Key chromophores in celluloses: analysis by 31P NMR spectroscopy

The three cellulosic key chromophores, 2,5-dihydroxy-[1,4]-benzoquinone, 5,8-dihydroxy-[1,4]-naphthoquinone and 2,5-dihydroxy-acetophenone, are nearly ubiquitous in cellulosic materials because of their special structures and resonance stabilization with highly delocalized double bonds that renders them thermodynamically very stable and resistant towards bleaching. All of these compounds possess acidic hydroxyl groups. The current work explores the derivatization of these OH groups by a phosphitylation agent followed by 31P NMR analysis, an approach that is very frequently used for quantitative OH group analysis and differentiation in lignins and lignin derivatives. The chemical shifts are reported and structural peculiarities of the chromophore derivatives are discussed. The 31P NMR approach adds to the toolbox of methods applicable in cellulose chromophore analysis, bleaching and aging studies.


Introduction
The "chromophore release and identification" (CRI) methods provided access to trace chromophores in cellulosic materials which have been analyzed for different cellulose I and cellulose II substrates and cellulose derivatives (Rosenau et al. 2004). It provides isolated chromophoric compounds for detailed analytical characterization where spectroscopic methods only report likely structural motifs (Loureiro et al. 2010;Piantanida et al. 2005;Missori et al. 2019;Wójciak et al. 2014). In later work, it was established that some cellulosic chromophores are nearly ubiquitous because of their special stabilization by resonance. This causes strongly delocalized double bonds which leads to extremely low reactivity towards oxidative bleaching agents and high thermodynamic stability. This structural peculiarity renders these compounds prime survivors of standard bleaching sequences on the one hand, and the first chromophore candidates to reappear in yellowing/brightness reversion processes on the other hand (Korntner et al. 2015a;Ahn et al. 2019). The term "key chromophores" has thus been coined for these compounds, 2,5-dihydroxy-[1,4]-benzoquinone (DHBQ, 1), 5,8-dihydroxy-[1,4]-naphthoquinone (DHNQ, 2) and 2,5-dihydroxy-acetophenone (DHAP, 3), and has become common terminology in the cellulose bleaching literature (Korntner et al. 2015a;Kumar et al. 2018;Hosoya et al. 2018). As the key chromophore structures, as a rule of thumb, make up between 50 and 75% of the overall isolable chromophores, there is a natural interest in methods to analyze and possibly quantify them in different cellulosic matrices. These analytical approaches are generally aggravated by the low concentration of the chromophores and often their presence in complex multi-component mixtures after CRI isolation.
The presence of relatively acidic hydroxyl groups is a common structural feature of the key chromophores. DHBQ with a pK a of 2.71 (Hosoya et al. 2013), for instance, is significantly more acidic than acetic acid (pK a 4.76). The proton release in these compounds is favored by the high stability (resonance) of the corresponding (di)anions (Scheme 1). The nature of the hydroxyl groups and the corresponding oxoanions resulting from deprotonation is hard to define formally. The resonance superposition of quinoid and aromatic canonic structures provides them with both quinoid and aromatic (phenolic) properties at the same time. Nevertheless, there is an obvious closeness to hydroxyl structures in lignins. In fact, DHBQ is a component in oxidized Kraft lignins (Musl et al. 2019). Apart from aliphatic OH groups in the lignins´ side chains, there are various aromatic (phenolic) hydroxyl groups in the differently substituted aromatic systems (e.g., Balakshin and Capanema 2015;Balakshin et al. 2020). Quantitative analysis of these hydroxyl groups is commonly performed by 31 P NMR after derivatization with special phosphitylation reagents, according to an approach originally introduced by Argyropoulos (Argyropoulos et al. 1993;Argyropoulos 1994Argyropoulos , 1995Faix et al. 1994). Since then, the method has been very frequently used, comprehensively optimized and tested, and has become a standard technique for in-depth structural characterization of native and technical lignins with regard to their acidic hydroxyl groups, i.e., aliphatic and aromatic OH as well as carboxylic acid functionalities (Meng et al. 2019;Korntner et al. 2015b). For derivatization, typically the chlorophospholane reagent 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane (TMDP, 4) is used (Granata and Argyropoulos 1995), which neatly affords phosphites upon reaction with acidic hydroxyl groups. The procedure is particularly easy, mostly done in situ in the NMR tube, and the spectra can be well interpreted due to the high sensitivity of the 31 P isotope in NMR and the broad chemical shift range of this nucleus. While the main focus of the approach has always been on lignin chemistry (Saake et al. 1996;Crestini and Argyropoulos 1997;Argyropoulos and Zhang 1998;Tohmura and Argyropoulos 2001;Jiang and Argyropoulos 1998;Akim et al. 2001;Guerra et al. 2008;Zinovyev et al. 2018), it was later transferred also to the analysis of non-lignin substrates, such as polysaccharide derivatives, modified wood, or the elucidation of active agents in bleaching or glycosidation (Spyros et al. 1997;Fu et al. 2007;Argyropoulos et al. 2006;King et al. 2009King et al. , 2010Hosoya et al. 2014;Sadeghifar et al. 2014). It was somehow obvious to test this derivatization also with the cellulosic key chromophores and to see whether the typical lignin 31 P NMR approach could become a useful tool also in the realm of cellulose analysis. For this, the respective 31 P NMR data of the standard key chromophore compounds-or, more correctly, their phosphite derivatives-were needed, which is the topic of the present communication.

