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Bioconjugate Chem. 2010, 21, 1404–1407
Photocleavable Polyethylene Glycol for the Light-Regulation of Protein Function Wesleigh E. Georgianna, Hrvoje Lusic, Andrew L. McIver, and Alexander Deiters* Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695. Received February 10, 2010; Revised Manuscript Received July 12, 2010
PEGylation is commonly employed to enhance the pharmacokinetic properties of proteins, but it can interfere with natural protein function. Protein activity can thus be abrogated through PEGylation, and a controllable means to remove the polyethylene glycol (PEG) group from the protein is desirable. As such, light affords a unique control over biomolecules through the application of photosensitive groups. Herein, we report the synthesis of a photocleavable PEG reagent (PhotoPEG) and its application to the light-regulation of enzyme activity.
Poly(ethylene glycol) (PEG) groups have been used extensively in biological and pharmaceutical applications to improve the pharmacokinetic properties of biomolecules (1-3). PEG bioconjugates provide enhanced in ViVo stability and are nontoxic and nonimmunogenic (4-6). They are typically generated from proteins through a process termed PEGylation, which involves the surface installation of PEG groups through a covalent reaction with amino functionalities, e.g., the ε-NH2 groups of reagent-accessible lysines. PEGylation greatly enhances the solubility, half-life, cellular uptake, and bioavailability of proteins in cells and organisms (1, 7, 8). A number of PEGylated therapeutics have been approved by the FDA and are currently on the market (9-11), demonstrating the growing interest in PEGylation chemistry toward drug delivery and protein stabilization. However, the PEGylation of proteins can negatively impact their biological activity and lead to complete inhibition of protein function, e.g., through steric blocking of the active site of an enzyme (12). The bulk of the PEG polymer wraps around the protein, thus providing the desired protective effects by sterically blocking it without perturbing the conformation and dynamics of the protein native fold (13, 14). For this reason, PEGs containing an ester linkage for tethering to the biomolecule have been developed that are susceptible to spontaneous hydrolysis and provide a slow release pathway for protein delivery (12, 15). While hydrolyzable linkers facilitate general release of the active therapeutic over time, a means to obtain precise spatial and temporal control over bioavailability without compromising stability or cellular uptake would be greatly advantageous. * E-mail
[email protected].
Due to our interest in the photochemical control of biological processes (16-23), we sought to accomplish such control over protein function using light. Light affords a noninvasive tool for modulating biological function, as it can be modified in amplitude, duration, and location to achieve a high level of spatiotemporal resolution. We reasoned that the installation of a photolabile PEG directly onto a protein would provide a PEG photocage that can inhibit protein activity (Figure 1A). Upon irradiation with non-phototoxic UV light, the PEG photocage will be removed from the protein, thus restoring its native activity. Since UV irradiation can be precisely controlled with high spatial and temporal resolution, this methodology provides a fundamentally new approach to achieve spatiotemporal control over protein activity. Herein, we describe the first photocleavable PEG reagent (PhotoPEG). In addition, we demonstrate that it provides photochemical control over the activity of a PhotoPEGylated enzyme. In order to demonstrate the applicability of our approach, we selected lysozyme as a target. Lysozyme is a 14.7 kDa protein that catalyzes the hydrolysis of 1,4-β-linkages in peptidoglycans and chitodextrins, thus destroying the cell walls of bacteria and protecting tissue from bacterial invasion (24). For this reason, lysozyme has been extensively studied (25-27), its crystal structure has been solved (28), and assays to assess its enzymatic activity have been developed (29, 30). Importantly, in addition to its N-terminus, lysozyme contains six solvent-accessible lysine residues (Figure 1B) and is thus readily PEGylated, which has previously been shown to inhibit its catalytic activity (30). We designed a lysozyme–photocleavable linker-PEG conjugate (Scheme 1) that is stable to hydrolysis and wholly abrogates enzymatic activity until a brief UV irradiation at 365
Figure 1. (A) Light-induced removal of PEG groups enables photochemical control of protein function. (B) X-ray structure of lysozyme with the six solvent-accessible lysine residues highlighted in yellow (PDB 2VB1).
