Amalie Solberg

Ingrid V Mo

Finn L Aachmann

Christophe Schatz

Bjørn E Christensen

Alginate-based diblock polymers: Preparation, characterization and Ca-induced self-assembly †

Renewable resources can provide a range of different polysaccharide blocks that can be used to prepare new types of stimuli-responsive polysaccharide-based block copolymers. Alginates are natural polysaccharides widely used as biomaterials. Functional properties depend on the content and distribution of the two 4-linked monomers (β-D-mannuronate (M) and α-L -guluronate (G)). Blocks of Lguluronate (Gn), are responsible for cooperative binding of calcium ions and hydrogel formation.

Incorporation of such blocks in block polysaccharide copolymers would represent a new class of engineered, Ca-sensitive biomacromolecules. Dioxyamines and dihydrazides have recently been shown to be well suited for preparation of block polysaccharides structures. Here we first show that when applied to alginate blocks (Gn and Mn) the two types are both very reactive, but the detailed distribution

Introduction

Alginates are linear, anionic polysaccharides of 4-linked β-D-mannuronic acid (M) and its C5-epimer, 4linked α-L-guluronic acid (G) (Fig. 1). They are produced by brown algae, some red algae, as well as some bacterial species. 1,2 Alginates have found numerous application areas in foods, biomaterials and pharmaceuticals due to their mild gelation properties in addition to being used as viscosifiers. 1 The monomers are located within homopolymeric M-blocks and G-blocks, as well as alternating (..MG..) blocks. The blocks coexist in alginate chains but in widely different proportions, depending on the source. 3 The G-blocks are largely responsible for the gel formation with divalent cations. [START_REF] Smidsrød | Molecular basis for some physical properties of alginates in the gel state[END_REF] Although alginates may themselves be classified as block polysaccharides, the length and distribution of the three block types vary due to the inherent compositional heterogeneity of alginates. 1 The relationship between the gelling properties of alginates with multivalent cations and the structure, sequence and chain length of alginates has been extensively investigated for decades. 1,2,[START_REF] Smidsrød | Molecular basis for some physical properties of alginates in the gel state[END_REF] In contrast, the properties of Author manuscript of article published in Polymer Chemistry 2021, 12, 5412 DOI: 10.1039/D1PY00727K 3 isolated M-and G-blocks and their incorporation in block polysaccharides have only been sporadically studied.

Fig. 1 Preparation of guluronate blocks (Gn) from alginates and their terminal conjugation to an activated polysaccharide, followed by the chain dimerization with Ca 2+ and Gn-b-Dexm.

Block polysaccharides consist of two or more oligo-or polysaccharide blocks connected at the chain termini through a suitable conjugation method. One of the main motivations for the study of diblock polysaccharides is to exploit their self-assembly properties under defined conditions. Blocks can be combined such that one block can develop short-range attractive interactions while the other develop long-range repulsive interactions. The resulting self-assembly is a spontaneous process leading to a great diversity of structures whose characteristics depend on the molecular parameters of the individual blocks. In the case of alginates, the strong and specific interactions of G-blocks with Ca 2+ , Sr 2+ and Ba 2+ could be balanced by repulsive interactions with a neutral polysaccharide block such as dextran when linearly conjugated to the G block.

We recently utilised click-like reactions based on a dihydrazide and a dioxyamine [START_REF] Novoa-Carballal | Synthesis of polysaccharide-b-PEG block copolymers by oxime click[END_REF][START_REF] Bondalapati | Rapid End-Group Modification of Polysaccharides for Biomaterial Applications in Regenerative Medicine[END_REF] to prepare chitinand chitosan-based [START_REF] Mo | 2, 5-Anhydro-d-Mannose End-Functionalized Chitin Oligomers Activated by Dioxyamines or Dihydrazides as Precursors of Diblock Oligosaccharides[END_REF][START_REF] Mo | Activation of enzymatically produced chitooligosaccharides by dioxyamines and dihydrazides[END_REF] diblock polysaccharides through conjugation at the reducing end. In the case of polyuronates such as alginates corresponding click reactions are particularly attractive alternatives to the conventional alkyne-azide cycloaddition catalysed by copper because of its strong complexation with polyuronates. [START_REF] Smidsrød | Dependence upon uronic acid composition of some ion-exchange properties of alginates[END_REF] For alginate blocks very little is known about the reactivity at the reducing ends, but it has been demonstrated that an aminoxy-peptide can conjugate to alginates by aniline-catalysed oxime click. [START_REF] Bondalapati | Rapid End-Group Modification of Polysaccharides for Biomaterial Applications in Regenerative Medicine[END_REF] Here we show that both oligoguluronates (G-blocks, Gn) and oligomannuronates (M-blocks, Mn), when compared to e.g. dextran and chitosan, react relatively fast at the reducing end with both PDHA (O,O'-1,3,-propanediylbishydroxylamine dihydrochloride) and ADH (adipic acid dihydrazide), with no or low dependence on the chain length. We further study the reactivity of Gn with several PDHA/ADH-activated oligosaccharides and oxyamine-functionalized PEGs. The oxime/hydrazone reduction with PB is also studied. Protocols for effective conjugations are provided. As proof-of-principle we prepare several diblock-polysaccharides based on oligoguluronate. We further demonstrate characteristic solution properties of an oligoguluronate-based diblocks associated with the flexible linker by means of multidetector SEC. Finally, we show that Gn-PDHA-dextran diblocks forms nanoparticles in the presence of Ca 2+ ion for certain combinations of chain lengths, in contrast to pure oligouronates, which form solid precipitates.

Experimental

Materials

Guluronic acid oligomers (G oligomer) with two different molecular weight distributions were prepared [START_REF] Nordgård | Oligosaccharides as modulators of rheology in complex mucous systems[END_REF] from partially acid hydrolysed, high guluronate alginate from Laminaria hyperborea stipes by acid precipitation to give oligomers with a number average degree of polymerization (DPn) of 21 and a fraction of guluronic acid (FG) of 0.90 (determined by NMR). Samples with DPn of 4 and 10 were prepared by further hydrolysis.

Trimannuronate was an inhouse sample prepared according to previously described methods. [START_REF] Gimmestad | The Pseudomonas fluorescens AlgG protein, but not its mannuronan C-5-epimerase activity, is needed for alginate polymer formation[END_REF][START_REF] Ertesvåg | Modification of alginate using mannuronan C-5-epimerases[END_REF] Dextran T-2000 (Mw = 2 000 000 g/mol) was purchased from Pharmacia Fine Chemicals. Maltotriose, trigalacturonic acid, adipic acid dihydrazide (ADH), O,O'-1,3,-propanediylbishydroxylamine dihydrochloride (PDHA) and 2-methylpyridine borane complex (α-picoline borane) was purchased from Sigma-Aldrich. PEG linkers were purchased from BroadPharma, US. All other chemicals were obtained from commercial sources and of analytical grade.

Semi-preparative gel filtration chromatograph (GFC)

Three Superdex 30 (preparative grade) columns (HiLoad 26/60, 26 mm x 60 cm, GE Healthcare Life sciences) were connected in series. The mobile phase (0.1 M ammonium acetate (AmAc), pH 6.9) was eluted at a flow rate of 0.8 ml/min. Samples (0.3 -65 mg/ml) were dissolved in the mobile phase and injected. The separation was monitored by an on-line RI detector (Shodex R1-101). Fractions were collected (4 -8 ml per fraction) and pooled according to elution volume. Fractions were dialysed (DPn < 7 with 100 -500 Da MWCO and DPn ≥ 7 with 3.5 kDa MWCO) against 50 mM NaCl (2 shifts) and MQ until the conductivity was below 2 μS and freeze-dried.

NMR spectroscopy

Alginate samples were dissolved in 470 -500 μl D2O (99.9% D; Sigma-Aldrich) (10 -12 mg/ml). For samples where pD was adjusted, DCl or NaOD was used. Samples were either analysed at the temperature 25 °C, 27 °C, or 82 °C.

Homo-and heteronuclear NMR were recorded on a Ascend 400 MHz Avance III HD instrument equipped with a 5 mm SmartProbe, Advance ultra-shield 600 MHz Avance III HD instrument equipped with 5 mm cryogenic CP-TCI, Bruker Ascend 600 MHz NEO instrument equipped with 5 mm iProbe or a Ascend 800 MHz Avance III HD instrument equipped with 5 mm cryogenic CP-TCI all from Bruker BioSpin AG, Fällanden, Switzerland.

Characterization of alginate oligomers was performed by obtaining 1D 1 H-NMR spectra at 25 °C or 82 °C on the 400 MHz or NEO 600 MHz, or at 27 °C on the 600 MHz with cryogenic probe. Time course NMR experiments was performed by obtaining 1D 1 H-NMR spectra at set time points over the course of the reaction on the 600 MHz with cryogenic probe at 27 °C or the NEO 600 MHz at 25 °C. Spectra were recorded every 30-40 min for the first 3 hours, then every 1 -3 hours until equilibrium was established. Reactions were performed in deuterated acetate buffer (500 mM, pD 4 and 5). TSP was added to a final concentration of 2 mM. The pD was adjusted with 1 M NaOD and the measured pH* (by the instrument calibrated by non-deuterated standards) was corrected as follows; pH = 0.9291 x pH* + 0.421. [START_REF] Baudendistel | Real-Time NMR Studies of Oxyamine Ligations of Reducing Carbohydrates under Equilibrium Conditions[END_REF] Guluronate oligomers (Gn), mannuronate oligomers (Mn), dextran oligomers (Dexm), maltotriose (Glc3) and galacturonic acid (GalA3) (7 -20.1 mM) and 2 or 10 equivalents ADH or PDHA was dissolved in NaAc-buffer. Reduction of conjugates was studied by adding PB (3 equivalents) to the NMR tube.

Integration was used to estimate the relative molar ratios of reactants and products (using either 1 H of the non-reducing end or TSP as internal standard).

Chemical shift assignment of equilibrium reaction mixtures was recorded on the NEO 600 MHz at 25 °C or 82 °C by acquiring the following spectra: 1D 1 H, 2D [START_REF] Baudendistel | Real-Time NMR Studies of Oxyamine Ligations of Reducing Carbohydrates under Equilibrium Conditions[END_REF] All data were recorded, processed and analysed using TopSpin software version 3.5pl7 or 3.6.1 (Bruker BioSpin).

SEC-MALS

Molar masses and intrinsic viscosities were analysed by Size Exclusion Chromatography (SEC) with

Multiangle Light Scattering (MALS). Samples were dissolved in the mobile phase (0.15 M NaNO3 with 10 mM EDTA) and filtered (0.45 μm) prior to injection. An Agilent Technologies 1260 IsoPump with a 1260 HiP degasser was used to maintain a flow of 0.5 ml/min during analyses. 50 -100 μl were injected from an Agiel Technologies Vialsampler. TKS Gel columns 4000 and 2500 PWXL were connected in series.

DAWN Heleos-II and ViscoStar II detectors from Wyatt Technology were connected in series with a Shodex refractive index detector (RI-5011). Astra 7.3.0 software was used for data collection and processing.

Preparation of oligouronate conjugates

For preparative purposes, guluronate (G) or mannuronate (M) oligomers (20.1 mM) were dissolved in NaAc-buffer (500 mM, pH 4) and 10 equivalents PDHA/ADH (201.0 mM) were added. After about 24 h, 3 equivalents (60.3 mM) of α-picoline borane (PB) were added. The mixture was left at room temperature for 120, 22, 39 and 12 hours for Gn-PDHA, Mn-PDHA, Gn-ADH and Mn-ADH, respectively. Samples were placed on shaking during reduction. The reaction was terminated by dialysis (for DPn < 7 with 100 -500 Da MWCO and for DPn ≥ 7 with 3.5 kDa MWCO) against 50 mM NaCl until all insoluble PB had been removed. The solution was subsequently dialysed against MQ water for 2 -5 h to remove high excess salt before the solution was freeze dried. The conjugates were subsequently purified by GFC.

