Bioconjugate Chem. 2010, 21, 807–810
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Solid-Phase Synthesis of PEGylated Lipopeptides Using Click Chemistry Rasmus I. Jølck, Rolf H. Berg, and Thomas L. Andresen* Technical University of Denmark, DTU Nanotech, Department of Micro- and Nanotechnology, Frederiksborgvej 399, 4000 Roskilde, Denmark. Received January 2, 2010; Revised Manuscript Received March 29, 2010
A versatile methodology for efficient synthesis of PEGylated lipopeptides via CuAAC “Click” conjugation between alkyne-bearing solid-supported lipopeptides and azido-functionalized PEGs is described. This new and very robust method offers a unique platform for synthesizing PEGylated lipopeptides with a high level of complexity. These molecules, obtained in a single purification step, are ideally suited for functionalization of solid-supported lipid bilayers and liposomal drug delivery systems and are particularly valuable in enzyme activation strategies.
We report for the first time a highly efficient and versatile methodology for the synthesis of PEGylated lipopeptides on solid-phase support by use of the Cu(I) catalyzed azide/alkyne Huisgen 1,3-dipolar cycloaddition (CuAAC “Click” conjugation) developed by the Meldal (1) and the Sharpless (2) laboratories. This approach allows for an efficient synthesis of highly functionalized PEGylated lipopeptides, which are particularly interesting in the field of drug delivery. Current research in advanced liposomal drug delivery systems is increasingly focused on materials with a high complexity and functionality. Functionalized lipids can be difficult and expensive to synthesize, which is highly problematic for their utilization in drug delivery systems in a clinical setting. This creates a crucial need for the development of new approaches for obtaining complex lipids efficiently. Conjugates with the general composition of lipid-peptide-PEG have drawn particular interest due to their use as triggering molecules in enzymatically sensitive drug delivery systems (3-7). Such conjugates are particularly difficult to synthesize due to the diverse properties of the peptidyl side chains, the high molecular mass of the poly(ethylene glycol) (PEG) polymer, and the amphiphilic character of the compounds, which results in difficult purification procedures. Currently, such molecules are predominantly synthesized by postassembly of the lipid, peptide, and PEG-polymer fragments using N-hydroxysuccinimide (NHS) and N,N′′-dicyclohexylcarboiimide (DCC) chemistry in solution (3, 4). This approach may result in unspecific binding if multiple binding sites are present within the peptide. Furthermore, it often results in moderate yields and time-consuming purification steps. In the present communication, we report a novel approach for the synthesis of complex PEGylated lipopeptides solely on solid-phase support. The method is based on the known advantages of solid phase peptide synthesis (SPPS) (8) and the unique features of the CuAAC reaction. By adopting SPPS methodology, excess reagents can easily be removed by washing and the required purification steps are greatly reduced. In addition, the CuAAC reaction is one of the most useful and robust ligation reactions available and offers unique flexibility due to the high level of orthogonality to other chemical functionalities. We have developed a general methodology for achieving PEGylated lipopeptides on solid-phase support by using the CuAAC reaction with the solvent, the choice of resin, and the reaction time as the optimization parameters. * Corresponding author. Tel: +45 46775480; Fax. +45 46774791; E-mail:
[email protected].
