Long Functionalized Poly(ethylene glycol) - American Chemical Society

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Bioconjugate Chem. 2008, 19, 973–981

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TECHNICAL NOTES Long Functionalized Poly(ethylene glycol)s of Defined Molecular Weight: Synthesis and Application in Solid-Phase Synthesis of Conjugates Dan Niculescu-Duvaz, Jayne Getaz, and Caroline J. Springer* Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, U.K. Received August 3, 2006; Revised Manuscript Received January 28, 2008

A concise synthesis of long-chain poly(ethylene glycol) (PEG) of defined molecular weight up to 29 ethyleneoxy units is described. These PEG diols were converted in a two-step synthesis into Fmoc-protected PEG amino acids, suitable as long linkers and compatible with solid-phase peptide synthesis. Long PEG chains (MW > 1000) can be readily synthesized with this method, which has the advantage of defined single molecular weight products over the comparable commercial polymers. The application of these PEG linkers to the synthesis of peptide-PEG-folate conjugates on a solid support was investigated. A method for the solid support synthesis of the targeting component of the conjugate, folic acid-cysteine, was developed, resulting in improved yields with respect to literature methods. The assembly of the peptide, PEG linker, and targeting group on solid support resulted in the synthesis of a conjugate of defined molecular weight and structure.

INTRODUCTION Poly(ethylene glycol) is a biologically inert polymer that has been used extensively to alter the physiological properties of drugs, therapeutic proteins, liposomes, and polyplexes. Conjugation with PEG has been reported to confer a number of beneficial properties including increased solubility, reduced interaction with serum and complement proteins and a significantly increased half-life in ViVo (1). PEGylation can improve the performance and dosing frequency of peptides, proteins, antibodies, oligonucleotides and many small molecules by optimizing pharmacokinetics (PK), increasing bioavailability and decreasing immunogenicity. PEGylated therapeutic agents with clinical approval include liposomes, proteins, and aptamers (2, 3). In addition, PEG chains can act as spacers. In bioconjugation, the separation between components is often necessary to preserve the affinity and activity of each component of the conjugate. For example, polyplexes containing targeting ligands for specific tissues have been shown to retain better ligand– receptor binding affinity when separated by a PEG spacer (4–6). Labeling of peptides with fluorophores and biotin often includes a PEG chain to improve the activity/affinity of the reporter group (7). The biological effects of PEG modification have been related to the molecular weight of the polyether used. In general, longer PEG chains are more efficient in improving the PK of conjugates and preserving the affinity of the targeting ligand for its receptor (8, 9). PEG of higher molecular weight is obtained by relatively uncontrolled polymerization techniques, leading to polydispersity in the molecular weight of the product. Advances in anionic polymerization techniques have led to the synthesis of relatively well-defined monobenzylated PEG (10) and het* Tel: +44 2087224214, Fax: +44 2087224046, E-mail: [email protected].

erofunctional PEG (11) with polydispersity indexes equal or below 1.09. However, the products of polymerization show a range in molecular weights, even at low polydispersity indexes. An example of the MALDI mass spectrometry analysis of commercially available PEG is shown in Figure 1. This heterogeneity in PEG molecular weight has an effect on the biological activity of the conjugates and compromises their characterization and quality control, which is important for clinical applications. Batch-to-batch variability could obscure the results and complicate investigations into the structure–activity relationship of these heterogeneous conjugates. The availability of intermediate and high molecular weight PEG of defined molecular weight (MW) is therefore highly desirable. One method for obtaining longer PEG chains of defined MW is to synthesize them starting from commercially available shorter chains of defined length. A few reports have described synthetic routes to PEG of intermediate length (up to 24 units). Here, we describe a simple route to PEG up to 29 units, with potential to be extended to higher MW. Another requirement for bioconjugation is that PEG chains need to be modified with functional groups that allow chemical coupling to biomolecules. The most common coupling strategies involve the formation of amide bonds (usually via activated esters of carboxylic acids) and thiol bonds (thioether or disulfide). One convenient functionalization of PEG is with an amino acid, which can be used in peptide synthesis on solid support, or it could be deprotected and coupled further to convert to a heterobifunctional linker. Here, we report a simple strategy for bifunctionalization of PEG diol to Fmoc-protected PEG amino acid. The utility of these linkers is exemplified by the synthesis of a folate-PEG-amphipathic peptide conjugate on solid support. Amphipathic peptides have been used in nonviral gene therapy approaches with some success in Vitro (12, 13). These peptides are able to facilitate endosomal escape by destabilizing the endosomal membrane directly as a result of a change in

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Figure 1. MALDI analysis of commercially available NHS-PEG3400-MAL. Polydispersity index 1.01.

conformation at the lower pH conditions within the endosomal compartment. Incorporation of such a peptide in a modular conjugate containing a PEG and a targeting ligand could lead to an effective nonviral gene vector for systemic application. Folic acid is the small molecule ligand for the R-folate receptor, and is overexpressed in pituitary adenomas (14) and ovarian (15) cancers. Folic acid has been conjugated to drugs (16), macromolecules (17), liposomes (5) and polyplexes (18), in order to achieve specific targeting and enhanced uptake of conjugates in R-folate-expressing tumor cells. We have attached the folic acid to the amphiphatic peptide-PEG on solid support, and demonstrated the synthesis of a complex conjugate of defined structure on solid support. This conjugate is the prototype for potential modular nonviral vectors for gene delivery.

