Post polymer modification of polyethylenimine with citrate esters: selectivity and hydrophobicity

Abstract

Citrate esters selectively react with primary (1°) amines on branched polyethylenimine (PEI). As the functionalization occurred, the water soluble PEI transformed into a thermoset. Adjusting stoichiometry and type of citrate ester allowed fine-tuning of hydrophobicity, modulus, and swelling behavior. Based on control experiments of citrate esters with model amines, FTIR analysis of thermosets, swelling experiments, and TGA data, molecular models were constructed to represent the thermosets. Quantifying the hydrophobicity of these molecular models with computational octanol–water (log P_oct) partition coefficients provided a method to assess how hydrophobicity changes during a post polymer modification (PPM) reaction.

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Introduction

Although most areas of science have unique terminology to describe interesting phenomena within sub-fields, the terms hydrophobic and hydrophilic cut across many disciplines. While these terms provide a great framework for understanding and explaining certain experimental observations, a more quantitative approach is frequently desired.^1,2

Given universal interest in “hydrophobic” surfaces,^2,3 monomers,^4–6 micelles,⁷ polymers,^8,9 peptides,¹⁰ proteins,¹¹ molecules,¹² solvents,¹³ and drugs,¹⁴ comparing one system to another is often necessary. While most researchers have developed an intuition about hydrophobicity, a great deal of experimental^2,15–18 and computational^19–21 effort has been done to quantify the qualitative nature of hydrophobic systems. In particular, HPLC assessment of hydrophobicity has been insightful for monomers¹⁶ and star polymers.²²

Recently, our efforts to predict hydrophobicity of polymers utilized octanol–water partition coefficients (log P_oct).²³ This method received inspiration from medicinal chemists who use computational log P_oct values to describe the hydrophobicity of drug-like molecules.^14,24 Since monomers and drug-like molecules share many functional groups, log P_oct values provide a convenient method for assessing hydrophobicity. To take into account the changes that monomers undergo during a polymerization, log P_oct values for oligomeric sequences of monomers helped quantify hydrophobicity of the corresponding polymer. As such, negative values indicated water solubility while positive values suggested hydrophobic polymers.

log P_oct values have an underutilized potential for quantifying hydrophobicity and could be very beneficial for post polymer modification (PPM) reactions. In recent years, attaching functional groups via a PPM strategy has received enormous attention.^25–27 A few of the many benefits of PPM include improving biocompatibility,²⁸ facilitating surface grafting,²⁹ and providing access to various polymer architectures.^30,31

To illustrate the concept of quantifying hydrophobicity during PPM reactions, we identified several citrate esters with a range of log P_oct values and envisioned the covalent attachment to a polymer. Our interest in citrate esters developed during use of citric acid as a monomer³² and interest in non-toxic molecules.^33,34 Although the benign nature of citrate esters makes them very suitable as plasticizers for PLA³⁵ and cellulose acetate,³⁶ they are rarely employed as monomers or crosslinkers.

Polyethylenimine (PEI) has amines that could potentially react with a variety of functional groups. For instance, PEI has been reacted with acid chlorides,³⁷ anhydrides,^38,39 aldehydes,⁴⁰ alkyl halides,³⁷ carbonates,⁴¹ carboxylic acids,⁴² and methacrylates.⁴³ Many of these hydrophobic modifications improve electrospinning, gene transfection, and alter LCST behavior. However, these studies are representative of the widespread empirical efforts and approaches to addressing hydrophobicity. At present, the reaction of PEI with citrate esters has not been reported and provides an opportunity to investigate and quantify hydrophobicity with partition coefficients.

