Mechanism of sulfonation-induced chain scission of selectively oxidized polysaccharides

Oxidation of polysaccharides to 2,3-dicarboxypolysaccharides is a two-stage process, where selective oxidation by periodate is followed by secondary oxidation by chlorite. Addition of sulfamic acid before the secondary oxidation influences the molecular weight and degree of oxidation of the product. Here, mechanism of sulfamic acid-catalysed chain scission is elucidated for selectively oxidized cellulose and dextrin. Initially, sulfamic acid sulfonates the aldehyde groups of 2,3-dialdehydepolysaccharide. Introduced -SO 3 H groups are in ideal position to protonate the oxygen atom of 1-4' glycosidic bond and to trigger acidic hydrolysis. This can be used to obtain a direct control over the molecular weight of the product. Observed slightly lower degree of oxidation was ascribed to the ability of sulfamic acid to scavenge the hypochlorite and thus protect the intermolecular hemiacetals from oxidation. Usually undesirable hypochlorite thus seems to be necessary for preparation of selectively oxidized polysaccharides with degree of oxidation above 90%.


Introduction
Functional derivatives of biopolymers are gaining increasing attention because they represent a sustainable, biocompatible, and biodegradable alternative to synthetic materials. Derivatives of cellulose, the most abundant naturally occurring biopolymer, are employed in a plethora of applications ranging from packaging materials and food additives to wound dressing (Doelker, 1993). Increasing attention has been dedicated to the selectively oxidized cellulose derivatives, such as 2,3dialdehydecellulose (DAC, Fig. 1a) (Kim, Kuga, Wada, Okano, & Kondo, 2000;Maekawa, 1991), or 2,3dicarboxycellulose (DCC, Fig. 1b) (Kim & Kuga, 2001;Maekawa & Koshijima, 1984;, which offer number of unique characteristics in comparison with other cellulose derivatives. The DAC is prepared by highly selective oxidation of cellulose hydroxyl groups at C2 and C3 to aldehydes by periodate (Kim et al., 2000;Maekawa, 1991). Raw insoluble DAC can be subsequently solubilized by hot water (Kim, Wada, & Kuga, 2004), which greatly expands its utilization in aqueous chemistry. Highly reactive aldehyde groups of DAC can be used to prepare microbeads and hollow Sulfamic acid (SAMAC) is usually added into the reaction mixture to enhance the effectivity of secondary oxidation of cellulose by scavenging of hypochlorite , which is generated in situ as a side product of secondary oxidation of DAC by chlorite, see Eq. (1).
Hypochlorite is a strong and non-selective oxidizing agent, which can react with remaining chlorite, Eq.
(2), and potentially also with residual -OH groups of DAC. It is therefore generally deemed undesirable because it decreases the efficiency of the secondary oxidation and causes unwanted side-reactions.
Hence, SAMAC is added to scavenge the hypochlorite and reduce the risk of side-reactions. One equivalent of SAMAC can react with two equivalents of hypochlorite, see Eqs. (3) and (4).
However, our recent study (Munster, Fojtů et al., 2019) revealed two previously unforeseen consequences of sulfamic acid addition to the reaction mixture. Firstly, the weight-average molecular weight of the product (Mw) was dependent on the molar amount of SAMAC -it decreased inversely with the increasing molar amount of SAMAC. Thus, Mw of DCC could be adjusted between ca. 20 and 100% of original material (DCC prepared without SAMAC), which allowed us to optimize the drug release rates. This increased the potency of drug-carrier conjugates against some cancer cell lines.
Notably, the SAMAC-mediated decrease of Mw did not lead to increase of the polydispersity index (PDI) of the resulting material and appearance of very low-molecular weight fractions resulting in low yields of oxidized polysaccharide, which happens with other methods of molecular weight control (e.g. heating) (Munster, Fojtů et al., 2019;Munster et al., 2017). On the other hand, the decrease of Mw of DCC was achieved at the price of 10-15% decreased maximum DO -from more than 95% obtained in the SAMAC absence to 80-85%. This effect is not desirable as it somewhat reduces the maximum drug loading capacity of the DCC for cisplatin (about 5%) (Munster, Fojtů et al., 2019).
Based on observed results it was assumed that i) mechanism of SAMAC-catalyzed chain scission is likely more complex than simple acidic hydrolysis of cellulose chain ii) the control over the Mw of the product should be applicable also to other polysaccharides besides cellulose. To confirm these hypotheses, the mechanism of SAMAC-in-duced chain scission is elucidated in this work on an example of oxidized cellulose and modulation of Mw by SAMAC is investigated also for dextrin, branched polysaccharide obtained from starch or glycogen, composed from anhydroglucose units (AGU) bonded with 1-4' and 1-6' glycosidic bonds. Furthermore, the factors influencing the maximum achievable DO, the role of the molar ratio of reactants, their concentration, and the length of secondary oxidation are investigated for both cellulose and dextrin.