The resin-bound diacylated lipopeptide 1 (Figure 1) was chosen as an immobilized model substrate for the on-resin CuAAC reaction. A bulky peptide with large side-chain protection groups was chosen in order to demonstrate the versatility of the developed method. The alkyne functionality at the C-terminal of the peptide serves as a site specific conjugation site, whereas the tryptophan residue was chosen as a UV-marker for HPLC monitoring at 280 nm. The model lipopeptide 1 was synthesized on both PS (1% DVB cross-linked) resin and NovaSyn TentaGel resin both equipped with the Rink amide linker in order to elucidate the effect of the solid-phase support. Lipopeptide 1 was synthesized manually by employing standard Fmoc chemistry (9) (see Supporting Information). The alkyne functionality was introduced at the C-terminal of the lipopeptide by using (S)-2-(Fmoc-amino)-4-pentynoic acid. The diacyl lipid anchor was achieved by conjugation of NR,Nβdi-Fmoc-L-2,3-diaminopropionic acid (Fmoc-dap(Fmoc)-OH) to the N-terminal of the peptide followed by diacylation with myristic acid (Scheme 1). After diacylation with myristic acid, a test cleavage of 1 from the resin was conducted in order to confirm the peptide sequence by MALDI-TOF MS and RPHPLC (see Supporting Information). The methoxy poly(ethylene glycol) azides 3a-c needed for the on-resin CuAAC reaction were synthesized from the commercially available corresponding monomethoxy poly(ethylene glycol)s by either tosylation or mesylation of the alcohol, followed by a nucleophilic substitution with sodium azide, as illustrated in Scheme 2. This approach was easily scaled up and resulted in good yields. Azide formation was confirmed by 1 H NMR, 13C NMR, MALDI-TOF MS, and FT-IR (see Supporting Information). The efficiency of the CuAAC reaction between the resinbound lipopeptide 1 and the monomethoxy poly(ethylene glycol) azides 3a-c was studied as a function of time, choice of resin, and solvent. The reactions were carried out in 2.5 mL reaction vessels by using 50 mg lipopeptide modified resin (12.5-15 µmol lipopeptide), 5 equiv of the azide functionalized polymer, 5 equiv copper(I) iodide, and 5 equiv sodium ascorbate in either DMF/piperidine (4:1) or CH2Cl2/piperidine (4:1). The total reaction volume was adjusted in all cases to give a final concentration of the above-mentioned reagents of 61.25 mM (Scheme 3). Small samples of resin were taken out after 1, 2, 5, 24, 48, and 72 h respectively. The PEGylated lipopeptides were cleaved from the resin with a mixture of TFA/TIS/H2O, lyophilized, and analyzed by analytical RP-HPLC, employing a Waters XTerra RP18 5 µm (4.6 × 150 mm) column with water/
10.1021/bc100002a 2010 American Chemical Society Published on Web 04/26/2010
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Figure 1. Fully protected diacylated lipopeptide 1 was employed as an immobilized model substrate for the on-resin CuAAC “Click” reaction with PEG. 1 was synthesized on both PS- and TentaGel resin equipped with Rink amide linker. Scheme 1. Synthetic Pathway to the Resin-Bound Lipopeptide 1
Scheme 2. Synthetic Pathway to the Methoxy Poly(Ethylene Glycol) Azides 3a-c Used for On-Resin CuAAC Reaction
acetonitrile as the mobile phase. The degree of PEGylation was calculated from the area under the curve (AUC) from the two peaks corresponding to the lipopeptide and the PEGylated lipopeptide, respectively, at 280 nm by the formula given in eq 1. PEGylation degree )
AUClipid-peptide-PEG × 100% AUClipid-peptide-PEG + AUClipid-peptide (1)
The obtained results from the on-resin CuAAC on PSRinkAmide resin with the synthesized PEG azides 3a-c are given in Figure 2a. As Figure 2a clearly illustrates, the average size of the PEG-azide used for the CuAAC reaction has a significant influence on both the reaction rate and the degree of conversion. Complete PEGylation with MeO-
PEG350-N3 (3c) was observed within the first hour, whereas MeO-PEG1000-N3 (3b) and MeO-PEG2000-N3 (3a) resulted in 80% and 50% PEGylation degree in 48 h, respectively. Since 99% of the available reaction sites on the resin are present inside the polystyrene matrix, diffusion of the reacting species is usually the limiting factor (10). This effect gets progressively more dominant with higher molecular weight polymers, which is clearly illustrated by the observed conversion. Similar observations have been reported by Klok et al. (11) and Mutter et al. (12), who studied N-terminal PEGylation with R-methoxy-ω-carbonyl functionalized PEG to PSsupported peptides. They reported quantitative conversion with MeO-PEG700-COOH, whereas only 40% conversion was achieved when the average molecular weight was increased to 1800 Da. The strong decrease in the conversion degree was explained by the lower reactivity of the polymer, by the
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Bioconjugate Chem., Vol. 21, No. 5, 2010 809
Scheme 3. Employed Reaction Conditions for the On-Resin PEGylation of 1, Followed by Deprotection and Cleavage from Solid Support