Reduction of conjugates at elevated temperatures

Oligoguluronate (20.1 mM) was dissolved in deuterated NaAc-buffer (500 mM, pD 4.0) and PDHA (40.2 mM) was added. After 24 h, 60.3 mM (3 equiv.) PB was added, and the reaction mixture was placed in a water bath (preheated to 40 °C). After 6, 10, 21 and 24 hours, samples (0.5 ml) were taken and analsd by 1 H 1D NMR, the yield (%) of reduced conjugates was obtained by integration.

Preparation of oligoguluronate diblocks (Gn-b-Gn)

A guluronate decamer (G10) (DP determined by 1 H 1D NMR) was dissolved in NaAc-buffer (500 mM, pH 4.0) to a concentration of 20.1 mM. 0.5 equivalents ADH was added to the solution and the reaction was placed on shaking for 24 h at room temperature. Six equivalents of (solid) PB (120.6 mM) were added. The mixture was further shaken for up to 40 hours. The reduction was terminated by dialysis against 50 mM NaCl (3.5 kDa MWCO), removing any solid PB from the reaction mixture. The solution was subsequently dialysed against MQ water for 2 -5 h and freeze dried. The diblocks were purified by GFC.

Preparation of guluronate-b-dextran (Gn-b-Dexm) block copolymers

Gn-b-Dexm were prepared in two steps. Hight molecular weight dextran (T-2000) was partially hydrolysed in 0.05 M HCl at 95 °C to obtain DPn (by NMR [START_REF] Paulo | Production, extraction and characterization of exopolysaccharides produced by the native Leuconostoc pseudomesenteroides R2 strain[END_REF][START_REF] Cheetham | Structure of the linear, low molecular weight dextran synthesized by a D-glucosyltransferase (GTF-S3) of Streptococcus sobrinus[END_REF][START_REF] Van Dijk-Wolthuis | Synthesis, characterization, and polymerization of glycidyl methacrylate derivatized dextran[END_REF] ) of ca. 34 (2.25 h) and 18 (6 h). Following cooling and neutralization (with dilute NaOH), the solution was dialysed (3.5 kDa MWCO) and freeze dried. They were further activated with PDHA prior to fractionation as described previously. [START_REF] Mo | 2, 5-Anhydro-d-Mannose End-Functionalized Chitin Oligomers Activated by Dioxyamines or Dihydrazides as Precursors of Diblock Oligosaccharides[END_REF] In brief, dextran was dissolved in NaAc-buffer (500 mM, pH 4.0) to a concentration of 10 mM and 10 equivalents of PDHA (100 mM) were added. After 24 h, 20 equivalents of PB (200 mM) were added and the reaction proceeded at 40 °C for 24 hours. The reaction was terminated by dialysis and freeze drying. Narrow molar mass fractions were further prepared by semi preparative GFC as described above. Fractions were freeze dried directly, removing volatile AmAc, and characterized by 1 H 1D NMR and SEC-MALS. In the second step, defined fractions of dextran-PDHA (7 mM) were dissolved in NaAc buffer (500 mM, pH 4.0) and oligoguluronate (21 mM) was added. The solution was shaken overnight before 3 equivalents of PB (63 mM) were added. The reaction was left on shaking for 120 hours before it was terminated by dialysis (3.5 kDa MWCO) against 50 mM NaCl and freeze dried. The block copolymer was purified by GFC.

Solubility and particle formation of oligoguluronate-b-dextran monitored by DLS

Gn-b-Dexm diblocks (n = 10, 40 and m = 100) (10 mg/ml) were dissolved in 1 ml 10 mM NaCl overnight before filtering (0.22 μm). After 24 h, the sample was dialysed (Float-A-Lyser 100 -500 Da) against 20 mM CaCl2 containing 10 mM NaCl (1 L). Samples (50 -100 μL) were taken regular time intervals. The scattering intensity (kilo counts per second, kcps) and intensity distribution were determined using a ZetaSizer Nano ZS (Malvern Instruments, UK) (25 °C, λ = 632.8) with back scattering detection (173°).

The number distribution was calculated assuming that the refractive index (1.590) and absorption factor (0.010) of polystyrene latex could be used. All samples were analysed using 8 runs with duration of 30 -40 sec. All samples were analysed by minimum 5 consecutive measurements. Data were acquired and analysed using Malvern Zetasizer Software Version 7.12.

Results and discussion

The chemistry and reaction kinetics of the reaction at the reducing ends of oligoguluronate and oligomannuronate with a dioxyamine (PDHA) and a dihydrazide (ADH) were first studied using trimers (G3 and M3) as model systems. Short oligomers ease the NMR characterisation, and the findings can further be compared to previously reported data for dextran, chitosan and chitins. [START_REF] Mo | 2, 5-Anhydro-d-Mannose End-Functionalized Chitin Oligomers Activated by Dioxyamines or Dihydrazides as Precursors of Diblock Oligosaccharides[END_REF][START_REF] Mo | Activation of enzymatically produced chitooligosaccharides by dioxyamines and dihydrazides[END_REF] The reaction schemes for conjugation with guluronate and PDHA/ADH, including the reduction of Schiff bases to the stable secondary amines, are shown in Fig. 2. Pure oligomannuronate and oligoguluronate blocks were prepared by conventional methods based on partial hydrolysis and pH-dependent fractional precipitation (Fig. S2 †). [START_REF] Ertesvåg | Modification of alginate using mannuronan C-5-epimerases[END_REF][START_REF] Haug | A study on the constitution of alginic acid by partial acid hydrolysis[END_REF][START_REF] Haug | Studies on the sequence of uronic acid residues in alginic acid[END_REF][START_REF] Holtan | Characterization of the hydrolysis mechanism of polyalternating alginate in weak acid and assignment of the resulting MGoligosaccharides by NMR spectroscopy and ESI-mass spectrometry[END_REF] Initial conditions for conjugation were otherwise identical to previous studies of dextran and chitin/chitosan oligomers, [START_REF] Mo | 2, 5-Anhydro-d-Mannose End-Functionalized Chitin Oligomers Activated by Dioxyamines or Dihydrazides as Precursors of Diblock Oligosaccharides[END_REF][START_REF] Mo | Activation of enzymatically produced chitooligosaccharides by dioxyamines and dihydrazides[END_REF] i.e. 500 mM acetate (strong buffering needed, especially for oxyamines [START_REF] Baudendistel | Real-Time NMR Studies of Oxyamine Ligations of Reducing Carbohydrates under Equilibrium Conditions[END_REF] ) pH 4.0, room temperature (RT). [START_REF] Mo | 2, 5-Anhydro-d-Mannose End-Functionalized Chitin Oligomers Activated by Dioxyamines or Dihydrazides as Precursors of Diblock Oligosaccharides[END_REF][START_REF] Mo | Activation of enzymatically produced chitooligosaccharides by dioxyamines and dihydrazides[END_REF][START_REF] Baudendistel | Real-Time NMR Studies of Oxyamine Ligations of Reducing Carbohydrates under Equilibrium Conditions[END_REF] The reactions were systematically monitored by recording NMR spectra at regular intervals until equilibrium was established. However, with previously used standard conditions (20 mM oligomer and 10 equivalents of PDHA/ADH) the reactions turned out to be too fast for accurate determination of the kinetics of reaction by time course NMR. Subsequent studies were mostly conducted at 7 mM oligomer with 2 equivalents PDHA/ADH.

Conjugation with PDHA

Results for the G3-PDHA system at equilibrium (with 2 equivalents PDHA) are shown in Fig. 3a. The 1 H-NMR spectrum includes annotations of the major resonances. Complete disappearance of reducing end signals is balanced by the appearance of E-and Z-oximes (2:1 molar ratio) (Fig. S4 -S7 †). N-pyranosides could hence not be found, which seems to be rare for hexoses, and to our knowledge only reported for a mannose-oxyamine system. [START_REF] Baudendistel | Real-Time NMR Studies of Oxyamine Ligations of Reducing Carbohydrates under Equilibrium Conditions[END_REF] The reaction was indeed rapid as equilibrium was established already after 90 minutes. The equilibrium reaction mixture was further analysed by 2D NMR (Fig. S5a †) to resolve and assign resonances that were partially overlapping in 1D NMR. Minor forms of E and Z oximes (approximately 5%) appeared at slightly higher chemical shifts than the main peaks (annotated (E')/(Z')oximes in Fig. 3a). When purifying unreduced G3-PDHA by GFC and dialysis, the relative amount of the minor forms increased considerably. Their presence was, however, independent of pH in the range 6 -11.8 (Fig. S5b †), precluding lactonization as a possible explanation. The detailed chemistry of the minor forms is hence not understood at this time, however, both forms do become reduced in the presence of

PB (see below).

A kinetic plot for the conjugation with G3 and PDHA (2 equiv.) is shown in the Supplementary Information (Fig. S6 †). The experimental data were fitted to a simplified kinetic model described in detail previously, [START_REF] Mo | Activation of enzymatically produced chitooligosaccharides by dioxyamines and dihydrazides[END_REF] providing rate constants (kT and k-T) for the combined yield (total oximes) of the reaction, i.e. by treating the reaction products as a single component. [START_REF] Mo | 2, 5-Anhydro-d-Mannose End-Functionalized Chitin Oligomers Activated by Dioxyamines or Dihydrazides as Precursors of Diblock Oligosaccharides[END_REF] The reaction rates were taken to be first order with respect to the concentrations of the reactants. [START_REF] Mo | 2, 5-Anhydro-d-Mannose End-Functionalized Chitin Oligomers Activated by Dioxyamines or Dihydrazides as Precursors of Diblock Oligosaccharides[END_REF] Indeed, as shown in the Supplementary Information (Fig. S6-S17 †), all experiments were well described by the model. It can in particular be used to predict the kinetics when concentrations have to be changed, for example for longer DPs. By the same model, the times for reaching 50% and 90% of the equilibrium yield (t0.5 and t0.9) were obtained. They turned out to be 10-15 times shorter than for a dextran trisaccharide conjugated to PDHA under identical concentrations and conditions (Fig. S9 †). Hence, the fast reaction and excellent yield both qualify for the term 'oxime click' in this case. Being in addition aniline free and copper-free the 'oxime click' method is well suited for oligoguluronate.

To investigate the role of DP the reaction was studied with an oligoguluronate with DP 8. Under otherwise equal conditions the octamer reacted about two times slower than the trimer (Fig. S8 †).

Higher DPs were investigated but gave progressively less quantifiable peak integrals in NMR (broad peaks being too close to the HDO resonance). However, complete PDHA-conjugated oligoguluronate with higher DP could indeed be prepared as shown below.