EXPERIMENTAL PROCEDURES All starting materials, reagents, and solvents for reactions were reagent grade and used as purchased. Chromatography solvents were HPLC grade and were used without further purification. Reactions were monitored by thin layer chromatography (TLC) analysis using Merck silica gel 60 F-254 thin layer plates. LCMS analyses were performed on a Micromass LCT/Water’s Alliance 2795 HPLC system with a Discovery 5 µm, C18, 50 mm × 4.6 mm i.d. column from Supelco at a temperature of 22 °C using the following solvent systems: solvent A, methanol; solvent B, 0.1% formic acid in water at a flow rate of 1 mL/min. Gradient starting with 10% A/90% B from 0 to 0.5 min, then 10% A/90% B to 90% A/10% B from 0.5 to 6.5 min and continuing at 90% A/10% B up to 10 min. From 10 to 10.5 min, the gradient reverted back to 10% A/90% B where the concentrations remained until 12 min. UV detection was at 254 nm and ionization was positive or negative ion electrospray. Molecular weight scan range is 50–1000. Samples were supplied as 1 mg/mL in DMSO or methanol with 3 µL injected on a partial loop fill. Preparative HPLC was carried out on an Agilent 1100 series system (Agilent Technologies, Wokingham, UK) using a Gemini C18 column (250 × 30.0 mm, 10 µm). Gradient (15 mL/min): 10% MeCN to 95% MeCN over 25 min, 5 min at 95% MeCN, 95% MeCN to 10% MeCN over 10 min. NMR spectra were recorded in DMSO-d6 on a

Bruker DPX 250 instrument operating at 250.13 MHz or on a Bruker Advance instrument 500 operating at 500.26 MHz. The signal of the deuterated solvent was used as internal reference; the chemical shifts are expressed in ppm (δ). MALDI analysis was carried out using either a Lasermat (Finnigan Mat, Hemel Hampstead, UK) or Dynamo system (Thermo Bioanalysis Ltd., Hemel Hampstead, UK). The samples were analyzed using a 0.4 µL matrix (5.1 mg R-cyano-4-hydroxy cinnamic acid in 100 µL TFA, 500 µL H2O, and 500 µL MeCN). 1. Synthesis of Diols. Preparation of BnO-PEG6-OH (2). a. Using NaH. A solution of hexa(ethylene glycol) (4.5 mL, 17.73 mmol) and NaH (60% in mineral oil) (0.85 g, 21.28 mmol) in dry THF (60 mL) was stirred under argon for 90 min. Benzyl bromide (2.1 mL, 17.73 mmol) was added dropwise, and the reaction was stirred at room temperature for 20 h. The reaction was quenched with H2O, evaporated, extracted with EtOAc, and dried over MgSO4. The residue was purified by column chromatography on silica (eluent EtOAc/hexane 5:1, then EtOAc) to afford the required product (2.37 g, 35%). b. Using Ag2O. Ag2O (6.16 g, 26.60 mmol) and KI (1.18 g, 7.10 mmol) were suspended in dry DCM (150 mL) under argon. Hexa(ethylene glycol) (4.5 mL, 17.73 mmol) was added dropwise, followed by benzyl bromide (2.3 mL, 19.50 mmol) after 5 min. The reaction was stirred at room temperature for 2 h, then filtered through Celite. The filtrate was evaporated, extracted with AcOEt, and dried over MgSO4. The residue was purified by column chromatography on silica (eluent EtOAc/ acetone 4:1) to afford the required product (3.49 g, 53%). LCMS (ES): 372 [M + H]+, 395 [M + Na]+. HPLC retention time (discovery column of LCMS): 5.59 min; the unsubstituted hexa(ethylene glycol) and disubstituted BnO-PEG6-OBn have retention times of 3.10 and 7.05 min, and are present only in traces in the purified BnO-PEG6-OH (2). 1H NMR (CDCl3): 7.35–7.34 (m, 5H, Harom), 4.58 (s, 2H, PhCH2), 3.73–3.60 (m, 25H, CH2,PEG, OH). 13C NMR (CDCl3): 138.32 (Carom), 128.33 (CHarom), 127.72 (CHarom), 127.55 (CHarom), 73.23 (PhCH2), 72.58 (OCH2CH2OH), 70.64 (CH2,PEG), 70.62 (CH2,PEG), 70.60 (CH2,PEG), 70.57 (CH2,PEG), 70.54 (CH2,PEG), 70.33 (CH2,PEG), 69.47 (CH2,PEG), 61.73 (CH2OH). Preparation of BnO-PEG17-OBn (4). In a solution of BnOPEG6-OH (2) (2.97 g, 7.97 mmol) in dry THF (50 mL) under

