TECHNICAL NOTE pubs.acs.org/bc
Trivalent PEGylated Platform for the Conjugation of Bioactive Compounds ngela Torres,‡,† Carlos Mas-Moruno,§,† Enrique Perez-Paya,||,^ Fernando Albericio,*,§,# and A Miriam Royo*,‡,# ‡
Combinatorial Chemistry Unit, Barcelona Science Park, University of Barcelona, Baldiri Reixac 10, 08028-Barcelona, Spain Institute for Research in Biomedicine, Barcelona Science Park, Baldiri Reixac 10, 08028-Barcelona, Spain Department of Medicinal Chemistry, Príncipe Felipe Research Centre, 46013-Valencia, Spain ^ IBV-CSIC, 46010-Valencia, Spain # CIBER-BBN, Networking Centre on Bioengineering Biomaterials and Nanomedicine, 08028-Barcelona, Spain
)
§
bS Supporting Information ABSTRACT:
PEGylated multivalent structures are a new class of platform for biological applications due to their biocompatibility properties. Here, we present the synthesis of a trivalent structure 1 based on poly(ethylene glycol) units (PEG) as potential synthetic multifunctional carrier molecule. To evaluate whether this PEGylated platform could be useful for the conjugation of bioactive compounds, a well-known lipopolysaccharide (LPS) inhibitor 2, developed in our laboratory, was selected to be conjugated to 1. The LPS-neutralizing activity of the resulted conjugates and precursors was established using the chromogenic Limulus amebocyte lysate (LAL) assay. The trivalent structure 1 did not show LPS-binding activity, nonconjugate LPS inhibitor 2 showed high LPSneutralizing activity, and the trivalent conjugate 4 displayed increased LPS-neutralizing activity and a reduced toxicity profile. These results prove the efficacy of this trivalent platform as a multivalent ligand scaffold for biological applications.
’ INTRODUCTION The conjugation of bioactive compounds to oligo- and polymeric materials constitutes a useful strategy of outstanding interest in the field of medicinal chemistry.1,2 These materials can be used either as carrier systems for drug delivery or as bioactive multivalent platforms for the presentation of several copies of a pharmacological agent with a specific topology. Overall, these systems should enable the administration of much lower doses of the drug with higher efficiency, thereby minimizing unwanted side effects. Additionally, for substances with poor water solubility, these systems afford the possibility to increase this property and hence the effectiveness of the drug. Dendrimer structures have emerged as a new class of biopolymers with structural properties35 and biological applications of interest.610 These compounds are highly branched polymers r 2011 American Chemical Society
with a well-defined chemical composition and structure. In this regard, PEGylated dendrimers, in which a multifunctional dendritic core is attached to polyethylene glycol (PEG) chains, are a subclass of dendrimers that maintain the special features of the dendrimers and amplify the properties of PEG.11,12 PEG is a highly flexible, water-soluble, nontoxic, and non-immunogenic polymer. Drug or protein conjugation to PEG increases its solubility in water, protects against degrading enzymes, and prevents immunogenicity, preserving the original biological functions of the conjugated bioactive molecules.13 In addition, the presence of PEG reduces kidney ultrafiltration, improves bioavailability, Received: September 22, 2010 Revised: July 18, 2011 Published: August 25, 2011 2172
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Bioconjugate Chemistry
TECHNICAL NOTE
Figure 1. Synthesis of conjugates 4 and 5. Trivalent platform 1 is depicted drawing multifunctional core NTA in red and the PEG chains in blue. The potent LPS-neutralizing agent 2 and the amphipathic monomer 3 are also shown. (i) TFA-DCM (1:1); (ii) TFA-H2O-TIS (95:2.5:2.5); (iii) PyBOP, HOBt, DIEA.
