A facile synthetic method to prepare fluorescently labeled ROMP polymers

Article abstract of DOI:10.1021/ol048935y

(Chemical Equation Presented) To probe the activities of sperm ADAM protein (fertilinβ), we devised a general synthetic strategy to generate fluorescently labeled fertilinβ oligopeptide polymers. Immunofluorescence studies with these polymers demonstrated that fertilinβ polymers bind specifically to a protein receptor on the mouse egg plasma membrane.

Full text of DOI:10.1021/ol048935y

ORGANIC
LETTERS
2004
Vol. 6, No. 19
3253-3255
A Facile Synthetic Method to Prepare
Fluorescently Labeled ROMP Polymers
Kenny S. Roberts and Nicole S. Sampson*
Department of Chemistry, State UniVersity of New York,
Stony Brook, New York 11794-3400
nicole.sampson@stonybrook.edu
Received June 7, 2004
ABSTRACT
To probe the activities of sperm ADAM protein (fertilinâ), we devised a general synthetic strategy to generate fluorescently labeled fertilinâ
oligopeptide polymers. Immunofluorescence studies with these polymers demonstrated that fertilinâ polymers bind specifically to a protein
receptor on the mouse egg plasma membrane.
The use of fluorescence microscopy is an extremely powerful
tool for studying cellular localization of biomolecules in cell
biology. The development of a wide array of dyes has pro-
vided detailed views of cellular exteriors and interiors and
allowed monitoring of biological activities. A wide variety
of processes involving protein-protein interactions, signal
transduction, or downstream intracellular responses can be
investigated selectively in vitro and in vivo using fluores-
cence techniques.1 The application of fluorescent probes in
studying membrane proteins and subcellular compartments
is rapidly expanding as a result. The types of probes available
cover a wide range that includes noncovalent reagents, e.g.,
fluorescent antibodies, and covalent reagents, e.g., GFP
fusion proteins or fluorophore-tagged small molecules.
We previously reported that norbornyl oligopeptide poly-
mers such as 1a are 50- to 70-fold improved inhibitors of in
vitro fertilization in mouse compared to their monomeric
counterpart.2 The IC50 for inhibition of in vitro fertilization
by 1a is 5.8 ( 0.3 µM. The sequence of the oligopeptides
was derived from the binding loop of fertilinâ. Fertilinâ is
a sperm transmembrane protein implicated in sperm-egg
binding.3,4 Its putative receptor is R6â1 integrin,5,6 although
genetic experiments suggest that its biological activity results
from binding another unidentified cell surface protein.7,8 We
required a fluorescently labeled polymer derivative to deter-
mine the cellular localization of the inhibitor. Here we report
a facile and versatile approach to synthesizing end-labeled
block copolymers that will be useful in many polymer
systems.
Our norbornyl monomers are polymerized using fully
protected peptides, e.g., 3 (Scheme 1). After polymerization,
side-chain protection is removed using trifluoroacetic acid.
We were concerned that commonly used fluorophores would
not survive the acidic deprotection conditions and chose a
(3) Primakoff, P.; Myles, D. G. Trends Genet. 2000, 16, 83-87.
(4) Cho, C.; Bunch, D. O. D.; Faure, J.-E.; Goulding, E. H.; Eddy, E.
M.; Primakoff, P.; Myles, D. G. Science 1998, 281, 1857-1859.
(5) Almeida, E. A. C.; Huovila, A. P. J.; Sutherland, A. E.; Stephens, L.
E.; Calarco, P. G.; Shaw, L. M.; Mercurio, A. M.; Sonnenberg, A.;
Primakoff, P.; Myles, D. G.; White, J. M. Cell 1995, 81, 1095-1104.
(6) Chen, H.; Sampson, N. S. Chem. Biol. 1999, 6, 1-10.
(7) He, Z.-Y.; Brakebusch, C.; Fa¨ssler, R.; Kreidberg, J. A.; Primakoff,
P.; Myles, D. G. DeV. Biol. 2003, 254, 226-237.
(1) Weijer, C. J. Science 2003, 300, 96-100.
(2) Roberts, S. K.; Konkar, S.; Sampson, N. S. ChemBioChem 2003, 4,
1229-1231.
(8) Miller, B. J.; Georges-Labouesse, E.; Primakoff, P.; Myles, D. G. J.
Cell Biol. 2000, 149, 1289-96.
10.1021/ol048935y CCC: $27.50 © 2004 American Chemical Society
Published on Web 08/21/2004

