Synthesis of Well-Defined Phthalimide Monofunctional

Article pubs.acs.org/Macromolecules

Synthesis of Well-Defined Phthalimide Monofunctional Hyperbranched Polyglycerols and Its Transformation to Various Conjugation Relevant Functionalities György Kasza,*,† Gergely Kali,§ Attila Domján,‡ Lilla Pethő,∥ Györgyi Szarka,† and Béla Iván*,† †

Polymer Chemistry Research Group, Institute of Materials and Environmental Chemistry and ‡NMR Laboratory, Research Centre for Natural Sciences, Hungarian Academy, Magyar tudósok körútja 2, H-1117 Budapest, Hungary § Organic Macromolecular Chemistry, Saarland University, Campus C4.2, 166123 Saarbrücken, Germany ∥ MTA-ELTE Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Pázmány Péter sétány 1/A, H-1117 Budapest, Hungary S Supporting Information *

ABSTRACT: Phthalimide monofunctional hyperbranched polyglycerols (HbPG) were successfully synthesized, for the first time, by applying a new, highly efficient phthalimide/potassium phthalimide (PhthIm/K-PhthIm) initiating system for the anionic ring-opening multibranching polymerization of glycidol. As the analyses of the resulting polymers by UV and NMR spectroscopies, vapor pressure osmometry, aqueous and organic phase GPCs and ESI-MS proved, well-defined HbPGs with one phthalimide moiety, predetermined average molar masses, and narrow molar mass distributions were formed. The phthaloyl group was quantitatively cleaved by hydrazinolysis to form a monoamine functional HbPG. The amine functionality of the HbPG molecules at the initiating site was transformed into carboxylic, maleimide, and chloroacetamide groups. All functionalization reactions were quantitative as proved by multidimensional NMR spectroscopy. These findings indicate that the PhthIm/KPhthIm combination can be utilized in the polymerization and subsequent derivatizations of other epoxides as well. In addition, the selectively modifiable reactive headgroup can be applied for obtaining various novel functionalized materials.

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mass.17,18 Nowadays, based on the above-mentioned advantageous properties, HbPG is also intensively investigated as biocompatible nanocarrier. This highly branched polymer has also been recommended to replace poly(ethylene glycol) (PEG) in various application fields.13,19,20 In the case of the linear PEG, several mono- and homo- or heterobifunctional derivatives with terminal functionalities have already been produced, and most of these are commercially available materials as well. End-functional PEGs with amine, carboxyl, maleimide, azide, alkyne, etc., end groups have been successfully applied in various conjugation reactions, called PEGylation, to produce stable biomaterials and drug delivery systems.21,22 The main advantage of HbPG, compared to PEG, in addition to the known disadvantages of this linear polymer,20 is related to the favorable physical and/or chemical properties of the hyperbranched polymer, which can be further tuned by derivatization of the high number of its hydroxyl functionalities.23 By copolymerization of glycidol with other monomers or transformation of the hydroxyl groups of HbPG, multifunctional hyperbranched macromolecules with various terminal

INTRODUCTION Hyperbranched polymers possessing relatively high branching densities and multiple terminal and/or pendant functionalities have been among the most intensively investigated macromolecular assemblies during the past decades.1−10 Their unique properties, for example, low solution/melt viscosity, compact volume, and high solubility compared to their linear analogues as well as the large number of functionalities per molecule make them attractive for common, specialty, and high added value applications in several fields. Such highly branched polymers are very promising candidates as components of a variety of advanced materials, e.g., specialty coatings, nanostructures, nanocomposites, smart materials, and delivery systems for bioactive molecules, such as drugs, nucleic acids, proteins, and enzymes, etc.1−16 Undoubtedly, hyperbranched polyglycerol (HbPG) is one of the most attractive highly branched polymers for such purposes. HbPG is a polyether polyol which contains a large number of secondary and primary hydroxyl groups.11−19 In addition to its outstanding water solubility and approved biocompatibility (blood compatible, nonimmunogenic, nontoxic),13−16 the simple and modular synthesis of HbPG is also among the useful advantages of this polymer. Ring-opening multibranching polymerization (ROMBP) of glycidol results in well-defined structure and predetermined average molar © XXXX American Chemical Society

Received: February 24, 2017 Revised: April 3, 2017

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DOI: 10.1021/acs.macromol.7b00413 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules

by sequential copolymerization of a phthalimide-functional epoxy monomer and finally the conversion of the phthalimide groups to amine multifunctionalized macromolecules. In this context, it is also noteworthy to mention that Beezer and Harth57 carried out postmodification of the hydroxyl groups in HbPG with N-hydroxyphthalimide followed by deprotection, which led to multiamino-oxy functionalized polyglycerol. In our approach, the synthesis of monophthalimide functional HbPGs was attempted by the phthalimide/potassium phthalimide initiating system followed by a simple modification reaction to obtain monoamine HbPGs. Subsequently, quantitative polymer analogous modifications of the amino group to obtain other widely applied functional moieties, namely carboxyl, maleimide, and chloroacetamide, on the HbPG molecule were also investigated and are reported in this study.

