Synthesis of Radiometal-Labeled and Fluorescent Cell-Permeating

Bioconjugate Chem. 2003, 14, 1083−1095

1083

Synthesis of Radiometal-Labeled and Fluorescent Cell-Permeating Peptide-PNA Conjugates for Targeting the bcl-2 Proto-oncogene Fabio Gallazzi,† Yi Wang,‡ Fang Jia,‡ Nalini Shenoy,§ Linda A. Landon,| Mark Hannink,| Susan Z. Lever,§,⊥ and Michael R. Lewis*,‡,X Molecular Biology Program, Department of Veterinary Medicine and Surgery, Department of Chemistry, Department of Biochemistry, University of Missouri Research Reactor, and Department of Radiology, University of MissourisColumbia, Columbia, Missouri 65211. Received May 16, 2003; Revised Manuscript Received September 18, 2003

The B-cell lymphoma/leukemia-2 (bcl-2) proto-oncogene has been associated with the transformation of benign lesions to malignancy, disease progression, poor prognosis, reduced survival, and development of resistance to radiation and chemotherapy in many types of cancer. The objective of this work was to synthesize an antisense peptide nucleic acid (PNA) complementary to the first six codons of the bcl-2 open reading frame, conjugated to a membrane-permeating peptide for intracellular delivery, and modified with a bifunctional chelating agent for targeting imaging and therapeutic radiometals to tumors overexpressing bcl-2. Four peptide-PNA constructs were synthesized by a combination of manual and automated stepwise elongation techniques, including bcl-2 antisense conjugates and nonsense conjugates with no complementarity to any known mammalian gene or DNA sequence. The PNA sequences were synthesized manually by solid-phase 9-fluorenylmethoxycarbonyl (Fmoc) techniques. Then a fully protected lysine monomer, modified with 1,4,7,10-tetraazacyclododecaneN,N′,N′′,N′′′-tetraacetic acid (DOTA) for radiometal chelation, was coupled manually to each PNA sequence. Synthesis of the DOTA-PNA conjugates was followed by automated elongation with a peptide sequence (PTD-4-glycine, PTD-4-G), known to mediate cellular internalization of impermeable effector molecules, or its retro-inverso analogue (ri-PTD-4-G). Preparation of the four conjugates required an innovative synthetic strategy, using mild acid conditions to generate hydrophobic, partially deprotected intermediates. These intermediates were purified by semipreparative reversed-phase HPLC and completely deprotected to yield pure peptide-PNA conjugates in 6% to 9% overall yield. Using modifications of this synthetic strategy, the ri-PTD-4-G conjugate of bcl-2 antisense PNA was prepared using a lysine derivative of tetramethylrhodamine (TMR) for fluorescence microscopy. Plasma stability studies showed that 111In-DOTA-labeled ri-PTD-4-G-anti-bcl-2 PNA was stable for 168 h at 37 °C, unlike the conjugate containing the parent peptide sequence. Scanning confocal fluorescence microscopy of TMR-labeled ri-PTD-4-G-anti-bcl-2 PNA in Raji lymphoma cells demonstrated that the retro-inverso peptide was active in membrane permeation and mediated cellular internalization of the antisense PNA into the cytoplasm, where high concentrations of bcl-2 mRNA are expected to be present.

INTRODUCTION

Apoptosis, or programmed cell death, is a major pathway by which both chemotherapeutic drugs and ionizing radiation kill tumor cells (1). The B-cell lymphoma/leukemia-2 (bcl-2)1 gene is a member of a relatively new category of proto-oncogenes involved in cell survival, by blocking apoptosis (2). The bcl-2 gene product is an inner mitochondrial membrane protein that heterodimerizes with and inhibits a conserved homolog, bax, which accelerates cell death in response to apoptotic * To whom correspondence should be addressed: Michael R. Lewis, Ph.D., Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, 379 E. Campus Drive, University of MissourisColumbia, Columbia, MO 65211. Phone: (573) 814-6000, ext. 3703. Fax: (573) 814-6551. E-mail: LewisMic@ missouri.edu. † Molecular Biology Program. ‡ Department of Veterinary Medicine and Surgery. § Department of Chemistry. | Department of Biochemistry. ⊥ University of Missouri Research Reactor. X Department of Radiology.

stimuli (3). Overexpression of bcl-2 in stably transfected cell lines has been implicated in the development of both drug (4-6) and radiation (7-9) resistance. The bcl-2 cellular oncogene was discovered by Croce and co-workers, after cloning the chromosome 18 breakpoint region in non-Hodgkin’s lymphoma (NHL) (10). Over 85% of follicular lymphomas and approximately 20% of large cell lymphomas carry a t(14;18) translocation (11-13), juxtaposing the bcl-2 gene with the IgH promoter and deregulating bcl-2 expression. However, mechanisms other than translocation may deregulate bcl-2 expression (14, 15) in lymphoma. In addition, 84% of acute myeloid leukemias (16), 80-100% of malignant cells in multiple myeloma (17), and 60-90% of many nonlymphoid malignancies, including breast (18), lung (19, 20), colon (21, 22), prostate (23, 24), neuroendocrine cancers (25), and malignant melanomas (26) overexpress bcl-2. In these cancers bcl-2 expression is often associated with transformation of benign lesions to malignancy, disease progression, and poor prognosis. Clinical understanding of the role of bcl-2 in disease progression and treatment response or failure will be-

10.1021/bc034084n CCC: $25.00 © 2003 American Chemical Society Published on Web 10/22/2003

1084 Bioconjugate Chem., Vol. 14, No. 6, 2003

come increasingly important as drugs that act to downregulate bcl-2 mRNA and protein are developed. While the majority of NHL patients respond well initially to conventional chemotherapy, bcl-2 overexpression has been identified as an independent predictor of increased relapse, shorter disease-free intervals, and poor causespecific survival (27, 28). The most important recent advances in the treatment of NHL have been the development of targeted immunotherapy and radioimmunotherapy (RIT). The pan-B cell, anti-CD20 monoclonal antibody rituximab down-regulates bcl-2 through a STAT3- and IL-10-mediated pathway (29, 30), sensitizing lymphoma cells in vitro to apoptotic killing by cytotoxic drugs (31). Data emerging from ongoing Phase III clinical trials suggest that addition of rituximab to combination chemotherapy may improve survival in patients with aggressive lymphoma. Yttrium-90-ibritumomab (Zevalin), the radiolabeled analogue of rituximab, has shown considerable efficacy for RIT of relapsed NHL (32). Zevalin RIT showed a significantly greater response rate than rituximab in a randomized Phase III trial and has effected responses in rituximab-refractory patients. Recent findings that tumor bcl-2 mRNA and protein levels decrease in response to RIT (33) suggest that downregulation of bcl-2 may play a significant role in response to low dose-rate radiation. Another alternative therapy involves the use of an antisense oligonucleotide, which is complementary to the first six codons of bcl-2 mRNA and is able to reduce bcl-2 gene expression. G3139, a DNA phosphorothioate oligonucleotide, has shown efficacy against B-cell lymphoma in tumor-bearing SCID mice (34) and is currently in clinical trials for antisense therapy of bcl-2-positive NHL (35). Preliminary results indicated that two of nine patients experienced a reduction in tumor burden. However, more encouraging was the fact that six of eight patients who went on to receive further chemotherapy achieved responses. Similarly, six of 14 patients with bcl2-positive metastatic melanoma in a Phase I/II trial of G3139 and dacarbazine achieved objective responses (36). Thus, bcl-2 antisense treatment may overcome drug resistance. Peptide nucleic acid (PNA) (37) is a DNA-like molecule in which the deoxyribose phosphodiester backbone of DNA has been replaced by (2-aminoethyl)glycine units, to which the nucleobases are attached by methylenecar1 Abbreviations: bcl-2, B-cell lymphoma/leukemia-2 gene; Bhoc, benzhydryloxycarbonyl; BLAST, Basic Local Alignment Search Tool; DCM, dichloromethane; DIEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; DOTA, 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid; DOTA(OtBu)3, DOTA tris(tert-butyl) ester; EDT, ethanedithiol; EDTA, ethylenediaminetetraacetic acid; ESI-MS, electrospray ionization mass spectrometry; FKD, N-R-(9-fluorenylmethoxycarbonyl)-N-[tris(tert-butyl)DOTA]-L-lysine; FKT, N-R-(9-fluorenylmethoxycarbonyl)-N--[tetramethylrhodamine-(5-carbonyl)]-L-lysine; Fmoc, 9-fluorenylmethoxycarbonyl; HATU, 2-(1H-7-azabenzotriazol1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HOAt, 1-hydroxy-7-azabenzotriazole; HOBt, 1-hydroxybenzotriazole; KD, N--(DOTA)-L-lysine; KT, N--[tetramethylrhodamine-(5-carbonyl)]-L-lysine; LC-MS, liquid chromatography-mass spectrometry; NHL, non-Hodgkin’s lymphoma; NMP, N-methylpyrrolidinone; OtBu, tert-butyloxy; PNA, peptide nucleic acid; PTD-4, synthetic protein transduction domain-4; ri-PTD-4, retro-inverso PTD-4; RP, reversed-phase; TA, thioanisole; TFA, trifluoroacetic acid; TIS, triisopropylsilane; TMR, tetramethylrhodamine; TNBS, 2,4,6-trinitrobenzenesulfonic acid; XAL PEG PS resin, xanthenylamide-poly(ethylene glycol)polystyrene.

