Evaluation of 5,6-cellulosene as a synthon for thiol–ene chemistry: NMR and computational study

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Koso, Tom Wirtanen, Katja T. Rinne-Garmston, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9335417/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract We report a detailed characterisation of the preparation of 5,6-cellulosene, a poorly investigated reactive cellulose derivative. A key literature procedure is followed, which utilises partially hydrolysed technical cellulose acetate, as a 6-OH rich starting material. Thorough NMR analysis, using well-established solution-state NMR protocols, is performed to identify mechanisms of reactivity. The major drawback and potential opportunity identified in this study is the propensity for 5,6-cellulosene linkages (containing an alkenyl-acetal moiety) to undergo hydrolysis, leading to depolymerisation. However, now we demonstrate, using modern theoretical and NMR methods, that hydrolysis of cellulosene is still controllable under room temperature photoinitiated thiol–ene reaction conditions, and aqueous workup. The suitability of 5,6-cellulosene as a synthon towards thiol–ene chemistry is finally assessed (under thermal and photoinitiated radical addition) using a model amine-appended thiol. Clear applications of interest are in biomedical science, due to the abundance of thiol-containing proteins utilised in selective protein labelling. cellulose acetate computational chemistry density functional theory (DFT) NMR spectroscopy regioselectivity thiol–ene click chemistry Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Cellulose is traditionally used in papermaking and textile manufacturing, as well as construction and packaging materials; however, there is additional potential in high-value applications, such as composite materials and pharmaceuticals (Arca et al. 2018; Thompson and Edgar 2025 ), and in far-term technologies, including optoelectronics and bioengineering (Li et al. 2021; Teodoro et al. 2021). The preparation of advanced functional materials is possible by targeted chemical modifications of cellulose. On a molecular level, cellulose is a linear biopolymer consisting of glucopyranose units linked by β-1,4-glycosidic bonds. It contains hydrolytically stable acetal linkages, with the low reactivity imparted by the conformational stability, especially in the crystalline state. Cellulose is structurally rich in hydroxyl groups in positions 2, 3 and 6 of the glucose residues, which provide a platform for introducing different chemical moieties, typically via nucleophilic substitution (S N 2) mechanisms. Thus, chemical modification of cellulose is a common strategy to prepare functional materials with desired properties, while maintaining cellulose’s sustainability advantage over petroleum-based products (Li et al. 2025 ; Li et al. 2021). The degree of substitution (DS), position of substitution, as well as the type of substituents are tightly linked to physical properties of cellulose-based materials, such as solubility in organic solvents and water. The ability to maintain control over the regioselectivity (position of substitution) is therefore advantageous, as it enables researchers to study the structure–property relationships of these cellulose-based polymers, and to fine-tune the material performance to match the anticipated requirements in specific applications (Edgar et al. 2001 ; Fox et al. 2011 ; Arca et al. 2018; Thompson and Edgar 2025 ). However, currently available low-cost heterogeneous strategies for modifications, such as acylation (including acetylation), carboxymethylation, methylation, epoxidation and etherification, are insufficient because they are essentially non-regioselective. Clearly, for high-value applications, there is a need to develop highly regioselective approaches for cellulose functionalization. Cellulose modification in solution state often yields some selectivity towards C6-substituted derivates, as the C6-OH is usually the most reactive position. However, these are usually not completely regioselective, since the secondary C2-OH and C3-OH groups tend to react before full displacement of C6-OH (Takahashi et al. 1986 ; Liu and Baumann 2002). Consequently, complete and exclusive substitution at the primary position 6 of unmodified cellulose is not synthetically straightforward and normally requires protection/deprotection strategies (e.g., 6-tritylation (Hearon, Hiatt, and Fordyce 1943 ; Heinze, Röttig, and Nehls 1994; Bui, Rosenau, and Hettegger 2023; Thompson and Edgar 2025 )). To our knowledge, only the Appel reaction, or variations thereof, allow for almost completely regioselective 6-substitution in a single-step synthesis (Furuhata, Chang, et al. 1992 ; Furuhata, Koganei, et al. 1992 ; Matsui et al. 2005 ). For example, the combination of triphenylphosphine and N -bromosuccinimide yields 6-bromo-6-deoxycellulose with a DS 6 value of 0.99, without any direct observation of 2- or 3-bromination (Dryś et al. 2024). However, this procedure requires hazardous and expensive direct-dissolution cellulose solvent systems, and the inclusion of heteroatoms in all reagents further increases costs. This also reduces overall sustainability, particularly through poor atom-efficiency. Recently, Prof. Kevin J. Edgar’s group presented an alternative strategy for highly chemo- and regioselective chlorination by using commercially available cellulose acetate (CA) with high hydroxyl content at position 6 (Liu and Edgar 2017; Gao, Liu, and Edgar 2018). This specific lower DS technical CA was chosen due to its availability of primary 6-OH groups, after a (6-OAc selective) hydrolytic post-ripening stage in the preparation of the CA (Steinmeier 2004 ). Position-specific DS 6−OAc was estimated to be 0.5 (maximum DS 6 is 1), which means that the remaining 50% of the unreacted primary 6-OH groups are available for modification, ideally under mild conditions that do not affect the secondary positions 2 and 3. The Edgar group have demonstrated that the 6-OH groups of CA can be selectively and completely chlorinated with methanesulfonyl chloride (mesyl chloride, MsCl) in DMF. The obtained chlorinated CA was subsequently reacted with a model azide, as well as with amines and thiols via iodinated CA generated in situ , achieving modest to good conversions for these S N 2 transformations (Gao, Liu, and Edgar 2018). In our previous work involving regioselective modification of microcrystalline cellulose, we investigated the reactivity of 6-halo-6-deoxycelluloses towards S N 2 transformations (Dryś et al. 2024). In addition to the main S N 2 reaction pathway, we observed the formation of a side product, resulting from the competing elimination (E2) mechanism (Vigo and Sachinvala 1999). Based on previously published literature (Ishii 1986 ), we determined that the obtained side product was 5,6-cellulosene. After a detailed investigation of literature, we found that the formation of 5,6-cellulosene (or ‘5,6-cellulose-ene’) actually goes back to (Kaverzneva, Ivanov, and Salova 1949 ). The interest in 5,6-cellulosene is related to the introduction of a carbon–carbon double bond (as an alkenyl-acetal). Previously, 5,6-cellulosene has shown reactivity towards addition reactions using CHCl 3 , CCl 4 , PCl 3 , HSiCl 3 , CH 3 COOH and CH 3 OH (Dimitrov et al. 1968 ; Dimitrov, Gal'braikh, and Rogovin 1965 ). From our perspective, the exocyclic double bond should promptly react in further addition reactions under radical initiation, potentially under aqueous conditions. This could expand the scope of high-value reagents in the formation of adducts. Considering the synthetic potential of 5,6-cellulosene, we now further probe the ‘CA method’ (Gao, Liu, and Edgar 2018), to assess the key challenges of this approach towards radical-mediated thiol–ene coupling, as a logical strategy for bioconjugation (Hoyle and Bowman 2010; Nolan and Scanlan 2020 ; Ahangarpour et al. 2021 ). During this process, we illustrate the potential of combining state-of-the-art computational methods with detailed NMR analysis for structural elucidation of the obtained products. Our aim is to show that it is possible to go beyond rudimentary analysis of cellulose derivatives, thanks to current complementary experimental and theoretical approaches. Materials and methods Materials Cellulose acetate CA-320S NF/EP (acetyl content 31.9%, DS Ac 1.8, DS OH 1.2) was provided free of charge by Eastman Chemical B.V.; methanesulfonyl chloride (mesyl chloride, MsCl, 99.5%) anhydrous N , N -dimethyformamide (DMF, 99.8%, extra dry over molecular sieve), anhydrous N , N -dimethylacetamide (DMA, 99.8%), anhydrous sodium thiosulfate (Na 2 S 2 O 3 , 98.5%) were purchased from Thermo Scientific; 2,5-hexanedione (99.57%) was obtained from BLD Pharm; 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, ≥ 99.0%), γ -valerolactone (GVL, ≥ 99%), β-methylglucopyranose (≥ 99%) were purchased from Sigma-Aldrich; 2-aminoethanethiol (2-AET, > 95.0%), 2,2-dimethoxy-2-phenylacetophenone (DMPA, > 98.0%), were purchased from TCI; sodium iodide (NaI, ≥ 99%), pyridine (≥ 99.7%), acetic anhydride (99.7%) were purchased from VWR (Finland); DMSO-d 6 (99.8% D), D 2 O (99.90% D) and CDCl 3 (99.8% D + 0.03% TMS v/v) were bought from Eurisotop. All reaction flasks were dried at 120°C for at least 16 h before syntheses. NMR measurements Unless otherwise indicated, NMR spectra were recorded in DMSO-d 6 at 65°C on a Bruker Avance NEO 600 spectrometer (600 MHz 1 H-frequency). Chemical shifts are referenced to DMSO-d 6 ( 1 H: δ = 2.50 ppm, 13 C: δ = 39.52 ppm), unless otherwise indicated. TopSpin 4.1.4 and MestReNova 14.3.1 were used for the processing of NMR spectra. Solubility tests Solubility of the raw cellulose acetate and synthesized materials was tested at 1% w/v concentration, in several organic solvents and water, at ambient temperature and, for samples insoluble at room temperature, at 50°C, under magnetic stirring. Computational details The computation of reaction Gibbs free energies via a supramolecular approach follows best DFT practices (Bursch et al. 2022 ). First, due to the expected flexibility of reactants and products, molecular conformations were sampled. Here, the GFN2-xTB/ALPB(H 2 O) level of theory (Bannwarth et al. 2021 ) was employed with a development version (> 3.0.2) of the CREST program (Pracht et al. 2024 ), where redundant conformations were sorted out under consideration of atom permutation (Pracht 2025 ). Conformational energies for sugar-like systems are prone to larger errors at this semiempirical level of theory and require postprocessing (Bannwarth et al. 2021 ; Pracht et al. 2024 ). Therefore, energies for all conformational ensembles were recomputed as single-points on the GFN2-xTB/ALPB geometries using the novel g-xTB method (Froitzheim et al. 2025 ). Since no implicit solvation model is yet parametrized for this Hamiltonian, a simple additive correction term, calculated as the difference \(\:{\partial\:G}_{solv}^{ALPB}=\:{E}_{ALPB\left(water\right)}^{GFN2-xTB}-\:{E}_{gas}^{GFN2-xTB}\) , was added to obtain \(\:{E}_{solv}^{g-xTB}\approx\:\:{E}_{gas}^{g-xTB}+\:{\partial\:G}_{solv}^{ALPB}\) . Boltzmann weights were calculated for all structures, and conformations with a population of > 0.5% were selected for subsequent DFT treatment. All DFT calculations were performed with version 6.0.2 of the ORCA program package (Neese et al. 2020 ; Neese 2025 ). Throughout, the ‘TightSCF’ setting was used and default thresholds for geometry, and frequency calculations, were employed. The selected geometries were optimized at the r 2 SCAN-3c level of theory (Grimme et al. 2021 ), employing the SMD implicit solvation model for water (Marenich, Cramer, and Truhlar 2009 ). The same level of theory was employed for calculating harmonic frequencies, where free energy contributions at 298.15 K and 373.15 K were obtained in the modified and scaled rigid-rotor harmonic-oscillator scheme ( \(\:{\varDelta\:G}_{msRRHO}^{T}\) ) (Grimme 2012 ; Pracht and Grimme 2021), using an entropy interpolation parameter of 50 cm − 1 and a frequency scaling factor of 0.9821 (Tikhonov et al. 2024 ). Finally, higher level electronic energies were computed on the r 2 SCAN-3c geometries at the ωB97X-V/def-QZVPP level of theory (Mardirossian and Head-Gordon 2014 ; Weigend and Ahlrichs 2005), employing the RIJCOSX approximation (Eichkorn et al. 1995 ; Neese 2003 ; Neese et al. 2009 ) and SMD(water) implicit solvation with dynamic radii adjustment for continuum solvation (Plett et al. 2024 ). The final free energy for all relevant conformers was obtained by adding the \(\:{\varDelta\:G}_{msRRHO}^{T}\) from r 2 SCAN-3c to the ωB97X-V electronic energies. A single average free energy ⟨G (T) ⟩ for each reactant and product was obtained as the Boltzmann-weighted average over the respective ensembles. Since the hydrolysis reactions studied here involve a change in the degrees of freedom (DOF) through (re)cyclization, an influence of conformational entropy may further contribute to the reaction free energies. To gauge the magnitude of that effect, conformational free energy contributions \(\:{\partial\:G}_{conf}^{\left(T\right)}\) were estimated using GFN2-xTB/ALPB(water) at 278.15 K, using the procedure described by (Pracht and Grimme 2021). The Ramachandran plots (Ramachandran, Ramakrishnan, and Sasisekharan 1963 ) were prepared using the python3 script collection ‘dihedral-plotter’, available from GitHub ( https://github.com/alistair-wt-king/dihedral-plotter ). This requires only the ‘crest.energies’ and ‘crest_conformers.xyz’ outputs from the CREST run. The GFN2-xTB optimised ensembles were utilised to visualise the relative populations between the hydrolysis structures. The atoms used for extraction of the dihedrals from the hexose geometries were C1-C2-C3-C4 (φ) and C3-C4-C5-O5 (ψ), as other dihedral options did not allow for clean separation of the conformational space (separate chair and twisted-boat regions). The chemical shifts were calculated using ORCA 6 (Neese et al. 2020 ; Neese 2025 ), using the PBE0 functional (Adamo and Barone 1998 ), pcSseg-3 basis set (Jensen 2015 ), and SMD implicit solvation (DMSO) (Marenich, Cramer, and Truhlar 2009 ). The calculations were performed on the r 2 SCAN-3c geometries. Boltzmann-weighting was performed based on the ωB97X-V/def2-QZVPP energies for those geometries. Calibration of the DFT chemical shifts was achieved by first measurement of the experimental HSQC spectrum for the β-methylglucopyranose in DMSO-d 6 at 25°C. The correct calibration values for 1 H and 13 C (for all computed values) was then determined by minimising the average error between the absolute values of the experimental vs computational chemical shifts, for the β-methylglucopyranose. Syntheses Synthesis of acetylated 6-chlorinated cellulose (6-Cl-CA) Regioselective chlorination was carried out as described in (Gao, Liu, and Edgar 2018). In a typical reaction, a 250 mL three-neck round-bottom flask was charged with CA (3.00 g) and DMF (50 mL), under argon flow. The reaction flask containing the suspension was placed in an oil bath pre-heated to 75°C, and the solid dissolved under stirring in ca 30 min. Subsequently, MsCl (9.5 mL) was added dropwise. The reaction mixture was stirred at 75°C for 3 h under argon flow. The clear colourless solution became yellow but remained clear through the course of the synthesis. The heating source was removed to allow the reaction mixture to cool down for ca 15 min and poured into ethanol (800 mL). The crude product was recovered by filtration, dissolved in acetone (50 mL) and reprecipitated in ethanol (600 mL), filtered, washed with ethanol and dried in vacuo at 60°C for 3 h. 1 H NMR (600 MHz, DMSO) δ 1.8–2.2 (acetyl C H 3 ), 2.8–5.3 (cellulose backbone C H and C H 2 -Cl), 7.9–8.4 (formate C H ). Yield: 2.69 g (white powder). Synthesis of acetylated 6-iodinated cellulose (6-I-CA) Conversion of chlorodeoxycellulose into deoxyiodocellulose was based on (Ishii 1986 ). 