Polysaccharide depolymerization from TEMPO-catalysis: Effect of TEMPO concentration

Polysaccharide TEMPO-oxidation was monitored using automatic continuous online monitoring of polymerization reactions (ACOMP). The products of oxidation, obtained at different pHs (9, 7 and 5) and different concentrations of catalyst TEMPO, were evaluated by Automatic Continuous Mixing (ACM) and Size Exclusion Chromatography (SEC).

Carbohydrate Polymers 170 (2017) 140–147 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Polysaccharide depolymerization from TEMPO-catalysis: Effect of TEMPO concentration Vivian C Spier a,b , Maria Rita Sierakowski a , Wayne F Reed b , Rilton A de Freitas a,∗ a b BioPol, Chemistry Department, Federal University of Paraná, Curitiba, Paraná, 81531-980, Brazil Tulane Center for Polymer Reaction Monitoring and Characterization (PolyRMC), Tulane University, New Orleans, LA, 70118, USA a r t i c l e i n f o Article history: Received February 2017 Received in revised form 11 April 2017 Accepted 23 April 2017 Available online 26 April 2017 Keyword: Xyloglucan N-oxil-2,2,6,6-tetramethylpiperidine (TEMPO) Automatic continuous online monitoring of polymerization reactions (ACOMP) Automatic continuous mixing (ACM) Size exclusion chromatography (SEC) Depolymerization a b s t r a c t Polysaccharide TEMPO-oxidation was monitored using automatic continuous online monitoring of polymerization reactions (ACOMP) The products of oxidation, obtained at different pHs (9, and 5) and different concentrations of catalyst TEMPO, were evaluated by Automatic Continuous Mixing (ACM) and Size Exclusion Chromatography (SEC) The degree of oxidation was higher at pH and polysaccharide degradation was observed under different pH conditions, but was much higher without catalyst TEMPO The rate constant (k) was dependent on reaction pH and TEMPO concentration The amount of −COOH per g of polysaccharide, at pH 9, in the presence and absence of TEMPO was different, 0.215 and 0.395 mmol g−1 , respectively This suggested a secondary and non-selective polysaccharide oxidation occurring at a lower rate in the absence of catalyst TEMPO protects the polysaccharide from degradation caused by secondary oxidant species, acting as a catalyst and “sacrificial molecule” at higher concentrations © 2017 Elsevier Ltd All rights reserved Introduction The compound 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) has been used as a catalyst in polysaccharide oxidation reactions, acting selectively on primary alcohols The main advantage of TEMPO-catalyzed oxidation, compared to other catalysts, is its selectivity, low reactivity in the presence of air, light, humidity, and storage without special conditions (Kato, Matsuo, & Isogai, 2003; Sierakowski, Milas, Desbrières, & Rinaudo, 2000; Sierakowski, de Freitas, Fujimoto, & Petri, 2002; Bragd, Besemer, & Van Bekkum, 2000; de Nooy, Besemer, & Van Bekkum, 1995a; de Nooy, Besemer, & Van Bekkum, 1995b; de Nooy, Besemer, Van Bekkum, Van Dijk, & Smit, 1996) Polysaccharide TEMPO-oxidation is usually performed under alkaline conditions However, there are few reports in the literature about the use of acid reaction conditions, affecting directly the selectivity and the degree of oxidation (Watanabe, Tamura, Saito, Habu, & Isobai, 2014) To selectively oxidize the primary alcohols, reactive oxygen species NaClO/NaBrO are used as a secondary oxidizing agent, promptly oxidizing TEMPO to nitrosonium ∗ Corresponding author E-mail addresses: rilton@quimica.ufpr.br, rilton@pq.cnpq.br (R.A de Freitas) http://dx.doi.org/10.1016/j.carbpol.2017.04.064 0144-8617/© 2017 Elsevier Ltd All rights reserved ions, the effective oxidant in the catalysis Nitrosonium ion acts as a catalyst, oxidizing the polysaccharide primary alcohols to aldehydes and is reduced to hydroxylamine The hydroxylamine can be re-oxidized to nitrosonium ion by the secondary oxidants, and a second round of oxidation converts the aldehyde groups to carboxylic acids (Isogai, Saito, & Fukuzumi, 2011; Bragd et al., 2000; de Nooy et al., 1995a, 1995b, 1996; Sierakowski et al., 2000; Sakakibara, Sierakowski, Lucyzyn, & de Freitas, 2016) The most common alkaline conditions, pH >9.0, used by several