functionalities, such as chloride, allyl, aldehyde, carboxyl, amine, azide, acrylate, tosyl, etc., were developed. These polymers were studied and applied in various areas, for instance, as nanocarriers or high-loaded support for organic syntheses.24−33 The biodistribution and accumulation of HbPG were followed after 3H and 64Cu radiolabel modifications, and it was found that these properties can be influenced by derivatization of the hydroxyl groups with sulfate moiety.34 In addition, HbPG-based drug delivery vehicles, decorated by targeting, imaging, and/or bioactive functionalities, were also exploited for cancer therapy.35 Numerous amphiphilic core− shell nanocarriers from HbPG are also reported in the literature. Unimolecular micelles with HbPG core were synthesized via functionalization by hydrophobic groups, linear polymers, or postpolymerization via the macroinitiator method.36−45 Such HbPGs were successfully applied to increase the solubilization efficiency of drugs and/or dyes. Preparation of unimolecular micelles was also described with the utilization of different hydrophobic initiators for the synthesis of HbPG, and these were applied as surfactants and drug delivery nanocapsules.46−49 While numerous studies have dealt with multifunctional HbPGs, it is surprising that despite the great potentials in monofunctional hyperbranched polyglycerols, investigations on such HbPGs are quite rare. There are many application possibilities for well-defined monofunctionalized HbPGs, e.g. for conjugation with other polymeric and nonpolymeric structures, such as surfaces, nanoparticles, and especially biomolecules (for instance, for targeting, labeling, and increasing biocompatibility, solubility, stability, etc.). For such purposes, amine- and carboxyl-functional HbPGs are among the most suitable substances. For obtaining amine-HbPG, Frey et al.50 prepared bis(2,3-dihydroxypropyl)-10-undecene-1amine initiator for the ROMBP of glycidol followed by the modification of the unsaturated functional group with cysteamine via thiol−ene click reaction. In another approach, Burakowska and Haag51 applied dibenzylamine as initiator for HbPG synthesis, and the monoamine functionality was prepared by subsequent palladium-catalyzed hydrogenation. Although this is an effective process, it has to be noted that due to the strict regulations on the palladium consumption and recovery,60,61 the application of this method is highly hampered. Other methods for the synthesis of hyperbranched or dendritic polyglycerol with clickable, orthogonally reactive monofunctionality have also been reported. Such HbPGs were prepared by Zimmerman and co-workers52−54 via an alkynecontaining initiator, or tert-butyldiphenylsilyl-protected polymer was synthesized by a multistep process followed by deprotection and functionalization to alkyne or azide groups. Herein, we report on a simple and highly efficient synthesis route for the preparation of hyperbranched polyglycerol containing one amine functionality per molecule, a versatile functional group for a variety of conjugation processes and subsequent highly efficient derivatization reactions. For achieving this goal, phthalimide/potassium phthalimide mixture was investigated by us as an initiating system for the bulk ROMBP of glycidol, for the first time according to the best of our knowledge. Then, since the resulting phthalimide headgroup can be easily converted by known methods,55 the preparation of monofunctional amine-HbPGs was attempted. Here, it has to be noted that Haag et al.56 described the synthesis of terminally functionalized multiamine HbPGs by a three-step method involving glycidol polymerization followed

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EXPERIMENTAL SECTION

Materials. Glycidol was freshly distilled from calcium hydride under reduced pressure prior to use. Phthalimide (99%+), potassium phthalimide (99%+), N-(3-hydroxypropyl)phthalimide (95%), hydrazine monohydrate (98%), succinic anhydride (97%), triethylamine (99%+), pentachlorophenol (97%), chloroacetic acid (98%), N,N′dicyclohexylcarbodiimide (DCC, 99%), 6-maleimidohexanoic acid (98%+), N-hydroxysuccinimide (NHS, 98%) (all from Sigma-Aldrich), acetic anhydride (99%), ethanol (EtOH), methanol (MeOH), diethyl ether, N,N-dimethylformamide (DMF), dichloromethane (DCM), hexane, pyridine (all from Molar Chemicals Ltd.), and magnesium sulfate (Reanal, Hungary) were used as received without purification. Tetrahydrofuran (THF, Molar Chemicals Ltd.) was refluxed over and distilled from potassium hydroxide to remove the peroxides before used as the eluent in gel permeation chromatography and was used without purification for precipitation. Synthesis of Hyperbranched Polyglycerol via Multibranching-Ring-Opening Polymerization of Glycidol with Phthalimide/Potassium Phthalimide Initiator. The hyperbranched polymers were synthesized via bulk ring-opening multibranching polymerization of glycidol by using various monomer (M)/initiator (I) ratios, i.e., M/I = 15, 19, 25, 33, and 70. The procedure of the synthesis of phthalimide monofunctional HbPG with theoretical molar mass of 1250 Da (PhthIm-HbPG_1: M/I = 15) is detailed below. The appropriate amounts of phthalimide (2.0048 g, 13.6 mmol) and potassium phthalimide (0.2804 g, 1.5 mmol) were placed into a dry 100 mL three-neck, round-bottom flask with a mechanical stirrer, connected to a vacuum line, and the third neck was sealed with a septum. The initiator mixture was heated to 95 °C under vacuum. The reaction flask was flushed with nitrogen flow, and the inert atmosphere was obtained by repeated five times pump−thaw cycles. Then 15 mL (16.7145 g, 225.6 mmol) of freshly distilled glycidol monomer was added slowly into the reaction flask by a syringe pump (feed rate: 2.5 mL/h). After the completed monomer addition, the stirring was continued for at least an additional 3 h. The cooled crude product was dissolved in 60 mL of EtOH, passed through a column filled with a cation exchange resin (Amberlite IR120, hydrogen form), and precipitated two times into large excess of diethyl ether. The collected polymer was dried in a vacuum oven at 50 °C until constant weight. The typical yield was around 90%. A series of HbPGs with various theoretical molar masses were prepared by the same way, varying the monomer/initiator ratio (PhthIm-HbPG_2: M/I = 19, Mtheor = 1525 Da; PhthIm-HbPG_3: M/I = 25, Mtheor = 2015 Da; PhthIm-HbPG_4: M/I = 33, Mtheor = 2610 Da; PhthIm-HbPG_5: M/I = 70, Mtheor = 5320 Da). Acetylation of HbPGs (PhthIm-HbPG-Ac). Acetylated HbPG derivatives were synthesized in order to investigate the molar mass and molar mass distribution of the PhthIm-HbPG samples by independent analytical methods. The acetylation was carried out by placing 200 mg of PhthIm-HbPG sample dissolved in 10 mL of pyridine in a 50 mL round-bottom flask equipped with a condenser, followed by the addition of 2.5 mL of acetic acid (10 equiv to repeating units). The B