6-Cl-CA (2.00 g) was dissolved in 2,5-hexanedione (50 mL), in a 250 mL three-neck round-bottom flask, under magnetic stirring, in an oil bath preheated to 80°C. Subsequently, NaI (6.6 g) was added to the clear solution, and the temperature of the oil bath was increased to 120°C. The reaction mixture was stirred at 120°C for 5 h, and after ca 15 min, the initially clear solution became turbid. Heating was switched off after 5 h and the mixture was allowed to cool down for ca 30 min. The reaction was quenched in ethanol (500 mL), filtered, suspended in aqueous sodium thiosulfate (0.1 M, 150 mL) and stirred for 1 h, filtered, washed with distilled water and dried in vacuo. 1 H NMR (600 MHz, DMSO) δ 1.8–2.2 (acetyl C H 3 ), 2.8–5.3 (cellulose backbone C H and C H 2 -I), 7.9–8.4 (formate C H ). Yield: 2.26 g (white powder). Synthesis of acetylated 5,6-cellulosene (5,6-ene-CA) Dehydroiodination was carried out according to (Ishii 1986 ). 6-I-CA (2.00 g) and anhydrous DMF (20 mL) were placed in a 100 mL round-bottom flask. The solid was dissolved under magnetic stirring at room temperature, yielding a clear light brown solution. Subsequently, DBU (2.0 mL) was added dropwise at room temperature, and the reaction mixture was heated in an oil bath preheated to 50°C. The reaction was stirred under heating for 2 h, and the initially clear light brown solution became dark brown. The heating source was subsequently removed and the reaction mixture was allowed to cool down for ca 30 min. The product was obtained by precipitation from cold ethanol (200 mL), followed by filtration. The crude product was resuspended in ethanol (200 mL), stirred overnight at room temperature, filtered, and dried in vacuo for 4 h, to give 1.3118 g of crude product as a brown powder. For purification, the crude product (300 mg) was suspended in distilled water, dialysed using dialysis tubing (CelluSep T1, Uptima, 3500 g/mol MWCO) for 3 days and freeze-dried. 1 H NMR (600 MHz, DMSO) δ 1.8–2.2 (acetyl C H 3 ), 2.8–5.3 (cellulose backbone C H and C H 2 ). Yield: 272.8 mg (light brown powder). Synthesis of acetylated (6-(2-aminoethane)thioether cellulose (6-AET-CA) In 20 mL screw cap septum vial, evacuated and flushed with argon thrice, 5,6-ene-CA (100 mg) was dissolved in anhydrous DMF (5 mL). Subsequently, 2-aminoethanethiol (260 mg) and DMPA (1.5 mg) were added under argon hood, and more DMF was added (5 mL) to completely dissolve the solid reagents. The obtained clear solution was stirred for 16 h, at ambient temperature, under UV-A irradiation (Kessil UVA LED PR160L 390 nm (max 52 W), at 25% power setting (13 W) 6 cm from light source). The product was recovered by precipitation from ethanol (100 mL), centrifugation, decanting the supernatant solution, washing with ethanol, and centrifugation/decantation. The obtained white solid was further washed with three portions of deionized water and freeze-dried for 1 day. The cloudy supernatant from water washes was pooled and freeze-died for 3 days. 1 H NMR (600 MHz, DMSO) δ 1.82 (amide acetyl C H 3 ), 1.88–2.08 (acetyl C H 3 ), 2.62–2.72 (C H 2 S and thiol chain C H 2 ), 2.85–5.10 (cellulose backbone C H and C H 2 and thiol chain C H 2 ). Yield: water-insoluble fraction: 59 mg (white powder), water-soluble fraction: 20 mg (white solid). Results and discussion Most strategies for obtaining unsaturated derivatives of mono- and polysaccharides are based on E2 elimination of certain functional groups under basic conditions (Nikologorskaya et al. 1970 ). In our case, the most efficient synthesis of 5,6-cellulosene (maximizing conversion and minimizing the number of discrete reaction steps) leads through 6-chloro and 6-iodocellulose derivatives (Fig. 1 a). The three-step sequence starts with 6-regioselective chlorination of cellulose acetate (CA), using mesyl chloride (MsCl) in DMF (Gao, Liu, and Edgar 2018). The isolated 6-chlorocellulose acetate (6-Cl-CA) is then treated with NaI in 2,5-hexanedione (Ishii 1986 ) to obtain the 6-iodo derivative (6-I-CA). Direct 6-iodination of CA is possible, with triphenylphosphine and N -iodosuccinimide as reagents (Usov, Krylova, and Suleimanova 1977 ); however, it is more common to perform a two-step sequence (Heinze and Liebert 2001 ; Vigo and Sachinvala 1999), thereby avoiding the need for these expensive reagents and ideally increasing atom efficiency. The last step involves stoichiometric treatment with a bulky non-nucleophilic base (DBU, ≈ 2 eq per sugar unit) to induce elimination of HI and formation of a double bond between C-5 and C-6, yielding partially acetylated 5,6-cellulosene (Ishii 1986 ). Direct conversion of chlorinated cellulose to 5,6-cellulosene is apparently possible, by treatment with KOH in MeOH or t -BuOK in DMSO (Srivastava, Harshe, and Gharia 1972 ); however, it is reported that alkali treatment of activated cellulose derivatives with good leaving groups in position C-6 may cause side reactions, forming 3,6-anhydroglucose moieties (Dimitrov et al. 1968 ; Makhsudov, Gal'braikh, and Rogovin 1967 ). Finally, to expand the applicability of 5,6-cellulosene as a synthon, it was reacted with a model thiol (2-aminoethanethiol) under radical-initiated conditions (Fig. 1 c), which is a common ‘click’ strategy for bioconjugation. Chlorination The chlorination of CA proceeded as described by (Gao, Liu, and Edgar 2018), achieving essentially full conversion of the C6 hydroxyl groups by the chloride moiety, as evidenced by diffusion-edited 1 H spectra (Fliri et al. 2023; King et al. 2018) and multiplicity-edited 1 H- 13 C heteronuclear single quantum coherence (ME-HSQC) correlation spectra (Fig. 1 b). In our experience, diffusion-edited 1 H and ME-HSQC experiments are quick and reliable analytical tools for initial (and in many cases sufficient) diagnosis of cellulose reactivity (Fliri et al. 2023). The diffusion-edited 1 H experiment removes fast-diffusing (low molecular weight) species from the spectra, allowing to tentatively confirm the presence of polymer-bound functionalities (Fliri et al. 2023; King et al. 2018). ME-HSQC spectra are phase-sensitive, with the multiplicity selections allowing for positive or negative signal intensity, after phasing; this helps to quickly resolve 6- CH 2 geminal (gem) correlations, using the standard multiplicity editing selection, since they are immediately apparent by the opposite phase to CH and CH 3 correlations. In our ME-HSQC dataset (Fig. 1 b), we identified 6- CH 2 -Cl gem cross-peaks at {3.90,43.6} and {3.70,43.6} ppm, and 6- CH 2 -OAc cross-peaks at {4.35,62.2} and {4.05,62.2} ppm. Unmodified 6- CH 2 -OH correlations are present in the NMR spectra of raw CA ( 1 ) at {3.71,59.2} and {3.55,59.2} ppm; these are no longer present in the NMR data for 6-Cl-CA ( 2 ), indicating that essentially all the 6-OH of CA (50% hydroxyl & 50% acetyl content at C6), are converted into 6-CH 2 -Cl. Consequently, the DS by chlorine at C6 is expected to be ≈ 0.5. More accurate calculation of DS 6 from 1 H NMR is not possible, since the protons attached to C6 resonate at frequencies that overlap with the cellulose backbone protons. Quantitative 13 C would be possible, but will contain significant error over short collection times, due to poor S/N. The full ME-HSQC spectrum ( Fig. S1 b ) shows additional correlations at {8.4–7.9;161.3} ppm, consistent with the presence of formate esters. The apparent formylation seems to arise from the transfer of formyl from DMF, according to the known reaction mechanism ( Scheme S1-S2 ) (Sato et al. 1990 ; Vigo, Daigle, and Welch 1972). In order to avoid the formylation side reaction, we investigated possibilities to replace DMF with an alternative ‘green’ dipolar aprotic solvent, capable of dissolving CA. Thus, informed by the results of solvent screening ( Table S1 ), we selected γ -valerolactone (GVL) due to its high dielectric constant (Kerkel et al. 2021 ). DMF could not be avoided completely, since the chlorination mechanism requires DMF to form the reactive (Vilsmeier) intermediate ( Scheme S1 ). However, by using 1.5 mol eq of DMF, we obtained almost completely 6-chlorinated derivative ( Fig. S2b ). In addition, we noted the incorporation of 4-hydroxyvaleric acid ester as a side chain of the cellulose backbone, albeit this was typically occurring only to a minor extent ( Fig. S2d ). For all experimental details, relevant NMR data, and extended discussion of the results, the reader is referred to the Supplementary Information . Iodination An essentially full displacement of the C-6 chloride by iodide was achieved using NaI in 2,5-hexanedione (Fig. 1 b), following optimized reaction conditions previously published by (Ishii 1986 ). The solvent requirement is due to the reaction being a modification of the classic Finkelstein reaction, where 2,5-hexanedione (used as a high boiling point alternative to acetone to yield faster kinetics or better solubility) dissolves NaI while favouring the precipitation of NaCl. Thus, the differential solubility of the two sodium salts is the driving force of the forward reaction, as with the classical Finkelstein reaction. However, there are drawbacks to this approach, namely, the use of iodine (requiring specialised disposal), the non-standard solvent and the additional synthetic step requiring isolation of 6-I-CA as an activated cellulose derivative. The Edgar group were apparently able to react 6-Cl-CA directly with different nucleophiles, via 6-I-CA generated in situ , in a one-pot reaction, with DMSO as the solvent (Gao, Liu, and Edgar 2018). We attempted to isolate the transient 6-I-CA under dipolar aprotic solvent conditions (DMSO, DMF and DMA). However, upon workup, we recovered mainly 6-chlorination product, with traces of 6-iodination, as evidenced by ME-HSQC data ( Fig. S4 ). The in-situ iodination does not seem to offer satisfactory (complete) displacement, which explains why Edgar’s group obtained products that still retained some 6-chloride moieties. Thus, it might be worth investigating if the additional step (isolation of 6-I-CA) would improve conversions in S N 2 reactions using less reactive nucleophiles, without including the additional elimination step to 5,6-cellulosene (Dryś et al. 2024). Dehydrohalogenation to 5,6-cellulosene acetate The unsaturated product, 5,6-cellulosene, can be synthesized from 6-iodinated cellulose by using a non-nucleophilic superbase, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), to eliminate HI over the 5,6 bond. The reaction conditions for converting 6-deoxy-6-iodocellulose to 5,6-cellulosene by DBU-mediated elimination mechanism were demonstrated by (Ishii 1986 ). In the present study, we utilize the acetylated analogues of 6-deoxy-6-halocellulose derivatives, thereby facilitating solubility in organic solvents (see Table S1 for information on the solubility of CA and CA derivatives in specific solvents). Complete conversion to 5,6-cellulosene, using a stoichiometric amount of DBU (≈ 1.5 mol eq/sugar unit), was apparent from ME-HSQC NMR data (Fig. 1 b, Fig. S6 ), as evidenced by the absence of 6- CH 2 -I gem correlations expected at ca {3.26,5.03} and {3.58,5.03} ppm. The formation of the 5,6-double bond was confirmed by a new set of gem cross-peaks observed at {4.57,94.0} and {4.64,94.0} ppm, attributable to terminal 6- CH 2 (Dryś et al. 2024). In addition, ME-HSQC data (Fig. 1 b, Fig. S6b ) suggest that some deacetylation had occurred (due to the presence of residual water), as evidenced by cross-peaks assignable to 6- CH 2 -OH ({3.55,59.2} and {3.71,59.2} ppm). Conversely, an apparent minimal drop in peak volumes attributable to 3- CH and 2- CH correlations of cellulose acetate ({4.59,71.0} and {5.11,71.7} ppm, respectively (Kono, Hashimoto, and Shimizu 2015 ), can be explained by the acetyl migration from positions 2 and 3 to position 6 (Lassfolk et al. 2019 ; Lassfolk et al. 2022 ). The discussion on the observed transacetylation is continued in the Supplementary Information . Hydrolysis of 5,6-cellulosene In the course of solvent screening for chemical modification of CA in solution state, we observed that the acetylated 5,6-cellulosene was soluble in water, under prolonged stirring at 100°C, while all other CA derivatives, as well as the CA itself, did not yield clear aqueous solutions ( Table S1 ). In order to confirm the apparent solubility of the unsaturated derivative, we obtained NMR spectra in D 2 O, after 1 day and 4 days (Fig. 2 ). The data clearly show progressive degradation of 5,6-cellulosene during solubilization at 100°C, indicated by the formation of sharp signals and reduction in the broader polymeric signals, observable in the 1 H spectral trace. The thermal degradation is accompanied by the conversion of 6-acetate to 6-hydroxyl groups for the residual polymeric glucose moieties, apparent from the ME-HSQC spectrum. Further, there seems to be a loss of the 5,6-cellulosene moiety, as indicated by the absence of the 6-CH 2 gem correlations in ME-HSQC, expected at approx. {4.59,94.5} ppm (Dryś et al. 2024). 