authors to induce alkoxide formation on polysaccharides, also demonstrated depolymerization using TEMPO as a catalyst, even at very low temperature (0–4 ◦ C) Such molar mass reduction was reported for different kinds of polysaccharides (Cunha, Maciel, Sierakowski, Paula, & Feitosa, 2007; de Freitas, Martin, Paula, Feitosa, & Sierakowski, 2004; Sakakibara et al., 2016 and de Souza, Lucyszyn, Ferraz, & Sierakowski, 2011) Some authors identify methods to avoid depolymerization during TEMPO reactions For example, Shibata & Isogai (2003) observed that hydroxyl radicals formed from NaBrO and TEMPO at pH 10–11 can cause depolymerization during oxidation, and that some scavengers of reactive species could only partially suppress the depolymerization Shinoda, Saito, Okita, and Isogai (2012) related the concentration of NaClO concentration with carboxylate content and the degree of polymerization (DP) of cellulose nanofibrils The authors assumed that C6-aldehyde formed as an interme- V.C Spier et al / Carbohydrate Polymers 170 (2017) 140–147 diate structure of TEMPO-oxidation could be associated with a ␤-elimination process, and that the elimination of such intermediates, due to post-oxidation or reduction of aldehydes formed, reduced such effect The alkaline oxidation medium suggests at least one main hypothesis to explain the polysaccharide degradation during TEMPO catalyzed oxidation, related to ␤-elimination Such reaction occurs mainly under alkaline conditions and in the presence of an aldehyde, acid or ester group at polysaccharide C6, contributes to increase the acidity of the hydrogen at C5, usually deprotonated during the alkaline reaction The reaction intermediate product is a double linkage between the carbohydrate C4-C5 The end products are related to an elimination of the group linked to carbon ␤ (C4), forming a reducing carbohydrate (aldehyde functional group) and an unsaturated carbohydrate (Anet, 1964) Another mechanism of depolymerization can be associated to non-specific oxidation of polysaccharides by the secondary oxidants The reactive oxygen species (NaClO/NaBrO), can promote an increase in the reducing carbohydrates, diminishing the polysaccharide molar mass Boruch (1985) observed hydrolytic degradation of starch molecules, with reduction of viscosity and increasing reducing sugars, during starch oxidation with NaClO Hiraoki, Ono, Saito, and Isogai (2015) also observed that the greater the amount of NaClO during TEMPO reaction conditions, the lower the molar mass of TEMPO-oxidized celluloses Based on the two hypotheses presented above, a secondary function is discussed, related to catalyst TEMPO, as a “sacrificial molecule”, protecting polysaccharides from depolymerization processes induced by the reactive oxygen species (NaClO/NaBrO) present in the reaction medium for catalysis This was confirmed using automatic continuous online monitoring of polymerization reactions (ACOMP), size exclusion chromatography (SEC), different TEMPO concentrations and pHs (9, and 5), to evaluate ␤-elimination and unselective oxidation and depolymerization, respectively Material and methods 2.1 Plant material and polysaccharide extraction Seeds of Tamarindus indica L were provided by Conceic¸ão de Almeida, Bahia state, Brazil The ground seeds were submitted to an extraction of pigments and lipids, and the isolation of the xyloglucan (XG) was performed as described Ground seeds were defatted and depigmented under ethyl ether reflux, using a Soxhlet apparatus, and dried in a fume hood at room temperature The XG was isolated after aqueous extraction using a blender, followed by nylon cloth filtration The filtered solution was centrifuged at 10,000g, 40 ◦ C for 30 After centrifugation, the supernatant was filtered sequentially by cellulose acetate membranes (3.0, 0.8, 0.45 e 0.22 ␮m) and followed by precipitation using ethanol After drying under vacuum at 40 ◦ C, the powder obtained was termed XG The extraction, chemical and oligosaccharide characterization of this polysaccharide was published elsewhere (Spier et al., 2015) 2.2 Selective oxidation of xyloglucan The TEMPO selective oxidation reaction was performed according to studies reported by de Nooy et al (1995a, 