276
Bioconjugate Chem. 2003, 14, 276−281
COMMUNICATIONS An Efficient and Versatile Synthesis of BisPNA-Peptide Conjugates Based on Chemoselective Oxime Formation Philippe Neuner,† Pasquale Gallo,† Laura Orsatti,‡ Laura Fontana,† and Paolo Monaci*,† Department of Molecular & Cell Biology and Department of Pharmacology, I.R.B.M. P. Angeletti, Pomezia (Roma), Italy. Received September 25, 2002
Oligomers with two identical peptide nucleic acid sequences joined by a flexible hairpin linker (bisPNA) can stably bind to specific DNA sequences without altering plasmid supercoiling, thus offering a unique opportunity to attach various functional entities to high molecular weight DNA. Current synthetic approaches, however, severely limit the possibility to link peptides or other chemical moieties (i.e., sugars, oligonucleotides, etc.) to bisPNA. Here we report a novel strategy for the synthesis of bisPNApeptide conjugates in which chemoselective ligation of bisPNA to peptides was accomplished through oxime formation between an oxy-amine-containing peptide and a bisPNA-methyl ketone (complementary modifications can also be used). The described synthesis is highly efficient, does not require a protection strategy, and is carried out under mild aqueous conditions. Through this methodology long peptide sequences in either C to N or N to C polarity can be linked to bisPNA. In addition, this protocol makes the conjugation of cysteine-containing peptides feasible and allows disulfide bond formation to be controlled. This same approach can be exploited to link oligonucleotides, sugars, or other chemical entities to bisPNA.
INTRODUCTION
Plasmid-based, nonviral gene delivery systems are a promising therapeutic approach for gene therapy and genetic immunization protocols (1-3). However, the poor efficiency and specificity of gene transduction obtained in vivo hampers the full exploitation of their potential in clinical settings. Several key steps can be identified along the route leading to gene expression: DNA condensation, efficient and specific targeting of cell surface receptors, cellular uptake, escape from the endosome/ lysosome, stability against nucleases, intracellular distribution, efficient recruitment of transcription factors, etc. In many cases specific peptide sequences have been shown to increase the efficiency of one or more of these steps (4-8). However, conjugating peptides to DNA is not a straightforward procedure. A stable modification of plasmid DNA in its supercoiled form is often required, multiple copies of the peptides with defined polarity need to be conjugated, and the modification must not interfere with plasmid transcriptional activity. Peptide nucleic acid (PNA) is a DNA analogue in which repeating N-(2-aminoethyl)glycine units replace the deoxyribose-phosphate backbone (9, 10). Due to the neutral charge of its backbone, PNA hybridizes to complementary DNA with affinity and specificity higher than the corre* Corresponding author: Paolo Monaci, IRBM P. Angeletti, Via Pontina km 30.600, 00040 - Pomezia (Roma), Italy. Tel. +39-06-91093-242, fax +39-06-91093-654, e-mail:
[email protected]. † Department of Molecular & Cell Biology. ‡ Department of Pharmacology.
sponding DNA-DNA hybrid (11). A bisPNA molecule is an oligomer with two identical PNA sequences joined by a flexible hairpin linker (11-13). Binding of bisPNA to its DNA target is sequence-specific, stoichiometric, extremely stable, and essentially irreversible under physiological conditions (11, 14). Of note is that a bisPNA molecule targets a specific site on DNA, invades duplex DNA, and displaces the complementary DNA strand to form a stable PNA:DNA:PNA triplex hybrid, without affecting the supercoiled form of plasmid DNA (11). These properties identify bisPNA as a preferential method to efficiently link molecules with various functional properties at specific sites on supercoiled plasmid DNA. For example, fluorescein or rhodamine can be linked to bisPNA and the ensuing conjugate used to bind irreversibly and specifically to a binding site cloned into a plasmid to track distribution in vivo (14). With the aim of improving plasmid-mediated gene transduction, peptides emerge as the most interesting class of molecules to link to bisPNA. Current methods for preparing bisPNA-peptide conjugates are based on collinear synthesis of the hybrid molecule, which present some drawbacks to its general application (15). For instance, synthesis is limited to C to N polarity of the peptide and is rather inefficient for long peptide sequences. In addition, the collinear synthesis of peptides containing cysteine residues is not feasible, since formation of disulfide bridges cannot be controlled. Finally, linking to bisPNA chemical moieties other than peptides, such as carbohydrates or oligonucleotide, clearly require alternative synthesis protocols.