Materials
SigmaCell type 20 (Sigma Aldrich Co.) was used as a source of cellulose and Dextrin from corn starch type I (Sigma Aldrich Co.) as a source of dextrin. Materials were characterized by FT-IR spectroscopy and X-ray diffraction, see Fig. S1 in Supporting Information. Cellulose has comparatively higher crystallinity than dextrin, which is reflected in different rates of primary oxidation, see below. Mw was estimated by GPC analysis to be 76kDa for cellulose and 51 kDa for dextrin (Engel, Hein, & Spiess, 2012;Ferguson et al., 2018;Munster et al., 2017). Sodium periodate (NaIO4) and ethylene glycol (Penta, Czech Republic) were employed in the primary oxidation of polysaccharides. Acetic acid (>99.8%), sulfamic acid (99.3%), sodium chlorite (80%) (Sigma Aldrich Co.), sodium hydroxide (Lachner, Czech Republic) and sulphuric acid (96%) (Penta, Czech Republic) were used in secondary oxidation. All chemicals were of analytical grade and were used without further purification. Demineralized water was used throughout the experiment.

Preparation of 2,3-dicarboxypolysaccharide derivatives
Preparation of 2,3-dicarboxypolysaccharide derivatives was achieved via two-step sequential oxidation. In the first step, polysaccharide (cellulose or dextrin) was oxidized by using sodium periodate. In order to achieve the highest degree of oxidation, the molar ratio of reactants was set to 1:1.2 (polysaccharide : NaIO4). Typically, 16.5 g of NaIO4 dissolved in 500 ml of water was used for oxidation of 10 g of source polysaccharide. The primary oxidation was performed at 30 °C in the absence of light to prevent spontaneous degradation of oxidation agent. Duration of oxidation step varied with the source of polysaccharide and was optimized by simple UV-VIS spectral analysis of periodate consumption (Maekawa & Koshijima, 1984). After there was no observable decrease in the intensity of the NaIO4 absorption peak (222 nm), the primary oxidation was considered complete. Primary oxidation of cellulose and dextrin was found to be almost completed after 72 and 8 h, respectively. The difference is caused by variances in crystallinity (see Fig. S1) of selected polysaccharides (slower oxidation of cellulose crystallite cores) (Calvini, Conio, Princi, Vicini, & Pedemonte, 2006) and aqueous solubility of dextrin. The oxidation reaction was stopped by an excess of ethylene glycol and the resulting product was purified. Insoluble DAC was subjected to the repeated centrifugation and mechanical homogenization using WiseTis homo-genizer HD-15 (Witeg, Germany). Partially soluble 2,3-dialdehydedex-trin, DAD, was purified by 48 -h dialysis using dialysis tubing (Sigma Aldrich Co.) of 14 kDa molecular weight cut-off (MWCO). Both products of primary oxidation were then analysed in the terms of weight concentration of 2,3-dialdehydepolysaccharide in given volume of suspension/solution. The aldehyde content (DO from primary oxidation) of both prepared 2,3dialdehydepolysaccharides was estimated via oxime reaction (Veelaert, de Wit, Gotlieb, & Verhé, 1997) to be 12.1 ± 0.6 (DO = 96.8 ± 4.8%). This was later verified by 1 H NMR spectra, where the intensity of the signals of non-oxidized anhy-droglucose units was found to be between 1-3% for all samples (relative to signals of corresponding 2,3-dicarboxypolysaccharides).
The secondary oxidation step involves oxidation of aldehydes by sodium chlorite in the presence of acetic acid. The reaction stoichiometry was set on the base of the molar ratio of reactants 1:4 (-CHO : NaClO2), assuming 12.5mmol/g of -CHO groups which corresponds to fully oxidized dialdehydes. The concentration of NaClO2 and acetic acid was set to 1 M and 0.5 M, respectively. The secondary oxidation starts by dropwise addition of the oxidation agent solution into the mixture containing 2,3dialdehydepolysaccharide and acetic acid. The reaction was carried out at 30 °C in the absence of light for 7 h. After this period, the reaction was stopped by setting the pH to 10 by the addition of 10 M NaOH. The solutions containing products of secondary oxidation (2,3-dicarboxycellulose, DCC and 2,3dicarboxydextrin, DCD) were placed in dialysis tubing (MWCO =14kDa) and dialysed for 5 days for desalination and purification. The volume of samples was then reduced by rotary evaporator; pH was set to 7.4 by diluted NaOH; the samples were filtered and lyophilised. The resulting products prepared in the form of their sodium salt are designated as 2,3-dicarboxycellulose (DCC) and 2,3dicarboxydextrine (DCD). Reaction yield was almost quantitative. The DO of the prepared 2,3dicarboxypolysaccharides was estimated from 1 H NMR spectra. It represents the amount of cellulose units which hydroxyl groups were converted to carboxylic acids during the entire two-stage oxidation process. It is calculated as DO [%] = 100 IntAGU -IntHEMI, where IntAGU is the average intensity of the signals of -CH groups of residual anhydroglucose units, which were not oxidized during the primary oxidation, and IntHEMI is the average intensity of the signals of -CH groups of intermolecular hemiacetals which were not (fully) oxidized during secondary oxidation. Because no other species were detected in the spectra, the DO represents the total percentage of polysaccharide units converted to 2,3-dicarboxypolysaccharides.