The reaction was also studied at pD 5 (otherwise identical conditions). Complete conversion was obtained also here (Fig. S10 †), but the rate of the reaction was about 3 times lower than at pD 4 (Fig. S11 †). Lowering pH is known to increase the reaction rate but reduce the yield. [START_REF] Mo | Activation of enzymatically produced chitooligosaccharides by dioxyamines and dihydrazides[END_REF][START_REF] Baudendistel | Real-Time NMR Studies of Oxyamine Ligations of Reducing Carbohydrates under Equilibrium Conditions[END_REF] Hence, pD 4 is preferred as it provides sufficiently rapid reaction without entering the range where longer oligoguluronates become protonated and insoluble. [START_REF] Haug | Composition and properties of alginates[END_REF] Corresponding results for trimannuronate (M3) with PDHA are shown in Fig. 3b. Oxime resonance structures analogous to those of G3-PDHA were observed. The E/Z ratio was 4:1, which is twice what has been observed for oligoguluronate. Also in this case, (E) and (Z)-oximes were almost exclusively formed, with minor forms (E'/Z') accounting for about 5%. A combination of 1D selective COSY, TOCSY, and 2D heteronuclear NMR spectra was used to verify the structure of purified M3-PDHA (Fig. S13-14 †). The kinetics was otherwise quite similar what was obtained for G3 (Table 1) (Fig. S15 †).

Conjugation with ADH

Conjugation with oligoguluronate and oligomannuronate with ADH was studied using a similar approach as described for PDHA. For oligoguluronates only small amounts (< 5%) corresponding to (E) or (Z) hydrazones were observed, whereas a major resonance appeared in 1 H-NMR as a doublet at 4.28 ppm ( 3 J(H1-H2) 9.54 Hz (Fig. 4a), in agreement with the formation of β-N-pyranosides. This is in line with conjugation of hydrazides and other reducing sugars, for example ADH-conjugated dextran. [START_REF] Mo | 2, 5-Anhydro-d-Mannose End-Functionalized Chitin Oligomers Activated by Dioxyamines or Dihydrazides as Precursors of Diblock Oligosaccharides[END_REF][START_REF] Mo | Activation of enzymatically produced chitooligosaccharides by dioxyamines and dihydrazides[END_REF][START_REF] Kwase | Protecting-Group-Free Glycoconjugate Synthesis: Hydrazide and Oxyamine Derivatives in N-Glycoside Formation[END_REF] The reaction reached equilibrium after 1.5 h (Fig. S16 †). However, the total conversion was only 48% with 7 mM oligoguluronate and 2 equiv. of ADH. This is also in agreement with previous findings for ADH conjugation to oligosaccharides. [START_REF] Mo | Activation of enzymatically produced chitooligosaccharides by dioxyamines and dihydrazides[END_REF] The rate constants are included in Table 2, and the kinetic plot is included in Supplementary Information (Fig. S17 †). To obtain higher conversion an experiment with G6 (20.1 mM) using 10 equiv. of ADH was conducted. A yield 88% was obtained after 8 h, which fits well with the predicted yield (87%) estimated by the kinetic model.

The conjugation of ADH to oligomannuronates turned out to be somewhat different from that of oligoguluronates. Contrary to oligoguluronates, significant amounts of hydrazones were in this case detected by characteristic resonances 7 at 7.3 and 7.6 ppm (Fig. 4 and Fig. S18 †). N-pyranosides normally have resonances in the 3.8 -4.5 ppm region, [START_REF] Mo | 2, 5-Anhydro-d-Mannose End-Functionalized Chitin Oligomers Activated by Dioxyamines or Dihydrazides as Precursors of Diblock Oligosaccharides[END_REF][START_REF] Kwase | Protecting-Group-Free Glycoconjugate Synthesis: Hydrazide and Oxyamine Derivatives in N-Glycoside Formation[END_REF] but due to peak overlap these resonances could not be directly identified in this case. However, their presence was estimated to account for about 20% based on difference in the decrease of the reducing end and the increase of the hydrazone signals. When increasing to 10 equivalents ADH the combined yield reached 92% (Fig. S19 †), which is higher than predicted by the model (81%). However, integration of signals was difficult and may have affected the estimated yield. Results are included in Table 2.

Attempts were made to purify the Mn-ADH conjugates by GFC (pH 6.9) followed by dialysis and freeze drying. However, this led unexpectedly to complete loss of linked ADH and recovery only of unconjugated oligomannuronate, most probably due to reversal when the excess ADH is removed in the dialysis. However, as will be shown below, stable Mn-ADH conjugates can be obtained in high yields when combined with a reduction step. In contrast, unreduced Gn-ADH conjugates seemed to be stable in this process.

The differences between oligoguluronates and oligomannuronates in terms of ADH conjugation raises the question how other uronates may react. To test this a conjugation reaction was performed with a trigalacturonate (GalA3). Results are included in Table 2 and in Supplementary Information (Fig. S20 †).

Trigalacturonate behaved quite similarly to the two others. The relative amount of the cyclic β-Npyranoside was higher than for oligomannuronate, but lower than for oligoguluronate. Taken together, these findings are unforeseen and indicate that the reaction with ADH, in contrast to PDHA, must depend on the detailed chemistry at some distance from the carbonyl group involved in the reactions provided the reacting sugars exist in the open chain form (Fig. 2). Specifically, the open chain forms of the reducing ends of M and G residues are identical at C1-C4, and at C5 only the stereochemistry is different. The data show that although having different reaction kinetics and distribution of conjugates (E-and Z-conjugates, and N-pyranosides) both ADH and PDHA can fully conjugate to oligouronates when proper conditions are met. It should be noted that for preparative purposes, 10 equivalents of PDHA/ADH is preferred in order to minimise the formation of doubly substituted conjugates. [START_REF] Mo | 2, 5-Anhydro-d-Mannose End-Functionalized Chitin Oligomers Activated by Dioxyamines or Dihydrazides as Precursors of Diblock Oligosaccharides[END_REF] However, even when using 10 equivalents, 5 -10% doubly substituted conjugates are formed. These can, however, be easily removed during purification (Fig. S21 †).

Reduction of oximes/hydrazones/N-pyranosides with α-picoline borane

Although the non-reduced oximes/hydrazones/N-pyranosides may sometime be useful, for instance by taking advantage of their reversibility, 5 many applications will require reduction for better stability.

Reduction also reduces the structural complexity when secondary amines are formed. Although only oximes and hydrazones are reduced directly, the principle of Le Châtelier ensures that the Npyranosides revert to the parent oximes/hydrazones and are therefore reduced (Fig. 2).

Reduction kinetics was initially studied by adding 3 equivalents of PB to the equilibrium reaction mixtures of four chosen oxime/hydrazone model systems (G2-PDHA, G3-ADH, M3-PDHA and M3-ADH).

Reduction was observed by the gradual disappearance of oxime/hydrazone/N-pyranoside resonances during time course NMR, and the corresponding emergence of methylene protons. [START_REF] Mo | Activation of enzymatically produced chitooligosaccharides by dioxyamines and dihydrazides[END_REF] For all systems, complete conversion to the secondary amine was obtained (Fig. S25-S28 †). Reduction data are given in Table 4.

For M3-PDHA the reduction was complete after ca. 22 hours, whereas G2-PDHA needed ca. 120 hours under the same conditions. Both samples were purified by GFC for further structural characterization.

For G2-PDHA, multiple forms were present at pH 5, as evident by multiple signals from the methylene protons in the range 2.8 -3.5 ppm. However, by adjusting to pH 10, only a single resonance from each methylene proton was observed (Fig. S32 †). 1D selective COSY and TOCSY NMR was used for chemical shift assignment of key resonances at pH 5 and 10 (Fig. S29-S30 † and Tables S3-S4 †). For M3-PDHA, the same trend was observed (Fig. S31 † and Table S5 †). The multiple forms observed at pH 5 could possibly be the result of lactonization involving the reducing ends. [START_REF] Spiro | Synthesis and characterization of tyramine-derivatized (1-->4)-linked alpha-D-oligogalacturonides[END_REF] For G3-PDHA, reduction was also investigated at 40 °C in order to possibly increase the reaction rate.

In this case PB was added in two portions (after 0 and 10 h), because the rate of decomposition of PB increases with temperature. [START_REF] Mo | Activation of enzymatically produced chitooligosaccharides by dioxyamines and dihydrazides[END_REF] After 24 h complete conversion to secondary amine was obtained. Such reduction times are considered acceptable for most protocols, and the alginate-based oximes are in any case reduced much faster than e.g. chitin/chitosan-based oximes, [START_REF] Mo | Activation of enzymatically produced chitooligosaccharides by dioxyamines and dihydrazides[END_REF] as well as dextran-PDHA conjugates. [START_REF] Mo | 2, 5-Anhydro-d-Mannose End-Functionalized Chitin Oligomers Activated by Dioxyamines or Dihydrazides as Precursors of Diblock Oligosaccharides[END_REF] It should be noted that at 40 C some reduction of the reducing end aldehyde of oligoguluronates themselves indeed takes place. This demonstrated using 20 equivalents of PB, which resulted in approximately 50 -60% reduction of the reducing end aldehyde after 24 h (Fig. S33 †). Therefore, when using elevated temperatures for the reduction, a general two-step protocol using first 10 equivalents of PDHA to obtain complete conversion to oximes (at room temperature) prior to the reduction step, is recommended.

Reduction of Mn-ADH was more rapid than for Gn-ADH. The former was fully reduced after 12 h and the latter after 39 h. This is reasonable since Mn-ADH has a higher proportion of directly reducible hydrazones. Both systems are reduced much faster than Dexm-ADH and chitosan-ADH (DnXA type). 8

Table 4. Reaction conditions for the reduction of equilibrium reaction mixtures of Mn-PDHA, Gn-PDHA, Gn-ADH and Mn-ADH with 3 equiv. PB to obtain complete conversion (%). The reduction was studied by monitoring the change in (E)/(Z)-oximes/hydrazones and N-pyranoside by 1 H-NMR. The 1 H-NMR spectra are included in Supplementary Information (Fig. S25-S28 †).

Reaction T [°C] Reduction time [h]

G2-PDHA The reduction was also studied for a system of G7 and aminooxy-PEG5-N3 (Fig. S34 †). The reduction followed the trend observed with PDHA, and fully reduced conjugates could be prepared. This shows the general applicability of reductive amination of alginates with oximes and also opens for (Cu-free) azidealkyne chemistry at the reducing end.

Alginate-based diblock polysaccharides

The results above show that fully reduced oligoguluronate based conjugates can be readily prepared.

We applied this approach to prepare different diblocks with oligoguluronate. We first prepared the symmetric G10-ADH-G10 diblock by employing 0.5 equivalents of ADH followed by reduction with PB. The diblock was purified by GFC (Fig. 6a) and the structure was verified by NMR (Fig. 6b). Integration show polymerization of ADH, a reaction that has previously been reported. [START_REF] Mo | Activation of enzymatically produced chitooligosaccharides by dioxyamines and dihydrazides[END_REF] The yield of the diblock was estimated to 50 -60% based on SEC chromatogram. The samples were subsequently studied by multidetector SEC (Fig. 6c) providing molar mass distributions as well as intrinsic viscosity distributions (Table 5), whereas radii of gyration cannot be obtained in this case because of the limited size compared to the wavelength of the laser. A symmetrical diblock was prepared with G10 and PDHA using the same approach. The SEC-MALS analysis provided intrinsic viscosity in agreement with a broken rod architecture in line with the findings for the G10-ADH-G10 block (Fig. S35 † and Table S6 †).

Table 5. Molar mass averages of a G10-b-G10 block (after purification by GFC) and the starting material (G10). G23 is included for comparison of intrinsic viscosities (see text). The data were obtained from SEC-MALS with an in-line viscosity detector. 5.