10.1021/bc020060p CCC: $25.00 © 2003 American Chemical Society Published on Web 02/26/2003
Communications
Bioconjugate Chem., Vol. 14, No. 2, 2003 277
Figure 1. Ligation of amino-oxy peptide to bisPNA-methyl ketone through oxime linkage. Table 1. Peptides Linked to BisPNAa peptide
length
polarity
sequence
NL1.1 NL4c (VP16)2 (VP16)3
37 aa 35 aa 28 aa 41 aa
C to N C to N N to C N to C
NH2-AEGEFMYWGDSHWLQYWYEGDPAKGGSGGGSGGGK(aox)G-CONH2 NH2-AEGEFFCVSSGGGSSCWPDPAKGGSGGGSGGGSK(aox)G-COHN2 (aox)NH-GGPADALDDFDLDMLPADALDDFDLDML-CONH2 (aox)NH-GGPADALDDFDLDMLPADALDDFDLDMLPADALDDFDLDML-CONH2
a
aox, amino-oxyacetyl.
Recently, Felgner and co-workers described an approach in which sulfhydryl-containing peptides are conjugated to streptavidin maleimide, and the resulting streptavidin conjugates are then bound to a biotinbisPNA-labeled plasmid (16). Alternatively, the same peptides can be directly coupled to a maleimide-bisPNA conjugate bound to plasmid DNA. This approach can also be extended to oligonucleotides containing free SH groups, but not to peptides containing cysteine residues. As a further step toward broadening the applications of bisPNA conjugates, we describe an alternative and more versatile synthetic approach to conjugate peptides and other molecules to bisPNA based on chemoselective ligation of an amino-oxy peptide to a bisPNA-methyl ketone which circumvents the limitations listed above. RESULTS AND DISCUSSION
We adopted a novel synthesis approach to link peptides to bisPNA. An amino-oxy peptide was conjugated to bisPNA-methyl ketone through an oxime forming ligation (Figure 1). The amino-oxy group could be displayed either at the C or at the N terminus of the peptide. Complementary modifications (i.e., amino-oxy group at the N-terminus of the bisPNA and methyl ketone group at the N or C terminus of the peptide) can also be used. Solid-Phase Synthesis of the Amino-oxy Peptides. Four different peptides were linked to a bisPNA molecule (Table 1). We chose peptides with specific biological properties, which differed in length, presence of cysteine residues, and polarity of conjugation to bisPNA. NL1.1 and NL4c are 24 and 22 aa long peptides, respectively, which specifically bind to HER2/neu, a tyrosine-kinase
receptor which is overexpressed in 30-50% of human breast, gastric, and colon tumors (17, 18). These peptides have been identified by screening phage-displayed random peptide libraries (19, unpublished data). Of note, NL4c contains two cysteine residues forming a disulfide bridge. Both peptides must be linked C to N to bisPNA. VP16 is 13 aa minimal transcription activator domain derived from herpes simplex virus protein 16 (20). Just two VP16 peptides bound to DNA through a GAL4 DNA binding domain are sufficient to activate transcription from a downstream promoter (20). We thus attempted to generate a minimal transcription activator by linking two or three VP16 peptides with the N to C polarity to bisPNA. Binding of this bisPNA chimera to plasmid DNA could enhance transcription from a downstream promoter without requiring the recruitment of intracellular transcription activators. Peptides NL1.1, NL4c, VP162, and VP163 were synthesized by standard Fmoc chemistry. GGGS linker sequences were added to the C-terminus of NL1.1 (11 residues) and NL4c (12 residues) to facilitate the interaction of the peptides with the target promoter (21). An amino-oxy group was incorporated at the C terminus of NL1.1 and NL4c peptide by coupling the (amino-oxy) acetyl functionality at the -amine side chain of an extra C-terminal Lys residue, as described by Canne et al. (15). Similarly, an amino-oxy group was incorporated at the N terminus of peptide VP162 and VP163. Peptides were purified by HPLC and their structure verified by mass spectroscopy. Solid-Phase Synthesis of the BisPNA-Methyl Ketone. All reagents including Boc-PNA-C(Z), Boc-PNAT, Boc-AEEA-ODCHA spacer (L ) 2-aminoethoxy-2-
278 Bioconjugate Chem., Vol. 14, No. 2, 2003
Neuner et al. Table 2. Experimental and Calculated Mass for the BisPNA-Peptide Chimera BisPNA-NL1.1 BisPNA-NL4 BisPNA-VP162 BisPNA-VP163
Figure 2. HPLC and mass analysis of the purified bisPNAmethyl ketone.