Gallazzi et al.

bonyl linkers. Compared to DNA oligonucleotides, the superior biological stability and greater affinity and specificity of PNA for complementary mRNA sequences (38) make it extremely attractive for antisense applications. However, poor cellular uptake of unmodified PNAs has limited their utility in vitro and in vivo. Several groups have demonstrated that PNA uptake in target cells can be increased substantially by conjugation to cell membrane-permeating peptides (39-43), such as Drosophila Antennapedia (43-58) (pAntp) (44) or an undecapeptide from the HIV Tat protein transduction domain (45). Using alanine residue substitution to identify the basic residues critical for cell membrane transduction, Dowdy and co-workers prepared a synthetic protein transduction domain, YARAAARQARA (PTD-4) (46), that was 33-fold more active than the Tat sequence. The results of these studies suggest that tumor protooncogenes such as bcl-2 might be targeted by radiolabeled, cell-permeating peptide-PNA constructs for in vivo imaging and targeted radiotherapy of cancer. Previously we demonstrated that a radiometal-labeled PTD4-anti-bcl-2 PNA conjugate bound to bcl-2 mRNA with high specificity and thermodynamic stability in cell-free systems (47). We report here the solid-phase synthesis of antisense peptide-PNA conjugates for targeting imaging and therapeutic radiometals to tumor cells expressing high levels of bcl-2 mRNA. The antisense PNA was based on the 18-residue sequence of G3139, while an 18-mer nonsense PNA did not correspond to any known mammalian gene or DNA sequence in a BLAST database search. The bcl-2 antisense and nonsense PNAs were conjugated with a new derivative of 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA), designed for chelation of radiometals and fully protected for solid-phase peptide and PNA synthesis. The DOTAPNA conjugates were then coupled by solid-phase techniques to glycine, a small amino acid spacer, followed by PTD-4 or its retro-inverso analogue (ri-PTD-4). Preparation of these DOTA conjugates required the development of an innovative solid-phase synthetic strategy, involving cleavage from the resin and partial deprotection of the peptide-PNA conjugates under mildly acidic conditions. This strategy allowed hydrophobic intermediates to be separated from unreacted PNA by semipreparative reversed-phase HPLC (RP-HPLC). In contrast, tetramethylrhodamine (TMR)-labeled PNA conjugates could be prepared in a straightforward manner by standard solid-phase synthesis, cleavage, deprotection, and purification techniques. Plasma stability studies demonstrated that ri-PTD-4 was much more stable to proteolytic degradation than the parent sequence, and scanning confocal fluorescence microscopy studies of the TMR conjugates showed that ri-PTD-4-mediated cellular internalization of the antisense PNA in NHL cells known to express high levels of bcl-2 mRNA. EXPERIMENTAL PROCEDURES

General. All reagents were HPLC or peptide synthesis grade. CDCl3, phenol, and EDT were obtained from Aldrich Chemical Co. (Milwaukee, WI). Anhydrous Nmethylpyrrolidinone (NMP), N,N-dimethylformamide (DMF), dichloromethane (DCM), acetonitrile, and methanol were obtained from Fisher Scientific, Inc. (Pittsburgh, PA). Trifluoroacetic acid (TFA) was obtained from VWR Scientific Products (St. Louis, MO). All standard, protected Fmoc amino acid derivatives, 2-(1H-benzotriazol1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), and 1-hydroxybenzotriazole (HOBt) were ob-

Cell-Permeating Anti-bcl-2 Peptide−PNA Conjugates

tained from Novabiochem (San Diego, CA). N-R-(9Fluorenylmethoxycarbonyl)-N--[tetramethylrhodamine(5-carbonyl)]-L-lysine (FKT) was purchased from Molecular Probes, Inc. (Eugene, OR). 2-(1H-7-Azabenzotriazol-1-yl)1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), 1-hyrdoxy-7-azabenzotriazole (HOAt), all Fmoc/Bhoc protected monomers for PNA synthesis, and Fmoc-XAL PEG PS resin were obtained from PerSeptive Biosystems (Framingham, MA). DOTA tris(tert-butyl) ester (DOTA(OtBu)3) was procured from Macrocyclics (Dallas, TX), and N-R-Fmoc-L-lysine was purchased from Advanced ChemTech (Louisville, KY). Piperidine, m-cresol, N,Ndiisopropylethylamine (DIEA), thioanisole (TA), triisopropylsilane (TIS), and 2,4,6-trinitrobenzenesulfonic acid (TNBS) were purchased from Fluka (Milwaukee, WI). 111 InCl3 was obtained from Mallinckrodt, Inc. (St. Louis, MO). Ultrapure water (18 MΩ-cm resistivity) was used for all procedures. All laboratory glassware was washed with a mixed acid solution (48) and thoroughly rinsed with ultrapure water. Manual reaction vessels were obtained from Chemglass, Inc. (Vineland, NJ). Normal phase TLC plates (Silica Gel 60 F254, plastic-backed) were purchased from EM Science (Gibbstown, NJ). An Advanced ChemTech ACT Model 396 Omega Multiple Biomolecular Synthesizer was used for automated solid-phase synthesis. 1H NMR analysis of purified N-R(9-fluorenylmethoxycarbonyl)-N--[tris(tert-butyl)DOTA]L-lysine was performed using a Bruker DRX 300 MHz spectrometer (Bruker BioSpin, Westmont, IL), and elemental analysis of the compound was performed by Atlantic Microlab (Norcross, GA). ESI-MS analyses were performed on a Finnigan TSQ7000 mass spectrometer (Thermo Finnigan, San Jose, CA). A Waters (Milford, MA) NovaPak C18 column (3.9 × 300 mm) was used for the LC-MS analysis. Analytical and semipreparative RP-HPLC were performed on a Beckman Coulter System Gold chromatograph equipped with a 168 diode array detector, a 507e auto-injector, and the 32 KARAT software package (Beckman Coulter, Fullerton, CA). A Keystone Scientific, Inc. (San Jose, CA) C-18 Kromosil column (4.6 × 150 mm, 5 µm, 100 Å) was used for analytical HPLC. For semipreparative HPLC, a Waters Prep NovaPak, HR-C18 column (7.8 × 300 mm, 6 µm, 60 Å) was used. The flow rate was maintained at 1.0 mL/min for analytical runs and at 4.0 mL/min for semipreparative purification. The wavelengths used for UV detection were 214 and 260 nm for analytical RP-HPLC and 235 and 245 nm for semipreparative RP-HPLC, respectively. Eluents used in all runs consisted of solvent A (0.1% TFA/H2O) and solvent B (0.1% TFA/CH3CN). Three different gradients were used: (1) “Full Gradient”: linear from 5% to 95% solvent B in 45 min, (2) “FKD Gradient” (optimized for semipreparative purification of N-R-(9-fluorenylmethoxycarbonyl)-N--[tris(tert-butyl)DOTA]-L-lysine): linear from 45% to 50% solvent B in 20 min, and (3) “Semiprep Gradient” (optimized for semipreparative purification of hydrophobic, partially deprotected peptide-PNA conjugate precursors): sequential linear from 10% to 20% solvent B in 20 min (step 1), 20% to 30% solvent B in 10 min (step 2), 30% to 50% solvent B in 5 min (step 3), and 50% to 60% solvent B in 20 min (step 4). Isocratic conditions were applied after steps 1 and 2 to permit all hydrophilic species and m-cresol to elute, prior to application of steps 3 and 4. Size exclusion HPLC was performed on a Waters Delta 600 chromatograph equipped with a manual Rheodyne injector, a 2487 dual wavelength UV detector, a Packard (Downers Grove, IL) 500TR Flow Scintillation Analyzer