Materials and methods
The used reagents and solvents were bought from commercial sources with a purity > 95% and, unless otherwise noted, used without further purification. NMR solvents were obtained from Sigma-Aldrich (Schnelldorf, Germany) or Eurisotop (Saint-Aubin, France). Glassware was dried in a vacuum oven. The derivatization agent TMDP was synthesized in-house.
The derivatization method (phosphitylation) was carried out based on literature (Meng et al. 2019) and our previous work (Korntner et al. 2015b). In a dry vial, the samples (10 mg) were dissolved in dry, perdeuterated pyridine (600 μL) under shaking and the vessel was closed with a septum. The phosphitylation reagent (2-chloro-4,4,5,5-tetramethyl-1,3,2dioxaphospholane, 50 μL) was added through the septum with a syringe to minimize any contact between reagents and moisture. The sample was shaken for 1 h at RT and transferred into an NMR tube.

Results and discussion
Upon derivatization of the three key chromophores with the phospholane chloride, prominent and clearly distinguishable product peaks were obtained in all cases. It was obvious that all three chromophores reacted neatly with the reagent to give phosphite products with 31 P NMR resonances in the expected range, i.e., 130-150 ppm for derivatized phenolic OH groups. Interestingly, also the derivatized DHBQ appeared in this range although its hydroxyl groups are not phenolic but quinoid. 31 P, 1 H-HMBC spectra proved the long-range coupling between the phosphorus and the methyl protons of the reagent as well as the ring protons of the chromophores, and thus the successful derivatization. These spectra, along with the 1 H and 13 C shift listings, are included in the Supplementary Information. Scheme 1 shows the structure of the cellulosic key chromophores and the 31 P NMR derivatization agent, Fig. 1 displays the 31 P NMR spectra of the reagent and the three derivatized compounds, and Table 1 lists the corresponding 31 P NMR chemical shift values (Zieher 2022). It can thus be concluded that the derivatization/ 31 P NMR approach is generally well suited to detect the chromophores in compound mixtures of chromophores.
The derivatization conditions were based on procedures to derivatize natural and technical lignins which are sterically more hindered than the easily accessible, small chromophore molecules. A threefold to fivefold molar excess of the reagent relative to the sum of the hydroxy groups to be derivatized was used. Perdeuterated pyridine was the solvent of choice because of the good solubility of the compounds and also for trapping of the HCl generated in the derivatization reaction yielding pyridinium hydrochloride as soluble byproduct. Evidently, protic solvents cannot be used as they would react with the derivatization agent. In addition, DHBQ's 3-H and 6-H would undergo H-D exchange so that the corresponding cross-peaks in correlated spectra would "disappear" (Hosoya et al. 2013).
Regarding the application of the analytical method in practice, two notes are warranted here. First, this analytical approach targets chromophore mixtures obtained according to the CRI chromophore isolation method or alternative extraction procedures developed for cellulosic materials containing no or very little lignin, such as highly bleached pulps, bacterial cellulose or cotton (Rosenau et al. 2007(Rosenau et al. , 2011. Chromophore mixtures from lignin-rich matrices can evidently not be analyzed with regard to the presence of individual components, because the chromophore resonances would be overrun from the predominant signals from the (residual) lignin: the 31 P NMR shift range around 140 ppm is typical of derivatized phenolic compounds. Instead, sum parameters for the different types of hydroxyl groups would be obtained, similar to the application of the 31 P NMR method in lignin chemistry. Second, the resonances of derivatized DHBQ and DHAP are too close to allow discrimination (Table 1). However, the shift of derivatized DHNQ, which differs significantly from those of the former (ca. 4 ppm), offers an interesting way to distinguish this chromophore in mixtures: hemicellulose precursors generate all three chromophores upon aging whereas cellulose forms only DHBQ and DHAP, but no DHNQ (Zwirchmayr et al. 2017). 31 P NMR could help to better define the "provenance" and "history" of chromophores in cellulosic matrices which has remained an unsolved problem.