Technical Notes

argon, NaH (60% in mineral oil, 0.43 g, 10.63 mmol) was added and stirred at room temperature for 2 h. Penta(ethylene glycol) di(p-toluenesulfonate) (3) (0.9 mL, 1.99 mmol) was added and the reaction was refluxed for 20 h. A second portion of NaH (0.43 g, 10.63 mmol) was added and the reaction mixture was stirred for 1 h. A second portion of penta(ethylene glycol) di(ptoluenesulfonate) (0.9 mL, 1.99 mmol) was then added and the reaction was refluxed for a further 20 h. The reaction was quenched with H2O, evaporated, extracted with EtOAc, and dried (MgSO4). The residue was purified by precipitation from DCM with cold diethyl ether, to afford the required product (3.16 g, 84%). LC-MS (ES): 948 [M + H]+, 474 [M + 2H]2+. MALDI +ve ion mode: 968 [M + Na]+, 984 [M + K]+. 1H NMR (CDCl3): 7.35–7.34 (m, 10H, Harom), 4.57 (s, 4H, PhCH2), 3.66–3.64 (m, 68 H, CH2,PEG). 13C NMR (CDCl3) 125 MHz: δ 128.30 (CHarom), 127.67 (CHarom), 127.52 (CHarom), 73.17 (PhCH2), 70.58 (CH2,PEG), 70.54 (CH2,PEG), 70.51 (CH2,PEG). Preparation of PEG17 Diol (5). A solution of BnO-PEG17OBn (4) (3.16 g, 3.34 mmol) containing 10% Pd/C (0.07 g) in EtOH (60 mL) was stirred under hydrogen for 4 days. The reaction was filtered through Celite and the filtrate evaporated. The residue was purified by precipitation from DCM with cold diethyl ether, to afford the required product (2.48 g, 97%). LCMS (ES): 767 [M + H]+, 334 [M + 2H]2+. 1H NMR (CDCl3) 500 MHz: δ 3.66–3.61 (m, 70 H, CH2,PEG, OH). 13C NMR (CDCl3) 125 MHz: δ 72.46 (HOCH2CH2O), 70.43 (CH2,PEG), 70.40 (CH2,PEG), 70.19 (CH2,PEG), 61.38 (HOCH2CH2O). Elemental analysis: (C34H70O18 · 0.5H2O): required C 52.63, H 9.22; found C 52.81, H 9.20. Preparation of BnO-PEG6-OTos (6). In a solution of BnOPEG6-OH (2) (2.00 g, 5.37 mmol) in dry DCM (100 mL), under argon, p-toluenesulfonyl chloride (1.54 g, 8.05 mmol), KI (0.36 g, 2.15 mmol), and Ag2O (1.87 g, 8.05 mmol) were added. The reaction was stirred at reflux for 20 h under argon and protected from light. The mixture was filtered through Celite and the filtrate evaporated. The residue was purified by column chromatography on silica (eluent EtOAc) to afford the required product (2.60 g, 92%). LC-MS (ES): 526 [M+H]+. 1H NMR (CDCl3): 7.81 (d, J ) 8.50, 2H, CHarom), 7.35–7.28 (m, 7H, CHarom), 4.57 (s, 2H, PhCH2), 3.70–3.58 (m, 24H, CH2,PEG), 2.45 (s, 3H, CH3). 13C NMR (CDCl3): 144.74 (Carom), 138.33 (Carom), 133.11 (Carom), 129.80 (CHarom), 128.32 (CHarom), 127.94 (CHarom), 127.69 (CHarom), 127.54 (CHarom), 73.20 (PhCH2), 70.72 (CH2,PEG), 70.64 (CH2,PEG), 70.59 (CH2,PEG), 70.55 (CH2,PEG), 70.51 (CH2PEG), 69.47 (OCH2CH2OTos), 68.65 (CH2OTos), 21.59 (CH3). Preparation of BnO-PEG29-OBn (7). To a solution of PEG17 diol (5) (0.50 g, 0.65 mmol) in dry THF (50 mL) under argon, NaH (0.52 g, 13.04 mmol) was added. The reaction was stirred under argon for 2 h at room temperature before a solution of BnO-PEG6-OTos (5) (0.69 g, 1.3 mmol) in dry THF (5 mL) was added. The reaction was refluxed for 20 h. The reaction was quenched with H2O, evaporated, extracted with EtOAc, and dried (MgSO4). The residue was purified by precipitation from DCM with cold diethyl ether, to afford the required product (0.80 g, 83%). LC-MS (ES): 1476 [M + H]+, 738 [M + 2H]2+, 493 [M + 3H]3+, MALDI +ve ion mode: 1495 [M + Na]+. 1 H NMR (CDCl3): δ 7.35–7.34 (m, 10H, Harom), 4.57 (s, 4H, PhCH2), 3.66–3.64 (m, 116 H, CH2,PEG). 13C NMR (CDCl3): δ 128.28 (CHarom), 127.64 (CHarom), 127.50 (CHarom), 73.16 (PhCH2), 70.61 (CH2,PEG), 70.56 (CH2,PEG), 70.54 (CH2,PEG). Preparation of PEG29 Diol (8). A solution of BnO-PEG29OBn (7) (0.60 g, 0.41 mmol) containing 10% Pd/C (0.01 g) in EtOH (10 mL) was stirred under hydrogen for 3 days. The reaction was filtered and the filtrate was evaporated. The residue was purified by precipitation from DCM with cold diethyl ether, to afford the required product (0.49 g, 93%). LC-MS (ES): 1296