increasing drug half-life, and, in general, facilitates its administration.14,15 In fact, various PEGylated dendrimers have been described and showed lower toxicity, fewer hemolytic properties, long blood circulation times, low organ accumulation, and high accumulation in tumor tissue.1618 Nevertheless, the synthesis of highly branched dendritic structures is often tedious and challenging and involves considerable synthetic effort, generally resulting in low yields of final dendrimers.19 Simple, easy-to-synthesize multivalent platforms (i.e., generation 0 dendrimers) that are able to maintain some of these unique dendrimer properties are the optimal scaffolds to construct new multivalent ligands. Multivalence is a phenomenon whereby multiple, simultaneous, energetically coupled target/ligand interactions enhance the overall activity and selectivity compared to the corresponding monomeric interaction. Here, we describe the synthesis of a very promising class of trivalent PEG-based platform 1, which consists of three copies of monodisperse PEG units attached to nitrilotriacetic acid (NTA) as trivalent ligand scaffold (Figure 1). In addition to the conjugation of multiple copies of bioactive compounds, such platforms may also increase water solubility and reduce toxicity of the conjugated molecules caused by the presence of PEG moieties. To evaluate whether this PEGylated platform could be useful in the conjugation of bioactive compounds, a LPS inhibitor developed in our laboratory, compound 2,20 was selected to be conjugated to 1 (Figure 1). LPS is a bacterial endotoxin present
in the outer leaflet of Gram-negative bacteria that plays a major role in Gram-negative sepsis.21 Sepsis is a serious systemic response to infection that represents the foremost cause of death in intensive care units (ICUs),22 accounting for 750 000 hospitalizations in the U.S. annually.23,24 To date, only one example of dendrimers with LPS-neutralizing activity has been reported.25,26 In these studies, David and co-workers examined a variety of amine-terminated poly(amidoamine) (PAMAM) dendrimers.27 The authors derivatized the surface amines of these dendrimers with lipopolyamines and obtained a multibranched dendritic structure that neutralized LPS-induced inflammatory responses in vitro and afforded protection against endotoxic shock in a murine model.26 No reports concerning other types of dendritic-like multivalent structures have been found in the literature. The selected LPS-inhibitor, compound 2, was successfully conjugated to the trivalent platform 1, thereby rendering a new construct 4 with an improved endotoxin-neutralizing activity and toxicity profile, confirming the utility of this new PEGylated platform as a multivalent scaffold.
’ EXPERIMENTAL PROCEDURES General Materials and Methods. All chemicals and solvents were purchased from Calbiochem-Novabiochem AG, Iris Biotech GmbH, Fluka Chemika, Albatross Chem, GL Biochem, SigmaAldrich, Panreac, KaliChemie, Merck KGaA, or Scharlau and 2173
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Bioconjugate Chemistry were used as received without further purification. Distilled and deionized water was used for the preparation and rinsing of all solutions. Endotoxin-free water, LPS from E. coli 055:B5 and Polymyxin B were purchased from Sigma. The LAL reagent and the chromogenic substrate were obtained from Cambrex. Analytical RP-HPLC was performed using an Alliance 2695 Waters chromatography system with a RP Symmetry column and a Waters 996 photodiode array detector, using different linear gradients (see SI). Semipreparative HPLC was carried out on a Waters chromatography system equipped with a Waters 2487 dual absorbance detector and ESI-MS Waters Micromass ZQ), performed on a Symmetry column with linear gradients of B, MeCN (with 0.1% TFA), in A, H2O (with 0.1% TFA), at a flow rate of 20 mL/min, using different linear gradients (see SI). Flash RP-LC was performed on a Combi Flash system from Isco Teledyne using RediSep C18 cartridges (4 g) with linear gradients of B, MeCN (with 0.1% TFA), in A, H2O (with 0.1% TFA), at a flow rate of 20 mL/min (see SI for elution conditions). HPLC-MS was performed on Waters Alliance 2695 chromatography system equipped with Waters 995 photodiode array detector and ESI-MS Waters Micromass ZQ), on a Symmetry column with linear gradients of B, MeCN (with 0.1% FA), in A, H2O (with 0.1% FA), at a flow rate of 20 mL/min (see SI for elution conditions). Mass spectra were recorded on a Voyager MALDI-TOF spectrometer using either ACH or SA matrices dissolved in H2O/MeCN/FA (1:1:0.1). 