Bioconjugate Chem., Vol. 14, No. 6, 2003 1085

with a GAMMA-C flow cell for 111In, a Waters busSAT/ IN analog-digital interface, and the Millennium 32 software package. A Phenomenex (Torrance, CA) BioSepSEC-S 3000 column (7.8 × 300 mm, 5 µm, 290 Å), an isocratic mobile phase of 100 mM NaH2PO4/0.05% NaN3, pH 6.8, and a flow rate of 1.0 mL/min were used. Molecular weights estimated by size exclusion HPLC were calculated from a calibration curve generated using a Bio-Rad (Hercules, CA) molecular weight standard. Raji Burkitt’s lymphoma cells were obtained from the American Type Culture Collection (Manassas, VA). Suspension cultures of cells were maintained in exponential growth phase in RPMI 1640 medium (Mediatech, Inc., Herndon, VA), supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 48 µg/mL gentamycin, at 37 °C and 5% CO2. Cell viability was determined to be >98% by trypan blue exclusion and hemacytometry. PNA Synthesis. Syntheses of anti-bcl-2 PNA (tctcccagcgtgcgccat) and nonsense PNA (tgtgttgcgaccctcttg) were performed in a manual reaction vessel. Fmoc/Bhoc chemistry was used with the following PNA monomers: Fmoc-A(Bhoc)-OH, Fmoc-C(Bhoc)-OH, Fmoc-G(Bhoc)OH, and Fmoc-T-OH. Fmoc-XAL PEG PS resin was used as a solid support at a substitution level of 0.18 mmol/g. Each cycle of elongation consisted of (1) Fmoc deprotection with 25% piperidine in DMF for 2 cycles of 1 min at room temperature, (2) washing with DMF, DCM, and twice with NMP for 1 min each at room temperature, (3) coupling using a molar ratio of resin:monomer:HATU: DIEA:2,6-lutidine ) 1.0:3.0:2.7:3.0:4.5; 5 min of preactivation followed by a 25-min coupling at room temperature, after which the process was repeated, (4) capping with 2 mL of 5% acetic anhydride/6% 2,6-lutidine in DMF for 5 min at room temperature, and (5) washing with NMP, DCM, and twice with DMF for 1 min each at room temperature. All chemical steps were followed by a TNBS test on the resin, and the resulting colorimetric reaction indicated the presence of free primary amines after Fmoc deprotection and the absence of primary amino groups after the coupling and capping steps. After 6, 12, and 18 cycles, aliquots of the resin-bound PNAs were cleaved and Bhoc-deprotected with m-cresol:TFA (1:19) for 30 min, and LC-MS analysis was performed to confirm that the observed masses were consistent with the calculated molecular weights of the Fmoc-protected intermediates. N-R-(9-Fluorenylmethoxycarbonyl)-N--[tris(tertbutyl)DOTA]-L-lysine (FKD, 1). To 1 mL of anhydrous NMP was added sequentially 218 mg (0.381 mmol) of DOTA(OtBu)3, 134 mg (0.352 mmol) of HATU, and 5.1 mg (0.038 mmol) of HOAt with continuous stirring. The reaction mixture was stirred at room temperature for 1.5 h, after which normal phase TLC, using acetone as the mobile phase, showed that formation of the DOTA(OtBu)3-OAt active ester was complete (Rf ) 0.66). Then 133 mg (0.360 mmol) of N-R-Fmoc-L-lysine was added, and the reaction mixture was stirred at room temperature for 10 min. After quenching the reaction with H2O, the reaction mixture was taken into ethyl acetate and extracted with H2O, while maintaining the pH at 6.0 with 5% NaHCO3. The organic phase was acidified with TFA and washed with water, after which the solvent was removed in vacuo. The residue was dissolved in 1 mL of ethyl acetate and added to 25 mL of diethyl ether with vigorous stirring and cooling to -20 °C. The thick oily precipitate was washed with cold diethyl ether and dried in vacuo, after which it was dissolved in acetonitrile. Unreacted N-R-Fmoc-L-lysine was precipitated by dropwise addition of 0.2% TFA, and this process was repeated four to five times. The supernatant was concentrated to


