S)-Guanidinylprolyl Collagen Peptides: On-Resin Synthesis

Mar 19, 2012 - ... 900 NCL Innovation Park, Dr Homi Bhabha Road, Pune 411008, India. J. Org. Chem. , 2012, 77 (8), pp 4131–4135. DOI: 10.1021/jo3000...
0 downloads 0 Views 3MB Size
Download PDF
Note pubs.acs.org/joc

4(R/S)-Guanidinylprolyl Collagen Peptides: On-Resin Synthesis, Complexation with Plasmid DNA, and the Role of Peptides in Enhancement of Transfection Manaswini Nanda and Krishna N. Ganesh*,† Organic Chemistry Division, National Chemical Laboratory, Pune 411008, India † Chemical Biology Unit, Indian Institute of Science Education and Research, 900 NCL Innovation Park, Dr Homi Bhabha Road, Pune 411008, India S Supporting Information *

ABSTRACT: Chimeric collagen peptides containing cationic 4(R/S)-guanidinylproline are synthesized by in situ amidinylation of 4(R/S)-aminoproline residues. These peptides uniquely enhance the transfection efficiency of GFP-encoded plasmid DNA (pRmHa3-GFP) into cells through efficient DNA condensation resulting from nonspecific electrostatic interactions of cationic guanidino groups and localize in subcytoplasmic organelles.

I

ntracellular drug delivery is a key component of contemporary drug development. Designing efficient mechanisms for delivering macromolecules, in particular DNA and peptides, to intracellular targets across impermeable cell membranes would create new therapeutic opportunities.1−4 Oligopeptides are attractive alternatives to cationic polymers and lipids for nonviral DNA delivery.5,6 Many cationic peptides induce translocation of DNA across the cellular membrane7 and deliver the attached cargo to the nucleus.8 In this context, polyproline peptides have been shown to possess cell penetrating ability.9,10 The proline-rich collagen was found to transport plasmid DNA and siRNA into cells,11,12 and theoretical studies have supported formation of efficient DNA−collagen complexes.13 We have previously shown that the cationic 4(R/S)-aminoproline collagen peptides P1 and P2 (Table 1) induce a higher thermal and pH-dependent stability of the derived triplexes compared to the natural, nonionic 4R-hydroxyproline peptides.14,15 The design of peptides was dictated by the established fact that in collagen peptide (X-Y-Gly)n, proline at the X-site prefers C4′-endo pucker, while that at the Y-site prefers C4′-exo pucker necessary for optimal packing of helical chains into a triplex. This is achieved by placing 4(S)-aminoproline at the X-site and 4(R)-aminoproline in Y-site. The ionizable 4(R/S)amino groups with pKa around 10.0 remain partially protonated at physiological pH. As a result mixed ring puckers exist for different proline rings giving rise to heterogeneous conformations for these peptides. Many cell-penetrating peptides have multiple cationic amino acids such as arginine/lysine16−19 or guanidines linked to peptoid20 or polyproline scaffolds.21 The guanidium groups in peptides are known to recognize the anionic © 2012 American Chemical Society

Table 1. Sequence of Peptides

peptide P1 P2 P3 P4 P5 (SAP)

sequence Ac-Phe-(Pro-Amp-Gly)6-NH2 X = H, Y = 4R-NH2 Ac-Phe-(amp-Pro-Gly)6-NH2 X = 4S-NH2, Y = H Ac-Phe-(Pro-AmpG-Gly)6-NH2 X = H, Y = 4R-NHC(NH)NH2 Ac-Phe-(ampG-Pro-Gly)6-NH2 X = 4S-NHC(NH)NH2, Y = H H2N-(Val-Arg-Leu-Pro-Pro-Pro)3-NH2

sulfate of heparin on the plasma membrane22 and efficiently translocate through cell membranes. These observations prompted us to fabricate the intrinsically cationic 4(R/S)-guanidinylproline collagen peptides P3 and P4 (Table 1) having guanidine groups directly linked to proline residues at Y and X position, respectively. The guanidine moiety with pKa ≈ 13.0 is likely to be completely protonated at pH 7.0, leading to single conformation for the Received: January 13, 2012 Published: March 19, 2012 4131