ethoxyacetic acid), and HBTU were obtained from PerSeptive Biosystems (Framingham, MA). MBHAresin was obtained from Calbiochem-Novabiochem Ag (Laufelfingen, Switzerland). A bisPNA-methyl ketone oligomer (mKetone-NH-LL-TCTCTCTC-LLL-JTJTJTJTCONH2) designed to bind the DNA sequence 5′GAGAGAGAGA-3′ was synthesized by hand on MBHAresin (215 mg; substitution ) 45.5 µmol/g; 10 µmol scale) essentially as described (22). Pseudoisocytosine J, an analogue of C which encourages a pH-independent specific formation of the Hoogsteen triplex hybrid, was synthesized as described by Egholm et al. (11). In a typical procedure, 40 µmol of monomers (4 equiv), N,N-diisopropylethylamine (6 equiv/monomer) and HBTU (0.9 equiv/monomer) were dissolved in 800 µL of a mixture N-methylpyrrolidinone/pyridine (1:1). After 3 min of preactivation, the reaction mixture was applied to the resin, coupling was allowed to proceed for 4 h, and resin was washed with DMF/DCM (twice for 3 min each). Prior to the next coupling step, the Boc-protected amino terminus was deprotected with a mixture of TFA/m-cresol (95:5; twice for 4 min each). After completion of the bisPNA sequence, the methyl ketone moiety was introduced by activating 4-acetylbutyric acid (100 µmol) as symmetric anhydride with DIC (50 µmol) in DCM. After
M (experimental)
M (calculated)
8886.00 8225.00 8046.00 9478.00
8887.65 8227.27 8047.32 9479.91
activation for 15 min, the mixture was diluted with DMF (1 mL), added to the PNA-resin, and coupled for 60 min. The resin was washed with DMF/DCM, dried, and transferred to a screw cap vial. The product bisPNA was deprotected and concomitantly cleaved from the resin by treatment with a mixture of TFMSA/TFA/m-cresol (18: 73:9; 1 mL) for 2 h at 0 °C. The solid was separated by filtration and the filtrate poured into 10 volumes of ice cold Et2O. Centrifugation yielded a pellet, which was purified by HPLC. M (calculated) for C204H282N78O73 ) 4995.00; M (experimental) ) 4994.00 (Figure 2). Synthesis of the BisPNA-Peptide Conjugate: Oxime-Forming Ligation. Purified bisPNA-methyl ketone was mixed with the amino-oxy peptide to generate the oxime-linked bisPNA-peptide conjugate. The aminooxy group was displayed either at the C (NL1.1 and NL4c) or at the N terminus (VP162 and VP163) of the peptide (Figure 1). In a typical experiment, bisPNA-methyl ketone 1 was coupled to the amino-oxy peptide 2 via oxime ligation (15). The bisPNA-methyl ketone (0.2 µmol) was dissolved in 250 µL of 0.1 M sodium acetate buffer (pH 4.2). Two equivalents (0.4 µmol) of the amino-oxy peptide and 20 µL of DMF were added to the resulting solution. Coupling reaction was allowed to proceed for 12 h, and the resulting conjugate was readily purified by semipreparative reverse-phase HPLC and lyophilized to give a white solid. Preparative RP C18 HPLC purification was performed using a SymmetryPrep C18 (7.8 × 150 mm) reverse-phase column (Waters, Milford, MA). Eluant A: 99.9% water, 0.1% TFA; eluant B: 10% water, 89.9% acetonitrile, 0.1% TFA; a linear gradient of 5%-30%
Figure 3. HPLC and mass analysis of the purified bisPNA-peptide chimeras. BisPNA-VP162 (A), bisPNA-VP163 (B), bisPNANL1.1 (C). and bisPNA-NL4c (D).