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Scheme 1. Synthesis of Monofunctional Phthalimide-HbPG by Ring-Opening Multibranching Polymerization of Glycidol with the Phthalimide/Potassium Phthalimide Initiating System

reaction mixture was stirred overnight at 75 °C. Then, the solvent and the byproducts were removed by a rotary evaporator. The crude product was dissolved in DCM and washed three times with water. The organic phase was dried over MgSO4, filtered, concentrated under reduced pressure, and precipitated into a large excess of hexane. The collected product was dried at 45 °C under vacuum until constant weight. Synthesis of Amine-Monofuctionalized HbPG (NH2-HbPG). One phthalimide monofunctional HbPG (PhthIm-HbPG_3, Mn ∼ 2100 Da, PDI ∼ 1.4) was selected to investigate the derivatization reactions of the single phthalimide functionality on the PhthIm-HbPG molecules. PhthIm-HbPG (1 g, 0.5 mmol) was dissolved in 20 mL of EtOH, followed by the addition of 0.46 mL of hydrazine monohydrate (0.4768 g, 9.5 mmol, 20 equiv) to the polymer solution. The reaction mixture was stirred for 40 h at room temperature. Subsequently, the reaction flask was cooled to 4 °C for 6 h, and the resulting solution was filtered. The crude product was precipitated into a large excess of THF−diethyl ether (2:1 V/V) mixture, and the product was collected by centrifugation. The purification process was repeated two times, and the amine-HbPG was dried until constant weight in vacuum at 45 °C (yield: 0.7165 g, 77%). Synthesis of Carboxylic-Monofunctionalized HbPG (COOHHbPG). Monoamine functionalized HbPG (100 mg, 0.05 mmol) was placed in a dry 10 mL round-bottom flask with a magnetic stirrer bar and dissolved in 3.6 mL of EtOH. Then, 0.8 mL of stock solution of succinic anhydride (10 mg/mL in EtOH, 8 mg, 0.08 mmol, 1.6 equiv) and 7 μL of triethylamine (5.1 mg, 0.05 mmol, 1 equiv) were added. The reaction mixture was stirred for 5 h at room temperature. The product was precipitated twice into a large excess of THF, collected by centrifugation, and dried in a vacuum oven at 35 °C until constant weight (yield: 0.0728 g, 71%). Synthesis of Maleimide-Monofunctionalized HbPG (MalHbPG). 100 mg of 6-maleimidohexanoic acid (0.47 mmol, 1 equiv) was placed in a dry 5 mL round-bottom flask with a magnetic stirrer bar and dissolved in 3 mL of DMF. Then N-hydroxysuccinimide (58.6 mg, 0.49 mmol, 1.05 equiv) and DCC (100.9 mg, 0.48 mmol, 1.01 equiv) were added. The reaction mixture was stirred for 2 h and filtered to remove the resulting dicyclohexylurea. Then, 0.4 mL of stock solution of the synthesized 6-maleimidohexanoic acid NHS ester (19.5 mg, 0.063 mmol, 1.25 equiv to NH2-HbPG) was added to the solution of NH2-HbPG (100 mg, 0.05 mmol in 4.6 mL of DMF) and stirred overnight at ambient temperature. The product was precipitated twice into a large excess of THF and diethyl ether (2:1 V/V) mixture, collected by centrifugation, and dried in a vacuum oven at 35 °C until constant weight (yield: 0.0876 g, 81%). Synthesis of Chloroacetamide Monofunctionalized HbPG (Cl-HbPG). Chloroacetylation of monoamine functionalized HbPG was performed in a 10 mL round-bottom flask equipped with a magnetic stirrer bar. In this reaction flask, 100 mg of NH2-HbPG (0.05 mmol) was added, and it was dissolved in 4 mL of DMF. After the complete dissolution, pentachlorophenyl chloroacetate (0.0223 g, 0.065 mmol, 1.3 equiv), prepared in our laboratory by the reaction of pentachlorophenol and chloroacetic acid assisted by DCC, was