a minimum volume, and the pH of the remaining aqueous solution was adjusted to 6.0 with 5% NaHCO3. The desired product was extracted into ethyl acetate, which was washed with H2O and evaporated to dryness in vacuo. The resulting oil was dissolved in 1 mL of ethyl acetate, to which was added 15 mL of diethyl ether with vigorous stirring to precipitate the product. After discarding the supernatant, the product was dried in vacuo to yield 308 mg (92.7%) of 1 as a white, microcrystalline solid. Normal phase TLC (CHCl3:CH3OH:concd NH4OH, 13:3:1) Rf ) 0.45; RP-HPLC, Full Gradient, tR ) 31.2 min (98.6% purity); ESI-MS m/z calcd for C49H74N6O11 (M + H)+ ) 924.2, found 923.9 (M + H)+, 462.5 (M + 2H)2+. Alternatively, DOTA(OtBu)3 (658 mg, 1.15 mmol), HATU (427 mg, 1.12 mmol), and HOAt (67.0 mg, 0.492 mmol) were dissolved in 2 mL of NMP at room temperature. The resulting pale yellow solution was stirred for 1.5 h. To this solution of the active ester was added 410 mg (1.11 mmol) of N-R-Fmoc-L-lysine. After dissolution, stirring was continued for an additional 2 h. The reaction was quenched by the addition of 1 mL of H2O and diluted with 40 mL of ethyl acetate. The organic phase was washed with 3 × 20 mL of H2O. The organic phase was then acidified with the addition of 100 µL of TFA. The organic layer was washed again with 2 × 20 mL of H2O and concentrated in vacuo, leaving a pale yellow, viscous oil (820 mg, 80.0%). Analytical RP-HPLC under Full Gradient conditions indicated the purity of the crude product to be >89%. The crude product was dissolved in 10 mL of CH3CN:H2O (1:1). The product was purified by semipreparative RP-HPLC, using the optimized FKD Gradient. The collected fractions, eluting at tR ) 26.5 min, were pooled and lyophilized, yielding 416 mg (40.6%) of 1 as a white solid. 1H NMR (300 MHz, CDCl3) δ 1.4 (s, 27 H), 1.5 (s, 6 H), 2.7 (s, 2 H), 2.7-3.8 (br complex m, 28 H), 4.0 (br s, 1H), 4.2 (t, 2H), 7.3 (dt, 4H), 7.5 (d, 2H), 7.6 (d, 2H); ESI-MS m/z calcd for C49H74N6O11 (M + H)+ ) 924.2, found 923.7 (M + H)+, 462.4 (M + 2H)2+. Anal. (C49H74N6O11‚4TFA‚2H2O): C, H; N: calcd 5.94; found 6.52.2 PTD-4-G-KD-anti-bcl-2 PNA (2). Fmoc-anti-bcl-2 PNA on XAL PEG PS resin was deprotected at the N-terminus with 25% piperidine in DMF for two cycles of 1 min at room temperature, followed by washing with DMF, DCM, and twice with NMP for 1 min each at room temperature. A solution containing 40 µmol of 1, 40 µmol of HOAt, 32 µmol of HATU, and 100 µmol of DIPEA in NMP was stirred for 15 min at room temperature. The resulting mixture was added to 9 µmol of anti-bcl-2 PNA on 100 mg of XAL PEG PS resin and mixed by nitrogen bubbling for 45 min in a reaction vessel for manual peptide synthesis. This process was repeated twice, after which the resin was reacted for 5 min with 5% acetic anhydride and 6% 2,6-lutidine in DMF at room temperature. After removal of the N-terminal Fmoc group with 25% piperidine in DMF for two cycles of 25 min at room temperature, KD-anti-bcl-2 PNA resin was then sequentially elongated with PTD-4-G, using the multiple automated synthesizer and standard Fmoc coupling chemistry. The peptide chain was assembled by sequential acylation with protected amino acids, which were activated in situ with HBTU/HOBt. Recoupling at each cycle with a minimal 15-fold molar excess of activated amino 2 Elemental analysis for N-R-(9-fluorenylmethoxycarbonyl)N--[tris(tert-butyl)DOTA]-L-lysine (FKD): Theoretical (C49H74N6O11‚4TFA‚2H2O): calcd C, 48.37%; H, 5.84%; N, 5.94%; found C, 48.52%; H, 6.14%; N, 6.52%.

Gallazzi et al.

acid derivative was performed, to minimize the production of truncated species. Each cycle of elongation involved the following steps: (1) Fmoc deprotection with 25% piperidine in DMF/NMP for one cycle of 1 min and three cycles of 10 min at room temperature, (2) washing with NMP for four cycles of 1 min at room temperature, (3) coupling with in situ activated amino acid (amino acid: HBTU:HOBt:DIEA molar ratio ) 1:0.9:1:2) in NMP for two cycles of 25 min at room temperature, and (4) washing with NMP for two cycles of 1 min at room temperature. To detach hydrophobic, partially deprotected intermediate products from the resin, two sequential procedures were evaluated in a reaction vessel: (1) reaction with 1% TFA/5% m-cresol in DCM for eight cycles of 15 min at room temperature, followed by filtration; and (2) reaction with 50% TFA/5% m-cresol in DCM for one cycle of 30 min at room temperature, followed by filtration. The partially deprotected intermediates were isolated by precipitation with cold diethyl ether and evaporation to dryness under a stream of nitrogen. Crude intermediates from both procedures were analyzed by RP-HPLC, using Full Gradient conditions, and then purified by semipreparative RP-HPLC, using Semiprep Gradient conditions. The purified intermediates, eluting from 37 to 41 min retention time for procedure 1 and from 33 to 38 min for procedure 2, were pooled, lyophilized, and then fully deprotected with 87.5% TFA containing 2.5% each of H2O, phenol, TA, EDT, and TIS for 4 h at room temperature. After this treatment, 4.5 mg (7.7%) of 2 was isolated by precipitation with cold diethyl ether and lyophilized to a white solid. ESI-MS: m/z 2190.0 (M + 3H)3+, 1642.5 (M + 4H)4+, 1314.2 (M + 5H)5+, 1095.1 (M + 6H)6+, 938.9 (M + 7H)7+. The observed molecular ions were consistent with the theoretical molecular weight of 2, calcd (M + H)+ ) 6569.1. PTD-4-G-KD-nonsense PNA (3). Fmoc-nonsense PNA on XAL PEG PS resin was deprotected at the N-terminus with 25% piperidine in DMF for two cycles of 1 min at room temperature, followed by washing with DMF, DCM, and twice with NMP for 1 min each at room temperature. A solution containing 20 µmol of 1, 20 µmol of HOAt, 18 µmol of HATU, and 60 µmol of DIPEA in NMP was stirred for 15 min at room temperature. The resulting mixture was added to 5 µmol of nonsense PNA on 45 mg of XAL PEG PS resin and mixed by nitrogen bubbling for 45 min in a reaction vessel for manual peptide synthesis. This process was repeated twice, after which the resin was reacted for 5 min with a large excess of acetic anhydride (5% v/v) and 2,6-lutidine (6% v/v) in DMF at room temperature. After Fmoc removal with 25% piperidine in DMF for two cycles of 25 min, KDnonsense-PNA resin was then sequentially elongated with PTD-4-G on the multiple automated synthesizer, as described for 2. To cleave hydrophobic, partially deprotected intermediates from the resin, the following three procedures were performed in sequence of increasing acidity: (1) reaction with 5% TFA, containing 5% each of H2O, m-cresol, and TIS in DCM, for four cycles of 15 min at room temperature, followed by filtration; (2) reaction with 25% TFA, containing 5% each of H2O, m-cresol, and TIS in DCM, for two cycles of 15 min at room temperature, followed by filtration; and (3) reaction with 50% TFA, containing 5% m-cresol and 2.5% each of H2O, TIS, TA, and phenol in DCM, for one cycle of 40 min at room temperature, followed by filtration. The partially deprotected intermediates were isolated by precipitation with cold diethyl ether and evaporation to dryness under nitrogen. These


