Cell penetrating agents based on a polyproline helix scaffold

Article abstract of DOI:10.1021/ja052377g

Cell penetrating agents were designed and synthesized that introduce cationic and hydrophobic moieties along the backbone of a polyproline helix (PPII) in an amphiphilic manner. The CD profile has the features that are expected for a PPII helix, demonstra

Full text of DOI:10.1021/ja052377g

Published on Web 07/30/2005
Cell Penetrating Agents Based on a Polyproline Helix Scaffold
Yannick A. Fillon, Jason P. Anderson, and Jean Chmielewski*
Contribution from the Department of Chemistry, Purdue UniVersity, 560 OVal DriVe,
West Lafayette, Indiana 47907
Received April 12, 2005; E-mail: chml@purdue.edu
Abstract: Cell penetrating agents were designed and synthesized that introduce cationic and hydrophobic
moieties along the backbone of a polyproline helix (PPII) in an amphiphilic manner. The CD profile has the
features that are expected for a PPII helix, demonstrating that the addition of these groups had little effect
on the backbone structure. Dramatic increases in uptake were found with MCF-7 cells when up to six
guanidinium groups were positioned on the polyproline helix, whereas only modest increases in cellular
uptake were observed with the amine-containing polyproline compounds as compared to their flexible
counterparts. Amphiphilicity played a key role in the enhanced cell translocation, as scrambled versions of
the designed agents, with hydrophobic and cationic groups on all faces of the helix, were only as effective
as their flexible peptide counterparts. Interestingly, the most potent agent, P11LRR, demonstrated almost
an order of magnitude more efficient cellular uptake as compared to that of the well-studied Tat peptide,
with minimal cytotoxicity.
Introduction
shown to deliver varied cargoes into cells from proteins and
oligonucleotides to magnetic nanoparticles.1,11
Numerous methods have been developed to achieve delivery
of therapeutic agents into cells. Cell penetrating peptides (CPPs)
that are rich in basic amino acids have become increasingly
used in this regard.1 The most well-studied CPPs are part of
transcription factors: the Tat peptide from the cationic domain
of HIV-1 Tat2 and the penetratin peptide from the Antennapedia
homeodomain.3 Polyarginine or polylysine peptides,4 cationic
nuclear localization signal sequences,5 and cationic moieties
linked to scaffolds, such as peptoids,6 â-amino acid peptides,7
oligocarbamates,8 loligomers,9 and PNA oligomers,10 have also
been studied for cell membrane translocation. CPPs have been
Although the cationic nature of CPPs is believed to play a
dominant role in cellular uptake, a recent study demonstrated
that hydrophobic residues contribute substantially to membrane
translocation.5b Amphiphilic, cationic peptides have been re-
ported to cross cell membranes; however, this activity is also
linked to membrane destabilization.12 In an effort to systemati-
cally investigate the role of hydrophobic residues in cell pen-
etration, we sought a scaffold that would orient both cationic
and hydrophobic moieties along a rigid backbone. This is com-
plicated within polyamide structures in that the close positioning
of cationic residues does not usually favor a well-folded
conformation. Indeed, the R-helical penetratin peptide undergoes
a conformational shift upon membrane translocation,13 and an
elegant study with a cationic, â-amino acid peptide indicated a
disorganized structure in water.7 With this in mind, we chose a
polyproline helix as the scaffold for our investigations.
(1) (a) Lindgren, M.; Hallbrink, M.; Prochiantz, A.; Langel, U. Trends
Pharmacol. Sci. 2000, 21, 99-103. (b) Langel, U. Cell Penetrating
Peptides: Processes and Applications; CRC Press: Boca Raton, FL, 2002.
(c) Lundberg, P.; Langel, U. J. Mol. Recognit. 2003, 16, 227-233. (d)
Snyder, E. L.; Dowdy, S. F. Pharm. Res. 2004, 21, 389-393.
(2) (a) Green, M.; Loewenstein, P. M. Cell 1988, 55, 1179-1188. (b) Frankel,
A. D.; Pabo, C. O. Cell 1988, 55, 1189-1193.
(3) (a) Derossi, D.; Joliot, A. H.; Chassaing, G.; Prochiantz, A. J. Biol. Chem.
1994, 269, 10444-10450. (b) Derossi, D.; Calvet, S.; Trembleau, A.;
Brunissen, A.; Chassaing, G.; Prochiantz, A. J. Biol. Chem. 1996, 271,
18188-18193. (c) Prochiantz, A. Curr. Opin. Neurobiol. 1996, 6, 629-
634.
In contrast with most of the currently existing CPPs, the
polyproline chain adopts a well-defined left-handed type II helix
(PPII) with trans-proline residues in polar solvents.14 PPII
contains three residues per turn aligning every third ring on the
same face of the helix with a pitch of approximately 10 Å per
turn. Previously, a proline oligomer was found to be internalized
in cells by microscopy, but the extent of cell penetration was
not quantitated.15 Fragments of naturally occurring peptides that
(4) (a) Ryser, H. J. P.; Shen, W. C. Proc. Natl. Acad. Sci. U.S.A. 1978, 75,
3867-3870. (b) Mitchell, D. J.; Kim, D. T.; Steinman, L.; Fathman, C.