Communications
Bioconjugate Chem., Vol. 14, No. 2, 2003 279
Figure 4. Iodoacetamide test reaction with reduced (path A) and oxidized (path B) bisPNA-NL4c chimera. Table 3. Nucleotides Sequence of Oligonucleotides Harboring BisPNA Binding Site
eluant B over 25 min at a flow rate of 3.0 mL/min monitored at 260 nm. The average yield was usually 1520% of purified chimeras. Final product was analyzed for purity by mass determination by LCQquadrupole ion trap mass spectrometer Finningan-MAT (San Jose, CA, Figure 3). Experimentally derived and calculated mass for the four bisPNA-peptides products are listed in Table 2. As for the bisPNA-NL4c chimera, the crude product was purified by HPLC after oxime ligation, and the desired fractions were lyophilized. The resulting solid was resuspended at a concentration of 1 mg/mL in distilled water and air-oxidized overnight to allow intramolecular disulfide bridge formation. Oxidation of the NL4c peptide cysteine residues was assessed by adding a small excess of iodoacetamide to an aliquot of the purified bisPNANL4c chimera before and after air oxidation (Figure 4, paths A and B, respectively). Mass analysis of the iodoacetamide reaction product derived from path A showed an increase in mass of 114 (2 × 57), corresponding to the two acetamide adducts of the reduced bisPNANL4c chimera, whereas no mass variation was detected for the oxidized bisPNA-NL4c.
Figure 5. Specific binding of bisPNA-VP16 chimeras to target sequence. Three and six pmoles of bisPNA-methyl ketone (lanes 2, 3 and 9, 10), bisPNA-VP162 (lanes 4, 5 and 11, 12) or bisPNA-VP163 (lanes 6, 7 and 13, 14) were incubated at 37 °C for 15 min with approximately 1.5 pmol of the [R-32P]-labeled double stranded oligonucleotide in 20 µL of HBS buffer (10 mM Hepes, 50 mM NaCl, pH 7.0). Lanes 2-7 and lanes 9-14 refer to reactions with target or control oligonucleotide, respectively. Additional control reactions were included in the absence of bisPNA (lanes 1 and 8, with target and control oligo, respectively). Five microliters of 20% glycerol was added, and the samples were fractionated on a 5% polyacrylamide gel in TBE buffer (45 mM Tris-borate, 1 mM EDTA, pH ) 7.5) at 10 V/cm for 3-4 h at 4 °C. Gel was dried and autoradiographed on Kodac XR5 film with intensifying screen.
280 Bioconjugate Chem., Vol. 14, No. 2, 2003
Neuner et al.
These results indicate that bisPNA-VP162 or bisPNAVP163 retains the binding properties of the reference bisPNA-methyl ketone and similarly prevented bisPNAfluorescein binding to its target site on supercoiled plasmid DNA. ACKNOWLEDGMENT
Figure 6. BisPNA-peptide chimeras bind the target sequence within supercoiled plasmid DNA. Increasing amounts (35, 70, 140, and 280 pmol) of bisPNA-methyl ketone (empty circles, lanes 4-7), bisPNA-VP162 (filled squares; lanes 8-11), or bisPNA-VP163 (filled triangles; lanes 12-15) were incubated with 1.12 pmol of pGGluc plasmid DNA in 40 µL of HBS buffer for 45 min. The binding of these bisPNA derivatives was challenged, adding 32 pmol of bisPNA-fluorescein to the reaction and incubating for additional 20 min. Control reactions with bisPNA fluorescein (lane1), pGGluc (lane 2), or bisPNAfluorescein conjugated with pGGluc (lane 3) were also included. Seven microliters of 20% glycerol were added, and the samples were fractionated on a 0.8% agarose gel in TBE buffer (90 mM Tris-borate, 2 mM EDTA, pH 7.5). The incubations were performed at 37 °C and the samples run at room temperature. Gels were scanned using a GS-700 Imaging Densitometer BIORAD (Hercules, CA). Quantitation of the PNA-fluorescein conjugated was performed with a MacIntosh G3 computer using the program Molecular Analyst.