1H NMR spectra were recorded on a Varian Mercury 400 spectrometer at 400 MHz in DO2. Peptide solid-phase synthesis was carried out following standard methods28 described in Supporting Information section. Synthesis of (TOTA)3NTA (1). NTA (12.0 mg, 0.063 mmol), was preactivated for 15 min with DIC (29.4 μL, 0.19 mmol) and HOBt (28.3 mg, 0.19 mmol) in 10 mL of DCMDMF (7:3). Then, Boc-TOTA (73.6 mg, 0.23 mmol) and TEA (52.74 μL, 0.38 mmol) dissolved in 10 mL of DCMDMF (7:3) were added to the preactivated NTA. The reaction was stirred for 48 h under N2 atmosphere at room and checked by RP-HPLC (conditions A). The solvent mixture was evaporated under vacuum, and the residue was dissolved in DCM; washed with saturated NaHCO3 and brine; dried over MgSO4 and evaporated to dryness under vacuum to obtain 1a as yellow oil (76.2 mg, 95% yield, 67% purity). The crude mixture 1a was purified using a flash chromatography system with a RP C18 column (condition G in SI) to afford 1a (83% of recovery yield). 1a: RP-HPLC (tR = 23.1 min, purity 97%, condition A in SI); MALDI-TOF (m/z calcd. for C51H99N7O18 1097.74, found 1098.6 [M+H]+). 1H NMR (400 MHz, D2O): δ ppm 1.43 (s, 27H), 1.711.84 (m, 12H), 3.21 (m, 6H), 3.26 (bs, 6H), 3.36 (dd, J = 6.8, 12.4 Hz, 6H), 3.53 (t, J = 6.02, 6.02 Hz, 12H), 3.563.66 (m, 24H), 5.12 (bs, 3H), 7.73 (bs, 3H). 13C NMR (400 MHz, MeOD): δ ppm 28.37, 29.15, 29.60, 37.23, 38.34, 50.49, 59.79, 69.34, 69.37, 70.08, 70.42, 76.68, 77.00, 77.32, 77.32, 156.06, 170.33. Then, compound 1a was treated with 15 mL of TFADCM (1:1) for 30 min. TFA was removed by evaporation to dryness yielding 1 (41.1 mg, 96% yield). 1: RP-HPLC (tR = 8.1 min, purity 88%, condition A in SI), MALDI-TOF (m/z calcd. for C36H75N7O12 797.55, found 798.6 [M+H]+). Synthesis of 2a. Fmoc-6-aminohexanoic acid (1 equiv) and DIEA (10 equiv) were sequentially added to 2-chlorotrityl chloride resin (300 mg, 1.00 mmol/g) and the resin was stirred for 1 h. The incorporation was followed by a 10 min capping step with MeOH (240 μL). After Fmoc removal, Fmoc-Arg(Pbf)OH (4 equiv) was incorporated into the resin using 2 45 min
TECHNICAL NOTE
consecutive coupling treatments with HATU (4 equiv) in DMF as coupling reagent in the presence of DIEA (8 equiv). Next, the Fmoc group was removed and Fmoc-protected N1-(Fmoc)1,12-diamino-4,9-dioxadodecan-succinamic acid (4 equiv) was coupled overnight using HOAt (4 equiv), PyBOP (4 equiv), and DIEA (12 equiv). Then, the Fmoc group was removed and Fmoc-Arg(Pbf)-OH was incorporated using the conditions explained above. Finally, palmitic acid (5 equiv) was added overnight with HOAt (5 equiv), PyBOP (5 equiv), and DIEA (15 equiv). The compound was then cleaved using TFADCM (1:99) (5 0.5 min). Solvents were evaporated and the residue was dissolved in H2OMeCN (1:1) and lyophilized to afford 2a (139.1 mg, 32% yield). The crude product was purified by semipreparative RP-HPLC (condition F in SI) to yield 2a with optimal purity. 2a RP-HPLC (tR = 14.9 min, purity 97%, condition D in SI), ES+MS (m/z calcd. for C74H125N11O15S2 1471.9, found 738 [M+H]+/2). Synthesis of 2. The synthesis of 2 has been described elsewhere.20 Fmoc-6-aminohexanoic acid (4 equiv) was coupled to Rink Amide MBHA resin with DIC (4 equiv) and HOAt (4 equiv) in DMF for 6 h. Subsequent steps in solid phase were carried out as explained for the synthesis of 2a. Finally, the cleavage of the compound from the solid support and deprotection of side-chain groups was carried out with TFAH2OTIS (95:2.5:2.5) for 2 h. TFA was then removed by evaporation with nitrogen and the crude compound was precipitated with cold anhydrous TBME, dissolved in H2OMeCN (1:1) and lyophilized. Then, it was purified by semipreparative RP-HPLC (condition E in SI) to yield the desired 2 with excellent purity. 