G.; Rothbard, J. B. J. Pept. Res. 2000, 56, 318-325. (c) Rothbard, J. B.;
Garlington, S.; Lin, Q.; Kirschberg, T.; Kreider, E.; McGrane, P. L.;
Wender, P. A.; Khavari, P. A. Nat. Med. 2000, 6, 1253-1257. (d) Futaki,
S.; Suzuki, T.; Ohashi, W.; Yagami, T.; Tanaka, S.; Ueda, K.; Sugiura, Y.
J. Biol. Chem. 2001, 276, 5836-5840. (e) Leonetti, J. P.; Degols, G.;
Lebleu, B. Bioconjugate Chem. 1990, 1, 149-153.
(5) (a) Ragin, A. D.; Morgan, R. A.; Chmielewski, J. Chem. Biol. 2002, 9,
943-948. (b) Ragin, A. D.; Chmielewski, J. J. Pept. Res. 2004, 63, 155-
160.
(10) Zhou, P.; Wang, M. M.; Du, L.; Fisher, G. W.; Waggoner, A.; Ly, D. H.
J. Am. Chem. Soc. 2003, 125, 6878-6879.
(6) Wender, P. A.; Mitchell, D. J.; Pattabiraman, K.; Pelkey, E. T.; Steinman,
L.; Rothbard, J. B. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 13003-13008.
(7) Umezawa, N.; Gelman, M. A.; Haigis, M. C.; Raines, R. T.; Gellman, S.
H. J. Am. Chem. Soc. 2002, 124, 368-369.
(11) Lewin, M.; Carlesso, N.; Tung, C. H.; Tang, X. W.; Cory, D.; Scadden, D.
T.; Weissleder, R. Nat. Biotechnol. 2000, 18, 410-414.
(12) Shai, Y. Biochim. Biophys. Acta 1999, 1462, 55-70.
(13) Letoha, T.; Gaal, S.; Somlai, C.; Czajlik, A.; Perczel, A.; Penke, B. J. Mol.
Recognit. 2003, 16, 272-279.
(8) Wender, P. A.; Rothbard, J. B.; Jessop, T. C.; Kreider, E. L.; Wylie, B. L.
J. Am. Chem. Soc. 2002, 124, 13382-13383.
(9) Singh, D.; Kiarash, R.; Kawamura, K.; LaCasse, E. C.; Gariepy, J.
Biochemistry 1998, 37, 5798-5809.
(14) (a) Cowan, P. M.; Mcgavin, S. Nature 1955, 176, 501-503. (b) Helbecque,
N.; Loucheuxlefebvre, M. H. Int. J. Pept. Protein Res. 1982, 19, 94-101.
9
11798
J. AM. CHEM. SOC. 2005, 127, 11798-11803
10.1021/ja052377g CCC: $30.25 © 2005 American Chemical Society

Cell Penetrating Polyprolines
A R T I C L E S
Figure 1. (a) Top view of an amphiphilic polyproline helix containing modified side chains (red, cationic; blue, hydrophobic), (b) sequences, and (c)
structures of modified polyproline oligomers containing amino (PK) or guanidinium (PR) functionality.
contain a high percentage of proline and arginine residues have
also been studied.16 Tam and co-workers observed cellular
uptake with a proline-rich peptide derived from the antimicrobial
peptide bactenecin 7.16a This peptide was 2-3-fold more
effective than the Tat peptide, although a hybrid R-helical and
PPII structure was observed. In a complementary study, Giralt
and co-workers found that cationic analogues of an amphiphilic,
proline-rich peptide, (VXLPPP)n, derived from the γ-Zein
protein were able to cross the cellular membrane, although the
best was 15-fold less effective than the Tat peptide.16b In our
design, we sought to maintain a continuous polyproline sequence
to ensure a well-folded structure with modifications to the side
chain of proline to incorporate the hydrophobic and charged
groups.
and six lysine or arginine mimics. Guanidinium groups within
CPPs have been shown to be superior to amino groups as the
cationic moiety needed for cellular uptake.4b This trend was also
evaluated with the PPII helical scaffold with the use of PK and
PR, in addition to investigating the role of the overall charge
on cellular uptake.
Two groups of control molecules were also designed to
address the importance of the PPII helical scaffold and am-
phiphilicity on cell membrane penetration. In this regard, we
used flexible tetramers and hexamers of lysine (K4: FL-K4-
NH2 and K6: FL-K6-NH2) and arginine (R4: FL-R4-NH2
and R6: FL-R6-NH2), and scrambled versions of the designed
agents with all compounds containing a fluorescein group (FL).
The latter were designed to remove amphiphilicity and contain
hydrophobic and cationic groups on all faces of the PPII helix
(P8KLr: FL-G(PKPL)3PK-CONH2; P11KLr: FL-GPK(PKPL)4-
PK-CONH2; P11RLr: FL-GPR(PRPL)4PR-CONH2). In addi-
tion, comparisons to the well-studied Tat peptide (FL-G4-
YGRKKRRQRRR-NH2) were performed.