Synthesis of DCC and DCD with different molecular weight
Modified secondary oxidation was employed to exemplify the role of SAMAC in molecular-weight control of DCC and DCD. Before the NaClO2 addition, different amounts of 0.1mg/ml solution of SAMAC were added dropwise to 2,3-dialdehydepolysaccharide solution containing acetic acid. The molar ratio of SAMAC to -CHO was set to 0.125, 0.25, 0.5 and 1 mol of SAMAC to 1 mol of -CHO. In order to investigate the influence of oxidizing agent concentration and duration of secondary oxidation on Mw of resulting 2,3-dicarboxypolysacchar-ides, the concentration of NaClO2 and duration of oxidation reaction was halved (i.e. to 0.5 M and to 3.5 h). The rest of the parameters and processes in DCC and DCD preparation remained the same.

Infrared analysis
FT-IR spectra were obtained by using Nicolet 6700 (Thermo Scientific, USA). Spectra were collected with ATR diamond crystal in the range of 4000 -400 cm -1 with 64 scans and a resolution of 2 cm -1 .

X-ray diffraction analysis
XRD diffractograms of source polysaccharides noted in Supporting Information were performed on a Rigaku MiniFlex 600 diffractometer in the diffraction 20 angle range 10-90°, CoKα (X =1.789 Å) radiation source was used with Kβ line filter.

Elemental analysis
Prepared oxidized polysaccharides were analysed in terms of elemental composition by the energydispersive X-ray spectroscopy (EDS) using Oxford INCA Energy 200 system (Oxford Instruments, UK) equipped within scanning electron microscope Vega II/LMU (Tescan, Czech Republic) operated at 30 kV.

Molecular weight analysis
Gel permeation chromatography (GPC) was performed on prepared DCC and DCD samples using a Waters HPLC Breeze chromatographic system equipped with a Waters 2414 refractive index detector (temperature of drift tube 60 °C), Tosoh TSKgel GMPWXL column (300 x 7.8 mm x13 pm, column T =30 °C). A mixture of 0.1 M NaNO3 and 0.05 M Na2HPO4 served as a mobile phase. Pullulan polysaccharide calibration kit SAC-10 (Agilent Technologies, Inc.) in a span of Mw 342-805 000 g/mol was used for calibration.

NMR analysis
All 1 H and 13 C NMR spectra were recorded using a Bruker Avance III HD 700 MHz NMR spectrometer equipped with a triple-resonance cryoprobe optimized for 13 C detection at 298 K in D2O. Besides the 1D experiments, the 1 H-13 C heteronuclear single quantum correlation (HSQC, JH-C =145 Hz) and 1H-13C heteronuclear multiple bond correlation (HMBC, nJH-C =10 Hz) experiments were performed (Munster et al., 2017).