intermediates were analyzed by RP-HPLC, using Full Gradient conditions, and intermediates from procedures 1 and 2 were purified by semipreparative RP-HPLC, using the Semiprep Gradient. The purified intermediates, eluting from 43 to 49 min retention time, were pooled and lyophilized, after which the resulting mixture was treated for 4 h at room temperature with 87.5% TFA containing 2.5% of each of H2O, phenol, TA, EDT, and TIS, to afford the completely deprotected product. After precipitation with cold diethyl ether and lyophilization, 3.0 mg (9.1%) of 3 was isolated as a white solid. ESIMS: m/z 2211.1 (M + 3H)3+, 1658.0 (M + 4H)4+, 1326.9 (M + 5H)5+, 1106.1 (M + 6H)6+, 948.1 (M + 7H)7+. The observed molecular ions were consistent with the theoretical molecular weight of 3, calcd (M + H)+ ) 6629.2. ri-PTD-4-G-KD-anti-bcl-2 PNA (4). ri-PTD-4-GKD-anti-bcl-2 PNA was prepared from 4 µmol of antibcl-2 PNA on 45 mg of XAL PEG PS resin. The synthetic procedure was identical to that employed for 2, except that after coupling of glycine, all D-amino acid analogues of the PTD-4 residues were added in the reverse sequence. Hydrophobic, partially deprotected crude intermediates were obtained from the three sequential cleavage procedures with increasing TFA concentrations, identical to those utilized for 3. The partially deprotected intermediates were isolated by precipitation with cold diethyl ether and evaporation to dryness under nitrogen. Crude intermediate products from procedures 1 and 2 were analyzed and purified by RP-HPLC, as described for 2, and the fraction eluting at 45 to 52 min retention time was collected. After full deprotection and isolation of the final product, using the procedures employed for 2, 1.7 mg (6.5%) of 4 was obtained as a white solid. ESIMS: m/z 2188.5 (M + 3H)3+, 1642.1 (M + 4H)4+, 1313.6 (M + 5H)5+, 1094.8 (M + 6H)6+, 938.7 (M + 7H)7+, 821.3 (M + 8H)8+, 730.4 (M + 9H)9+. The observed molecular ions were consistent with the theoretical molecular weight of 4, calcd (M + H)+ ) 6569.1. ri-PTD-4-G-KD-nonsense PNA (5). ri-PTD-4-GKD-nonsense-PNA was synthesized from 9 µmol of nonsense PNA on 104 mg of XAL PEG PS resin, using a procedure identical to that employed for 3, except that the ri-PTD-4 sequence was elongated after coupling of glycine. Hydrophobic, partially deprotected crude intermediates were obtained from the three sequential cleavage procedures using increasing TFA concentrations, identical to those employed for 3. The partially deprotected intermediates were isolated by precipitation with cold diethyl ether and evaporation to dryness under nitrogen. Crude intermediate products from procedures 1 and 2 were analyzed and purified by RP-HPLC, as described for 3, and the fraction eluting at 42 to 52 min retention time was collected. After full deprotection and isolation of the final product, using the procedures employed for 3, 3.6 mg (6.1%) of 5 was obtained as a white solid. ESI-MS: m/z 2209.8 (M + 3H)3+, 1657.9 (M + 4H)4+, 1326.4 (M + 5H)5+, 1104.8 (M + 6H)6+, and 947.6 (M + 7H)7+. The observed molecular ions were consistent with the theoretical molecular weight of 5, calcd (M + H)+ ) 6629.2. KT-anti-bcl-2 PNA (6). Fmoc-anti-bcl-2 PNA on XAL PEG PS resin was deprotected at the N-terminus using a procedure identical to that employed for 2. A solution containing 20 µmol of FKT, 20 µmol of HOAt, 18 µmol of HATU, and 60 µmol of DIPEA in NMP was stirred for 15 min at room temperature. The resulting mixture was added to 8 µmol of anti-bcl-2 PNA on 90 mg of XAL PEG PS resin and mixed by nitrogen bubbling for 45 min in a reaction vessel for manual peptide synthesis. This process


was repeated twice, after which the resin was reacted for 5 min with 5% acetic anhydride and 6% 2,6-lutidine in DMF at room temperature. The crude product was cleaved from 45 mg (4 µmol) of the resin and deprotected with 87.5% TFA containing 2.5% H2O, phenol, TA, EDT, and TIS each for 2 h at room temperature, after which it was purified by semipreparative RP-HPLC, using Semiprep Gradient conditions. The peak eluting at tR ) 34.0 min was collected, and 1.3 mg (6.1%) of 6 was isolated as a magenta solid, after precipitation with cold diethyl ether and lyophilization. RP-HPLC, Full Gradient, tR ) 16.5 min (>99.5% purity); ESI-MS m/z 1784.0 (M + 3H)3+, 1338.2 (M + 4H)4+, 1070.8 (M + 5H)5+. The observed molecular ions were consistent with the theoretical molecular weight of 6, calcd (M + H)+ ) 5333.1. ri-PTD-4-G-KT-anti-bcl-2 PNA (7). ri-PTD-4-GKT-anti-bcl-2 PNA was synthesized from 4 µmol of Fmoc-deprotected KT-anti-bcl-2 PNA on 45 mg of XAL PEG PS resin, using a procedure for elongation of the ri-PTD-4-G sequence identical to that employed for 4. The crude product was cleaved from the resin, deprotected, and purified by semipreparative RP-HPLC using the procedures employed for 6. The peak eluting at tR ) 35.5 min was collected, and 1.8 mg (6.8%) of 7 was obtained as a magenta solid, after precipitation with cold diethyl ether and lyophilization. RP-HPLC, Full Gradient, tR ) 16.1 min (>99.5% purity); ESI-MS m/z 2197.3 (M + 3H)3+, 1648.4 (M + 4H)4+, 1319.4 (M + 5H)5+, 1099.1 (M + 6H)6+, 942.2 (M + 7H)7+, 824.6 (M + 8H)8+, 733.3 (M + 9H)9+. The observed molecular ions were consistent with the molecular weight of 7, calcd (M + H)+ ) 6593.5. Plasma Stability Studies. Compounds 2 and 4 were labeled with 111In as described previously (47). An aliquot of 948 µCi of 111In-labeled 2 (4.89 µg) or 915 µCi of 111Inlabeled 4 (4.47 µg) was added to 1 mL of mouse plasma. The resulting mixtures were incubated at 37 °C for 168 h. Aliquots of 100 µL of the mixtures were analyzed by size exclusion HPLC at 0, 0.25, 1, 4, 24, 48, 96, and 168 h of incubation, to determine conjugate and chelate stability. Scanning Confocal Fluorescence Microscopy Studies. An aliquot of 6 (0.18 mg/mL, 34 µM) or 7 (0.72 mg/mL, 109 µM) in 0.2 M ammonium acetate, pH 5.0, was added to a final concentration of 2.5 µM in a suspension of Raji cells (1 × 107 cells/mL) in 2.0 mL of RPMI 1640 medium at 37 °C and 5% CO2. Cells and supernatants from 100 µL of the suspension were separated by centrifugation at 1, 5, and 30 min and at 2 h of incubation. After removal of the supernatant, the cells were fixed by addition of 100 µL of 4% formaldehyde and washed with 1% formaldehyde to remove any fluorescent probe in solution. Formaldehyde was not added for fluorescence microscopy of live Raji cells. Live Raji cells were incubated with 6 or 7 as described above and immediately analyzed by fluorescence microscopy. Digital microscopy was performed with an Olympus IX70 microscope/Bio-Rad Radiance 2000 scanning confocal fluorescence system, interfaced to a photomultiplier tube array and computer workstation. Excitation of TMR was accomplished at 568 nm, followed by image acquisition using a 580 nm long path emission filter. All fluorescence microscopy studies were performed using identical microscope settings, and all images were acquired in the medial plane. RESULTS AND DISCUSSION

The optimized procedure for the preparation of cellpermeating peptide-PNA conjugates (Figure 1) for


Figure 1. Structures of DOTA- and TMR-conjugated PTD-4-G and ri-PTD-4-G bcl-2 antisense and nonsense PNA conjugates. PNA residues are indicated in lower case letters, and peptide residues are indicated in upper case letters.

radiometal targeting of the bcl-2 proto-oncogene consisted of a combination of manual solid-phase synthesis of PNA sequences and automated solid-phase synthesis of peptide sequences, using standard Fmoc chemistry. Manual syntheses of PNAs were undertaken in order to perform sequential Fmoc deprotection of the growing PNA oligomers under carefully controlled conditions. Unlike peptides, upon Fmoc deprotection, PNA can undergo an alkaline pH-dependent N-acyl transfer rearrangement that converts the primary N-terminus into an unreactive amide (49). When the automated multiple biomolecular synthesizer was used for PNA synthesis, truncated PNA species or degraded products were often obtained. This problem was likely mechanical in nature and probably resulted from exposure of the PNA to piperidine for periods longer than 1 min during Fmoc deprotection. When PNAs were synthesized manually, piperidine reactions were carried out in precise 1-min cycles, and no evidence of N-acyl transfer rearrangement was observed. Using manual solid-phase synthesis techniques, the desired sequences could be prepared in their entirety. To incorporate a macrocyclic chelating agent into any sequence position of a peptide-PNA conjugate, a new Scheme 1