Studies were initiated with the synthesis of the appropriate
hydroxyproline derivatives containing protecting groups ap-
propriate for solid-phase synthesis (Scheme 1, compounds 1,
2, and 3). For the synthesis of the protected leucine mimic (1),
Cbz-hydroxyproline was deprotonated and treated with 3-bromo-
2-methylpropene to provide the desired ether 4 after purification.
Hydrogenation of compound 4 simultaneously removed the Cbz
protecting group and reduced the double bond, and further
treatment with Fmoc-OSu provided the desired compound 1
(Scheme 1a). The synthesis of the protected lysine mimic (2)
was initiated by deprotonation of Cbz-hydroxyproline, followed
by Michael addition with acrylonitrile to provide ether 5.
Protection of 5 as a benzyl ester provided compound 6, and
reduction of the nitrile in the presence of di-tert-butyl dicar-
bonate generated 7.17 Compound 7 was hydrogenated, followed
Results and Discussion
Functionality was incorporated into the PPII helix by O-
alkylation of a hydroxyproline monomer to yield a scaffold that
displays both hydrophobic (blue) and cationic (red) moieties
(Figure 1a). Introduction of an isobutyl group onto hydroxy-
proline led to a proline-based mimic of leucine (PL, Figure 1b
and c), whereas functionalization with amino or guanidinium
groups led to proline-based mimics of lysine (PK) or arginine
(PR), respectively. Amphiphilic agents were obtained by taking
advantage of the 3-fold repeating unit of the PPII helix. In our
design, one face of the PPII helix was hydrophobic, while the
other two faces contained cationic groups, resulting in agents
with a repeating PLXX (X ) PK or PR) unit (Figure 1). On the
basis of this repeating unit, we developed 7-mer and 10-mer
PPII helicessthe 7-mer contains three leucine mimics and four
basic moieties, whereas the 10-mer contains four leucine mimics
(15) Crespo, L.; Sanclimens, G.; Montaner, B.; Pe´rez-Toma´s, R.; Royo, M.;
Pons, M.; Albericio, F.; Giralt, E. J. Am. Chem. Soc. 2002, 124, 8876-
8883.
(16) (a) Sadler, K.; Eom, K. D.; Yang, J. L.; Dimitrova, Y.; Tam, J. P.
Biochemistry 2002, 41, 14150-14157. (b) Fernandez-Carneado, J.; Kogan,
M. J.; Castel, S.; Giralt, E. Angew. Chem., Int. Ed. 2004, 43, 1811-1814.
(17) McGeary, R. P.; Jablonkai, I.; Toth, I. Tetrahedron 2001, 41, 8733-8742.
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A R T I C L E S
Fillon et al.
Scheme 1. (a) Synthesis of Fmoc-PL (1), (b) Fmoc-PK(Boc) (2),
and (c) Fmoc-PR(Boc)2 (3)
Figure 2. Circular dichroism spectra of P8LKK (solid line) and P11LRR
(dashed line) lacking the fluorescein group at 100 µM in PBS buffer (pH
7.4).
by Fmoc protection, to provide the desired compound 2 (Scheme
1b). Last, the protected arginine mimic (3) was prepared from
compound 2 by treatment with TFA, followed by reaction with
N,N′-bis(tert-butoxycarbony)-N′′-triflylguanidine to yield the
desired compound 3 (Scheme 1c).18
All polyproline derivatives were synthesized on solid support
using the Rink resin. An Fmoc-based strategy was employed
using HATU as the coupling reagent.19 Each coupling step was
monitored for completion using the chloranil test.20 The N-
terminus of each compound was fluorescently labeled with
N-hydroxysuccinimide (NHS)-fluorescein while on the solid
support. The agents were cleaved from the resin with simulta-
neous deprotection by treatment with a TFA/triisopropylsilane/
water mixture. All compounds were purified to homogeneity
by reverse phase HPLC and characterized by mass spectrometry.
The series of peptides containing proteinogenic amino acids (K4,
K6, R4, R6, and Tat) was synthesized as described above, except
that HBTU/HOBT was used as the coupling reagent.
Prior to adding fluorescein to the N-terminus of polyproline
derivatives P8LKK and P11LRR, samples of these compounds
were cleaved from the resin and purified to homogeneity for
analysis by circular dichroism. Polyproline oligomers are known
to adopt a type II (PPII) helical conformation in polar solvents
with characteristic CD signals that include a strong negative
band at 206 nm and a weak positive band at 226 nm.14b Analysis
of P8LKK and P11LRR lacking fluorescein (P8LKK-FL and
P11LRR-FL) by CD provided spectra that corresponded well
with the PPII helical structure (Figure 2). These data confirm
that side chain extensions to proline do not affect the fold of
the backbone, and that the polyproline scaffold maintains its
structure even with cationic groups along the same face of the
helix.
