RNA-inspired phosphate diester dynamic covalent networks

Neighboring group assisted rearrangement substantially increases relaxation rates in dynamic covalent networks, allowing easier (re)processing of these materials. In this work, we introduce a dynamic covalent network with anionic phosphate diesters as the sole dynamic group, incorporating β-hydroxy groups as a neighboring group, mimicking the self-cleaving backbone structure of RNA. The diester-based networks have slightly slower dynamics, but significantly better hydrolytic (and thermal) stability than analogous phosphate triester-based networks. Catalysis by the β-hydroxy group is vital for fast network rearrangement to occur, while the nature of the counterion has a negligible effect on the relaxation rate. Variable temperature 31P solid-state NMR demonstrated a dissociative bond rearrangement mechanism to be operative.

A phosphate triester-containing polycaprolactone network (PCL-PX) was synthesized from CAPA® 3201 and ethylene glycol chlorophosphate as described by our group before. 1

Synthesis of diethyl phosphate (DEP)
DEP (1) was made from [EMIM][DEP] as described before in the literature. 2 Scheme S2: Synthesis of diethyl phosphate 80-90 g thoroughly washed Amberlite® IRC120 hydrogen form and 150 mL water was added to a 250 mL Erlenmeyer flask together with 10 g 1-ethyl-3-methylimidazolium diethyl phosphate ([EMIM] [DEP]).This was left in the fridge for 24 hours.The mixture was filtered through filter paper and washed with fresh water into a 250 mL round bottom flask.Water was removed from the filtrate by means of reduced pressure, followed by threefold co-evaporation with toluene.This yielded protonated diethyl phosphate (DEP) in quantitative conversion (5.78 g, 99%) in the form of a yellow liquid.In the 31 P NMR spectrum (Figure S1), a shift is visible towards lower chemical shift, indicating the protonation.pg. 4

Scheme S3: Synthesis of BDDE-dcP and BDDE-dP-TEA
2 g DEP (13.0 mmol) was dissolved in 400 mL dry toluene that was dried over 4 Å molecular sieves, together with an equimolar amount (6.5 mmol, 1.31 g) 1,4-butanediol diglycidyl ether (BDDE).A 20 mL Dean-Stark apparatus was attached, of which the burette was filled with 4 Å molecular sieves.The mixture was refluxed overnight.The reaction mixture was cooled down and filtered into a 500 mL separation funnel to filter off orange solid side-products.Only one peak was visible in the 31 P NMR spectrum (Figure S3 top), which was attributed to the ring-closed BDDE-dcP.20 mL Triethylamine (TEA) was added and the mixture was mixed well.25 mL water was added, the mixture was shaken multiple times, and left to equilibrate.The aqueous layer was added to a 100 mL round bottom flask and the organic layer was washed with water two more times before being discarded.The combined aqueous layers were freed from water by means of reduced pressure to yield BDDE-dP-TEA as a colorless to slightly yellow ionic liquid in 72% conversion (3.24 g).From 31 P NMR, (Figure S3 bottom), it was visible that the two positional isomers of the first hydrolysis product are formed. 1 Replacing TEA in this synthetic approach for pyridine (Pyr) or methyl imidazole (MIM) yields BDDE-dP-Pyr or BDDE-dP-MIM, respectively.For MIM, the aqueous layer after hydrolysis was washed with toluene for a total of 8 times to remove all unreacted MIM.pg.6

Network synthesis
For network synthesis, an equimolar amount (with respect to end groups) of BDDE-dP-TEA and a three-functional, OH-terminated polymer (PEG-3f, PCL-3f) were combined in a 100 mL round bottom flask.The mixture was heated to 60 °C and stirred to combine the fractions.Next, the mixture was freed from any residual water by means of lyophilization in case of PEG, and by means of threefold coevaporation with toluene in case of PCL.Afterwards, the mixture was stirred well, and under high vacuum (< 1 mbar) the temperature was increased to 150 °C until network formation was complete.Vacuum was necessary to remove ethanol.Afterwards, the material was collected in a glass petri dish and left in a vacuum oven at 180 °C to ensure further curing

Reprocessing via compression molding
Synthesized networks were pressed into 1 -1.2 mm thick squares, from which 8 mm disk were punched for rheology experiments, and rectangles were cut for DMTA experiments.For all other characterization methods, pressed scraps were used.Compression molding was performed in a Fontijne LabEcon 300 hot press using a set method.Material was placed in a mold and sandwiched between 0.05 mm thick Teflon sheets and 4 mm thick stainless steel sheets.This was placed under 100 kN at 150 °C for 30 minutes.The temperature was reduced to 100 °C over the course of 30 minutes (under pressure) and then the samples were actively cooled to room temperature.
Small molecule experiments for catalysis study EGE (5) was phosphorylised into EGE-dP-TEA (7) in a comparable manner to BDDE. 2 g DEP (13.0 mmol) was dissolved in 300 mL toluene that was dried over 4 Å molecular sieves, together with an equimolar amount (13.0 mmol, 1.4 mL) EGE.A 20 mL Dean-Stark apparatus was attached, of which the burette was filled with 4 Å molecular sieves.The mixture was refluxed overnight.The reaction mixture was cooled down and filtered into a 500 mL separation funnel to filter off solid side-products.pg.7 One major peak was visible in the 31 P NMR spectrum (Figure S4 top), which was attributed to the ringclosed EGE-dcP (6), as well as one minor peak, which was attributed to an unknown side product.20 mL triethylamine (TEA) was added and the mixture was mixed well.25 mL water was added, the mixture was mixed multiple times, and left to equilibrate.The aqueous layer was added to a 100 mL round bottom flask and the organic layer was washed with water two more times before being discarded.The combined aqueous layers were dried by means of reduced pressure to obtain EGE-dP-TEA as a colorless ionic liquid in 47% conversion (2.00 g).From 31 P NMR (Figure S4 bottom), it was visible that the two positional isomers of the first hydrolysis product are formed.