dx.doi.org/10.1021/jo300070p | J. Org. Chem. 2012, 77, 4131−4135

The Journal of Organic Chemistry

Note

reagent on the 4(R/S)-aminoproline peptides in solution failed to give the desired products. The guanidinylated peptide was released from the resin by treatment with TFA-TFMSA.25 The 4(S)-guanidinylprolyl peptide (P4) was obtained similarly from resin-bound 4(S)-aminoprolyl peptide (5). The sweet arrow peptide (SAP, P5) (Table 1) having arginine was synthesized as a reference peptide26 for biological experiments. The purity of the synthesized peptides P1−P5 was ascertained (>95%) by RP-HPLC and characterized by MALDI-TOF data. The secondary structures adapted by the 4-guanidinylproline peptides P3 and P4 and the SAP peptide P5 were determined from their CD spectra measured at different concentrations, temperatures, and solvents. These peptides exhibited CD characteristic of single chain PPII-like conformation.27 Unlike the 4-aminoprolyl collagen peptides P1 and P2,14,15 no change in CD profile of P3 and P4 was seen with an increase in peptide concentration (Supporting Information), suggesting that they remain in a single-chain helix form. The nonformation of collagen-like triplexes14,15 is perhaps a consequence of both interstrand charge repulsion and unfavorable steric factor in interchain association. Temperature-dependent CD spectroscopy showed that in aqueous buffer the melting temperature (Tm) of the single chain helix for 4(R)-peptide P3 was higher than that of 4(S)-peptide P4 by 8.6 °C (Table 2). In a relatively nonpolar

proline ring and therefore well-defined conformation for the derived peptides. Here we demonstrate that the 4(R/S)-guanidinylproline collagen peptides (P3 and P4) can function as efficient enhancers in transfection of plasmid DNA encoding the green fluorescent protein in S2 cells and the fluorescent-tagged 4-guanidinylproline peptides are localized in specific cytoplasmic organelles. The 4(R/S)-aminoproline collagen peptides (P1 and P2) were synthesized by a solid-phase method (Scheme 1) on MBHA Scheme 1. On-Resin Synthesis of 4(R)-Guanidinylprolyl (P3) and 4(S)-Guanidinylprolyl (P4) Collagen Peptides Using Amidinylating Reagent 4

Table 2. Tm of 4(R/S)-Guanidineproline Peptides Tma (oC) peptide sequence Ac-Phe-(Pro-AmpG-Gly)6-NH2 Ac-Phe-(ampG-Pro-Gly)6-NH2 a

P3 P4

pH 7.2

EG:W

58.0 50.6

41.6 49.0

Phosphate buffer (10 mM), NaCl (10 mM); EG:W (3:1, v/v).

medium of ethylene glycol/water (EG:H2O, 3:1), the reverse was noticed: the 4(S)-peptide P4 had a higher Tm than the 4(R)-peptide P3 by 7.4 °C. The Tm of 4(R)-peptide P3 in aqueous medium is considerably higher than in EG:H2O (ΔTm, +17.4 °C), compared to the relative Tm difference for 4(S)-peptide P4 (ΔTm, +1.6 °C). The lower stability of ionizable peptides in EG:H2O is perhaps a consequence of repulsive positive charges that remain unscreened in the nonpolar system. However, the possibility of intramolecular H-bonding of 4(S)-substituent with the Cα-amide carbonyl27 in a relatively nonpolar EG:H2O system favors a trans geometry of the peptide bond,27,28 thereby promoting PPII-like form. This results in a higher Tm for the 4(S)-peptide P4 compared to 4(R)-peptide P3 in which such H-bond formation is not possible. In experiments to evaluate the DNA-binding properties of the peptides by agarose gel electrophoresis, the peptide P4 showed a relatively stronger binding with plasmid DNA in comparison to peptide P3 as seen by greater mobility retardation of its complexes (Figure 1). At a plasmid DNA:peptide concentration of 1:10, the retardation of complex was almost complete in the case of 4(S)peptide P4 (lane 7) but only partial in the case of 4(R)-peptide P3 (lane 3). At a 1:25 ratio, both peptides (P3 and P4, lanes 4 and 8, respectively) completely retarded the DNA in the well, and hence, this ratio was used in DNA transfection experiments; a binding ratio of 1:50 has been reported for the control peptide P5 (SAP).26 The transfection efficiency of plasmid DNA (pRmHa3-GFP) encoding green fluorescent protein GFP expressed in Drosophilia S2 cells was examined using the Qiagen transfection reagent kit,29 which contains two reagents, “effectene” and “enhancer”, along with EC buffer. The cationic enhancer