2 RP-HPLC (tR = 10.4 min, purity 98%, condition C in SI), MALDI-TOF (m/z calcd. for C48H94N12O8 966.73, found 967.81 [M+H]+). Synthesis of 3. Fmoc-Arg(Pbf)-OH (1 equiv) in DCM and DIEA (10 equiv) were sequentially added to CTC resin (218 mg, 1.00 mmol/g), and the resin was stirred for 1 h. At the end of the coupling, a treatment with MeOH (175 μL) for 10 min was carried out to cap the free chloride groups of the resin. The Fmoc group was removed and palmitic acid (5 equiv) was added overnight with HOAt (5 equiv), PyBOP (5 equiv), and DIEA (15 equiv) as coupling reagents. Cleavage of the final compound from the resin was afforded by mild acidic treatments with TFADCM (1:99; 5 0.5 min). The solvents were evaporated, the compound dissolved in H2OMeCN (1:1), and lyophilized to obtain 3a (102.7 mg, 71% yield), which was used without purification. 3a HPLC (tR = 16.8 min, purity 95%, condition D in SI). Then, 3a was treated with TFAH2OTIS (95:2.5:2.5) for 1 h. TFA was evaporated and the residue was dissolved in MeOH, precipitated with cold TBME, dissolved in H2OMeCN (1:1), and lyophilized to obtain 4.2 mg of 3 with 86% of yield. 3 RPHPLC (tR = 19.5 min, purity 89%, condition B in SI), MALDI-TOF (m/z calcd. for C22H44N4O3 412.34, found 413.01 [M+H]+). Synthesis of Compound (2-TOTA)3NTA (4). Compound 2a (20.0 mg, 0.0136 mmol) dissolved in 10 mL of DCMDMF (7:3) was preactivated for 15 min by adding PyBOP (7.1 mg, 0.0136 mmol) and HOBt (2.0 mg, 0.0136). Then, 1 (3.30 mg, 0.0041) and DIEA (4.6 μL, 0.0272 mmol) dissolved in 10 mL of DCMDMF (7:3) were added. The mixture was stirred under N2 atmosphere at room temperature for 72 h; the reaction was controlled by RP-HPLC (condition B in SI). The solvent was evaporated under vacuum and the residue dissolved in DCM; washed with saturated NaHCO3, 10% HCl, and brine; dried over MgSO4; and evaporated to dryness. The crude product was dissolved in MeOH and precipitated with cold TBME to obtain 4a (18.8 mg, 70% yield). 4a: RP-HPLC (tR = 26.3 min, purity 2174
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Bioconjugate Chemistry 80%, condition B in SI); MALDI-TOF (m/z calcd. for C258H444N40O54S6 5162.91, found 5180.02 [M+18]+). Then, 4a (18.8 mg) was treated with TFAH2OTIS (95:2.5:2.5) for 3 h and TFA was evaporated. The residue was dissolved in MeOH and precipitated with cold TBME. After centrifugationdecantation, the compound was dissolved in H2OMeCN (1:1) and lyophilized to afford 4 as a white solid (6.1 mg, 86% yield). 4: RP-HPLC (tR = 17.2 min, purity 83%, condition B in SI); MALDI-TOF (m/z calcd. for C180H348N40O36 3648.94, found 3649.93 [M+H]+). Synthesis of (3-TOTA)3NTA (5). 3a (20.0 mg, 0.030 mmol) was preactivated for 15 min with PyBOP (15.7 mg, 0.030 mmol) and HOBt (4.5 mg, 0.030 mmol) in 10 mL of DCMDMF (7:3). Compound 1 (7.26 mg, 0.0091 mmol) and DIEA (10.2 μL, 0.060 mmol) were dissolved in 10 mL of DCMDMF (7:3) and after stirring for 10 min were added to the preactivated compound 3a. The mixture reaction was stirred under N2 atmosphere at room temperature for 72 h and controlled by RP-HPLC (condition B). Then, the solvent was evaporated under vacuum and the residue was dissolved in DCM; washed with saturated NaHCO3, 10% HCl, and brine; dried over MgSO4; filtered; and evaporated to dryness to obtain 5a. This compound was dissolved in MeOH and precipitated by the slow addition of cold MeCN (20.2 mg, 81% yield). 5a: RP-HPLC (tR = 26.6 min, purity 84%, condition B in SI); MALDI-TOF (m/z calcd. for C141H249N19O27S3 2738.79, found 2762.01 [M+Na]+). The protected 5a (20.2 mg, 0.0073 mmol) was treated with TFA, as previously explained for compound 4. Workup yielded 5 as a white solid (8.6 mg, 88% yield). 5 RP-HPLC (tR = 14.9 min, purity 92%, condition B in SI); MALDI-TOF (m/z calcd. for C102H201N19O18 1981.8, found 1981.78 [M+H]+). LPS Neutralizing Activity. All solutions used in the LPSneutralizing activity assay were tested to ensure they were endotoxinfree, and the material was sterilized by heating for 3 h at 180 C. LPS-neutralizing activity was measured using the chromogenic Limulus Amebocyte Lysate (LAL) test, following the manufacturer’s instructions (Cambrex). The LAL reagent contains a clottable protein that is activated in the presence of non-neutralized LPS, being an extremely sensitive indicator of the presence of endotoxin. When activated, this enzyme catalyzes the release of p-nitroaniline (pNA) from the colorless chromogenic substrate Ac-Ile-Glu-Ala-Arg-pNA. The released pNA can be measured photometrically at 405 nm. Compounds were initially dissolved in sterile phosphate buffered saline, PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.0) at a concentration of 100 μM and preincubated