Evaluation of a cell penetrating prenylated peptide lacking an intrinsic fluorophore via in situ click reaction

Article abstract of DOI:10.1016/j.bmcl.2011.04.138

Protein prenylation involves the addition of either a farnesyl (C ₁₅) or geranylgeranyl (C₂₀) isoprenoid moiety onto the C-terminus of many proteins. This natural modification serves to direct a protein to the plasma membrane of the

Full text of DOI:10.1016/j.bmcl.2011.04.138

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4998–5001
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Bioorganic & Medicinal Chemistry Letters
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Evaluation of a cell penetrating prenylated peptide lacking an intrinsic
fluorophore via in situ click reaction
Joshua D. Ochocki a, Daniel G. Mullen a, Elizabeth V. Wattenberg b, Mark D. Distefano a,
⇑
a Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA
b Division of Environmental Health Sciences, University of Minnesota, Minneapolis, MN 55455, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 6 May 2011
Protein prenylation involves the addition of either a farnesyl (C15) or geranylgeranyl (C20) isoprenoid moi-
ety onto the C-terminus of many proteins. This natural modification serves to direct a protein to the
plasma membrane of the cell. A recently discovered application of prenylated peptides is that they have
inherent cell-penetrating ability, and are hence termed cell penetrating prenylated peptides. These pep-
tides are able to efficiently cross the cell membrane in an ATP independent, non-endocytotic manner and
it was found that the sequence of the peptide does not affect uptake, so long as the geranylgeranyl group
is still present [Wollack, J. W.; Zeliadt, N. A.; Mullen, D. G.; Amundson, G.; Geier, S.; Falkum, S.; Watten-
berg, E. V.; Barany, G.; Distefano, M. D. Multifunctional Prenylated Peptides for Live Cell Analysis. J. Am.
Chem. Soc. 2009, 131, 7293–7303]. The present study investigates the effect of removing the fluorophore
from the peptides and investigating the uptake by confocal microscopy and flow cytometry. Our results
show that the fluorophore is not necessary for uptake of these peptides. This information is significant
because it indicates that the prenyl group is the major determinant in allowing these peptides to enter
cells; the hydrophobic fluorophore has little effect. Moreover, these studies demonstrate the utility of
the Cu-catalyzed click reaction for monitoring the entry of nonfluorescent peptides into cells.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Peptide
Lipid modification
Post-translational modification
Prenylation
Farnesyl
Cell-penetrating peptide
One particularly prevalent post-translational modification is
known as protein prenylation, which occurs on approximately 2%
of all mammalian proteins.1 This modification involves the addi-
tion of a C15 (farnesyl) or C20 (geranylgeranyl) isoprenoid moiety
onto a cysteine residue near the C-terminus of proteins that bear
a ‘CAAX’ box motif, catalyzed by either the farnesyltransferase or
geranylgeranyltransferase enzyme.2–4 In the ‘CAAX’ box, A repre-
sents any aliphatic amino acid and X represents the residue that
controls farnesylation or geranylgeranylation.5,6 The prenylation
modification serves to direct membrane association of many pro-
teins including members of the Ras superfamily of proteins.7 It
has been previously shown that the K-Ras protein is involved in
approximately 30% of all human cancers and inhibition of the farn-
esylation of Ras abolishes its oncogenic activity.8–10 Hence, consid-
erable work has been aimed at developing farnesyltransferase
inhibitors, many of which are in clinical trials.11–13
up into cells by an energy-dependent endocytotic process, which
leads to a majority of the cargo being transported to endosomes
and eventually lysosomes for degradation.18 Designing CPPs that
are able to escape lysosomal degradation has become a high
priority.
Our group has previously developed a series of peptides that are
based on the C-terminus of the naturally geranylgeranylated pro-
tein CDC42. These peptides were functionalized with a fluorophore
and a geranylgeranyl group and found to have intrinsic cell-pene-
trating ability, by a mechanism that was energy independent.19
This study also demonstrated that without the geranylgeranyl
group, the peptides were unable to enter cells; related experiments
demonstrated that farnesylated peptides derived from CDC42 can
also enter a wide variety of cell types.20 A further study examining
the role of the positively charged amino acids in the sequence
found that these amino acids were not necessary for uptake, and
that a peptide as short as two amino acids was able to penetrate
cells, provided the geranylgeranyl group was present.21 Our cur-
rent study serves to elucidate the role of the 5-carboxyfluorescein
(5-Fam) fluorophore on the uptake of these peptides. The benefit of
this class of cell penetrating prenylated peptides is that they enter
cells by a non-endocytotic, ATP independent mechanism that al-
lows accumulation into the cytosol of cells.19,21 These peptides
may prove useful in cell-penetrating applications in which the car-
go can be easily released in the cytosol.
Several important molecules for therapeutic consideration are
not able to naturally traverse the cell membrane and are thus being
attached to cell penetrating peptides (CPPs) to facilitate membrane
crossing.14 The majority of the current CPPs range from 5 to 25
amino acids and often contain several charged amino acid resi-
dues.15–17 The drawback of most current CPPs is that they are taken
⇑
Corresponding author.
E-mail address: diste001@umn.edu (M.D. Distefano).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.138