dissolved in 1 mL of DMF and was added to the polymer solution. The reaction mixture was stirred overnight at room temperature. Then, the product was precipitated two times into large excess of THF and was collected by centrifugation. The Cl-HbPG was dried under vacuum at 35 °C (yield: 0.0689 g, 68%). Characterizations. Gel Permeation Chromatography (GPC). The PhthIm-HbPG samples were analyzed by aqueous GPC at 25 °C. The GPC equipment was composed of a Waters 515 HPLC pump, a suprema lux column set (PSS), and Bischoff 8110 RI detector. The mobile phase was aqueous 0.1 M NaNO3 with a flow rate of 1 mL/min. Average molar masses and dispersities (PDI) were determined on the basis of calibration with seven linear PEG standards in the range of 430−26 100 Da (PSS). The acetyl derivatives (PhthIm-HbPG-Ac) were characterized by GPC equipped with differential refractive index detector (Agilent 390), Waters Styragel columns (HR1, HR2, and HR4), and a Waters Styragel HR guard column connected in series and thermostated at 35 °C. The eluent was THF with a flow rate of 1 mL/min. The evaluation of the chromatograms was made on the basis of calibration with linear polystyrene standards (PSS). NMR Spectroscopy. For the determination of the number-average degree of polymerization (DPn), and thus the number-average molar mass of the PhthIm-HbPGs and the acetyl derivatives of HbPGs, 1H NMR spectroscopy measurements were performed on a Bruker Avance 500 equipment operating at 500 MHz 1H frequency with a 5 mm inverse detection tunable dual-broadband {1H−19F}/{31P−15N}probe in deuterated dimethyl sulfoxide (DMSO-d6) at 30 °C. For the quantitative determination of the degree of branching and numberaverage molar mass of the HbPG samples, solution state NMR spectra were obtained by a Varian NMR system spectrometer operating at the 1 H frequency of 600 MHz (150 MHz for 13C) with a 5 mm direct detection tunable dual-broadband {1H−19F}/{31P−15N} probe. The samples were dissolved in deuterated dimethyl sulfoxide (DMSO-d6) with the addition of 20 mg of cobalt(II) acetylacetonate paramagnetic relaxation agent. The signal of the solvent (39.50 ppm) was used as a reference for the chemical shift. For the quantitative 13C spectra, 10 s delay time, and inverse gated 1H decoupling was used,58 the temperature of the measurements was 25 °C. The monofunctional modifications were confirmed by multidimensional NMR analysis. Qualitative solution state NMR spectra were obtained by a Varian NMR system spectrometer operating at the 1H frequency of 400 MHz (100 MHz for 13C) with a 5 mm inverse detection tunable dual-broadband {1H−19F}/{31P−15N} probe equipped with Z-gradient. The samples were dissolved in deuterated dimethyl sulfoxide (DMSO-d6). The signals of the solvent (2.50 ppm on the 1H scale and 39.50 ppm on the 13C scale) were used as a reference for the chemical shift. For the full assignation, single pulse (1H, 13C), heteronuclear (heteronuclear single-quantum correlation: HSQC; heteronuclear multiple bond correlation: HMBC), and homonuclear (zero quantum suppressed total correlation spectroscopy: zTOCSY) spectra were recorded under standard condition at 25 °C. C


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Macromolecules UV Spectroscopy. The phthalimide content of the PhthIm-HbPG samples was determined by a UV−vis spectrophotometer (Jasco V650) equipped with Jasco MCB-100 mini circulation bath and Peltier thermostat heating and cooling system. The samples were dissolved in absolute EtOH, and the UV spectra were recorded with standard 1 × 1 cm quartz cuvettes thermostated at 23 °C with absolute EtOH as a reference. HbPG synthesized with 1,1,1-tris(hydroxymethyl)propane initiator according to the previous literature8 was used as a control sample for the determination of the appropriate wavelength for quantitative determination of the phthalimide content. The phthalimide group content was determined at 290 nm based on the calibration curve prepared from various concentrations of N-(3hydroxypropyl)phthalimide in absolute EtOH. The number-average molar mass of the PhthIm-HbPG samples was calculated from the phthalimide content and the mass concentration of the polymers. Vapor Pressure Osmometry. Vapor pressure osmometry measurements were carried out by using a Gonotec Osmomat 070 vapor pressure osmometer in methanol at 45 °C in a concentration range of 1−7 g/kg. A methanolic solution of benzil, in the same concentration region, was used for calibration. Electrospray Ionization Mass Spectrometry (ESI-MS). The mass spectrometric measurements of the phthalimide- and aminemonofunctional HbPG samples were carried out by electrospray ionization mass spectrometry (ESI-MS) on a Bruker Daltonics Esquire 3000 Plus (Bremen, Germany) ion trap mass spectrometer, operating with continuous sample injection at 4 μL/min flow rate. Samples were dissolved in acetonitrile−water mixture (1:1 V/V %) with 0.1% acetic acid. The ESI-MS spectra were recorded in positive ion mode in the m/z 200−2200 range, with m/z 1000 as target mass.

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Figure 2. 1H NMR spectra of unmodified (bottom) and peracetylated (top) hyperbranched polyglycerol (sample PhthIm-HbPG_1; see Table 1) synthesized by the phthalimide/potassium phthalimide initiating system (DMSO-d6, room temperature).

RESULTS AND DISCUSSION The synthesis of monofunctional, highly branched macromolecules and subsequent, efficient chemical modification of

Table 1. Theoretical (Mtheor) and Experimental (Mn) Number-Average Molar Masses by UV and 1H NMR Spectroscopies, Vapor Pressure Osmometry (VPO), the Average Initiating Efficiencies (Chain End Functionalities) (F), and the Degree of Branching (DB) of Hyperbranched Polyglycerols Synthesized by the PhthIm/K-PhthIm Initiating System Mn (Da)

PhthImHbPG_1 PhthImHbPG_2 PhthImHbPG_3 PhthImHbPG_4 PhthImHbPG_5

Figure 1. UV spectra of the synthesized monophthalimide-functional HbPG samples compared with that of N-(3-hydroxypropyl)phthalimide as calibration standard and HbPG* synthesized with TMP initiator.

