Correction: Amphiphilic and double hydrophilic block copolymers containing a polydehydroalanine block

Correction for ‘Amphiphilic and double hydrophilic block copolymers containing a polydehydroalanine block’ by Mark Billing et al., Polym. Chem., 2017, DOI: 10.1039/c6py02076c.


Introduction
Polymers carrying charged groups are generally referred to as polyelectrolytes 1,2 and, depending on the nature of the functional groups, can be further subdivided into strong and weak polycations and polyanions. 3Whereas strong polyelectrolytes remain permanently charged irrespective of the pH value of the surrounding medium, weak polyelectrolytes exhibit pHdependent charge characteristics.][10] As a result, a wide range of applications has been reported for polyelectrolytes and related materials, including sewage treatment, as metal binders (e.g.Cd) in oil recovery, water purification, or as flocculants and emulsifiers. 5In case of polyzwitterions, specific appli-cations in antifouling coatings 11 or membranes 12 are constantly discussed.Even their use as protective layers for sensors and membranes to extend their lifetime in medical applications or to improve their sensitivity has been reported. 12,13xamples for polyampholytes synthesized by free radical polymerization are copolymers of quaternized poly((N,N-dimethylaminoethyl acrylate)-co-(sodium styrene sulfonic acid)) P(DMAEAq-co-NaSS) 14 or systems composed of N-(3-aminopropyl) methacrylamide hydrochloride and acrylic acid P(APMco-MAA). 15In another example, polyampholyte membranes composed of 2-acrylamide-2-methyl propane sulfonic acid (AMPS) and [2-(methacryloyloxy)ethyl] trimethylammonium chloride (DMC) were reported. 16Reversible deactivation radical polymerization (RDRP) techniques have also been used to synthesize well-defined polyampholytes, as shown for poly (N,N-dimethylaminoethyl methacrylate) (PDMAEMA)/poly (methacrylic acid) (PMAA) block copolymer brushes using ARGET ATRP, 17 or the copolymerization of DMAEMA and MAA 18 or 4-vinylbenzoic acid and 4-vinylbenzyl(triphenylphosphonium) chloride via reversible addition fragmentation chain transfer polymerization (RAFT). 19egarding block polyampholytes, first examples consisting of poly(2-vinylpyridine) (P2VP) and different acrylates/methacrylates were presented by Kamachi et al. 20 Giebeler and Stadler later studied the aqueous solution behavior of polyampholytic triblock terpolymers containing both a PMAA and a P2VP segment. 21Such materials were afterwards used for the preparation of multicompartment micelles and, starting from that, the synthesis of nanoscopic hybrid materials, 22,23 compartmentalized interpolyelectrolyte complexes, 24 and as non-viral vectors in gene delivery applications. 25The temperature responsive self-assembly of zwitterionic block copolymers was recently investigated by the group of Yoshida. 26Here, block copolymers containing N,N-dimethyl-N-(3-(methacrylamido) propyl)aminopropanesulfonate and N-isopropylacrylamide were synthesized via RAFT polymerization and their self-assembly was investigated.In another example, zwitterionic poly(2-methoxyethyl acrylate)-block-poly(3-(N-(2-methacryloyloxyethyl)-N,N-dimethylamino)propane sulfonate) (PMEA-b-PDMAPS) block copolymers were synthesized using ATRP. 27n this context, we recently reported on tert-butoxycarbonylaminomethyl acrylate (tBAMA) as versatile building block for the preparation of polyampholytes and its polymerization using either free radical polymerization or atom transfer radical polymerization (ATRP). 28,29The use of (in principle) orthogonal protective groups for the amino function (tertbutoxy carbonyl) and the carboxylic acid (methyl ester) allows for selective deprotection of either the two functionalities. 29he formed polymers can be subsequently converted into weak polycations, polyanions, or zwitterionic polydehydroalanine (PDha) featuring high charge densities. 29,30][33] In this contribution, we report on the preparation and solution properties of block copolymers containing a polydehydroalanine block (PDha).Starting from previously synthesized PS-b-PtBAMA and PnBA-b-PtBAMA block copolymers, different deprotection reactions led to amphiphilic or double hydrophilic block copolymers.Thereby, we discovered that our previous assumption that either the -COOH or -NH 2 moiety of PtBAMA can be selectively deprotected has to be reconsidered.In both cases, considerable amounts of the other protective group (Boc in the case of -NH 2 and methyl ester for -COOH) is cleaved off as well.Nevertheless, we show first investigations of the solution properties of the resulting amphiphilic PS-b-PDha or double hydrophilic PAA-b-PDha block copolymers in aqueous media.