Sample

Data show the expected doubling of molar mass and retention of the narrow molar mass distribution of the starting materials. Interestingly, the intrinsic viscosity of the diblock was only about 1.5 times higher than that of G10, and only 2/3 of that of G23. Alginates have under the SEC-MALS conditions a persistence length of about 15 nm. [START_REF] Vold | A study of the chain stiffness and extension of alginates, in vitro epimerized alginates, and periodate-oxidized alginates using size-exclusion chromatography combined with light scattering and viscosity detectors[END_REF] With an estimated length of each G residue of 4.35 nm, 24 G10 becomes relatively rod-like, whereas the flexible spacer (ADH) and the two terminal guluronates (in the open chain form) have single bonds with large conformational freedom. The diblock therefore approaches a broken rod type geometry. Future studies will therefore try to clarify the role of such architectures in gel formation, Ca 2+ binding etc. in comparison to the known roles of pure oligoguluronates as calcium alginate gel modifiers. [START_REF] Yuguchi | Local structure of Ca2+ induced hydrogels of alginate-oligoguluronate blends determined by small-angle-X-ray scattering[END_REF][START_REF] Nakauma | Calcium binding and calcium-induced gelation of sodium alginate modified by low molecular-weight polyuronate[END_REF] To demonstrate the applicability of the conjugation protocols to long chains the G12-PDHA-Dex100 diblock was prepared by reacting free G12 with purified PDHA-dextran with DPn of 100. Three equivalents of G12 were here chosen to ensure quantitative substitution of the PDHA-dextran. Residual (unreacted) G12 was selectively removed by GFC (Fig. 7a). SEC-MALS data for the diblock showed a clear shift in elution profile compared to the free blocks (Fig. 7b). The molar masses obtained by SEC-MALS (Table 6) agreed well with the theoretical values. 6.

Table 6. Mn, Mw, and DPn from SEC-MALS analyses of G12-PDHA-Dex100 block copolymer (after purification by GFC) and the starting material (G12 and Dex100-PDHA).

Calcium induced self-assembly of a Gn-b-Dexm diblock

As a first step towards the study of the self-assembly properties of Gn-b-Dexm, the solution behaviour was studied by dynamic light scattering (DLS) when CaCl2 (20 mM) was introduced by dialysis. A membrane with a cut-off of 100 -500 Da was used minimize the formation of out-of-equilibrium aggregates. We were initially interested in the self-assembly of the G40-b-Dex100 copolymer. Two relaxation modes were observed before dialysis, a fast mode corresponding to the relaxation of free chains and a slower mode corresponding to the presence of electrostatic aggregates (Fig. 8a). The scattering intensity is a good indicator of the progress of the self-assembly process. After 3 days of dialysis the intensity increased sharply and stabilized on day 7 which suggests that equilibrium or steady state had been reached (Fig. 8c). The increase in scattering intensity coincided with the disappearance of the fast mode and the transient appearance of a population with diameter around 60 nm (Fig. 8a). The final state is characterized by the presence of two populations with diameters around 25 nm and 150 nm, respectively, in the intensity size distribution. However, the second population was not detected in the number distribution (Fig. 8b), suggesting that it represented only a small fraction of the sample. The population at 25 nm probably corresponds to micellar structures consisting of an alginate-based core hydrogel stabilized by dextran blocks. The hypothesis of a core-shell morphology is supported by the fact that that G40 blocks alone precipitate under similar conditions. Therefore, the diblock structure enabled a strict phase separation between the G-based core and the dextran corona. Deeper characterization by small angle scattering techniques (X-ray or neutron) should allow to access the micellar characteristics in terms of radius of the core, thickness of the corona, and aggregation number.

Interestingly, the G11-b-Dex100 diblock has a markedly different behavior under similar conditions.

Initially, the scattering profiles differ. In G11-b-Dex100 the scattering is more dominated by the larger dextran block compared to G40-b-Dex100. Hence, the fast mode peak [START_REF] Sedlák | What can be seen by static and dynamic light scattering in polyelectrolyte solutions and mixtures?[END_REF] becomes in comparison weaker.

The broad peak above 100 nm also indicates some chain aggregation which disappears at the onset of the reaction since the size distribution sharpens around 100 nm and the gradually moves to higher diameters. Large changes could however be observed upon addition of calcium. The G11-b-Dex100 block copolymer tended to form very large structures in solution (> 1 µm) after a few days of dialysis (Fig. 8d).

Furthermore, the scattering intensity did not increase significantly (data not shown), suggesting the formation of rather loose aggregates. From a thermodynamic point of view, this could mean that the loss of entropy associated with the formation of a dextran corona is not balanced by a sufficient gain in enthalpy through the gelling of G blocks as they are probably too short. Therefore, the ratio of the two blocks lengths must be carefully considered to have self-assembly properties in presence of calcium ions.

Preparation and characterization of guluronate oligomers (G n )

guluronate oligomers (G n ) were prepared by acid precipitation of guluronate rich alginates, removing any oligomers containing one or more M-residue(s).

A study using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) was performed, monitoring the acid hydrolysis of a guluronate with DP n 20, enabling tailoring of the resulting DP n . Oligomers with defined DP n were prepared by GFC, and DP n was verified by 1 H-NMR (Fig. S3). Isolated triguluronate was annotated according to literature 1 . Fig. S2 GFC fractionation of guluronate oligomers (guluronate with DPn 20 hydrolyzed for 9 h at 95°, pH 3.61).

Fig. S3 1 H-NMR (82 °C, 400 MHz) spectrum of isolated triguluronate, peaks were assigned according to literature 1 .

The equilibrium reaction mixture with G 3 and PDHA was characterized by heteronuclear NMR. The reaction mixture with M 3 and PDHA (24 h reaction time, 10 equiv. PDHA, 500 mM NaAc-buffer pH 4) was purified by GFC, dialysis and freeze drying. H1 of the oxime conjugates were assigned based on literature 2, 3 . H2 and H3 of the (E)-and (Z)-oxime was assigned using 1D selective COSY and TOCY NMR (Fig. S13). Complete assignment was obtained by heteronuclear NMR, Fig. S14.

Attachment of the second block: A-b-B diblock polysaccharides

Alginate-based diblock polysaccharides

A symmetrical diblock was prepared with G 10 and PDHA (using 0.5 equiv.) and by reduction with PB. Excess PB was removed by dialysis and the sample was freeze dried. The reaction mixture was analysed by SEC-MALS (the sample was not purified by GFC) with an in-line viscosity detector.

13 C

 13 C Heteronuclear Single Quantum Coherence (HSQC) with multiplicity editing, 2D Double Quantum Filtered Correlation Spectroscopy (DQF-COSY), 2D Heteronuclear 2 Bond Correlation (H2BC) and 2D 13 C heteronuclear multiple bond correlation (HMBC). 1D selective pulse program with gradient selection COSY (pulse program: selcogp) and Total Correlation Spectrocopy TOCSY (pulse program: seldigpzs) was acquired on the 800 MHz at 25 °C. Chemical shift assignment of purified conjugates was done using the 800 MHz at 25 °C by acquiring the following spectra: 1D 1 H, DQF-COSY, HSQC with multiplicity editing, 2D 13 C HSQC-[ 1 H, 1 H]TOCSY, 2D H2BC and 2D HMBC.

Fig. 2

 2 Fig. 2 Schematic overview of the reducing modifications of oligoguluronate (Gn) with a) O,O'-1,3,-

Fig. 3

 3 Fig.3 Reaction schemes and 1 H-NMR spectra of the equilibrium reaction mixture for the reaction with

Fig. 4

 4 Fig.4 Reaction schemes and 1 H-NMR spectra of the equilibrium reaction mixture for the reaction of a)

Fig. 5 1

 5 Fig. 5 1 H-NMR spectra of the equilibrium reaction mixture with G3 and PDHA-Dex10 (1:1) in 500 mM

Fig. 6 A

 6 Fig.6A symmetrical G10-ADH-G10 diblock was prepared and purfied by GFC (a, hatched area) and analysed by1 H NMR (600 MHz, 25 °C) (b) and SEC-MALS (c). The latter included G10 (black) and G23 (not

Fig. 7

 7 Fig. 7 G12-PDHA-Dex100 diblock polysaccharides were purified by GFC (hatched area in a) and analysed

Fig. 8

 8 Fig. 8 Self-assembly of Gn-b-Dex100 copolymers by dialysis (MWCO 100 -500 Da) against Ca 2+ (20 mM)

Fig. S1

 S1 Fig. S1 HPAEC-PADH chromatograms for guluronate oligomers taken at different timepoints during hydrolysis (95°C, pH 3.62).

Fig.

  Fig. S5 a,b) 13 C HSQC of the reaction mixture obtained for the conjugation with G 3 (7mM) and PDHA (2 equiv.) (500 mM AcOH[d 4 ] pD 4) recorded at t > 24 h (82 °C, NEO 600 MHz magnet). c) 1 H-NMR spectra of G 3 -PDHA after purification by GFC and dialysis in D 2 O at pH 6.5 and 11.8 (27 °C, 600 MHz)

Fig. S7 1

 1 Fig.S71 H-NMR spectra of the equilibrium reaction mixture with G 3 (7mM) and PDHA (2 equiv.) (in 500 mM AcOHd4 pD 4 at 27 °C, 600 MHz) recorded after 4 h. Integration using H1 of the non-reducing end used to estimate yield is included.

Fig. S8

 S8 Fig. S8 Plot with combined yield for conjugation with a) G 8 (7mM) and b) G 6 (20 mM) and PDHA (2 equiv.) (500 mM AcOHd4 pD 4) studied by time course NMR. Data obtained by integration using H1 of the non-reducing ends as internal standard. A simple model assuming first order kinetics based on combined yield is included.

Fig. S9

 S9 Fig. S9 Plot with combined yield for conjugation with a) Dextran DP 3 (7mM) and b) Maltotriose (7 mM) and PDHA (2 equiv.) (500 mM AcOHd4 pD 4) studied by time course NMR (27 °C, 600 MHz). Data obtained by integration using H1 of the non-reducing ends or TSP as internal standard. A simple model assuming first order kinetics based on combined yield is included.

Fig. S10

 S10 Fig. S10 Stack of 1 H-NMR spectra obtained at different time points for the reaction with G 3 (7mM) and PDHA (2 equiv.) at pD 5 (500 mM AcOHd4, 27 °C, 600 MHz).

Fig. S11

 S11 Fig. S11 Conjugation with G 3 (7mM) and PDHA (2 equiv.) at pD 5 (500 mM AcOHd4) studied by time

Fig. S12

 S12 Fig. S12 Stack of 1D 1 H-NMR spectra obtained at different timepoints for the reaction with M 3 (7mM) and PDHA (2 equiv.) in 500 mM AcOH[d4] (27 °C, 600 MHz) (M 3 is included for comparison). Key resonances indicative of the reaction are annotated.

Fig. S13

 S13 Fig. S13 1D selective COSY and TOCSY NMR spectra of M 3 -PDHA (after purification) in D 2 O, pH 4 (25 °C, 800 MHz). a) Assignment of H1-H3 of the (E)-oxime, and b) assignment of H1-H3 of the (Z)-oxime. The ppm of the pulse is indicated to the left.

Fig. S14 1 H- 1 H

 11 Fig. S14 1 H-1 H COSY spectra of the oxyamine modified trimannuronate (M 3 -PDHA) (after purification by GFC, dialysis and freeze drying) in D 2 O pH 4 (25 °C, 800 MHz).

Fig. S15

 S15 Fig. S15Plot with combined yield for conjugation with M 3 (7 mM) and PDHA (2 equiv.) (500 mM AcOHd4 pD 4) studied by time course NMR (at 27 °C using the 600 MHz). Data obtained by integration using TSP as internal standard. A simple model assuming first order kinetics based on combined yield is included.