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[M + H]+, 648 [M + 2H]2+, 432 [M + 3H]3+. MALDI +ve ion mode: 1318 [M + Na]+. 1H NMR (CDCl3): δ 3.66–3.61 (m, 118 H, CH2,PEG, OH). 13C NMR (CDCl3): 72.52 (HOCH2CH2O), 70.61 (CH2,PEG), 70.57 (CH2,PEG), 70.36 (CH2,PEG), 61.71 (HOCH2CH2O). Elemental analysis (C58H118O30 · 0.5H2O): required C 53.40, H 9.19; found C 53.28, H 9.17. 2. Functionalization to Fmoc-Protected Amino Acid. Preparation of PEG17 Diacid (9). Potassium tert-butoxide (2.93 g, 26.08 mmol) was added to a solution of PEG17 diol (5) (1.00 g, 1.30 mmol) in tert-butanol (46 mL). The mixture was stirred at 50 °C under argon for 8 h, protected from light. Ethyl bromoacetate (2.9 mL, 26.08 mmol) was then added and a precipitate formed. The reaction was stirred for 24 h before being filtered and the filtrate evaporated. The residue was taken in 30 mL 1.0 M NaOH and stirred at room temperature for 8 h. The reaction was adjusted to pH 3 with 1.0 M HCl and extracted with chloroform. The organic layer was collected and dried with MgSO4. The residue was purified by precipitation from DCM with cold diethyl ether, to afford the required product (0.91 g, 79%). LC-MS (ES): 883 [M + H]+, 442 [M + 2H]2+, 295 [M + 3H]3+. MALDI +ve ion mode: 903 [M + Na]+, 919 ([M + K]+. 1H NMR (CDCl3): δ 4.08 (s, 4H, OCH2COOH), 3.66–3.58 (m, 68 H, CH2,PEG). 13C NMR (CDCl3): δ 172.00 (CO), 71.20 (CH2,PEG), 70.64 (CH2,PEG), 70.61 (CH2,PEG), 70.54 (CH2,PEG), 70.50 (CH2,PEG), 70.46 (CH2,PEG), 70.39 (CH2,PEG), 68.81 (COCH2O). Preparation of PEG29 Diacid (10). The title compound was prepared starting from PEG29 diol (8) (0.4 g, 0.31 mmol) using the same method as described for compound 9 (0.39 g, 88%). LC-MS (ES): 1412 [M + H]+, 706 [M + 2H]2+, 471 [M + 3H]3+. MALDI +ve ion mode: 1433 [M+Na]+. 1H NMR (CDCl3): δ 4.09 (s, 4H, OCH2COOH), 3.60–3.58 (m, 116 H, CH2,PEG). 13C NMR (CDCl3): δ 71.08 (CH2,PEG), 70.61 (CH2,PEG), 70.59 (CH2,PEG), 70.54 (CH2,PEG), 70.47 (CH2,PEG), 70.44 (CH2,PEG), 70.36 (CH2,PEG), 68.86 (COCH2O). Preparation of Fmoc-PEG17-COOH (11). N-Hydroxysuccinimide (0.05 g, 0.45 mmol) was added to a solution of PEG17 diacid (9) (0.40 g, 0.45 mmol) in dry DCM (40 mL) under argon. The reaction mixture was cooled in an ice bath, and a solution of DCC (0.09 g, 0.45 mmol) in dry DCM (5 mL) was added dropwise. The reaction was allowed to warm to room temperature and stirred for 4 h. A solution containing 1.0 equivalent of mono-Fmoc 1,3 diaminopropane hydrochloride (0.15 g, 0.45 mmol) in dry NMP (1 mL) was then added, followed by 4.0 equiv of triethylamine (0.3 mL, 1.81 mmol). The reaction was stirred at room temperature for 20 h before being evaporated. The residue was purified by preparative HPLC to afford the required product (0.12 g, 23%). LC-MS (ES): 1162 [M + H]+, 581 [M + 2H]2+. MALDI +ve ion mode: 1183 [M + Na]+. HPLC retention time (min): 12.95. Purity by HPLC: 99%. 1H NMR (CDCl3): δ 7.75 (d, 2H, CHarom, J ) 7.45 Hz), 7.61 (d, 2H, CHarom, J ) 7.35 Hz), 7.39 (t, 2H, CHarom, J ) 7.45 Hz), 7.31 (t, 2H, CHarom, J ) 7.45 Hz), 5.79 (t, 2H, J ) 6.35 Hz), 4.40 (d, 2H, FmocCH2O, J ) 7.25 Hz), 4.20 (t, 1H, FmocCH, J ) 6.90 Hz), 4.14 (s, 2H, CH2COOH), 4.00 (s, 2H, COCH2O), 3.73–3.58 (m, 68H, CH2, PEG), 3.35 (d, J ) 6.20, 2H, CH2NHCO), 3.21 (d, 2H, OCONHCH2, J ) 6.20), 1.69 (t, 2H, NHCH2CH2, J ) 6.30 Hz). 13C NMR (CDCl3): δ 144.05 (Carom), 141.29 (Carom), 127.63 (CHarom), 127.05 (CHarom), 125.10 (CHarom), 119.91 (CHarom), 71.01 (CH2,PEG), 70.93 (CH2,PEG), 70.53 (CH2,PEG), 70.46 (CH2,PEG), 70.40 (CH2,PEG), 70.29 (CH2,PEG), 70.24 (CH2,PEG), 70.13 (CH2,PEG), 68.99 (CH2CO), 66.35 (CH2,Fmoc), 47.28 (CHFmoc), 37.72 (CH2), 35.70 (CH2), 29.71 (CH2). Elemental analysis (C52H92N2O23 · 2H2O): required C 56.17, H 8.08, N 2.34; found C 56.25, H 8.01, N 2.11. Preparation of Fmoc-PEG29-COOH (12). The title compound was prepared starting from PEG29 diacid (10) (0.30 g, 0.21

