Switchable Coacervate Formation via Amino Acid Functionalization of Poly(dehydroalanine)

Our group recently developed a family of side-chain amino acid-functionalized poly(S-alkyl-l-homocysteines), Xaa-CH (Xaa = generic amino acid), which possess the ability to form environmentally responsive coacervates in water. In an effort to further study how the molecular structure affects polypeptide coacervate formation, we prepared side-chain amino acid-functionalized poly(S-alkyl-rac-cysteines), Xaa-rac-C, via post-polymerization modification of poly(dehydroalanine), ADH. The use of the ADH platform allowed straightforward synthesis of a diverse range of side-chain amino acid-functionalized polypeptides via direct reaction of unprotected l-amino acid 2-mercaptoethylamides with ADH. Despite their differences in the main-chain structure, we found that Xaa-rac-C can form coacervates with properties similar to those seen with Xaa-CH. These results suggest that the incorporation of side-chain amino acids onto polypeptides may be a way to generally favor coacervation. The incorporation of l-methionine in Met-rac-C allowed the preparation of coacervates with improved stability against high ionic strength media. Further, the presence of additional thioether groups in Met-rac-C resulted in an increased solubility change upon oxidation allowing facile reversible redox switching of coacervate formation in aqueous media.


Synthetic Procedures
S-(tert-butylcarboxymethyl)-L-cysteine L-cysteine hydrochloride monohydrate (5.0 g, 28 mmol, 1.0 eq) and NaOH (2.5 g, 57 mmol, 2.0 eq) were dissolved in DI H2O (40 mL) with stirring.The reaction mixture was cooled to 0 °C in an ice-water bath before the dropwise addition of tert-butyl bromoacetate (2.8 mL, 31 mmol, 1.1 eq) over 30 min.The ice bath was then removed and 10 mL of THF was added.The reaction mixture was stirred at room temperature overnight resulting in formation of a white solid precipitate.The while solid was collected via filtration and washed with a 3:7 mixture of 95% EtOH and diethyl ether.The solids were placed in a 50 mL centrifuge tube and washed 3 times with a 95:5 mixture of diethyl ether and methanol to remove residual impurities.The sticky white solid was then dried under vacuum to give the product.(4.1 g, 61%).Spectra data were in agreement with previously reported values. 1 1,2 S-(tertbutylcarboxymethyl)-L-cysteine (2.0 g, 8.5 mmol, 1.0 eq) was suspended in analytical grade THF (40 mL) in a heavy walled glass container.Epichlorohydrin (6.7 mL, 85 mmol 10 eq) was then added.In a well-ventilated fume hood, triphosgene (1.3 g, 4.6 mmol, 0.55 eq) was added, and the vessel was sealed and allowed to react for 2 h at room temperature.WARNING: Triphosgene is an extremely dangerous chemical and proper precautions must be taken to avoid exposure.The resulting turbid mixture was then cooled to 4 o C and 10 mL of cold DI H2O was added.The reaction mixture was stirred for 1 minute before extraction with ethyl acetate, which was then washed with brine and dried with anhydrous sodium sulfate.The resulting solution was transferred to an oven dried Schlenk flask and the solvent was removed under vacuum.The crude product was transferred to a N2 filled glove box, resuspended in minimal THF, and layered under 5x hexanes (v/v) to give white or slightly off-white crystals.(1.1 g, 50%).Spectral data were in agreement with previously reported values. 1

Procedure for synthesis of poly(S-(tert-butylcarboxymethyl)-L-cysteine) (C BCM 65)
Samples of C BCM 65 were prepared at ca. 500 mg scale in a N2 filled glove box.bpyNiCOD initiator solution (3.1 mL, 20 mg/mL in THF) was quickly added to a solution of tBuCM-Cys NCA (10 mL, 50 mg/mL in THF).After ca.90 min, complete consumption of NCA was confirmed by FTIR spectroscopy.In order to determine polypeptide chain lengths a small aliquot of the reaction mixture (ca.200 μL) was removed for end-group analysis where active chain-ends were reacted with mPEG-NCO (vide infra).The remaining polypeptide solution was then end capped with excess acetic anhydride, precipitated by addition to DI H2O (50 mL), and isolated by centrifugation.The solid was then washed two additional times with DI H2O and dried under reduced pressure to yield C BCM 65 as a lightly purple colored powder.(460 mg, 92%) Spectral data were in agreement with previously reported values. 1 General procedure for determination of polypeptide chain length using end-group analysis after reaction with mPEG-NCO 3 The procedure for synthesis of C BCM 65 was followed.Once the polymerization reaction was determined to be complete by FTIR, a solution of mPEG-NCO (Mn = 1000 Da, 50 mg/mL in THF, 4 eq per bpyNiCOD) was added in a N2 filled glove box to a ca.200 μL aliquot of active polymerization reaction mixture.The sample was let stand overnight, and then removed from the glove box and the polypeptide was precipitated by addition to DI H2O.The sample was centrifuged at 3000 rpm and the supernatant was discarded.The pellet was washed 3 times with DI H2O and centrifuged to remove unconjugated mPEG-NCO, and the resulting pellet was then lyophilized to yield the PEG-polypeptide conjugate as a white solid (typical yields = 90 to 95%).To determine the molecular weight (Mn) of the polypeptide, a 1 H NMR spectrum was obtained in deuterated trifluoroacetic acid (TFA-d).The ratio of the integral of the methylene unit closest to the polypeptide backbone to the integral of the PEG methylene resonance was used to calculate polypeptide length (see spectral data section).
General procedure for synthesis of poly(S-carboxymethyl-L-cysteine), sodium salt (C CM 65) 1 A sample of C BCM 65 (450 mg) was dissolved in TFA (20 mg/mL) and allowed to stand for 5 h.The reaction mixture was diluted to 10 mg/mL by slowly adding 50 mM NaHCO3 and then transferred to a 1000 Da MWCO dialysis bag and dialyzed against aqueous 50 mM NaHCO3 (24 h, 3 dialyzate changes) followed by DI H2O (24 h, 4 dialyzate changes).The retentate was then lyophilized to give the product as a white fluffy solid.(340 mg, 89%).Spectral data were in agreement with previously reported values. 1 General procedure for synthesis of poly(dehydroalanine) (A DH 65) A sample of C CM 65 (340 mg) was dissolved in 150 mM sodium phosphate buffer (pH 8) at a concentration of 20 mg/mL.Iodomethane (25 eq per S-(carboxymethyl)-L-cysteine residue) was added, and the reaction flask was sealed and placed in a heating block at 37 °C, covered in foil and allowed to react for 3 days.The resulting suspension was mixed with a solution of BHT in dinitrogen sparged DMSO (0.2 mg/mL) to give a final 0.01 mol% ratio of BHT to dehydroalanine residues and then transferred to a 2000 Da MWCO dialysis bag and dialyzed against dinitrogen sparged DI H2O (48 h, 4 dialyzate changes, dialysis jar covered in foil).The retentate was then lyophilized to give the product as a white solid (130 mg, 100%).Spectral data were in agreement with previously reported values.