Fig. S17

 S17 Fig. S17Plot with combined yield for conjugation with G 2 (7 mM) and ADH (2 equiv.) (500 mM AcOHd4 pD 4) studied by time course NMR (at 27 °C using a 600 MHz magnet). Data obtained by integration using H1 of the non-reducing end as internal standard. A simple model assuming first order kinetics based on combined yield is included.

Fig. S18

 S18 Fig. S18 Stack of 1 H-NMR (27 °C, 600 MHz) spectra obtained at different time points for the reaction with M 3 (7mM) and ADH (2 equiv.) at pD 4 (500 mM AcOHd4).

Fig. S19 1

 1 Fig. S19 1 H-NMR (27 °C, 600 MHz) spectrum of the equilibrium reaction mixture (t > 20 h) for the reaction with M 3 (7mM) and ADH (10 equiv.) (500 mM AcOHd4 pD 4).

Fig. S20

 S20 Fig. S20 Stack of 1 H-NMR (27 °C, 600 MHz) spectra obtained at different time points for the reaction with Galacturonic acid DP 3 (TriGalA 3 ) (7mM) and ADH (2 equiv.) at pD 4 (500 mM AcOHd4). A spectrum of pure TriGalA is included for comparison.

Fig. S22 1

 1 Fig. S22 1 H-NMR (27 °C, 600 MHz) spectrum of the reaction mixture with G 3 (7mM) and maltotriose DP 3 with PDHA (Glc 3 -PDHA) (2 equiv.) (500 mM AcOHd4 pD 4). Purified Glc 3 -PDHA (in 500 mM AcOHd4 pD 4) is included for comparison.

Fig. S23

 S23 Fig. S23 Stack of 1 H-NMR (27 °C, 600 MHz) spectra obtained at different time points for the reaction with G 7 (20.1 mM) and aminoox-PEG 5 -N 3 (2 equiv.) (500 mM AcOHd4, pD 4).

Fig. S24

 S24 Fig. S24 Reaction scheme for the reaction with G 7 and aminoox-PEG 5 -N 3 .

Fig. S26 1

 1 Fig. S26 1 H-NMR (27 °C, 600 MHz) spectra of equilibrium reaction mixture with M 3 (7mM) and PDHA (2 equiv.) before reduction (t red = 0). PB (3 equiv.) was added to the reaction mixture and the spectrum obtained after 22 h is shown with annotation of key resonances.

Fig. S27 1

 1 Fig. S27 1 H-NMR (27 °C, 600 MHz) spectra of equilibrium reaction mixture with G 2 (7mM) and ADH (2 equiv.) before reduction (t red = 0). PB (3 equiv.) was added to the reaction mixture and the spectrum obtained after 39 h is shown with annotation of key resonances.

Fig. S28 1

 1 Fig. S28 1 H-NMR (27 °C, 600 MHz) spectra of equilibrium reaction mixture with M 3 (7mM) and ADH (10 equiv.) before reduction (t red = 0). PB (3 equiv.) was added to the reaction mixture and the spectrum obtained after 12 h is shown with annotation of key resonances.

Fig. S29

 S29 Fig. S29 Assignment of key reducing end resonances from the oxyamine modified reducing end of G 2 -PDAH (with reduction, after purification by GFC, dialysis and freeze drying) in D 2 O at pH 5.1 (25 °C,

Fig. S30

 S30 Fig. S30 Assignment of key reducing end resonances from the oxyamine modified reducing end of G 2 -PDAH (with reduction, after purification by GFC, dialysis and freeze drying) in D 2 O at pH 10.2 (25 °C, 800 MHz) using 1D selective COSY and TOCSY NMR. Red arrows indicate the ppm of the pulse.

Fig. S31

 S31 Fig. S31 Assignment of key reducing end resonances from the oxyamine modified reducing end of M 3 -PDAH (with reduction, after purification by GFC, dialysis and freeze drying) in D 2 O at pH 10.2 (25°C, 800 MHz) using 1D selective COSY and TOCSY NMR. Red arrows indicate the ppm of the pulse.

Fig. S32 1

 1 Fig. S32 1 H-NMR (27 °C, 600 MHz) spectra of G 2 -PDHA after purification by GFC and dialysis. The sample was dissolved in D 2 O and pH was adjusted from 5.1 to 10.2 with NaOD.

Fig. S33 1

 1 Fig. S33 1 H-NMR spectra of G 6 (20.1 mM) with PB (20 equiv.) in 500 mM AcOHd4 pD 4 at incubated in water bath at 40°C, recorded after 24 h (82°C, 400 MHz).

Fig. S34 1 H

 1 Fig.S341 H-NMR (27 °C, 600 MHz) spectra of the reduction of the equilibrium rx. mixture with G7 (20.1 mM) and aminoox-PEG 5 -N 3 after addition of PB (3 equiv. added at t0, another 3 equiv. added after 72 h).

Fig. S35 A

  Fig. S35 A symmetrical G 10 was reacted with 0.5 equivalents PDHA, and the reaction mixture was analysed by SEC-MALS (red). A G 10 was included for comparison (blue). The coupling to form a diblock (G 10 -PDHA-G 10 ) is manifested by a significant shift in elution volume (peak 1). Unreacted G 10 and G 10 -PDHA comprise approximately 50 -60 % of the sample (peak 2), and elutes close to pure G 10. Data are summarized in tableS6.

Table 1 .

 1 Reactions with alginate oligomers with 2 equivalents PDHA studied by time course NMR. Rate constant (kT) is based on a first order kinetics model for combined yield (%). The reactions were studied at room temperature. Dextran (Dexm) and maltotriose (Glcm) is included as a comparison. (n.d.: Could not be accurately determined because of yields close to 100%). Values marked with an asterisk (*) are recalculated from kT and k-T values obtained at 20.1 mM.

	Oligomer Oligomer	pD AcOH[d4]	Ratio	t0.5[h]	t0.9[h]	kT	k-T	Equil.
		conc. [mM]		[mM]	E:Z:β-N-pyr.					conversion (%)
	G3	7.0	4	500	2.2:1:0	0.29	1.06	8.7x10 -2	7.7x10 -2	92
	M3	7.0	4	500	2.6:1:0	0.17	0.62	1.6x10 -1	n.d.	94
	G8	7.0	4	500	2.1:1:0	0.40	1.45	6.4x10 -2	4.1x10 -2	97
	G3	7.0	5	500	2.6:1:0	0.75	2.67	3.1x10 -2	8.7x10 -2	93
	G6	20.1	4	500	2.2:1:0	0.38	1.39	2.4x10 -2	n.d.	92
		7.0*				1.10*	4.01*
	Dex3	7.0	4	500	4.1:1:1	4.55	14.76	4.4x10 -3	2.4x10 -2	77
	Glc3	7.0	4	500	3.1:1:1.6	4.02	13.26	4.0x10 -3	5.1x10 -2

Table 2 .

 2 Reactions of alginate oligomers with ADH studied by time course NMR. The rate constants (kT and k-T) are based on the first order kinetics model for the combined yield. Values marked with an asterisk (*) are recalculated from kT and k-T values obtained for 20.1 mM of oligomer and 2 equivalents of ADH. n.d.: the reaction was too fast to accurately monitor by NMR.

	Oligomer	ADH	Oligomer	pD	AcOH[d4]	Ratio	t0.5[h]	t0.9[h]	kT	k-T	Eq.
		equiv.	conc. [mM]		[mM]	E:Z:β-N-					yield (%)
						pyr.
	G2	2	7.0	4.0	500	1:0.2:32	0.39	1.33	3.0x10 -2	8.2x10 -1	48
	G6	10	20.1	4.0	500	1:0.1:26	0.10	0.34	3.1x10 -2	7.7x10 -1	88
		2*	7.0*				0.40*	1.36*
	M3	2	7.0	4.0	500	1:0.3:1.6	0.55	1.85	1.6x10 -2	7.4x10 -1	36**
	M3	10	20.1	4.0	500	1:0.1:1.1	n.d.	n.d.	n.d.	n.d.	92**
	GalA3	2	7.0	4.0	500	1:0.2:8	0.23	0.84	4.95x10 -2	7.76x10 -1

Table 3 .

 3 Coupling of PDHA-activated oligosaccharides to oligoguluronates (G3).

	Activated block (A*) Second	Ratio	Conc. of	Equ. yield	kT	k-T	t0.5 [h] t0.9 [h]
		block (B)	(A*-B)	B [mM]	(%)
	G10-PDHA	G3	1:1	7.0	45	3.3x10 -2	1.6x10 -1	1.4	5.1
	Dex10-PDHA	G3	1:1	7.0	42	7.1x10 -2	3,8x10 -1	0.6	2.2
	A4M-PDHA	G2	1:1	20.1	57	8.0x10 -3	5.1x10 -2	2.7	10.1
	b-1,3-glucan-PDHA	G3	1:1	7.0	61	8.0x10 -2	1.4x10 -1	0.9	3.3
	G7	aminooxy-	1:2	20.1	94	4.4x10 -2	3.3x10 -3	0.16	1.79
		PEG-N3

Table S2 :

 S2 Assignment of chemical shifts for oxyamine modified trimannuronate (M 3 -PDHA) (after purification by GFC, dialysis and freeze drying) in D 2 O pH 4 obtained from 1D selective and heteronuclear NMR experiments. Prime indicates the minor form.

	H/C 1	H/C 1'	H/C 2	H/C 3	H/C 4	H/C 5	C 6
	(E)-oxime 7.61; 151.7	7.65; 151.7	4.40; 68.04	3.79; 75.65 4.11; 77.6	4.25; 71.31	178.1
	(Z)-oxime 6.96; 151.6	7.00; 151.3	4.98; 63.1	3.79; 75.65 4.11; 77.6	4.25; 71.31	178.1

Table S3 :

 S3 Assignment of proton resonances of G 2 -PDHA (with reduction, purified by GFC, dialysis and freeze drying). Dissolved in D 2 O, pH 5.1 by 1D selective COSY and TOCSY. RT is the modified reducing termini of G2-PDHA.

		H1	H2	H3	H4	H5
	RT pH 5.1	2.96/3.38	3.90	3.65	4.08	4.37
		H1/H1'	H2/H2'
	NRT pH 5.1	5.08/5.17	3.94/3.98	3.92	4.11	4.53

Table S4 :

 S4 Assignment of proton resonances of G 2 -PDHA (with reduction, purified by GFC, dialysis and freeze drying. Dissolved in D 2 O, pH 5.1) by 1D selective COSY and TOCSY. RE denotes is the oxyamine modified reducing end of G 2 -PDHA. N.d. denotes not determined.