1995b, 1996) Briefly, a XG solution (0.001 g cm−3 ) was dissolved overnight in ultrapure water (MilliQ system, USA) at 25 ◦ C, and filtered through 0.45 ␮m cellulose acetate filters (Millipore, Merck KGaA, Germany) The polymer solution was cooled and the reaction was performed at temperature of ± ◦ C under continuous flow of N2 In a reactor under stirring, 8.61 mg mL−1 of a 10% (v/v) sodium 141 ® hypochlorite solution (NaClO – SIGMA ) and 0.077 mg mL−1 NaBr ® ® (SIGMA ) were added together with TEMPO (SIGMA ), at concen−1 trations of 0, 0.015, 0.030, 0.060 and 0.150 mol L Previously to adding the oxidant mixture to the reaction, the secondary oxidants (NaClO/NaBrO) and TEMPO mixture were mixed, and the solution filtered through cellulose acetate of 0.45 ␮m The oxidation was performed at different pH values; 9, 7, and 5, adjusted with mol L−1 HCl solution During the reaction, the pH values were maintained using 0.05 mol L−1 NaOH solution After the oxidation process, the reaction was stopped using an alcoholic solution of NaBH4 (0.0015 g cm−3 ) Then, the pH was adjusted to The end products of oxidation were purified over 48 h dialysis against ultrapure water, precipitated in ethanol and dried at room temperature The oxidation products were analyzed through automatic continuous mixing (ACM) and size exclusion chromatography (SEC) to evaluate the polyelectrolyte effect and the oxidation effect on molar mass (Bayly, Brousseau, & Reed, 2002; Sorci & Reed, 2002) 2.3 Reaction monitoring and characterization of the end product 2.3.1 Real time reaction monitoring During the XG selective oxidation (item 2.2) the reaction was continuously monitored using an ACOMP system, with a Shimadzu LC-10AD pump and Shimadzu quaternary mixing module FCV10AL with 1.0 mL min−1 flow rate The pump continuously extracted sample from the reactor and flowed it through the following detector train: a Brookhaven BI-MwA multi-angle light scattering detector (MALS), a Shimadzu RI detector (RID 10A), and a custom built single capillary viscometer was a custom built reviewed previously (Reed, 2003) Additionally, this work presents first time polarimetric detection (AUTOPOL VI Automatic Polarimeter, Rudolph Research Analytical), and DLS detection (NanoDLS Particle Size Analyzer Brookhaven Instruments) in the ACOMP platform It is noted that using DLS in a flow cell requires stop-flow capability, since there is a velocity dependent term in the autocorrelation function not related to diffusion, so that motion of the scattering liquid must stop during the DLS measurement The BI Nanosizer is equipped with stop flow Measures of pH and conductivity were also monitored (Jenway 3540 pH & Conductivity meter) 2.3.2 Reaction end product characterization The XG end products of oxidation after purification (item 2.2), were characterized by Automatic continuous mixing (ACM) and SEC ACM experiments were performed to observe the polyelectrolyte properties of the oxidized products XG was evaluated at 0.001 g cm−3 , and two solutions were prepared for the same sample, one in purified water and the other in NaNO3 0.1 mol L−1 Both samples were filtered through cellulose acetate filters of 0.45 ␮m Using a Shimadzu LC-10AD pump and Shimadzu quaternary mixing module FCV10AL with 1.0 mL min−1 flow rate, a gradient was created from to 0.1 mol L−1 of NaNO3 The samples passed through a light scattering detector at ␭ = 660 nm (Brookhaven Instrument MwA), and the static light scattering signal was followed during the salt ramp with constant XG concentration SEC analyses were also carried out using a Shimadzu LC-10AD pump, Brookhaven Instruments Corp BI-MwA MALS detector, a Shimadzu RI, a custom-built viscometer, and Shodex OHpak SB806 HQ columm, using 0.1 mol L−1 NaNO3 with 0.02% (w/v) NaN3 as an eluent, and 0.8 mL min−1 flow rate Solutions (0.001 g cm−3 ) were prepared in the mobile-phase, during 16 h and passed through a 0.22 ␮m cellulose acetate filter (Millipore) 142 V.C Spier et al / Carbohydrate Polymers 170 (2017) 140–147 XGoxipH7 and XGoxipH5 are shown in the Supplementary material (Fig S1) To obtain the Zimm data a diluted polysaccharide concentration (c = 0.001 g cm−3 ) and q2 z 1, was used, according to Eq (1) Kc = R␪ Mw 1+ q2 < S > z + 2A2 c (1) where R is the Rayleigh scattering ratio, at a scattering vector amplitude defined as q = (4 n/ )sin(Â/2), where  is the scattering angle, c is the polymer concentration, z the z-averaged square of the radius of gyration and A2 is the second virial coefficient (obs: A2 effect was considered small enough and ignored), and Mw the weight average molar mass K is an optical constant (Eq (2)) given for vertically polarized incident light by 2n Fig mmol −COOH.g−1 of polymer as a function of time, at pH values of 9, 7, and in the presence of catalyst TEMPO (0.015 mol L−1 ), and also without TEMPO at pH (XG oxipH9,NT ) 2.4 1H and 13 C-1 H NMR analysis of xyloglucan The native and oxidized XG were analyzed by monodimensional NMR spectrum (hydrogen – H) and bidimensional (HSQC – heteronuclear single quantum coherence), in a BRUKER, DRX400 model, AVANCE series A mm inverse probe was utilized, with deuterated water (D2 O) as solvent and TMS-p (2,2,3,3-tetradeuterium-3-trimethysilyl sodium propionate salt) as reference for the calibration spectra (␦ = ppm) All analyses were performed at 60 ◦ C K= NA (2) n0 is the refraction index of the solvent, dn⁄dc is the differential index of refraction, NA is Avogadro’s number and the laser wavelength The reduced viscosity (Áred ) was obtained by VSample − VSolvent Áred = VSolvent − VZero Flow /cSample (3) where VSample , VSolvent and Vzeroflow are the voltage signals from the viscometer (differential of pressure) for the sample, solvent and zero flow, respectively Assuming, at diluted concentrations that intrinsic viscosity ([Á]) is ∼ = Áred , it was possible to determine the viscometric radius ␩ from Flory-Fox (1953), by Results and discussion 3⁄2 [Á] = TEMPO-catalyzed polysaccharide oxidation (mmol −COOH) was monitored from the amount of NaOH solution titrated during the reaction As observed in Fig 1, comparing the reactions at pH values of 9, 7, and 5, the amount in mmols −COOH per g of polymer was reduced, respectively from basic to acid conditions The rate, using first order kinetics, was 1.62 × 10−4 s−1 at pH 9, and at pHs and the rates are almost the same, ∼0.52 × 10−4 s−1 These three first reactions were made using the secondary oxidants (NaClO/NaBrO) and TEMPO at concentration of 0.015 mol L−1 However, when reactions were performed without TEMPO (XGoxipH9,NT ), the oxidation process occurred at a rate of 0.58 × 10−4 s−1 , with a total degree of polysaccharide oxidation higher than the reactions catalyzed by TEMPO, suggesting some non-selective oxidation in absence of TEMPO (Table 1) The first observation is that apparently the secondary oxidants present in the medium, NaClO/NaBrO, can be responsible for a nonselective oxidation of the polysaccharide In fact, Table shows that at pH = over 1.8x more oxidation occurs without TEMPO than with TEMPO as catalyst The XG oxidation products at pH with the system TEMPO/NaClO/NaBrO will be termed XGoxipH9 and for pHs and 5, XGoxipH7 and XGoxipH5 , respectively The reaction without TEMPO as catalyst, but in presence of the secondary oxidants NaClO/NaBrO at pH 9, will be termed XGoxipH9,NT The ACOMP reaction end product values are presented in Table 2, as a function of the degree of oxidation (mmol -COOH.g−1 of polymer), monitoring the results of oxidation from native to oxidized XG at different pHs: 9, and 5, with and without TEMPO as catalyst In Table dn/dc is the differential index of refraction of XG in solution In Fig 2, only the results of XGoxipH9 in the presence of TEMPO 0.015 mol L−1 and in the absence of TEMPO are shown For dn⁄dc ϕ0 S2 3⁄2 Á MW (4) where ϕ0 = 2.56 × 1023 is the Flory constant Because of solubility issues, reactions could only be carried out at low concentrations of XG ≤0.001 g cm−3 Hence, this presents an unusual context for ACOMP, which normally dilutes concentrated media from reactor by factors ranging from 10 to over 1000 times Here, XG dilution was unnecessary and so it was possible to simply circulate the reactor contents directly through the detector train The recirculation also permits full recovery of the final product, whereas conventional ACOMP normally loses a fraction of the material in the extraction