Figure S1. 1 H
Figure S1. 1 H NMR spectra of pegylated polypeptide intermediates in the synthesis of Leu-rac-C showing negligible polypeptide chain cleavage during modifications. 1 H NMR spectra and structures of A) PEG22-b-C BCM 50, B) PEG22-b-A DH 50, and C) PEG22-b-(Leu-rac-C)50.Solvents are noted in upper right of each spectrum.S = solvent resonances.Degree of polymerization of the polypeptide segments was designated as an average value of 50.

Figure S4 .
Figure S4.Temperature dependent coacervate formation in 3.0 mg/mL solutions of Val-rac-C65 as a function of pH.Panel shows optical transmittance at 500 nm for 3.0 mg/mL solutions of Valrac-C65 in 150 mM PBS buffer measured over a range of temperature at different pH.

Figure S6 .
Figure S6.A) Temperature dependent coacervate formation in a 3.0 mg/mL solution of Leu-rac-C O 65 at pH 7.0.Plot shows optical transmittance at 500 nm for a 3.0 mg/mL solution of Leu-rac-C O 65 in 150 mM PBS buffer measured over a range of temperature at pH 7.0.B) Optical micrograph of Leu-rac-C O 65 mixed with TPP.A solution of Leu-rac-C O 65 at 3.0 mg/mL in 150 mM NaCl at 20 °C and pH 8.5 was mixed with TPP (12 mM final concentration) and the resulting turbid suspension was allowed to settle onto a glass slide before imaging.Scale bar = 20 µm.

Figure S7 .
Figure S7.Optical micrograph of Leu-rac-C O 65 mixed with polyA.A solution of Leu-rac-C O 65 at 5.0 mg/mL in 150 mM NaCl at 20 °C and pH 7.0 was mixed with polyA (0.015 mM final concentration) and the resulting turbid suspension was allowed to settle onto a glass slide before imaging.Scale bar = 20 µm.

Figure S8 .
Figure S8.Size distributions from dynamic light scattering analysis of polypeptide samples over time.Intensity size distributions of samples of (A) 3.0 mg/mL Met O -rac-C O 65 prepared in 150 mM NaCl at pH 7.0 with 12 mM TPP, and (B) 5.0 mg/mL Met O -rac-C O 65 prepared in 150 mM NaCl at pH 7.0 with 0.015 mM polyA.Number size distributions of samples of (C) 3.0 mg/mL Met Orac-C O 65 prepared in 150 mM NaCl at pH 7.0 with 12 mM TPP, and (D) 5.0 mg/mL Met O -rac-C O 65 prepared in 150 mM NaCl at pH 7.0 with 0.015 mM polyA.All samples were diluted to 0.1 mg/mL of Met O -rac-C O 65 by addition of aqueous 150 mM NaCl at pH 7.0, and passed through a 0.45 µm pore size PTFE syringe filter before analysis using a Malvern Zetasizer Nano ZS at 20 °C.d.nm = average hydrodynamic diameter in nanometers.

Figure S9 :
Figure S9: Size distributions from dynamic light scattering analysis of TPP, polyA, and Met Orac-C O 65 solutions.(A) Number and (B) intensity size distributions of a solution of 12 mM TPP in 150 mM NaCl at pH 7.0.(C) Number and (D) intensity size distributions of a solution of 5.0 mg/mL polyA in 150 mM NaCl at pH 7.0.(E) Number and (F) intensity size distributions of a solution of 3.0 mg/mL Met O -rac-C O 65 in 150 mM NaCl at pH 7.0.d.nm = average hydrodynamic diameter in nanometers.