Gel fraction
Gel fractions of the networks were determined with tetrahydrofuran (THF) as the extraction solvent.A piece of (dry) sample was weighed (  ) and then swollen in the extraction solvent for 24 hours.The swollen network was then washed with fresh solvent and allowed to dry in a vacuum oven at 100 °C overnight.The dried sample was weighed (  ) and the gel fraction was calculated following equation S1.

Swelling ratio
Swelling ratio was determined using THF as solvent.A piece of (dry) sample was weighed (  ) and allowed to swell in the solvent for 24 hours.The swollen sample was pat dry with a piece of paper and weighed (  ).Swelling ratio () was calculated flowing equation S2.
Nuclear magnetic resonance (NMR) spectroscopy 1 H, 13 C and 31 P NMR spectroscopy measurements were performed on a 400 MHz Bruker Avance III spectrometer at 25 °C.
pg. 10 (Variable temperature) solid-state NMR (SSNMR) spectroscopy VT 31 P SSNMR spectra were obtained using an 11.7 Tesla Bruker Avance Neo 500 MHz spectrometer operating at 31 P resonance frequency of 202 MHz.The measurements were performed under static conditions, without magic angle spinning, using a one pulse sequence with a 90° pulse of 5 μs and an inter-scan delay of 10 s.A 0.0485 M triphenylphosphate (TPhP) solution in CDCl3 was used as a chemical shift reference at 17.70 ppm.The variable temperature experiment was performed from 60 °C to 120 °C at intervals of 20 °C up and down.The system was allowed to equilibrate until equilibrium was reached at 60 °C, as well as for 15 min at each temperature.

Determination of ΔH Ѳ
Scheme S5: Equilibrium between the ring-opened and the ring-closed state of a phosphate diester DCN.
Given the equilibrium as shown in Scheme S5, the equilibrium constant  for the equilibrium between the ring-opened state and the ring-closed state in phosphate diester DCNs can be determined as represented in Equation S3.

𝐾𝐾 = [𝐶𝐶𝐶𝐶][−𝑂𝑂𝑂𝑂] [𝑂𝑂𝐶𝐶] Equation S3
Since 1:1 stoichiometry was used in the network synthesis, the networks will contain dissociated phosphate diesters and polymer -OH functionalities in a 1:1 ratio.All processes involved are transesterification reactions, hence the OH content in the network remains constant.The concentration ratio between the dissociated and associated phosphate esters can be calculated from the SSNMR spectra, as the ratio between areas of the corresponding peaks.Using Gibbs' fundamental equation and the Van 't Hoff's law, an expression can be derived for the [CP]/[OP] dependence of  Ѳ (Equation S4).
Thermogravimetric analysis (TGA) Thermal stability of the networks were probed using a TGA 550 (TA instruments) under N2 flow.Temperature ramps were performed by ramping from 20 °C to 100 °C at 20 °C per minute, allowing an isothermal at 100 °C for 30 minutes, followed by a ramp to 600 °C at 10 °C per minute.Temperature calibration was performed using the Curie points of high purity ferromagnetic standards.

Differential scanning calorimetry (DSC)
DSC was performed in a Q2000 DSC (TA instruments).Measurements were performed using standard aluminum pans.The temperature range used was -80 °C until 200 °C.The material was cooled to the minimum temperature at 5 °C per minute.Heating runs were performed at 10 °C per minute.

Dynamic mechanical thermal analysis (DMTA)
DMTA was performed on rectangular shaped compression molded samples using the film tension setup in a Discovery DMA 850 (TA instruments).Samples were heated to 180 °C and left to equilibrate for 2 hours.The sample was cooled at 1° C/min under oscillation to -50 °C and left to equilibrate for 1 hour.Then, the temperature ramp was performed at 3 °C/min from -50 °C to 200 °C.A preload force of 0.01 N and a force track of 125% was used.The storage-and loss modulus were recorded as a function of temperature.

Shear rheology
All shear rheology studies were performed using a Discovery HR 20 (TA instruments) with environmental temperature control (ETC) setup and 8 mm parallel-plate geometry.All experiments were performed with an axial force range of 1 ± 0.1 N. In general, all samples were equilibrated at 180 °C for 2.5 hours before measurement (unless stated otherwise).

