CHEM. SOC. VOL. 133, NO. 3, 2011 563

A R T I C L E S
Johnson et al.
Scheme 3. Synthesis of DOX-Nitrobenzyloxycarbonyl (NBOC)-Alkyne Derivative 9 for Click Chemistry and Photorelease of DOX
Scheme 4. CuAAC Coupling of 9 to nN3 Brush Using
Tris(hydroxypropyltriazolylmethyl)amine (THPTA) Ligand,
Copper(II) Sulfate, and Sodium Ascorbate As an in Situ Reductant
groups. After initial attempts without accelerating ligand, which
led to <20% conversion after several days, we were pleased to
find that addition of THPTA to the reaction mixture gave
quantitative conversions. In a typical reaction, the polymer,
THPTA (20 equiv. to azide), and alkyne 9 (1.05 equiv. to azide)
were dissolved in 95% H2O/5% DMSO such that the concentra-
tion of alkyne 9 was 2 mM. Aqueous sodium ascorbate (1.0
M) was added followed by copper(II) sulfate (1.0 M); two more
aliquots of aqueous copper sulfate and sodium ascorbate were
added over 2 days such that their final amounts were 10 and 50
equiv., respectively, relative to azide. The reactions were
performed at 40 °C to achieve the maximum rate possible
without inducing potential azide-olefin cycloaddition side reac-
tions.84
Figure 4A (inset) depicts the CuAAC coupling of 9 to 100N3
as monitored by liquid-chromatography mass-spectrometry (LC-
MS). Compound 9 eluted at ∼4.2 min while DOX-loaded
polymer eluted from ∼3.5 - ∼4 min. The unique absorbance
of DOX (∼500 nm) enables facile monitoring of the CuAAC
reaction; the 100N3 polymer does not absorb at 500 nm and
therefore the growth of a new, broad polymer peak is attributed
to CuAAC coupling of 9 to the polymer. The time-dependent
LC-MS traces (Figure 4A, inset) show an increasing polymer
absorbance at the expense of the alkyne 9 absorbance until a
final, very high (>97%) conversion is reached. As for the case
with chloride-azide exchange, one may expect the CuAAC
reaction progress to depend on DP. Figure 4A shows preparatory
high-performance liquid chromatography (prep-HPLC) 500 nm
absorbance traces for several crude nDOX polymers; the total
ratio of nDOX:9 is the same for each DP which again suggests
that DP has little effect on the efficiency of reactions to these
structures. The retention times for the nDOX polymers increase
slightly for each DP indicating that polymer-column interactions
are dependent on DP. The pure polymer fractions were collected
and either lyophilized to dryness or concentrated before use in
subsequent experiments. Figure 4B shows the FTIR spectrum
of purified 50DOX compared to that of 50N3. The azide
antisymmetric stretch at ∼2100 cm-1, present in the spectrum
of 50N3, is completely absent from the 50DOX spectrum which
suggests very high consumption of the azide group. Integration
bioconjugation where the requisite azide and alkyne functional
groups are present in very low concentration among a number
of other reactive groups.108-111 The Finn laboratory recently
reported optimized CuAAC conditions for bioconjugation which
utilize a new accelerating ligand THPTA (Scheme 4).112
Aqueous CuAAC was found to proceed orders of magnitude
faster with THPTA than in the absence of a ligand. Since these
nN3 bivalent-brush polymers are nanoscopic, narrowly dis-
persed, water-soluble materials with buried functional groups,
we envisioned CuAAC reactions to them as similar to biocon-
jugation reactions to proteins bearing multiple azide or alkyne
(108) Ngo, J. T.; Champion, J. A.; Mahdavi, A.; Tanrikulu, I. C.; Beatty,
K. E.; Connor, R. E.; Yoo, T. H.; Dieterich, D. C.; Schuman, E. M.;
Tirrell, D. A. Nat. Chem. Biol. 2009, 5, 715–717.
(109) Dieterich, D. C.; Link, A. J.; Graumann, J.; Tirrell, D. A.; Schuman,
E. M. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 9482–9487.
(110) Speers, A. E.; Cravatt, B. F. Chem. Biol. 2004, 11, 535–546.
(111) Prescher, J. A.; Bertozzi, C. R. Nat. Chem. Biol. 2005, 1, 13–21.
(112) Hong, V.; Presolski, S. I.; Ma, C.; Finn, M. G. Angew. Chem., Int.
Ed. 2009, 48, 9879–9883.
1
of the broad aromatic resonances in the H NMR spectrum of
50DOX compared to the PEG methylene groups is in close
agreement to that expected for fully DOX-functionalized
polymer (Figure S5). The final DOX weight percentage for the
9
564 J. AM. CHEM. SOC. VOL. 133, NO. 3, 2011

