Enzymatic synthesis of novel aromatic‐aliphatic polyesters with increased hydroxyl group density

Polyesters with pendant hydroxyl groups are attractive materials which offer additional functionalization points in the polymer chain. In contrast to chemical polycondensation, lipase regioselectivity enables the synthesis of these materials as certain hydroxyl groups remain unaffected during the enzymatic process.

biodegradability of the catalyst. In addition to green polymerization routes, lipases provide a selectivity specific to the individual lipase. This property can be used to synthesize polyesters possessing different functional groups without undesirable side reactions, [8][9][10][11][12][13][14] a task for which laborious protection/deprotection steps would be required in conventional synthesis. These functional groups are available for further functionalization or crosslinking. The sn1 and sn3 regioselectivity of lipase B from Candida antarctica in its native reaction of fatty acid ester hydrolysis can be used in the reverse reaction, the esterification, to synthesize polyesters with pendant secondary hydroxyl groups. Routinely, immobilized lipase biocatalysts are used, such as the commercial biocatalyst Novozyme-435 which supplies lipase B from Candida antarctica (CAL-B) on microporous poly(methyl methacrylate) beads. Glycerol, [10,[15][16][17] sorbitol, [10,17] L-malic acid, [11,12] 1,2,4-butanetriol, and 1,2,6-trihydroxyhexane [15] have been successfully used for the synthesis of aliphatic polyesters with pendant hydroxyl groups by lipase catalysis.
Aliphatic-aromatic polyesters have excellent physical and mechanical properties and a favorable cost structure compared to purely aliphatic polyesters and are thus indispensable materials for a large variety of applications such as packaging, adhesives and organic coatings. Yet, the aliphatic-aromatic polyesters applied to date require harsh synthetic reaction conditions with temperatures above 200 • C [18,19] and the biodegradability of these materials is often low.
Hence, aromatic-aliphatic polyesters such as poly(butylene succinatecoterephthalate) (PBST), poly(ethylene succinate-coterephthalate) (PEST) and poly(butylene adipate-coterephthalate) (PBAT), which are readily hydrolyzed by enzymes, [20] have received much interest and are produced on industrial scale. On the other hand, enzymes are being developed that are able to degrade polyesters with classical chemical aromatic building blocks as well as emerging biobased aromatic monomers. [21] The degradation of poly(ethylene terephthalate) (PET) and poly(ethylene furonoate) (PEF) by enzymes has been shown recently. [22][23][24][25][26][27] The direct enzymatic synthesis of aliphatic-aromatic polyesters is an interesting alternative as it provides inherently biodegradability and mild reaction conditions. [28,29] Yet, the acceptance of the common aromatic building blocks as phthalic acids as substrates by lipases is limited. [30,31] In contrast, the sustainable aromatic building block furanoate has been successfully applied in lipase catalyzed polyester synthesis. [32,33] Recently, aromatic diols have been included into the enzymatic synthesis of polyesters. [34] Yet, biodegradable aliphaticaromatic polyester with pendant hydroxyl group in the chain remain a challenge.
In this study, we present an enzymatic pathway to aliphaticaromatic oligoesters with pendant hydroxyl groups that are not accessible by chemical polycondensation (Scheme 1). The diol 2,6bishydroxymethyl-p-cresol was used as aromatic building block with a phenolic hydroxyl group that is preserved in the polymerization.
Reaction conditions were explored in order to obtain an oligoester with a high pendant phenolic group content. A lipase-catalyzed postpolymerization reaction with glycerol was developed to increase the hydroxyl group density and decrease the acid value for further applications.

Synthesis of poly(cresyl adipate-co-hexyl adipate) in the reactor
A 500 mL reaction vessel with cooling jacket was used as reactor. The vessel was filled with 0.35 mol adipic acid (51.4 g), 0.11 mol 1,6-hexane diol (12.5 g) and 0.25 mol 2,6-bis(hydroxymethyl)-p-cresol (41.4 g), 10% w/w molecular sieves (10.5 g) and diphenyl ether (210.7 g, 200% w/w). A blade agitator with a reservoir for the enzyme was manufactured of chlorinated polyethylene with a three dimensional-printer and enclosed with an aluminum mesh. The agitator and the breakers were designed according to German Industry Standard (DIN, 1992). [35] The outer dimensions of the agitator were 36 × 36 × 36 mm and the wall thickness was 5 mm. (s. CAD model Figure S1). A vessel lid with breakers was also produced with a three dimensional printer. The temperature was 52 • C and the agitator was set to the maximum feasible stirring rate of 400 min -1 .
For evaluation of the reaction progress, 20 mL samples were withdrawn. The samples and the final product were subjected to the precipitation steps described above.

