N-Hydroxyethyl acrylamide as a functional eROP initiator for the preparation of nanoparticles under “greener” reaction conditions

N-Hydroxyethyl acrylamide was used as a functional initiator for the enzymatic ring-opening polymerisation of ε-caprolactone and δ-valerolactone. N-Hydroxyethyl acrylamide was found not to undergo self-reaction in the presence of Lipase B from Candida antarctica under the reaction conditions employed. By contrast, this is a major problem for 2-hydroxyethyl methacrylate and 2-hydroxyethyl acrylate which both show significant transesterification issues leading to unwanted branching and cross-linking. Surprisingly, N-hydroxyethyl acrylamide did not react fully during enzymatic ring-opening polymerisation. Computational docking studies helped us understand that the initiated polymer chains have a higher affinity for the enzyme active site than the initiator alone, leading to polymer propagation proceeding at a faster rate than polymer initiation leading to incomplete initiator consumption. Hydroxyl end group fidelity was confirmed by organocatalytic chain extension with lactide. N-Hydroxyethyl acrylamide initiated polycaprolactones were free-radical copolymerised with PEGMA to produce a small set of amphiphilic copolymers. The amphiphilic polymers were shown to self-assemble into nanoparticles, and to display low cytotoxicity in 2D in vitro experiments. To increase the green credentials of the synthetic strategies, all reactions were carried out in 2-methyl tetrahydrofuran, a solvent derived from renewable resources and an alternative for the more traditionally used fossil-based solvents tetrahydrofuran, dichloromethane, and toluene.


Plotting First-Order Kinetics
Ratio of HEAA:Lactone was taken to be constant throughout the reaction, as such HEAA can be used as an internal 1 H-NMR standard. The ratio of monomer to initiator/ internal standard, M, was calculated following Equation S6. In the case of HEAA: caprolactone and HEAA: valerolactone this equation takes the forms of Equation S7 and Equation S8 respectively. Using the value of M obtained the natural logarithm of M at time zero (M 0 ) over M can be taken and subsequently plotted against time. To account for HEAA being consumed both the unreacted and reacted HEAA was incorporated into the internal standard integral.

Equation S8 Ratio of monomer to initiator / internal standard in the case of valerolactone (monomer) and HEAA (initiator / internal standard).
Data obtained for HEAA initiated eROP of CL and VL following Equation S6 can be seen in SI Table 5 and SI Table 6.  Table 7 shows the kinetic constant (k) associated with monomer conversion for different initiator:monomer ratios based on the first-order kinetic model. The analytical solution for a firstorder model (Equation S10) expressed in terms of monomer conversion (Equation S9) successfully describes the conversion profiles of caprolactone throughout enzymatic reaction (SI Figure 12).

SI
Where M is the monomer concentration, M 0 is the initial monomer concentration, t corresponds to reaction time and k stands for the kinetic constant associated to monomer consumption throughout enzymatic reaction. Where x corresponds to the dimensionless conversion, k is the first-order kinetic constant, and t is the reaction time.
Considering the similar behaviour of monomer conversion (SI Figure 12), it is crucial to evaluate if the estimated constants can be considered different from a statistical point of view. According to SI Figure  13A, we can assume that the kinetic constants are statistically different with a level of confidence of 95.0%, except for a particular comparison of k estimated for 1:5 HEAA:CL and 1:10 HEAA:CL conditions, where it is not reasonable to state unequivocally that the estimated parameters are statistically different. As an additional and important piece of information, SI Figure 13A shows that the estimated parameter k can be expressed as a linear function (k=1.05•10 -4 (HEAA:CL)+0.015611, R 2 =0.991) of the caprolactone units (HEAA:CL) added to the polymerisation system at the beginning of the enzymatic reaction. On the other hand, when both Equation S9 and statistical inference are applied to VL, the experimental data are not well explained by the first-order model (SI Figure 14). Equation S9 also overestimates the monomer conversion after 120 min reaction time independent of the HEAA:VL ratio evaluated. A comparative analysis of the estimated values for the kinetic constants (SI Figure 15A) strongly indicates that, from a statistical point of view only, the value of k associated with the 1:5 HEAA:VL condition may be considered different from the one related to 1:20 HEAA:VL. Additionally, (SI Figure 15A), a linear relation between k and the initiator: monomer ratio cannot be established, as a result of the kinetic behaviour of the enzymatic reaction carried out with 1:40 HEAA:VL, exhibiting in the first hour of reaction a conversion profile very similar to the reaction performed at a 1:5 HEAA:VL ratio. Alternatively, a second-order kinetic model can be used to describe the conversion profile of VL, as illustrated in SI Figure 16. According to the model predictions (Equations S11 and S12), the second-order model fits better to the VL conversion data, but it loses reliability in predicting conversion within the first 60 minutes of reaction. Reactivity of lactones in the presence of N435 is known to increase with ring size. 1 Kinetic constants for VL were found to be significantly lower in comparison than those of CL (SI Table 7), demonstrating lower reactivity in the studied reactions. Discussed in the main text as a result of differing ring-strain. Regardless, both VL and CL were able to be ring-opened enzymatically using HEAA and N435 in the observed timeframe.

Recyclability of N435 in 2-MeTHF
Enzyme recyclability is key from cost saving and green chemistry perspectives. The stability of N435 was investigated by repeatedly subjecting the same enzyme beads to reaction conditions, only replacing the liquid phase of the reaction with fresh monomer, initiator, and solvent between cycles. Three experiments were run in parallel, and the average (mean) conversion is plotted in SI Figure 19. Only a minimal decrease in the conversion of CL is detectable by 1 H-NMR, dropping from 94.8% for virgin N435 to 93.2% during the tenth recycle. This decrease of 1.6 % is minimal, and this decrease is still within the acceptable error for 1 H-NMR. From literature N435 has been recycled up to 15 times with minimal decrease in reactivity, varying with temperature and solvent used during the reaction. 2,3 It is expected for N435 to lose activity with each recycle due to factors such as: enzyme leeching into the reaction media, removal of essential water, support degradation, amongst other factors. 3 The data presented here demonstrates that the N435 is a robust enzymatic catalyst capable of being reused up to ten times in the reaction conditions employed without reduction in catalytic activity. CL and HEAA conversion are consistent throughout the enzyme recycles and the maintained activity also demonstrates the suitability of 2-MeTHF as a solvent for N435 catalysed reactions. The excellent recyclability of the immobilized catalyst demonstrated here significantly contributes to the economic and environmental credentials of the reactions.
SI Table 9 Tabulated results of the N435 recyclability study seen in SI Figure 19. Some error in the HEAA conversion can be attributed to the NH signal shifting due to hydrogen bonding effects. As a result, some integrations could potentially lose accuracy, see SI Figure

Alternative initiator Species
N-Hydroxyethyl methacrylamide (HEMAM) was synthesised, following a known procedure, 5 to observe the effect of increasing initiator hydrophobicity. It was hypothesised that an increase in hydrophobicity would lead to an improvement in initiator conversion by increasing the affinity for the active site of CALB. It was found however that HEMAM and HEAA had similar reactivity in eROP of CL. CL conversion reaches 99.7% conversion, HEMAM conversion reaches a conversion of 66.9% (SI Figure  21). The final initiator conversion reached does not vary significantly between HEMAM and HEAA. These experimental findings were also backed up by computational docking studies, which found that HEAA and HEMAM have very similar affinities for the active site of CALB (SI Table 9), explaining the very similar behaviour observed.

DLS Plots
SI Figure 28 DLS Plots of polymeric nanoparticles in water (1 mgmL -1 ). "Entry" codes refer to Table 3 of the main text.