Figure 3. Flow cytometry data for compounds containing (a) amino groups
and (b) guanidinium groups and Tat (blue, 4 cationic moieties; pink, 6
cationic moieties).
compounds (50 µM). After a 6 h incubation period, the
fluorescent agents were removed from the media, the cells were
dislodged, resuspended in media, and analyzed by flow cytom-
etry. Cellular uptake was demonstrated with all compounds
analyzed, but a variety of trends could be discerned from the
data (Figure 3). More general trends include increased fluores-
cence of cells treated with the guanidinium-containing agents
as compared to their corresponding amine-containing com-
pounds (P8LRR/P8LKK, P11LRR/P11LKK, and P11RLr/
P11KLr), and an increased cellular fluorescence with an
increasing number of cationic groups (P8LKK/P11LKK and
P8LRR/P11LRR), both effects that have been previously
observed.4b
All cationic agents were evaluated for cellular uptake via flow
cytometry using adherent MCF-7 breast cancer cells. For this
assay, the cells were incubated with the fluorescently labeled
Trends are also observed that are more specific to the
polyproline scaffold. The amine-containing compounds show
relatively small variations in uptake (Figure 3a). For instance,
(18) Feichtinger, K.; Zapf, C.; Sings, H. L.; Goodman, M. J. Org. Chem. 1998,
63, 3804-3805.
(19) Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4397-4398.
(20) Christensen, T. Acta Chem. Scand. B 1979, 33, 763-766.
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Cell Penetrating Polyprolines
A R T I C L E S
Figure 4. Confocal microscope images of MeOH-fixed MCF-7 cells treated with polyproline derivatives and Tat; (top) transmission images and (bottom)
fluorescence images.
the rigid and amphiphilic P8LKK is internalized within MCF-7
cells only slightly (1.3-fold) more effectively than its flexible
peptide counterpart K4. However, this small effect is lost when
the hydrophobic and cationic moieties are scrambled to remove
the amphiphilic character of the polyproline helix (P8KLr).
Interestingly, this trend is more significant for the longer amine-
containing sequences; cells treated with P11LKK are 1.7-fold
more fluorescent than cells treated with the K6 peptide, and
removing the amphiphilic character of the polyproline compound
(P11KLr) eliminates this enhancement. When the side chain
contains a guanidinium moiety, the shorter sequence containing
four cationic groups (P8LRR) is only 1.5-fold more effective
Figure 5. Confocal microscope images of live MCF-7 cells treated with
polyproline derivative P11LRR and Tat; (top) transmission images and
(bottom) fluorescence images.
than the flexible R4 (Figure 3b). A more dramatic difference,
however, is noted with the longer sequences; a 7.7-fold increase
in cellular uptake is observed with P11LRR as compared to
R6, and this effect is lost upon randomizing the amphiphilic
character of the agent (P11RLr). Indeed, P11LRR is almost an
order of magnitude more effective at cellular penetration than
the well-studied Tat peptide, with two less cationic moieties.
These data demonstrate the essential roles of rigidity and
amphiphilicity for effective cellular uptake, although differences
are more easily noted when there are six guanidinium groups
within the agents.
cells as P11LRR, with punctate fluorescence in the cytoplasm,
but no penetration of the nucleus was observed.21
To determine if the polyproline compounds were toxic to
cells, an MTT assay22 was performed. Under the conditions used
for confocal analysis, all polyproline compounds displayed
minimal cytotoxicity to MCF-7 cells (84-99% viability), values
that are comparable to those of the polylysine and polyarginine
compounds (89-99% viability). These data confirm that the
polyproline compounds have the possibility to be safely used
to deliver cargo to cells.
To determine the subcellular localization of these compounds,
a subset of polyproline compounds and controls was examined
by confocal microscopy (Figure 4). MCF-7 cells were treated
in a similar fashion as in the flow cytometry experiments, with
the exception that the cells were incubated for 4 h with the
fluorescein-labeled compounds in LabTek chambered slides. All
compounds were found to be localized in both the nucleus and
cytoplasm of MeOH-fixed MCF-7 cells. As predicted from flow
cytometry, the guanidinium-containing compounds show in-
creased fluorescence as compared to that of the amino series.
Conclusion
We have demonstrated that functionality may be easily
incorporated into a polyproline helix by O-alkylation of the side
chain of hydroxyproline. A range of modifications can be made,
including introduction of hydrophobic and cationic (amino and
guanidinium) moieties. These added groups have little effect
on the backbone structure since the CD profile has the features
that are expected for a PPII helix. Whereas only modest in-
creases in cellular uptake are observed with the amine-containing
polyproline compounds as compared to that of their flexible
counterparts, more dramatic increases in uptake are found with
To verify that these compounds enter living cells, live
confocal images of selected compounds were also obtained
(Figure 5). The results of live studies indicate that fluorescence
is not due to external binding followed by influx upon fixation,
which has been recently reported.21 Interestingly, nuclear and
cytoplasmic staining is evident for P11LRR (Figure 5, left), but
a punctate pattern of fluorescence is also observed. The Tat
peptide (Figure 5, right) displays a similar uptake profile in live
(21) (a) Lundberg, M.; Johansson, M. Biochem. Biophys. Res. Commun. 2002,
291, 367-371. (b) Richard, J. P.; Melikov, K.; Vives, E.; Ramos, C.;
Verbeure, B.; Gait, M. J.; Chernomordik, L. V.; Lebleu, B. J. Biol. Chem.