J. D. Ochocki et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4998–5001
4999
For this study, the peptides (Fig. 1) were synthesized using stan-
dard Fmoc coupling conditions on an automated synthesizer with
Rink-amide resin to afford the C-terminal amide peptides. While
served. The Mander’s overlap coefficient of the cells is 0.901 when
the nucleus is included in the analysis and 0.960 when it is ex-
cluded, indicating there is significant overlap of green and red fluo-
rescence, even when the nucleus is considered (a Mander’s
coefficient of 1 indicates perfect overlap23). It is important to note
that it appears there is some nuclear labeling when HeLa cells are
incubated with 1. To explore this issue, we incubated peptide 1
with HeLa cells, followed by a subsequent click reaction to either
TAMRA-N3 or 5-Fam-PEG-N3. This will help establish if the nuclear
localization is an artifact of the fixation or of the fluorophore used.
Regardless of the fluorophore used, nuclear staining is observed
when incubating HeLa cells with 1 (Fig. 3). This effect is absent
when performing the click reaction on cells that were not treated
with alkyne peptide (see Fig. 2A), and is also absent in cells treated
with 2 (see Fig. 2B). This suggests that peptide 1 that lacks the fluo-
rophore partitions differently within cells and is able to enter the
nucleus whereas peptide 2 does not; however, it should be noted
that the amount of nuclear localization is small compared to the
amount of peptide distributed throughout the remainder of the
cell. Overall, the localization patterns of 1 and 2 are quite similar.
Having visually established that peptide 1 can enter HeLa cells,
we next sought to quantify the differences in uptake between 1
and 2. Accordingly, a reagent containing 5-Fam linked to an azide
moiety was synthesized for this purpose. This reagent was de-
signed so that the fluorescence of the two peptides could be more
directly compared since both peptides would be labeled by the
same fluorophore. A succinimidyl ester of 5-Fam was reacted with
11-azido-3,6,9-trioxaundecan-1-amine in the presence of diisopro-
pylethylamine overnight at room temperature. Following purifica-
tion by reverse phase HPLC, 5-Fam-PEG-N3 was isolated and used
as the subsequent azide for click chemistry. HeLa cells were incu-
bated with 1 or 2 at various concentrations for 1 h. Both sets of
cells were rinsed, fixed and permeabilized. Cells treated with pep-
tide 1 were subjected to the click reaction with 5-Fam-PEG-N3 for
1 h followed by several rinses, and subsequent flow cytometry
analysis. Cells treated with peptide 2 were analyzed directly by
flow cytometry without click reaction. Based on those experi-
ments, peptide 1 appears to enter the cells to a lesser extent (2-
still on resin, the N-terminal lysine side chain (e amine) was acet-
ylated, followed by acylation with an alkyne-containing acid (4-
pentynoic acid) on the amino group to give the resulting N-termi-
nal alkyne. Geranylgeranylation of that peptide was performed in
solution using acidic Zn(OAc)2 coupling conditions22 to afford pep-
tide 1. Peptide 2 was prepared in an analogous manner except that
the e-amino group of the N-terminal lysine was acylated with 5-
Fam. Thus peptide 1 contains an alkyne but no fluorophore
whereas peptide 2 contains both an alkyne and a fluorophore.
The ability of both of these peptides to enter cells was first
established using confocal laser scanning microscopy (CLSM). Be-
cause peptide 2 contains the 5-Fam fluorophore, visualization after
uptake in HeLa cells is easily accomplished after a 2 h incubation of
the peptide at 1 lM (Fig. 2B). To investigate whether the 5-Fam
fluorophore is necessary for uptake, peptide 1 was synthesized
lacking the fluorophore. Following cellular incubation of 1 at
1 lM for 2 h, HeLa cells were rinsed three times with PBS and fixed
with 4% paraformaldehyde, followed by permeabilization with
0.1% Triton X-100. A copper catalyzed click reaction was then con-
ducted using tetramethylrhodamine azide (TAMRA-N3) to form a
covalent triazole linkage between the peptide-alkyne and the
tetramethylrhodamine (TAMRA) fluorophore; when 1 enters cells,
it can be visualized through the TAMRA fluorophore using CLSM
(Fig. 2C). Thus, it can be seen that the peptide lacking a fluorophore
is still able to efficiently cross the membrane of HeLa cells. While
control experiments in which HeLa cells were treated with TAM-
RA-N3 (without prior peptide treatment) manifested no back-
ground labeling (Fig. 2A), we wanted to compare the localization
pattern obtained by direct visualization of 5-Fam with that ob-
served from the TAMRA-N3 labeling. Incubation of 2 with HeLa
cells and subsequent click reaction to TAMRA-N3 resulted in strong
co-localized fluorescence in the cells (yellow color, Fig. 2D); this
observation also confirms that little background reaction occurs
in the TAMRA-N3 labeling process since the red TAMRA fluoro-
phore is only present where the green 5-Fam of the peptide is ob-
Figure 1. The sequences of the peptides used in this study.

