Synthesis of fluorogenic polymers for visualizing cellular internalization

Article abstract of DOI:10.1021/ol800932w

(Chemical Equation Presented) The binding of a polymeric ligand to a cell surface receptor can promote its internalization. Methods to track and visualize multivalent ligands within a cell can give rise to new therapeutic strategies and illuminate signaling processes. We have used the features of the ring-opening metathesis polymerization (ROMP) to develop a general strategy for synthesizing multivalent ligands equipped with a latent fluorophore. The utility of ligands of this type is highlighted by visualizing multivalent antigen internalization in live B cells.

Full text of DOI:10.1021/ol800932w

ORGANIC
LETTERS
2008
Vol. 10, No. 14
2997-3000
Synthesis of Fluorogenic Polymers for
Visualizing Cellular Internalization
Shane L. Mangold,† Rachael T. Carpenter,‡ and Laura L. Kiessling*,†,‡
Departments of Chemistry and Biochemistry, UniVersity of WisconsinsMadison,
Madison, Wisconsin 53706
kiessling@chem.wisc.edu
Received April 25, 2008
ABSTRACT
The binding of a polymeric ligand to a cell surface receptor can promote its internalization. Methods to track and visualize multivalent ligands
within a cell can give rise to new therapeutic strategies and illuminate signaling processes. We have used the features of the ring-opening
metathesis polymerization (ROMP) to develop a general strategy for synthesizing multivalent ligands equipped with a latent fluorophore. The
utility of ligands of this type is highlighted by visualizing multivalent antigen internalization in live B cells.
Advances in organic and polymer chemistry are providing
access to new classes of bioactive polymers.1 With the ability
to generate synthetic macromolecules that vary in structure
and physical properties, the applications of polymers have
expanded rapidly. Some uses include as substrates for cell
growth and differentiation,2 vehicles for drug delivery,3
therapeutics,4 and molecules for studying cell signaling.5 For
some biological applications, bioactive polymers must be
taken up into cells. Still, little is known about the cellular
internalization and trafficking of polymers. Here, we present
an approach for following these processes.
The bioactive polymers that we focused upon were those
generated by ruthenium carbene-initiated ring-opening me-
tathesis polymerization (ROMP). It was shown that ROMP
could be used to generate biologically active polymers over
10 years ago.6 Since that time, ROMP has been used produce
materials with a variety of biological activities.7 Continuous
advances in catalyst development and polymerization tech-
† Department of Chemistry.
(5) (a) Kiessling, L. L.; Gestwicki, J. E.; Strong, L. E. Angew. Chem.,
Int. Ed. 2006, 45, 2342–2368. (b) Borrok, M. J.; Kolonko, E. M. ACS Chem.
Biol. 2008, 3, 101–109. (c) Mammen, M.; Choi, S-K.; Whitesides, G. M.
Angew. Chem., Int. Ed. 1998, 37, 2754–2794.
‡ Department of Biochemistry.
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Bryant, S. J. Annu. ReV. Biomed. Eng. 2004, 6, 41–75. (d) Drury, J. L.;
Mooney, D. J. Biomaterials 2003, 24, 4337–4351.
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1994, 116, 12053–12054.
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10.1021/ol800932w CCC: $40.75
Published on Web 06/19/2008
2008 American Chemical Society