Computational details
Structures were optimized by using B3LYP functional (

Mechanism of DCC chain scission in the presence of SAMAC
There are several possibilities for how SAMAC could cause chain scission. Firstly, the addition of SAMAC decreases the pH of the solution, which may trigger spontaneous acidic hydrolysis of 1-4' glycosidic bonds of DAC as is often exploited for cellulose (Lelekakis, Wijaya, Martin, & Susa, 2014; Negahdar, Delidovich, & Palkovits, 2016). To examine this possibility, series of secondary oxidations of DAC was performed using various amounts of SAMAC (from 1:0 to 1:1 -CHO: SAMAC molar ratio, see Table 1 and Section 2.3 for experimental details). Results were compared with the analogical set of reactions in which SAMAC was replaced by sulphuric acid. The acidity of the solutions was kept in the same range of pH values (1.6-0.9, see Table 1).
Presumably, if the DCC chain scission is indeed caused purely by acidic hydrolysis of 1-4' glycosidic bonds, both series should lead to similar results. Reactions were terminated after 7 h, and products were dialysed and lyophilised. Mw of all samples was estimated by GPC and degree of oxidation was determined from 1 H NMR spectra. To rule-out the possible buffering effect of acetic acid in the solution, the same reactions were repeated in its absence. The reaction performed without acetic acid however led to the destruction of DAC macromolecules (products of brown colour with Mw between 3-9 kDa). The presence of acetic acid in the reaction mixture is thus clearly essential for successful secondary oxidation. Most likely, acetic acid buffers the solution and sustains the acidic pH even after addition of basic NaClO2 salt, which stabilizes the dialdehydes and prevents their degradation in basic pH (Munster et al., 2017).
Considerable differences in both Mw and DO were observed between the SAMAC and H2SO4 series. The addition of SAMAC causes an expected decrease of Mw already for DCC-0.125 sample (-CHO : SAMAC ratio of 1:0.125) and reaches maximum for DCC-0.5. Note, that Mw of DCC-1 prepared using 1:1 ratio of -CHO : SAMAC is higher than in case of DCC-0.5. The DO of DCC follows the same V-shaped trend. The DO established from 1H NMR spectra ranges between 96% (DCC-0) and 84% (DCC-0.25).
Reasons behind DO modulation are discussed in the following Section 3.2, here we focus exlusively on the modulation of Mw.
In contrast to the SAMAC series, the addition of H2SO4 has no observable effect on the Mw of DCC. In other words, no sign of acidic hydrolysis of DCC chains was observed in given range of pH. The DCC chain scission is thus more effective for SAMAC than for sulphuric acid solution. This is rather surprising because acidic hydrolysis of cellulose is known to strongly depend on the strength (pKa) of the acid (Rinaldi, Meine, vom Stein, Palkovits, & Schuth, 2010). Another difference can be found in the comparison of polydispersity indexes (PDI), see Table 1. The PDI of the products prepared by using H2SO4 remain roughly the same (1.7 ± 0.1). For comparison, the PDI of samples prepared by using SAMAC is steadily decreasing from 1.95 to 1.65. The mechanism of SAMAC-induced DCC chain scission is thus not based on a simple mechanism of acidic hydrolysis.
To answer the question of what causes the high effectivity of SAMAC in DCC chain scission, we have considered known differences between chemistry of SAMAC and H2SO4. One of the key differences is the effectivity of sulfonation reactions. Sulphuric acid is a much weaker sulfonating agent than SAMAC (Benson & Spillane, 1980;Wagenknecht, Nehls, & Philipp, 1993). Moreover, SAMAC is also known to react readily with both aldehydes and ketones at normal temperature (Chen,  . Therefore, SAMAC may be able to directly sulfonate aldehydic/gem-diol groups (hydrated aldehydes) at C2 and C3 of DAC even at mild conditions. On the other hand, sulfo-nation of hydroxyl groups of DAC/DCC by SAMAC is unlikely, because high temperature, organic solvent, and urea as a catalyst are necessary for sulfonation of -OH groups of cellulose (Wagenknecht et al., 1993).
Let us now consider possible consequences of sulfonation of aldehyde groups at C2 and C3 of DAC. Sulfonation of aldehyde group(s) will bring highly acidic -SO3H group to the close vicinity of the oxygen atom of 1-4' glycosidic bond. It can thus effectively protonate O4', which may trigger the acidic hydrolysis. The -SO3-group is then eliminated from aldehyde groups during their oxidation to the carboxylic acid by ClO2 -(no signals of sulfonated C2 or C3 were detected in NMR spectra of DCC, see below). The suggested mechanism of this "sulfonation-induced scission" is shown in Scheme 1. To a certain extent, it resembles the action of acidic solid-state catalysts used in depolymerisation of cellulose (Shrotri, Kobayashi, & Fukuoka, 2018).