Scheme 1. Synthesis of Labeled Fertilinâ Oligopeptide-Substituted Polymer
strategy to introduce the fluorophore after deprotection of
the polymer. We incorporated N-hydroxysuccinimide nor-
bornyl carboxylate monomer 2 into the oligopeptide polymer
in a sequential fashion. The polymerization is initiated with
a 1:1 ratio of 2 and catalyst. After ring-opening metathesis
is complete, 10 equiv of 3 is added to synthesize the
oligopolymer block. The average DP determined by 1H NMR
was consistent with the [M]0/[C]0 of 11/1 in the reaction
mixture. The N-hydroxysuccinimide ester withstands treat-
ment with trifluoroacetic acid. After deprotection, treatment
with the appropriate nucleophilic fluorophore and base yields
the end-labeled fluorescent polymers. The fluorophore was
incorporated into the polymer as determined by gel filtration
chromatography with detection at 220 nm (peptide backbone)
and 495 nm (fluorophore). Moreover, the polymers show
the expected fluorescence excitation and emission charac-
teristics (Figure 1).
This method is efficient because a single preparation of
polymer may be conjugated with a range of fluorophores to
tune the fluorescence properties of the polymer for individual
applications. Alternative strategies have been reported for
end-labeling polymers synthesized by ROMP, for example,
terminating the polymerization with an ethylene glycol based
trimethylsilyl ester, hydrolysis of the ester, electrophilic
activation of the unmasked carboxylate, and conjugation to
a nucleophilic fluorophore.9 Like ours, this method also
allows substitution of a single preparation of polymer with
a variety of fluorophores or other molecular tags. However,
it was not suitable for use with carboxylic oligopeptide
norbornyl polymers such as 5. Electrophilic activation of the
terminal carboxylic acid in the presence of many peptide
side chain carboxylates is not synthetically feasible. In
Figure 1. (Top) Representative emission spectrum of 5a (0.5 µM)
with excitation at 495 nm. (Bottom) Representative excitation
spectrum of 5a (0.5 µM) with emission at 520 nm.
addition, our synthetic route is convergent; the monomer is
activated before polymerization and no synthetic elaboration
of the polymer is required before conjugation with the desired
fluorophore. This reduces the number of synthetic manipula-
(9) Owen, R. M.; Gestwicki, J. E.; Young, T.; Kiessling, L. L. Org. Lett.
2002, 4, 2293-2296.
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Org. Lett., Vol. 6, No. 19, 2004

tions of polymer required for preparation. Our method has
the additional advantage of amplification of signal. The
number of fluorophores or other reporter groups such as
affinity handles incorporated into a single polymer may be
increased by increasing the ratio of N-hydroxysuccinimide
monomer 2 to catalyst.
We chose to conjugate Oregon green 488 cadaverine to
our polymer because it is pH-insensitive in the physiological
pH range and contains a primary amine linker. We prepared
a fluorescent polymer with fertilinâ oligopeptide 5a and a
control with a scrambled sequence 5b. The fluorescent
polymers 5a and 5b were purified by precipitation with 1 N
HCl and extensive washing. We stained live zona-free
oocytes with 5a and 5b to determine whether the polymer
5a specifically interacts with the cell surface; 10% goat serum
was used to block non specific binding. Following incuba-
tion, oocytes were washed in 3 mL of M16 buffer and fixed
using 3.7% paraformaldehyde. The oocytes were washed and
mounted with Vecta Shield.
Figure 2. DIC (A, C, and E) and epi-fluorescence images (B, D,
and F) of zona-free oocytes stained with 5a (A, B), zona-free
oocytes with 5b (C, D), and no polymer (E, F) with a 40×/0.75
NA objective. Eggs were incubated with 50 µm (in peptide) 5a or
5b for 35 min, washed, fixed, and mounted. Exposure times for D
and F were 4 times that of B.
We imaged the stained oocytes using epi-fluorescence and
DIC microscopy (Figure 2). Our staining experiments
demonstrated that binding of the polymers to the cell surface
is dependent on the oligopeptide sequence. Polymer 5a
bearing the fertilinâ binding sequence stains the egg plasma
membrane (Figure 2A and B), and 5b bearing the scrambled
sequence does not even if the image is overexposed (Figure
2C and D). Control eggs with no polymer show that all of
the fluorescence observed with 5b is due to autofluorescence
of the egg (Figure 2E and F). Moreover, 5a did not stain the
oocyte if the cell surface proteins were fixed with paraform-
aldehyde before staining. This experiment implies that native
receptor is required for oligopeptide polymer binding. These
studies demonstrate that inhibition of in vitro fertilization
by polymer 1a is due to specific binding at the egg plasma
membrane surface. Future experiments will employ 5a as a
tool to probe polymer-receptor interactions on the egg
surface.
Acknowledgment. Financial support from NIH (HD38519,
N.S.S.) and the NSF (CHE0131146 and CHE9709164, NMR
spectrometers and fluorimeter) is gratefully acknowledged.
Supporting Information Available: Detailed descrip-
tions of experimental procedures and spectra. This material
is available free of charge via the Internet at http://pubs.acs.org.
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