		H1	H2	H3	H4	H5
	RE pH 10.2	3.20/2.76	3.84	3.63	4.07	n.d.
	NRT pH 10.2	5.04	3.91	3.88	4.09	4.52

Author manuscript of article published in Polymer Chemistry 2021, 12, 5412 DOI: 10.1039/D1PY00727K 20

Attachment of the second block: A-b-B diblock polysaccharides:

The high reactivity of oligouronates with PDHA implies that reaction with PDHA-activated oligosaccharides to obtain diblock oligo-or polysaccharides would proceed equally well. This was tested in kinetic studies with dextran-PDHA (DP 10), maltotriose-PDHA (DP 3), β-1,3-glucan-PDHA (DP 9) and chitin-PDHA (AnM type) [START_REF] Mo | 2, 5-Anhydro-d-Mannose End-Functionalized Chitin Oligomers Activated by Dioxyamines or Dihydrazides as Precursors of Diblock Oligosaccharides[END_REF] (DP 5). Here, A stands for N-acetyl-D-glucosamine and M signifies the reactive terminal 2,5-anhydro-D-mannose (generally denoted M in the literature) at the reducing end. In addition, the reaction was also studied with Gn-PDHA for preparation of symmetrical blocks. All conjugates had been fully reduced with PB prior to coupling with oligoguluronate. [START_REF] Mo | 2, 5-Anhydro-d-Mannose End-Functionalized Chitin Oligomers Activated by Dioxyamines or Dihydrazides as Precursors of Diblock Oligosaccharides[END_REF][START_REF] Mo | Activation of enzymatically produced chitooligosaccharides by dioxyamines and dihydrazides[END_REF] These PDHAactivated oligosaccharides represent widely different chemistries (Table 3): Dextrans are neutral chains with high chain flexibility due to α-1,6 linkages. Chitins are uncharged but more rigid and hydrophobic and can self-assemble (crystallise) at higher DPs. Amylose (α-1,4-linked glucans) and β-1,3-glucans are both semi-rigid, neutral chains with the ability to form higher order structures. Collectively they illustrate the versatility of the approach towards almost any type of diblock polysaccharides.

The conjugations with oligoguluronates were initially studied using a 1:1 or 2:1 molar ratio between the reactants. Fig. 5 shows NMR spectra of Dex10-PDHA (PDHA conjugated dextran decamer) before and after further conjugation to G3. Results for all PDHA-activated oligosaccharides are summarized in Table 4. Several PDHA-oligosaccharides react with Gn with rates similar to those of free PDHA, except for PDHA-activated chitin oligomer, which is a factor two slower (Table 1). Another exception was PDHAmaltotriose, which for presently unknown reasons did not seem to react (Fig. S22 † The reaction kinetics was determined by integrating the 1 H-NMR spectra, using H1 of the non-reducing end and TSP as internal standards. A simple model based on first order kinetics was used to determine the rate constants (k T and k -T ) for the combined yield.


Ingrid Vikøren Mo

Christophe Schatz

Bjørn E Christensen

† Nobipol

Functionalisation of the non-reducing end of chitin: A new approach to form complex block polysaccharides and soluble chitin-based block polymers

Bullet points

• Chitin oligomers prepared by nitrous acid degradation of chitosan only possess a single vicinal diol prone to periodate oxidation at the non-reducing end (NRE) residue

• Both aldehydes formed at the NRE can subsequently be activated by dioxyamines or dihydrazides

• The NRE aldehydes are equally or more reactive towards dioxyamines and dihydrazides than the pending aldehyde of the 2,5-anhydro-D-mannose (M) reducing end residue

• The free end of the dioxyamines/dihydrazides after conjugation can react with aldehydes from the same (intra) or from a different oligosaccharide (inter)

• NRE functionalised and activated chitin oligomers can act as precursors for block polysaccharides or form water-soluble chitin-based block polymers

Abstract

Most polysaccharides used in polysaccharide-based block copolymers are attached to the second block through the reducing end, due to the few and highly polysaccharide specific non-reducing end (NRE) functionalisation methods available. Chitin oligomers, prepared by nitrous acid degradation of chitosan (AnM) can, however, be selectively oxidised by periodate since they only possess a single vicinal diol in the NRE residue. Here, we show that both aldehydes formed after oxidation are highly reactive towards activation by bifunctional oxyamines and hydrazides. The reactivity was shown to be even higher than for the aldehyde of the already reactive M reducing end residue of such oligomers. Fully activated oxidised AnM oligomers was obtained using an excess of bifunctional linkers, enabling the preparation of more complex block polysaccharides such as ABA-or ABC-type. A sub-stochiometric amount of linkers resulted in conjugation of the blocks through the reducing and NRE to form a discrete distribution of 'polymerised' AnM oligomers. Such chitin-based block polymers were, in contrast to chitins of the same chain lengths, water-soluble.

Introduction

AB-type diblock polysaccharides represent a new class of engineered block polysaccharides, utilising only natural polysaccharide blocks 1,[START_REF] Breitenbach | Double stimuli-responsive polysaccharide block copolymers as green macrosurfactants for near-infrared photodynamic therapy[END_REF] . Coupling of the polysaccharide blocks through their reducing ends, result in antiparallel chains. Preparation of more complex block polysaccharides, such as linear ABA or ABC triblock copolymers, requires reactions at the non-reducing end (NRE) of the B block and only a few polysaccharide specific methods for functionalisation of the NRE have been published. However, NRE reactivity has been demonstrated for dextran [START_REF] Chen | Regioselective Bromination of the Dextran Nonreducing End Creates a Pathway to Dextran-Based Block Copolymers[END_REF] and lyase degraded heparin [START_REF] Wang | Efficient access to the non-reducing end of low molecular weight heparin for fluorescent labeling[END_REF] after intermediate functionalisation steps.

Periodate oxidation of polysaccharides converts vicinal diols (and closely related structures) to the corresponding dialdehydes. Hence, the ability of different polysaccharides to be oxidised depends on their chemical composition and linkage geometry [START_REF] Kristiansen | Periodate oxidation of polysaccharides for modification of chemical and physical properties[END_REF] . In the special case of fully N-acetylated chitin oligomers prepared by nitrous acid degradation of chitosan (AnM) [START_REF] Tømmeraas | Preparation and characterisation of oligosaccharides produced by nitrous acid depolymerisation of chitosans[END_REF] , only the non-reducing end residue contains oxidisable, vicinal diols (Figure 1a). Therefore, periodate oxidation of such oligomers should in theory provide oligomers with two aldehydes (C3 and C4) in the NRE residue, in addition to the highly reactive pending aldehyde of the 2,5-anhydro-D-mannose (M) residue (Figure 1b). Only a few studies describing the functionalisation of the NRE of chitin by periodate oxidation is reported in the literature [START_REF] Hirano | Periodate oxidation of the non-reducing end-groups of substrates increases the rates of enzymic hydrolyses by chitinase and by lysozyme[END_REF][START_REF] Huang | Chemo-enzymatic synthesis of 1,4-oxazepanyl sugar as potent inhibitor of chitinase[END_REF][START_REF] Imai | Directional degradation of β-chitin by chitinase A1 revealed by a novel reducing end labelling technique[END_REF] , and to our knowledge, subsequent reactions with the aldehydes have not been investigated. Hence, it is unknown whether both the aldehydes at the NRE are reactive, but the question is pertinent as only one of the dialdehydes obtained by lateral periodate oxidation of alginate has shown to react with amines through reductive amination [START_REF] Kristiansen | An evaluation of tritium and fluorescence labelling combined with multi-detector SEC for the detection of carbonyl groups in polysaccharides[END_REF][START_REF] Dalheim | Efficient functionalization of alginate biomaterials[END_REF] .

AnM oligosaccharides have previously been subjected to reducing end activation by a dioxyamine (PDHA) and a dihydrazide (ADH) to form precursors for AB-type block polysaccharides [START_REF] Mo | 2,5-Anhydro-d-Mannose End-Functionalized Chitin Oligomers Activated by Dioxyamines or Dihydrazides as Precursors of Diblock Oligosaccharides[END_REF] . Such activated AnM oligomers were used to prepare AnM-b-dextran diblocks. Reaction of oxidised AnM oligomers with such dioxyamines or dihydrazides may form a range of new complex structures. In the present work we first study the reaction of periodate oxidised AnM with an excess PDHA or ADH using the previously developed two-step reductive amination protocol [START_REF] Mo | 2,5-Anhydro-d-Mannose End-Functionalized Chitin Oligomers Activated by Dioxyamines or Dihydrazides as Precursors of Diblock Oligosaccharides[END_REF] to assess the relative reactivities of aldehydes at the reducing and non-reducing end (Figure 1c). Secondly, we reacted oxidised AnM oligomers with a sub-stoichiometric amount of PDHA to obtain a discrete distribution of a new type of water-soluble chitin block polysaccharides composed of a progressively increasing number of AnM blocks reacted through both ends.

The introduction of a dialdehyde at the NRE combined with the highly reactive aldehyde at the reducing of AnM oligomers enables the preparation of a range of new glycoconjugates, including complex block polysaccharides.

Materials and methods

Materials

High molecular weight chitosan (FA = 0.48, [ɳ] = 1210 mL/g) was obtained from Advanced Biopolymers ABC. Adipic acid dihydrazide (ADH), O,O'-1,3-propanediylbishydroxylamine dihydrochloride (PDHA) and 2-methylpyridine borane complex (α-Picoline borane, PB) were obtained from Sigma-Aldrich. All other chemicals were obtained from commercial sources and were of analytical grade.

Gel filtration chromatography (GFC)

Preparative and analytical gel filtration chromatography (GFC) were used for fractionation of chitin oligosaccharides and fractionation of products, respectively, as described earlier [START_REF] Mo | 2,5-Anhydro-d-Mannose End-Functionalized Chitin Oligomers Activated by Dioxyamines or Dihydrazides as Precursors of Diblock Oligosaccharides[END_REF] . In brief, both systems were composed of Superdex 30 columns (BPG 140/950 (140 x 950 mm) and HiLoad 26/600 (26 x 600 mm), respectively) connected in series, continuously eluting ammonium acetate (AmAc) buffer (0.15 M, pH 4.5 and 0.1 M, pH 6.9, respectively). Fractionation was monitored on-line using a refractive index (RI) detector and fractions were collected and pooled according to elution times. The pooled fractions were reduced to appropriate volumes, dialyzed (MWCO = 100-500 Da) against ultrapure Milli-Q (MQ) water until the measured conductivity of the water was < 2 µS/cm and freeze-dried or freeze-dried directly without dialysis to remove the volatile mobile phase (AmAc-buffer).

NMR spectroscopy

Water-soluble samples for NMR characterization were dissolved in D2O (450-600 µL, approx.10 mg/mL).

For some samples, 1% deuterated sodium 3-(trimethylsilyl)-propionate (TSP-d4, 3 µL) was added as an internal standard. Water-insoluble samples were dissolved in deuterated hexafluoro isopropanol (HFIP-d2).

Characterization was performed by obtaining 1D 1 H NMR spectra at 300K on a Bruker Ascend 14.1 Tesla 600 MHz equipped with Avance III HD electronics and a 5 mm Z-gradient CP-TCI cryogenic probe. All spectra were recorded, processed and analysed using TopSpin 3.5pl7 software (Bruker BioSpin).

Mass spectrometry (MS)

MS characterisation was performed using flow injection analysis (FIA) coupled with quadrupole time of flight (gTOF) MS as described earlier [START_REF] Vikøren Mo | Activation of enzymatically produced chitooligosaccharides by dioxyamines and dihydrazides[END_REF] . The mass range was set to 50-2000 Da which limited the characterisation of larger structures.

Preparation of chitin oligomers by nitrous acid degradation

Chitin oligomers (AnM) were prepared by nitrous acid (HONO) degradation as described earlier [START_REF] Mo | 2,5-Anhydro-d-Mannose End-Functionalized Chitin Oligomers Activated by Dioxyamines or Dihydrazides as Precursors of Diblock Oligosaccharides[END_REF] . In brief, chitosan (FA = 0.48) was degraded using an excess of HONO (1.3 moles of HONO per mole GlcN) overnight in the dark at 4 °C. Washing and centrifugation steps were performed to remove the insoluble fraction, assumed to be water-insoluble chitin oligomers. The water-soluble low molecular weight chitin oligomers were fractionated according to degree of polymerization (DP) by GFC (DP < 10). Isolated oligomers were purified by dialysis, freeze-dried and characterized by 1 H-NMR.