resin by site-specific incorporation of the protected monomers N1(t-Boc)-4(R/S)NH-Fmoc-proline (1a, 1b) synthesized by a reported procedure.14,15 At the end of the synthesis, the 4(R)NHFmoc group of the peptide on-resin (2) was removed by piperidine treatment (Scheme 1). The liberated 4-NH2 groups on the prolyl residues of the peptide on-resin (3) were reacted with the amidinylating agent N,N′-bis-Boc-1H-pyrazole-1-carboxamidine23,24 (4) to transform them into the analogous resin-linked guanidinium peptides P3. A similar reaction carried out with the 4132

dx.doi.org/10.1021/jo300070p | J. Org. Chem. 2012, 77, 4131−4135

The Journal of Organic Chemistry

Note

Figure 1. Gel agarose electrophoresis of peptides binding to plasmid DNA at different ratios (w/w) in EDTA (10 μM), buffer (1X-TAE), 2 h, 90 V: lane 1, only pDNA; lanes 2−5, pDNA + P3 (1:5, 1:10, 1:25, 1:50 w/w); lanes 6−9, pDNA + P4 (1:5, 1:10, 1:25, 1:50 w/w); lane 10, pDNA + P5 (1:50 w/w).

condenses the plasmid DNA, while the surfactant effectene assists in the internalization of the complex into cells. The transfection experiments were done by replacement of either or both of the reagents with the individual peptides P1−P5 at a plasmid/peptide ratio of 1:25 (w/w) to investigate the role of the guanidine peptides in the transfection process. The results of the expression of GFP are shown in Figures 2 and 3.

Figure 3. Relative transfection efficiency of 4(R/S)-amino/guanidine proline (P1−P4) and SAP (P5) peptides in the presence and absence of enhancer in Qiagen Effectene transfection kit.

plasmid DNA by more than 2-fold compared to control (green bars), and (iii) transfection efficiencies for peptides P1, P2, and P5 are negligible in the absence of enhancer (red bars). Most importantly, the guanidinyl peptides P3 and P4 show high transfection efficiency even in the absence of enhancer (red bars), about 12 times more than the control. Thus, not only is the transfection efficiency of Qiagen reagents boosted in the presence of cationic 4-aminoprolyl (P1,P2)/4-guanidinyl (P3,P4) peptides but also the guanidinyl peptides exhibited a better enhancer effect in the absence of the equivalent Qiagen reagent. The 4(S)-guanidino peptide P4 was better than the 4(R)-guanidinyl peptide P3 in the GFP expression in the cells. Further, the 4(R/S)-guanidinylproline peptides P3 and P4 transfected the plasmid DNA (red bars) in the absence of enhancer with efficiency appreciably higher by 2 to 2.5 fold compared to that with Qiagen kit. These results clearly suggest that the 4(R/S)-guanidinylproline peptides are efficient functional enhancers in transfection of DNA. The Texas Red-tagged cationic peptides P6−P8 were used to examine the postuptake localization sites of peptides in S2 cells. These were obtained by reacting the resin-bound peptides with fluorescent Texas Red-X succinimidyl ester reagent followed by cleavage (Supporting Information). The fluorescent guanidinyl peptides P6 and P7 exhibited a punctated pattern in cytoplasm, while the fluorescent peptide SAP P8 showed a red diffused spread in cytoplasm (see the Supporting Information). Such punctation may suggest that the peptides P6 and P7 localize themselves into cytoplasmic organelles/endosomes,30 while the fluorescent SAP peptide P8 destabilizes the endosomes with the peptide spread all over the cytoplasm. In an experiment involving transfection of GFP encoding plasmid with fluorescent peptide, the expressed GFP protein could be visualized amidst the excess red fluorescent peptide in the cytoplasm (Figure 2D). The fluorescent peptides taken up by the cells induced no significant toxicity since the size distribution of the S2 cells remained essentially identical with that of the untreated cells. In conclusion, it is shown here that the 4(R/S)-aminoprolyl (P1, P2) and 4(R/S)-guanidinylproline (P3, P4) cationic collagen peptides boost the transfection efficiencies and the highly cationic guanidinyl peptides P3 and P4 are functional enhancers in transfecting the gene-encoded plasmids. Unlike the