1

Mtheor (Da)

UV

H NMRa

1 H NMRb

VPO

Fc

DBd

1250

1280

1360

1390

1370

0.93

0.54

1525

1650

1640

1680

1530

0.94

0.52

2015

2070

2080

2080

2160

0.96

0.53

2610

2860

2640

2630

2750

0.96

0.53

5320

5820

5620

5730

5190

0.95

0.56

a

Mn determined by 1H NMR spectra of the unmodified HbPG. bMn determined by 1H NMR spectra of the acetylated HbPG. cInitiating efficiencies obtained by the averages of the Mn values determined with the different methods. dDegree of branching determined by 13C NMR spectroscopy via eq 1.

the monofuctionality are still significant challenges in polymer science. Because amine functionality is one of the most versatile functional groups not only for small molecules but also on polymer chains, especially for (bio)conjugations, we have intended to develop a simple process to obtain monoamine functionalized hyperbranched polyglycerol (HbPG), a watersoluble biocompatible macromolecule. Because the N-alkylphthalimides can be easily transformed to alkylamines,55 systematic experiments have been carried out by us on the ring-opening multibranching polymerization (ROMBP) of glycidol in bulk by the phthalimide/potassium phthalimide (PhthIm/K-PhthIm) initiating system, which has not been

reported for such purpose so far. Based on this concept, the PhthIm/K-PhthIm mixture can PhthIm/K-PhthIm mixture can be applied to the synthesis of HbPG with phthalimide monofunctionality (PhthIm-HbPG) under suitable conditions. Such a process would have several advantages, for instance, the commercial availability of the ingredients with high purity and the unnecessary additional deprotonation of the initiator, a D


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Table 2. Theoretical (Mtheor) and Experimental NumberAverage Molecular Masses, and the Corresponding Dispersity (PDI = Mw/Mn) Values of the Peracetylated (Mn,Ac) and Unmodified (Mn(calc) and Mn,aq) Hyperbranched Polyglycerol Samples by GPCs with THF and Water Eluents, Respectively unmodified HbPGc

acetyl derivativesa

PhthImHbPG_1 PhthImHbPG_2 PhthImHbPG_3 PhthImHbPG_4 PhthImHbPG_5

Figure 3. Results of vapor pressure osmometry measurements of phthalimide monofunctional hyperbranched polyglycerols in methanol at 45 °C, calibrated with benzil.

Mtheor (Da)

Mn,Aca (Da)

Mn(calc)b (Da)

PDIa

Mn,aq (Da)

PDIc

1250

1550

1020

1.28

770

1.41

1525

1870

1220

1.39

880

1.35

2015

1960

1280

1.44

910

1.39

2610

2290

1490

1.47

1100

1.50

5320

3400

2190

1.60

2000

1.55

a

Obtained by organic GPC, eluent: THF, conventional polystyrene calibration. bDetermined by eq 2. cObtained by aqueous GPC, eluent: 0.1 M NaNO3, conventional linear PEG calibration.

Figure 4. Molar mass distribution curves of the hyperbranched polyglycerol samples obtained by aqueous GPC with PEG calibration (top) and acetylated HbPGs measured by organic phase GPC (THF eluent) with PSt calibration (bottom).

usual process with most of the currently applied initiators for ROMBP of glycidol. The synthetic route for obtaining HbPG bearing one phthalimide headgroup with the PhthIm/K-PhthIm initiating system is shown in Scheme 1. Several aspects should be considered for reaching high initiating efficiency by using such a functional initiator for ROMBP. First, the proton/potassium exchange between the initiator molecules and also the potassium alkoxide chain ends and the reaction of glycidol with the deprotonated initiator and alkoxide chain ends should be sufficiently fast to provide the consumption of all the functional initiating molecules in initiating glycidol polymerization. Second, the rate of polymerization should be slower than that of the initiation in order to provide conditions for

Figure 5. Experimental Mn values determined by UV and 1H NMR spectroscopies, VPO and GPC (A), the dispersities (PDI = Mw/Mn) obtained by GPC (B), and the initiating efficiencies F (C) as a function of the theoretical number-average molar masses (Mtheor) of the phthalimide monofunctional hyperbranched polyglycerols.

E


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Scheme 2. Transformation of the Phthalimide Group of the Monofunctional HbPG to Amine and Then the Amine to Carboxyl, Maleimide, and Chloroacetamide Functionalitiesa

a

Reagents and conditions: (A) hydrazine monohydrate, DMF, RT, 40 h; (B) succinic anhydride, TEA, EtOH, RT, 6 h; (C) 6-maleimidohexanioic acid, NHS, DCC, DMF, RT, overnight; (D) pentachlorophenyl chloroacetate, DMF, RT, overnight.

The PhthIm-HbPG samples were also investigated by 1H NMR spectroscopy in DMSO-d6. The recorded 1H NMR spectrum of one representative sample (PhthIm-HbPG_1) is shown in Figure 2 (bottom). The signals of the main chain of the polymer overlap with the signals of water; therefore, this region cannot be used for quantitative evaluations. However, the chemical shifts of the phthalimide group observed in the 7.82−7.95 ppm region and that of the hydroxyl groups in the range of 4.25−4.82 ppm are well separated from the signals of the branched polymer backbone (3.10−3.85 ppm). Therefore, the DPn can be calculated from the ratio of the integrals of the NMR signals of the aromatic phthalimide initiator moiety and the hydroxyl groups. This ratio gives one unit higher result than the DPn because every incorporated monomer increases the number of the hydroxyl groups by one, regardless of the forming branched structure. The calculated number-average molar masses (see Table 1) based on the DPn values from the 1 H NMR spectra agree well with the Mtheor and also with the Mn values determined by UV spectroscopy. This is an additional strong evidence that the initiating efficiency of the PhthIm/K-PhthIm combination in the ROMBP of glycidol under the conditions used by us is near to 100%, that is phthalimide-monofunctional HbPGs are formed with high yields. Acetylation of the HbPG samples was carried out for additional analysis purposes, mainly for GPC measurements in an organic solvent, such as THF. Figure 2 (top) shows the 1H NMR spectrum of an acetylated HbPG sample (PhthImHbPG_1). The acetylation of the HbPG macromolecules in all cases was quantitative, proved by the 1H NMR measurements (see Figure 2, top, and Figures S1−S4). Namely, the chemical shifts of acetyl protons are around 1.9 ppm, and methyl and methylene protons next to the carbonyl group appear in the 4.78−5.14 and 3.86−4.22 ppm regions, respectively. Evidently, the signals belonging to the hydroxyl protons in the range of 4.25−4.82 ppm do not appear, which proves the conversion of the hydroxyl groups to acetate with high, practically quantitative yields. The number-average molar masses of the PhthIm-HbPG samples were calculated based on the 1H NMR spectrum of the acetyl derivatives analogously to the nonmodified polymers. The ratios of the integral values of the methyl protons from the acetyl groups and phthalimide were used for this calculation. As shown in Table 1, the resulting Mn values are in good agreement with the theoretical Mtheor and also with that of obtained by UV and 1H NMR spectroscopies of the unmodified polymers. These indicate not only the high