10.1021/bc100084n 2010 American Chemical Society Published on Web 07/22/2010
Communications
Bioconjugate Chem., Vol. 21, No. 8, 2010 1405
Scheme 1. Synthesis of the 5000 Da, Lysine-Reactive, Photocleavable PEG Reagent 6 (PhotoPEG), and Its Application in the PEGylation of Lysozyme (Only One NH2 Group Is Shown)
nm cleaves the linker and releases free, active lysozyme. The photolabile linker is covalently attached to PEG (MW 5000 Da) and is comprised of an ortho-nitrobenzyl core that confers light sensitivity. The ortho-nitrobenzyl functionality has traditionally been used to provide photocontrol over proteins, nucleic acids, and small molecules (16-23). Reagent 6 also bears a terminal N-hydroxysuccinimide (NHS) ester that makes the PEG-linker moiety susceptible to nucleophilic attack by a primary amine. The PhotoPEG 6 was synthesized by reacting mPEG5000-NH2 (1, Laysan Bio, Inc.) with the alkynyl N-hydroxysuccinimide ester 2 to give the alkyne PEG reagent 3. Using Cu(I)-catalyzed “click” chemistry (31, 32), the alkyne PEG 3 was then connected to the novel azido-bearing caging group 4. The difunctional molecule 4 (see Supporting Information for a detailed synthesis) represents a photocleavable linker that can be used in a wide range of applications, since its two functional groups, hydroxyl and azido, have completely orthogonal reactivity and thus can be employed in the linking of a wide range of substrates. Here, the alcohol 5 was subsequently activated as an NHS ester using NHS carbonate in the presence of DMAP to yield 6. Hen egg white lysozyme (EMD Chemicals) was first incubated with increasing equivalents of the non light-cleavable N-hydroxysuccinimide (NHS)-PEG5000 7 (Jenkem Technology) overnight at 4 °C in phosphate buffered saline (PBS), pH 7.5. One equivalent of 7 (Figure 2, lane 2) produced a singly PEGylated protein; however, a substantial amount of nonPEGylated wild-type enzyme remained. Increasing the amount of 7 from 5 to 25 equiv led to a total consumption of wild-type lysozyme (lanes 3-5) but produced multiple PEGylated protein species which still displayed enzymatic activity (data not shown). PEGylation with 50 equiv of 7 (lane 6) completely abolished lysozyme activity. Thus, we employed 50 equiv of the PhotoPEG 6 in the PEGylation of lysozyme (lane 7), followed by dialysis into PBS (pH 7.5) using a dialysis membrane with a cutoff of 50 kDa in order to remove excess PEG reagent. Irradiation of the PhotoPEG-modified enzyme with UV light of 365 nm (25 W, 30 min) released all PEG groups,
regenerating wild-type lysozyme as shown by SDS PAGE analysis (lane 8).
Lysozyme assays were performed as previously reported (29). The amount of purified PEGylated lysozyme was determined by a Bradford assay (33). The lyophilized gram-positive substrate Micrococcus lysodeikticus (Sigma) was suspended in 66 mM sodium phosphate, pH 6.24, to achieve a starting optical density of approximately OD450 ) 1. Lysozyme or PEGylated lysozyme in 100 µL PBS was added to 100 µL of cell suspension for a final enzyme concentration of 3 µM. The OD450 was measured after a 20 min reaction time. Active wild-type
Figure 2. SDS-PAGE analysis of lysozyme PEGylation. M, protein maker (in kDa); lane 1, wild-type lysozyme; lanes 2-6, PEGylation with 1, 5, 10, 25, and 50 equiv of NHS-PEG 7, respectively; lane 7, PEGylation with 50 equiv PhotoPEG 6; lane 8, PhotoPEG-modified lysozyme from lane 7 after UV exposure (365 nm, 25 W, 30 min).