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mmol) using the same method as described for compound 11 (0.39 g, 88%). 0.13 g of required product was obtained. Yield: 35%. LC-MS (ES):1690 [M + H]+, 845 [M + 2H]2+, 564 [M + 3H]3+. MALDI +ve ion mode: 1712 [M + Na]+. HPLC retention time (min): 12.56. Purity by HPLC: 99%. 1H NMR (CDCl3): δ 7.84 (d, 2H, CHarom, J ) 7.60 Hz), 7.68 (d, 2H, CHarom, J ) 7.25 Hz), 7.51 (m, 1H, OCONH), 7.40 (t, 2H, CHarom, J ) 7.40 Hz), 7.32 (t, 2H, CHarom, J ) 7.25 Hz), 6.53 (m, 1 H, CH2NHCO), 4.36 (d, 2H, FmocCH2O, J ) 7.25 Hz), 4.22 (t, 1H, FmocCH, J ) 6.77 Hz), 4.10 (s, 2H, CH2COOH), 3.92 (s, 2H, COCH2O), 3.72–3.52 (m, 116H, CH2, PEG), 3.32–3.28 (m, 2H, CH2NHCO), 3.20–3.17 (m, 2H, OCONHCH2), 1.70–1.67 (m, 2H, NHCH2CH2). 13C NMR (CDCl3): δ 170.68 (Carom), 157.21 (Carom), 145.07 (CHarom), 142.00 (CHarom), 128.38 (CHarom), 127.82 (CHarom), 125.94 (CHarom), 120.68 (CHarom), 71.66 (CH2,PEG), 71.30 (CH2,PEG), 71.13 (CH2,PEG), 71.00 (CH2,PEG), 70.94 (CH2,PEG), 68.77 (CH2CO), 66.62 (CH2,Fmoc), 48.06 (CHFmoc), 38.64 (CH2), 36.38 (CH2), 29.71 (CH2). Elemental analysis (C80H140N2O35 · H2O): required C 56.26, H 8.38, N 1.64; found C 55.97, H 8.35, N 1.42. 3. Synthesis of the Peptide-PEG-Folate Conjugate. Synthesis of Folic Acid-Cysteine Targeting Ligand (13). A Glu(tBu)Cys(Trt) dipeptide was synthesized on 0.65 g of Fmoc-Cys(Trt) HMP resin (loading capacity 0.58 mmol/g) using automated peptide synthesis. 4.0 equiv of Fmoc-Glu-OtBu (Bachem, UK) was utilized in each coupling step. The peptideo-resin was washed in DCM and MeOH and dried in a desiccator. Pteroic acid (0.10 g, 0.32 mmol) was taken up in dry DMF (3 mL) and dry DMSO (1.3 mL) and added to 0.19 g of peptideo-resin under argon. A 0.5 mM HOBt/HBTU solution in DMF (0.6 mL, 0.32 mmol) was added, followed by a 2.0 mM DIPEA solution in NMP (0.3 mL, 0.64 mmol). The reaction mixture was protected from light and shaken at room temperature for 24 h. The peptido-resin was filtered and washed in DMSO (4 mL) four times, DMSO/DMF 1:1 (4 mL) once, DMF (4 mL) once, and two alternating washes of DCM and MeOH (4 mL). The resin was then dried in a desiccator. 0.10 g of peptido-resin was cleaved using 3.0 mL cleavage mixture (0.75 g phenol, 0.5 mL thioanisole, 0.5 mL H2O, 0.3 mL EDT, 10.0 mL TFA). The reaction was shaken at room temperature for 3 h. The filtrate was precipitated in cold diethyl ether and centrifuged to pellet. The pellet was washed in diethyl ether four times before drying in a desiccator 0.02 g of required product was obtained. Yield: 62% LC-MS (ES): 544 [M + H]+. HPLC retention (min): 7.38. Purity by HPLC: 70%. 1H NMR (CD3)2SO 500 MHz: δ 8.66 (s, 1H, AromH1), 8.21–8.17 (m, 2H, NH amide), 7.66 (d, 2H, J ) 8.50, AromH4), 7.33–7.23 (m, 3H, PhNH, NH2), 6.64 (d, 2H, J ) 8.85, AromH3), 4.50 (s, 2H, H2), 4.42–4.37 (m, 1H, H5), 4.31–4.28 (m, 1H, H8), 2.85–2.68 (2m, 2H, H9), 2.31–2.19 (m, 2H, H7), 2.09–1.86 (2m, 2H, H6). Preparation of Amphipathic Peptide KKALLALALHHLAHLALHLALALKKA (14) (19). The peptide was synthesized using an ABI 433A peptide synthesizer (Applied Biosystems, California, USA) equipped with conductivity monitoring. The activation solution used in each coupling step contained HOBt/ HBTU in DMF (0.5 mM) and DIPEA in NMP (2 mM). Each coupling step was carried out over approximately 4.5 min and was followed by a wash in NMP. Fmoc deprotection was carried out using 22% piperidine in DMF over 2 min and was followed by conductivity monitoring. This step was repeated up to 6 times. The peptide was synthesized on 149 mg of Fmoc-Ala HMP resin (loading capacity 0.7 mmol/g) using 10.0 equiv of amino acid in each coupling step. 462 mg of peptido-resin was obtained from the synthesis. Theoretical yield: 88%. The peptide from 100 mg peptido-resin was cleaved using a cleavage mixture: 0.75 g crystalline phenol, 0.25 mL EDT, 0.50 mL thioanisole, 0.50 mL H2O, and 10.00 mL TFA. Cleavage