and dilution stream, which is normally wasted The normal advantage of dilution is that the supporting solvent under which detection occurs can be modified at will; e.g ionic strength (IS) and pH can be changed, solvent mixtures made, etc In the case of XG, any dilution of the already very dilute reactor content degrades detector signals The disadvantage of the recirculation with no dilution is that light scattering cannot be lowered, which negatively affects the ability to monitor reaction kinetics due to the build-up of polyelectrolyte properties As observed by ACOMP experiments (Fig and Table 2) the sample XGoxipH9 presented a Mw reduction of 8.3% and z of 24%, suggesting that some depolymerization and some increasing in chain flexibility was observed during oxidation Sakakibara et al (2016) also observed that at pH there is some reduction in the molar mass of galactomannans and an increase in chain flexibility, due to reduction of persistence length of oxidized products at moderate ionic strength The acid groups formed during XG oxidation were continuously neutralized by titration with NaOH solution and, as expected, the polysaccharide even during oxidation processes was maintained V.C Spier et al / Carbohydrate Polymers 170 (2017) 140–147 143 Table First order rate constant (k) of oxidation and oxidation degree in presence and absence of TEMPO at 0.015 mol L−1 Sample k/10−4 (s−1 ) Oxidation degree (mmol −COOH.g−1 )* R Oxidation with TEMPO XGoxipH9 XGoxipH7 XGoxipH5 1.62 0.53 0.52 0.215 0.062 0.046 0.998 0.997 0.995 Oxidation without TEMPO XGoxipH9,NT 0.58 0.395 0.997 *at 1.1 × 104 s of reaction Table End product values from ACOMP; dn/dc, weight average molar mass (Mw ), radius of gyration (z ), intrinsic viscosity ([␩]), Flory-Fox radius of gyration (␩ ), hydrodynamic radius (Rh ) and optical rotation ([∝]) of Xyloglucan (XG) native and oxidized, after oxidation time of 1.1 × 104 s Sample dn/dc (cm3 g−1 ) Mw (105 g mol−1 ) z (nm) [␩] (cm3 g−1 ) ␩ (nm) Rh (nm) 25 [∝]D (◦ ) Native sample XG 0.140 4.8 120 517 39 194 +121 Oxidation with TEMPO 0.154 XGoxipH9 0.153 XGoxipH7 0.153 XGoxipH5 4.4 4.3 4.3 91 95 96 265 300 370 33 35 37 146 118 127 +83 +95 +101 Oxidation without TEMPO XGoxipH9,NT 0.153 1.6 46 165 10 35 +96 continuously in a low excluded volume state The change of the light scattering signal during the oxidation was not large, and small decreases are associated with small molar mass reduction Samples monitored by ACOMP at pH values of and yielded almost the same value of Mw and z (Table 2) However, for the sample XGoxipH9 , NT the Mw and z reduced 66.7% and 74%, respectively, confirming that in the absence of catalyst TEMPO, the degradation of XG was much more significant Two mechanisms can be used here to explain the Mw reduction observed by ACOMP experiments The first one was related to ␤-elimination and the second one due to reactive oxygen species (NaClO/NaBrO) present in the reaction medium Some authors previously observed that an increase in the amount of NaClO can be related to an increase in the degree of oxidation of the products (Milanovic, Kostic, Milanovic, & Skundric, 2012; Xu, Li, Cheng, Yang, & Qin, 2014) de Freitas et al (2004), Milanovic et al (2012) and Sakakibara et al (2016) observed a molar mass reduction during TEMPO mediated oxidations, for different polysaccharides, almost in the same experimental conditions ␤-elimination was minimized due to reduction of pH, however, our results proved that even at acid pH, the Mw reduction was also observed This clearly suggested that ␤-elimination is not the only mechanism of polysaccharide depolymerization from TEMPO oxidation reactions using the experimental conditions here (Isogai et al., 2011) In parallel, non-selective oxidation reactions from the reactive oxygen species present in the medium can be responsible for some depolymerization This hypothesis was confirmed in the experiments without TEMPO This is the first report that finds a function for TEMPO besides catalysis Here, it is deduced that TEMPO acts as a “sacrificial molecule” In the absence of TEMPO that is promptly oxidized by NaClO/NaBrO to nitrozonium ion, the secondary oxidants reacted with the polysaccharide, promoting