Nuclear magnetic resonance spectroscopy (NMR)
1 H-NMR and 13 C-NMR spectra were recorded in CDCl 3 , CD 2 Cl 2 , d-TFA, DMSO-d 6 , or D 2 O on a Bruker Fourier spectrometer equipped with a direct observe probehead operating at a proton frequency of 300 MHz.Sample temperature was set to 298 K.Chemical shifts are given in parts per million ( ppm, [δ]) and were referenced by using the residual signal of the deuterated solvent.

Solid state NMR
13 C solid-state magic angle spinning (ssMAS) NMR spectra were either acquired utilizing cross polarization with a contact time of 2 ms and a spinning frequency of 15 kHz or in the case of PAA-b-PtBAA utilizing direct excitation and proton decoupling and a spinning frequency of 5 kHz.All data were collected on a Bruker Avance III HD 400 MHz spectrometer equipped with a 4 mm dual channel probe.Sample temperature was set to 303 K.The carbon chemical shifts were referenced externally, setting the high-frequency (methylene) signal of adamantane to 38.5 ppm. 34

Zeta-potential measurements
The samples for the zeta-potential measurements were prepared by titration of a 1 g L −1 solution of PAA 25 -b-PDha 50 in 0.1 N HCl with 0.1 N NaOH.For the titration and pH detection, a Metrohm 765 Dosimat titrator with a Greisinger electronic GMH3539 digital pH-/mV-electrode with thermometer was used. 1 mL samples for the measurements were taken at the desired pH values.The ζ-potentials were measured using a Zetasizer Nano ZS from Malvern (Malvern Instruments GmbH Gültstein, Germany) via M3-PALS technique with a He-Ne laser operating at 633 nm.The detection angle was 13°.The electrophoretic mobilities (u) were converted into ζ-potentials via the Smoluchowski equation. 35¼ uη ε where η denotes the viscosity and ε the permittivity of the solution.
Synthesis of tert-butoxycarbonylaminomethyl acrylate (tBAMA) N-(tert-Butoxycarbonyl)-D-serine methyl ester (10 g, 45.6 mmol) was dissolved in dichloromethane (200 mL).Methanesulfonyl chloride (Ms-Cl; 6 mL, 77.5 mmol) was added to the solution under vigorous stirring.The reaction mixture was cooled to 0 °C and triethylamine (TEA, 23 mL, 165.9 mmol) was added drop-wise.The solution was stirred at 0 °C for 1 h, and further 2 h at room temperature.Then the reaction mixture was washed with a potassium bisulfate solution (1%) to neutrality.The organic phase was dried over Na 2 SO 4 , filtered and the solvent removed under reduced pressure.The product was further purified via column chromatography with silica gel (hexane/ethyl acetate v/v 8/2).The product was dried under reduced pressure obtaining a colorless oil in a yield of 87% (8 g, 39 mmol).PS 30 -b-PtBAMA 40 (300 mg) was dissolved in dichloromethane (5 mL).Afterwards, trifluoroacetic acid (1.15 mL, 0.0149 mol, 10 eq. per monomer unit) was added and the reaction was stirred at RT for 19 h.Then, the reaction mixture was precipitated in 40 mL hexane.The isolated block copolymer (257 mg) was dried in vacuo. .per monomer unit) was suspended in the solution and the reaction mixture was heated to 100 °C for 22 h.After cooling down to RT and centrifugation, the precipitated block copolymer was isolated and mixed with water (10 mL), before the solution was neutralized with HCl aq .For further purifi-cation, the solution was dialyzed against water.Afterwards, the block copolymer was dried in vacuo (210 mg).PnBA 25 -b-PtBAMA 50 (300 mg) was dissolved in dichloromethane (5 mL).Afterwards, trifluoroacetic acid (1.15 mL, 0.0149 mol, 10 eq. per monomer unit) was added and the reaction was stirred at 50 °C for 1 h.After cooling down to RT, the reaction mixture was precipitated in 40 mL hexane.The isolated block copolymer (176 mg) was dried in vacuo.Afterwards, LiOH•H 2 O (1.32 g, 21 eq.per monomer unit) was suspended in the solution and the reaction mixture was heated to 80 °C for 3 h.After cooling down to RT the precipitated block copolymer was mixed with water (10 mL), and the dispersion was neutralized with HCl aq .After dialysis against water, the block copolymer was dried in vacuo (159 mg).