The main peak from the derivatized DHBQ was accompanied by minor resonances. These come from rearrangement and polymerization side products in small amounts (< 5%) which are almost ubiquitous companions of DHBQ unless it is strictly kept under an inert atmosphere. DHAP gave always rise to two signals, one from the mono-derivatized and one from the bis-derivatized product. Even at high excess of the derivatization reagent, the mono-substituted product was still present. Mono-derivatization occurred regioselectively at OH-5 because OH-2 is engaged in a very stable hydrogen bond to the carbonyl oxygen of the adjacent acetyl group and consequently showed rather a low acidity and reactivity in the derivatization. This behavior is well known from polyphenols with ortho-acyl substituents, e.g., flavonoids or vitamin E derivatives (Rosenau et al. 2005), and can-by using low temperatures and suitable solvents-even be exploited in chemical synthesis for a highly selective distinction of the hydroxyl functions.
The key chromophores should not be converted into their anions (salts) in an attempt to increase the rate of the derivatization, for three reasons. First, the around 17.0 For formation mechanism, see text derivatization process is quite fast anyway and reliably completed after 30 min. The used reaction time of 1 h adds additional time for complete derivatization. Second, the corresponding salts would be difficult to dry and much harder to dissolve in the NMR solvent. Third, derivatization of the anions causes byproduct formation, in particular reactions under C-P coupling, which becomes understandable from the resonance structures with their increased C-nucleophilicity (see Scheme 1). One by-product with a resonance at about 17 ppm (Table 1) deserves some attention since it was found in all spectra of the derivatized compounds without exception. The compound, 4,4,5,5-tetramethyl-1,3,2λ 5 -dioxaphospholan-2-one, possesses the five-membered 1,3,2-dioxaphospholane ring of the derivatization reagent, but-in addition-a doublebonded oxygen and a hydrogen bound to the phosphorus atom (Skarżyńska et al. 2011). The 1 H/ 31 P-HMBC spectrum in Fig. 2 illustrates the coupling between the H atom (orange) and the P-atom (blue) of the compound, with a characteristic coupling constant of 1 J = 704 Hz. This 31 P doublet appears also in the non-1 H-decoupled 31 P spectra with the same coupling constant.
It should be noted that this compound is not a true oxidation product of the reagent, formed by the unavoidable contact with atmospheric oxygen, as it might appear at first glance from the P=O motif. Instead, it is simply the hydrolysis product: contact of the reagent with trace amounts of water results in the formation of 2-hydroxy-4,4,5,5-tetramethyl-1,3,2dioxaphospholane, which immediately forms 4,4,5,5-tetramethyl-1,3,2λ 5 -dioxaphospholan-2-one (5) by a [1,2]-sigmatropic proton shift (Scheme 2). Formally, both compounds can be considered as Fig. 2 Hydrolysis product 5 and its 1 H (x-axis)/ 31 P (y-axis) HMBC-spectrum, showing the 1 J coupling of 704 Hz between the H atom (orange) and the P atom (blue). This spectrum originates from the phosphitylation of DHAP (3), but 5 was also found in all other reaction mixtures Scheme 2 Formation of the hydrolysis product 4,4,5,5-tetramethyl-1,3,2λ 5 -dioxaphospholan-2-one (5) from derivatization reagent 4 tautomers (Janesko et al. 2015), with the equilibrium between the two compounds far on the side of the pentavalent phosphorus (K = 2 × 10 10 , Guthrie 1979). However, the O,P-sigmatropic H-shift also implies an intramolecular redox process in which phosphorus is oxidized from + III to + V, while H is being reduced from + 1 to − 1. The compound is an organic diester of phosphorous acid, which is a diprotic acid (H 3 PO 3 , with one H being bound directly to P as O = PH(OH) 2 ). This is different from its P(V) counterpart, the triprotic phosphoric acid (H 3 PO 4 , corresponding to O = P(OH) 3 ).

Conclusions
The three cellulosic key chromophores can be neatly derivatized using the phosphitylation reagent 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane (4), which is very well-known from lignin analysis. The reaction is carried out in the NMR tube in perdeuterated pyridine, which serves simultaneously as the derivatization medium, catalyst, HCl trap and NMR solvent. The presence of water or other protic solvents would interfere with the derivatization. We hope that this easy derivatization in combination with 31 P NMR will be well accepted as a useful tool in chromophore research and become as widely used as in lignin analysis.