5,6-Cellulosene appears to be unstable in water, at least at elevated temperatures, since it seems to be stable under dialysis conditions at RT. Due to the clear instability at 100°C, in D 2 O, we can speculate about potential hydrolysis mechanisms; where water is, logically, added to the 1, 5 or 6 positions. From appearance of many sharp signals in the 1 H spectra (Fig. 2 ), the mechanism is clearly multistep—however, the initial steps must involve hydrolysis. To assess the relative stabilities of the different hydrolysis products, we calculated Gibbs free energies for products resulting from water addition at the 5 & 6 positions in a 5,6-glucosene β-methylglycoside model compound (Scheme 1 ). Gibbs free energies were calculated taking into account conformational entropy contributions afforded through entropy calculations (Pracht and Grimme 2021), using the Conformer-Rotamer Ensemble Sampling Tool (CREST) (Pracht et al. 2024 ). The initial ensemble generation was carried out using the GFN2-xTB/ALPB(H 2 O) method (Bannwarth et al. 2021 ). The ensemble energies were recalculated using the more accurate g-xTB method (Froitzheim et al. 2025 ), adding ALPB(H 2 O) solvation contributions from the initial GFN2-xTB calculations. Conformers which had a Boltzmann population > 0.5% were then reoptimized (r 2 SCAN-3c/SMD(water)) and final weighted energies were calculated at two levels, using ORCA(Neese 2025 ; Neese et al. 2020 ): 1) the robust but lower accuracy r 2 SCAN-3c/SMD(water) level, 2) the more accurate ωB97X-V/def2-QZVPP/SMD(water)/DRACO level. The conformational Gibbs free energy contributions (based on the GFN2-xTB ensemble extrapolation of the conformational entropy), δG conf , and internal, non-conformational, free energy contributions (based on the r 2 SCAN-3c/SMD(water) calculations in a modified and scaled rigid-rotor harmonic-oscillator approximation), δG msRRHO (Pracht and Grimme 2021), were added to the final electronic energies (Table 1 ). Table 1 Gibbs free energies, including the conformational entropy contributions, calculated at two DFT levels using implicit water solvation (with opposing computational cost vs energy accuracy superpositions), with values referenced against the starting glucosene glycoside and water. The conformational contribution \(\:{\varDelta\:\partial\:{\varvec{G}}_{\varvec{c}\varvec{o}\varvec{n}\varvec{f}}^{\left(\varvec{T}\right)}}^{\:}\) is additive to the DFT computed values \(\:{\varDelta\:\varvec{G}}^{\left(\varvec{T}\right)}\) . Products r 2 SCAN-3c/SMD [kcal mol -1 ] ωB97X-V/def2-QZVPP/SMD [kcal mol -1 ] [kcal mol -1 ] \(\:{\varDelta\:\varvec{G}}^{\varvec{R}\varvec{T}}\) \(\:{\varDelta\:\varvec{G}}^{100\:{}_{\:}{}^{\varvec{o}}\varvec{C}}\) \(\:{\varDelta\:\varvec{G}}^{\varvec{R}\varvec{T}}\) \(\:{\varDelta\:\varvec{G}}^{100\:{}_{\:}{}^{\varvec{o}}\varvec{C}}\) \(\:{\varDelta\:\partial\:{\varvec{G}}_{\varvec{c}\varvec{o}\varvec{n}\varvec{f}}^{\varvec{R}\varvec{T}}}^{\:}\) \(\:\varDelta\:\partial\:{\varvec{G}}_{\varvec{c}\varvec{o}\varvec{n}\varvec{f}}^{100\:{}_{\:}{}^{\varvec{o}}\varvec{C}}\) Glucoside 2.69 5.30 -0.51 2.10 -0.50 -0.64 Open-chain ketose -7.80 -5.79 -8.19 -6.17 -0.97 -1.43 Closed-chain ketose 1 -4.98 -1.94 -6.71 -3.67 0.03 0.14 Closed-chain ketose 2 -7.31 -4.42 -9.00 -6.14 -0.03 0.03 The theoretical results in Table 1 indicate that the ketose forms are thermodynamically favoured over the glucoside. Considering conformational contributions to the free energy \(\:\varDelta\:\partial\:{G}_{conf}^{\left(T\right)}\) , the open-chain ketose form is predicted as favored, even at RT, despite the enthalpic stabilisation of the cyclic ketoses. This observation is consistent with both chemical intuition and previous observations, as ring closure generally leads to significantly reduced conformational degrees of freedom compared to the corresponding open form of a molecule (Pracht and Grimme 2021). Note that sub-kcal mol − 1 free energy differences observed for the ketose forms fall below the expected error margin, even when employing high-level methods, such as ωB97X-V. Consequently, no definitive conclusions regarding the preference for open- vs closed-chain ketose forms should be drawn from these results alone. When translating these values to the context of polymer chain breakages, using simpler glycoside models as a reference, a small increase in entropy is expected during the conversion of polymers to oligomers. This is primarily driven by gains in rotational and translational entropy. Associated formation of globular or aggregated species is not expected, as: 1) cellulose is substituted, i.e ., it will not crystallise and low DS cellulose acetates (≈ 0.5) are known to be water soluble (Todorov, King, and Kilpeläinen 2023), which is consistent with our NMR data; and 2) the sample dissolves upon degradation at 100°C, with sharp NMR signals retained at RT (Fig. 2 ). The additional free energy gain will then clearly result from addition of water to any position, resulting in chain-scission. The differences in configurational entropy between the models will then become more important, as molecular weight decreases. As there are considerable differences between the conformational degrees of freedom of the different saccharides (Table 1 ), this conformational flexibility can be visually illustrated by following the dihedral angle space (through Ramachandran plots) in the ring and open-chain forms in the hydrolysis models, from the conformer ensemble outputs of CREST (GFN2-xTB/ALPB(H 2 O)). Ramachandran plots were prepared for the majority of the conformers (Fig. 3 ), to assess the preferred conformations for each structure ensemble, contributing to the major Boltzmann populations. This is relevant for understanding further reactivity of the different species and for more accurate calculation of Boltzmann-weighted NMR spectra. As illustrated in Fig. 3 , the open-chain ketose model exhibits a wide range of dihedral angles, φ & ψ, following water addition at the 5-position, subsequent ring-opening and tautomerisation to the ketone form (Scheme 1 ). This behaviour is consistent with the largest calculated increase in configurational entropy across all models (Table 1 ). In contrast, the glucoside model adopts mainly a single stable chair conformation, with a significant population of twisted boat conformations, as anticipated. Lowest energy conformers (LC1) for GFN2-xTB vs ωB97X-V differ only in the rotation around the C5-C6 bond. Relative to the glucoside model, the glucosene glycoside reference model shows mainly twisted boat conformations at the GFN2-xTB level, with the adoption of some small population of chair conformations. This might result from the energetic preference towards a more planar ring conformation, due to the preference for preserving a nearly planar C4-C5 = C6-O5 bonding motif. The majority of φ values, which correspond to the C4-C5 = C6-O5 bonding motif and are expected to exhibit significant dihedral variation, are distributed around 0°. Such distribution implies some limitation to ring planarity, albeit substantially less pronounced than in the glucoside. This undoubtedly affects the total energies ( Table S5 ) and the driving force for adoption of new chemical species that do not adopt this ring strain, i.e ., with sp 3 hybridisation at C5. However, the ωB97X-V DFT level does place the two lowest energy conformers in one preferred chair conformation ( Fig. S5 ), where the planar C4-C5 = C6-O5 bonding motif is preserved, even without a more planar 6-membered ring. The cumulative Boltzmann weighting for these two lowest conformers amounts to a considerable population of 89.5%. Thus, there are subtle differences between the geometry optimisation and energy calculation methods that may yield significant changes in conformational entropy. For the present study, this is not unexpected, as the xTB methods are known to perform poorly for conformational energy calculations of sugar isomers (Bannwarth et al. 2021 ; Pracht et al. 2024 ), and range-separated hybrid functionals in a large basis, such as ωB97X-V/def2-QZVPP, performing significantly better on the corresponding benchmarks. Nevertheless, the glucoside model is bordering on higher Gibbs free energies, relative to the glucosene glycoside, with some variation in theory and temperature. The other ketose hydrolysis products, resulting from 5-addition, are clearly thermodynamically favoured for all tested theories and temperatures. The lowest energy cyclised ketose GFN2-xTB ensembles mainly populate the same interconverting (A & B) chair-twisted boat conformation space, while the DFT results show a preference for chair conformations for the lowest energy conformers. Their final configurational entropies are rather similar to that of the starting glucosene glycoside, but with favourable Gibbs energies. To determine if any of these decomposition products are formed, indicating the preferred reactivity, further NMR studies were performed. In subsequent experiments, we dissolved 5,6-cellulosene in DMSO-d 6 with the addition of 10 vol% of D 2 O. The sample remained in solution, with no apparent precipitation. By comparing ME-HSQC spectra of the sample in DMSO-d 6 , before and after the addition of water, we noted that the gem signals characteristic for the 5,6-double bond essentially disappeared after heating at 90°C for 1 day. Simultaneously, two sets of cross peaks appeared at around {1.36–1.54;14.7–15.3} ppm and {2.26–2.05;27.8–27.5} ppm, with correlations to two different C5 resonances in HMBC, corresponding to two unique species bearing quaternary (non-protonated) C5 species (Fig. 4 , Fig. S9 ). These could conceivably be resulting from formation of the open-chain ketose (containing a 5-ketone to 6-CH 3 spin-system) and closed-chain ketose anomers (containing a 5-acetal to 6-aliphatic CH 3 ). Since such ketose structures are not available in the literature, calculation of the chemical shifts, using DFT-based theory and Boltzmann weighting based on the calculated energies, was performed. Boltzmann weighting was applied using the ωB97X-V/def2-QZVPP energies. The chemical shifts are displayed as scatter plots (Fig. 4 b) and tabulated against the assigned spin-systems from the experimental NMR measurements ( Table S6 ). Clearly, strong similarities exist between the new experimental correlations for both the open-chain and closed-chain ketose structures, including the chemical shifts for the quaternary 5-position. There seems to be more than two specific anomers, based on the proton HSQC and HMBC correlations, which may be related to the adoption of further stable conformers and degradation products. However, full assignment requires more in-depth studies. Therefore, based on the synergistic combination of current experimental NMR and theoretical methodologies, it is apparent that hydrolysis of 5,6-cellulosene is occurring through water addition at the 5-position (Fig. 4 c), resulting in the formation of equilibrating open-chain ketose and its closed-chain hexose anomers. The addition of water at the 6-position ( Scheme S7 ) is not energetically favoured and there is no literature support for this reactivity under non-catalytic conditions. The NMR data presented earlier (Fig. 2 ) clearly suggest fragmentation of the polymer, initiated by hydrolysis. Two speculative mechanisms have been identified that may allow for the fragmentation of the linking units, resulting in depolymerization. The first includes an initial β-elimination on the open-chain (ketone-containing) ketose, which could potentially lead to further reverse-aldol-based ring fragmentations. However, this degradation is only known to occur experimentally under alkaline conditions. The second is demonstrated by (Kuznetsova, Kaverzneva, and Ivanov 1957 ) ( Scheme S8 ), where the keto group in the α position, with respect to the glycosidic linkage of model compounds, makes the C1 position of the adjacent sugar unit prone to hydrolysis under acidic and basic conditions. By analogy, in cellulosene-containing polymer, the C1-position on a linking unit (where the ketone functionality is on the adjacent sugar unit) would be prone to hydrolysis. This would directly result in chain-scissions. In our case, the fragmentation occurs under neutral conditions, so the likelihood of these latter mechanisms is rather low. Further work is needed to probe the source of this reactivity. Model radical-mediated thiol–ene reaction As such, 5,6-cellulosene appears to be an attractive platform for different modification reactions. In particular, it can serve as a double bond donor for radical-mediated thiol–ene chemistries, which, to our knowledge, have not yet been reported in literature. In this context, we set out to explore the reactivity of the obtained 5,6-ene-CA ( 4 ) towards radical thiol–ene coupling with biologically relevant thiols, using 2-aminoethanethiol (2-AET, commonly referred to as cysteamine). The choice of this model thiol reagent was further motivated by its additional terminal amine group, and the possibility for investigating further reactivity of this bifunctional reagent. Following a series of screening reactions, we determined that the highest conversion of the 5,6-cellulosene to the corresponding thioether is achieved under photoinitiation conditions at RT ( Fig. S10 ). The molecular structure of the anticipated product of the thiol–ene coupling of 5,6-ene-CA ( 4 ) with 2-AET, i.e., thioether with amine terminus, contains three methylene groups. Correspondingly, we expected to find three pairs of gem cross-peaks in ME-HSQC, attributable to 6- CH 2 -S and S- CH 2 - CH 2 -NH 2 of the thiol chain. However, contrary to our predictions, ME-HSQC shows more gem correlations than expected (Fig. 1 c). We initially considered the possibility of a side reaction to the main thiol–ene transformation, namely, linkage via C6-NH. This hypothesis was, however, inconsistent with the proposed radical mechanism, which is not selective for nitrogen addition. Furthermore, we found no signals in the region characteristic for 6- C H 2 -NH, typically present at 47 ppm in 13 C NMR (Gao, Liu, and Edgar 2018). We also noted a new ME-HSQC cross-peak present in the acetyl region at {1.83, 22.2} ppm, which is also inconsistent with the C6-NH connectivity. Importantly, the formation of the new acetyl resonance is already clearly visible in the diffusion-edited 1 H NMR spectrum at 1.83 ppm ( Fig. S10 ). These observations led to further characterization work with 2D NMR to confirm the source of this resonance. Based on complementary 1 H- 15 N HSQC and 1 H- 15 N HMBC measurements (Fig. 5 ), it is clear that the radical thiol–ene conditions yielded the expected thioether with terminal amine group and, additionally, a corresponding moiety with terminal acetamide. Conclusive evidence was gained from 1 H- 15 N HMBC spectrum, where we traced the new acetate cross-peak to N H-C(O)C H 3 correlation. The acetamide moiety is likely generated via acetyl group migration from C2-OAc or C3-OAc positions, in a manner similar to acetyl transfer over glycosidic bonds across polysaccharide units (between two non-neighbouring positions) (Lassfolk et al. 2019 ; Lassfolk et al. 2022 ). The NMR analysis was concluded with quantitative 1 H- 13 C HSQC (Heikkinen et al. 2003 ; Koskela, Kilpeläinen, and Heikkinen 2005 ), used to estimate the ratio of the equilibrated thioether amine/amide products (Fig. 5 c). By comparing the peak volumes of cross-peaks assigned to 6-CH 2 -S of the two species ({2.75,40.8} and {2.63,35.0} ppm), we concluded that, upon reaching equilibrium in DMSO-d 6 , the acetate and acetamide forms are present in approximately equal amounts. Further, the quantitative experiment allowed us to estimate that the conversion from the unsaturated 5,6-cellulosene to the thioether, under mild conditions, was essentially quantitative (≈ 90%), irrespective of the transacetylation. (The residual C6 of 5,6-ene-CA was ≈ 10%, see Table S8 ). Considering the thermal instability of the 5,6-cellulosene units, at least in the presence of water, it is likely that the photoinitiated reactions are more effective due to the use of RT, as opposed to thermal-radical initiation. Conclusion In this study, we synthesized acetylated 5,6-cellulosene and investigated its reactivity towards thiol–ene click reactions. Based on solution-state NMR, 5,6-cellulosene undergoes nearly quantitative conversion to the thioether under photochemical thiol–ene conditions at RT. By application of current NMR and theoretical methods, an in-depth understanding of the limitations of cellulosene chemistry is now available. The proposed hydrolysis mechanism of 5,6-cellulosene puts some limitations on reaction conditions; however, the use of a photoinitiator at RT is favourable. Due to the hydrolytic (in)stability, retention of some cellulosene linkages may afford suitable biodegradability, similar to oxy-cellulose, which might enable in vivo clinical use. It is also clear that current NMR and theoretical methods can be effectively combined to give considerable insights into poorly understood reaction mechanisms involving cellulose, a traditionally difficult polymer to work with. In future work, priority should be given to improving the synthesis of 5,6-cellulosene, aiming at minimizing the need for sequential isolations and purifications. Declarations Electronic supplementary material The data supporting this article is included in the Supplementary Information . The code for the dihedral plotter can be found at GitHub ( https://github.com/alistair-wt-king/dihedral-plotter ). Author Contribution M.D., T.K. & A.K. designed the study. M.D. performed chemical syntheses and solubility tests, acquired NMR data and wrote the original draft. A.M.C. contributed to chemical syntheses. M.D., T.K., A.K. & I.K. analysed NMR results. A.K., P.P. & T.W. performed computational analysis. K.R.G. acquired funding and administered the project. 