a non-selective oxidation and depolymerization Based on that, TEMPO competes with the polysaccharide during oxidation, protecting it from depolymerization and non-selective oxidations Even catalytic amounts of TEMPO partially protected the extensive depolymerization of the polysaccharide The viscometer and DLS detectors also showed the same tendency of degradation, with [␩] and Rh decreasing during oxidation In both cases, the higher values of z are associated to a z-average, Table Determination of molar mass (Mw ), viscometric radius of gyration Á , dispersion (Ð = Mw /Mn ) and recovery of XG and XGoxi from SEC experiments SAMPLE Mw (105 g mol−1 ) XG 4.8 With TEMPO XGoxipH9 XGoxipH7 XGoxipH5 3.1 2.9 3.0 Without TEMPO 1.3 XGoxipH9,NT S Ð Recovery (%) 39 1.4 95 12 20 18 1.8 2.0 2.1 97 93 96 1.7 92 Á (nm) suggesting that large aggregates formed during the secondary oxidation/depolymerization weight the ACOMP light scattering measurements towards higher values Viscometry is much less sensitive to the presence of aggregates This technique was also used to provide S Á , which is a more reliable size parameter than light scattering z when aggregates are present (Table 2, Fig 2) The same contamination by aggregation was observed in experiments of de Freitas, Drenski, Alb, and Reed (2010) analyzing chitosan carboxymethylation, by Spier et al (2015) analyzing XG enzymatic depolymerization, due to self-association of the fragments of XG and by Mkedder et al (2013) studding the cellulase depolymerization of xyloglucan The values of [∝]25 D measured from ACOMP were less positive for all samples (Table 2, Fig 2), and apparently, were much more affected at higher pH or in the absence of TEMPO The optical rotation was modified, compared to native polymer, due to formation of glucuronic acid (GlcA) and galacturonic acid (GalA) Isbell & Frush (1943) observed an optical rotation reduction of 31%, at pH 9.0, from ␤-d-galactose to ␤-d-galacturonic acid, confirming that oxidation was occurring during real time measurements Depolymerization and mutarotation of the reducing carbohydrate have a non-negligible effect on optical rotation To confirm the aggregation the end products of oxidation at different pHs and in presence or absence of TEMPO were precipitated, purified and characterized by SEC (Table and Fig 3A and B) As clearly observed by SEC all the pH values led to Mw reduction, in the presence and absence of TEMPO, respectively This confirms that, V.C Spier et al / Carbohydrate Polymers 170 (2017) 140–147 4.5 10 10 3.5 10 10 2.5 10 10 A1 10 4.5 10 140 B1 140 120 -1 120 MOLAR MASS (g.mol ) 10 100 80 40 20 1.5 105 10 0.05 0.1 0.15 0.2 3.5 10 10 2.5 10 10 100 80 z z 60 105 60 40 20 1.5 105 0.25 10 0 -1 0.1 0.2 0.3 -1 0.4 0.5 mmol COOH.g polymer mmol COOH.g polymer A2 560 560 B2 -3 35 30 320 25 15 160 10 80 0 0.05 0.1 0.15 0.2 0.25 35 30 400 25 320 η η 20 240 480 20 240 15 160 10 80 0 -1 A3 0.2 0.3 0.4 300 120 B3 140 120 250 100 100 200 h h 60 100 80 150 60 [α]D (°) D [α] (°) 80 150 R (nm) 200 R (nm) 0.5 mmol COOH.g polymer 140 250 0.1 -1 mmol COOH.g polymer 300 (nm) 400 Reduced Viscosity (g.cm ) 40 (nm) Reduced Viscosity (g.cm-3) 40 480 (nm) (nm) MOLAR MASS (g.mol-1) 144 100 40 50 20 0 0.05 0.1 0.15 0.2 0.25 -1 mmol COOH.g polymer 40 50 20 0 0.1 0.2 0.3 -1 0.4 0.5 mmol COOH.g polymer Fig ACOMP of XGoxipH9 in presence of 0.015 mol L−1 TEMPO (A) and XGoxipH9 , NT (B) 1–Data from Zimm equation (Eq (1)), 2–data from viscometer (Eqs (3) and (4)) and 3- data from DLS and optical rotation, all as a function of mmol COOH.g−1 polymer during oxidation and online monitoring by ACOMP the presence of aggregates are affecting the end values of Mw and S z The end-products of oxidation were much better characterized using SEC analysis than from ACOMP experiments, mainly due to the presence of aggregates in solution The polyelectrolyte behavior of XG oxidized samples was determined by ACM experiments, as presented in Fig 4, confirming the oxidation of the polysaccharide due to increasing of the dependence of Kc/R␪ as a function of NaNO3 concentration In the Supplementary material (Fig S2.1) presented the H NMR characterization of the anomeric hydrogen for native and oxidized XGs obtained