Post-polymerization
The equivalent amount of diol for postpolymerization reactions was calculated according to the acid value given in Equation (1).

Polymer characterization
The acid value was determined by titration. A polyester sample was dissolved in a 2:1 toluene/ethanol phenolphthalein solution, which was alkalized with three drops of ethanolic potassium hydroxide solution (c = 0.1 mol L -1 ). Titration was performed with ethanolic potassium hydroxide solution. The acid value was calculated with Equation (2).
Fourier transform infrared spectroscopy was performed with a Nicolet IS5 spectrometer (Thermo Fisher Scientific) between 4000 and 500 cm -1 with a resolution of 4 cm -1 . Gel permeation chromatography (GPC) data was obtained from service analytics. Here, an Alliance 2695e (Waters) GPC system with a refractive index detector (2414, Waters) equipped with a highly networked, porous polystyrene-divinylbenzene column (PIgel 20 μm mixed-A, 7.5 mm × 300 mm, 2400-40,000,000 g mol -1 ) in tetrahydro-furan containing 200 ppm BHT at a flow rate of 1 mL min -1 was used.

Synthesis development of a cresolic oligoester
2,6-bis(hydroxymethyl)-p-cresol was polymerized with adipic acid. The neat polycondensation at elevated temperatures, which is required for melting the reaction mixture, showed no product formation (Table 1).
Prior melting of the reaction mixture without enzyme at 130 • C did not result in polymer formation. This also holds for polymerization in diphenyl ether (DPE). It was only upon the addition of hexane diol and diphenyl ether that polycondensation occurred at even low temperatures (Table 1). This indicates that the solubility of the oligoesters formed by hexane diol and adipic acid in the reaction mixture is a prerequisite for the incorporation of the aromatic alcohol in the oligoester. The relevance of solubility for enzymatic polymerization performance is in accordance with previous studies with different monomers. [31,37,38] Interestingly, 2,6-bis(hydroxymethyl)-p-cresol is not completely soluble in diphenyl ether but slowly dissolves in the reaction mixture upon the progress of the reaction which transforms the heterogeneous slurry at the beginning of the synthesis to a homogeneous mixture. It is already known that aromatic acids as the prominent building blocks phthalic are challenging monomers for enzymatic polymerization reactions. Pellis et al. explains the low reactivity of phthalic acid monomers with sterical constraints of the binding pocket and solubility issues. [31] There, only minor conversion of isophthalic acid leading to dimers and trimers was observed whereas no conversion of the other isomers was found. Yet, the respective methyl esters were successfully converted to polyesters of similar molar masses as obtained in this study. [31] This bolsters the conclusion that the intermediate heptanoyl ester formation is a prerequisite for the incorporation of the cresol.
Increasing the molar fraction of hexane diol from 30% to 60% did not further increase the molar mass as judged by NMR spectroscopy (Table 1). Hence, a mixture of 70% of 2,6-bishydroxymethyl-p-cresol and 30% of 1,6-hexane diol was used in further experiments.
In order to examine the influence of temperature on the polymerization product, polymerization was performed at 50, 70, and 90 • C.
No changes in the acid value were observed. NMR analysis reveals that increasing the temperature from 50 to 70 • C slightly improves the molar weight, but the synthesis at 90 • C fails in terms of cresol content and molecular weight. This finding might indicate the formation of cyclic polyesters, which limit the molar mass of the product. The formation of cyclic polymers in enzymatic synthesis of oligo(2-hydroxyethoxy benzoate) has been analyzed recently in detail by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. [18] Interestingly, the molar mass of the obtained aliphatic-aromatic oligoester had a similar molecular weight of ca. 2000 g mol -1 . Skoczinski et al. showed that TA B L E 1 Analysis of the oligomerization of adipic acid (A) with 2,6-bis(hydroxymethyl)-p-cresol (C) and hexane diol (H) in a flask (72 h) neat and in diphenyl ether (DPE) via end group titration and 1 H-NMR spectroscopy. The number of repeat units, n, and the molecular weight, M n , were determined by 1 H-NMR spectroscopy (Equation (3)). % hexane diol refers to the molar percentage of monomer diol provided. Each reaction was performed at least in triplicate. The standard deviations of the molecular weights are < 10% higher temperatures favor the formation of cyclic polyesters in enzymatic synthesis. [32] Hence, the lowest feasible temperature was chosen for the following scale-up synthesis.