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Figure 3. Micrococcus lysodeikticus exposed to different lysozymes for 20 min, followed by an optical density (OD450) measurement. Wildtype lysozyme leads to complete cell lysis. In contrast, lysozymes PEGylated with the NHS-PEG 7 or the PhotoPEG 6 are completely inactive, thus inducing no cell lysis. Error bars represent standard deviations from three independent experiments.
Communications
increasing the amount of the caged biological molecule and/or extending the time of the process under study. Importantly, the PhotoPEG-modified lysozyme fulfills the crucial criterion of complete inactivity prior to irradiation. In summary, we have developed a photocleavable PEG reagent (PhotoPEG) and applied it successfully in the photochemical regulation of enzyme activity. This light-removable PEGylation represents the first example of a literal protein photocage, as the polyethylene glycol chains surround the enzyme and prohibit any interaction with its substrate (34). The PEGylated enzyme is completely inactive, and irradiation with UV light of 365 nm removes the otherwise stable PEG groups and restores enzymatic activity. Lysine PEGylation with the developed reagent is rapid and facile; the installation of cagingPEG groups on the protein can be easily assessed by standard gel electrophoresis, which, if necessary, also facilitates purification of the caged protein from noncaged protein. This methodology combines the advantages of protein PEGylation (increased protein solubility, intracellular stability, and circulation time) with the advantages of light-activation of protein function (spatial and temporal activity control with high precision). Due to the simplicity of the necessary manipulations, the developed methodology has a wide range of potential applications in the study of protein function in cell culture and whole organisms, as well as activation of protein therapeutics, with the only requirement being the presence of a suitable number of reactive lysine residues on the protein of interest.
ACKNOWLEDGMENT
Figure 4. Micrococcus lysodeikticus exposed to lysozyme modified with Photo-PEG 6, followed by an optical density (OD450) measurement after 20 min. The Photo-PEGylated lysozyme is completely inactive before irradiation (0 min); however, activity was restored to the level of wild-type lysozyme (wt) through UV irradiation (365 nm; 25 W; 5-30 min). Error bars represent standard deviations from three independent experiments.
lysozyme induces cell lysis, leading to a reduction in optical density (Figure 3). In contrast, the lysozyme PEGylated with the classical NHS-PEG reagent 7 (from Figure 2, lane 6), and the lysozyme modified with the photocleavable PEG 6 (from Figure 2, lane 7) were completely inactive, since no change in the OD450 was observed, as in the absence of lysozyme (Figure 3). Next, we investigated the photochemical activation of enzyme function through a light-induced removal of the PEG groups. The completely inactive PhotoPEG-modified lysozyme was irradiated for 0-30 min, and substrate lysis was determined as described above. As expected, in the absence of UV irradiation (0 min) no enzymatic activity was observed, resulting in an OD450 of 0.98 after 20 min, identical to the OD450 at the beginning of the assay. However, UV irradiation for 5, 10, 15, and 20 min led to increasing enzymatic activity resulting in the reduction of the final optical density of the cell suspension due to cell lysis. Optimal light-activation of the enzyme was observed after irradiation for 25 and 30 min, since the measured OD450 values were identical to the value for wild-type lysozyme (Figure 4). This observation is in agreement with the gel-based assessment of complete photochemical PEG removal after 30 min of UV irradiation (Figure 2, lane 8). Furthermore, we conducted time-course studies of the lysis assay (see Supporting Information) and found that the 30 min UV irradiation of the PhotoPEGylated lysozyme restored 50% of the maximal enzymatic velocity of wild-type lysozyme. An incomplete restoration of enzymatic activity is not uncommon in the light-activation of biological processes (22) and can be easily augmented by
This work was financially supported by the Research Corporation (Cottrell Scholar Award to AD) and the Beckman Foundation (Beckman Young Investigator Award to AD). Supporting Information Available: Synthesis of the azide 4 and the PhotoPEG 6, lysozyme PEGylation and assay protocols, assay timecourse. This material is available free of charge via the Internet at http://pubs.acs.org.
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