Experimental

Materials

Triethyl citrate (TEC, >98%, Sigma Aldrich), tributyl citrate (TBC, ≥97.0%, Sigma Aldrich), triethyl 2-acetylcitrate (TEC-OAc, 99%, Sigma Aldrich), tributyl O-acetylcitrate (TBC-OAc, 98%, Sigma Aldrich), allyl amine (98+%, Acros Organics), benzyl amine (99%, Acros Organics), n-butylamine (99%, Acros Organics), diethyl amine (>99.5%, Sigma Aldrich), di-n-butylamine (99%, Acros Organics), diisopropyl amine (99+%, Acros Organics), diisopropylethyl amine (99.5+%, Acros Organics), N,N-dimethylformamide (DMF, 99.8%, anhydrous, Sigma Aldrich) hexamethyleneimine (99%, Acros Organics), hexanes (HPLC grade, Fisher), morpholine (99+%, Acros Organics), oleyl amine (technical grade, primary amine content > 98%), n-tripropyl amine (98%, Acros Organics), N,N,N-trimethylethylene diamine (98%, Acros Organics), poly(ethyleneimine) (PEI) (M_n ∼ 10 000 g mol⁻¹ and M_n ∼ 600 g mol⁻¹, Sigma Aldrich), and water (HPLC grade, Sigma Aldrich) were used as received.

Characterization

FTIR spectra were recorded with a diamond ATR crystal on a ThermoFisher Nicolet iS10 FTIR spectrometer. FTIR spectra were averaged from 64 scans at a 4 cm⁻¹ resolution. The thermal stability and decomposition temperature (T_d) was analysed with a TA Instruments thermogravimetric analyzer (TGA) Q500 at 20 °C min⁻¹ under nitrogen.

Young's modulus and elongation at break was obtained by Dynamic Mechanical Analysis (DMA) using a TA Instruments Q800. Samples were ∼3–4 mm wide, ∼1 mm thick and ∼25 mm long. After attaching the sample to a film clamp, data was collected at ambient temperature and 1 N min⁻¹. The Young's modulus was calculated from the slope of stress versus strain graphs between 0 and 2% strain. The reported values for modulus value and elongation at break were determined by averaging results from four samples.

Swelling studies were conducted with water or hexanes at ambient temperature. Samples (∼200–240 mg) were cut (∼1 mm × ∼4 mm × ∼25 mm) and immersed in water. The mass increase was measured gravimetrically over a 24 h period. The swelling ratio (SR) was calculated from the following equation: SR (%) = [[(mass of swelled polymer) − (mass of dry polymer)]/(mass of dry polymer)] × 100.

Polymerization method

All polymerizations were heated in air using aluminium pans (Fisher Scientific, volume ∼ 70 mL). The stoichiometry was adjusted to produce polymer films with diameter of ∼6.5 cm and thickness of ∼1 mm.

General procedure for kinetic experiments in Fig. 1

PEI (M_n ∼ 10 000 g mol⁻¹) (2.000 g, ∼11.6 mmol 1° NH₂ groups) was mixed with citrate ester (1.45 mmol) ([1° NH₂]/[TEC] = 8) in an aluminum pan while heating at 60 °C for several minutes. After mixing, the film was reacted at 80–100 °C on a temperature controlled hotplate and periodically monitored by FTIR spectroscopy.

Reaction of PEI and TEC for DMA and swelling experiments

PEI (M_n ∼ 10 000 g mol⁻¹) (2.000 g, ∼11.6 mmol 1° NH₂ groups) was mixed with TEC (1.613 g, 5.8 mmol, [1° NH₂]/[TEC] = 2) in an aluminum pan while heating at 60 °C for several minutes. After mixing, the film was reacted at 100 °C on a temperature controlled hotplate. After 2 h, the mass loss via evaporation of ethanol was 0.327 g.

Reaction of PEI and TBC for DMA and swelling experiments

PEI (M_n ∼ 10 000 g mol⁻¹) (2.000 g, ∼11.6 mmol 1° NH₂ groups) was mixed with TBC (2.09 g, 5.8 mmol, [1° NH₂]/[TBC] = 2) in an aluminum pan while heating at 60 °C for several minutes. After mixing, the film was reacted at 100 °C on a temperature controlled hotplate. After 2 h, the mass loss via evaporation of butanol was 0.382 g.