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was carried out in an Isolute SPE single fritted reservoir (Argonaut Technologies Inc., Mid Glamorgan UK). The resin was filtered and the filtrate was collected and precipitated by adding cold diethyl ether. The precipitate was collected by centrifugation and washed with diethyl ether. The precipitate was dried in a desiccator. The peptide was purified by semipreparative HPLC on a Jupiter C18 column (250 × 10 mm, 5 µm). A gradient was run at 3 mL/min from 95% H2O (0.1% TFA), 5% MeCN (0.1% TFA) to 5% H2O (0.1% TFA), 95% MeCN (0.1% TFA) over four column volumes. 38 mg of required product was obtained. Yield of purified peptide: 54%. MALDI +ve ion mode: 2779 [M + H]+, 2801 [M + Na]+. HPLC retention (min): 12.84. Purity by HPLC: 98%. Preparation of Amphipathic Peptide-PEG17-NH-Fmoc (15). 1.5 equiv each of FmocNHPEG17-COOH (7 mg, 6.24 µmol) and HCTU (3 mg, 6.24 µmol) were taken up in dry NMP/DCM 9:1 (1 mL) and added to 25 mg amphipathic peptido-resin under argon. 3.0 equiv of DIPEA in NMP (2 mM) (0.01 mL, 12.0 µmol) were added and the reaction was shaken for 72 h. The reaction was filtered, rinsed in DMF, DCM, and MeOH, and dried in a desiccator. Cleavage was carried out as previously described. MALDI, +ve ion mode: 3923 [M + H]+,3944 [M + Na]+. HPLC retention (min): 13.47. Product in crude by HPLC: 66%. Preparation of Amphipathic Peptide-PEG17-NH2 (16). 65 mg of amphipathic peptide-PEG17-NHFmoc peptido-resin was taken up in 1.5 mL of 20% piperidine in DMF and shaken for 3 h. The reaction was filtered, rinsed in DMF, DCM, and MeOH, and dried in a desiccator. Cleavage was carried out as previously described. MALDI, +ve ion mode: 3700 [M + H]+, 3722 [M + Na]+. HPLC retention (min): 12.37. Preparation of Amphipathic Peptide-PEG17-NH-Maleimide (17). 10.0 equiv of 6-maleimidohexanoic acid N-hydroxysuccinimide ester (26 mg, 83.00 µmol) was taken up in dry DMF (2 mL) and added to 54 mg amphipathic peptide-PEG17-NH2 peptido-resin. The reaction was shaken for 72 h. The reaction was filtered, rinsed in DMF, DCM, and MeOH, and dried in a desiccator. Cleavage was carried out as previously described. MALDI, +ve ion mode: 3891 [M + H]+, 3913 [M + Na]+. HPLC retention (min): 12.62. Preparation of Amphipathic Peptide-PEG17-Folate Cysteine (18). 9 mg folate cysteine was taken up in dry DMF (1 mL) and added to 35 mg amphipathic peptide-PEG17-maleimide peptido-resin under argon. The reaction was protected from light and shaken at room temperature for 72 h. The reaction was filtered, rinsed in DMSO, DMF, DCM, and MeOH, and dried in a desiccator. Cleavage was carried out as previously described. The crude product was purified by semipreparative HPLC on a Jupiter C18 column, followed by repeat purification on a Luna CN (250 × 6.4 mm, 5 µm) column. 1.2 mg of required product was obtained. Yield: 7% over four synthetic steps and two purification steps. HPLC retention time (standard conditions) (min): 10.90. HPLC retention time (Luna CN column, 250 × 4.60 mm, 5 µm, Phenomenex Ltd.) (min): 10.86. Purity by HPLC (standard conditions): 92%. MALDI: 4438 [M + H]+.

RESULTS AND DISCUSSION 1. Synthesis of PEG17 and PEG29. The controlled synthesis of PEG is achieved by the Williamson ether formation; a suitably activated PEG chain is attacked by another PEG chain with a nucleophilic alkoxide ion. The use of protecting groups enables the controlled synthesis of extended PEG chains of defined MW. Orthogonal protection with two different protecting groups has been used in the synthesis of smaller PEG oligomers (up to

Technical Notes

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Scheme 1. Synthesis of PEG17 and PEG29

24 glycol units) (20, 21). Our strategy is a quicker, more efficient route to PEG oligomers of defined MW, achieved through the simultaneous extension of both terminal hydroxyls of a PEG diol, using a single protecting group (Scheme 1). Similar approaches were investigated previously using monotritylated and ditosylated PEG oligomers (22, 23). The oligomers synthesized were 12 and 16 units and synthesis was initiated with monoprotected ethylene and diethylene glycol. Hexa(ethylene glycol) (1) and penta(ethylene glycol) ditosylate (3) were used as starting materials for the synthesis of PEG oligomers of 17 glycol units. Benzyl ether was chosen as the protecting group due to its stability under the basic conditions required to generate the alkoxide ion and ease of removal by hydrogenation. The first step in this synthetic route required the generation of a monoprotected hexa(ethylene glycol) (2). The initial approach utilized sodium hydride to generate the alkoxide ion, followed by reaction with benzyl bromide. Monoprotection was achieved by carrying out the reaction in diluted solution. Yields from this approach were modest: at or below 35%. An alternative approach to the monoprotection of diols involves the use of silver oxide. This method is milder, since there is no need for excess base to deprotonate the hydroxyl group, and it has been reported to result in monoprotection (24). The silver atom is hypothesized to act as a Lewis acid and complex with one hydroxyl group and the other terminal oxygen atom within a single PEG chain, which may make the proton that is not involved in the intramolecular bond more labile (25). Yields using this approach were improved to 52%. The monoprotected hexa(ethylene glycol) (2) was reacted with ditosylated penta(ethylene glycol) (3) to afford 4. An excess of sodium hydride was used to generate the monoprotected