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Steinmeier, Hans. 2004. '3. Acetate manufacturing, process and technology 3.1 Chemistry of cellulose acetylation', Macromolecular symposia. , 208: 49–60. Takahashi, Shin-Ichi, Tetsuya Fujimoto, Basu M. Barua, Takeaki Miyamoto, and Hiroshi Inagaki. 1986. '13C-NMR spectral studies on the distribution of substituents in some cellulose derivatives', Journal of Polymer Science Part A: Polymer Chemistry , 24: 2981–93. Teodoro, Kelcilene B. R., Rafaela C. Sanfelice, Fernanda L. Migliorini, Adriana Pavinatto, Murilo H. M. Facure, and Daniel S. Correa. 2021. 'A Review on the Role and Performance of Cellulose Nanomaterials in Sensors', ACS Sensors , 6: 2473–96. Thompson, Jeffrey E., and Kevin J. Edgar. 2025. 'Efficient, Regioselective Design of Mixed Cellulose Esters and Macroinitiators', Biomacromolecules , 26: 5680–93. Tikhonov, Denis S., Igor Gordiy, Danila A. Iakovlev, Alisa A. Gorislav, Mikhail A. Kalinin, Sergei A. Nikolenko, Ksenia M. Malaskeevich, Karina Yureva, Nikita A. Matsokin, and Melanie Schnell. 2024. 'Harmonic Scale Factors of Fundamental Transitions for Dispersion-corrected Quantum Chemical Methods', ChemPhysChem , 25: e202400547. Todorov, Aleksandar R., Alistair W. T. King, and Ilkka Kilpeläinen. 2023. 'Transesterification of cellulose with unactivated esters in superbase–acid conjugate ionic liquids', RSC Advances , 13: 5983–92. Usov, A. I., R. G. Krylova, and F. R. Suleimanova. 1977. 'Preparation of 6-chloro-6-desoxy- and 6-iodo-6-desoxycellulose', Bulletin of the Academy of Sciences of the USSR Division of Chemical Science , 26: 2000–02. Vigo, Tyrone L., Donald J. Daigle, and Clark M. Welch. 1972. 'Reaction of cellulose with chlorodimethylformiminium chloride and subsequent reaction with halide ions', Journal of Polymer Science Part B: Polymer Letters , 10: 397–406. Vigo, Tyrone L., and Navzer Sachinvala. 1999. 'Deoxycelluloses and related structures', Polymers for Advanced Technologies , 10: 311–20. Weigend, Florian, and Reinhart Ahlrichs. 2005. 'Balanced basis sets of split valence, triple zeta valence and quadruple zeta valence quality for H to Rn: Design and assessment of accuracy', Physical Chemistry Chemical Physics , 7: 3297–305. Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files CellulosenemanuscriptSIforCellulosejournal.docx SC1.png Scheme 1 Stoichiometry and geometries used for calculations of Gibbs free energies for water addition products at the 1, 5 or 6 positions of 5,6-cellulosene. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9335417","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":618643151,"identity":"8b2bdf90-5178-4379-b09f-36e1f7760093","order_by":0,"name":"Magdalena Dryś","email":"","orcid":"","institution":"University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Magdalena","middleName":"","lastName":"Dryś","suffix":""},{"id":618643152,"identity":"f895c090-c374-44ba-8c4f-656f4d59c5f7","order_by":1,"name":"Tetyana V. Koso","email":"","orcid":"","institution":"VTT Technical Research Centre of Finland","correspondingAuthor":false,"prefix":"","firstName":"Tetyana","middleName":"V.","lastName":"Koso","suffix":""},{"id":618643154,"identity":"2173bc1d-1aaa-4e4c-955f-432b8c951826","order_by":2,"name":"Tom Wirtanen","email":"","orcid":"","institution":"VTT Technical Research Centre of Finland","correspondingAuthor":false,"prefix":"","firstName":"Tom","middleName":"","lastName":"Wirtanen","suffix":""},{"id":618643155,"identity":"5b3d37ff-681e-4f83-a513-b64b6a138ef6","order_by":3,"name":"Katja T. Rinne-Garmston","email":"","orcid":"","institution":"Natural Resources Institute Finland","correspondingAuthor":false,"prefix":"","firstName":"Katja","middleName":"T.","lastName":"Rinne-Garmston","suffix":""},{"id":618643157,"identity":"34eb7cc3-f6c1-47cd-ad39-665282c5f8cb","order_by":4,"name":"Andrés Mollar-Cuni","email":"","orcid":"","institution":"University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Andrés","middleName":"","lastName":"Mollar-Cuni","suffix":""},{"id":618643165,"identity":"4cb2434e-1e3d-4e70-8e7d-9e90a30072a5","order_by":5,"name":"Ilkka Kilpeläinen","email":"","orcid":"","institution":"University of Helsinki","correspondingAuthor":false,"prefix":"","firstName":"Ilkka","middleName":"","lastName":"Kilpeläinen","suffix":""},{"id":618643171,"identity":"22ff03d2-d14e-48e7-883f-7caa3e49b32e","order_by":6,"name":"Philipp Pracht","email":"","orcid":"","institution":"Heidelberg University","correspondingAuthor":false,"prefix":"","firstName":"Philipp","middleName":"","lastName":"Pracht","suffix":""},{"id":618643176,"identity":"0dd90d0e-c4aa-4a09-9741-dfc0ac437d4c","order_by":7,"name":"Alistair W. T. King","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIie2RsQrCMBCGLwTqEsl6RdRXiLgoFHwbHV0dRCqFdBGfQHwM50igLvoOiuBcEcVJTBQdU90E88GFEO7Ln3AAHs8vUiKxWRGA2c3AHpGtcirUdKq3sgZkQEWBYurRwez98qk4DZ7Q5JQPW31gWu7Ocz3qmGBnCmoiUWXYjsvjtFldaJsC7ocZBVSAAjiRlXDRNQpXTqWuSZKr20uZdYtThCYxLqVRymMZHuOoWGnYv2ymKAK2lBXIonCiC5TaKt3ng8tIcNY7hNch8lKa0NylvAlMUTubx6Q+hly/6fZ4PJ6/4Q5kikHAL+2irwAAAABJRU5ErkJggg==","orcid":"","institution":"VTT Technical Research Centre of Finland","correspondingAuthor":true,"prefix":"","firstName":"Alistair","middleName":"W. T.","lastName":"King","suffix":""}],"badges":[],"createdAt":"2026-04-06 15:38:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9335417/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9335417/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107412711,"identity":"a8286398-72cd-489d-8413-e29857e4a904","added_by":"auto","created_at":"2026-04-21 09:22:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":302801,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e) Full scheme for the synthesis of acetylated 5,6-cellulosene from cellulose acetate (high 6-OH availability, CA); reaction conditions (A) MsCl, DMF, 75\u0026nbsp;°C, 3 h; (B) NaI, 2,5-hexanedione, 120\u0026nbsp;°C, 5\u0026nbsp;h; (C) DBU (≈2 eq/sugar unit), DMF, 50\u0026nbsp;°C, 2\u0026nbsp;h; R = OAc or OH. \u003cstrong\u003eb\u003c/strong\u003e)\u0026nbsp;ME-HSQC NMR spectra of CA and its derivatives, with annotations of 6-functionalities apparent from the opposite phase (600 MHz, DMSO-d\u003csub\u003e6\u003c/sub\u003e, 65\u0026nbsp;°C). \u003cstrong\u003ec\u003c/strong\u003e) General scheme for the radical-initiated thiol addition. R = OAc or OH, R’ = aliphatic chain. The presented structures of CA derivatives are simplified for clarity and do not imply complete regioselective substitution at position 6.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9335417/v1/fe5f575e54a56c93af3649dd.png"},{"id":107412714,"identity":"147763f7-7634-48fe-a0f1-f21529144fbd","added_by":"auto","created_at":"2026-04-21 09:22:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":65544,"visible":true,"origin":"","legend":"\u003cp\u003eME-HSQC\u0026nbsp;NMR spectra of 5,6-ene-CA (crude product before dialysis), acquired after stirring the sample (10 mg mL\u003csup\u003e-1\u003c/sup\u003e) in a sealed vial at 100\u0026nbsp;°C for 1 day \u0026amp; 4 days (500 MHz, D\u003csub\u003e2\u003c/sub\u003eO, 25\u0026nbsp;°C). Changes in key correlations are annotated. The \u003csup\u003e1\u003c/sup\u003eH spectra clearly show fragmentation of the polymer over time.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9335417/v1/7d3fe10302e1c0ed32a28df2.png"},{"id":107412717,"identity":"e64a6b76-734f-4bb3-aa41-1496cb7a9d95","added_by":"auto","created_at":"2026-04-21 09:22:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":590981,"visible":true,"origin":"","legend":"\u003cp\u003eRamachandran plots for the GFN2-xTB ensembles: separation of chair (A \u0026amp; D) vs twisted-boat (C \u0026amp; D) conformer space is best achieved by plotting C3-C4-C5-O5 (ψ) vs C1-C2-C3-C4 (φ) for all hexose species.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9335417/v1/b4f80ceb7ff04319606ff675.png"},{"id":107487803,"identity":"2acef1c7-6265-4bdc-91b6-94d11ff37660","added_by":"auto","created_at":"2026-04-22 02:42:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":349238,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e) HMBC spectrum (with ME-HSQC overlay) of 5,6-ene-CA hydrolysed in DMSO-d\u003csub\u003e6\u003c/sub\u003e with 10 vol% D\u003csub\u003e2\u003c/sub\u003eO, compared with \u003cstrong\u003eb\u003c/strong\u003e) PBE0/pcSseg-3 Boltzmann-weighted (using r\u003csup\u003e2\u003c/sup\u003eSCAN-3c geometries \u0026amp; ωB97X-V/def2-QZVPP energies) chemical shifts for the ketoses (referenced against DMSO). \u003cstrong\u003ec\u003c/strong\u003e) Scheme for the proposed water addition to the double bond of 5,6-cellulosene at position C5, and the resulting keto-enol species.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9335417/v1/9050b4303168a05b70e3df67.png"},{"id":107488916,"identity":"143149dc-044d-4561-b01a-57e2cdcd062a","added_by":"auto","created_at":"2026-04-22 02:46:10","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":212358,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e) Structural representations of two species obtained from the radical thiol–ene coupling of 5,6-ene-CA (4) with 2-aminoethanethiol (2-AET); R = OAc or OH. \u003cstrong\u003eb\u003c/strong\u003e) Overlay of \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN HSQC \u0026amp; HMBC, confirming the formation of acetamide cellulose thioether species (600 MHz, DMSO-d\u003csub\u003e6\u003c/sub\u003e, 65 °C). \u003cstrong\u003ec\u003c/strong\u003e) 3D projection of quantitative \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC HSQC spectrum, with indicated cross-peaks used for estimating relative ratios of the amine (\u003cstrong\u003e5a\u003c/strong\u003e) vs amide (\u003cstrong\u003e5b\u003c/strong\u003e) thioether forms.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9335417/v1/95697b0a3efe06c3fc97913e.png"},{"id":107489994,"identity":"9f4fe537-64e3-42d5-8d6e-ce5423a0d4c5","added_by":"auto","created_at":"2026-04-22 02:49:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2087190,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9335417/v1/bba57f2f-921b-42d4-8bdb-4e41ef044434.pdf"},{"id":107412712,"identity":"41b65dc0-3090-4e28-967f-05eadeebf6e3","added_by":"auto","created_at":"2026-04-21 09:22:35","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1715844,"visible":true,"origin":"","legend":"","description":"","filename":"CellulosenemanuscriptSIforCellulosejournal.docx","url":"https://assets-eu.researchsquare.com/files/rs-9335417/v1/ad6b4b25e1a8abe2b378d9db.docx"},{"id":107412716,"identity":"27781088-5650-430d-a1cd-5c863c11b6e6","added_by":"auto","created_at":"2026-04-21 09:22:35","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":124281,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1\u003c/strong\u003e Stoichiometry and geometries used for calculations of Gibbs free energies for water addition products at the 1, 5 or 6 positions of 5,6-cellulosene.\u003c/p\u003e","description":"","filename":"SC1.png","url":"https://assets-eu.researchsquare.com/files/rs-9335417/v1/dc3b6bc03dda50015853300e.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluation of 5,6-cellulosene as a synthon for thiol–ene chemistry: NMR and computational study","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCellulose is traditionally used in papermaking and textile manufacturing, as well as construction and packaging materials; however, there is additional potential in high-value applications, such as composite materials and pharmaceuticals (Arca et al. 2018; Thompson and Edgar \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), and in far-term technologies, including optoelectronics and bioengineering (Li et al. 2021; Teodoro et al. 2021). The preparation of advanced functional materials is possible by targeted chemical modifications of cellulose.\u003c/p\u003e \u003cp\u003eOn a molecular level, cellulose is a linear biopolymer consisting of glucopyranose units linked by β-1,4-glycosidic bonds. It contains hydrolytically stable acetal linkages, with the low reactivity imparted by the conformational stability, especially in the crystalline state. Cellulose is structurally rich in hydroxyl groups in positions 2, 3 and 6 of the glucose residues, which provide a platform for introducing different chemical moieties, typically \u003cem\u003evia\u003c/em\u003e nucleophilic substitution (S\u003csub\u003eN\u003c/sub\u003e2) mechanisms. Thus, chemical modification of cellulose is a common strategy to prepare functional materials with desired properties, while maintaining cellulose\u0026rsquo;s sustainability advantage over petroleum-based products (Li et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Li et al. 2021).\u003c/p\u003e \u003cp\u003eThe degree of substitution (DS), position of substitution, as well as the type of substituents are tightly linked to physical properties of cellulose-based materials, such as solubility in organic solvents and water. The ability to maintain control over the regioselectivity (position of substitution) is therefore advantageous, as it enables researchers to study the structure\u0026ndash;property relationships of these cellulose-based polymers, and to fine-tune the material performance to match the anticipated requirements in specific applications (Edgar et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Fox et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Arca et al. 2018; Thompson and Edgar \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). However, currently available low-cost heterogeneous strategies for modifications, such as acylation (including acetylation), carboxymethylation, methylation, epoxidation and etherification, are insufficient because they are essentially non-regioselective. Clearly, for high-value applications, there is a need to develop highly regioselective approaches for cellulose functionalization.\u003c/p\u003e \u003cp\u003eCellulose modification in solution state often yields some selectivity towards C6-substituted derivates, as the C6-OH is usually the most reactive position. However, these are usually not completely regioselective, since the secondary C2-OH and C3-OH groups tend to react before full displacement of C6-OH (Takahashi et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e1986\u003c/span\u003e; Liu and Baumann 2002). Consequently, complete and exclusive substitution at the primary position 6 of unmodified cellulose is not synthetically straightforward and normally requires protection/deprotection strategies (e.g., 6-tritylation (Hearon, Hiatt, and Fordyce \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1943\u003c/span\u003e; Heinze, R\u0026ouml;ttig, and Nehls 1994; Bui, Rosenau, and Hettegger 2023; Thompson and Edgar \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2025\u003c/span\u003e)). To our knowledge, only the Appel reaction, or variations thereof, allow for almost completely regioselective 6-substitution in a single-step synthesis (Furuhata, Chang, et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Furuhata, Koganei, et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Matsui et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). For example, the combination of triphenylphosphine and \u003cem\u003eN\u003c/em\u003e-bromosuccinimide yields 6-bromo-6-deoxycellulose with a DS\u003csub\u003e6\u003c/sub\u003e value of 0.99, without any direct observation of 2- or 3-bromination (Dryś et al. 2024). However, this procedure requires hazardous and expensive direct-dissolution cellulose solvent systems, and the inclusion of heteroatoms in all reagents further increases costs. This also reduces overall sustainability, particularly through poor atom-efficiency.