Elucidating enzymatic polymerisations: Chain-length selectivity of Candida antarctica lipase B towards various aliphatic diols and dicarboxylic acid diesters

Abstract The sustainable synthesis of polymers is a field with growing interest due to the need of modern society to preserve the environment whilst making used products and food sustainable for the future generations. In this work we investigate the possibility of synthesizing aliphatic polyesters derived from various dicarboxylic acid diesters and diols in a solvent-free reaction system. Candida antarctica lipase B was selected as biocatalyst and its selectivity towards the carbon and ester chain length were elucidated. The selected enzyme was able to synthesize various polyesters combining C4-C10 diesters and C4-C8 diols. All combinations led to monomer conversions above 90% in 24 h with the best number average molecular weights (Mn) being obtained through the combination of dimethyl adipate and 1,8-octanediol leading to a Mn of 7141 Da. Differential scanning calorimetry analysis shows a clear trend with an increase in melting temperature of the polymers that correlates with both the increase of the Mn or of the polymer’s constitutional repeat unit carbon chain length. Thermogravimetric analysis and rheology measurements performed on selected samples also confirm the trend showing a variation of the polymer’s degradation temperatures and viscosity profiles.

calorimetry analysis shows a clear trend with an increase in melting temperature of the 27 polymers that correlates with both the increase of the M n or of the polymer's constitutional 28 repeat unit carbon chain length. Thermogravimetric analysis and rheology measurements Introduction 33 The application of biocatalysts in organic synthesis offers several advantages compared 34 with traditional chemo-catalysts such as milder reaction conditions with regards to 35 temperature (usually T<100 ºC), pressure and pH (normally 3-8). Such conditions often 36 lead to remarkable energy efficiency, high enantio-, regio-and chemo-selectivities as well 37 as controlled stereochemistry. These features allow the development of new functional 38 compounds for pharmaceuticals, agrochemicals and polymers using nontoxic natural 39 catalysts with a significant "green" appeal having commercial benefits and satisfying 40 ecological requirements [1]. 41 Despite studies focused on the use of glycosidases for the synthesis of natural and 42 unnatural polysaccharides [2], as well as oxidoreductases for the polymerisation of phenol 43 derivatives [3] and vinyl monomers [1] (mainly using laccases and peroxidases), the most 44 investigated area of enzymatic synthesis is the production of polyesters via both 45 polycondensation (transesterification) and ring opening polymerisations (ROPs) [4,5]. 46 These areas have predominantly emerged thanks to the discovery and commercial availability of Candida antarctica lipase B (CaLB). Over recent years the extraordinary 48 properties of this enzyme were brought to light from several research teams in the kinetic 49 resolution of organofluorine rac-alcohols [6], the synthesis of glucoside esters [7] and the 50 enantioselective synthesis of a -amino acid ester via a solvent-free chemo-enzymatic 51 reaction among others [8].
Further to this, CaLB has been shown to be an active catalyst for the synthesis of a wide 53 range of aliphatic [9], aliphatic functional [10,11] (e.g. polyesters containing lateral 54 functionalities such as vinyl and hydroxy groups) and aliphatic-aromatic polyesters [12,13] 55 and polyamides [14]. In recent years these polyesters and polyamides have been derived 56 preferentially from renewable monomers such as 2,5-furandicarboxylic, adipic and succinic 57 acids and 1,4-butanediol among others [5]. biocatalyst has been shown to be active and stable in several different conditions ranging 65 from water-based to anhydrous organic media and up to temperatures of ~100 ºC.

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Among the many synthesis studies both on polycondensation and ROPs that were 67 performed over the years using this enzyme, we found there is a lack of comparative 68 studies on the range of monomers shown in Scheme 1 (most of which bio-based) [20] for 69 the synthesis of aliphatic polyesters. 70 synthesis of aliphatic polyesters in solvent-free systems using Candida antarctica lipase B as biocatalyst.