General procedure with TBC-OAc

PEI (M_n ∼ 600 g mol⁻¹) (2.000 g, ∼11.6 mmol 1° NH₂ groups) was mixed with TBC-OAc (1.167 g, 2.9 mmol) at 60 °C. After 30 minutes, the temperature was increased to 100 °C on a temperature controlled hotplate. After 3 h at 100 °C, film formation was noted. The reaction continued for 24 h to promote evaporation of butanol. Then, the film was cooled to ambient temperature and analysed by FTIR spectroscopy (Abs_amide/Abs_ester = 3.2). The SR after 2 h in water was 329%.

Control experiments with amines

In a vial (4 mL), citrate ester (TEC or TBC) (0.2 mL) and a small molecule amine (0.3 mL) (Table S1†) were combined at ambient temperature. The vial was capped and heated for 2 h at 80 °C while stirring. Then, the reaction was cooled to ambient temperature and analysed by FTIR spectroscopy.

Hydrophobicity calculations

Molecular models were constructed using Chem3D Pro version 13.0.2.3021 and Materials Studio 7.0. After building each structure, the model was minimized using the MM2 forcefield (Chem3D) and geometry optimization in Forcite module (Materials Studio). The Forcite module used a Universal forcefield and Smart algorithm. Afterwards, the partition coefficients (log P_oct for Chem3D and A log P for Materials Studio) were extracted from the chemical properties module. The Connolly molecular surface area was calculated using a probe of 1.4 Å.

Results and discussion

Structure and reactivity of PEI

The ring opening polymerization of aziridine produces branched PEI (Scheme 1) with primary (1°), secondary (2°), and tertiary (3°) amine groups.⁴⁴ Although the theoretical ratio of 1° : 2° : 3° amines has been reported to be 1 : 2 : 1, some variability exists depending on synthetic procedures.⁴⁵

Scheme 1 Structure of branched polyethyleneimine (PEI).

Since hexamethylene diamine [H₂N(CH₂)₆NH₂] reacts with triethyl citrate,⁴⁶ we hypothesized that the 1° amines on PEI would also be reactive towards commercially available citrate esters. Indeed, based on FTIR spectroscopy (Fig. S1†), amines on PEI reacted with citrate esters in Scheme 2 and converted the esters to amides via nucleophilic addition. Reactions of PEI and citrate esters were also accompanied by mass loss due to evaporation of ethanol or butanol.

Scheme 2 Select examples of citrate esters and corresponding log P_oct values.

Furthermore, experiments with TEC, TEC-OAc, and TBC mixed well with PEI (M_n ∼ 10 000 g mol⁻¹) and provided homogeneous films without the need for organic solvents. In contrast, the larger log P_oct value for TBC-OAc resulted in mixing and phase separation issues. Reducing the molecular weight of PEI to M_n ∼ 600 g mol⁻¹ or adding a small quantity of DMF improved homogeneity. The large disparity in hydrophobicity between PEI and TBC-OAc highlights a challenge that exists for hydrophobic modification of water soluble polymers.

Kinetics

After determining the feasibility of reacting PEI with citrate esters in Scheme 2, FTIR spectroscopy (Fig. 1) confirmed that a range of temperatures allowed the ester groups in citrate esters to react with PEI. In fact, these polymerizations underwent gelation after ∼10 min at 100 °C and ∼20 min at 80 °C.

Fig. 1 FTIR spectroscopy data for reaction of PEI (M_n = 10k) and TEC at 80 °C () and 100 °C (). Abs_amide/Abs_ester ratio calculated from height of amide (1662 cm⁻¹) and ester (1734 cm⁻¹) absorbances.

In Fig. 1, the formation of the amide groups occurred with simultaneous decrease in the ester absorbance for TEC. At moderate conversions, the amide : ester ratio gave predictable and reproducible results for a constant film thickness. Based on regression analysis of FTIR data (Fig. S2†), the larger butyl groups on TBC lowered the reactivity (k_rel = 1) compared to the less sterically hindered TEC (k_rel = 1.8).

Selectivity of primary amines

While analysis of Fig. 1 indicated formation of amide bonds, further experiments were necessary to determine reactivity differences between 1°, 2°, and 3° amines in PEI (Scheme 1).