2003, 278, 585-590. (c) Thoren, P. E. G.; Persson, D.; Isakson, P.; Goksor,
M.; Onfelt, A.; Norden, B. Biochem. Biophys. Res. Commun. 2003, 307,
100-107.
(22) Mosmann, T. J. Immunol. Methods 1983, 65, 55-63.
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A R T I C L E S
Fillon et al.
was removed in vacuo. The desired product was purified by silica gel
column chromatography (95% CH2Cl2, 4% MeOH, 1% AcOH) to
provide 5 as a colorless oil in 70% yield. MS (EI/CI): 319 m/z (M +
H+). 1H NMR (300 MHz, CDCl3): δ 7.34 (m, 5H), 5.15 (m, 2H), 4.54
(m,1H), 3.65 (m, 3H), 2.59 (t, J ) 5.6 Hz, 2H), 2.40 (m, 1H), 2.15 (m,
2H), 2.39 (m, 2H).
MCF-7 cells when up to six guanidinium groups are positioned
on the polyproline helix. Amphiphilicity plays a key role in the
enhanced cell translocation since scrambled versions of the
designed agents, with hydrophobic and cationic groups on all
faces of the helix, are only as effective as their flexible peptide
counterparts. Interestingly, the most potent agent, P11LRR,
demonstrates almost an order of magnitude more efficient cell
uptake as compared to that of the well-studied Tat peptide, with
minimal cytotoxicity. These data bode well for the use of the
polyproline scaffold in the delivery of therapeutic agents into
cells, an avenue under current exploration.
Compound 6. To a solution of 5 (3.82 g, 12.0 mmol) in DMF (50
mL) were added potassium carbonate (4.15 g, 30.0 mmol) and benzyl
bromide (1.43 mL, 12.0 mmol). The mixture was stirred for 2 h at 0
°C, allowed to warm to room temperature, and filtered through Celite.
To the filtrate was added a saturated aqueous solution of LiCl (100
mL), and the mixture was extracted with diethyl ether. The organic
layer was dried over anhydrous MgSO4, and the solvent was removed
in vacuo. The desired product was purified by silica gel column
chromatography (97% CH2Cl2, 3% MeOH) to provide 6 as a colorless
oil in 85% yield. MS (EI/CI): 409 m/z (M + H+). 1H NMR (300 MHz,
CDCl3): δ 7.40 (m, 10H), 5.10 (m, 4H), 4.60 (m, 2H), 3.75 (m, 3H),
3.00 (m, 2H), 2.60 (m, 2H), 2.34 (m, 1H), 2.15 (m, 1H).
Experimental Section
Amino acids were obtained from Advanced ChemTech, and reagents
were purchased from Aldrich. All starting materials were used without
purification.
Compound 4. To a solution of Cbz-hydroxyproline (1.0 g, 3.68
mmol) in THF (20 mL) at -40 °C and under N2 was added KHMDS
(15 mL, 0.5 M in toluene, 7.73 mmol), and the mixture was stirred for
30 min. To this mixture was added 3-bromo-2-methylpropene (1.5 mL,
14.7 mmol), and the reaction was allowed to stir for 30 min at -40
°C. The reaction mixture was allowed to warm to room temperature
and stirred for an additional 2.5 h. The mixture was cooled to 0 °C,
and 10% HCl was added to bring the pH to 1. The resulting solution
was extracted with CH2Cl2, the organic layer was dried over anhydrous
MgSO4, and the solvent was removed in vacuo. The desired product
was purified by silica gel column chromatography (95% CH2Cl2, 4%
MeOH, 1% AcOH) to provide 4 as a colorless oil in 95% yield. MS
(EI/CI): 320 m/z (M + H+). 1H NMR (300 MHz, CDCl3): δ 7.32 (m,
5H), 5.20 (dd, J ) 8, 4 Hz, 2H), 4.95 (d, J ) 15 Hz, 2H), 4.55 (m,
1H), 4.15 (m, 1H), 3.89 (m, 2H), 3.66 (m, 2H), 2.45 (m, 1H), 2.30 (m,
H), 1.74 (s, 3H).
Compound 7. Sodium borohydride (3.89 g, 0.103 mol) was added
portion-wise to a stirred solution of the nitrile 6 (4.20 g, 10.3 mmol),
CoCl2‚6H2O (4.90 g, 20.6 mmol), and di-tert-butyl dicarbonate (3.55
mL, 0.12 mmol) in methanol (110 mL) at 0 °C. The ice bath was
removed from the reaction, and stirring was continued for 4 h. The
reaction mixture was filtered through Celite, water (100 mL) was added
to the filtrate, and the methanol was removed in vacuo. The resulting
solution was extracted with EtOAc, the organic layer was dried over
anhydrous MgSO4, and the solvent was removed in vacuo. The desired
product was purified by silica gel column chromatography (97% CH2-
Cl2, 3% MeOH) to provide 7 as a colorless oil in 70% yield. MS (EI/
1
CI): 513 m/z (M + H+). H NMR (300 MHz, CDCl3): δ 7.30 (m,
10H), 5.08 (m, 4H), 4.50 (m, 2H), 4.09 (m, 2H), 3.60 (m, 3H), 3.19
(m, 2H), 2.40 (m, 1H), 2.15 (m, 1H), 1.75 (m,2H), 1.46 (s, 9H).