Scale up in a three dimensional printed reactor
The synthesis was scaled up from the 1-10 g scale, which was per-  Figure S1). An aluminum tissue was used to jacket the stirrer and confine the biocatalyst. Whereas the idea of a rotating bed reactor has been developed and commercialized previously, [39,40] the optimizing of the mixing conditions is the focus of the present set-up. Mixing of the reactants was visibly improved with respect to the flask and oligomerization was already obtained within 24 h at 50 • C. The analysis of the products obtained during the synthesis by end group titration and 1 H-NMR spectroscopy reveals that the reactor outperformed the standard synthesis in the flask. In the reactor a polymer with higher molecular weight and cresol content can be obtained at low temperatures ( Table 2). This proves the efficacy of the designed stirred reactor with optimized mixing conditions as specified in the technical guidelines for mixer design for the successful synthesis of the desired aromatic aliphatic polyester. Table 2, in the first 24 h hexane diol is predominantly incorporated into the polymer. It is only at longer reaction times that cresol is integrated according to the molar ratio provided and the molecular weight increases ( 1 H-NMR spectra: Figure S2). This implies that cresol is a poorer substrate than hexane diol. Yet, the enhanced incorporation of cresol in the reactor reveals that the incorporation of cresol is also significantly limited by solubility. This is supported by the fact that low stirring rates and the removal of flow breakers reduces the relative amount of cresol incorporated into the oligoester (Table   S1).

As depicted in
Lipase catalyzed polycondensation reactions are often performed at elevated temperatures. Typically, a temperature of 80-120 • C is used, the maximum temperature at which Novozyme-435 retains activity. [18,32,38] The need of high temperatures is typically attributed to high activation energies, the formation of a homogeneous reaction mixture and diffusion constraints. Our finding of improved polyester synthesis in the stirred reactor emphasizes the importance of solubility for polymerization to take place.
The 1 H-NMR and 13 C-NMR spectra of the final oligoester produced in the reactor are shown in Figure 1. Due to the selectivity of lipase catalysis, the phenolic hydroxyl groups are expected to remain unaffected. [11,12] The lower intensity of the hydroxyl signal (J) in the 1 H-NMR spectrum is assigned to the deprotonation equilibrium of the oligoester and hydrogen bonding which is in line with the splitting of the respective 13 C signal (M). There are no signals of esterification of the phenolic group present in the 13 C NMR spectrum. It has to be mentioned that a significant retention of phenolic groups is not surprising under the moderate reaction conditions used herein. Phenolic esters are labile toward hydrolysis although the continuous removal of water can shift the equilibrium to the ester side. [41] However, a nonenzymatic approach would need to be carried out at high temperatures and with acid/base catalysis, where a number of side reactions as oxidative polymerization and ether formation of the phenolic OH groups would take place. [42,43] The final oligoester was analyzed by GPC and a molecular weight (M w ) of (5580 ± 70) g mol -1 , a number average (M n ) of (2680 ± 99) g mol -1 and a dispersity of 2.1 were determined. The molecular weight determined via NMR analysis greatly varies with the precipitant. Whereas precipitation in hexane only removed diphenyl ether, the precipitation in methanol provided a polyester with a very high molecular weight (9630 g mol -1 ) but a low content of cresol (n C = 10, n H = 33).
Hence, the unfractionated product as obtained by hexane precipitation which might include residual diol monomers was analyzed herein.
For further applications in industry α,ω-telechelic diol oligomers are targeted due to the feasibility of the hydroxyl end groups to react with TA B L E 2 Analysis of the polymerization process at 50 • C in the stirred reactor by end group titration and NMR analysis in comparison to the product obtained in flask. The composition of the co-oligoester was determined from the 1 H-NMR spectra ( Figure S2) giving the molar fraction x of the respective alcohols 2,6-bis(hydroxymethyl)-p-cresol (C) and hexane diol (H) and the molecular weight (M n ) as calculated by Equation (3) different functional groups. In order to increase the number of hydroxyl groups the oligomers were reacted with glycerol in a solvent free postpolymerization step.