complete initiator consumption. Third, chain transfer to monomer should be suppressed for avoiding the formation of HbPG chains without functionality. To ensure high initiating efficiency, namely the quantitative incorporation of the phthalimide functional group into the polymer, besides the regular conditions, i.e., relatively high polymerization temperature (95 °C) and inert atmosphere, slower than usual rate of monomer addition, that is, 2.5 mL/h instead of 4−6 mL/h, feeding rate was applied during the polymerizations. To verify the feasibility of this process, the synthesis of HbPGs with theoretical average molar masses in the range of 1−6 kDa was attempted by using different monomer/initiator ratios. The resulting polymers were analyzed by various independent analytical methods, such as UV and NMR spectroscopies, aqueous and organic phase GPCs, and vapor pressure osmometry (VPO). Both the UV (Figure 1) and 1H NMR spectra (Figure 2) clearly indicate the incorporation of the phthalimide moieties in the polymer chains. As displayed in Figure 1, all the HbPG samples prepared by the PhthIm/K-PhthIm initiating system show absorption peak in the aromatic region (270−310 nm), similar to a model compound, N-(3-hydroxypropyl)phthalimide (N-HPPhthIm). In contrast, a control sample, that is HbPG* synthesized by 1,1,1-tris(hydroxymethyl)propane, an initiator without an aromatic group, does not have any absorption in this region. In order to determine the amount of phthalimide incorporated in the PhthIm-HbPG samples, calibration was made with N-HPPhthIm on the basis of the absorption values at 290 nm. The concentrations and absorptions of N-HPPhthIm and the hyperbranched HbPG samples together with the calibration equation are shown in Table S1 of the Supporting Information. Assuming that each HbPG molecule contains one phthalimide functional group, the number-average molar mass (Mn) can be obtained from the UV absorption data. As shown in Table 1, the Mn values determined from the UV spectra of the PhthIm-HbPG samples fit quite well with the theoretical number-average molecular masses (Mtheor) calculated from the monomer/initiator ratios in the feed. These results confirm near to quantitative initiation with the PhthIm/K-PhthIm initiating system under the applied condition, i.e., every HbPG bears one phthalimide functionality. Should the Mn obtained by UV spectroscopy be considerably lower than Mtheor would indicate that a significant number of hyperbranched polyglycerols is formed either by initiation from or by chain transfer to the monomer. F


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Figure 6. Insets of the 1H−13C HSQC spectra of functional group signals of phthalimide (a), amine (b), carboxylic (c), maleimide (d), and chloroacetamide (e) monofunctional HbPGs. The signal of CH2 next to the amine group is marked with dashed ellipsoids. Besides the solvent peak (2.50, 39.5 ppm), residues of solvents used for the reactions can be seen (DMF: 2.89, 35.73; 2.73, 30.73 ppm, diethyl ether: 1.09, 15.12 ppm, methanol: 3.16, 48.59 ppm).

The average molar mass determination by VPO is based on the decrease in the vapor pressure of the solvent, in our case methanol, which depends only on the concentration of the polymer. Based on the results of the VPO measurements (see Figure 3 and Table 1), it can be observed that the Mn values increase with increasing monomer/initiator ratio and agree well with the Mns determined by the spectroscopic methods and with the theoretical number-average molar masses as well. Evidently, this means that no chain transfer occurs during glycidol polymerization with the phthalimide/potassium phthalimide initiating system, on the one hand. On the other hand, these findings corroborate that nearly all polymer chains possess one phthalimide functional group. The initiating efficiencies (F), i.e., the average phthalimide functionalities,

efficiency of the acetylation process but also the phthalimide monofunctionality of the HbPG molecules. In order to obtain number-average molar mass with a direct method, the Mn of the HbPG samples were measured by vapor pressure osmometry (VPO). VPO determines Mn independently of the structural parameters, such as the presence or absence of the initiator moiety in the chains, and also of the hydrodynamic volumes, which is the basis of GPC measurements (see later). The presence of the initiating phthalimide group in the HbPG samples was verified by UV and 1H NMR spectroscopies above, and this was used for average molar mass determination. Since the Mn values were calculated by assuming one initiator moiety per chain by using NMR and UV spectroscopies, chain transfer to monomer cannot be excluded. G