decrease in diffusion into the PS-matrix, and by the increasing incompatibility of the polymer with the solid support. We have therefore compared the coupling efficiency of PEG on the PS-RinkAmide resin with the efficiency on NovaSyn TentaGel resin. The TentaGel resin was chosen because of its hydrophilic character due to the high content of PEG (approximately 70 mass%), which should improve the compatibility of the PEG-azides with the solid support. Furthermore, enzymes such as porcine pancreatic trypsin (23.5 kDa) and various lipases (30-40 kDa) have been reported to be able to penetrate to the core of TentaGel resins (13, 14), which clearly illustrates the easy accessibility of this class of resins. However, although the TentaGel resin should be more compatible and accessible to the reacting PEG-azides, no significant improvement of the PEGylation degree was observed (Figure 2b). Again, complete PEGylation with MeO-PEG350-N3 (3c) was observed within the first hour, whereas MeO-PEG1000-N3 (3b) and MeO-PEG2000-N3 (3a) resulted in 85% and 55% PEGylation degree in 48 h, respectively. Due to the lack of success in increasing the yield for the longer PEGs by choice of resin, we attempted to improve the degree of conversion by changing the solvent mixture for the on-resin CuAAC reaction. The swelling behavior of the resin is highly dependent on the solvent polarity, which will affect the diffusion of solute molecules within the matrix (10, 15). By changing the solvent system from DMF/piperidine (4:1) to CH2Cl2/piperidine (4:1), the degree of conversion was improved from 85% to quantitative yield for MeO-PEG1000-N3 (3b) and from 55% to 85% for MeO-PEG2000-N3 (3a) (Figure 2c). Precipitation of a small amount of 1-(chloromethyl)-piperidine was observed, but did not effect the CuAAC reaction. Furthermore, addition of the Cu(I)-stabilizing ligand bathophenanthroline disulfonic acid (16) or an increase of temperature to 50 °C did not result in an improved conversion degree (data not shown). No evidence of alkyne-alkyne homocoupling (Glaser-coupling) was observed by MALDI-TOF MS at any time during the reaction, most likely due to the pseudo dilution effect (17). Excess of reagents and Cu(I) was easily washed away resulting in high purity of the crude products. In conclusion, we have demonstrated a novel approach for the synthesis of PEGylated lipopeptides via the CuAAC
“Click” reaction between an alkyne-bearing solid-supported diacylated lipopeptide and R-methoxy-ω-azido functionalized PEGs. The alkyne functionality was easily inserted into the peptide and served as a site-specific conjugation site, without the need for complex protective group strategies. The R-methoxy-ω-azido functionalized PEGs were readily available from the corresponding alcohols, and the on-resin CuAAC with the large polymers proved to be fully compatible with Fmoc-SPPS methodology. HPLC analysis of the cycloaddition products demonstrated a clear polymer size dependent conversion degree. Small polymers such as MeOPEG350-N3 (3c) reacted readily and resulted in full conversion regardless of the choice of resin and solvent mixture. Full conversion was also achieved after 24 h with MeO-PEG1000N3 (3b) when applying the NovaSyn TentaGel resin in a mixture of CH2Cl2/piperidine (4:1), whereas 85% conversion was achieved with MeO-PEG2000-N3 (3a) under the same conditions. The employed strategy allows for PEG insertion (and other molecules) in the final step of the synthesis to any position that is desired in the peptide, and importantly provides the possibility of verifying possible amino acid deletions before PEGylation, which can otherwise be very difficult to detect. The method described here enables easy synthesis of molecules with a high level of complexity which, after a single purification step, is ideally suited for functionalization of solid-supported lipid bilayers and liposomal drug delivery systems and is particularly valuable in enzyme activation strategies. Additional targeting ligands or functional groups can easily be attached to the distal end of the PEG-azide, resulting in an even higher level of complexity, if needed. Furthermore, this method also enables easy synthesis of asymmetric diacyl lipopeptides by choosing a 2,3-diaminopropionic acid derivate with two orthogonal amine protection groups. This allows for optimization of the lipopeptide properties with respect to its integration and compatibility with lipid bilayers, which has been shown to be a crucial parameter in controlling the release of encapsulated content in liposomal drug delivery systems (18).
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C NMR, IR, MALDI-TOF MS). This material is available free of charge via the Internet at http://pubs.acs.org.
LITERATURE CITED
Figure 2. Measured degree of conversion of the CuAAC reaction between the resin bound lipopeptide 1 and the methoxy poly(ethylene glycol) azides (0) MeO-PEG2000-N3 (3a), (O) MeO-PEG1000-N3 (3b), and ()) MeO-PEG350-N3 (3c). (a) PS-resin in DMF/piperidine (4:1), (b) TentaGel-resin in DMF/piperidine (4:1), and (c) TentaGel-resin in CH2Cl2/piperidine (4:1).
ACKNOWLEDGMENT Financial support was kindly provided by the Danish Strategic Research Council (NABIIT) ref. 2106-07-0033 and the Technical University of Denmark (DTU). Supporting Information Available: Detailed description of the synthetic approach to compound 1, 3a-3c, and 4a-c, HPLC data, and characterization of the synthetic compounds (1H NMR,
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