Figure 2. Expession of GFP in Drosophila S2 cells in the presence of peptide P4: (A) DIC image of cells; (B) cells transfected with pRmHa3-GFP plasmid using Qiagen reagents and induced with CuSO4 (1 mM), (C) cells transfected in the presence of Peptide P4 and absence of enhancer; (D) superimposed confocal images of Texas red labeled fluorescent peptide P7 along with DAPI stained nuclei (blue) and the expressed green fluorescence (encircled).

In comparison to the GFP fluorescence obtained by the Qiagen kit with both reagents (Figure 2B), the cationic guanidine peptides P4 (Figure 2C) and P3 (Supporting Information) are able to specifically replace the “enhancer” component of the Qiagen kit in transfection of the GFP reporter vector (pRmHa3GFP). The higher intensities of the green fluorescence in the presence of P4 indicated improved transfection efficiencies with the guanidinyl peptides. The transfection efficiencies quantitated from the count of green fluorescent cells are shown in Figure 3. It is seen that (i) in the absence of Qiagen enhancer, the transfection efficiency of plasmid DNA decreases by 8 fold (control), (ii) peptides P1−P5 considerably enhance the transfection efficiencies of 4133

dx.doi.org/10.1021/jo300070p | J. Org. Chem. 2012, 77, 4131−4135

The Journal of Organic Chemistry

Note

Cleavage of Peptides from Resin, Purification and Characterization of Peptides. A mixture of trifluoroacetic acid and trifluoromethane sulfonic acid (90% TFA, 10% TFMSA, 4 mL) was added to the resin, and the mixture was allowed to stir for 2 h followed by filteration through glass wool into a 15 mL centrifuge tube. The resin was washed with TFA (50% in DCM, 3 × 2 mL), and the combined filtrate was concentrated in vacuo. The residue was dissolved in cold diethyl ether (4 mL) 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 by reversed-phase HPLC. All peptides were purified to homogeneity by reversed-phase HPLC and characterized by MALDI-TOF mass spectrometry: peptide P3, m/z 2056.8967 (2056.2589, calcd for C89H134N38O20); peptide P4, m/z 2056.8789 (2056.2589, calcd for C89H134N38O20); peptide P6, m/z 2717.8298 (2716.0736, calcd for C124H171N41O26S2); peptide P7, m/z 2716.3008 (2716.0736, calcd for C124H171N41O26S2); peptide P8, m/z 2699.1646 (2698.3417, calcd for C133H201N31O25S2). Circular Dichroism (CD) Spectroscopy. CD spectra were recorded on a CD spectropolarimeter using a cylindrical, jacketed quartz cell (10 mm path length), connected to a cold water circulator. Spectra were recorded with a spectral resolution of 0.05 nm, bandwidth 1 nm at a scan speed of 100 nm/min and a response time 1 s. All of the spectra were corrected for respective buffer conditions and are typically averaged over 6−12 scans. The samples were annealed in a water bath at 90 °C and slowly cooled to rt over a period of 6 h and kept at 4 °C for 12 h. The CD spectra were recorded at different temperatures in steps of 5 °C. The data processing was done using Origin 8.0 software. The Tm (melting temperature) values are derived from the first derivative curve of the temperature−CD plots. Plasmid DNA Isolation and Purification. E. coli cells were transformed with pRmHa3-GFP plasmid DNA and grown overnight in Luria−Bertani (LB) media with 50 μg/mL of ampicillin. DNA was isolated using standard Maxiprep protocol. Purified plasmid DNA was resuspended in deionized water. The concentration (1626 ng/μL) and purity were determined on a spectrophotometer at 260/280 (∼1.8). Agarose Gel Electrophoresis. pRmHa3-GFP plasmid DNA (1 μg; diluted to a final concentration of 0.1 μg DNA/μL) with increasing amounts of cationic peptides (P3, P4 and P5; 50 μg each in EDTA buffer; 1 mM, 15 μL) at ratios of 1:5, 1:10, 1: 25, 1:50 (w/w) were electrophoresed on agarose gel (1% with 50 ng/mL EtBr for visualization) at 90 V for 120 min in TAE buffer. Loading dye = 2 μL, 40% w/v sucrose and 0.25% bromophenol blue in deionized H2O. The bands were observed with a transilluminator and images were captured using a G:BOX digital camera. Cell Culture and Transfection. Drosophila melanogaster Schneider 2 (S2) cells were grown at 25 °C under normal atmosphere (100% air without CO2) and S2 cells medium was supplemented with 10% heat-inactivated FBS. Cells were grown to a density of 1 to 5 × 106 cells/ml, with splitting done into fresh medium at a 1:4 or 1:5 dilutions every 3 days. Qiagen’s transfection protocol29 followed for optimized transfection using Qiagen’s transfection kit and CuSO4 on a 24-well culture cluster. The cluster plate was analyzed by fluorescence light microscopy. Green fluorescent cells in addition to nonfluorescent cells were counted manually by focusing in different areas of the well, and average transfection efficiency was calculated. The cells were adhered onto coverslips with poly-L-lysine and fixed with 3% paraformaldehyde in PBS; MeOH free fixing solution and mounted to glass-slides with DAPI-Fluoromount-G for confocal images (experimental details in the Supporting Information).