\u003c/p\u003e \u003cp\u003eRecently, Prof. Kevin J. Edgar\u0026rsquo;s group presented an alternative strategy for highly chemo- and regioselective chlorination by using commercially available cellulose acetate (CA) with high hydroxyl content at position 6 (Liu and Edgar 2017; Gao, Liu, and Edgar 2018). This specific lower DS technical CA was chosen due to its availability of primary 6-OH groups, after a (6-OAc selective) hydrolytic post-ripening stage in the preparation of the CA (Steinmeier \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Position-specific DS\u003csub\u003e6\u0026minus;OAc\u003c/sub\u003e was estimated to be 0.5 (maximum DS\u003csub\u003e6\u003c/sub\u003e is 1), which means that the remaining 50% of the unreacted primary 6-OH groups are available for modification, ideally under mild conditions that do not affect the secondary positions 2 and 3. The Edgar group have demonstrated that the 6-OH groups of CA can be selectively and completely chlorinated with methanesulfonyl chloride (mesyl chloride, MsCl) in DMF. The obtained chlorinated CA was subsequently reacted with a model azide, as well as with amines and thiols \u003cem\u003evia\u003c/em\u003e iodinated CA generated \u003cem\u003ein situ\u003c/em\u003e, achieving modest to good conversions for these S\u003csub\u003eN\u003c/sub\u003e2 transformations (Gao, Liu, and Edgar 2018).\u003c/p\u003e \u003cp\u003eIn our previous work involving regioselective modification of microcrystalline cellulose, we investigated the reactivity of 6-halo-6-deoxycelluloses towards S\u003csub\u003eN\u003c/sub\u003e2 transformations (Dryś et al. 2024). In addition to the main S\u003csub\u003eN\u003c/sub\u003e2 reaction pathway, we observed the formation of a side product, resulting from the competing elimination (E2) mechanism (Vigo and Sachinvala 1999). Based on previously published literature (Ishii \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1986\u003c/span\u003e), we determined that the obtained side product was 5,6-cellulosene. After a detailed investigation of literature, we found that the formation of 5,6-cellulosene (or \u0026lsquo;5,6-cellulose-ene\u0026rsquo;) actually goes back to (Kaverzneva, Ivanov, and Salova \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1949\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe interest in 5,6-cellulosene is related to the introduction of a carbon\u0026ndash;carbon double bond (as an alkenyl-acetal). Previously, 5,6-cellulosene has shown reactivity towards addition reactions using CHCl\u003csub\u003e3\u003c/sub\u003e, CCl\u003csub\u003e4\u003c/sub\u003e, PCl\u003csub\u003e3\u003c/sub\u003e, HSiCl\u003csub\u003e3\u003c/sub\u003e, CH\u003csub\u003e3\u003c/sub\u003eCOOH and CH\u003csub\u003e3\u003c/sub\u003eOH (Dimitrov et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1968\u003c/span\u003e; Dimitrov, Gal'braikh, and Rogovin \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1965\u003c/span\u003e). From our perspective, the exocyclic double bond should promptly react in further addition reactions under radical initiation, potentially under aqueous conditions. This could expand the scope of high-value reagents in the formation of adducts. Considering the synthetic potential of 5,6-cellulosene, we now further probe the \u0026lsquo;CA method\u0026rsquo; (Gao, Liu, and Edgar 2018), to assess the key challenges of this approach towards radical-mediated thiol\u0026ndash;ene coupling, as a logical strategy for bioconjugation (Hoyle and Bowman 2010; Nolan and Scanlan \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ahangarpour et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). During this process, we illustrate the potential of combining state-of-the-art computational methods with detailed NMR analysis for structural elucidation of the obtained products. Our aim is to show that it is possible to go beyond rudimentary analysis of cellulose derivatives, thanks to current complementary experimental and theoretical approaches.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eCellulose acetate CA-320S NF/EP (acetyl content 31.9%, DS\u003csub\u003eAc\u003c/sub\u003e 1.8, DS\u003csub\u003eOH\u003c/sub\u003e 1.2) was provided free of charge by Eastman Chemical B.V.; methanesulfonyl chloride (mesyl chloride, MsCl, 99.5%) anhydrous \u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e-dimethyformamide (DMF, 99.8%, extra dry over molecular sieve), anhydrous \u003cem\u003eN\u003c/em\u003e,\u003cem\u003eN\u003c/em\u003e-dimethylacetamide (DMA, 99.8%), anhydrous sodium thiosulfate (Na\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e, 98.5%) were purchased from Thermo Scientific; 2,5-hexanedione (99.57%) was obtained from BLD Pharm; 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, \u0026ge;\u0026thinsp;99.0%), \u003cb\u003eγ\u003c/b\u003e-valerolactone (GVL, \u0026ge;\u0026thinsp;99%), β-methylglucopyranose (\u0026ge;\u0026thinsp;99%) were purchased from Sigma-Aldrich; 2-aminoethanethiol (2-AET, \u0026gt;\u0026thinsp;95.0%), 2,2-dimethoxy-2-phenylacetophenone (DMPA, \u0026gt;\u0026thinsp;98.0%), were purchased from TCI; sodium iodide (NaI, \u0026ge;\u0026thinsp;99%), pyridine (\u0026ge;\u0026thinsp;99.7%), acetic anhydride (99.7%) were purchased from VWR (Finland); DMSO-d\u003csub\u003e6\u003c/sub\u003e (99.8% D), D\u003csub\u003e2\u003c/sub\u003eO (99.90% D) and CDCl\u003csub\u003e3\u003c/sub\u003e (99.8% D\u0026thinsp;+\u0026thinsp;0.03% TMS v/v) were bought from Eurisotop. All reaction flasks were dried at 120\u0026deg;C for at least 16 h before syntheses.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eNMR measurements\u003c/h3\u003e\n\u003cp\u003eUnless otherwise indicated, NMR spectra were recorded in DMSO-d\u003csub\u003e6\u003c/sub\u003e at 65\u0026deg;C on a Bruker Avance NEO 600 spectrometer (600 MHz \u003csup\u003e1\u003c/sup\u003eH-frequency). Chemical shifts are referenced to DMSO-d\u003csub\u003e6\u003c/sub\u003e (\u003csup\u003e1\u003c/sup\u003eH: δ\u0026thinsp;=\u0026thinsp;2.50 ppm, \u003csup\u003e13\u003c/sup\u003eC: δ\u0026thinsp;=\u0026thinsp;39.52 ppm), unless otherwise indicated. TopSpin 4.1.4 and MestReNova 14.3.1 were used for the processing of NMR spectra.\u003c/p\u003e\n\u003ch3\u003eSolubility tests\u003c/h3\u003e\n\u003cp\u003eSolubility of the raw cellulose acetate and synthesized materials was tested at 1% w/v concentration, in several organic solvents and water, at ambient temperature and, for samples insoluble at room temperature, at 50\u0026deg;C, under magnetic stirring.\u003c/p\u003e\n\u003ch3\u003eComputational details\u003c/h3\u003e\n\u003cp\u003eThe computation of reaction Gibbs free energies via a supramolecular approach follows best DFT practices (Bursch et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). First, due to the expected flexibility of reactants and products, molecular conformations were sampled. Here, the GFN2-xTB/ALPB(H\u003csub\u003e2\u003c/sub\u003eO) level of theory (Bannwarth et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) was employed with a development version (\u0026gt;\u0026thinsp;3.0.2) of the CREST program (Pracht et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), where redundant conformations were sorted out under consideration of atom permutation (Pracht \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Conformational energies for sugar-like systems are prone to larger errors at this semiempirical level of theory and require postprocessing (Bannwarth et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Pracht et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Therefore, energies for all conformational ensembles were recomputed as single-points on the GFN2-xTB/ALPB geometries using the novel g-xTB method (Froitzheim et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Since no implicit solvation model is yet parametrized for this Hamiltonian, a simple additive correction term, calculated as the difference \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\partial\\:G}_{solv}^{ALPB}=\\:{E}_{ALPB\\left(water\\right)}^{GFN2-xTB}-\\:{E}_{gas}^{GFN2-xTB}\\)\u003c/span\u003e\u003c/span\u003e, was added to obtain \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{E}_{solv}^{g-xTB}\\approx\\:\\:{E}_{gas}^{g-xTB}+\\:{\\partial\\:G}_{solv}^{ALPB}\\)\u003c/span\u003e\u003c/span\u003e. Boltzmann weights were calculated for all structures, and conformations with a population of \u0026gt;\u0026thinsp;0.5% were selected for subsequent DFT treatment. All DFT calculations were performed with version 6.0.2 of the ORCA program package (Neese et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Neese \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Throughout, the \u0026lsquo;TightSCF\u0026rsquo; setting was used and default thresholds for geometry, and frequency calculations, were employed. The selected geometries were optimized at the r\u003csup\u003e2\u003c/sup\u003eSCAN-3c level of theory (Grimme et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), employing the SMD implicit solvation model for water (Marenich, Cramer, and Truhlar \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The same level of theory was employed for calculating harmonic frequencies, where free energy contributions at 298.15 K and 373.15 K were obtained in the modified and scaled rigid-rotor harmonic-oscillator scheme (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\varDelta\\:G}_{msRRHO}^{T}\\)\u003c/span\u003e\u003c/span\u003e) (Grimme \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Pracht and Grimme 2021), using an entropy interpolation parameter of 50 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and a frequency scaling factor of 0.9821 (Tikhonov et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Finally, higher level electronic energies were computed on the r\u003csup\u003e2\u003c/sup\u003eSCAN-3c geometries at the ωB97X-V/def-QZVPP level of theory (Mardirossian and Head-Gordon \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Weigend and Ahlrichs 2005), employing the RIJCOSX approximation (Eichkorn et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Neese \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Neese et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) and SMD(water) implicit solvation with dynamic radii adjustment for continuum solvation (Plett et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The final free energy for all relevant conformers was obtained by adding the \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\varDelta\\:G}_{msRRHO}^{T}\\)\u003c/span\u003e\u003c/span\u003e from r\u003csup\u003e2\u003c/sup\u003eSCAN-3c to the ωB97X-V electronic energies. A single average free energy ⟨G\u003csup\u003e(T)\u003c/sup\u003e⟩ for each reactant and product was obtained as the Boltzmann-weighted average over the respective ensembles. Since the hydrolysis reactions studied here involve a change in the degrees of freedom (DOF) through (re)cyclization, an influence of conformational entropy may further contribute to the reaction free energies. To gauge the magnitude of that effect, conformational free energy contributions \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\partial\\:G}_{conf}^{\\left(T\\right)}\\)\u003c/span\u003e\u003c/span\u003e were estimated using GFN2-xTB/ALPB(water) at 278.15 K, using the procedure described by (Pracht and Grimme 2021).\u003c/p\u003e \u003cp\u003eThe Ramachandran plots (Ramachandran, Ramakrishnan, and Sasisekharan \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1963\u003c/span\u003e) were prepared using the python3 script collection \u0026lsquo;dihedral-plotter\u0026rsquo;, available from GitHub (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/alistair-wt-king/dihedral-plotter\u003c/span\u003e\u003cspan address=\"https://github.com/alistair-wt-king/dihedral-plotter\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). This requires only the \u0026lsquo;crest.energies\u0026rsquo; and \u0026lsquo;crest_conformers.xyz\u0026rsquo; outputs from the CREST run. The GFN2-xTB optimised ensembles were utilised to visualise the relative populations between the hydrolysis structures. The atoms used for extraction of the dihedrals from the hexose geometries were C1-C2-C3-C4 (φ) and C3-C4-C5-O5 (ψ), as other dihedral options did not allow for clean separation of the conformational space (separate chair and twisted-boat regions).\u003c/p\u003e \u003cp\u003eThe chemical shifts were calculated using ORCA 6 (Neese et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Neese \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), using the PBE0 functional (Adamo and Barone \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1998\u003c/span\u003e), pcSseg-3 basis set (Jensen \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), and SMD implicit solvation (DMSO) (Marenich, Cramer, and Truhlar \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The calculations were performed on the r\u003csup\u003e2\u003c/sup\u003eSCAN-3c geometries. Boltzmann-weighting was performed based on the ωB97X-V/def2-QZVPP energies for those geometries. Calibration of the DFT chemical shifts was achieved by first measurement of the experimental HSQC spectrum for the β-methylglucopyranose in DMSO-d\u003csub\u003e6\u003c/sub\u003e at 25\u0026deg;C. The correct calibration values for \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC (for all computed values) was then determined by minimising the average error between the absolute values of the experimental vs computational chemical shifts, for the β-methylglucopyranose.\u003c/p\u003e\n\u003ch3\u003eSyntheses\u003c/h3\u003e\n\u003cp\u003eSynthesis of acetylated 6-chlorinated cellulose (6-Cl-CA)\u003c/p\u003e \u003cp\u003eRegioselective chlorination was carried out as described in (Gao, Liu, and Edgar 2018). In a typical reaction, a 250 mL three-neck round-bottom flask was charged with CA (3.00 g) and DMF (50 mL), under argon flow. The reaction flask containing the suspension was placed in an oil bath pre-heated to 75\u0026deg;C, and the solid dissolved under stirring in ca 30 min. Subsequently, MsCl (9.5 mL) was added dropwise. The reaction mixture was stirred at 75\u0026deg;C for 3 h under argon flow. The clear colourless solution became yellow but remained clear through the course of the synthesis. The heating source was removed to allow the reaction mixture to cool down for \u003cem\u003eca\u003c/em\u003e 15 min and poured into ethanol (800 mL). The crude product was recovered by filtration, dissolved in acetone (50 mL) and reprecipitated in ethanol (600 mL), filtered, washed with ethanol and dried in vacuo at 60\u0026deg;C for 3 h. \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, DMSO) δ 1.8\u0026ndash;2.2 (acetyl C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3\u003c/span\u003e\u003c/sub\u003e), 2.8\u0026ndash;5.3 (cellulose backbone C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e and C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2\u003c/span\u003e\u003c/sub\u003e-Cl), 7.9\u0026ndash;8.4 (formate C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e). Yield: 2.69 g (white powder).