Results and discussion
We herein report the synthesis and solution behavior of block copolymers containing a polydehydroalanine (PDha) segment and (at that stage) either a hydrophobic PS or a hydrophilic PAA block.We are interested in such materials as, depending on the co-block, either amphiphilic or double hydrophilic block copolymers with pH-dependent charge characteristics are obtained and these could serve as interesting building blocks in interpolyelectrolyte complexes (IPECs).The materials are synthesized using sequential atom transfer radical polymerization (ATRP) of styrene/n-butyl acrylate, followed by tBAMA, as reported recently. 28Subsequently, deprotection of either the carboxyl moiety or the amino functionality is carried out, followed by conversion to polyampholytic PDha-containing block copolymers (Schemes 1 and 2).First, macroinitiators of PS 30 -Br and PnBA 25 -Br were synthesized (the subscripts denote the corresponding degree of polymerization).

Synthesis of PAA 25 -b-PDha 50
We have earlier reported on the (selective) deprotection of either the Boc protective group using TFA at 50 °C or the hydrolysis of the methyl ester of PtBAMA homopolymers at 100 °C using LiOH•H 2 O. 29,30 However, during the course of these studies we observed that both for PnBA 25 -b-PtBAMA 50 and PS 30 -b-PtBAMA 40 also significant deprotection of the other moiety occurred, e.g.treatment with TFA at RT removed both Boc group and the methyl ester.We therefore varied the conditions (50 °C and 1 h reaction time) under which these reactions have been carried out, although without obtaining a truly orthogonal procedure far.Nevertheless, in our opinion the final amphiphilic and double hydrophilic block copolymers featuring PDha segments represent the most interesting materials and for selective deprotection of either -COOH or -NH 2 presumably another combination of protective groups might be favorable, at least in the case of block copolymers.Below we describe the so far most selective reaction conditions whereas more detailed descriptions including 1 H-NMR and Starting from PnBA 25 -b-PtBAMA 50 , the Boc protective group was cleaved by adding TFA (10 eq. per monomer unit) in dichloromethane at 50 °C for 1 h. 36After precipitation in hexane, the block copolymer was characterized by NMR (Fig. 1).As can be seen, the signal of the Boc group at 1.5 ppm disappears (∼100%), indicating complete cleavage.A new broad signal ranging from 2.4 to 3.4 ppm can be found, indicating the presence of a protonated amine functionality.Further, the alkyl chain of the nBA ester  The results obtained by 1 H-NMR were confirmed by 13 C-NMR (Fig. 2).For PnBA-b-PtBAMA, the signals of the backbone (61 and 41 ppm), the carbonyl group of PnBA (172 ppm), the methyl ester of PtBAMA (172 ppm) and the Boc protective  distribution, indicating an intact polymer backbone after the different reaction steps (Table 1, Fig. 3A).We were interested in the solution behavior of the above described amphiphilic and double hydrophilic block copolymers.Starting with PAA 25 -b-PDha 50 , we investigated the pHdependent solubility and net charge using a combination of zeta-potential measurements and dynamic light scattering (Fig. 3B).Starting under basic conditions ( pH 14), the block copolymer shows a negative zeta potential (approximately −25 mV) and this value remains more or less constant until pH 5, presumably due to the presence of deprotonated -COO − moieties.Upon acidification, at pH 4 the zeta potential starts to increase up to −5 mV at pH 2. This hints towards both the protonation of the carboxylates as well as the amino moiety.We explain the fact that the zeta potential remains slightly negative mainly by partial aggregation of the block copolymer at lower pH values.These findings were supported by DLS experiments at different pH values in aqueous solution at a concentration of 1 g L −1 (Fig. 3B and 4).As can be seen, below pH values of 4, a significant increase in apparent count rate occurs, hinting towards aggregation of PAA 25 -b-PDha 50 .This process is accompanied by increasing turbidity.DLS CONTIN plots of PAA 25 -b-PDha 50 at different pH values show mainly unimers at pH 10, as well as at pH 6 and 4 (Fig. 4, hydrodynamic radii of 1.5 to 3 nm are detected, hinting towards the presence of mainly block copolymer unimers).Under acidic conditions ( pH 3), distinct differences were observed as the degree of protonation for both the carboxylic acid groups of PAA and PDha increases.This leads to a decreased solubility, followed by aggregation and the respective DLS experiments revealed an apparent hydrodynamic radius of 220 nm, pointing towards the presence of loosely defined block copolymer aggregates (Fig. 4).We also carried out potentiometric titrations of PAA 25 -b-PDha 50 at a concentration 3 g L −1 , starting under basic conditions (pH 13, 0.1 M NaOH, double negatively charged block copolymer unimers are present as discussed above) by stepwise addition of HCl (0.1 M, Fig. 5).Here, the first steps (pH 13.0-11.0)mainly correspond to the neutralization of excess NaOH.According to previous studies, the pK B of the -NH 2 moiety in PDha is around 9.2, indicating a pH-range of increasing protonation from pH 11-7. 29In that region, the block copolymer consists of a negatively charged segment (PAA) and a zwitterionic block (PDha).Further decrease of the pH value leads to continuous protonation of the amino functionality.For the -COOH groups, typical pK A values are reported around 6-7. [40][41][42][43][44] Therefore, at a pH value of 5 the majority of -COOH moieties is protonated, leading to a charge neutral PAA segment and mainly positive charges within the PDha block.This then leads to aggregation at lower pH values (pH < 4), accompanied by increasing turbidity and precipitation at pH 2.3 (see also DLS experiments).