Oscillatory time sweep experiments
Oscillatory time sweep experiments were performed before each stress relaxation experiments at the same temperature to ensure proper curing at the experiment temperature.The experiments were performed under 1% oscillatory strain and at an angular frequency of 10 rad/s.

Stress relaxation experiments
Stress relaxation experiments were performed between 110 °C and 170 °C.The relaxation modulus () was followed as a function of step time under a constant strain of 5%.All plots in main text have been normalized to the relaxation modulus at 1 s ((1)), in order to compare the relaxation rates solely based on the kinetics of bond exchange.
Fits of the normalized stress relaxation plots were made following a stretched exponential function normalized to 1 s, as seen in Equation S5, yielding the characteristic relaxation times () and the stretch parameter ().The obtained characteristic relaxation times were plotted against the reciprocal temperatures via an Arrhenius plot.Slopes from these plots can be used to determine the apparent activation energy of viscous flow for the system (Equation S6).

Repeated stress relaxation experiments for thermal stability
Stress relaxation experiments were repeated on single samples at equal temperatures four times in order to assess the thermal stability of the material.The same method was used as described above.

Frequency sweep experiments
Frequency sweep experiments from 10 -2 rad/s to 10 2 rad/s were performed on the networks between 110 °C and 170 °C.The oscillation amplitude was set to 1% strain.The storage modulus, loss modulus and tan delta were followed as a function of frequency.

Relaxation modes via Tikhonov regularization
Stress relaxation spectra of PEG-dP-TEA and PCL-dP-TEA were checked for plurality in relaxation modes via Tikhonov regularization.The used method has been described before, 4 and is based on solving the stress relaxation function for the distribution function () as described in: Here, () is defined as gradual stress relaxation to the equilibrium stress   .Assuming stress is fully relaxed in the measurements (  = 0), the integral in Equation S7 can be written in the generic form of a Fredholm equation of the first kind: Where () is the measured signal, (, ) the kernel function exp �−   � where  depicts  between 0 and +∞; and () the unknown solution to the integral which needs be solved for.This is an example of an ill-posed problem, which thus requires regularization.Here, it is chosen to utilize Hansen's algorithms in MATLAB to solve the problem. 5These employ the L-curve criterion to determine the optimum regularization parameter , and Tikhonov regularization for the computation of the relaxation spectra.

Degradation experiments
Samples of PCL-dP-TEA and PCL-PX (Majumdar et al. 1 ) were weighed and left in open vials in a desiccator containing saturated NaCl solution, maintaining a humidity of 75% at room temperature.For 5 days, three samples per network were removed per day, and the gel content was determined as described before.
H, 13 C, and 31 P NMR of BDDE-dP-TEA are shown below in section BDDE-dP-TEA NMR spectra.

Figure S5: 1 H
Figure S5: 1 H NMR of (top) 2-ethyl hexanol, (middle) DEP, (bottom) reaction mixture of toluene, DEP, and 2-ethyl hexanol.From integration of the marked areas was determined that no exchange reaction occurred.

Figure S11 :
Figure S11: Stress relaxation experiments on PEG-dP-TEA.(a) Normalized stress relaxation data on different temperatures.(b) Arrhenius plot using characteristic relaxation times derived from (a).

Figure S12 :
Figure S12: Stress relaxation experiments on PEG-dP-Pyr.(a) Normalized stress relaxation data on different temperatures.(b) Arrhenius plot using characteristic relaxation times derived from (a).

Figure S13 :
Figure S13: Stress relaxation experiments on PEG-dP-MIM.(a) Normalized stress relaxation data on different temperatures.(b) Arrhenius plot using characteristic relaxation times derived from (a).

Figure S14 :
Figure S14: Stress relaxation experiments on PCL-dP-TEA.(a) Normalized stress relaxation data on different temperatures.(b) Arrhenius plot using characteristic relaxation times derived from (a).

Figure S15 :
Figure S15: Relaxation spectrum of stress relaxation on PEG-dP-TEA at different temperatures.

Figure S16 :
Figure S16: Relaxation spectrum of stress relaxation on PCL-dP-TEA at different temperatures.

Figure S18 :
Figure S18: Four cycles of repeated stress relaxation experiments on PCL-dP-TEA at 140 °C.

Figure S19 :
Figure S19: Four cycles of repeated stress relaxation experiments on PCL-dP-TEA at 160 °C.

Figure S25 :
Figure S25: DSC thermogram of the second heating and cooling run for PEG-dP-Pyr (exo up).

Figure S26 :
Figure S26: DSC thermogram of the second heating and cooling run for PEG-dP-MIM (exo up).

Figure S27 :
Figure S27: DSC thermogram of the second heating and cooling run for PCL-dP-TEA (exo up).

Figure S29 :
Figure S29: Van 't Hoff plot as derived from data of the heating run of VT 31 P SSNMR measurements on PEG-dP-TEA.

Table S1 :
Characteristic relaxation times () and stretch parameters () as derived from plotting the normalized stress relaxation data above with the stretched exponential function.

Table S2 :
Ratios between [OP] and [CP] during heating and cooling cycles of VT 31 P SSNMR experiments on PEG-dP-TEA.All normalized to [CP]