with LPS (100 pg/mL in sterile, endotoxin-free water) in a 96-well microplate for 60 min at 37 C. Polymyxin B (10 μg/mL in sterile PBS) was used as a positive control. The colorimetric reaction was started by adding 12.5 μL of LAL reagent (reconstituted by adding 1.4 mL of LAL reagent water) for an incubation period of 10 min at 37 C. After this time, non-neutralized LPS was detected by the addition of 25 μL of the chromogenic substrate (reconstituted with 6.5 mL of LAL reagent water) for 5 to 8 min at 37 C. Acetic acid (25% v/v final concentration) was added to stop the reaction, and the absorbance was monitored at 405 nm in a Multiskan Ascent microplate plate reader (ThermoLabsystems). At this concentration, compounds that showed an LPS neutralization above 75% were tested to determine their IC50 (the concentration required to neutralize 50% of LPS in vitro). IC50 values were determined by a serial dilution assay using 100 pg/mL of LPS and a range of compound concentrations (50 to 0.001 μM). All assays
TECHNICAL NOTE
were run in triplicate, and the curves were automatically adjusted by nonlinear regression using Prism 4 (GraphPad) software. Cell Culture. Mouse macrophages (RAW 264.7) were obtained from ATCC (American Type Culture Collection, USA). The cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco BRI) supplemented with 10% fetal bovine serum (FBSGibco BRI) and 1% L-glutamine. The cultures were incubated at 37 C in a humidified atmosphere of 5% CO295% air. Subcultures of macrophages were prepared every 23 days by scraping cells into fresh medium. MTT Cell Viability Assays. Cell viability was evaluated by a MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay. RAW 264.7 cells were seeded in sterile 96-well microtiter plates at a seeding density of 6 104 cells/mL in DMEM supplemented with 10% FBS and allowed to settle for 24 h. Compounds were added at a 10 μM concentration to the plates and the cells were further incubated for 24 h. After removal of the medium, the precipitated formazan crystals were dissolved in optical grade DMSO (100 μL), and the plates were read at 570 nm using a Wallac 1420 Workstation.
’ RESULTS AND DISCUSSION Design of Drug Conjugates with LPS-Neutralizing Activity. The trivalent PEGylated platform (compound 1, Figure 1)
was obtained by convergent synthesis from the condensation of a commercially available propylene glycol (TOTA) and a trifunctional core (NTA). This and other types of polycarboxylic acids have been used in vivo as chelating agents for metals and their complexes as contrast agents for magnetic resonance imaging (MRI).29,30 Since their physiological removal was confirmed in studies,31 they have been considered excellent cores for the design of further biological applications. As a first step and in order to evaluate whether our trivalent platform would be useful for the conjugation of biologically active compounds, we conjugated 2 to the trivalent structure 1 (Figure 1). In an ongoing research program currently underway in our laboratory, compound 2 has recently been reported to show high LPS-neutralizing activity.20 This compound displays unique chemical features for optimal LPS binding: two Arg residues conveniently separated by a PEG linker and a fatty acid linked at the N-terminus. In fact, the cationic residues interact with the negative phosphate groups of lipid A32 which is the endotoxic moiety of the LPS,33,34 while the palmitic acid promotes hydrophobic interactions with the lipophilic part of the endotoxin35 and confers amphipathicity to the whole molecule, a prerequisite for LPS neutralization.36 Moreover, the presence of a PEG spacer is of interest in terms of improved water solubility and pharmacokinetic profiles.14 In addition, the palmitoyl Arg monomer 3 was included in the design to further evaluate the relevance of the topology of positive charges in terms of LPS interaction (Figure 1). Synthesis of Trivalent PEGylated Platforms. The synthesis of the trivalent platform 1 was achieved after conjugation of conveniently monoprotected TOTA into the NTA core (see Scheme 1 in Supporting Information). From diverse reaction conditions evaluated, the use of DIC in the presence of HOBt and TEA in DMFDCM (7:3) for 48 h gave the best results. To ensure an optimal yield, an excess of base was required to avoid amine proton capture. The use of Fmoc as the protecting group of TOTA was precluded in this design because of partial Fmoc elimination during the coupling reaction. Hence, Boc-TOTA was 2175