To prove the suggested mechanism, the DAC was dissolved in the mixture containing acetic acid and 1 molar equivalent of SAMAC (relative to -CHO groups of DAC), which was stirred in the absence of light for 7 h. This setup mimics conditions used for secondary oxidation of DAC, albeit in the absence of an oxidizing agent. Obtained sample, DAC-S (abbreviation from DAC-SAMAC), was subsequently dialyzed against demineralized water for 5 days in order to remove all traces of unbound SAMAC and lyophilised. The amount of sulphur in resulting material was then determined by EDS analysis. Results were compared with DCC-1 sample prepared using standard oxidation scheme with the same amount of SAMAC as DAC-S. Presumably, if the -CHO groups of DAC are sulfonated according to Scheme 1, the content of sulphur should be much higher in the DAC-S than in DCC-1. Indeed, the measured content of sulphur in DAC-S was 4.2 ± 0.8 at%, while in case of DCC-1 it was 20-times less with 0.2 ± 0.04 at%. Assuming that aldehyde carbons C2 and C3 form about 15 at% of each hydrated DAC unit (consisting of six carbon and seven oxygen atoms) and about one half of them is involved in variety of hemiacetal bonds (Munster et al., 2017), the significant portion of remaining hydrated aldehyde (gemdiol) groups is sulfonated. Lower content of sulphur in DCC-1 sample agrees with presumed elimination of -SO3H groups from DAC during secondary oxidation. These findings support suggested reaction mechanism, Scheme 1.
At this point, it is necessary to note that there are two possible pathways of SAMAC addition to hydrated aldehyde groups found in DAC solutions (gem-diols) (Munster et al., 2017). The -SO3H may either attach directly to aldehyde carbon (assuming simultaneous elimination of water from gem-diol) (Liimatainen, Visanko, Sirvio, Hormi, & Niinimaki, 2013) or via one of the hydroxyl groups of gem-diol (Benson & Spillane, 1980), forming essentially a DAC ester of sulphuric acid. Although the latter mechanism seems to be more likely in an aqueous environment, neither pathway could be ruled out immediately. Therefore, the FT-IR spectra of DCC-0 (0 at% of sulphur, prepared without SAMAC) and DCC-1 (with 0.2 ± 0.04 at.% of S) were measured, subtracted and analysed, see Fig. 2. Observed bands were assigned to vibrations of O=S=O and S-O groups (621 and 1117 cm -1 ). No bands corresponding to S-C bonds vibration modes were detected in the spectra, which supports the predominant formation of DAC-sulphuric acid ester structure. The analysis was performed using DCC FT-IR spectra because direct comparison of DAC and DAC-S was not feasible due to spectral changes (hemiacetal breaking) caused by dissolving the latter in acidic SAMAC solution.
Moreover, DFT calculations were used to simulate the structures of DAC sulfonated at C2 (C2-SO3H) and DAC sulphuric acid ester (C2-O-SO3H) to further investigate the mode of binding of -SO3H and to determine the distances between the -SO3H group and the oxygen atom of 1-4' glycosidic bond. The C2 of DAC was selected as the most likely target for sulfonation reaction because C3 of DAC is in solution mostly bound in C3-O-C6 hemiacetal (Fig. 1c). Besides, the C2 is known to be a preferred target for sulfonation reactions in cellulose (after C6) ( The calculated distance between the proton of -SO3H group and the oxygen atom of 1-4' glycosidic bond for C2-O-SO3H (ester structure) is only 1.61 A, see Fig. 3. This is much shorter than the sum of vdW radii of H and O. One may expect a protonation equilibrium between -SO3H and O4' to form rather easily. On the other hand, the distance between O4' and -SO3H in C2-SO3H structure is 2.25 A, which is likely caused by the formation of second, 1.84 A long hydrogen bond between -SO3H group and O6' from nearby C3-O-C6 intermolecular hemiacetal ring, Fig. 3. The C2-SO3H structure thus seems less suitable for protonation of O4', but may be partly responsible for the absence of C3-O-C6 intramolecular hemiacetal rings in the final product despite their high abundance in DAC.
Based on observed data and supporting calculations, it is assumed that addition of SAMAC during secondary oxidation causes sulfonation of aldehyde/gem-diols groups of DAC, with the -SO3H groups bound most likely via oxygen atom of gem-diol group (see Scheme 1). The -SO3H groups are then in an ideal position to protonate the oxygen atom of 1-4' glycosidic bonds, which may trigger acidic hydrolysis, causing DAC chain scission. The -SO3H groups are subsequently eliminated from the material during oxidation of DAC to DCC.