triplex forming 4(R/S)-aminoproline peptides (P1 and P2), the highly cationic 4(R/S)-guanidinylproline peptides (P3 and P4) remain as single-chain PPII helices and condense DNA very efficiently, leading to enhanced DNA transfection. These interesting results prompt elaborate studies involving temperature and time dependence of the peptide uptake and their effects in different cell lines to understand the mechanistic aspects. Conjugation with lipid chains may also impart hybrid functions of both DNA compaction and cell permeability to a single reagent leading to rational design of much needed nonviral DNA transfection agents,31 and as probes for delineating the mechanisms of cell penetration.

■

EXPERIMENTAL SECTION

Resins for solid-phase peptide synthesis and Fmoc-protected amino acids, fluorescent molecular probe Texas Red-X, succinimidyl ester, transfection kit, and general reagents and chemicals were obtained from commercial sources. All starting materials were used without purification. The plasmid DNA (pRmHa3-GFP) was received as a gift from A. J. Courey’s Laboratory at ULCA. Salts and reagents used in buffer preparation were of molecular biology reagent grade. Synthesis of Monomers 1a and 1b. Synthesis of N1-t-Boc- and N4-Fmoc-protected (2S,4R)- and (2S,4S)-4-aminoproline monomers (1a and 1b) were achieved in six steps from the naturally occurring trans-4(R)-hydroxyproline as per previously reported procedure.14,15 General Synthesis of Cationic Peptides (P1−P5). These peptides were synthesized by manual solid phase synthesis on MBHA (4-methylbenzhydryl amine) resin (loading value = 0.8 g/mol) using the standard t-Boc protocol from the C-terminus to the N-terminus using orthogonally protected N4-Fmoc and N1-t-Boc monomeric units (1a and 1b), which upon cleavage directly yielded the peptide-Cterminal amide. MBHA (HCl salt) resin (100 mg) was taken in a peptide synthesis flask (10 mL), washed with DCM (2 × 4 mL), and neutralized with DIPEA (50% in DCM, 3 × 4 mL). The resin was further washed with DCM (2 × 4 mL) and DMF (2 × 4 mL). Boc-protected amino acids or monomers (3 equiv, 0.18 mmol) in DMF (2 mL) were added to the reaction flask with HBTU (3 equiv, 0.18 mmol) and DIPEA (3 equiv, 0.18 mmol), and the flask was agitated for 4 h. The resin was washed successively with DMF, DCM, MeOH, DCM, and DMF (2 × 3 mL each). TFA (50% in DCM, 3 × 4 mL) was added to the reaction flask and agitated for 5 min followed by draining the solution. The resin was then neutralized with DIPEA (5% in DCM, 3 × 4 mL) and washed with DCM and DMF (2 × 3 mL each).This procedure was repeated until all amino acids were sequentially coupled to the resin. The terminal amino group of the final peptide was capped with Ac2O (50% in DCM−DIPEA mixture, 3 mL) and the side-chain Fmoc protection removed by stirring with piperidine (20% in DMF, 4 mL) for 30 min. The deprotection and coupling reactions were monitored using qualitative Ninhydrin (Kaiser) test32 for Gly and Chloranil test33 for imino acids. The control peptide P5 (SAP) was synthesized on an automated synthesizer pursuing Fmoc-chemistry and using commercially available Fmoc-protected amino acids as per the literature protocol.26 On-Resin Guanidylation. Guanidylation was carried out directly on the peptides bound to resin. N,N′-Bis-Boc-1H-pyrazole1-carboxamidine23,24 (30 equiv, 1.8 mmol) and DIPEA (30 equiv, 1.8 mmol) in DMF (4 mL) were added to the resin-bound peptides and stirred for 4 h at rt. The resin was washed with DMF, DCM, and MeOH (2 × 4 mL each) and dried in vacuo for 3 h. Synthesis of Texas Red Labeled Peptides (P6−P8). The resin after synthesis of peptides was washed with DMF (2 × 4 mL) and NMP (1 × 4 mL). N-Succinimidyl ester of Texas Red-X reagent (1.1 equiv, 0.06 mmol) and DIPEA (1.1 equiv, 0.06 mmol) were added, along with 4 mL of NMP. The reaction was kept stirred at rt for 24 h in the dark, washed successively with NMP, DMF, and MeOH (2 × 4 mL each), and dried in vacuo for 3 h.

