A Single Amino Acid Able to Promote High‐Temperature Ring‐Opening Polymerization by Dual Activation

Abstract Amino acids are indispensable compounds in the body, performing several biological processes that enable proper functioning. In this work, it is demonstrated that a single amino acid, taurine, is also able to promote the ring‐opening polymerization (ROP) of several cyclic monomers under industrially relevant conditions. It is shown that the unique zwitterionic structure of taurine, where the negatively charged sulfonic acid group and the protonated amine group are separated by two methylene groups, not only provides high thermal stability but also leads to a dual activation mechanism, which is corroborated by quantum mechanical calculations. This unique mechanism allows for the synthesis of polylactide of up to 50 kDa in bulk at 180 °C with good end‐group fidelity using a highly abundant catalyst. Furthermore, cytotoxicity tests confirm that PLLA synthesized with taurine is non‐toxic. Moreover, it is demonstrated that the presence of taurine does not have any detrimental effect on the thermal stability of polylactide, and therefore polymers can be used directly without any post‐polymerization purification. It is believed that the demonstration that a simple structure composed of a single amino acid can promote polymerization can bring a paradigm shift in the preparation of polymers.


Differential Scanning Calorimetry (DSC)
Differential scanning calorimetry (DSC) measurements were performed using a DSC8500 from Perkin Elmer, Inc. calibrated with indium and tin standards.The DSC scans were performed with approximately 5 mg of sample at a heating rate of 20 °C/min from -20 °C to 180 °C under a nitrogen flow rate of 20 mL/min.Thermogravimetric analysis (TGA) Thermogravimetric analysis (TGA) was carried out using a Q500 Thermogravimetric Analyzer from TA Instruments.Samples were heated from room temperature to 600 °C at a rate of 10 °C/min under a constant N2 flow.
Matrix Assisted Laser Desorption Ionization -Time of Flight (MALDI-TOF) analysis MALDI-TOF measurements were performed on a Bruker Autoflex Speed system (Bruker, Germany) instrument, equipped with a 355 nm NdYAG laser using chloroform as solvent and DCTB-NaTFA substrate.

Size Exclusion Chromatography (SEC)
Size Exclusion Chromatography (SEC) was performed in chloroform at 30 °C using a Waters chromatograph equipped with four 5 mm Waters columns (300 mm x 7.7 mm) connected in series with increasing pore sizes.Toluene was used as a marker and the calibration was done using polystyrene standards.

Pm value
Pm value is the probability of meso linkage between monomer units, and it was calculated from the methine region of the 13

X-ray diffraction
Single crystal diffractometer (SuperNova Cu) with four-circle goniometer, Kappa geometry and microfocus Cu source was used equipped with a large Atlas model two-dimensional CCD detector.
The measurements were done at standards conditions, at 100 K.

Toxicity test
To assess the cytotoxicity of the developed materials, ISO/EN 10993 protocols were followed 4 .
Extracts of the materials were obtained by incubating the polymer/the sample with complete medium (DMEM + 10 % FBS + 1 % P/S) at a ratio of sample mass to extraction medium of 200 mg/ml.They were incubated for 24 h at 37 °C in humidified atmosphere containing 5 % CO2.HeLa cells were seeded at a concentration of 5000 cells/well on a 96 well-plate in complete medium.
After 24 h in culture, complete media was replaced by previously filtered extracts.Cell cytotoxicity was then evaluated using the Alamar Blue assay after 24 h and 72 h.

