Set of small molecule polyurethane (PU) model substrates: Ecotoxicity evaluation and identification of PU degrading biocatalysts

Polyurethanes (PUs) are an exceedingly heterogeneous group of plastic polymers, widely used in a variety of industries from construction to medical implants. In the past decades, we have witnessed the accumulation of PU waste and its detrimental environmental impacts. PUs have been identified as one of the most toxic polymers leaching hazardous compounds derived both from the polymer itself and the additives used in production. Further environmental impact assessment, identification and characterization of substances derived from PU materials and establishing efficient degradation strategies are crucial. Thus, a selection of eight synthetic model compounds which represent partial PU hydrolysis products were synthesized and characterized both in terms of toxicity and suitability to be used as substrates for the identification of novel biocatalysts for PU biodegradation. Overall, the compounds exhibited low in vitro cytotoxicity against a healthy human fibroblast cell line and virtually no toxic effect on the nematode Caenorhabditis elegans up to 500 µg ml−1, and two of the substrates showed moderate aquatic ecotoxicity with EC50 values 53 µg ml−1 and 45 µg ml−1, respectively, on Aliivibrio fischeri. The compounds were successfully applied to study the mechanism of ester and urethane bond cleaving preference of known plastic-degrading enzymes and were used to single out a novel PU-degrading biocatalyst, Amycolatopsis mediterranei ISP5501, among 220 microbial strains. A. mediterranei ISP5501 can also degrade commercially available polyether and polyester PU materials, reducing the average molecular number of the polymer up to 13.5%. This study uncovered a biocatalyst capable of degrading different types of PUs and identified potential enzymes responsible as a key step in developing biotechnological process for PU waste treatment options.


Solvents and Reagents
Solvents for dry-flash chromatography and MS analysis, as well as commercial materials and other solvents were purchased at the highest commercial quality from the providers Acros Organics, Alfa Aesar, Merck, Sigma Aldrich, and Thermo Fisher Scientific, in a purity of over 99% (HPLC-grade).

Chromatography
Thin-layer chromatography (TLC) was performed on precoated plates of silica gel F254 (Merck) with UV detection at 254 and 365 nm.Column chromatography was performed on silica gel Silica 10e18, 60 Å, ICN Biomedicals.

HR-MS
For high resolution mass spectrometry mass spectra were obtained on MS LTQ Orbitrap XL with heated ESI ionization (HESI).
NMR 1 H and 13 C Nuclear Magnetic Resonance Spectra (NMR) were recorded on Varian/Agilent NMR 400 MHz ( 1 H at 400 MHz, 13 C at 100 MHz).Chemical shifts (δ) are expressed in ppm and coupling constant (J) in Hz.TMS was used as an internal standard.The following abbreviation were used for signal multiplicities (s as singlet, t as triplet, q as quartet, dd as doublet of doublets, tt as triplet of triplets, m as multiplet).
Bis(2-hydroxyethyl) (4-methyl-1,3-phenylene)dicarbamate (PU-6): Toluene diisocyanate (1 eq) was added drop-wisely to the solution of ethylene glycol (25 eq) and ethyl-acetate (50 ml) under Ar atmosphere, and refluxed overnight at 77°C.The next day, the reaction was stopped, cooled down to room temperature, and extracted with ethyl-acetate and water.The organic layer was washed with sodium bicarbonate and Brine solution.The obtained yellow oil was dissolved in a minimal amount of hot ethyl-acetate and cooled in the fridge overnight to obtained white crystals.

Table S1 .
Solubility of PU model compounds in selection of common organic solvents.