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Bioconjugate Chemistry coupled to NTA under the conditions described above to yield the Boc-protected compound 1a (95% yield, 67% purity). The protected compound was then purified using an automated flash chromatography system with a reverse-phase C18 column under acid conditions. It is essential to purify the compound while it is being protected to separate it efficiently from other byproduct. Finally, the Boc group was removed by treatment with TFA 50% in DCM for 30 min to obtain 1. Conjugation of Bioactive Compounds to the Trivalent Platform. To achieve proper conjugation, the carboxamide group of compound 2 was replaced by a free carboxylic acid and Arg side chains were kept protected with the Pbf group (see Scheme 2 in Supporting Information). The synthesis of 2 has been reported in detail elsewhere.20 CTC resin was used as solid support in order to obtain the free carboxylic acid compound 2 required for amide bond conjugation. After the addition of Fmoc6-aminohexanoic acid (Fmoc-Ahx-OH) using DIEA as a base, Fmoc-Arg(Pbf)-OH was coupled with two consecutive treatments with the reactive aminium salt HATU.37 Next, N1-(Fmoc)-1, 12-diamino-4,9-dioxadodecan-succinamic acid was coupled overnight using PyBOP instead of HATU to prevent N-terminus guanidylation.38 After the second Arg coupling, palmitic acid was added overnight. The compound was finally cleaved from the solid support with mild acid treatments to yield 2a. In turn, 3a was easily obtained on solid phase after incorporation of FmocArg(Pbf)-OH into CTC, and subsequent Fmoc removal and palmitic acid coupling. Thus, 2a and 3a were conjugated to 1 with the coupling reagent PyBOP in the presence of HOBt and DIEA under anhydrous conditions (Figure 1). The reactions were monitored by HPLC (see Supporting Information). The conjugation HPLC traces of 2a with 1 showed that the conjugated compound 4a polarity did not diminish drastically compared with its precursor 2a, even though 4a contained three palmitoyl residues and six protected side chain arginines (Arg(Pbf)). The same effect was observed for the conjugate 5a (data not shown). This behavior illustrates how PEG-containing platforms are useful molecules to solubilize hydrophobic compounds. After completion of the conjugation, a series of workup methods were evaluated, and we found that simple extraction and precipitation procedures were useful to obtain the protected conjugates with optimal purities, thus avoiding tedious and both compound and time-consuming RP-HPLC purifications. Finally, the protected conjugates were treated with TFA to yield the desired conjugates 4 and 5 with purities higher than 80% without any purification steps. LPS-Neutralizing Activity. The LPS-neutralizing activity of the conjugated constructs 4 and 5 was assayed using the chromogenic Limulus amebocyte lysate (LAL) assay.39 The activity of these compounds was compared against the activity of unconjugated 2 and 3 to study the effect of the conjugation. Finally, trivalent platform 1 was also included to determine whether this structure displayed on its own anti-LPS activity. All compounds were tested at 100 μM, and the assay was performed as described in the Experimental Procedures. The anti-LPS peptide polymyxin B (PMB)40 was used as a positive control in this assay (Figure 2). The trivalent structure 1 did not display LPS-binding activity. This result correlates well with other studies that describe how, although necessary, the presence of positive charges is not enough for effective LPS binding and neutralization.35 Moreover, the inability of this platform to neutralize LPS is relevant, since it ensures that these structures will not interfere with the biological activity of bioactive conjugated molecules and will only act as
TECHNICAL NOTE
Figure 2. Inhibitory activity of the compounds was determined using the chromogenic LAL assay. PMB was included as a positive control. Compounds were tested at a 100 μM concentration in the presence of 100 pg/mL of LPS. The LPS-binding assay was performed in three independent assays as described in the Experimental Procedures. Data are represented with ( standard deviation (SD).