niques have provided methods to control key features of
polymer structure, including their length and the functional
groups they present.8 Ruthenium carbene initiators tolerate
a diverse array of functional groups; therefore, they can be
used synthesize highly functionalized polymers with tailored
biological activities.
The presence of the phenolic acetate group within the
trimethyl lock dictates that the chemical reactions used to
append the latent fluorophore to the polymer occur under
mild conditions. We envisioned that the Cu(I)-catalyzed
azide-alkyne cycloaddition (CuAAC), discovered indepen-
dently by the Sharpless and Meldal groups,12 might offer
an attractive solution. The reactive partners (azide and copper
acetylide) are stable under many conditions yet react with
each other with excellent chemoselectivity. Moreover, this
reaction process has been used to modify polymers13 and to
attach fluorophores to species that have undergone affinity
labeling.14 To synthesize the target compounds, the best
disconnection appeared to be an azide-substituted polymer
and an alkyne-bearing fluorogenic label, such that the azide,
not the alkyne, was appended to the polymer backbone.15
This dissection should avoid unwanted metathesis side
reactions of the alkyne.16
Our strategy for generating the azide-substituted polymer
relies on employing a selective end-labeling strategy.17 When
ruthenium carbenes are used as initiators in ROMP, the
polymer terminus can be modified selectively by terminating
the polymerization reaction with a functionalized enol
ether.17a,b,18 An enol ether capping agent with the requisite
azide could be readily synthesized from dihydropyran
(Scheme 1). Exposure to aqueous acidic media generated
ROMP initially was used to generate polymers that act
on the outside of the cell, but more recently, it has been
employed to assemble compounds that can be internalized
by cells. Our interest in polymer uptake was prompted by
our studies of B cell signaling.9 For example, polymers
generated by ROMP that display antigenic epitopes can
promote antibody production in vivo. The ability of these
polymers to activate this process depends upon their interac-
tions with the antigen-specific B cell receptor (BCR), a
membrane-bound immunoglobulin on the surface of B cells.
These polymers bind to the specific B cell receptor to activate
signaling, but they also promote its internalization. We
therefore anticipated that, like other antigens, these polymers
would be taken up by endocytosis. To investigate polymer
internalization, we envisioned using antigenic polymers
equipped with a group that would report directly on inter-
nalization.
Most approaches to following ligand internalization rely
on appending a fluorescent dye to the molecule of interest.
Uptake is detected using a discontinuous assay (typically
fluorescence microscopy). Because extensive cell washing
steps are required to remove background fluorescence,
internalization cannot be tracked in real time. The need for
multiple washing steps is exacerbated when the object of
the study is a macromolecule, such as a polymer. Macro-
molecules are more likely to engage in nonspecific interac-
tions with cells, thereby increasing the assay background.
We reasoned that polymers bearing a label, whose fluores-
cence is unmasked only upon endocytosis, could illuminate
the processes of internalization and trafficking.
Scheme 1. Route to Azide-Containing Capping Agent
the incipient aldehyde, which was subjected to Wittig
reaction with (methoxymethyl)triphenylphosphonium chlo-
ride to afford enol ether 1 (3:1 mixture of E/Z isomers). When
this species was treated with diphenylphosphoryl azide
The latent fluorophore developed by Raines and co-
workers can provide a sensitive measure of biomolecular
entry into cells.10 The reporter dye possesses a trimethyl
lock11 that masks fluorescence until it is released by cellular
esterases (Figure 1). While a strategy to link a profluorophore
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Figure 1. Upon cellular internalization of the labeled compound,
esterases liberate the fluorophore.
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of this type to proteins has been developed,10c the process
used relies on a thiol and is not readily applicable to
functionalizing compounds such as the polymers generated
by ROMP. Thus, we sought to develop an alternative general
strategy to attach a latent fluorophore.
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Org. Lett., Vol. 10, No. 14, 2008