Computational methods
The studies of complex formation, the reaction mechanism, and their corresponding kinetics were performed using the Gaussian 09 package, version D.01. 5 The ωB97XD 6,7 and the M06-2X 8 functionals were employed.The ωB97XD was applied to keep consistency with previous theoretical studies conducted on similar systems 9 while the use of Minessota functional was dictated by its high performance for main group thermochemistry, kinetics, and non-covalent interactions 10 , as well as by our own long experience in applying this functional in studying diverse bio-organic reactions [11][12][13][14][15] with many successful outcomes.The 6-31+G(d,p) basis set was employed for all atoms in both cases.To mimic the influence of the environment, the conductorlike polarizable continuum model (CPCM) [16][17][18] with a dielectric constant, ε = 12.0 was applied, which corresponds to the value of permittivity of the ethyl lactate experimentally determined in previous studies at T = 358 K 19 .To characterize optimized stationary points, diagonalized Hessian matrices were computed at the same levels of theory confirming that the localized structures correspond to minima (all positive eigenvalues) or transition states (one negative eigenvalue).
Subsequently, the zero-point vibrational energy (ZPE) and the thermal vibrational corrections obtained at T = 403 K (the temperature employed in the experiments of the present study) were added to the electronic energy.The IRC method has been used to verify that the obtained transition states are related to the desired minima.A note of caution must be introduced at this point since, because the obtained reaction mechanisms correspond to a multistep process, the intermediate obtained from the IRC traced down from TS1 can be geometrically different from the INT1 obtained from TS2.However, the conformational differences between structures of the same state are not associated with a relevant energy cost.Moreover, r.d.s. is not affected since it corresponds to the same chemical transformation related to lactide ring-opening.
The vial was then submerged into a pre-heated oil bath at 180 °C and the conversion was followed by 1 H NMR in deuterated chloroform.After reaction completion, the formed polylactide was let to cool down to room temperature naturally.For purification, the sample was dissolved in chloroform and precipitated in cold methanol.The resulted polyester was filtrated and dried under vacuum at RT for 24 h before its characterization.
The vial was heated up at 180 °C and the evolution was followed by 1 H NMR in deuterated chloroform.After that, the same purification process as for the ROP of L-lactide was followed.
The vial was heated up to 180 °C and the conversion was followed by 1 H NMR in deuterated chloroform.After 4 h of reaction, the polycarbonate was purified by the use of the same procedure as for the synthesis of polylactide.Table S1.Key distances (in Å) of optimized and characterized stationary points along the uncatalyzed processes of the ring-opening polymerization of L-lactide(L-LA) with benzyl alcohol (BnOH) and butylamine (BuNH2) as initiators computed at ωB97XD and M06-2X level at T = 403 K.The ROP of the L-LA reaction catalyzed by taurine The established H-bond interaction between reactants and catalyst ensures the reactive orientation required for the first step of the reaction, i.e. the nucleophilic attack, by bringing close together the nucleophilic group and electrophilic center (at a distance (dNu:-C=O) of 3.32 and 3.11 Å, and a Bürgi-Dunitz 14,20 (αBD) angle of 109.7° and 104.8° defined as an angle between the nucleophile, the carbonyl atom, and the carbonyl oxygen, for structures optimized at ωB97XD and M06-2X level, respectively).The value of 105 ± 5° determined by Bürgi et al. for small-molecule substrates is the angle that ensures a reliable position for the nucleophile to attack 20 .Anyway, the starting orientation of the reactants in the uncatalyzed process is characterized by similar short distance dNu:-C=O of 3.08 and 2.79 Å and favorable αBD angle of 101.7° and 99.5° determined at ωB97XD and M06-2X level, respectively, suggesting that the relative position of both reactants is not responsible in this case for the observed meaningful reduction of the energy barrier for this step in the Tau assisted process.Our calculations show that both DFT functionals provide the same molecular mechanism of taurine-assisted ROP of L-LA, being step two the r.d.s., and with very similar geometries of optimized stationary structures.Nevertheless, the values of the computed energy barriers differ significantly from each other.In the case of the ωB97XD functional, the computed energy of activation was 22.9 kcal/mol while at M06-2X it was 16.8 kcal/mol.Considering the value of experimentally determined activation energy of 17.4 kcal/mol calculated for this process using Arrhenius plots and in the framework of the Transition State Theory, it was concluded that M06-2X provides results in better agreement with experiments.Moreover, only in the case of results computed at M06-2X the final product complex was found to be thermodynamically more stable (by 1.1 kcal/mol) than the reactant complex.This was not the case for ωB97XD.Therefore, the remaining computational simulations and analyses presented in this work were conducted based on results obtained with the Minnesota functional.

Figure S3. 1 H
Figure S3. 1 H NMR spectrum of the ROP of L-lactide initiated by MSA:butylamine salt.

Figure
Figure S4.a) Conditions and results for the ROP of L-Lactide with unimolecular acid-base naturally occurring catalyst, b) SEC curves and c) 1 H NMR spectra of the catalysts mixed with L-lactide.

Figure S5. 13 C
Figure S5. 13C NMR spectra of L-LA in contact with taurine (L-LA + Taurine) and L-LA.

Figure S8 .
Figure S8.Structure of taurine confirmed by X-ray analysis.

Figure S10 .
Figure S10.Energy profiles of the uncatalyzed initiation step of the ring-opening polymerization of L-lactide(L-LA) with A. benzyl alcohol (BnOH) and B. butylamine (BuNH2) as initiators computed at ωB97XD (in orange) and M06-2X (in blue) level at T = 403 K.

Figure S11 .
Figure S11.Structure and relative energies of optimized and characterized stationary points along the taurine-assisted initiation step of ROP of L-LA and BnOH playing the role of the initiator at M06-2X level.

Figure
Figure S13.ROP of a) TMC, b) CL and c) synthesis of PLA-b-PCL block copolymer and their SEC and DSC results.

Table S5 .
Key distances (in Å) of optimized and characterized stationary points along ROP progress in the presence of taurine molecule as a catalyst and BnOH playing the role of initiator computed at ωB97XD and M06-2X level at T = 403 K.

Table S6 .
Mulliken charges (in a.u.) on key atoms of optimized and characterized stationary points along ROP progress in the presence of taurine molecule as a catalyst and BnOH playing the role of initiator computed at ωB97XD and M06-2X level at T = 403 K.

Table S7 .
Key distances (in Å) of optimized and characterized stationary points along ROP progress in the presence of butylamine-methane sulfonic acid salt as a catalyst and BnOH/BuNH2 playing the role of initiator computed at M06-2X level at T = 403 K.

Table S8 .
Mulliken charges (in a.u.) on key atoms optimized and characterized stationary points along ROP progress in the presence of butylamine-methane sulfonic acid salt as a catalyst and BnOH/BuNH2 playing the role of initiator computed at M06-2X level at T = 403 K.