■

ASSOCIATED CONTENT

S Supporting Information *

Procedures for solid-phase synthesis and characterization with HPLC, MALDI-TOF spectra of peptides P1−P8, 1H and 13C NMR of monomers, CD spectra, Tm data, cell transfection, and 4134

dx.doi.org/10.1021/jo300070p | J. Org. Chem. 2012, 77, 4131−4135

The Journal of Organic Chemistry

Note

(26) Pozo-Rodríguez, A. D.; Pujals, S.; Delgado, D.; Solinís, M. A.; Gascón, A. R.; Giralt, E.; Pedraz, J. L. J. Controlled Release 2009, 133, 52−59. (27) Sonar, M. V.; Ganesh, K. N. Org. Lett. 2010, 12, 5390−5393. (28) Erdmann, R. S.; Wennemers, H. Angew. Chem., Int. Ed. 2011, 50, 6835−6838. (29) Effectene Transfection Reagent Handbook, Qiagen, May 2002, www.qiagen.com/products/transfection/transfectionreagents/ effectenetransfectionreagent.aspx#Tabs=t2. (30) Geisler, I. M.; Chmielewski, J. Pharm. Res. 2011, 28, 2797− 2807. (31) Masotti, A.; Mossa, G.; Cametti, C.; Ortaggi, G.; Bianco, A.; Grosso, N. D.; Malizia, D.; Esposito, C. Colloids Surf., B 2009, 68, 136−144. (32) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal. Biochem. 1970, 34, 595−598. (33) Dörwald, F. Z. Organic Synthesis on Solid Phase: Supports, Linkers, Reactions, 2nd ed.; Wiley-VCH: Weinheim, 2002; p 7.

permeation results. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Tel: 91 (20) 2590 8021. Fax: 91 (20) 2589 9790. E-mail: kn. [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS M.N. thanks CSIR (New Delhi) for the award of a research fellowship. K.N.G. is a recipient of a JC Bose Fellowship from DST (New Delhi). We thank Dr. Girish Ratnaparkhi (IISER Pune) for his helpful discussions and assistance in the biological experiments. We acknowledge Dr. Richa Ricky (IISER Pune) for expert assistance in confocal microscopy.

■

■

NOTE ADDED AFTER ASAP PUBLICATION Minor text changes throughout the text and a change to the journal name in ref 14 were made to the version published ASAP on March 28, 2012; the corrected version was reposted on March 30, 2012.