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Despite the many studies on the topic, there remains an absence of any detailed 76 systematic study into the implications of changing the diester and/or diol whilst applying 77 identical methodologies for the polymerisation. In the present study we investigated the 78 CaLB-catalyzed polycondensation of various methyl-, ethyl-and butyl-dicarboxylic acid 79 esters with various linear diols having a carbon chain length of 4, 6 or 8, shedding light on 80 how reactive different size diesters are when employing enzymatic catalysis in a solvent-81 free reaction system (Scheme 2). These results will/have help(ed) us to understand the 82 strengths and weaknesses of using this, until now, sporadically investigated enzyme for 83 polyester synthesis.   were performed in duplicates. Statistical analysis reporting the mean value ± standard deviation for all 157 reactions can be found in ESI, Figure S1 and the complete spectra assignment in Figure S5.

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In addition to monomer conversion and molecular weight analysis, differential scanning 239 calorimetry analysis for the determination of the polymers' melting points and 240 thermogravimetric analysis for the determination of the mass loss were performed. Figure   241 3 reports the melting temperatures of all the synthesized polymers when the standard 242 protocol (6 h at 1000 mbar, 18 h at 20 mbar, 85 ºC, 10% w w -1 CaLB, 24 h total reaction 243 time) was applied. It is possible to observe a general trend where an increase in polymer 244 melting point aligns with increasing carbon chain length of the diester, whereC 4 -based polymers have lower melting points than the C 10 -based polymers. This is due to the 246 crystallinity of the C 4 -based polymers as discussed above. A similar increase is also 247 observed when increasing the diol carbon chain length from C 4 (BDO, blue) to C 8 (ODO, 248 green). In this case, increasing the dicarboxylic ester length from C 4 to C 8 reduces the 249 melting point differences among the produced polymers, analogous to number average 250 molecular weight observations plotted in Figure 2.  C10 5677 g mol -1 * 100% 4908 g mol -1 * 14% 4478 g mol -1 * 21% * Calculated via GPC using a -Da polystyrene calibration curve and toluene as internal standard.

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In Table 2 we reported the % of M n reduction for any diethyl ester chain in relation to the 254 alkyl group in relation to 1,8-octanediol. As we can see from the collected data, the methyl 255 ester (Me) (taken as 100%) is always the best performing one, followed by the diethyl (Et) 256 and the dibutyl (Bu) ones. Also to notice that the differences in the M n s decrease for the 257 Bu end group are lower with the increase of the carbon chain length of the diester for the 258 considered reaction.

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Further to this, the difference in polymer melting temperature between the different diol 260 chain lengths becomes less pronounced when increasing the diester chain length ( Figure   261 3).  Table S1 for details).

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The inert atmosphere (N 2 ) thermogravimetric profile of the polymers presented in Figure 4 267 is also consistent with the previously discussed molecular mass data. In fact, with the 268 increase of the polymer's M n and the increase of the diol's carbon chain length (see Figure   269 4a insert) the degradation temperature increases accordingly (Figure 4a).  Table S2 in ESI.

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Rheology was used to further characterise the polymers and identify differences in their 288 viscosity. Figure 5 shows the viscosity change of the polymer melts based on a decrease 289 of temperature for poly(1,6-hexylene adipate) synthesized from dimethyl adipate (blue), 290 diethyl adipate (red) and dibutyl adipate (green). In all cases, viscosity steadily rose with 291 decreasing temperature until the onset of freezing was reached, at which point viscosity

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Conclusions 310 From the collected data we can conclude that CaLB is an effective catalyst for the 311 synthesis of a range of aliphatic polyesters using a solventless reaction system. Polyesters 312 based on diesters with an internal carbon chain length between 4 (succinate) and 10 313 (sebacate) and diols with a carbon chain length between 4 (1,4-butanediol) and 8 (1, octanediol) were successfully synthesized via transesterification. A strong effect of the 315 selected alkyl group of the diester (dimethyl, diethyl and dibutyl) was observed for all 316 polyesters. Lower molecular weights and monomer conversions were obtained using 317 dibutyl esters since it proved more difficult to remove the butanol by-product during the reaction due to its higher boiling point (relative to methanol and ethanol). DSC, TGA and