Periodate oxidation

Chitin oligomers (AnM) or chitin oligomer conjugates (A6M-PDHA or A2M-ADH-MA2) (10 mM) were dissolved in MQ-water. Oxygen was removed by bubbling the solution with N2 gas for 10 minutes. Freshy prepared and N2 bubbled NaIO4 (100 mM) was added to give a final molar ratio of 2:1 or 4:1 (NaIO4: AnM or conjugates). Reactions were performed in the dark at 4 °C for 24 hours on a shaking device. Ethylene glycol (10 molar equivalents) was added to the reaction mixture and stirred for 30 minutes to terminate the reaction. The reaction mixture was dialyzed (MWCO = 100-500 Da) against MQ-water until the measured conductivity was < 2 µS/cm and freeze-dried.

Conjugation of oxidised AnM oligomers to ADH and PDHA

Oxidised AnM oligomers (6.5 mM) and ADH or PDHA (0.5 -10 equivalents) were dissolved in NaAcbuffer (500 mM, pH 4.0) and reacted for 24 hours at room temperature. PB (20 equivalents) was added to the reaction mixture, and the reduction was allowed to proceed for 72 hours at room temperature. The reaction mixtures were dialysed (MWCO = 100-500 Da) against 0.05 M NaCl until the partly insoluble PB dissolved and subsequently freeze-dried. The mixtures were further fractionated by GFC. Isolated fractions were purified by direct freeze-drying and characterised by NMR or MS.

Results and discussion

Periodate oxidation of AnM oligomers

AnM oligomers, where n corresponds to DP-1, were prepared by nitrous acid depolymerisation as described earlier [START_REF] Mo | 2,5-Anhydro-d-Mannose End-Functionalized Chitin Oligomers Activated by Dioxyamines or Dihydrazides as Precursors of Diblock Oligosaccharides[END_REF] . The water-soluble oligomers (n = 0-8) were isolated by GFC fractionation to obtain oligomers of specific DP. Initially, A2M oligomers were oxidised using 2 equivalents of periodate, purified by dialysis and characterised by 1 H-NMR (Figure 2b). New resonances appeared in the anomeric region (4.8-5.6 ppm), but the spectrum was otherwise too complex for a complete determination of structure and degree of oxidation. The high complexity can be attributed to inter-or intra-residue hemiacetals readily formed with the dialdehydes after oxidation of the NRE [START_REF] Ishak | Formation of interresidue hemiacetals during the oxidation of polysaccharides by periodate ion[END_REF][START_REF] Sharon | Complex carbohydrates[END_REF] . Such hemiacetals are acid stable and difficult to structurally elucidate. A reduced intensity was observed for the resonance resulting from H1, M (gem-diol) relative to other resonances in the spectrum after oxidation. The reduced intensity may be caused by the formation of inter-residue hemiacetals with the aldehydes in the NRE. The M residue seemed otherwise unaltered as predicted from its structure. Hence, the oxidation only took place at the NRE. Oxidation with 4 equivalents of periodate gave essentially the same result, however, with an even lower intensity of the H1, M resonance suggesting a higher degree of oxidation and more inter-residue hemiacetals formed (Figure 2c). Unless otherwise stated, oligomers oxidised with 2 equivalents periodate were used in the following experiments.

Activation of oxidised A2M oligomers by PDHA and ADH

Even though oxidised AnM preparations were rather complex mixtures, we decided to proceed without further purification as this most likely could be better obtained after conjugation to PDHA or ADH. Thus, oxidised A2M oligomers were reacted with 10 equivalents PDHA or ADH using the two-step reductive amination protocols recently developed for AnM oligomers [START_REF] Mo | 2,5-Anhydro-d-Mannose End-Functionalized Chitin Oligomers Activated by Dioxyamines or Dihydrazides as Precursors of Diblock Oligosaccharides[END_REF] . Interestingly, the two reaction systems behaved very differently. With ADH an insoluble fraction was formed, while this was not observed for PDHA. The reaction mixtures (soluble fractions) were characterized by 1 H-NMR (Supporting information, S1). Compared to the corresponding conjugates prepared with unoxidised oligomers, the NMR spectra of the reaction mixtures were more complex. However, resonances resulting from the characteristic secondary amine protons (around 3.0 ppm) appeared, indicating the formation of conjugates. Importantly, the H1 resonances for unreacted M disappeared, confirming complete reaction at the reducing end, as expected [START_REF] Mo | 2,5-Anhydro-d-Mannose End-Functionalized Chitin Oligomers Activated by Dioxyamines or Dihydrazides as Precursors of Diblock Oligosaccharides[END_REF] .

Incomplete reduction of conjugates formed with PDHA was indicated by the presence of resonances in the Schiff base area (6-8 ppm). Conjugates formed with ADH were in contrast completely reduced (Supporting information, S1). The reaction mixtures were further fractionated by GFC (Figure 3). Purified fractions were characterised by 1 H-NMR and MS (Supporting information, S2 and S3 for PDHA and ADH, respectively). Fraction C consisted predominantly of 9, which was the structure aimed for when using an excess of the diamine linkers. The presence of 9 confirms the reactivity of both aldehydes at the NRE. This is in contrast to lateral periodate oxidation of alginate, where only one of the aldehydes is reactive towards substitution [START_REF] Kristiansen | An evaluation of tritium and fluorescence labelling combined with multi-detector SEC for the detection of carbonyl groups in polysaccharides[END_REF][START_REF] Dalheim | Degradation kinetics of peptidecoupled alginates prepared via the periodate oxidation reductive amination route[END_REF] . The absence of conjugates containing unreacted aldehydes (2-4, 7 and 8), indicates high reactivity of all aldehydes, including the possibility to rapidly form intramolecular (cyclic) structures. The presence of 1 confirmed incomplete oxidation of the oligomers under the given conditions. For structures with one oligomer (1-9) all possible combinations are presented (transparent structures were not identified). For products with two or three oligomers (10-14 and 15, respectively), only the identified structures are presented. The symbols used are defined in Figure 1.

The presence of fractions corresponding to a hexamer and a nonamer (Fraction A and B, respectively in Figure 3b based on the calibration with AnM oligomers in Figure 3c) indicated conjugation of PDHA to both the RE and NRE of the oligomers to form chitin block structures. 1 H-NMR characterisation confirmed the presence of oligomer and PDHA linked by Schiff base or secondary amine bonds (Supporting information, S2). Due to the incomplete oxidation, the block structures were composed of a mixture of oxidised and unoxidised A2M oligomers. MS characterisation revealed a complex mixture of structures due to the multiple ways of combining oligomers and PDHA. For the block structures composed of two oligomers (Fraction B, Figure 3b), diblocks containing unoxidised oligomers linked by a single PDHA were identified (10 in Figure 5). Interestingly, diblocks composed of two oxidised oligomers linked by two PDHA molecules through the NRE (11) were also identified. Their presence suggests similar or even higher reactivities of the NRE aldehydes compared to the RE aldehyde. In the opposite case, one would expect predominantly diblocks attached only at the reducing ends. Importantly, such diblocks could not be identified. Diblocks with intramolecular (cyclic) structures (12 or 13) were also detected, in addition to fully substituted diblocks attached through the NRE (14). For the block structures composed of three oligomers (Fraction A, Figure 3b) we identified block structures composed of two oxidised oligomers and one unoxidised oligomer linked through the RE and NRE (15). Details regarding the 1 H-NMR and MS characterisation are given in Supporting information, S2. The presence of non-reduced Schiff bases, especially for conjugates linked through the NRE suggest the need for longer reduction times and/or higher concentrations of PB for PDHA based reactions with oxidised oligomers.

Fractionation of the water-soluble fraction obtained with ADH revealed a more complex mixture of structures (Figure 3c). The three main fractions (II, III and IV) were confirmed to contain oligomers and ADH linked secondary amine bonds by 1 H-NMR (Supporting information, S3). Fraction IV (Figure 3c) was mainly composed of A2M-ADH conjugates formed with unoxidised oligomers (16, Figure 5), confirming incomplete oxidation discussed above. The presence of intramolecular cyclic structures (17 or 18) in fraction III, analogous to those observed with PDHA, confirmed the high reactivity of all the aldehydes. Structures containing multiple ('polymerised') ADH (19 or 20) were identified in fraction II, in line with previous experiments with ADH [START_REF] Vikøren Mo | Activation of enzymatically produced chitooligosaccharides by dioxyamines and dihydrazides[END_REF] . Fraction VI and V contained free 'polymerised' ADH (21 and 22, respectively). In contrast to the PDHA case, complete reduction was obtained for all the conjugates with ADH. This supports our previous observations of faster reduction of hydrazones compared to oximes with PB [START_REF] Mo | 2,5-Anhydro-d-Mannose End-Functionalized Chitin Oligomers Activated by Dioxyamines or Dihydrazides as Precursors of Diblock Oligosaccharides[END_REF] .

The water-insoluble fraction was dissolved in hexafluoro isopropanol (HFIP) and shown to only contain 'polymerised' ADH by 1 H-NMR (Supporting information, S4). The mechanism for the polymerisation is unknown but seems to be enhanced by the pre-treatment of the oligomers by periodate due to the extensive formation of 'polymerised' insoluble ADH formed after only 96 hours at RT. In a corresponding reaction where 'polymerised' ADH was observed, the reaction proceeded for 40 days at RT without forming an insoluble fraction. The reactions were performed under the same conditions only differing in reaction time and type of oligomer (N,N'-diacetyl chitobiose (AA) instead of oxidised A2M) [START_REF] Vikøren Mo | Activation of enzymatically produced chitooligosaccharides by dioxyamines and dihydrazides[END_REF] . Oxidised oligomers were only purified by dialysis prior to activation and traces of periodate or ethylene glycol may have enhanced the 'polymerisation' mechanism. We therefore assume that a more thorough purification by e.g. GFC fractionation of the oligomers prior to activation will reduce the formation of 'polymerised' ADH.

Fully ADH activated oxidised A2M conjugates (corresponding to 9 in Figure 4) were not identified in the reaction mixture, in contrast to the reaction with PDHA were a large fraction of this structure was formed.

This can be explained by the fast and extensive formation of 'polymerised' ADH.

Soluble A2M-based block polymers

Based on the results from the initial conjugation experiment with oxidised A2M and PDHA, we attempted to form longer chitin block polymers by conjugating oxidised A2M using a sub-stoichiometric amount of PDHA (0.5 equivalents). The resulting reaction mixture was completely water soluble. 1 H-NMR characterisation confirmed the presence of secondary amines (Supporting information, S5). The reaction mixture was subsequently fractionated by GFC (Figure 6a). The reaction resulted in the formation of longer chitin block polymers (up to > DP 24 based on calibration with AnM oligomers). Interestingly, all fractions were completely water-soluble, whereas AnM oligomers only are water-soluble up to DP 9 (Figure 6b). Fractions (E-I) were purified and characterised by 1 H-NMR and MS (Supporting information, S6). Fraction I contained oxidised oligomers activated with one PDHA molecule forming intramolecular cyclic structures (3 or 4, Figure 4). This fraction also contained a large portion of reduced unreacted AnM oligomers. Interestingly, no unreacted oxidised oligomers were detected, confirming that the NRE aldehydes are more reactive than the M aldehyde. Fractions G and H corresponded to fraction A and B in Figure 3 with short chitin block structures (DP 6 and 9, respectively). However, none of the structures were fully substituted with PDHA as a consequence of the lower equivalence of PDHA used. Due to limitation in the mass detection for larger ions by the MS method, identification of specific block structures in fraction E and F was difficult. However, the obtained 1 H-NMR spectra were identical to the spectrum obtained for fraction G, indicating that these are longer block polymers with otherwise similar structure.