REFERENCES

(1) Fonseca, S. B.; Pereira, M. P.; Kelley, S. O. Adv. Drug Delivery Rev. 2009, 61, 953−964. (2) Said Hassane, F.; Saleh, A. F.; Abes, R.; Gait, M. J.; Lebleu, B. Cell. Mol. Life Sci. 2010, 67, 715−726. (3) Torchilin, V.; Sawant, R. Mol. BioSyst. 2010, 6, 628−640. (4) Heitz, F.; Morris, M. C.; Divita, G. Br. J. Pharmacol. 2009, 157, 195−206. (5) Wagner, E. Adv. Drug Delivery Rev. 1999, 38, 279−289. (6) Luo, D.; Saltzman, W. M. Nature Biotechnol. 2000, 18, 33−7. (7) Joliot, A.; Prochiantz, A. Nat. Cell Biol. 2004, 6, 189−196. (8) Escriou, V.; Carrière, M.; Scherman, D.; Wils, P. Adv. Drug Delivery Rev. 2003, 55, 295−306. (9) Pujals, S.; Giralt, E. Adv. Drug Delivery Rev. 2008, 60, 473−84. (10) Fillon, Y. A.; Anderson, J. P.; Chmielewski, J.; Lafayette, W. Synthesis 2005, 127, 11798−11803. (11) Sano, A.; Maeda, M.; Nagahara, S.; Ochiya, T.; Honma, K.; Itoh, H.; Miyata, T.; Fujioka, K. Adv. Drug Delivery Rev. 2003, 55, 1651− 1677. (12) Takeshita, F.; Minakuchi, Y.; Nagahara, S.; Honma, K.; Sasaki, H.; Hirai, K.; Teratani, T.; Namatame, N.; Yamamoto, Y.; Hanai, K.; Kato, T.; Sano, A. Pharmacia 2005, 102, 1−6. (13) Svintradze, D. V.; Mrevlishvili, G. M.; Metreveli, N.; Jariashvili, K.; Namicheishvili, L.; Skopinska, J.; Sionkowska, A. Biomacromolecules 2008, 9, 21−28. (14) Babu, I. R.; Ganesh, K. N. J. Am. Chem. Soc. 2001, 123, 2079− 2080. (15) Umashankara, M.; Babu, I. R.; Ganesh, K. N. Chem. Commun. 2003, 9, 2606−2607. (16) McKenzie, D. L.; Collard, W. T.; Rice, K. G. J. Peptide Res. 1999, 54 (4), 311−318. (17) Futaki, S.; Nakase, I.; Tadokoro, A.; Takeuchi, T.; Jones, A. T. Biochem. Soc. Trans. 2007, 35, 784−787. (18) Rothbard, J. B.; Kreider, E.; VanDeusen, C. L.; Wright, L.; Wylie, B. L.; Wender, P. A. J. Med. Chem. 2002, 45, 3612−3618. (19) Schmidt, N.; Mishra, A.; Lai, G. H.; Wong, G. C. L. FEBS Lett. 2010, 584, 1806−1813. (20) Wender, P. A.; Galliher, W. C.; Goun, E. A.; Jones, L. R.; Pillow, T. H. Adv. Drug Delivery Rev. 2008, 60, 452−472. (21) Geisler, I.; Chmielewski, J. Chem. Biol. Drug Des. 2009, 73, 39− 45. (22) Gonçalves, E.; Kitas, E.; Seelig, J. Biochemistry 2005, 44, 2692− 2702. (23) Drake, B.; Patek, M.; Lebl, M. Synthesis 1994, 1994, 579−582. (24) Olsen, C. A.; Bonke, G.; Vedel, L.; Adsersen, A.; Witt, M.; Franzyk, H.; Jaroszewski, J. W. Org. Lett. 2007, 9, 1549−1552. (25) Jubilut, G. N.; Cilli, E. M.; Tominaga, M.; Miranda, A.; Okada, Y.; Nakaie, C. R. Chem. Pharm. Bull. 2001, 49, 1089−1092. 4135

dx.doi.org/10.1021/jo300070p | J. Org. Chem. 2012, 77, 4131−4135