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M n = Mi + M mon(M n,Ac − Mi − MAc)/(M mon + MAc) (2)

where Mn, Mi, Mn,Ac, MAc, and Mmon stand for the numberaverage molar mass of the starting (non-acetylated) polymer, the molar mass of the phthalimide initiator, the determined number-average molar mass of the acetylated polymer, the molar mass of the acetylated end groups (42 Da), and the molar mass of the monomer, respectively. Comparing the Mn values of the unmodified samples in Table 2, it can be seen that the Mns obtained by the GPCs with aqueous and THF eluents are close to each other, following the same trend, i.e., the Mn increases with increasing monomer/initiator ratios, but the Mns with aqueous GPC are somewhat lower than that of determined by the GPC with THF as eluent. This might be due to either the differences in the hydrodynamic properties of the polystyrene and PEG calibration standards or certain interaction of the HbPG samples with the columns in the case of the aqueous GPC, which may delay the elution of the polymers from the chromatographic column. However, as shown by the data in Table 2, the most valuable findings by the GPC measurements are related to the fact that the Mns measured by GPCs are significantly lower than the theoretical ones and also lower than the Mns determined by the other methods, i.e., UV and NMR spectroscopies and VPO. Since the average molar mass obtained by GPC is based on the hydrodynamic volume (Vh) of the polymer chains, and branched polymers have lower V h than that of their corresponding linear analogues with the same molar mass,62 the results of the GPC measurements determined by using calibration made with linear polymer standards clearly indicate the compact branched structure of the synthesized polyglycerols. This is in good agreement with the results of the degrees of branching obtained by 13C NMR spectroscopy (Table 1) and the large number of hydroxyl groups in these polymers. Furthermore, the observed PDI values, measured by both aqueous and organic GPCs, are all relatively low; that is, these are in the range 1.3−1.6, which is an expectation for the applied ring-opening multibranching polymerization method. Based on the comparison of the GPC result of the PhthIm-HbPG samples and their acetylated derivatives, it can be concluded that the acetylation is a suitable way for preparing HbPGs soluble in organic solvents, and these are suitable for Mn and PDI determination of the starting unmodified macromolecules in an organic solvent by a combination of GPC and 1H NMR spectroscopy. Figure 5 summarizes the major results, i.e., Mn, PDI, and F values, of the analyses of the monophthalimide HbPGs (see Table S2 for details). Figure 5A clearly shows that the numberaverage molar masses determined by UV and NMR spectroscopies as well as the ones obtained by VPO are in good agreement with the theoretical ones. In contrast, the Mns measured by either aqueous or organic phase GPCs are significantly lower than the theoretical values and those obtained by spectroscopic methods or VPO. This provides strong evidence for the formation of the compact, highly branched HbPGs with one phthalimide moiety per chain. This is further proved by the initiating efficiency data in Figure 5C, indicating that the F values are close to the theoretical values and are also independent of the monomer/initiator ratios. It is also noteworthy to mention that HbPGs with relatively narrow MMDs are formed as the PDI data, falling in the 1.28−1.60 range, indicate (Figure 5B).

Figure 7. ESI-MS spectra of phthalimide-HbPG (a) and amine-HbPG (b).

were calculated by using the averages of the Mn values obtained by the UV, 1H NMR, and VPO methods. As shown in Table 1, the initiating efficiencies obtained this way are in the range of 0.93−0.96, i.e., above 90% in all cases. In addition to the Mn and functionality values, the degree of branching (DB) is also a substantial characteristic of hyperbranched polymers. The DBs of the synthesized HbPGs were determined according to previous literature,59 i.e., by the integrals of the 13C NMR signals of the hyperbranched polymers (see Figures S5−S9) from the fraction of structural units using the equation DB = 2D/(2D + L13 + L14)

(1)

where D, L13, and L14 represent the fractions of dendritic, linear 1,3-units, and linear 1,4-units, respectively. The DB values obtained by using this equation are between 0.52 and 0.56 (Table 1), and there is no significant correlation with average molar mass. Thus, it can be concluded that all the synthesized polyglycerol samples have randomly branched structure, and their branching frequencies are similar to each other. The average molar masses and the dispersities (PDI = Mw/ Mn) of the HbPG samples and their acetylated derivatives were measured by aqueous and organic GPCs, respectively. The molar mass distribution curves (MMD) are displayed in Figure 4, and the Mn and PDI results are presented in Table 2. As shown in Figure 4, the MMD curves of the acetylated HbPGs are located at higher molecular weight regions than that of the MMDs obtained by aqueous GPC of the unmodified watersoluble polymer samples. This is attributed to the presence of the acetyl termini on the modified HbPGs which increases the molecular weight of these samples. The number-average molar masses of the unmodified HbPGs can be calculated by the equation H


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analyzed samples because of the low ionization efficacy in the higher molecular mass ranges and the multiple ionization. Furthermore, it must be noted that due to the molecular weight of the phthalimide (M = 147.13 Da) and the mass of two glycerol monomer units (M = 148.16 Da), the m/z values would nearly coincide for polymer chains formed by the phthalimide initiator and chain transfer by one proton loss during the multibranching polymerization. Therefore, the mass spectrum of the phthalimide initiated polyglycerol alone cannot be regarded as a convincing proof of the incorporation of the phthalimide initiator. However, the removal of the phthalimide moiety decreases the m/z by 130 Da, and indeed, this difference can be detected in the spectrum of the amino functionalized HbPG, on the one hand. On the other hand, the m/z values for the amine-HbPG sample (Figure 7b) are equivalent with polyglycerols having one amine group per chain. Thus, these results provide additional structural evidence that the phthalimide/K-phthalimide initiating system results in phthalimide functional HbPG, and its hydrazinolysis leads to monoamine functional hyperbranched polyglycerol. This means that the ESI-MS results undisputedly confirm the incorporation of one phthalimide group into the polymer in the initiating process and the formation of the amine functional HbPG after the cleavage of the protecting group.