Post-polymerization
Glycerol was used in the postpolymerization reaction as it is a source of additional aliphatic pendant hydroxyl groups. Moreover, it does not significantly displace cresol in the polyester in contrast to an excess of hexane diol or ethane diol (Table S2). The influence of the molar ratio of oligomer and glycerol on the product composition of the postpolymerization step was analyzed by 1 H-NMR ( Figure S3) and 13 C NMR spectroscopy ( Figure 2). As depicted in Table 3 Hence, a moderate (five-fold) excess of glycerol was used for the analysis of the impact of reaction temperature. As depicted in Table 3, glycerol incorporation can be amplified by slowly increasing the temperature from 50 to 90 • C during the reaction. Yet, here a star polyester is obtained as proven by the appearance of glycerol triester signals in the 1 H NMR spectrum, which consummate pendant OH groups. This also impedes the estimation of molecular weight by NMR analysis.
Remarkably, no signal of the glycerol triester is found in the spectra of the products formed at 50 • C.
This finding can be bolstered by 13 C-NMR analysis of the glycerol substitution ( Figure 2). The signals of the glycerol adducts are assigned with small variations according to Kumar et al. [10] The CH signals Hence, a postpolymerization temperature of 50 • C is favorable in order to retain lipase selectivity and obtain a linear polyester with maximum OH group density. This is in line with our previous study on the biocatalytic esterification of glycerol carbonate emphasizing that low reaction temperatures are required for the full exploitation of the unique lipase selectivity. [44] 1 HNMR analysis indicates a slight increase of the number average molecular weight by postpolymerization ( Table 3). The weight average molecular weight M W as determined by GPC analysis does not change (M w = 5490 g mol -1 ) and the dispersity is slightly reduced to 2.0, which is in the common range for commercial polyester polyols. This suggests that enzymatic postpolymerization adds glycerol to acidic end groups and links short oligomers thus reducing the difference between M n and M w but does not build up much larger chains. These findings can be explained by the competition of transesterification and esterification at moderate temperatures where the lipase is still fully active. [45] The lower mobility, solubility and the increasing bulkiness with respect to the substrate cleft [46,47] of larger chains are suggested to reduce the Additionally, the formation of cyclic oligoesters, which would further limit the molar mass, cannot be excluded.

DISCUSSION
Lipase catalysis in polyester synthesis introduces not only new functionalities but also goes along with new constraints due to the substrate specific activity of the enzyme. It is well known that long chain diols provide a high reactivity and thus polymers of higher molecular weights in lipase catalyzed polyester synthesis whereas alcohol groups in the chain reduce the activity. [8,9,14,38,[47][48][49] The lipase active site is hydrophobic [50,51] and natively not designed for aromatic substrates.
Hence, the low reactivity of aromatic monomers in lipase catalyzed polymerization is commonly assigned to sterical constraints of the substrate cleft. This problem is currently addressed by protein engineering studies, which should contribute to synthesis optimization in the future. [52] For the purpose of a potential economic applicability, diacids available on large scale have to be used instead of activated diester. Their low solubility in a number of diols and high melting points already add a general challenge to the enzymatic polymerization process. [10][11][12]14,37] We show that an inhomogeneous reaction mixture is an even more severe obstacle in the chemo-enzymatic polymerization of aromatic polyols. We suggest that optimizing the mixing conditions in enzymatic reactions is another step in developing enzymatic polymerization with a broad set of building blocks as required for material design.
Post-polymerization of prepolymers synthesized by lipase catalysis has previously been performed chemically at high temperatures. [18,46,47] This excludes the presence of pendant hydroxyl groups as these would be consumed in the process. The enzymatic postpolymerization with glycerol under mild conditions performed herein further increases the hydroxyl content of the oligoester and optimizes the polymer properties for technical applications requiring pendant hydroxyl groups in the chain and at the chain ends.
We hope that the present study providing the process development for polyester design with diverse building blocks according to technical requirements by lipase catalysis prompts future attempts in enzyme engineering so that reaction times can be reduced for the goal of an industrial implementation in the future.

CONCLUSION
A synthesis of an aliphatic-aromatic oligoester with pendant hydroxyl groups by lipase-catalysis at low reaction temperatures is presented.
The bottleneck of the successful polymerization lies in the realization of a well dispersed reaction mixture which is effectuated by the use of a solvent, a ternary monomer mixture and an adapted stirred reactor design. We show that efficient stirring and the reaction time are key parameters for the incorporation of the aromatic monomer into the polymer. Enzymatic postpolymerization with glycerol at low temperatures further increases the hydroxyl content by aliphatic OH groups. We believe that the described synthesis route and reaction parameters allow the design of aliphatic-aromatic polyesters with pendant hydroxyl groups according to individual requirements. Hence, we hope that our study will provoke the development of new materials for various applications such as adhesives and coatings where performance characteristics are tuned by the hydroxyl group density and aromatic/aliphatic monomer ratio.

ACKNOWLEDGMENTS
This work was supported by the German "Bundesministerium für Bildung und Forschung (BMBF)" in the framework of the program "FHPro- Open access funding enabled and organized by Projekt DEAL.