PEG-Branch-Azide Bivalent-Bottle-Brush Polymers by ROMP
A R T I C L E S
Figure 4. A: Prep-HPLC traces of crude CuAAC mixtures which depict similar conversion for each n value. A (inset): Progress of CuAAC coupling of
9 to 100N3 monitored by LC-MS. B: FTIR spectra of 50N3 and 50DOX confirming complete loss of azide after CuAAC.
Figure 5. A: Time-dependent UV photolysis of 100DOX monitored by LC-MS; after 10 min irradiation ∼70% of free DOX was released. A minor
product (labeled “*”, see Scheme 5) was also observed at longer irradiation times. B: Viability of MCF-7 human breast cancer cells treated with free DOX,
100N3 bivalent-brush polymer, and drug-loaded 100DOX polymer both with and without UV irradiation. Data points were fit to a sigmoidal function and
the half-maximum inhibitory concentrations (IC50) are shown for DOX and the photocleaved 100DOX. The x-axis labels refer to the concentration of both
free and polymer-conjugated drug.
functionalized polymers is 12.6%. As mentioned above, this
value is independent of DP; it could be adjusted by varying the
PEG length or the degree of branching in the MM precursor.
Photorelease of DOX and Cell Culture Studies. Having
successfully coupled 9 to nN3 we next studied the release of
free DOX from the brush polymer in response to UV light
(∼365 nm). An aqueous solution of purified nDOX polymer
was irradiated at 365 nm for a given time and subjected to
LC-MS analysis. The progress of photorelease was monitored
over 10 min by repeated irradiation and LC-MS; the data for
100DOX are shown in Figure 5A. As irradiation time increased,
we observed a new peak at ∼3 min that increased in intensity
at the expense of the 100DOX polymer peak. The new peak
corresponds to the retention time and mass of free DOX. We
also observed a small side product at ∼3.8 min with a mass of
569 Da (labeled “*” in Figure 5A), which is likely the result of
intramolecular trapping of carbamic acid 10 to give cyclic
carbamate 11 (Scheme 5). Nevertheless, the major product of
UV photolysis was free DOX; the photolysis yield after 10 min
irradiation was ∼70% for all nDOX polymers which led us to
examine the effectiveness of these materials against human
cancer cells in cell culture.
We performed cell viability experiments using MCF-7 human
breast cancer cells. Cells were treated with aqueous solutions
of either free DOX or the corresponding nDOX polymer at
various concentrations and irradiated for 10 min using 365 nm
light or kept in the dark. The cells were then incubated in the
dark for 24 h, washed twice, and incubated for another 24 h in
fresh, drug-free growth medium. Cell viability was assessed
using the MTT assay (see Methods and Materials for details).
Data for n ) 100 polymers (100N3 and 100DOX) are shown
in Figure 5B (cell viability data for other n values is given in
the Supporting Information). Free DOX, with and without UV
irradiation, effectively killed the cells at 3-5 µM while azide
polymer 100N3 was nontoxic with and without light up to 100
µM; these data taken together suggest that under our conditions
10 min of 365 nm UV irradiation has no adverse affect on cell
viability nor does it interrupt the cellular toxicity of DOX. None
of the nDOX bivalent-brush polymers studied were toxic up to
50 µM in the absence of UV irradiation; irradiation for 10 min
yielded IC50 values ∼7-10 µM which confirms that free DOX
generated by photorelease is therapeutically active. The IC50
values were not DP-dependent. This observation suggests that
the nDOX polymers remain in the extracellular environment
9
J. AM. CHEM. SOC. VOL. 133, NO. 3, 2011 565

A R T I C L E S
Johnson et al.
Scheme 5. Photolysis of nDOX Polymers Gives Carbamic Acid 10a
a Decarboxylation of 10 yields free DOX as the major product while intramolecular trapping provides DOX-carbamate derivative 11 as a minor product.
Reactive nitrosobenzaldehyde photoproducts remain bound to the polymer core after photolysis.
and upon photolysis free DOX is released and diffuses into the
cell nucleus where it induces apopotosis. Although in vitro
experiments do not show DP-dependent cytotoxicity, we expect
that DP will affect in vivo trafficking and thus the overall utility
of these materials in drug delivery. Targeting groups could be
appended to the periphery of these materials to promote
trafficking into the cell.113
pH-, and redox-cleavable), attachment of targeting moieties
at the PEG periphery, incorporation of degradable units within
the polymer backbone, and use of chain-transfer agents to
append orthogonal groups selectively to the ends of these
polymers are being studied. Furthermore, copolymerization
of MMs like 6 with other monomers (such as a norbornene-
alkyl halide small molecule) will provide access to new
multiblock polymer structures with the potential for higher
drug loadings.
Conclusions
Here we have demonstrated the utility of a combined graft-
through/click-to strategy for the synthesis of a new class of
clickable, branched nanostructures: PEG-branch-azide biva-
lent-brush polymers. This approach makes use of the remark-
able efficiency of ROMP for graft-through polymerization
and the modular coupling power of click chemistry for
polymer functionalization. A wide range of nanoscale sizes
is accessible which will enable detailed structure/function
correlation in biological settings. We are currently exploring
applications for these materials in drug delivery. Design and
synthesis of novel clickable linkers (e.g., longer wavelength-,
Acknowledgment. We thank Dr. S. Virgil and Mr. S. Presolski
for helpful advice. This work was supported by the National
Institutes of Health (NIH, R01-GM31332), the Beckman Institute
at Caltech (postdoctoral fellowship for J.A.J.), and the MRSEC
program of the National Science Foundation (NSF) under Award
No. DMR-0520565.
Supporting Information Available: Synthetic procedures,
spectral data, liquid chromatography methods, and details of
cell viability studies. This material is available free of charge
via the Internet at http://pubs.acs.org.
(113) Kolonko, E. M.; Kiessling, L. L. J. Am. Chem. Soc. 2008, 130, 5626–
5627.
JA108441D
9
566 J. AM. CHEM. SOC. VOL. 133, NO. 3, 2011