Characterization of the BisPNA-Peptide Conjugates. We wanted to assess the DNA-binding properties of these bisPNA-peptide chimeras. To this end we synthesized two double-stranded oligonucleotides containing the bisPNA binding sequence (target oligo) and the same sequence with two mutations (control oligo) (Table 3; binding sequence is indicated in bold and mutation are underlined). Two different amounts of bisPNA-methyl ketone, bisPNA-VP162, or bisPNAVP163 were incubated with target or control oligonucleotide (2 and 4 molar ratios). Formation of the DNA:bisPNA-VP16 complex was detected by a gel mobility shift assay (Figure 5). Both bisPNA-VP162 and bisPNA-VP163 chimeras bound the target oligonucleotide in a manner indistinguishable from that of the reference bisPNA-methyl ketone molecule (Figure 5, lanes 2-7). By contrast, no binding could be detected to control oligonucleotide in the same conditions (Figure 5, lanes 9-14). Next, we tested the ability of the same chimeras to bind the target binding sequence in the context of a supercoiled plasmid DNA. We used plasmid pGGluc (Gene Therapy Systems, San Diego, CA) which bears a CMV immediate early promoter driving the expression of a luciferase reporter gene. This plasmid contains 10 binding sites for bisPNA molecule immediately after the translational termination site. We preincubated pGGluc plasmid DNA with increasing amounts of bisPNAmethyl ketone, bisPNA-VP162, or bisPNA-VP163. Then, a fixed amount of bisPNA-fluorescein was allowed to bind plasmid DNA. Increasing the amount of either bisPNA derivatives reduced fluorescence-labeling of the plasmid (Figure 6).
In summary, the data we report in this paper demonstrate that bisPNA-peptide conjugate can be prepared by post-assembly conjugation of an amino-oxy peptide with a bisPNA-methyl ketone molecule. The reaction is simple, efficient, and versatile, since it can be applied to linking long peptides, with or without cysteine residues, in both polarities, to bisPNA. The same approach can be extended to linking other chemical moieties to bisPNA, namely to oligonucleotides. The synthetic approach we describe makes generating several different bisPNApeptide conjugates limited only by the efficiency of synthesis of the individual components. Indeed, bisPNApeptide chimeras appear particularly suited for providing plasmid DNA with new functions (e.g., targeting specific cell types, subcellular localization, transcription activating domain, etc.) which could improve the efficiency of gene expression, in vitro and in vivo. ACKNOWLEDGMENT
We wish to thank Antonello Pessi for useful discussion, Marco Finotto for the synthesis of the modified peptides, and Janet Clench for proofreading the manuscript. LITERATURE CITED (1) Spack E. G., and Sorgi, F. L. (2001) Developing nonviral DNA delivery systems for cancer and infectious disease. Drug Discov. Today 6, 186-197. (2) Luo, D., and Saltzman, W. M. (2000) Synthetic DNA delivery systems. Nat. Biotechnol. 18, 33-37. (3) Donnelly, J. J., Ulmer, J. B., Shiver, J. W., and Liu, M. A. (1997) DNA vaccines. Annu. Rev. Immunol. 15, 617-648. (4) Branden, L. J., Christensson, B., and Smith, C. I. (2001) In vivo nuclear delivery of oligonucleotides via hybridizing bifunctional peptides. Gene Ther. 8, 84-87. (5) Good, L., Awasthi, S. K., Dryselius, R., Larsson, O., Nielsen, P. E. (2001) Bactericidal antisense effects of peptide-PNA conjugates. Nat. Biotechnol. 