Table 1. LPS Neutralization Activity (IC50) and Cell Viability of Compounds 2 and 4 compound
IC50 (μM)a
% of cell viabilityb
2
18 ( 1
109 ( 18
4
9 (1
110 ( 14
a
Inhibition of the compounds was determined using the chromogenic LAL assay. The inhibitory activity is represented as IC50. Standard deviations (SD) are also included. The assay was performed as described in the Experimental Procedures. b Cell viability was evaluated using RAW 264.7 cells, after 24 h of incubation in the presence of 10 μM concentration of compounds by MTT assay. The assay is described in the Experimental Procedures.
multivalent scaffolds. At the evaluated concentration, 2 showed high LPS-neutralizing activity, which is consistent with published data.20 In contrast, monomeric compound 3 was inactive, thereby suggesting that two positively charged residues were required to interact with lipid A, regardless of amphipathicity. Finally, conjugates 4 and 5 showed interesting behaviors. Construct 4 retained the neutralizing activity of inhibitor 2 and totally neutralized LPS at 100 μM. In contrast, compound 5 had poor activity, even though three copies of the acyl-Arg monomer were exposed to LPS. Hence, as previously observed with other LPS-binders, even though these compounds are usually cationic their LPS neutralizing properties lie on the appropriate geometrical distribution of the positive charges on the binder chemical structure rather than on their number.35,41 To further analyze the effect of conjugation and multivalency, compounds 2 and 4 were subjected to serial dilutions, and their IC50 values (i.e., the concentration required to neutralize 50% of LPS in vitro) were calculated (Table 1). The experimental IC50 values obtained revealed that the conjugation of 2 to the PEGylated trivalent structure involved a twofold enhancement of its LPS-neutralizing activity. Such improvement correlates with the presence of three equal copies of bioactive compound 2 in construct 4, suggesting an almost full exposure of 2 in the multivalent construct. The trivalent structures proposed herein are cationic in nature, and it is known that some amphipathic cationic compounds could present basal toxicity associated to cell membrane activity.42,43 Thus, we examined the toxicity, if any, of these conjugates in cells. In order to choose an appropriate cell model, we selected RAW 264.7 murine macrophages. These cells are a powerful model to 2176
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Bioconjugate Chemistry study LPS-induced cell signaling as well as the efficacy of LPS inhibitors, given that they are critical members of the innate immune system and play a major role in the pathogenesis of sepsis.21 In fact, the inhibition of inflammatory mediators such as tumor necrosis factor-α (TNF-α) from LPS-challenged macrophages44 is usually measured to determine the efficacy of LPS-neutralizers.33,45 However, cationic dendrimers have recently been described to induce apoptosis in this cell model.46 Thus, in a first attempt to determine whether these multivalent platforms are viable agents for future in vitro and in vivo studies, the cytotoxicity of compounds 2 and 4 was evaluated using MTT assays. These compounds were tested at a concentration close to their IC50 values, or 10 μM. At this concentration, both compounds were nontoxic (Table 1). It has already been reported that these cells can tolerate 2 at 10 μM, although it becomes moderately toxic at higher concentrations.20 Interestingly, 4 was devoid of any toxic effect even though it contained three copies of 2. These findings are consistent and support the notion that both PEGylation and the conjugation of molecules to cationic dendrimers is a viable strategy to reduce or even remove their intrinsic toxicity while maintaining the bioactive properties of the drug.16
’ CONCLUSIONS Here, we have presented the synthesis of a novel trivalent PEGylated structure. This platform was obtained by convergent synthesis from the condensation of a commercially available propylene glycol (TOTA) and NTA as the multifunctional core. The value of this PEGylated platform for the conjugation of bioactive compounds was assayed with compound 2, a previously described LPS-neutralizer. As a proof of concept, compound 2 was conjugated to the trivalent structure 1 to render construct 4. The conjugated compound displayed improved LPS-neutralizing activity and a reduced toxicity profile over the parent compound, thus proving the efficacy of this platform as a multivalent ligand scaffold for biological applications. ’ ASSOCIATED CONTENT
bS
Supporting Information. Detailed chromatographic elution methods used for characterization and purification, peptide solid-phase synthesis method used, scheme of synthesis of trivalent platform 1, compounds 2a and 3a, analytical HPLC traces of the conjugate 4a synthesis, 1H and 13C NMR of compound 1a, and HPLC traces and MS spectra of compounds 4 and 5. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*F.A. E-mail:
[email protected]. M.R. Phone: 0034 934037122. Fax: 0034 934037126. E-mail:
[email protected] Author Contributions †
These authors contributed equally to the present study.