Compound 2-Fmoc-PK(Boc). To a solution of 7 (2.77 g, 5.4 mmol)
in MeOH (50 mL) was added Pd/C (0.28 g, 10 wt %), and the solution
was stirred under 1 atm of hydrogen for 12 h. The solution was filtered
through Celite, the solvent was removed in vacuo, and the residue was
used in the next step without further purification. A solution of the
resulting material (1.33 g, 4.62 mmol) in water/acetone (40 mL, 1:1)
was cooled to 0 °C, and sodium bicarbonate (1.36 g, 16.2 mmol) was
added. To this mixture was added Fmoc-OSu (2.03 g, 6.0 mmol),
and the reaction was stirred for 1 h at 0 °C and 2 h at 25 °C. The
reaction was treated with 10% HCl to a pH of 4 and extracted with
CH2Cl2. The organic layer was dried over anhydrous MgSO4, and the
solvent was removed in vacuo. The desired product was purified by
silica gel column chromatography (97% CH2Cl2, 3% MeOH) to provide
Compound 1-Fmoc-PL. To a solution of 4 (2.0 g, 6.26 mmol) in
MeOH (50 mL) was added Pd/C (0.20 g, 10 wt %), and the solution
was stirred under 1 atm of hydrogen for 12 h. The solution was filtered
through Celite, the solvent was removed in vacuo, and the residue was
used in the next step without further purification. A solution of the
resulting material (0.88 g, 4.71 mmol) in water/dioxane (18 mL, 1:2)
was cooled to 0 °C, and a solution of sodium bicarbonate (1.0 g, 11.8
mmol) in water/dioxane (24 mL, 1:1) was added. To this mixture was
added Fmoc-OSu (2.07 g, 6.12 mmol), and the resulting slurry was
stirred for 1 h at 0 °C and 3 h at 25 °C. The reaction was treated with
10% HCl to a pH of 1 and extracted with CH2Cl2. The organic layer
was dried over anhydrous MgSO4, and the solvent was removed in
vacuo. The desired product was purified by silica gel column chro-
matography (97% CH2Cl2, 2% MeOH, 1% AcOH) to provide 1 as a
22
1
2 as a white foam in 60% yield. [R]D ) -40.0 (c 0.58 CHCl3). H
NMR (300 MHz, CDCl3): δ 7.79 (t, J ) 7.5 Hz, 2H), 7.62 (t, J ) 7.5
Hz, 2H), 7.37 (m, 5H), 4.48 (m, 3H), 4.13 (m, 2H), 3.57 (m, 3H), 3.25
(m, 2H), 2.34 (m, 1H), 2.15 (m, 1H), 1.78 (m, 2H), 1.50 (s, 9H). 13C
NMR (75 MHz, CDCl3): δ 176.4*, 175.6, 156.2, 155.5, 154.9*, 144.2*,
144.0, 141.5, 128.7*, 128.1, 127.4, 125.4, 120.4, 81.7, 80.1, 68.7*,
68.1, 67.2, 59.0, 58.6*, 52.7, 48.1, 39.4, 37.8, 36.3, 31.1, 29.6
(*indicates minor rotamer). HRMS calcd for C28H34N2O7 [M + Na+]
533.2264, found 533.2272.
22
1
white foam in 60% yield. [R]D ) -49.2 (c 0.89 CHCl3). H NMR
(300 MHz, CDCl3): δ 7.90 (t, J ) 6.9 Hz, 2H), 7.75 (t, J ) 6.9 Hz,
2H), 7.40 (m, 4H), 4.10 (m, 4H), 3.52 (m, 2H), 3.04 (m, 2H), 2.30 (m,
1H), 2.15 (m, 2H), 1.70 (m, 1H), 0.78 (d, J ) 6.9 Hz, 6H). 13C NMR
(75 MHz, CDCl3): δ 177.7*, 176.4, 155.8, 154.7*, 144.2*, 144.0, 143.9,
141.5, 128.7*, 128.1, 128.0, 127.4, 125.4, 120.4, 120.3, 77.0, 68.8,
68.6*, 59.1, 58.5*, 53.0*, 52.8, 48.1, 37.9, 36.3, 29.8, 20.6 (*indicates
minor rotamer). HRMS calcd for C24H27NO5 [M + H+]: 410.1967,
found 410.1965.
Compound 3-Fmoc-PR(Boc)2. A solution of 2 (0.56 g, 1.09 mmol)
was treated with a solution of TFA in CH2Cl2 (10 mL, 1:1), and the
mixture was allowed to stir for 2 h at room temperature. The TFA and
CH2Cl2 were removed in vacuo. The resulting residue was dissolved
in dry CH2Cl2 (5 mL), and a solution of N,N′-bis(tert-butoxycarbony)-
N′′-triflylguanidine (0.41 g, 1.04 mmol) and triethylamine (0.45 mL,
3.27 mmol) in CH2Cl2 (5 mL) was added. The reaction was stirred at
room temperature for 5 h, and the mixture was washed with saturated
sodium bicarbonate and brine. The organic layer was dried over
anhydrous MgSO4, and the solvent was removed in vacuo. The desired
product was purified by silica gel column chromatography (95% CH2-
Cl2, 4% MeOH, 1% AcOH) to provide 3 as a white foam in 75% yield.