hexa(ethylene glycol) alkoxide, which was refluxed in dry THF in the presence of 2 equiv of penta(ethylene glycol) ditosylate. The reaction proceeded in good yield at 83%, with no contaminating monoprotected hexa(ethylene glycol) (2) as detected by HPLC. The product (4) was isolated by extraction and precipitation in cold ether and did not require purification by column chromatography, in contrast to the orthogonal protection approach (21). Hydrogenation of the product obtained proceeded quantitatively to yield the PEG17 diol (5) (Scheme 1). The next step in PEG extension was the synthesis of PEG29 utilizing the PEG17 diol (5) and monoprotected hexa(ethylene glycol) tosylate (6). The monoprotected hexa(ethylene glycol) tosylate 6 was prepared from monobenzyl ether hexa(ethylene glycol) (2) using Ag2O and tosyl chloride (21). The tosylate (6) (2 equiv) was reacted with the dialkoxide of PEG17 (5), to afford the dibenzylated PEG29 (7). Hydrogenation over palladium catalyst produced the PEG29 diol (8) (Scheme 1). The reaction proceeded in 70% yield over three steps from 5 to 8. The product was isolated by precipitation from diethyl ether and did not require further purification. This novel approach has demonstrated a controlled, straightforward, and high-yielding synthesis of PEG chains of higher molecular weight in relatively few synthetic steps. The MALDI analysis of the product showed a single peak (Figure 2A), unlike the distribution of molecular weights in commercial products (compare to Figure 1). 2. Functionalization of PEG17 and PEG29 as FmocProtected Amino Acids. Methods to convert PEG diols to amino acids have been described (26, 27). Here, we describe a short (two-step) synthetic approach to obtain a PEG amino acid directly in the Fmoc-protected form. This method generates the

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Figure 2. MALDI analysis. (A) PEG29 diol (8); (B) Fmoc-PEG17-COOH (11); (C) Fmoc-PEG29-COOH (12); (D) peptide-PEG17-folate conjugate (18).

dicarboxylic acids of PEG (9) and (10), followed by the conversion of a single carboxyl group to mono-Fmoc 1,3 diamino propane to obtain the required mono-Fmoc PEG amino acids 11 and 12 (Scheme 2). Veronese et al. synthesized a PEG carboxylic acid using ethyl bromoacetate and potassium tert-butoxide in tert-butanol, followed by the hydrolysis of the ethyl ester (28). This approach was successfully incorporated into the synthesis of the PEG diacids. The diacids were precipitation from DCM with diethyl

ether, without need for further purification. Yields of up to 79% and 88% were obtained for PEG17 diacid (9) and PEG29 diacid (10), respectively. The PEG diacids were coupled with 1 equiv of mono-Fmoc 1,3 diamino propane to obtain the required mono-Fmoc PEG amino acids 11 and 12 alongside the corresponding di-Fmoc amino modified PEG. Purification of this mixture was carried out using preparative HPLC on reverse-phase silica. Yields of the products were 23% and 35% for PEG17 amino acid (11)

Technical Notes Scheme 2. Synthesis of Fmoc-Protected PEG Amino Acids

Scheme 3. Solid-Phase Synthesis of Folate-Cysteine Ligand

and PEG29 amino acid (12), respectively. The MALDI analysis of 11 and 12 is shown in Figure 2B,C, respectively. The main Scheme 4. Synthesis of Peptide-PEG17-Folate Conjugate

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peaks correspond to the product; the extra peaks at 942 (Figure 2B) and 1469 (Figure 2C) are due to the loss of Fmoc under the MALDI conditions. 3. Synthesis of the Peptide-PEG-folate Conjugate. The PEG17 amino acid (11) was used in the solid-phase synthesis of a conjugate comprising an amphiphatic peptide and a targeting moiety (folate) separated by the PEG17 linker. This conjugate is the prototype of a potential nonviral gene delivery vector, where the peptide achieves DNA condensation and endosomal escape, the folate is specifically targeting tumors overexpressing the R-folate receptor and contributes to conjugate internalization, and the PEG chain improves the PK properties of the conjugate and contributes to the DNA protection. An amphiphatic histidine-rich peptide (KKALLALALHHLAHLALHLALALKKA) (14), reported to have high transfection efficiency (19), was chosen for the conjugate. The peptide was synthesized on solid support using Fmoc chemistry. A portion of the peptide was cleaved, purified, and analyzed; the bulk of the peptide was left on the resin and used for conjugate synthesis. The folate-cysteine targeting ligand (13) was synthesized on a solid support. Advantages of this approach include higher purity of the final product and conjugation at only the preferred γ-carboxyl of the glutamic moiety, since suitable protected components such as Fmoc-Glu-OtBu are commercially available. Previous work by Zhang et al. described the synthesis of the folate-cysteine conjugate on a solid support utilizing the expensive N10-trifluoroacetyl pteroic acid group, which improves the solubility of pteroic acid, but affords low yields due to its incomplete deprotection with base (29). We carried out the solid-phase synthesis of folate-cysteine using the less expensive pteroic acid rather than its N10-