5000
J. D. Ochocki et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4998–5001
Figure 2. HeLa cells incubated with peptides 1 or 2 at 1 lM for 2 h. Panel (A) Click reaction performed on cells that were not treated with an alkyne peptide, showing no
background labeling by TAMRA-N3. Panel (B) Peptide 2 visualized with green 5-Fam fluorescence. Panel (C) Peptide 1 monitored by click reaction with TAMRA-N3 on fixed
cells. Panel (D) Peptide 2 monitored by click reaction with TAMRA-N3 on fixed cells and green 5-Fam fluorescence; the yellow color indicates co-localization of 5-Fam and
TAMRA fluorophores, now present on the same peptide. The size bar represents a distance of 25 lm.
Figure 3. HeLa cells incubated with peptide 1 at 1
by click reaction with TAMRA-N3 on fixed cells. Regardless of the fluorophore used, nuclear staining is observed in both cases. This effect is not seen in cells that are subjected
to the click reaction but not treated with peptide; this indicates that peptide 1 has a small amount of nuclear localization. The size bar represents a distance of 25 m.
lM for 2 h. Panel (A) Peptide 1 visualized by click reaction with 5-Fam-PEG-N3 on fixed cells. Panel (B) Peptide 1 visualized
l
to 3-fold) than 2 (Fig. 4) at all concentrations tested. This could
indicate that 1 is indeed not as efficient at penetrating the cellular
membrane compared to 2 since it lacks the fluorophore. Alterna-
tively, this result could be an artifact of the analysis; the click reac-
tion may not quantitatively label all of the available alkyne-
functionalized peptide during the course of the reaction. It is also
plausible that if 1 enters the cells and localizes to an internal mem-
brane (endoplasmic reticulum localization is natural for geranyl-
buried in the membrane and is inaccessible for the click reaction.
These latter two possibilities would both lead to an artificially low-
er mean fluorescence value. Regardless of which explanation is
correct for the reduced fluorescence of cells treated with 1 versus
2, the important conclusion remains—peptide 1 lacking a fluoro-
phore is able to efficiently enter HeLa cells. This further substanti-
ates the importance of the hydrophobic geranylgeranyl group for
peptide uptake; without that moiety, the peptide is unable to enter
cells.19
geranylated proteins24
) that the N-terminal alkyne becomes

J. D. Ochocki et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4998–5001
5001
fers by only approximately 0.5 units, indicating the addition of the
alkyne is a rather benign change to the hydrophobicity of the pep-
tide (see Figure S4). However, the presence of the alkyne allows for
facile visualization via click-mediated fluorescent labeling when
desired. Furthermore, the alkyne group may be used in a variety
of click reactions beyond fluorophore attachment. This approach
should be quite useful for a variety of studies of cell penetrating
molecules.
Acknowledgments
The authors would like to thank Professor G.B. for use of his
automated peptide synthesizer and lyophilizer. We would also like
to acknowledge the assistance of the Flow Cytometry Core Facility
of the Masonic Cancer Center, a comprehensive cancer center des-
ignated by the National Cancer Institute, supported in part by P30
CA77598. This work was supported by the National Institutes of
Health (GM 58442).
Figure 4. Flow cytometry analysis of peptides 1 and 2 incubated for 1 h at various
concentrations. Each bar represents the geometric mean fluorescence of 10,000
cells, with the background fluorescence subtracted from each sample. Each
experiment was performed in at least triplicate, expressed as the geometric
mean standard deviation.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.04.138.
References and notes
In previous work, we have shown that peptide sequences con-
taining a geranylgeranyl group are efficiently taken into cells in
an energy-independent manner, regardless of positive charge in
the sequence. The findings reported here demonstrate that the
presence of the hydrophobic fluorophore 5-Fam in such peptides
has minimal effect on their cell penetrating ability, further under-
scoring the importance of the isoprenoid moiety. This information
may be useful for applications utilizing cell-penetrating peptides to
deliver cargo across membranes. Using the smallest molecule for a
CPP has the benefit of facile synthesis as well as minimum distur-
bance of the cell during cargo delivery. The non-endocytotic mech-
anism that functions in the uptake of these peptides may also
prove to be useful since it avoids potential endosomal localiza-
tion/degradation of cargo. For instance, Medintz and coworkers at-
tached a CPP to a quantum dot and a fluorescent protein to study
the internalization of cargo, finding that microinjection of the con-
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ly, we note the novel method for visualizing cell penetrating
peptides described herein. To date, most studies of cell penetrating
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