Synthesis of PS
We were also interested in the solution properties of amphiphilic PS 30 -b-PDha 40 and therefore dissolved the block copolymer in water (D 2 O) by short heating to 80 °C at a concentration of c = 0.1 g L −1 .After cooling down, a turbid solution was obtained, which we ascribed to the micellization of PS 30 -b-PDha 40 (Fig. 6).The core of these aggregates is formed by the hydrophobic PS block and the corona is formed by zwitterionic PDha under these conditions.Subsequent DLS measurements revealed the presence of micelles with a hydrodynamic radius of 51 nm, as well as some larger aggregates above 200 nm in radius (Fig. 6A).Additional cryo-TEM measurements revealed the presence of aggregates with a diameter of 67 nm ± 10 nm (measurement of 50 particles), being in relatively good agreement with the size regime obtained from DLS experiments as here only the PS core is directly visible (Fig. 6B).

Conclusion
We have demonstrated the transformation of previously synthesized PS-b-PtBAMA and PnBA-b-PtBAMA diblock copolymers into double hydrophilic PAA-b-PDha and amphiphilic PS-b-PDha materials by sequential deprotection steps.Thereby, we discovered that neither the TFA-mediated deprotection of the Boc moiety nor the hydrolysis of the methyl ester under basic conditions using LiOH are truly selective.Instead, in both cases already considerable amounts of PDha are generated directlyas evidenced mainly by in-depth NMR investigations.Although the focus of this work here was mainly put on the final PDha-based block copolymers, future work will also be directed towards different monomer substitution patterns which can be orthogonally addressed.Nevertheless, the resulting block copolymers revealed interesting solution properties in aqueous media, as shown for example by pH-dependent DLS and zeta potential measurements in the case of PAA-b-PDha or first micellization studies of PS-b-PDha.Such materials in our opinion are of interest as building blocks for micellar interpolyelectrolyte complexes (IPECs) or for the preparation of membranes with charge-tunable separation layers.
Scheme 2 Structure of PS 30 -b-PtBAMA 40 and the corresponding block copolymers after different deprotection steps.