(DPPA)19 under Mitsunobu-like conditions,20 the azide-
terminated capping agent 2 was obtained.
with an alkyne handle. For this purpose, we used a
rhodamine derivative bearing a urea linker.10c Previous
studies had shown that a urea substituent has only a minor
effect on the fluorescence intensity of the dye. The
modified dye was generated by desymmetrization to afford
Boc-protected rhodamine 5. The alkyne was introduced
using an isocyanate generated in situ from the Curtius
rearrangement of 6-heptynoic acid. The electrophilic
intermediate was captured with amine 5 to afford the
alkyne-terminated fluorophore 6 (Scheme 3). The Boc
protecting group was cleaved to yield a fluorescent
The new capping agent was used to append a single azide
group to a polymeric antigen that can promote B cell receptor
internalization. We had previously used ROMP to synthesize
multivalent ligands that bear antigenic dinitrophenyl (DNP)
epitopes, which bind to a cell line expressing a DNP-specific
B cell receptor.20 Thus, we modified the pre-existing
synthetic route to generate DNP-substituted polymers bearing
a terminal azide group. A norbornene derivative that bears
a succinimidyl ester was polymerized using a ruthenium
initiator21 to generate polymers of defined lengths (Scheme
2). The degree of polymerization was controlled by the ratio
Scheme 3. Synthesis of Alkyne-Terminated Latent Fluorophore
Scheme 2. Synthesis of DNP-Substituted Polymers by ROMP
rhodamine derivative 7, which was condensed with
trimethyl lock derivative 710c to generate the pro-fluoro-
phore 8.
To attach the latent fluorophore to the polymer, we
examined several conditions. The combination of CuI and
CuBr in water-DMSO solvent afforded the desired triazole
product but in low yield. To generate Cu(I) from Cu(II),
sodium ascorbate can be used as a reducing agent.22 The
Cu(I) oxidation state can be stabilized by the addition of
ligands such as (benzyltriazolylmethyl)amine (TBTA), and
this additive has been used to promote many bioconjugation
reactions.23 We found that this reagent combination also
facilitates attachment of the fluorogenic label to the polymer.
Specifically, the conjugation of 4 and 8 was achieved in the
presence of CuSO4, sodium ascorbate, and TBTA (Scheme
4). Importantly, no unmasking of the latent fluorophore was
observed under these reaction conditions.
of the ruthenium initiator to monomer (1:10 or 1:500). The
reaction was terminated using an excess of the azide-
containing enol ether 2. To convert the polymer into an
antigen that can bind to the DNP-specific B cell receptor,
we conjugated DNP-substituted lysine to the backbone.
Previous reports from our laboratory indicated that substitu-
tion with 0.4 equiv of DNP-lysine (relative to the succin-
imidyl ester groups) afforded polymers that could activate
B cell signaling; therefore, we used reaction conditions to
generate polymers with this level of backbone substitution.
Glucamine was added to quench any remaining succinimidyl
esters and to promote water solubility of the final product
polymers.
With access to the labeled polymers, we tested whether a
model esterase (pig liver esterase) could liberate the fluo-
rophore. When polymer 9a was exposed to esterase (5 µM),
the fluorophore was liberated with a kcat/KM value of 5.8 ×
106 M-1 s-1 (see the Supporting Information). These kinetic
For conjugation of the latent fluorophore to the azide-
terminated polymers 4a and 4b, we needed a masked dye
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was apparent, indicating that the fluorogenic label serves as
a sensitive reporter of polymer uptake (Figure 2C).
The observed polymeric antigen internalization is consis-
tent with B cell receptor-mediated uptake. We had shown
previously that DNP-substituted polymers promote B cell
receptor internalization.9 The labeled polymers reveal that
the B cell receptor and polymer 9b (M:I ) 500) colocalize
(Figure 2E). In contrast, polymers lacking DNP groups were
not taken up (see the Supporting Information). Together, the
results suggest that receptor and polymer are internalized
together. Most significantly, the fluorescence arising from
unmasking of the fluorogenic label is present only in the
interior of the cells. This result underscores the utility of
the latent fluorophore for monitoring multivalent ligand
uptake.
In summary, we have developed a strategy for synthesizing
multivalent ligands equipped with a fluorogenic label. Our
approach relies upon the attachment of a profluorophore to
a polymer using the Cu(I)-catalyzed azide-alkyne cycload-
dition. This approach is general; it can facilitate the conjuga-
tion of fluorophores or other prodrugs to a variety of azide-
containing species. We have demonstrated its utility for
generating reagents to track and visualize B cell receptor-
mediated antigen uptake. This new approach provides a
means to visualize the uptake and position of multivalent
ligands within the cell. We anticipate that our method of
fluorogenic label installation will facilitate studies of cellular
internalization and trafficking of both multivalent and
monovalent compounds.
Scheme 4
.
Profluorophore Attachment via a Cu(I)-Catalyzed
Azide-Alkyne Cycloaddition
parameters suggest that the fluorogenic label can be used to
monitor uptake processes that occur on the minute time scale.
We next employed fluorescence microscopy to test whether
polymer uptake could be detected in live cells. To label the
DNP-specific B cell receptor without engaging multiple
receptors, we used a fluorescent monovalent Cy3-conjugated
anti-IgM fragment. Images collected immediately after
polymer addition reveal diffuse staining of the B cell
Acknowledgment. This research was supported by the
NIH (R01 AI055258). The UWsMadison Chemistry NMR
facility is supported by the NSF (CHE-0342998 and CHE-
9629688) and the NIH (1-S10-RR13866). R.T.C. was sup-
ported by the Chemistry-Biology Interface Training Pro-
gram (T32 GM008505) The authors thank E. M. Kolonko,
L. D. Lavis, and R. T. Raines for helpful discussions. TBTA
was a generous gift of K. B. Sharpless. The A20.2J/HLTNP
cells were a generous gift of A. Ochi.
Figure 2. Images of polymer-treated live A20.2J/HLTNP cells. Cells
were treated for 30 min with Cy3-conjugated anti-IgM to stain the
DNP-specific B cell receptor. Polymer 9b was added at 37 °C, and
images were acquired without washing immediately (A, B) and
after 15 min (C, D). Panel E depicts data from the emission of
both the Cy3 and rhodamine probes. The scale bar represents 39
µm in panels A and B and 10 µm in C-F.
Supporting Information Available: Experimental pro-
cedures and characterization data for compounds 1-4, 6, 8,
and 9. This material is available free of charge via the Internet
at http://pubs.acs.org.
receptor, indicating that it is distributed across the cell surface
(Figure 2B). No signal from the polymer was detected
(Figure 2A). After 15 min, however, rhodamine fluorescence
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Org. Lett., Vol. 10, No. 14, 2008