The degree of oxidation of DCC in the presence of SAMAC
As mentioned above, the addition of SAMAC influences also the DO. Let us first recall the trends in DO from Table 1. DO of DCC does not decrease linearly with the increasing amount of SAMAC as one may expect, but the minimum (84%) is reached for DCC-0.25, with DCC-0.5 only marginally better (86%). No such dependence is observed for materials prepared in the presence of sulphuric acid (DO 97%). Observed minima are thus likely related to another feature of SAMAC -the hypochlorite scavenging, which is not possible with H2SO4. To investigate, the 1 H, 13 C and 1 H-13 C HSQC and HMBC correlation spectra of DCC-0 (DO = 96%) and DCC-0.25 (DO = 84%) were recorded and analyzed, see Table 2 and Fig. 4.
Beside DCC itself, two minor species were detected in both spectra. The first set of signals was assigned to the residual anhydroglucose units of cellulose (AGU), which were not oxidized during primary oxidation. The second set was identified as former C6-O-C2' hemiacetals ( Fig. 1d), partially oxidized in positions 2, 3 and 3' during the secondary oxidation, see Fig. 4. This molecule was unequivocally identified based on distinct correlation signals between C6/H6 and C2'/H2' (Munster, Fojtů et al., 2019;Munster et al., 2017). Its signals are marked with asterisks in Fig. 4 and signal assignment is given in Table 2. Fig. 3. DFT optimized structures of DAC (2,3-dialdehydecellulose) sulphuric acid ester (left) and sulfonate (right). The distance between proton of -SO3H group and closest oxygen atom(s) is highlighted. More distant DAC residues are omitted for clarity. The signal intensity of residual AGUs is approximately 1% relative to signals of DCC in both cases, which illustrates the high effectivity and good reproducibility of primary oxidation. On the other side, the signals of C6-O-C2' hemiacetal are much stronger in DCC-0.25 (15% intensity relative to DCC) than in DCC-0 (3%). For comparison, the amount of C6-O-C2' hemiacetals in neat DAC solution was estimated to ca. 20% (Munster et al., 2017). The presence of SAMAC in the reaction mixture (above 0.25:1 molar ratio) apparently "protects" inermolecular hemi-acetals from secondary oxidation and thus reduces maximum achievable DO.
A possible explanation can be found in differences between mechanisms of oxidation of aldehydes by chlorite and hypochlorite, respectively, in combination with hypochlorite scavenging ability of SAMAC. The mechanism of oxidation of the aldehyde group by chlorite is shown in Scheme 2a . The reaction starts with the addition of O-Cl = O -to the aldehyde carbon in an acidic environment with the simultaneous breaking of the C=O double bond (Sirvio et al., 2014). However, this mode of action could not be directly employed for hemiacetals. In such a case, the hemiacetal bond needs to be first hydrolyzed and an aldehyde group re-formed. Because intermolecular hemiacetals are stabilized by inductive effects of large polysaccharide residues, not mentioning possible steric hindrance, their hydrolysis will likely become the rate-determining step. The efficiency of oxidation of these groups by chlorite will thus be significantly reduced.
On the other hand, hypochlorite can oxidize hemiacetal directly to the ester of carboxylic acid, because the reaction runs on the available hydroxyl oxygen atom, Scheme 2b. Breaking of hemiacetal bond is thus not required. The resulting DCC C6-O-C2' ester could hydrolyze easily in the acidic environment of the reaction mixture, which results in the formation of DCC, Scheme 2b.
The indices supporting suggested mechanism can be deduced from the observed trend of DO in different concentrations of SAMAC. It seems to be a result of an interplay of two factors: i) the hypochlorite scavenging which protects intermolecular hemiacetals from oxidation according to Scheme 2b; ii) increased rate of chlorite oxidation of hemiacetals (Scheme 2a) due to more acidic environment (increasing concentration of SAMAC). In theory, each SAMAC molecule is capable of reaction with two molecules of hypochlorite, see Eq.
(3) and (4). Hence, the SAMAC : -CHO ratio of 0.5 : 1 ensures complete scavenging of generated hypochlorite. In the real system, part of the hypochlorite is however bound to react with other targets, particularly with the highly abundant ClO2 -, see Eq.
(2). As a result, already the 0.25 molar equivalent of SAMAC seems to be capable of scavenging the remaining hypochlorite and thus protecting C6-O-C2' hemiacetals. Lower pH of DCC-0.5 and particularly DCC-1 solution causes faster hydrolysis in comparison with DCC-0.25, which leads to subsequent oxidation of hemiacetals according to Scheme 2 and to the increase of DO.
We attempted to prove the importance of hypochlorite for the oxidation of hemiacetals (Scheme 2b) by performing tertiary oxidation of DCC-0.25 with 15% of DCC C2'-O-C6 hemiacetals using solely hypochlorite. Up to two equivalents of hypochlorite (relative to the amount of residual DCC C2'-O-C6 hemiacetal) were added to the mixture in the presence of acetic acid and stirred for 7 h in the dark to mimic the conditions of secondary oxidation. However, these attempts did not bring the desired results. Hypochlorite caused a partial degradation of DCC and resulting complex NMR spectra of the product prohibited analysis of residual hemiacetal content (signal overlap). Most likely, we were unable to replicate a relatively slow generation of hypochlorite in situ. The proposed mechanism thus cannot be unequivocally proven at the moment.
The role of SAMAC for the Mw and DO of the product was investigated for cellulose derivatives. However, will these results be valid also for other polysaccharides? Is the fine-tunning of Mw possible, while keeping the DO above 90% at the same time? To find out, the optimization of secondary oxidation of both cellulose and dextrin was performed to obtain products with variable Mw and with highest possible DO.