at different values of pH (9, and 5) with TEMPO and also the sample at pH without TEMPO At 5.43 ppm the chemical shift of the ␣-d-Xyl substituted for ␤d-Gal or ␤-d-GalA was observed, and at 5.23 ppm the ␣-d-Xyl not substituted by ␤-d-Gal or ␤-d-GalA was observed The ␦ for ␤-d-Glc was observed at 4.85 ppm and at 4.84 ppm for ␤-d-Gal For higher degree of oxidation another ␦ was observed at 4.77–4.78 ppm (XG oxipH9.0 and XG oxipH9.0,NT ) For XG oxipH5.0 and XG oxipH7.0 it can be observed only as a shoulder Based on the chemical shift above, the ratio Glc: Xyl: Gal was determined for native XG as 2.6: 2.1: 1.0, respectively No reducing sugar was observed, since the polysaccharide was purified by dialysis previously to characterization, as described in the item 2.2 The ␤-d-Gal and ␤-d-Glc chemical shift at 4.85 ppm and 4.84 ppm were used to estimate the amount of Gal units still V.C Spier et al / Carbohydrate Polymers 170 (2017) 140–147 145 mol.L-1 0.015 mol.L-1 0.030 mol.L-1 0.060 mol.L-1 0.150 mol.L-1 mmol COOH/ g polymer mmol COOH/g polymer 0.4 0.35 0.3 0.25 0.1 0.08 0.06 0.04 0.02 0 500 1000 1500 2000 2500 3000 Time (s) 0.2 0.15 0.1 0.05 ◦ Fig Light scattering (LS) @ 90 elution profile from SEC of XG native and TEMPO oxidized products XGoxipH9 , XGoxipH7 , XGoxipH5 and XGoxipH9,NT 0 2000 4000 6000 8000 10 Time (s) Fig mmol COOH/g of polymer as a function of time of XG oxidation at pH and TEMPO concentrations of 0, 0.015, 0.030, 0.060 and 0.150 mol L−1 , compared with XG oxidation at pH without TEMPO (XGoxipH9,NT ) The first 3000 s is inserted, for TEMPO concentrations of 0.030, 0.060 and 0.150 mol L−1 Fig Kc/R␪ @ 90◦ of XG and oxidized products XGoxipH9 , XGoxipH7 , XGoxipH5 with tempo, and XGOXIpH9,NT , versus NaNO3 concentration, by ACM remaining in the XG The deconvoluted spectra, Supplementary material (Fig S2.2), were used to determine the total area related to ␤-d-Gal + ␤-d-Glc of XG oxi samples Such values were normalized to ␤-d-Gal and ␤-d-Glc in native XG From this approach, the amount of ␤-d-Gal in XG from 16.7% in native XG reduced to 15,6%, 15.5%, 13.4% and 12.8%, respectively, to XGoxipH5.0 , XGoxipH7.0 , XGoxipH9.0 and XGoxipH9.0,NT Comparing the amount of -COOH per gram of polysaccharide determined by titration, and the amount of Gal reduction, a very interesting correlation can be observed, with approximately the same amount of oxidation observed per gram of polysaccharide (Table 1), except to XGoxipH9.0,NT This suggested that ␤-d-Gal units were oxidized partially, to ␤-d-GalA in TEMPO samples, however, other free units of Glc and non-selective oxidation sites can not be discarded, during TEMPO reaction Using the HSQC spectra (13 C-1 H), supplementary material (Fig S2.3), were possible to identify at anomeric H and 13 C region, the ␤-d-Gal chemical shifts at 4.84/104.63 ppm and ␤-d-Glc at 4.83/103.34 ppm At 5.42 ppm the chemical shift of the ␣-d-Xyl substituted for ␤-d-Gal and at 5.22 ppm the ␣-d-Xyl not substituted All these chemical shifts are compatible with the hydrogen mono-dimensional spectra For XG oxidized samples, it was possible to observe a new correlation at, approximately, 4.78/103.6 ppm, attributed by Lucyszyn et al (2009) to the chemical shift of ␤-dGalA The chemical shift at 4.22/68.95 ppm was the C6 of ␤-d-Glc and at 4.06/61.41 ppm the C6 of ␤-d-Glc The 3.85/61.75 ppm and 4.01/61.73 ppm are related to H5 and C5 of xylose The other chemical shifts are attributed to C2, C3, C4 and C5 of XG, as described by Arruda et al (2015) To confirm the second effect of TEMPO, protecting the polysaccharide from degradation, different concentrations of the catalyst were used, keeping constant the amount of the secondary oxidants NaClO/NaBrO The concentrations used were, 0.030, 0.060 and 0.150 mol L−1 , corresponding to 2, and 10 times of the initial amount of TEMPO (0.015 mol L−1 ) All these TEMPO concentrations were compared to experiments in the absence of TEMPO (0 mol L−1 ) Table (Fig 5) SEC analysis of the XG oxidized products at pH using different TEMPO concentrations, are presented in Table and Fig As clearly observed on Fig an increase in the amount of the catalyst TEMPO, reduced the degree of depolymerization Only increasing 10 