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derivative of DOTA, N-R-(9-fluorenylmethoxycarbonyl)N--[tris(tert-butyl)DOTA]-L-lysine (FKD, 1), was prepared as a fully protected building block for solid-phase Fmoc chemistry. DOTA chelates a wide variety of radiometals for diagnostic imaging and targeted radiotherapy, including 111In (T1/2 ) 67.4 h; EC 849 keV (100%); γ 173 keV (89%), 247 keV (94%)) and 90Y (T1/2 ) 64.0 h; β- 2.27 MeV (100%)), respectively, with extremely high in vivo stability (50). The synthesis of 1 is shown in Scheme 1. Compound 1 was prepared in a one-pot, two-step process, by activating commercially available DOTA tris(tertbutyl) ester with HATU and HOAt and reacting the resulting active ester with commercially available N-RFmoc-L-lysine. An extensive solvent extraction workup under carefully controlled pH conditions afforded 1 in high yield and 98.6% purity. The only impurity detected by analytical RP-HPLC was the dilysine adduct of DOTA tris(tert-butyl) ester (1.4%). Alternatively, compound 1 was obtained in somewhat lower yield using a simplified solvent extraction procedure, followed by semipreparative RP-HPLC. Using a mobile phase gradient optimized for purification of 1, the bifunctional DOTA derivative was obtained in analytically pure form. Furthermore, this procedure greatly simplified scale-up of the reaction and purification of the desired product. To date, over 1.7 g of 1 have been prepared using these two procedures. Coupling of 1 to bcl-2 antisense and nonsense PNAs was followed by sequential automated elongation of the PNA conjugate with the cell-permeating PTD-4-G peptide sequence. Cleavage of the constructs from the resin, with concomitant deprotection, was followed by attempts to purify the crude products by semipreparative RP-HPLC and characterize them by LC-MS. It was discovered that neither the final products nor the unmodified PNA oligomers were retained on RP-HPLC, and both unmodified PNA and DOTA-derivatized PTD-4-G-PNA conjugates coeluted at the solvent front. Therefore, direct separation of the fully deprotected peptide-PNA conjugates from unmodified PNA byproducts was not possible. To solve this problem, an innovative strategy was devised to detach each conjugate from the resin under mildly acidic conditions, using dilute TFA and a partial cocktail of radical scavengers, to leave most of the protecting groups on the peptide residues. While it was predicted that the crude intermediates obtained by this process would consist of a mixture of different compounds, corresponding to various protecting groups remaining on the peptide, it was hypothesized that these partially protected conjugates would be very hydrophobic and retained on RP-HPLC. This strategy offered the possibility of separating the hydrophobic, partially deprotected intermediates from the very hydrophilic unreacted PNA. By collecting the hydrophobic components and subsequently treating them with a high concentration of TFA in full radical scavenger cocktail, the fully deprotected final products could be obtained.


Figure 2. (Top) Semipreparative RP-HPLC chromatogram, using Semiprep Gradient conditions, of intermediate products obtained after cleavage of 2 with 1% TFA containing 5% m-cresol. (Bottom) Analytical RP-HPLC chromatogram, using Full Gradient conditions, of hydrophobic, partially deprotected species purified after cleavage of 2 with 1% TFA containing 5% m-cresol.

This strategy was ultimately successful for the preparation of PTD-4-G-KD-anti-bcl-2 PNA (2), after developing procedures for cleavage and partial deprotection. Initially two sets of conditions were evaluated, where the concentration of TFA was varied. It was surmised that 1% TFA would be sufficiently acidic to detach the conjugate from the resin without any risk of removing protecting groups, as is the case for the similarly labile 2-chlorotrityl chloride resin. However, after eight cycles of reaction with 1% TFA containing 5% m-cresol, only a small amount of crude intermediate product was obtained. After diethyl ether precipitation, the partially deprotected peptide-PNA was purified by semipreparative RP-HPLC, eluting at 37 to 41 min retention time (Figure 2, top). Analytical RP-HPLC (Figure 2, bottom) of the main fraction collected showed that it consisted of a relatively pure hydrophobic species. The resin was then treated with 50% TFA containing 5% m-cresol to ensure full detachment of the conjugate from the resin. Under these conditions, it was anticipated that most of the protecting groups would probably be removed, possibly complicating the subsequent HPLC purification. A much larger quantity of crude intermediate product was obtained and purified by semipreparative RP-HPLC (Figure 3, top). The chromatogram showed that this mixture contained a large amount of hydrophilic species, which had to be discarded because this fraction, eluting at 21 to 29 min retention time, was contaminated with unmodified PNA. The major hydrophobic fraction, eluting at 33 to 38 min retention time, was collected, subjected to analytical RP-HPLC (Figure 3, bottom), and lyophilized. To remove the protecting groups still present, the lyophilized samples obtained by cleavage with 1% and 50% TFA were treated with concentrated TFA and a full cocktail of radical scavengers, in each case yielding a single product, for which ESI-MS analysis showed a pattern of multiply charged molecular ions consistent with the calculated molecular weight of 2.


Figure 3. (Top) Semipreparative RP-HPLC chromatogram, using Semiprep Gradient conditions, of intermediate products obtained after cleavage of 2 with 50% TFA containing 5% m-cresol. (Bottom) Analytical RP-HPLC chromatogram, using Full Gradient conditions, of hydrophobic, partially deprotected species purified after cleavage of 2 with 50% TFA containing 5% m-cresol.

As observed previously during the synthesis of 2, the final deprotected bcl-2 antisense peptide-PNA conjugate was not retained upon subsequent RP-HPLC analysis and eluted with the solvent front. This result confirmed that the procedure for purification of the partially protected precursor was necessary to separate the PTD-4 conjugate from unreacted PNA and obtain the desired product in pure form. In addition, it was concluded that neither 1% TFA nor 50% TFA was an optimal treatment for generating a relatively small number of hydrophobic, partially deprotected precursors in the highest possible yield. Cleavage with 1% TFA gave a relatively pure hydrophobic product, but in low yield, while cleavage with 50% TFA afforded an excessive amount of fully deprotected conjugate, which could not be isolated in pure form by semipreparative RP-HPLC. Therefore, intermediate acid concentrations were then evaluated to optimize the cleavage, deprotection, and purification conditions. In the synthesis of PTD-4-G-KD-nonsense PNA (3), once the protected conjugate had been assembled, the resin was reacted with increasing concentrations of TFA in partial radical scavenger cocktail, until detachment of the partially deprotected intermediates occurred. First, cleavage with four cycles of 5% TFA containing 5% H2O, m-cresol, and TIS each was attempted, followed by reaction with two cycles of 25% TFA containing 5% H2O, m-cresol, and TIS each, and finally reaction with one cycle of 50% TFA containing 5% m-cresol and 2.5% H2O, TIS, TA, and phenol each. Using 5% TFA, a modest amount of product was obtained, but the conjugate was not completely cleaved from the resin. When 25% TFA was employed, a substantially larger quantity of crude intermediate was obtained. Semipreparative RP-HPLC purification (Figure 4, top) afforded mainly very hydrophobic species, eluting at 43 to 49 min retention time, and a relatively small hydrophilic fraction, eluting at 17 to 24 min retention time. Analytical RP-HPLC of the hydrophobic fraction collected revealed that it was com-


Figure 4. (Top) Semipreparative RP-HPLC chromatogram, using Semiprep Gradient conditions, of intermediate products obtained after cleavage of 3 with 25% TFA containing 5% H2O, m-cresol, and TIS each. (Bottom) Analytical RP-HPLC chromatogram, using Full Gradient conditions, of hydrophobic, partially deprotected species purified after cleavage of 3 with 25% TFA containing 5% H2O, m-cresol, and TIS each.