980 Bioconjugate Chem., Vol. 19, No. 4, 2008

trifluoroacetyl protected form. Synthesis of the Glu-Cys dipeptide was carried out using automated peptide synthesis. The final coupling of pteroic acid onto the dipeptide resin was carried out manually due to the low solubility of pteroic acid in NMP, which blocked the peptide synthesizer filters and prevented delivery to the reaction vessel. The manual coupling of pteroic acid utilized 3.0 equiv of pteroic acid in a mixture of DMF and DMSO and was carried out over 24 h. Despite the low solubility of pteroic acid, coupling to the dipeptide sequence proceeded efficiently and cleanly. No coupling of the unprotected N10 amine of pteroic acid, leading to large multipteroate conjugates, was detected by MALDI analysis. This indicated that unprotected pteroic acid could be utilized in a viable route to the synthesis of folate-cysteine conjugates. This synthetic route is shown below in Scheme 3. The yield obtained by using pteroic acid in the synthesis of the folate-cysteine conjugate was 64%, in comparison with yields of 38% obtained using the N10-trifluoroacetyl pteroic acid as reported by Zhang et al. (29). Therefore, this approach was been shown to be a more efficient, less expensive synthesis of folate cysteine on a solid support. The first step in the multicomponent vector assembly was the coupling of the FmocNH-PEG17-COOH chain to the N-terminus of the resin-bound peptide. The coupling was carried out using the aminium-based coupling reagent HCTU. Optimization of the reaction conditions was required. The best conditions for coupling were found to be 1.5 equiv FmocNHPEG17-COOH, 1.5 equiv of HCTU, 3.0 equiv of DIPEA, dry DMF, 72 h. The conversion to the required product was 66%, as determined by HPLC analysis. Standard Fmoc deprotection conditions using 20% piperidine in DMF was quantitative and was followed by coupling to a commercially available NHSactivated ester of maleimide. The success of this series of reactions validated the functionalization of PEG on solid support as an achievable step in the synthesis of multicomponent vectors. This approach is versatile and can be used to incorporate alternative coupling reagents. This could be of interest, as it has been reported that certain linkers such as maleimide can initiate linker-specific immune responses (30), and therefore, the ability to change the conjugation chemistry could be of benefit. The maleimide functionalized peptide-PEG conjugate was coupled to the targeting ligand folate-cysteine. Folate-cysteine was dissolved in dry DMF, added to the peptido-resin and shaken for 72 h. The synthetic scheme demonstrating PEG and ligand coupling on solid support is shown in Scheme 4. Coupling of folate-cysteine proceeded in good yield. Following cleavage from the resin, the multicomponent vector, amphipathic peptide-PEG17-folate-cysteine, was purified twice by semipreparative HPLC. The first purification used a Jupiter C18 column; however, the required product was contaminated by a side product, amphipathic peptide-folate-cysteine, obtained due to the incomplete coupling of FmocNH-PEG17COOH. Therefore, the mixture obtained was further purified on a Luna CN column, which was able to separate the two products. The global yield of the complete vector was calculated from the amphipathic peptide resin over the four modification steps including FmocNH-PEG17-COOH coupling, deprotection, 6-maleimidohexanoic acid NHS ester coupling, and folatecysteine coupling and two purification steps. The yield of purified amphipathic peptide-PEG17-folate-cysteine vector was 7%. This calculation is based on a theoretical amount of resinbound peptide. The yield could be improved by acetylation of the unreacted resin-bound amphipathic peptide prior to Fmoc deprotection of the PEG oligomer. This would prevent the

Niculescu-Duvaz et al.

formation of the contaminating side product and reduce the number of purification steps required. The MALDI analysis of the conjugate confirms the single molecular weight of the product (Figure 2D; the large peak at 5734 is the calibration standard).

SUMMARY AND CONCLUSION An efficient synthetic route to long PEG oligomers was designed. This utilized only a benzyl ether protecting group which avoided the tedious purification and low overall yields of methods reported previously (20, 21). The availability of starting materials longer than hexa(ethylene glycol) would make this route a practical method to obtain PEG of comparable length to polymeric commercial products, but with the added advantage of a single MW composition. The functionalization as PEG amino acids suitable for solidphase synthesis and heterobifunctional linkers was accomplished through the corresponding diacids and the coupling of a single equivalent of a monoprotected diamine. The preparation of two mono-Fmoc PEG amino acids of 17 and 29 units was achieved. This simple method also allows the rapid synthesis of a variety of PEG linkers. Using diamines with one amino group modified with other moieties instead of Fmoc (for example, biotin, maleimide, targeting ligands), the range of bifunctional PEG linkers can be greatly extended. The application of these linkers was demonstrated by the synthesis of a complex conjugate on a solid support. An advantageous method for the solid support synthesis of the targeting component of the conjugate, folic acid-cysteine, was found, resulting in improved yields with respect to literature methods. The assembly of the peptide, PEG linker, and targeting group on a solid support was successful, resulting in a conjugate of defined molecular weight and structure. The successful synthesis of a complex multicomponent conjugate on a solid support demonstrates the power and versatility of this approach, and opens the way to the generation of a combinatorial library of peptide-PEG-targeting ligands with defined structure and molecular weight as potential nonviral vectors and conjugates for biological applications.

ACKNOWLEDGMENT This work was supported by Cancer Research UK (CUK) grants number: C309/A2187, C107/A3096, and C1364/A3118 and the Institute of Cancer Research. We would like to acknowledge the Structural Chemistry Team: Amin Mirza and Meirion Richards for their support with MS and NMR analyses, and Jacky Metcalfe for support with peptide synthesis and MALDI analysis.

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