Compound 5. To a solution of Cbz-hydroxyproline (1.5 g, 5.65
mmol) in THF (20 mL) at 0 °C and under N2 was added an ice-cooled
solution of NaH (0.75 g, 19.8 mmol) in THF (20 mL), and the mixture
was stirred at 0 °C for 1.5 h. To this mixture was added acrylonitrile
(1.5 mL, 22.6 mmol), and the reaction was allowed to warm to room
temperature and stirred for 24 h. The reaction mixture was cooled to
0 °C, and water (100 mL) was added to quench the excess NaH. The
THF was removed in vacuo, and 10% HCl was added to bring the
solution to a pH of 1. The resulting solution was extracted with EtOAc,
the organic layer was dried over anhydrous MgSO4, and the solvent
9
11802 J. AM. CHEM. SOC. VOL. 127, NO. 33, 2005

Cell Penetrating Polyprolines
A R T I C L E S
Table 1. Purification and Mass Spectral Data for Polyproline
Compounds
residues in the peptide. CD spectra of P8LKK and P11LRR (100 µM)
lacking the fluorescent label were taken in PBS buffer, pH 7.4 (140
mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, and 1.5 mM KH2PO4).
Cell Culture Experiments. Screening of cellular uptake was carried
out on cancerous MCF-7 (breast) cells grown continuously as adherent
monolayers in RPMI (Rosewell Park Memorial Institute) culture media
supplemented with 10% HIFCS and PS (100 units/mL penicillin and
0.1 mg/mL streptomycin). The cells were grown in a humidified 5%
CO2 atmosphere at 37 °C. The cells were passaged biweekly and
discarded after six passages to ensure consistency in uptake.
Flow Cytometry Analysis. Cellular uptake of the fluorescently
labeled agents was verified using flow cytometry. The cells were seeded
into 24-well plates at a density of 100 000 cells/well in 1 mL of media
and grown in a humidified 5% CO2 atmosphere at 37 °C for 48 h. The
cells were then incubated for 6 h in the presence of 84 µL of
fluorescently labeled peptides (in sterile water) at a concentration of
50 µM in culture media (170 µL). Following incubation, the cells were
washed three times with 100 µL of PD (Ca2+- and Mg2+-deficient
phosphate-buffered saline). The cells were gently dislodged from the
wells with 100 µL of trypsin (0.25%) and resuspended in 300 µL of
the culture media. For each experiment, a control of cells that were
not incubated with fluorescent compound was also analyzed. The
samples were run in triplicate, and each experiment was repeated twice
within the same week. Mean fluorescence values were measured on a
BD FACS Caliber Flow Cytometer using an air-cooled laser for
excitation of fluorescein at 488 nm.
Confocal Laser Scanning Microscopy. The cells were seeded into
LabTek chambered slides at a density of 50 000 cells/well in 1 mL of
media and grown for 48 h as described above. The cells were incubated
for 4 h with the fluorescent agents using conditions and concentrations
described above. The cells were then washed three times with 100 µL
of PD (Ca2+- and Mg2+-deficient phosphate-buffered saline). The cells
were analyzed live in PBS buffer (250 µL) or by fixation with 100 µL
of cold methanol. Fluorescent images were recorded on a Biorad
Confocal Laser Scanning Microscope at slow speed using a 60X oil
objective and a 488 nm laser line for fluorescein excitation. Images
were overlayed using the Thumbs Plus software.
MTT Viability Assay. Cellular toxicity of the fluorescent agents
was examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-
razolium bromide (MTT) cell viability assay. The cells were seeded
into 24-well plates at a density of 100 000 cells/well in 1 mL of media
and grown in a humidified 5% CO2 atmosphere at 37 °C for 48 h. The
cells were then incubated for 4 h in the presence of 84 µL of
fluorescently labeled peptides (in sterile water) at a concentration of
50 µM in culture media (170 µL). Following this incubation, the cells
were washed three times with 100 µL of PD (Ca2+- and Mg2+-deficient
phosphate-buffered saline) and incubated for 1 h with 0.5 mg/mL of
MTT in media. After 1 h, the MTT/media solution was removed, and
the precipitated crystals were dissolved in 2-propanol (500 µL). The
solution absorbance was read at 580 nm. For each experiment, a control
of cells that were not incubated with fluorescent compound was also
analyzed. The samples were run in triplicate, and each experiment was
repeated twice within the same week. The average absorbance for each
sample was calculated and percent viability determined using the
following equation: % cell viability ) A580 treated cells/A580 untreated
cells × 100.
retention
mass (exp)
(M
H+)
compound
timea (min)
% purity
+
P8LKK
P11LKK
P8KLr
P11KLr
P8LRR
P11LRR
P11RLr
26.9
27.2
21.3
21.5
18.7
28.5
24.8
98
99
98
98
97
99
99
1624
2132
1624
2132
1789
2384
2384
a HPLC conditions: 5-70% solvent A using a 40 min gradient on a
Vydac C8 analytical column at 1.0 mL/min.