times the initial TEMPO concentration (from 0.0150 mol L−1 to 0.150 mol L−1 ) the Mw reduction was of only of 8% Further increase in TEMPO concentration did not provided any significant modification of Mw values, and can be related to ␤-elimination process Table Rate constant of oxidation (k) and degree of oxidation measured at pH 9, using different concentration of TEMPO Sample TEMPO (mol L−1 ) k/10−4 (s−1 ) Degree of oxidation (mmol g−1 )* R XGoxipH9,NT XGoxipH9 XGoxipH9 XGoxipH9 XGoxipH9 0.015 0.030 0.060 0.150 0.58 1.62 18.8 34.2 45.7 0.395 0.215 0.092 0.097 0.095 0.997 0.998 0.976 0.998 0.998 *at 1.1 × 104 s of reaction 146 V.C Spier et al / Carbohydrate Polymers 170 (2017) 140–147 Table Determination of molar mass (Mw ), viscometric radius of gyration Á , dispersion (Ð = Mw /Mn ) and recovery from SEC experiments of XG and XG oxidized at pH at different concentrations of TEMPO AMOSTRA TEMPO (mol L−1 ) Mw (105 g mol−1 ) XG XGoxipH9,NT XGoxipH9 XGoxipH9 XGoxipH9 XGoxipH9 0 0.015 0.030 0.060 0.150 4.8 1.3 3.1 3.6 4.1 4.4 S 39 12 18 26 30 Á (nm) Ð Rec (%) 1.4 1.8 1.4 1.6 1.5 1.5 95 92 97 96 94 97 Acknowledgments Support for this work was provided by Brazilian funding agencies CNPq (Conselho Nacional de Pesquisa), process no 477275/2012-5 and 306245/2014-0 Rede Nanobiotec/CapesBrazil, project 34 and Nanoglicobiotec-Ministry of Science and Technology/CNPq no 564741/2010-8 and no.555169/2005-7 Vivian C Spier was a beneficiary of a doctoral fellowship from CAPES and by collaboration of the Tulane Center for Polymer Reaction Monitoring and Characterization (PolyRMC) Appendix A Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.carbpol.2017.04 064 References Fig SLS @ 90◦ elution profile from SEC of XG native and TEMPO oxidized products XGoxipH9 using TEMPO concentration of 0, 0.015, 0.030, 0.060 and 0.150 mol L−1 These results demonstrate that one interesting approach to reduce the depolymerization of polysaccharides, during TEMPO/NaClO/NaBrO oxidation is to increase the amount of primary catalyst, not affecting the ␤-elimination process elimination process, but reducing depolymerization induced by secondary antioxidants as NaClO and NaBrO Conclusion In this manuscript, for the first time, TEMPO reactions, used to selectively oxidize polysaccharide, were monitored through real time analysis, measuring molar mass, radius of gyration, viscosity, hydrodynamic radius and optical rotation The last two are used here for the first time during ACOMP reactions The optical rotation confirms the formation of uronic acids in XG, and can be considered an important tool to monitor real time modification of polysaccharides Independently of the pH (9, or 5), using 0.015 mol L−1 of TEMPO, some depolymerization was observed, proving that ␤-elimination is not the only mechanism allowing molar mass reduction In experiments without TEMPO as a catalyst, a much more significant molar mass reduction was observed, and at this point attributed to the secondary oxidants presented in the medium (NaClO/NaBrO) TEMPO could protect XG from nonselective oxidations that culminate with molar mass reduction, since depolymerization reduced with crescent amounts of TEMPO During XG oxidation, TEMPO acts not only as a catalyst, but also as a “sacrificial molecule”, reacting with the secondary oxidants The nitrosonium ion formed, the effective catalyst, was responsible for the selective primary alcohol oxidation and its oxidation protected the polysaccharide from non-selective degradative oxidations Based on this manuscript, the amount of TEMPO 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amount of the catalyst TEMPO, reduced the degree of depolymerization Only increasing 10 times the initial TEMPO concentration (from 0.0150 mol L−1 to 0.150 mol L−1 ) the Mw reduction was of only of. .. C2, C3, C4 and C5 of XG, as described by Arruda et al (2015) To confirm the second effect of TEMPO, protecting the polysaccharide from degradation, different concentrations of the catalyst were... that, TEMPO competes with the polysaccharide during oxidation, protecting it from depolymerization and non-selective oxidations Even catalytic amounts of TEMPO partially protected the extensive depolymerization