\u003c/p\u003e \u003cp\u003eSynthesis of acetylated 6-iodinated cellulose (6-I-CA)\u003c/p\u003e \u003cp\u003eConversion of chlorodeoxycellulose into deoxyiodocellulose was based on (Ishii \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). 6-Cl-CA (2.00 g) was dissolved in 2,5-hexanedione (50 mL), in a 250 mL three-neck round-bottom flask, under magnetic stirring, in an oil bath preheated to 80\u0026deg;C. Subsequently, NaI (6.6 g) was added to the clear solution, and the temperature of the oil bath was increased to 120\u0026deg;C. The reaction mixture was stirred at 120\u0026deg;C for 5 h, and after \u003cem\u003eca\u003c/em\u003e 15 min, the initially clear solution became turbid. Heating was switched off after 5 h and the mixture was allowed to cool down for \u003cem\u003eca\u003c/em\u003e 30 min. The reaction was quenched in ethanol (500 mL), filtered, suspended in aqueous sodium thiosulfate (0.1 M, 150 mL) and stirred for 1 h, filtered, washed with distilled water and dried in vacuo. \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, DMSO) δ 1.8\u0026ndash;2.2 (acetyl C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3\u003c/span\u003e\u003c/sub\u003e), 2.8\u0026ndash;5.3 (cellulose backbone C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e and C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2\u003c/span\u003e\u003c/sub\u003e-I), 7.9\u0026ndash;8.4 (formate C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e). Yield: 2.26 g (white powder).\u003c/p\u003e \u003cp\u003eSynthesis of acetylated 5,6-cellulosene (5,6-ene-CA)\u003c/p\u003e \u003cp\u003eDehydroiodination was carried out according to (Ishii \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). 6-I-CA (2.00 g) and anhydrous DMF (20 mL) were placed in a 100 mL round-bottom flask. The solid was dissolved under magnetic stirring at room temperature, yielding a clear light brown solution. Subsequently, DBU (2.0 mL) was added dropwise at room temperature, and the reaction mixture was heated in an oil bath preheated to 50\u0026deg;C. The reaction was stirred under heating for 2 h, and the initially clear light brown solution became dark brown. The heating source was subsequently removed and the reaction mixture was allowed to cool down for \u003cem\u003eca\u003c/em\u003e 30 min. The product was obtained by precipitation from cold ethanol (200 mL), followed by filtration. The crude product was resuspended in ethanol (200 mL), stirred overnight at room temperature, filtered, and dried in vacuo for 4 h, to give 1.3118 g of crude product as a brown powder. For purification, the crude product (300 mg) was suspended in distilled water, dialysed using dialysis tubing (CelluSep T1, Uptima, 3500 g/mol MWCO) for 3 days and freeze-dried. \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, DMSO) δ 1.8\u0026ndash;2.2 (acetyl C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3\u003c/span\u003e\u003c/sub\u003e), 2.8\u0026ndash;5.3 (cellulose backbone C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e and C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2\u003c/span\u003e\u003c/sub\u003e). Yield: 272.8 mg (light brown powder).\u003c/p\u003e \u003cp\u003eSynthesis of acetylated (6-(2-aminoethane)thioether cellulose (6-AET-CA)\u003c/p\u003e \u003cp\u003eIn 20 mL screw cap septum vial, evacuated and flushed with argon thrice, 5,6-ene-CA (100 mg) was dissolved in anhydrous DMF (5 mL). Subsequently, 2-aminoethanethiol (260 mg) and DMPA (1.5 mg) were added under argon hood, and more DMF was added (5 mL) to completely dissolve the solid reagents. The obtained clear solution was stirred for 16 h, at ambient temperature, under UV-A irradiation (Kessil UVA LED PR160L 390 nm (max 52 W), at 25% power setting (13 W) 6 cm from light source). The product was recovered by precipitation from ethanol (100 mL), centrifugation, decanting the supernatant solution, washing with ethanol, and centrifugation/decantation. The obtained white solid was further washed with three portions of deionized water and freeze-dried for 1 day. The cloudy supernatant from water washes was pooled and freeze-died for 3 days. \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, DMSO) δ 1.82 (amide acetyl C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3\u003c/span\u003e\u003c/sub\u003e), 1.88\u0026ndash;2.08 (acetyl C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3\u003c/span\u003e\u003c/sub\u003e), 2.62\u0026ndash;2.72 (C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2\u003c/span\u003e\u003c/sub\u003eS and thiol chain C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2\u003c/span\u003e\u003c/sub\u003e), 2.85\u0026ndash;5.10 (cellulose backbone C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e and C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2\u003c/span\u003e\u003c/sub\u003e and thiol chain C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2\u003c/span\u003e\u003c/sub\u003e). Yield: water-insoluble fraction: 59 mg (white powder), water-soluble fraction: 20 mg (white solid).\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003eMost strategies for obtaining unsaturated derivatives of mono- and polysaccharides are based on E2 elimination of certain functional groups under basic conditions (Nikologorskaya et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e1970\u003c/span\u003e). In our case, the most efficient synthesis of 5,6-cellulosene (maximizing conversion and minimizing the number of discrete reaction steps) leads through 6-chloro and 6-iodocellulose derivatives (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The three-step sequence starts with 6-regioselective chlorination of cellulose acetate (CA), using mesyl chloride (MsCl) in DMF (Gao, Liu, and Edgar 2018). The isolated 6-chlorocellulose acetate (6-Cl-CA) is then treated with NaI in 2,5-hexanedione (Ishii \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1986\u003c/span\u003e) to obtain the 6-iodo derivative (6-I-CA). Direct 6-iodination of CA is possible, with triphenylphosphine and \u003cem\u003eN\u003c/em\u003e-iodosuccinimide as reagents (Usov, Krylova, and Suleimanova \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e1977\u003c/span\u003e); however, it is more common to perform a two-step sequence (Heinze and Liebert \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Vigo and Sachinvala 1999), thereby avoiding the need for these expensive reagents and ideally increasing atom efficiency. The last step involves stoichiometric treatment with a bulky non-nucleophilic base (DBU, \u0026asymp;\u0026thinsp;2 eq per sugar unit) to induce elimination of HI and formation of a double bond between C-5 and C-6, yielding partially acetylated 5,6-cellulosene (Ishii \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). Direct conversion of chlorinated cellulose to 5,6-cellulosene is apparently possible, by treatment with KOH in MeOH or \u003cem\u003et\u003c/em\u003e-BuOK in DMSO (Srivastava, Harshe, and Gharia \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e1972\u003c/span\u003e); however, it is reported that alkali treatment of activated cellulose derivatives with good leaving groups in position C-6 may cause side reactions, forming 3,6-anhydroglucose moieties (Dimitrov et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1968\u003c/span\u003e; Makhsudov, Gal'braikh, and Rogovin \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1967\u003c/span\u003e). Finally, to expand the applicability of 5,6-cellulosene as a synthon, it was reacted with a model thiol (2-aminoethanethiol) under radical-initiated conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec), which is a common \u0026lsquo;click\u0026rsquo; strategy for bioconjugation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eChlorination\u003c/h3\u003e\n\u003cp\u003eThe chlorination of CA proceeded as described by (Gao, Liu, and Edgar 2018), achieving essentially full conversion of the C6 hydroxyl groups by the chloride moiety, as evidenced by diffusion-edited \u003csup\u003e1\u003c/sup\u003eH spectra (Fliri et al. 2023; King et al. 2018) and multiplicity-edited \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC heteronuclear single quantum coherence (ME-HSQC) correlation spectra (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). In our experience, diffusion-edited \u003csup\u003e1\u003c/sup\u003eH and ME-HSQC experiments are quick and reliable analytical tools for initial (and in many cases sufficient) diagnosis of cellulose reactivity (Fliri et al. 2023). The diffusion-edited \u003csup\u003e1\u003c/sup\u003eH experiment removes fast-diffusing (low molecular weight) species from the spectra, allowing to tentatively confirm the presence of polymer-bound functionalities (Fliri et al. 2023; King et al. 2018). ME-HSQC spectra are phase-sensitive, with the multiplicity selections allowing for positive or negative signal intensity, after phasing; this helps to quickly resolve 6-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eCH\u003c/span\u003e\u003csub\u003e2\u003c/sub\u003e geminal (gem) correlations, using the standard multiplicity editing selection, since they are immediately apparent by the opposite phase to CH and CH\u003csub\u003e3\u003c/sub\u003e correlations.\u003c/p\u003e \u003cp\u003eIn our ME-HSQC dataset (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), we identified 6-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eCH\u003c/span\u003e\u003csub\u003e2\u003c/sub\u003e-Cl gem cross-peaks at {3.90,43.6} and {3.70,43.6} ppm, and 6-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eCH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2\u003c/span\u003e\u003c/sub\u003e-OAc cross-peaks at {4.35,62.2} and {4.05,62.2} ppm. Unmodified 6-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eCH\u003c/span\u003e\u003csub\u003e2\u003c/sub\u003e-OH correlations are present in the NMR spectra of raw CA (\u003cb\u003e1\u003c/b\u003e) at {3.71,59.2} and {3.55,59.2} ppm; these are no longer present in the NMR data for 6-Cl-CA (\u003cb\u003e2\u003c/b\u003e), indicating that essentially all the 6-OH of CA (50% hydroxyl \u0026amp; 50% acetyl content at C6), are converted into 6-CH\u003csub\u003e2\u003c/sub\u003e-Cl. Consequently, the DS by chlorine at C6 is expected to be \u0026asymp;\u0026thinsp;0.5. More accurate calculation of DS\u003csub\u003e6\u003c/sub\u003e from \u003csup\u003e1\u003c/sup\u003eH NMR is not possible, since the protons attached to C6 resonate at frequencies that overlap with the cellulose backbone protons. Quantitative \u003csup\u003e13\u003c/sup\u003eC would be possible, but will contain significant error over short collection times, due to poor S/N.\u003c/p\u003e \u003cp\u003eThe full ME-HSQC spectrum (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb\u003c/b\u003e) shows additional correlations at {8.4\u0026ndash;7.9;161.3} ppm, consistent with the presence of formate esters. The apparent formylation seems to arise from the transfer of formyl from DMF, according to the known reaction mechanism (\u003cb\u003eScheme S1-S2\u003c/b\u003e) (Sato et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Vigo, Daigle, and Welch 1972). In order to avoid the formylation side reaction, we investigated possibilities to replace DMF with an alternative \u0026lsquo;green\u0026rsquo; dipolar aprotic solvent, capable of dissolving CA. Thus, informed by the results of solvent screening (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e), we selected \u003cb\u003eγ\u003c/b\u003e-valerolactone (GVL) due to its high dielectric constant (Kerkel et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). DMF could not be avoided completely, since the chlorination mechanism requires DMF to form the reactive (Vilsmeier) intermediate (\u003cb\u003eScheme S1\u003c/b\u003e). However, by using 1.5 mol eq of DMF, we obtained almost completely 6-chlorinated derivative (\u003cb\u003eFig. S2b\u003c/b\u003e). In addition, we noted the incorporation of 4-hydroxyvaleric acid ester as a side chain of the cellulose backbone, albeit this was typically occurring only to a minor extent (\u003cb\u003eFig. S2d\u003c/b\u003e). For all experimental details, relevant NMR data, and extended discussion of the results, the reader is referred to the \u003cb\u003eSupplementary Information\u003c/b\u003e.\u003c/p\u003e\n\u003ch3\u003eIodination\u003c/h3\u003e\n\u003cp\u003eAn essentially full displacement of the C-6 chloride by iodide was achieved using NaI in 2,5-hexanedione (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), following optimized reaction conditions previously published by (Ishii \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). The solvent requirement is due to the reaction being a modification of the classic Finkelstein reaction, where 2,5-hexanedione (used as a high boiling point alternative to acetone to yield faster kinetics or better solubility) dissolves NaI while favouring the precipitation of NaCl. Thus, the differential solubility of the two sodium salts is the driving force of the forward reaction, as with the classical Finkelstein reaction. However, there are drawbacks to this approach, namely, the use of iodine (requiring specialised disposal), the non-standard solvent and the additional synthetic step requiring isolation of 6-I-CA as an activated cellulose derivative.\u003c/p\u003e \u003cp\u003eThe Edgar group were apparently able to react 6-Cl-CA directly with different nucleophiles, \u003cem\u003evia\u003c/em\u003e 6-I-CA generated \u003cem\u003ein situ\u003c/em\u003e, in a one-pot reaction, with DMSO as the solvent (Gao, Liu, and Edgar 2018). We attempted to isolate the transient 6-I-CA under dipolar aprotic solvent conditions (DMSO, DMF and DMA). However, upon workup, we recovered mainly 6-chlorination product, with traces of 6-iodination, as evidenced by ME-HSQC data (\u003cb\u003eFig. S4\u003c/b\u003e). The \u003cem\u003ein-situ\u003c/em\u003e iodination does not seem to offer satisfactory (complete) displacement, which explains why Edgar\u0026rsquo;s group obtained products that still retained some 6-chloride moieties. Thus, it might be worth investigating if the additional step (isolation of 6-I-CA) would improve conversions in S\u003csub\u003eN\u003c/sub\u003e2 reactions using less reactive nucleophiles, without including the additional elimination step to 5,6-cellulosene (Dryś et al. 2024).