Optimization of secondary oxidation of cellulose and dextrin
Cellulose and dextrin were oxidized to DCC and DCD, respectively, by the two-stage oxidation process, see Sections 2.2 and 2.3 for more details. While the primary oxidation of cellulose is well-documented (Kim et al., 2000;Munster, Fojtů et al., 2019;Munster et al., 2017), data about primary oxidation of dextrin lacks. Therefore, the kinetics of primary oxidation of both materials was briefly investigated by observing the consumption of periodate over time (Maekawa & Koshijima, 1984). While DAC with DO > 95% is obtained after 72 h of primary oxidation, for DAD it is only 8 h. This difference is due to better solubility and lower crystallinity of dextrin compared to cellulose (see Fig. S1) (Calvini et al., 2006).  Table 2 for assignment.
Scheme 2. a) the mechanism of chlorite oxidation of aldehyde, b) the mechanism of hypochlorite oxidation of partially oxidized C2'-O-C6 hemiacetal.
Resulting 2,3-dialdehydepolysaccharides were subsequently oxidized by chlorite. Secondary oxidation of both materials was performed using different conditions: i) various molar equivalents of SAMAC (up to 1:1 SAMAC : -CHO ratio, see Table 1), ii) different concentrations of the reactants (0.5 and 1 mol/L of chlorite) and iii) oxidation times (3.5 and 7 h). The DO and Mw of the products were investigated by using NMR and GPC analysis and results are given in Supporting Information, Tables S1 and S2 and Figs. S2-S8. Different conditions of secondary oxidation are emphasized by upper and lower index in sample notation. For instance, DCC prepared without SAMAC and by using shorter oxidation time is noted as DCC-0 3.5 , while the same sample prepared by using 0.5 mol/L concentration of chlorite is noted as DCC-00.5.
The Mw of DCC, DCC 3.5 , and DCC0.5 as a function of the SAMAC molar ratio is given in Fig. 5a. Addition of SAMAC causes a decrease of Mw of DCC from ~70kDa to ~18kDa, i.e. to approximately one-quarter of the original value. This agrees well with previous observations (Munster, Fojtů et al., 2019). Increase of Mw observable in all DCC-1 samples can be attributed to increased effectivity of chlorite oxidation in lower pH, which partially prohibits the sulfonation and thus also the chain scission.
Interestingly, shortening the secondary oxidation to 3.5 h has minimal effect on the Mw of the product, although it slightly decreases the maximum DO of some samples, see Fig. 5 and the corresponding discussion below it. Lower concentration of reactants, on the other hand, influences the Mw of DCC considerably and leads to the preparation of DCC0 5 samples with Mw 30-70 kDa, see Fig. 5a and Table  S1. The largest difference in Mw caused by different concentration of reactants is observed for DCC-0.25 and DCC-0.250.5, respectively. Lower concentrations of chlorite salt leads to 2.5-times higher Mw in the latter (21 kDa vs. 52 kDa).
The degree of oxidation of DCC follows the trends described in the previous section, i.e. minimum is found in all DCC-0.25 samples, see Table S1. While the decrease of the duration of oxidation has a relatively small effect to DO (see Table S1), lower concentration of reactants allows preparing of DCC with DO above 90% in all cases. Most likely, SAMAC is less effective in scavenging of hypochlorite at lower concentrations, and the hemiacetals are thus oxidized more efficiently according to Scheme 2b.
Dependences of Mw of DCD samples on SAMAC molar ratio are given in Fig. 5b. Observed trends in of Mw of DCD resemble those of DCC. The Mw of DCD prepared using standard conditions, i.e. 1 mol/L concentration of chlorite and 7 -h oxidation duration, decreases from 43 kDa to 15 kDa (35%). However, secondary oxidation of DCD at these conditions led to partial degradation of the product, hence lower Mw of DCD-0. This is best observable in NMR spectra of DCD series, see Fig. S5. Note that NMR spectra were improving with the increasing amount of SAMAC (lower pH).  On the other hand, DCD05 materials are of a good purity and of Mw between 53 and 38 kDa (70%). Hence, to avoid degradation, the role of oxidation time was studied for more diluted solutions of 0.5 mol/L concentration of chlorite (DCD0.5 series). The resulting material is designated as DCD0.53.5. Decrease of duration of secondary oxidation did not influence minimal achieved Mw of DCD, which is the same as for DCD05 (38 kDa). However, the decrease is continuous and more linear than in DCD05 series, and the lowest Mw is achieved for SAMAC : -CHO ratio of 1:1, not 0.5:1 as before, see Fig. 5b.
The smaller influence of SAMAC to Mw of DCD (in comparison with DCC) may be related to 1-6' branching of dextrin, which is from principle less susceptible to SAMAC-induced chain scission.
To determine the DO of DCD, its 1 H NMR spectra were measured and assigned. 1 H NMR spectrum of DCD-00.5 (prepared without SAMAC) is shown in Fig. 6, and the assignment is given in Table S3 in Supporting Information. For the other NMR 1 H spectra see Figs. S5, S6, and S7 in Supporting Information. Minor signals in the spectra were identified based on multiplicity edited 1 H-13 C HSQC spectra (Fig. S8) as a 1-6' branching of DCD units (Petersen, Motawie, M0ller, Hindsgaul, & Meier,  2015). These units form about 10% of DCD (relative to the signal intensity of major DCD signals).