’ ACKNOWLEDGMENT This work was partially supported by the “Fundacio La Marato de TV3” (TV3 telethon), the Spanish Ministry of Science and Innovation (MICINN) (CTQ2005-00315, CTQ2008-00177, BIO2007-60666, and CSD2008-00005) and the CICYT
TECHNICAL NOTE
(CTQ2006-03794/BQU), the “Generalitat de Catalunya” (2005SGR 00662), the Institute for Research in Biomedicine, and the Barcelona Science Park. A.T. and C.M.M. are IRB and FPU (MEC) fellows, respectively.
’ ABBREVIATIONS ACH, cyano-4-hydroxycinnamic acid; Boc-TOTA, 1-(t-butyloxycarbonyl-amino)-4,7,10-trioxa-13-tridecanamine; CTC, 2-chlorotrityl chloride resin; D2O, deuterated water; DCM, dichloromethane; DIC, N,N0 -diisopropylcarbodiimide; DIPEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; FA, formic acid; HATU, O-(7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate; HOAt, 1-hydroxy-7-azabenzotriazole; HOBt, 1-hydroxybenzotriazole; MALDI-TOF, matrix-assisted laser-desorption time-of-flight; MeCN, acetonitrile; MeOH, methanol; NTA, nitrilotriacetic acid; SA, sinapinic acid; PyBOP, benzotriazole-1yl-oxy-tris(pyrrolidino)phosphonium hexafluorophosphate; TBME, t-butylmethyl ether; TEA, triethylamine; TFA, trifluoroacetic acid; TIS, triisopropylsilane ’ REFERENCES (1) Lutz, J. F., and B€orner, H. G. (2008) Modern trends in polymer bioconjugate design. Prog. Polym. Sci. 33, 1–39. (2) Khandare, J., and Minko, T. (2006) Polymer-drug conjugates: Progress in polymeric prodrugs. Prog. Polym. Sci. 31, 359–397. (3) Crespo, L., Sanclimens, G., Pons, M., Giralt, E., Royo, M., and Albericio, F. (2005) Peptide and amide bond-containing dendrimers. Chem. Rev. 105, 1663–1681. (4) Seiler, M. (2002) Dendritic polymers-interdisciplinary research and emerging applications for unique structural properties. Chem. Eng. Technol. 25, 237–253. (5) Frechet, J. M. J. (2002) Supramolecular chemistry and selfassembly special feature: dendrimers and supremolecular chemistry. Proc. Natl. Acad. Sci. U.S.A. 99, 4782–4787. (6) Svenson, S., and Tomalia, D. A. (2005) Dendrimers in biomedical applications-reflections on the field. Adv. Drug Delivery Rev. 57, 2106–2129. (7) Boas, U., and Heegaard, M. H. (2004) Dendrimers in drug research. Chem. Soc. Rev. 33, 43–63. (8) Lee, C. C., Mackay, J. A., Frechet, J. M. J., and Szoka, F. C. (2005) Designing dendrimers for biological applications. Nat. Biotechnol. 23, 1517–1526. (9) Niederhafner, P., Sebestik, J., and Jezek, J. (2005) Peptide dendrimers. J. Pept. Sci. 11, 757–788. (10) Sanclimens, G., Shen, H., Giralt, E., Albericio, F., Saltzman, M. W., and Royo, M. (2005) Synthesis and screening of a small library of proline-based biodendrimers for use as delivery agents. Biopolymers 80, 800–814. (11) Gajbhiye, V., Kumar, P. V., Tekade, R. K., and Jain, N. K. (2007) Pharmaceutical and biomedical potential of PEGylated dendrimers. Curr. Pharm. Des. 13, 415–429. (12) Guillaudeu, S. J., Fox, M. E., Haidar, Y. M., Dy, E. E., Szoka, F. C., and Frechet, J. M. J. (2008) PEGylated dendrimers with core functionality for biological applications. Bioconjugate Chem. 19, 461–469. (13) Zhao, H., Rubio, B., Sapra, P., Wu, D., Reddy, P., Sai, P., Martinez, A., Gao, Y., Lozanguiez, Y., Longley, C., Greenberger, L. M., and Horak, I. (2008) Novel prodrugs of SN38 using multiarm poly(ethylenglycol) linker. Bioconjugate Chem. 19, 849–859. (14) Greenwald, R. B., and Zhao, H. (2007) Poly (ethylene glycol) prodrugs: altered pharmacokinetics and pharmacodynamics. Prodrugs: Challenges and Rewards. Part 1 (Stella, V. J., Borchardt, R. T., Hageman, M. J., Oliyai, R., Maag, H., and Tilley, J. W., Eds.) pp 283338, Chapter 2.3.1, Springer, Boston. 2177
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