19, 360-364. (6) Cutrona, G., Carpaneto, E. M., Ulivi, M., Roncella, S., Landt, O., Ferrarini, M., and Boffa, L. C. (2000) Effects in live cells of a c-myc anti-gene PNA linked to a nuclear localization signal. Nat. Biotechnol. 18, 300-303. (7) Scarfi, S., Giovine, M., Gasparini, A., Damonte, G., Millo, E., Pozzolini, M., and Benatti, U. (1999) Modified peptide nucleic acids are internalized in mouse macrophages RAW 264.7 and inhibit inducible nitric oxide synthase. FEBS Lett. 451, 264-268. (8) Pooga, M., Soomets, U., Hallbrink, M., Valkna, A., Saar, K., Rezaei, K., Kahl, U., Hao, J. X., Xu, X. J., Wiesenfeld-Hallin, Z., et al. (1998) Cell penetrating PNA constructs regulate galanin receptor levels and modify pain transmission in vivo. Nat. Biotechnol. 16, 857-861. (9) Nielsen, P. E., Egholm, M., Berg, R. H., and Buchardt, O. (1991) Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science 254, 1497-1500. (10) Egholm, M., Buchardt, O., Christensen, L., Behrens, C., Freier, S. M., Driver, D. A., Berg, R. H., Kim, S. K., Norden, B., and Nielsen, P. E. (1993) PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogenbonding rules. Nature 365, 566-568. (11) Egholm, M., Christensen, L., Dueholm, K. L., Buchardt, O., Coull, J., and Nielsen, P. E. (1995) Efficient pHindependent sequence-specific DNA binding by pseudoisocytosine-containing bis-PNA. Nucleic Acids Res. 23, 217-22.
Communications (12) Vickers, T. A., Griffith, M. C., Ramasamy, K., Risen, L. M., and Freier, S. M. (1995) Inhibition of NF-kappa B specific transcriptional activation by PNA strand invasion. Nucleic Acids Res. 23, 3003-3008. (13) Kuhn, H., Demidov, V. V., Nielsen, P. E., and FrankKamenetskii, M. D. (1999) An experimental study of mechanism and specificity of peptide nucleic acid (PNA) binding to duplex DNA. J. Mol. Biol. 286, 1337-45. (14) Zelphati, O., Liang, X., Hobart, P., and Felgner, P. L. (1999) Gene chemistry: functionally and conformationally intact fluorescent plasmid DNA. Hum. Gene Ther. 10, 15-24. (15) Canne, L. E., Ferre-D’Amare, A. R., Burley, S. K., and Kent, S. B. H. (1995) Total Chemical Synthesis of a Unique Transcription Factor-Related Protein: cMyc-Max. J. Am. Chem. Soc. 117, 2998-3007. (16) Zelphati, O., Liang, X., Nguyen, C., Barlow, S., Sheng, S., Shao, Z., and Felgner, P. L. (2000) PNA-dependent gene chemistry: stable coupling of peptides and oligonucleotides to plasmid DNA. Biotechniques 28, 304-310, 12-14, 16. (17) Slamon, D. J., and Clark, G. M. (1988) Amplification of c-erbB-2 and aggressive human breast tumors? Science 240, 1795-1798.
Bioconjugate Chem., Vol. 14, No. 2, 2003 281 (18) Klapper, L. N., Kirschbaum, M. H., Sela, M., and Yarden, Y. (2000) Biochemical and clinical implications of the ErbB/ HER signaling network of growth factor receptors. Adv. Cancer Res. 77, 25-79. (19) Urbanelli, L., Ronchini, C., Fontana, L., Menard, S., Orlandi, R., and Monaci, P. (2001) Targeted gene transduction of mammalian cells expressing the HER2/neu receptor by filamentous phage. J. Mol. Biol. 313, 965-76. (20) Seipel, K., Georgiev, O., and Schaffner, W. (1992) Different activation domains stimulate transcription from remote (‘enhancer’) and proximal (‘promoter’) positions. EMBO J. 11, 4961-8. (21) Argos, P. (1990) An investigation of oligopeptides linking domains in protein tertiary structures and possible candidates for general gene fusion. J. Mol. Biol. 211, 943-58. (22) Christensen, L., Fitzpatrick, R., Gildea, B., Petersen, K. H., Hansen, H. F., Koch, T., Egholm, M., Buchardt, O., Nielsen, P. E., et al. (1995) Solid-phase synthesis of peptide nucleic acids. J. Pept. Sci. 1, 185-83.
BC020060P