Conclusions
Sulfamic acid (SAMAC) can be used to control the weight-average molecular weight (Mw) of various 2,3-dicarboxypolysaccharides containing 1-4' glycosidic bonds, which was demonstrated for oxidized cellulose and dextrin. Initial hypothesis suggesting a rather complex mechanism of action was confirmed, because scission of 2,3-dicarbox-ypolysaccharide chains is not caused by simple acidic hydrolysis. Instead, it is initiated by sulfonation of hydrated aldehyde groups (gem-diols) of corresponding 2,3-dialdehydepolysaccharides. The -SO3H groups bound to gem-diols via available hydroxyl groups can effectively protonate the oxygen atoms of 1-4' glycosidic bond and thus trigger the chain scission. The -SO3H-groups are subsequently eliminated during oxidation of aldehydes to carboxylic acids. The suggested reaction mechanism is supported by a comparative study with sulfuric acid (not capable of sulfonation), NMR, GPC, EDS, FT-IR analyses, and DFT calculations.
Additional investigation revealed that the Mw of 2,3-dicarbox-ycellulose (DCC), which contains exclusively 1-4' glycosidic bonds, can be modulated in between 25-100% of DCC prepared without SAMAC. Finer control over the Mw can be achieved by reducing the concentration of chlorite. The control over the Mw of 2,3-dicarboxydextrin (DCD) is also possible, but the effect of SAMAC is less pronounced, as the Mw of DCD can be reduced only by 30%, probably due to presence of 1-6' glycosidic bonds in dextrin structure. Further decrease of Mw may however be possible by increasing the SAMAC :-CHO of dextrin molar ratio, while simultaneously shortening the duration of the oxidation and keeping the concentration of chlorite <0.5 mol/L in order to avoid degradation.
Slightly lower maximum achievable degree of oxidation (DO) in the presence of SAMAC was traced to the persistence of intermolecular hemiacetal bonds. The reason being that SAMAC is, at least in larger concentrations, capable of scavenging the in situ generated hypochlorite before it can oxidize hemiacetals to the esters of carboxylic acids. Suggested mechanism is supported by indirect evidence (dependence of DO on molar amount and concertation of SAMAC), because attempts to perform tertiary oxidation of hemiacetals using hypochlorite resulted in the mixture of products and very complex NMR spectra. Subsequent optimization of reaction conditions revealed that to obtain DCC with adjustable Mw and DO above 90%, one should use longer secondary oxidation in combination with a lower concentration of reactants. No special treatment is required for dextrin, where only trace amounts of hemiacetals persist after secondary oxidation, probably due to 1-6' branching.
To summarize, the use of SAMAC allows to effectively control the Mw of the 2,3dicarboxypolysaccharides without risk of uncontrolled degradation of the material and appearance of very low-molecular weight fraction, which is associated with other methods of molecular weight reduction like solubilization or heating (Munster et al., 2017). Resulting product can be obtained in quantitative yields, has a high purity and molecular weight distribution (expressed by polydispersity index, PDI) comparable or better than that of original material. To the best of our knowledge, presented mechanism of sulfonation-catalyzed chain scission is unique and might find applications in the preparation of drug delivery systems with molecular weight-controlled drug release rates or it may inspire the development of new synthetic approaches in the future.