posed of two major intermediates (Figure 4, bottom). No additional product was recovered from reaction with 50% TFA. The purified hydrophobic species generated by cleavage and slight deprotection with 5% and 25% TFA were pooled and lyophilized, after which they were completely deprotected with concentrated TFA and full radical scavenger cocktail. A single product was isolated, and ESI-MS analysis revealed a pattern of multiply charged molecular ions consistent with the calculated molecular weight of 3. Conjugates 2 and 3 were previously labeled to high specific activities with 111In and 90Y, and the specificity and stability of hybridization of 90Y-labeled 2 with bcl-2 mRNA in cell-free systems was compared to that of the corresponding 32P-labeled anti-bcl-2-DNA (47). While 90Ylabeled 2 showed equivalent specificity and superior thermodynamic stability in bcl-2 mRNA binding, the plasma stability of 111In-labeled 2 was suboptimal, as proteolytic degradation of the PTD-4 sequence was evident after 24 h at 37 °C. Therefore, the ri-PTD-4 analogues of 2 and 3 were synthesized in order to improve plasma stability. The retro-inverso peptide, which consists of all D-amino acids in the reverse sequence, places the basic residues of PTD-4 critical for cell permeation in approximately the same three-dimensional spatial configuration as the parent peptide. Thus, ri-PTD-4 should retain biological activity mediating cellular internalization of the PNA conjugates and show greater proteolytic stability than the parent peptide. AldrianHerrada et al. (39) demonstrated that the retro-inverso counterpart of pAntp was active in causing PNA conjugates to permeate target cells and presumed that it was much more stable to proteolysis. However, before now ri-PTD-4 had not been synthesized and evaluated for biological activity and stability. DOTA-conjugated ri-PTD-4 constructs of bcl-2 antisense (4) and nonsense (5) PNAs were prepared based on the methods developed for the syntheses of the parent PTD-4 conjugates 2 and 3. As in the case of conjugate 3, optimized sequential cleavage and slight deprotection using 5% TFA, followed by 25% TFA, in partial radical scavenger cocktail, was employed. These reaction conditions readily afforded hydrophobic precursors. These precursors were purified by semipreparative RP-HPLC,

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completely deprotected in concentrated TFA and full radical scavenger cocktail, precipitated with diethyl ether, and lyophilized to yield 4 and 5. ESI-MS analysis of 4 and 5 were consistent with the calculated molecular weights of the desired products. Conjugates 2 and 4 were labeled with 111In at specific activities of 1298 Ci/mmol and 1370 Ci/mmol, respectively, using a previously published procedure (47). The radiolabeled conjugates were incubated in mouse plasma for 168 h at 37 °C (Figure 5). The retention time of 111Inlabeled 2 shifted reproducibly from 11.4 min immediately upon mixing to 11.5 min at 168 h. In addition, the radiolabeled conjugate peak, initially sharp, showed considerable tailing by 168 h of incubation in plasma. After 168 h at 37 °C, an aliquot of the plasma mixture was spiked with pure 111In-KD-anti-bcl-2 PNA and analyzed by size exclusion HPLC. The species corresponding to 11.5 min retention time coeluted with the radiolabeled PNA lacking the PTD-4 sequence, with no observed changes in the chromatographic profile. These results suggested that considerable degradation of PTD-4 by plasma proteases and peptidases had occurred. In contrast, the peak corresponding to 111In-labeled 4 remained sharp during the 168-h incubation in plasma at 37 °C, its retention time changed by less than 0.02 min, and the lack of lower molecular weight species indicated that ri-PTD-4 was stable to plasma proteases and peptidases. During the course of these experiments, the protein-bound fraction of 111In-labeled 2 changed relatively little, from 28.8% at 0 h to 34.5% at 168 h. By comparison, the protein-bound fraction of 111In-labeled 4 increased over 4-fold, from 13.7% to 60.2%, during the 168-h incubation in plasma. In the case of conjugate 4, the major component of protein-associated 111In (41.7%) eluted at 9.24 min, consistent with the molecular weight of immunoglobulin G (158 kDa). Since transferrin (80 kDa) is the main indium-binding protein in plasma, this result suggested that little, if any, dissociation of 111In from the DOTA chelate occurred. Instead the proteinassociated fractions of 111In likely resulted from nonspecific binding of the radiolabeled peptide-PNA conjugate. Under the size exclusion HPLC conditions employed, transferrin could not be separated from albumin (69 kDa). However, the peak eluting in this molecular weight range, at 10.5 min retention time, corresponded to approximately 6% of the total radioactivity. In addition, no low molecular species corresponding to 111In transchelated by EDTA in the plasma was observed, a further indication of the kinetic stability of the DOTA chelate. The superior proteolytic stability of 111In-labeled 4 rendered it the more suitable peptide-antisense-PNA conjugate to evaluate for tumor bcl-2 targeting in NHL cells in culture and lymphoma-bearing SCID-hu mouse models. However, prior to initiating these studies, the ability of ri-PTD-4 to mediate cellular internalization of anti-bcl-2 PNA required validation. Therefore, anti-bcl-2 PNA was conjugated to a lysine derivative of the red fluorescent dye tetramethylrhodamine (TMR) in the same sequence position as DOTA, after which the ri-PTD-4-G sequence was added. KT-anti-bcl-2 PNA (6) was synthesized manually using modifications of the procedures for synthesizing the DOTA-conjugated PNAs. Unlike the DOTA conjugates, the hydrophobic nature of the tetramethylrhodamine residue allowed for isolation of 6 by standard peptide and PNA cleavage, deprotection, and purification conditions. Following assembly, the crude product was cleaved from the resin and completely deprotected, using concentrated



Figure 5. (Left) Size exclusion HPLC chromatograms of 111In-labeled 2 after 0 h (top) and 168 h (bottom) of incubation in mouse plasma at 37 °C. (Right) Size exclusion HPLC chromatograms of 111In-labeled 4 after 0 h (top) and 168 h (bottom) of incubation in mouse plasma at 37 °C.

TFA and full radical scavenger cocktail, and then purified by semipreparative RP-HPLC. The 33.5 min retention time of 6 allowed for easy separation from the unreacted PNA byproduct. Compound 6 was also conjugated to riPTD-4-G by automated, stepwise elongation with the peptide sequence using standard Fmoc chemistry. The ri-PTD-4-G-KT-anti-bcl-2 PNA conjugate (7) had a retention time of 35.5 min on semipreparative RP-HPLC, also affording easy separation from unmodified PNA. Following RP-HPLC purification, conjugates 6 and 7 were isolated by diethyl ether precipitation and lyophilization. ESI-MS analyses showed patterns of molecular ions that were consistent with the calculated molecular weights of the conjugates. Analytical RP-HPLC analysis of 6 and 7 indicated that the purity of both TMR conjugates was >99.5%. To determine whether ri-PTD-4 was active in cell permeation and mediated cellular internalization of antibcl-2 PNA, scanning confocal microscopy studies were performed with conjugates 6 and 7 in Raji Burkitt’s lymphoma cells, after fixation with 4% formaldehyde at various incubation times. Raji cells express high levels of homogeneous 6.5-kb bcl-2 mRNA (51). During the course of these studies, fluorescence in Raji cells was much more intense with 7 than with 6 at all time points from 0 to 2 h of incubation, but the fluorescence intensity from each conjugate was normalized to the value at 5 min, to show qualitative differences between the two compounds. As shown in Figure 6, at 1 to 5 min, fluorescence from both conjugates was confined largely to the plasma membrane, and a small amount of cytoplasmic staining observed with 7. By 30 min, cellassociated fluorescence from 7 had decreased considerably, and that from 6 had nearly disappeared. However, after 2 h of incubation, widespread, intense fluorescence was seen in the cytoplasm of essentially all cells exposed to 7, while little to no fluorescence was observed from 6 at the same time point. The fluorescence microscopy studies demonstrated that ri-PTD-4 was active in permeation of the plasma membrane and mediated cellular internalization of the bcl-2 antisense PNA. Furthermore,