22
1
[R]D ) -30.6 (c 1.0 CHCl3). H NMR (300 MHz, CDCl3): δ 7.79
(t, J ) 7.5 Hz, 2H), 7.62 (t, J ) 7.5 Hz, 2H), 7.37 (m, 5H), 4.48 (m,
3H), 4.13 (m, 2H), 3.57 (m, 3H), 3.25 (m, 2H), 2.46-2.20 (m, 2H),
1.78 (m, 2H), 1.50 (s, 18H). 13C NMR (75 MHz, CDCl3): δ 177.2*,
175.9, 163.0, 156.0, 155.8*, 154.75, 153.1, 144.0, 143.8*, 143.7, 141.3,
141.2*, 128.3, 127.8, 127.7*, 127.2, 127.1, 125,1, 120.0, 119.9*, 83.4,
79.8, 68.2, 68.1*, 68.0, 58.2, 57.6*, 51.6*, 51.3, 47.1, 39.8, 36.8, 35.1,
28.8, 28.3, 28.1* (*indicates minor rotamer). HRMS calcd for
C34H44N2O7 [M + H+] 653.3187, found 653.3185.
General Synthesis of Polyproline Compounds. A 10 mL peptide
synthesis flask was loaded with 100 mg (0.06 mmol) of Rink amide
resin. The resin was washed with CH2Cl2 (2 × 4 mL) and DMF (2 ×
4 mL). Piperidine (20% in DMF, 4 mL) was added to the reaction
flask, the flask was agitated for 30 min, and the piperidine solution
was drained. The resin was washed with DMF, CH2Cl2, MeOH, CH2-
Cl2, and DMF (2 × 4 mL each). Fmoc-protected amino acids (2.5 equiv,
0.15 mmol) in DMF (4 mL) were added to the reaction flask with
HATU (2.5 equiv, 0.15 mmol) and DIEA (2.5 equiv, 0.15 mmol), and
the flask was agitated for 4 h. The resin was washed with DMF, CH2-
Cl2, MeOH, CH2Cl2, and DMF (2 × 3 mL each). Piperidine (20% in
DMF, 4 mL) was added to the reaction flask, the flask was agitated
for 30 min, and the piperidine solution was drained. The resin was
washed with DMF, CH2Cl2, and MeOH (2 × 4 mL each). This
procedure was repeated until all amino acids were coupled to the resin.
Coupling of NHS-Fluorescein to Polyproline Compounds. The
resin was washed with DMF (2 × 4 mL) and NMP (1 × 4 mL). NHS-
fluorescein (1.1 equiv, 0.06 mmol) and DIEA (1.1 equiv, 0.06 mmol)
were added, along with 4 mL of NMP. The reaction flask was protected
from light with aluminum foil and agitated for 24 h. The resin was
washed with NMP, DMF, and MeOH (2 × 4 mL each) and dried in
vacuo for 3 h.
Cleavage of Polyproline Compounds from Resin and Purifica-
tion. A trifluoroacetic acid (TFA) cocktail solution (95% TFA, 2.5%
triisopropylsilane, 2.5% water, 4 mL) was added to the resin, and the
mixture was agitated for 2 h. The solution was filtered through glass
wool into a 50 mL centrifuged tube. The resin was washed with CH2-
Cl2 (3 × 4 mL), and the filtrate was collected into the same tube. The
resulting solution was concentrated in vacuo to remove the TFA. The
residue was dissolved in cold diethyl ether and placed in the freezer to
precipitate the desired compound. The precipitate was collected by
centrifugation and washed with cold diethyl ether. The collected
precipitate was purified to homogeneity by reverse phase HPLC using
a linear 60 min gradient of a solvent system consisting of solvent A
(acetonitrile/0.1% TFA) and solvent B (water/0.1% TFA) with a flow
rate of 8 mL/min (λ214 nm and λ254 nm). All peptides were characterized
by MALDI-TOF mass spectrometry, and stock solutions were quan-
titated by amino acid analysis.
Acknowledgment. This work is supported by the Purdue
Research Foundation. The authors are grateful for the early work
of D. Roberts and E. Chang.
Circular Dichroism Analysis. CD spectra were recorded on a Jasco
circular dichroism spectropolarimeter (Model J810) at 25 °C using a
0.1 cm path length quartz cell. The spectra were averaged over three
scans taken from 250 to 196 nm with a resolution of 0.1 nm at a scan
rate of 50 nm/min. The CD data obtained were processed to convert
the data from degrees of rotation to mean residue ellipticity by dividing
by the appropriate path length, peptide concentration, and number of
Supporting Information Available: 1H and 13C NMR spectra
for compounds 1, 2, and 3, peptide purity, and cell viability
data. This material is available free of charge via the Internet
at http://pubs.acs.org.
JA052377G
9
J. AM. CHEM. SOC. VOL. 127, NO. 33, 2005 11803