After conducting control experiments (Table S1†) with a large number of 1°, 2°, and 3° amines to simulate the chemical environment of PEI, FTIR spectroscopy (Fig. S3 and S4†) confirmed citrate esters selectively react with primary amines. Generally, 1° amines were probably at least two orders of magnitude more reactive than 2° amines. For instance, 2° amines, like diethyl amine and dibutyl amine, did not react with TEC and produce detectable amide absorbances (Fig. 2) after 2 h at 80 °C or 100 °C. Dibutyl amine was also unreactive with TBC under similar conditions. In contrast, 1° amines reacted without difficulty under similar conditions and gave a reactivity profile that decreased as follows: butyl > allyl > benzyl. A very small amount of reactivity occurred with cyclic secondary amines (i.e. morpholine and hexamethyleneimine). However, compared to butyl amine, the magnitude of this reactivity was insignificant. Tertiary amines, like diisopropylethyl amine and n-tripropyl amine were also unreactive.

Fig. 2 FTIR spectra for the product of TEC and butyl amine (solid line) after 2 h at 80 °C compared to dibutyl amine (dashed line) and TEC after 2 h at 100 °C.

Often, the focus of PPM reactions with amines or thiols involves increasing the reactivity of activated esters to obtain quantitative yield^47,48 or selectively preparing block copolymers.⁴⁹ Surprisingly, PPM reactions that allow 1° amines to selectivity react with esters, like TEC and TBC, have not been reported. Interestingly, secondary amines will react with activated esters,⁵⁰ epoxides,⁵¹ urethanes⁵² dithiocarbonates,⁵³ and acrylates,^54,55 but do not react with carbonates.⁵¹ Since 2° amines are very unlikely to react with citrate esters, the observed selectively benefits the goal of obtaining hydrophobic thermosets and allows the existence of terminal units in Scheme 3.

Scheme 3 Representation of terminal and crosslinked citrate esters (R = ethyl, butyl; R′ = H, acetate).

Based on control experiments, Scheme 3 depicts several amide bonds that result from the reaction of 1° amines and citrate esters. Since the structure in Scheme 3 formed the basis for constructing computational models (Schemes S1–S7†) to quantify hydrophobicity, a combination of reaction kinetics (Fig. 1), control experiments with model amines (Fig. 2), thermal analysis, and swelling studies provided a synergistic perspective. Depending on experimental parameters, both terminal and crosslinked citrate groups are expected.

Thermal analysis

As the 1° amines selectively react with citrate esters, the thermoplastic PEI becomes functionalized with amide groups and begins to crosslink into a thermoset (Scheme 3). During the functionalization reaction, the formation of each amide group produces an alcohol (i.e. ethanol or butanol) that will potentially evaporate given appropriate time and temperature.

To investigate the propensity for crosslinking and evaporation of ethanol or butanol, a series of polymerizations were monitored by TGA. Based on the onset of decomposition (∼340–370 °C) from weight loss profiles, crosslinking increased with increasing time (Fig. S5†) and temperature (Fig. S6†). Additionally, reactivity of the citrate ester played a role and TEC gave more crosslinking than TBC after 1 h at 100 °C (Fig. S7†). This observation that TEC is more reactive than TBC correlates with FTIR analysis (Fig. S2†). Considering the mass loss below 300 °C, TGA data indicates short reaction times (i.e. < 2 h) and lower reaction temperatures (i.e. < 100 °C) produced films that contained alcohol as well as unreacted citrate ester.

Mechanical properties

To further expand upon the perspective established by TGA and FTIR spectroscopy, a series of flexible films were investigated by DMA. After reacting PEI and citrate esters for 2 h at 100 °C, the Young's Modulus (YM) value for [1° NH₂]/[TEC] = 8 was 10.6 ± 0.34 MPa. When changing the [1° NH₂]/[TEC] ratio from 8 to 4, larger elongation at break (Fig. S8†) and higher YM values (Fig. S9†) were observed relative to TBC.