internalization of the ri-PTD-4 conjugate 7 in Raji cells delivered the antisense PNA predominantly into the cytoplasm, where concentrations of mRNA, including bcl2, are highest. Recently, Richard et al. (52) demonstrated that cell fixation with 3.7% formaldehyde caused subcellular redistribution artifacts during fluorescence microscopy studies of cell-penetrating peptides. In that report, fixation of HeLa and CHO cells led to redistribution of fluorescein- and Alexa 488-conjugated Tat peptide into the nucleus, whereas no nuclear uptake was observed in live cells. When Dowdy and co-workers (46) examined the uptake of fluorescein-conjugated PTD-4 in fixed Jurkat cells, both nuclear and cytoplasmic localization were visualized by fluorescence microscopy. To test whether cell fixation led to redistribution artifacts in the present studies, scanning confocal fluorescence microscopy of conjugates 6 and 7 in live Raji cells was performed. Association of 6 and 7 with live cells from 0 to 2 h of incubation was qualitatively similar to the results obtained with fixed cells (data not shown). At 2 h, very little membrane-bound fluorescence from 6 was observed in live Raji cells (Figure 7, left), and essentially all cells incubated with 7 showed internalization of the compound and widespread, intense cytoplasmic staining (Figure 7, right). Surprisingly, though, approximately 50% of live Raji cells incubated with the ri-PTD-4 conjugate 7 exhibited nuclear uptake after 2 h of incubation. This result was contrary to the findings of Richard et al. (52), but they studied different cell-permeating peptides in cells of epithelial origin. The nuclear localization of 7 in live lymphoma cells, but not in fixed cells, suggests that the uptake mechanisms of cell-permeating peptide conjugates are complex and remain poorly understood. As reviewed by Moulton and Moulton (53) and Vives et al. (54), most publications on cell-permeating peptides highlighted energy-independent processes that occurred at low temperatures and in the presence of drugs that inhibit active transport. However, these reviews pointed to recent reports strongly suggesting that arginine-rich peptide


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Figure 6. Scanning confocal fluorescence microscopy images of Raji cells incubated with 7 (top) or 6 (bottom) at 37 °C and 5% CO2, then fixed with 4% formaldehyde. Transmission images of cells with little or no fluorescence are shown in the lower right corner of some panels.

Figure 7. Scanning confocal fluorescence microscopy images of live Raji cells incubated with 6 (left) or 7 (right) at 37 °C and 5% CO2 for 2 h. Arrows indicate cells with nuclear accumulation of 7. Transmission images of the cells are shown in the lower right corner insets.

conjugates are internalized by an energy-dependent endocytotic pathway. Furthermore, early assay techniques produced artifacts leading to widespread acceptance of the nonendocytotic, membrane-permeating model as the mechanism of uptake. Using fluorescein conjugates and live COS7 cells, Dom et al. (55) demonstrated that cellular uptake of pAntp is a two-step process. The first step is phase transfer from a hydrophilic to a hydrophobic environment, which is probably mediated by charge neutralization in the presence of negatively charged lipids. Phase transfer of pAntp is then followed by further translocation across the membrane bilayer, a process dependent on a critical tryptophan residue at position 6. Thoreń and colleagues (56) showed that pAntp was internalized by PC-12 and V79 cells via an endocytotic mechanism that could be abrogated at 4 °C or by depleting ATP. In contrast, they observed only an energyindependent, nonendocytotic pathway for R7W in the

same cell lines. Behavior of the Tat peptide in those studies was more complex. It showed substantial endocytosis in PC-12 cells, as well as energy-independent uptake. However, in V79 cells, Tat peptide internalization was exclusively energy-independent and led to uniform uptake in the cytoplasm and nuclear accumulation, similar to the subcellular distribution of conjugate 7 in the present studies. Piwnica-Worms and co-workers (57) found evidence for a membrane permeability barrier to Tat uptake in welldifferentiated epithelial cells that form tight junctions in monolayer culture. They observed a complete lack of internalization of fluorescein-conjugated Tat peptide in monolayers of MDCK and CaCo-2 cells, but treatment with membrane permeabilizing agents resulted in immediate translocation. In contrast, epithelial cells that do not form tight junctions in monolayer culture, such as HeLa and KB 3-1, showed facile cytoplasmic and nucleolar accumulation of the fluorescent Tat conjugate.


However, it should be noted that these fluorescence microscopy studies were performed on paraformaldehydefixed cells. Piwnica-Worms’s group have also examined the effects of sequence and chirality on cell permeation activity (58). Screening a library of 42 99mTc-labeled basic domain peptides in live Jurkat cells, Piwnica-Worms and colleagues found that D-peptides were more active in cell permeation, a result not attributable to decreased proteolytic stability of the corresponding L-sequences. Furthermore, peptide uptake was dependent on length, sequence, and the type of radiometal chelate, with D-Tat-Orn6 and (D-Arg)9 having the greatest activity. Taken together, the results of these and the present studies indicate that further investigation of the uptake mechanisms of cell-permeating peptides is clearly warranted. CONCLUSION

In the present work, an innovative solid-phase synthesis strategy was developed to prepare, purify, and characterize a variety of cell-permeating peptide-PNA conjugates for targeting imaging and therapeutic radiometals to the bcl-2 cellular oncogene. The strategy was based on cleavage and partial deprotection of resin-bound DOTA-conjugated peptide-PNA constructs, followed by RP-HPLC separation of the hydrophobic precursors from the hydrophilic unreacted PNA. Using this approach, bcl-2 antisense and nonsense PNA sequences were conjugated to a new bifunctional derivative of DOTA, which can be incorporated into any position of a peptidePNA conjugate, and to peptides that mediate cell permeation and internalization of the PNA. Peptide-PNA conjugates for both radiometal and fluorophore labeling were obtained in high purity and 6% to 9% overall yield by this synthetic strategy and modifications of the procedure. The 111In-labeled retro-inverso PTD-4-G-KDanti-bcl-2 PNA conjugate showed superior plasma stability to the parent PTD-4-G construct. Moreover, scanning confocal fluorescence microscopy studies with the analogous tetramethylrhodamine conjugate demonstrated that retro-inverso PTD-4 was active in mediating internalization of anti-bcl-2 PNA in Raji NHL cells, which are known to express high basal levels of bcl-2 mRNA. Retro-inverso PTD-4-G-KT-anti-bcl-2 PNA showed nuclear accumulation in approximately 50% of live cells, but formaldehyde fixation resulted in artifactual redistribution into the cytoplasm only. Further optimization of the design and synthesis of internalizing peptide-PNA conjugates for in vivo tumor uptake could generate powerful oncogenetargeting tools for both diagnostic imaging and targeted radiotherapy of cancer. ACKNOWLEDGMENT

This work was supported by NIH Grant CA86290 (W.A. Volkert, PI) and a grant from the University of Missouri Research Reactor Research Partnerships Initiative (to M.R.L.). The authors would like to thank Dr. Mayandi Sivaguru of the University of Missouris Columbia Molecular Cytology Core, for valuable assistance in performing the fluorescence microscopy studies. We also acknowledge the Department of Veterans Affairs, for providing resources and the use of facilities at the Harry S Truman Memorial Veterans’ Hospital in Columbia, MO, and the University of Missouri Research Reactor, for purchasing the Advanced ChemTech ACT Model 396 Omega Multiple Biomolecular Synthesizer used in this research.

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BC034084N

Synthesis of Radiometal-Labeled and Fluorescent Cell-Permeating

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