Table 1 3 Pa
Characteristics of PS 30 -b-PtBAMA 40 and PnBA 25 -b-PtBAMA 50 and the resulting block copolymers after different deprotection steps (the subscripts denote the degree of polymerization of the corresponding block) Entry (Block co-) polymer M n [g mol −1 ] M n, theo [g mol −1 ] nBA 25 -b-(PAMA 15 -co-PDha 35 ) -18 500 -4 P nBA 25 -b-(PtBAA 20 -co-PDha 30 ) -Determined by SEC (CHCl 3 /TEA/i-PrOH: 94/4/2).PS-calibration.b Determined by SEC 0.1 M Na 2 HPO 4 pH 9. Pullulan calibration.tooharsh for the methyl ester, leading to partial cleavage.Compared to earlier studies, here the second block can be used as internal reference, thereby improving the accuracy of NMR characterization.The cleavage of the methyl ester (and the butyl ester of PnBA) was carried out at 80 °C for 3 h and using 21 eq.LiOH•H 2 O.29,37After neutralization using HCl aq and subsequent dialysis against water,1 H-NMR in deuterated TFA showed typical signals for PnBA (4.5, 2.45, 2.20, 1.98, 1.85 and 1.2 ppm) and PtBAA (3 ppm and 1.65 ppm).intensity of the Boc group at 1.5 ppm decreased to 30%, again indicating unselective cleavage (PnBA 25 -b-(PtBAA 20 -co-PDha 30 )).The removal of the Boc protective group during treatment with LiOH•H 2 O was already observed.In further studies, simultaneous cleavage of the Boc and methyl ester in N-Boc alanine methyl ester was justified by the possibility of water to act as dual acid/base catalyst. 38,39Subsequent full deprotection was achieved by treatment of PnBA 25 -b-(PtBAA 20 -co-PDha 30 ) with TFA at RT in water for 96 h.The resulting PAA 25 -b-PDha 50 block copolymer is well soluble in water and 1 H-NMR in D 2 O shows only signals for the polymer backbone (3.2-1.2 ppm), with the exception that a small fraction of remaining PnBA can still be observed (below 10%).
group (153 ppm, 79 ppm, 28 ppm) can be observed.The signals of the butyl group of PnBA can be detected at 64, 30, 19 and 13 ppm and the methyl ester at 52 ppm.For PnBA 25 -b-(PAMA 15 -co-PDha 35 , measured in d-TFA) the Boc group (153, 79, 28 ppm) is absent and the methyl ester (65 ppm) can still be detected.For PnBA 25 -b-(PtBAA 20 -co-PDha 30 ), the signals for the carbonyl moieties could be detected (179.24 ppm and 179.14 ppm) and even the methyl groups of the Boc group (37 ppm) are visible.Also, both PnBA (70, 65, 42, 28, 19 and 15 ppm) and PtBAA (161, 92, 70 and 42 ppm) can be clearly identified.For PAA-b-PDha, mainly carbon atoms of the backbone (61, 59, 45 and 43 ppm) as well as the carbonyl groups (184 and 179 ppm) of the acid functions are observed (NMR in D 2 O).The end group of PnBA-b-PtBAMA (and also for PS-b-PtBAMA) could not be detected in NMR spectroscopy.However, almost complete re-initiation of PtBAMA prepared via ATRP was possible, hinting to a good endgroup fidelity. 28During the deprotection steps, we expect to hydrolyze both the terminal bromine as well as the ester moiety of the ATRP initiator.Differences in solubility of the purified intermediate stages are discussed in the ESI.† It was possible to measure NMR of PnBA 25 -b-(PAMA 15 -co-PDha 35 ) in DCM and TFA as well as PnBA 25 -b-(PtBAA 20 -co-PDha 30 ) in TFA and in D 2 O (Fig. S3-S6 †).After complete deprotection, SEC (0.1 M Na 2 HPO 4 /NaN 3 pH 9, pullulan calibration) of PAA 25 -b-PDha 50 showed a monomodal