According to our concept, the phthalimide group of the monofunctional HbPG can be further converted to a versatile amine functionality which can subsequently be utilized in conjugation reactions and also in further modifications. The schematic representation of the targeted functionalization reactions is depicted in Scheme 2. One selected sample (PhthIm-HbPG_3, Mn ∼ 2100 Da) was reacted with hydrazine to cleave the phthaloyl group and obtain monoamino-functional macromolecule (H2N-HbPG). As shown in Scheme 2, this product was further modified by succinic anhydride, 6-maleimidohexanoic acid, and pentachlorophenyl chloroacetate to obtain the carboxyl, maleimide, and chloroacetamide functional groups, respectively. Such functional groups can be broadly applied for modification of biomolecules and a large variety of organic and inorganic substances via conjugation reactions. Although simple 1H NMR spectra could have been applied to prove the modifications in the case of well-purified low molecular mass HbPG samples, due to the broad and low-intensity functional group signals, the success of the functional group modifications was followed by solution state multidimensional NMR spectroscopy in our study. The functionalities were undoubtedly identified by HSQC spectra which can be obtained regardless of the molecular weight of the investigated HbPG samples, the presence of remaining reagents, byproducts, or solvents. The extended HSQC spectra of the resulting samples are displayed in Figure 6. The full assignations were carried out by using the correlation spectra (see Figures S10−S29 and captions in the Supporting Information for the detailed assignations in all the recorded 1H, 13C, and full HSQC spectra of the functionalized HbPG samples). The NMR signal of the CH2 group next to the amine/amide group (signed with dashed ellipsoids in Figure 6) is composed of two 1H signals because the nonbonding electron pair of the N distinguishes the protons, which are sensitive to the changes in the chemical environment of the N. Shift of this signal clearly shows the changes in the structure of the functional group. Transformation of the phthalimide group to amine causes ∼1 ppm shift on the 1H scale, which corresponds to the removal of the aromatic phthaloyl group. No traces of the phthalimide group were found in the amine spectra, and we can conclude that this transformation step was successful. Further transformations of the amine group to different functionalities by the formation of the amide bond leads to a higher chemical shift on the 1H scale, and the amine signal disappears, indicating full transformations of the amine functions. Here, we have to note that signals belonging to the HbPG branched main chain did not suffer any detected changes. On the basis of the obtained results, we can conclude that the performed modifications proceed nearly quantitatively, and as a consequence, monofunctional HbPGs were successfully produced with amine, carboxylic, maleimide, and chloroacetamide (bio)conjugation relevant groups. ESI-MS measurements of the phthalimide and amine functionalized HbPGs were also performed. The recorded spectra are presented in Figure 7 (for the expanded ESI-MS spectra see Figure S30). The mass differences between the peaks represent the molar mass of glycidol (M = 74.08 Da). The exact masses of the peaks are the sum of the phthalimide initiator mass of 147.13 Da (Figure 7a) and 17.03 Da of ammonia (Figure 7b), the respective molar masses of the glycidol repeating units, and the counterions (H+, Na+). It should be emphasized that the ESI-MS measurements cannot provide direct information about the Mn and PDI of the

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CONCLUSIONS

Phthalimide monofunctional hyperbranched polyglycerols were synthesized by anionic ring-opening multibranching polymerization of glycidol with a new phthalimide/potassium phthalimide initiating system. We have found that this initiator system is suitable for the synthesis of well-defined phthalimide monofunctional HbPGs with high, close to 100% initiating efficiencies, i.e., with quantitative monofunctionality and predetermined average molecular weights. The resulting polymers possess degree of branching in the range 0.52−0.56, which is independent of the molecular weight, indicating a randomly branched structure. GPC measurements confirmed the compact, branched structure and revealed that HbPGs with relatively narrow MMDs were formed. The transformation of the phthalimide functional group of HbPG to a primary amine by hydrazinolysis was successfully carried out. The incorporation of the phthalimide functional initiator into the HbPG and the cleavage of the phthaloyl group were also proved by ESI-MS measurements. The modification of the resulting monoamine HbPG to produce carboxylic, maleimide and chloroacetamide functionalities was performed quantitatively as proved by 2D NMR spectroscopic analysis. On the basis of our results, i.e., the successful polymerization of glycidol with the PhthIm/K-PhthIm initiating system, it can be concluded that this mixture can most likely be applied as initiator for polymerization of a large variety of other epoxies and other cyclic monomers to obtain polymers with phthalimide head functionality. In addition to that, the novel monofunctional HbPGs are expected to open new routes for the synthesis of a variety of materials by leading to increased water solubility, stability, and/or biocompatibility of bioactive molecules, such as proteins, drugs or dyes, and other materials, e.g., catalysts which can be linked to the functional groups of the polymer, modified surfaces to obtain 2D materials or coated nanoparticles, etc. I


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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.7b00413. Determination of average molar mass of HbPGs by UV spectroscopy, 1H NMR spectra of hyperbranched polyglycerols synthesized by phthalimide initiator and acetyl derivatives,13C NMR spectra of hyperbranched polyglycerols synthesized by phthalimide initiator; determination of the average molar mass of the synthesized HbPGs, the initiating efficiency; solution state 1H, 13C and 1H−13C HSQC spectra and functional group assignations of monofunctional HbPGs and ESIMS spectra of phthalimide and amine monofunctional HbPGs (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (G.K.). *E-mail: [email protected] (B.I.). ORCID

György Kasza: 0000-0003-3335-8730 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful for the technical assistance in the GPC analyses to Blandine Boßmann with water and Tamás Ignáth with THF as eluent. Support by the Protein Science Research Synergy Program (MedInProt) and the National Research, Development and Innovation Office, Hungary (K115939 and K112094), is also acknowledged. Attila Domján acknowledges the support of the Bolyai Fellowship. Lilla Pethő thanks for the support of the Hungarian Templeton Program (a grant from Templeton World Charity Foundation, Inc.).

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Synthesis of Well-Defined Phthalimide Monofunctional

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