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDehydrohalogenation to 5,6-cellulosene acetate\u003c/h2\u003e \u003cp\u003eThe unsaturated product, 5,6-cellulosene, can be synthesized from 6-iodinated cellulose by using a non-nucleophilic superbase, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), to eliminate HI over the 5,6 bond. The reaction conditions for converting 6-deoxy-6-iodocellulose to 5,6-cellulosene by DBU-mediated elimination mechanism were demonstrated by (Ishii \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). In the present study, we utilize the acetylated analogues of 6-deoxy-6-halocellulose derivatives, thereby facilitating solubility in organic solvents (see \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e for information on the solubility of CA and CA derivatives in specific solvents). Complete conversion to 5,6-cellulosene, using a stoichiometric amount of DBU (\u0026asymp;\u0026thinsp;1.5 mol eq/sugar unit), was apparent from ME-HSQC NMR data (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, \u003cb\u003eFig. S6\u003c/b\u003e), as evidenced by the absence of 6-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eCH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2\u003c/span\u003e\u003c/sub\u003e-I gem correlations expected at \u003cem\u003eca\u003c/em\u003e {3.26,5.03} and {3.58,5.03} ppm. The formation of the 5,6-double bond was confirmed by a new set of gem cross-peaks observed at {4.57,94.0} and {4.64,94.0} ppm, attributable to terminal 6-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eCH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2\u003c/span\u003e\u003c/sub\u003e (Dryś et al. 2024).\u003c/p\u003e \u003cp\u003eIn addition, ME-HSQC data (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, \u003cb\u003eFig. S6b\u003c/b\u003e) suggest that some deacetylation had occurred (due to the presence of residual water), as evidenced by cross-peaks assignable to 6-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eCH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2\u003c/span\u003e\u003c/sub\u003e-OH ({3.55,59.2} and {3.71,59.2} ppm). Conversely, an apparent minimal drop in peak volumes attributable to 3-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eCH\u003c/span\u003e and 2-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eCH\u003c/span\u003e correlations of cellulose acetate ({4.59,71.0} and {5.11,71.7} ppm, respectively (Kono, Hashimoto, and Shimizu \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), can be explained by the acetyl migration from positions 2 and 3 to position 6 (Lassfolk et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Lassfolk et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The discussion on the observed transacetylation is continued in the \u003cb\u003eSupplementary Information\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eHydrolysis of 5,6-cellulosene\u003c/h2\u003e \u003cp\u003eIn the course of solvent screening for chemical modification of CA in solution state, we observed that the acetylated 5,6-cellulosene was soluble in water, under prolonged stirring at 100\u0026deg;C, while all other CA derivatives, as well as the CA itself, did not yield clear aqueous solutions (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). In order to confirm the apparent solubility of the unsaturated derivative, we obtained NMR spectra in D\u003csub\u003e2\u003c/sub\u003eO, after 1 day and 4 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The data clearly show progressive degradation of 5,6-cellulosene during solubilization at 100\u0026deg;C, indicated by the formation of sharp signals and reduction in the broader polymeric signals, observable in the \u003csup\u003e1\u003c/sup\u003eH spectral trace. The thermal degradation is accompanied by the conversion of 6-acetate to 6-hydroxyl groups for the residual polymeric glucose moieties, apparent from the ME-HSQC spectrum. Further, there seems to be a loss of the 5,6-cellulosene moiety, as indicated by the absence of the 6-CH\u003csub\u003e2\u003c/sub\u003e gem correlations in ME-HSQC, expected at approx. {4.59,94.5} ppm (Dryś et al. 2024). 5,6-Cellulosene appears to be unstable in water, at least at elevated temperatures, since it seems to be stable under dialysis conditions at RT.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDue to the clear instability at 100\u0026deg;C, in D\u003csub\u003e2\u003c/sub\u003eO, we can speculate about potential hydrolysis mechanisms; where water is, logically, added to the 1, 5 or 6 positions. From appearance of many sharp signals in the \u003csup\u003e1\u003c/sup\u003eH spectra (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), the mechanism is clearly multistep\u0026mdash;however, the initial steps must involve hydrolysis. To assess the relative stabilities of the different hydrolysis products, we calculated Gibbs free energies for products resulting from water addition at the 5 \u0026amp; 6 positions in a 5,6-glucosene β-methylglycoside model compound (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Gibbs free energies were calculated taking into account conformational entropy contributions afforded through entropy calculations (Pracht and Grimme 2021), using the Conformer-Rotamer Ensemble Sampling Tool (CREST) (Pracht et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The initial ensemble generation was carried out using the GFN2-xTB/ALPB(H\u003csub\u003e2\u003c/sub\u003eO) method (Bannwarth et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The ensemble energies were recalculated using the more accurate g-xTB method (Froitzheim et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), adding ALPB(H\u003csub\u003e2\u003c/sub\u003eO) solvation contributions from the initial GFN2-xTB calculations. Conformers which had a Boltzmann population\u0026thinsp;\u0026gt;\u0026thinsp;0.5% were then reoptimized (r\u003csup\u003e2\u003c/sup\u003eSCAN-3c/SMD(water)) and final weighted energies were calculated at two levels, using ORCA(Neese \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Neese et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e): 1) the robust but lower accuracy r\u003csup\u003e2\u003c/sup\u003eSCAN-3c/SMD(water) level, 2) the more accurate ωB97X-V/def2-QZVPP/SMD(water)/DRACO level. The conformational Gibbs free energy contributions (based on the GFN2-xTB ensemble extrapolation of the conformational entropy), δG\u003csub\u003econf\u003c/sub\u003e, and internal, non-conformational, free energy contributions (based on the r\u003csup\u003e2\u003c/sup\u003eSCAN-3c/SMD(water) calculations in a modified and scaled rigid-rotor harmonic-oscillator approximation), δG\u003csub\u003emsRRHO\u003c/sub\u003e (Pracht and Grimme 2021), were added to the final electronic energies (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGibbs free energies, including the conformational entropy contributions, calculated at two DFT levels using implicit water solvation (with opposing computational cost vs energy accuracy superpositions), with values referenced against the starting glucosene glycoside and water. The conformational contribution \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\varDelta\\:\\partial\\:{\\varvec{G}}_{\\varvec{c}\\varvec{o}\\varvec{n}\\varvec{f}}^{\\left(\\varvec{T}\\right)}}^{\\:}\\)\u003c/span\u003e\u003c/span\u003e is additive to the DFT computed values \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\varDelta\\:\\varvec{G}}^{\\left(\\varvec{T}\\right)}\\)\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eProducts\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003er\u003csup\u003e2\u003c/sup\u003eSCAN-3c/SMD [kcal\u0026nbsp;mol\u003csup\u003e-1\u003c/sup\u003e]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eωB97X-V/def2-QZVPP/SMD [kcal\u0026nbsp;mol\u003csup\u003e-1\u003c/sup\u003e]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e[kcal mol\u003csup\u003e-1\u003c/sup\u003e]\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\varDelta\\:\\varvec{G}}^{\\varvec{R}\\varvec{T}}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\varDelta\\:\\varvec{G}}^{100\\:{}_{\\:}{}^{\\varvec{o}}\\varvec{C}}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\varDelta\\:\\varvec{G}}^{\\varvec{R}\\varvec{T}}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\varDelta\\:\\varvec{G}}^{100\\:{}_{\\:}{}^{\\varvec{o}}\\varvec{C}}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\varDelta\\:\\partial\\:{\\varvec{G}}_{\\varvec{c}\\varvec{o}\\varvec{n}\\varvec{f}}^{\\varvec{R}\\varvec{T}}}^{\\:}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\varDelta\\:\\partial\\:{\\varvec{G}}_{\\varvec{c}\\varvec{o}\\varvec{n}\\varvec{f}}^{100\\:{}_{\\:}{}^{\\varvec{o}}\\varvec{C}}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlucoside\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOpen-chain ketose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-7.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-5.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-8.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-6.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-0.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-1.43\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClosed-chain ketose 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-4.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-1.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-6.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-3.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClosed-chain ketose 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-7.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e-4.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-9.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-6.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e-0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe theoretical results in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e indicate that the ketose forms are thermodynamically favoured over the glucoside. Considering conformational contributions to the free energy \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\varDelta\\:\\partial\\:{G}_{conf}^{\\left(T\\right)}\\)\u003c/span\u003e\u003c/span\u003e, the open-chain ketose form is predicted as favored, even at RT, despite the enthalpic stabilisation of the cyclic ketoses. This observation is consistent with both chemical intuition and previous observations, as ring closure generally leads to significantly reduced conformational degrees of freedom compared to the corresponding open form of a molecule (Pracht and Grimme 2021). Note that sub-kcal mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e free energy differences observed for the ketose forms fall below the expected error margin, even when employing high-level methods, such as ωB97X-V. Consequently, no definitive conclusions regarding the preference for open- vs closed-chain ketose forms should be drawn from these results alone.\u003c/p\u003e \u003cp\u003eWhen translating these values to the context of polymer chain breakages, using simpler glycoside models as a reference, a small increase in entropy is expected during the conversion of polymers to oligomers. This is primarily driven by gains in rotational and translational entropy. Associated formation of globular or aggregated species is not expected, as: 1) cellulose is substituted, \u003cem\u003ei.e\u003c/em\u003e., it will not crystallise and low DS cellulose acetates (\u0026asymp;\u0026thinsp;0.5) are known to be water soluble (Todorov, King, and Kilpel\u0026auml;inen 2023), which is consistent with our NMR data; and 2) the sample dissolves upon degradation at 100\u0026deg;C, with sharp NMR signals retained at RT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The additional free energy gain will then clearly result from addition of water to any position, resulting in chain-scission. The differences in configurational entropy between the models will then become more important, as molecular weight decreases.\u003c/p\u003e \u003cp\u003eAs there are considerable differences between the conformational degrees of freedom of the different saccharides (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), this conformational flexibility can be visually illustrated by following the dihedral angle space (through Ramachandran plots) in the ring and open-chain forms in the hydrolysis models, from the conformer ensemble outputs of CREST (GFN2-xTB/ALPB(H\u003csub\u003e2\u003c/sub\u003eO)). Ramachandran plots were prepared for the majority of the conformers (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), to assess the preferred conformations for each structure ensemble, contributing to the major Boltzmann populations. This is relevant for understanding further reactivity of the different species and for more accurate calculation of Boltzmann-weighted NMR spectra.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the open-chain ketose model exhibits a wide range of dihedral angles, φ \u0026amp; ψ, following water addition at the 5-position, subsequent ring-opening and tautomerisation to the ketone form (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This behaviour is consistent with the largest calculated increase in configurational entropy across all models (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In contrast, the glucoside model adopts mainly a single stable chair conformation, with a significant population of twisted boat conformations, as anticipated. Lowest energy conformers (LC1) for GFN2-xTB vs ωB97X-V differ only in the rotation around the C5-C6 bond. Relative to the glucoside model, the glucosene glycoside reference model shows mainly twisted boat conformations at the GFN2-xTB level, with the adoption of some small population of chair conformations. This \u003cem\u003emight\u003c/em\u003e result from the energetic preference towards a more planar ring conformation, due to the preference for preserving a nearly planar C4-C5\u0026thinsp;=\u0026thinsp;C6-O5 bonding motif. The majority of φ values, which correspond to the C4-C5\u0026thinsp;=\u0026thinsp;C6-O5 bonding motif and are expected to exhibit significant dihedral variation, are distributed around 0\u0026deg;. Such distribution implies some limitation to ring planarity, albeit substantially less pronounced than in the glucoside. This undoubtedly affects the total energies (\u003cb\u003eTable S5\u003c/b\u003e) and the driving force for adoption of new chemical species that do not adopt this ring strain, \u003cem\u003ei.e\u003c/em\u003e., with sp\u003csup\u003e3\u003c/sup\u003e hybridisation at C5. However, the ωB97X-V DFT level does place the two lowest energy conformers in one preferred chair conformation (\u003cb\u003eFig. S5\u003c/b\u003e), where the planar C4-C5\u0026thinsp;=\u0026thinsp;C6-O5 bonding motif is preserved, even without a more planar 6-membered ring. The cumulative Boltzmann weighting for these two lowest conformers amounts to a considerable population of 89.5%. Thus, there are subtle differences between the geometry optimisation and energy calculation methods that may yield significant changes in conformational entropy. For the present study, this is not unexpected, as the xTB methods are known to perform poorly for conformational energy calculations of sugar isomers (Bannwarth et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Pracht et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), and range-separated hybrid functionals in a large basis, such as ωB97X-V/def2-QZVPP, performing significantly better on the corresponding benchmarks. Nevertheless, the glucoside model is bordering on higher Gibbs free energies, relative to the glucosene glycoside, with some variation in theory and temperature. The other ketose hydrolysis products, resulting from 5-addition, are clearly thermodynamically favoured for all tested theories and temperatures. The lowest energy cyclised ketose GFN2-xTB ensembles mainly populate the same interconverting (A \u0026amp; B) chair-twisted boat conformation space, while the DFT results show a preference for chair conformations for the lowest energy conformers. Their final configurational entropies are rather similar to that of the starting glucosene glycoside, but with favourable Gibbs energies.