Hydrophobicity calculations

In order to determine the effect of attaching citrate esters to PEI, a series of molecular models replicated the local environment of PEI thermosets. The models, which were based on FTIR and TGA data, considered scenarios without crosslinks (Scheme S1†) and with crosslinks (Schemes S2–S7†). As shown in Fig. 3 and S10,† these oligomeric models allowed a theoretical calculation of log P_oct values for various [1° amine]/[citrate ester] ratios. In fact, the range of stoichiometry in Fig. 3 provided an excess of 1° amines as well as an excess of esters. Previously, solvatochromatic dye experiments with Nile Red validated calculation of hydrophobicity for homopolymers and copolymers using log P_oct values.²³ To facilitate comparison between different sized citrate esters, the log P_oct values were normalized with surface area (SA).

Fig. 3 Hydrophobicity calculations of PEI oligomers based on octanol–water partition coefficients. The data represents the influence of terminal citrate esters on PEI without crosslinks using TEC (), TBC (), and TBC-OAc () groups. See Tables S2 and S3† for additional information and data. The solid lines represent logarithmic regression.

In regards to Fig. 3, several observations are worth mentioning. First, the amount of hydrophobicity is initially small for an excess of 1° amines. As reflected by log P_oct/SA values, attachment of citrate esters to 1° amines during PPM increases hydrophobicity. For a given conversion, the theoretical increase in hydrophobicity for terminal citrate esters would decrease as follows: TBC-OAc > TBC > TEC. Second, if all 1° amine groups were reacted with TEC or TBC groups, the hydrophilic nature of PEI would still result in negative log P_oct/SA values. Consequently, these materials with TEC or TBC groups will be expected to swell in water. Third, terminal citrate groups, which only have 1 ester replaced with an amide group, would contribute a larger increase in hydrophobicity than crosslinked groups. For example, the addition of crosslinking to molecular models (Schemes S2–S7†) lowers the number of alkyl esters. Consequently, slightly lower log P_oct/SA values (Fig. S10†) were observed compared to models (Scheme S1†) without crosslinking (Fig. 3).

Swelling studies

Although branched PEI is a viscous polymer that easily dissolves in water at room temperature, the reaction with trifunctional citrate esters resulted in crosslinked films that swelled in water (Fig. 4). Since these films did not swell in hexanes, the negative log P_oct/SA values in Fig. 3 are very reasonable. Additionally, Fig. S11† suggests a relationship between the amount of alkyl esters and the swelling ratio.

Fig. 4 Swelling ratios in water for polymer films made from reaction of PEI (M_n = 10k) with TBC () and TEC (). The films were heated at 100 °C for 2 h before immersion in water for 24 h. The solid line represents linear regression. For comparison, swelling data in hexanes after 18 h at ambient temperature is shown for TBC (circles) and TEC ().

Conclusion

The reaction of water soluble PEI with citrate esters appeared quite facile and occurred with a variety of alkyl substituted citrate esters. Based on FTIR spectroscopy, model reactions with 1°, 2°, and 3° amines confirmed the 1° amines on branched PEI preferentially react with citrate esters. This selectivity of 1° amines with citrate esters provided an avenue for thermosets with tunable hydrophobicity, modulus, and thermal stability.

Computational log P_oct values and experimental swelling data confirm PPM reactions between PEI and citrates esters experience a dramatic increase in hydrophobicity as the conversion increases. This change in hydrophobicity during PPM reactions would be expected to occur in other systems when a disparity exists between the hydrophobicity of a polymer and the functional group.

log P_oct values provided a less empirical roadmap for designing and conducting synthetic experiments. In addition, this approach offers clear direction for obtaining certain amounts of hydrophobicity and may reduce the need for exhaustive experimentation. As such, the concept of creating polymer libraries as well as hydrophobically modified water-soluble polymers based on acrylamides,⁵⁶ oxazolines,⁵⁷ acrylates,⁵⁸ and aliphatic amines⁵⁹ could benefit from this strategy with log P_oct values.

Acknowledgements

R. T. M. thanks the National Science Foundation (CHE-MSN and DMR-Polymers) for support under Grant CHE 1308247.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra14953g

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