\u003c/p\u003e \u003cp\u003eTo determine if any of these decomposition products are formed, indicating the preferred reactivity, further NMR studies were performed. In subsequent experiments, we dissolved 5,6-cellulosene in DMSO-d\u003csub\u003e6\u003c/sub\u003e with the addition of 10 vol% of D\u003csub\u003e2\u003c/sub\u003eO. The sample remained in solution, with no apparent precipitation. By comparing ME-HSQC spectra of the sample in DMSO-d\u003csub\u003e6\u003c/sub\u003e, before and after the addition of water, we noted that the gem signals characteristic for the 5,6-double bond essentially disappeared after heating at 90\u0026deg;C for 1 day. Simultaneously, two sets of cross peaks appeared at around {1.36\u0026ndash;1.54;14.7\u0026ndash;15.3} ppm and {2.26\u0026ndash;2.05;27.8\u0026ndash;27.5} ppm, with correlations to two different C5 resonances in HMBC, corresponding to two unique species bearing quaternary (non-protonated) C5 species (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cb\u003eFig. S9\u003c/b\u003e). These could conceivably be resulting from formation of the open-chain ketose (containing a 5-ketone to 6-CH\u003csub\u003e3\u003c/sub\u003e spin-system) and closed-chain ketose anomers (containing a 5-acetal to 6-aliphatic CH\u003csub\u003e3\u003c/sub\u003e).\u003c/p\u003e \u003cp\u003eSince such ketose structures are not available in the literature, calculation of the chemical shifts, using DFT-based theory and Boltzmann weighting based on the calculated energies, was performed. Boltzmann weighting was applied using the ωB97X-V/def2-QZVPP energies. The chemical shifts are displayed as scatter plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb) and tabulated against the assigned spin-systems from the experimental NMR measurements (\u003cb\u003eTable S6\u003c/b\u003e). Clearly, strong similarities exist between the new experimental correlations for both the open-chain and closed-chain ketose structures, including the chemical shifts for the quaternary 5-position. There seems to be more than two specific anomers, based on the proton HSQC and HMBC correlations, which may be related to the adoption of further stable conformers and degradation products. However, full assignment requires more in-depth studies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTherefore, based on the synergistic combination of current experimental NMR and theoretical methodologies, it is apparent that hydrolysis of 5,6-cellulosene is occurring through water addition at the 5-position (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec), resulting in the formation of equilibrating open-chain ketose and its closed-chain hexose anomers. The addition of water at the 6-position (\u003cb\u003eScheme S7\u003c/b\u003e) is not energetically favoured and there is no literature support for this reactivity under non-catalytic conditions.\u003c/p\u003e \u003cp\u003eThe NMR data presented earlier (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) clearly suggest fragmentation of the polymer, initiated by hydrolysis. Two speculative mechanisms have been identified that may allow for the fragmentation of the linking units, resulting in depolymerization. The first includes an initial β-elimination on the open-chain (ketone-containing) ketose, which could potentially lead to further reverse-aldol-based ring fragmentations. However, this degradation is only known to occur experimentally under alkaline conditions. The second is demonstrated by (Kuznetsova, Kaverzneva, and Ivanov \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1957\u003c/span\u003e) (\u003cb\u003eScheme S8\u003c/b\u003e), where the keto group in the α position, with respect to the glycosidic linkage of model compounds, makes the C1 position of the adjacent sugar unit prone to hydrolysis under acidic and basic conditions. By analogy, in cellulosene-containing polymer, the C1-position on a linking unit (where the ketone functionality is on the adjacent sugar unit) would be prone to hydrolysis. This would directly result in chain-scissions. In our case, the fragmentation occurs under neutral conditions, so the likelihood of these latter mechanisms is rather low. Further work is needed to probe the source of this reactivity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eModel radical-mediated thiol\u0026ndash;ene reaction\u003c/h2\u003e \u003cp\u003eAs such, 5,6-cellulosene appears to be an attractive platform for different modification reactions. In particular, it can serve as a double bond donor for radical-mediated thiol\u0026ndash;ene chemistries, which, to our knowledge, have not yet been reported in literature. In this context, we set out to explore the reactivity of the obtained 5,6-ene-CA (\u003cb\u003e4\u003c/b\u003e) towards radical thiol\u0026ndash;ene coupling with biologically relevant thiols, using 2-aminoethanethiol (2-AET, commonly referred to as cysteamine). The choice of this model thiol reagent was further motivated by its additional terminal amine group, and the possibility for investigating further reactivity of this bifunctional reagent.\u003c/p\u003e \u003cp\u003eFollowing a series of screening reactions, we determined that the highest conversion of the 5,6-cellulosene to the corresponding thioether is achieved under photoinitiation conditions at RT (\u003cb\u003eFig. S10\u003c/b\u003e). The molecular structure of the anticipated product of the thiol\u0026ndash;ene coupling of 5,6-ene-CA (\u003cb\u003e4\u003c/b\u003e) with 2-AET, i.e., thioether with amine terminus, contains three methylene groups. Correspondingly, we expected to find three pairs of gem cross-peaks in ME-HSQC, attributable to 6-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eCH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2\u003c/span\u003e\u003c/sub\u003e-S and S-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eCH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2\u003c/span\u003e\u003c/sub\u003e-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eCH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2\u003c/span\u003e\u003c/sub\u003e-NH\u003csub\u003e2\u003c/sub\u003e of the thiol chain. However, contrary to our predictions, ME-HSQC shows more gem correlations than expected (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). We initially considered the possibility of a side reaction to the main thiol\u0026ndash;ene transformation, namely, linkage \u003cem\u003evia\u003c/em\u003e C6-NH. This hypothesis was, however, inconsistent with the proposed radical mechanism, which is not selective for nitrogen addition. Furthermore, we found no signals in the region characteristic for 6-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003e-NH, typically present at 47 ppm in \u003csup\u003e13\u003c/sup\u003eC NMR (Gao, Liu, and Edgar 2018). We also noted a new ME-HSQC cross-peak present in the acetyl region at {1.83, 22.2} ppm, which is also inconsistent with the C6-NH connectivity. Importantly, the formation of the new acetyl resonance is already clearly visible in the diffusion-edited \u003csup\u003e1\u003c/sup\u003eH NMR spectrum at 1.83 ppm (\u003cb\u003eFig. S10\u003c/b\u003e). These observations led to further characterization work with 2D NMR to confirm the source of this resonance.\u003c/p\u003e \u003cp\u003eBased on complementary \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN HSQC and \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN HMBC measurements (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), it is clear that the radical thiol\u0026ndash;ene conditions yielded the expected thioether with terminal amine group and, additionally, a corresponding moiety with terminal acetamide. Conclusive evidence was gained from \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e15\u003c/sup\u003eN HMBC spectrum, where we traced the new acetate cross-peak to \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eN\u003c/span\u003eH-C(O)C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3\u003c/span\u003e\u003c/sub\u003e correlation. The acetamide moiety is likely generated \u003cem\u003evia\u003c/em\u003e acetyl group migration from C2-OAc or C3-OAc positions, in a manner similar to acetyl transfer over glycosidic bonds across polysaccharide units (between two non-neighbouring positions) (Lassfolk et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Lassfolk et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe NMR analysis was concluded with quantitative \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC HSQC (Heikkinen et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Koskela, Kilpel\u0026auml;inen, and Heikkinen \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), used to estimate the ratio of the equilibrated thioether amine/amide products (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). By comparing the peak volumes of cross-peaks assigned to 6-CH\u003csub\u003e2\u003c/sub\u003e-S of the two species ({2.75,40.8} and {2.63,35.0} ppm), we concluded that, upon reaching equilibrium in DMSO-d\u003csub\u003e6\u003c/sub\u003e, the acetate and acetamide forms are present in approximately equal amounts. Further, the quantitative experiment allowed us to estimate that the conversion from the unsaturated 5,6-cellulosene to the thioether, under mild conditions, was essentially quantitative (\u0026asymp;\u0026thinsp;90%), irrespective of the transacetylation. (The residual C6 of 5,6-ene-CA was \u0026asymp;\u0026thinsp;10%, see \u003cb\u003eTable S8\u003c/b\u003e). Considering the thermal instability of the 5,6-cellulosene units, at least in the presence of water, it is likely that the photoinitiated reactions are more effective due to the use of RT, as opposed to thermal-radical initiation.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we synthesized acetylated 5,6-cellulosene and investigated its reactivity towards thiol\u0026ndash;ene click reactions. Based on solution-state NMR, 5,6-cellulosene undergoes nearly quantitative conversion to the thioether under photochemical thiol\u0026ndash;ene conditions at RT. By application of current NMR and theoretical methods, an in-depth understanding of the limitations of cellulosene chemistry is now available. The proposed hydrolysis mechanism of 5,6-cellulosene puts some limitations on reaction conditions; however, the use of a photoinitiator at RT is favourable. Due to the hydrolytic (in)stability, retention of some cellulosene linkages may afford suitable biodegradability, similar to oxy-cellulose, which might enable \u003cem\u003ein vivo\u003c/em\u003e clinical use. It is also clear that current NMR and theoretical methods can be effectively combined to give considerable insights into poorly understood reaction mechanisms involving cellulose, a traditionally difficult polymer to work with. In future work, priority should be given to improving the synthesis of 5,6-cellulosene, aiming at minimizing the need for sequential isolations and purifications.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eElectronic supplementary material\u003c/h2\u003e \u003cp\u003eThe data supporting this article is included in the \u003cb\u003eSupplementary Information\u003c/b\u003e. The code for the dihedral plotter can be found at GitHub (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/alistair-wt-king/dihedral-plotter\u003c/span\u003e\u003cspan address=\"https://github.com/alistair-wt-king/dihedral-plotter\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM.D., T.K. \u0026amp; A.K. designed the study. M.D. performed chemical syntheses and solubility tests, acquired NMR data and wrote the original draft. A.M.C. contributed to chemical syntheses. M.D., T.K., A.K. \u0026amp; I.K. analysed NMR results. A.K., P.P. \u0026amp; T.W. performed computational analysis. K.R.G. acquired funding and administered the project. All authors reviewed and edited the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank Eastman Chemical B.V. for their donation of the commercial cellulose acetate used in this work. We also thank Benedikt Gocht from Prof. Jan Deska\u0026rsquo;s group at the University of Helsinki for providing the UVA LED source.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data supporting this article is included in the Supplementary Information. The code for the dihedral plotter can be found at GitHub (https://github.com/alistair-wt-king/dihedral-plotter).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdamo, Carlo, and Vincenzo Barone. 1998. 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'Balanced basis sets of split valence, triple zeta valence and quadruple zeta valence quality for H to Rn: Design and assessment of accuracy', \u003cem\u003ePhysical Chemistry Chemical Physics\u003c/em\u003e, 7: 3297\u0026ndash;305.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"cellulose acetate, computational chemistry, density functional theory (DFT), NMR spectroscopy, regioselectivity, thiol–ene click chemistry","lastPublishedDoi":"10.21203/rs.3.rs-9335417/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9335417/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWe report a detailed characterisation of the preparation of 5,6-cellulosene, a poorly investigated reactive cellulose derivative. A key literature procedure is followed, which utilises partially hydrolysed technical cellulose acetate, as a 6-OH rich starting material. Thorough NMR analysis, using well-established solution-state NMR protocols, is performed to identify mechanisms of reactivity. The major drawback and potential opportunity identified in this study is the propensity for 5,6-cellulosene linkages (containing an alkenyl-acetal moiety) to undergo hydrolysis, leading to depolymerisation. However, now we demonstrate, using modern theoretical and NMR methods, that hydrolysis of cellulosene is still controllable under room temperature photoinitiated thiol\u0026ndash;ene reaction conditions, and aqueous workup. The suitability of 5,6-cellulosene as a synthon towards thiol\u0026ndash;ene chemistry is finally assessed (under thermal and photoinitiated radical addition) using a model amine-appended thiol. Clear applications of interest are in biomedical science, due to the abundance of thiol-containing proteins utilised in selective protein labelling.\u003c/p\u003e","manuscriptTitle":"Evaluation of 5,6-cellulosene as a synthon for thiol–ene chemistry: NMR and computational study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-21 09:22:31","doi":"10.21203/rs.3.rs-9335417/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"380baa5e-c921-4b1c-8adf-151c93a07ca2","owner":[],"postedDate":"April 21st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-21T09:22:31+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-21 09:22:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9335417","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9335417","identity":"rs-9335417","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}