The large amount of reduced unreacted unoxidised oligomers indicates slow kinetics for the periodate oxidation of the AnM oligomers. Less unreacted oligomers were obtained when oxidised A2M were prepared using 4 equivalents periodate and reacted under the same conditions (Supporting information, S7).

Hence, the degree of oxidation can be increased by using a higher excess of periodate and/or longer reaction (oxidation) time.

Soluble chitin-based block polymers using longer oxidised AnM oligomers

Oxidised A5M oligomers were reacted with 0.5 equivalents PDHA and fractionated by GFC (Figure 7a).

The resulting chromatogram showed a series of distinct fractions with DP (by extrapolation of the GFC calibration) corresponding to block structures composed of multiple oxidised A5M blocks linked by PDHA.

Both the unfractionated sample and the fractions were soluble in water (buffer) even though A5M oligomers are close to the solubility limit for chitin. The longest soluble block polymers detected had an apparent DP above 36 (Figure 7). 1 H-NMR characterisation confirmed the presence of oligomers linked through both the RE and NRE by PDHA in all fractions (Supporting information, S8). Due to limitation in mass detection for larger ions by the MS method and the complex mixture of structures formed, the possibilities for precise structure determination of the longer block polymers was limited. However, MS characterisation of the fraction eluting at approx. same time as the parent oligomer turned out to be a mixture of unoxidised oligomers and oxidised oligomers activated with a single PDHA molecule forming an intramolecular cyclic structure, confirming the high reactivity of the NRE aldehydes. Hence, the oxidised A5M oligomers behaved in the same way as the oxidised A2M. We therefore assume the longer block structures to be of the same composition as the structures identified above.

We suggest that the increased solubility is primarily associated with the flexibility of the PDHA linker, contributing to conformational entropy of the block structures as compared to homogenous chitin chains By increasing the degree of oxidation of the AnM oligomers and optimising the conjugation protocol, this method can be used to prepare a new type of soluble chitin block polymers of longer chain length with potential for pharmaceutical and biomaterial applications.

An alternative approach to NRE functionalisation of chitin oligomers

Reaction with periodate oxidised AnM oligomers resulted in a complex mixture of structures. We therefore investigated the oxidation of chitin conjugates (AnM-ADH/PDHA) as an alternative approach to functionalise the NRE of chitin oligomers. This approach requires stability of the secondary amine linkage between the oligomers and PDHA or ADH throughout the oxidation step. AnM conjugates (A6M-PDHA and the diblock A2M-ADH-MA2) were oxidised using 2 equivalents periodate. 1 H-NMR characterisation after dialysis showed complete disappearance of the resonances resulting from the secondary amines and a drastic change in the proton resonances resulting from the reducing end M residue (Figure 8 for A6M-PDHA and Supporting information, S9 for A2M-ADH-MA2). Hence, the results indicate that the secondary amine linkages are destroyed during the oxidation. Interestingly, a minor resonance from the reducing end reappeared after the treatment with periodate for the A6M-PDHA conjugate (Figure 8c). This was not observed for the periodate treatment of A2M-ADH-MA2 (Supporting information, S9), suggesting different mechanisms for the degradation of the conjugates. In any case, this approach is unfortunately not applicable for the preparation of precursors for ABA-or ABC-type block polysaccharides.

Conclusions

The vicinal diol in the NRE end residue of AnM oligomers can be selectively oxidised by periodate, forming the corresponding dialdehydes adding to the aldehyde of the M reducing end residue. The dialdehydes are even more reactive towards dioxyamines and dihydrazides than the already reactive aldehyde of the M residue. Reactions with an excess of these divalent linkers can provide AnM oligomers fully substituted at the RE and NRE. Such oxidised oligomers are therefore potential precursors for a range of new glycoconjugates, including complex block polysaccharides. The latter was demonstrated by the formation of a discrete distribution of 'polymerised' AnM oligomers through reactions with sub-stoichiometric amounts of linkers. Importantly, these block polysaccharides were, in contrast to chitins of the same chain lengths, water-soluble. Such soluble chitin-based polymers may be relevant for biomedical and biomaterial applications.

S1 Characterisation of reaction mixtures with oxidised A2M oligomers and an excess PDHA or ADH

The reaction mixtures formed when oxidised A2M oligomers were reacted with 10 equivalents PDHA using the two-pot reductive amination procedure (pH 4.0, 20 equivalents PB added after 24 hours, with a total reduction time of 72 hours) and the water-soluble fraction from the corresponding experiment with ADH were characterized by 1 H-NMR and compared to purified A2M-PDHA and A2M-ADH conjugates prepared using unoxidised oligomers (Figure S1 and S2, respectively). Compared to the latter, the 1 H-NMR spectra of the reaction mixtures formed with oxidised A2M oligomers were more complex.

However, resonances resulting from the characteristic secondary amine protons (around 3.0 ppm) were present in the samples, confirming formation of conjugates. The resonances were, however, more complex than for the conjugates with unoxidised A2M oligomers suggesting more than one type of conjugates formed. Incomplete reduction of conjugates formed with PDHA (Figure S1) was observed by the presence of resonances in the Schiff base area (6-8 ppm). Conjugates formed with ADH were in contrast completely reduced (Figure S2).

S2 Characterisation of fractions from the reaction with oxidised A2M oligomers and an excess PDHA

The reaction mixture obtained when oxidised A2M oligomers were reacted with 10 equivalents PDHA was fractionated by GFC (Figure S3). Collected fractions (A-D) were purified and characterised by 1 H-NMR and MS. The 1 H-NMR characterisations of the fractions are given in Figure S4-S7. The structures identified by MS and their theoretical mass (Da) are included in the figures. The incomplete reduction of Schiff bases did, however, influence the observed mass of some of the structures. The results from the MS characterisation of the fractions are presented in Table S1.

S3 Characterisation of fractions from the reaction with oxidised A2M oligomers and an excess ADH (water-soluble fraction)

The water-soluble fraction from the reaction of oxidised A2M oligomers and 10 equivalents ADH was separated by GFC (Figure S8). Collected fractions (I.-VI.) were purified and characterised by 1 S2. The water-insoluble fraction from the reaction of oxidised A2M oligomers with 10 equivalents ADH was dissolved in hexafluoro isopropanol (HFIP) and characterised by 1 H-NMR (Figure S15a). S1. S7 Reaction of oxidised A2M oligomers, obtained using 4 equivalents periodate, with a substoichiometric amount of PDHA Oxidised A2M oligomers, obtained using 4 equivalents periodate, were reacted with 0.5 equivalents PDHA. GFC fractionation of the reaction mixture was compared to the fractionation of the reaction mixture obtained when oxidised A2M oligomers, obtained using 2 equivalents periodate, were reacted with PDHA under the same conditions (Figure S23). The fractionation revealed a smaller relative fraction of unreacted oligomers when a higher concentration of periodate was used, indirectly confirming that a higher degree of oxidation was obtained using 4 equivalents periodate. The results also confirm that oxidised oligomers react more rapidly than unoxidised oligomers.

S9 Periodate oxidation of A2M-ADH-MA2 diblocks

The A2M-ADH-MA2 diblock was subjected to oxidation using 2 equivalents periodate.

Figure 1 :

 1 Figure 1: a) chemical structure of an A2M oligomer, b) chemical structure of a periodate oxidised A2M oligomer, c) conjugation of PDHA or ADH to the reducing and non-reducing end of oxidised A2M oligomer.

Figure 2 :

 2 Figure 2: 1 H-NMR characterisation of a) A2M (unoxidised) b) oxidised A2M prepared using 2 equivalents NaIO4 c) oxidised A2M prepared using 4 equivalents NaIO4.

Figure 3 :

 3 Figure 3: Analytical GFC fractionation of a) a standard mixture of AnM oligomers for GFC calibration, b) the mixture following reaction of oxidised A2M with 10 equivalents PDHA + PB and c) the watersoluble fraction from the reaction of oxidised A2M with 10 equivalents ADH + PB.

Figure 4 :

 4 Figure 4: Graphical presentation of possible structures formed when unoxidised and oxidised A2M oligomers were reacted with an excess PDHA and the theoretical mass of each structure (fully reduced).For structures with one oligomer (1-9) all possible combinations are presented (transparent structures were not identified). For products with two or three oligomers (10-14 and 15, respectively), only the identified structures are presented. The symbols used are defined in Figure1.

Figure 5 :

 5 Figure 5: Graphical presentation of identified structures from the reaction of oxidised (and unoxidised) A2M oligomers with an excess ADH and the mass of each structure (fully reduced).

Figure 6 :

 6 Figure 6: a) GFC fractionation following reaction of oxidised A2M with 0.5 equivalents PDHA. b) fractionation of a standard mixture of AnM oligomers for GFC calibration.

Figure 7 :

 7 Figure 7: a) GFC fractionation following reaction of oxidised A5M, prepared using 2 equivalents periodate, with 0.5 equivalents PDHA b) fractionation of a standard mixture of AnM oligomers for GFC calibration.

Figure 8 :

 8 Figure 8: 1 H-NMR characterisation of a) a purified A3M oligomer, b) the purified A6M-PDHA conjugate prior to oxidation and c) the resulting mixture after oxidation and dialysis.

Figure S1: 1

 1 Figure S1: 1 H-NMR characterisation of a) the mixture formed upon reaction of oxidised A2M oligomers with 10 equivalents PDHA and b) the purified A2M-PDHA conjugate with annotations of identified resonances 1 .

Figure S2: 1

 1 Figure S2: 1 H-NMR characterisation of a) the mixture formed upon reaction of oxidised A2M oligomers with 10 equivalents ADH, b) purified A2M-ADH conjugate with annotations of identified resonances 1 .

Figure S3 :

 S3 Figure S3: GFC fractionation of a) a standard mixture of AnM oligomers for calibration and b) the reaction mixture obtained for oxidised A2M oligomers reacted with 10 equivalents PDHA using the two-pot reductive amination protocol.

Figure S4: 1

 1 Figure S4: 1 H-NMR characterisation of fraction A from Figure S3. The structure identified by MS is included in the figure.

Figure S5: 1

 1 Figure S5: 1 H-NMR characterisation of fraction B from Figure S3. The structures identified by MS are included in the figure.

Figure S6: 1

 1 Figure S6: 1 H-NMR characterisation of fraction C from Figure S3. The structure identified by MS is included in the figure.

Figure S7: 1

 1 Figure S7: 1 H-NMR characterisation of fraction D from Figure S3. The structures identified by MS are included in the figure.

Table S1 :

 S1 Results of the MS characterisation of fractions obtained in the reactions with oxidised A2M oligomers and 10 (fraction A-D) or 0.5 equivalents (fraction E-I) PDHA. Structures were either identified as fully reduced or with x number (#) of Schiff bases (oximes) due to incomplete reduction. The m/z ions in bold represent ions identified in the fractions. Semi-transparent structures were not identified in any of the fractions. Fractions E-I are discussed in chapter S6.

  H-NMR and MS. The 1 H-NMR characterisations of the fractions are given in Figure S9-S14. The structures identified by MS and their theoretical mass (Da) are included in the figures. All structures were completely reduced as in contrast to the structures from the corresponding reaction with PDHA. The results from the MS characterisation are presented in Table

Figure S8 :

 S8 Figure S8: GFC fractionation of a) a standard mixture of AnM oligomers for calibration and b) the water-soluble fraction obtained for oxidised A2M oligomers reacted with 10 equivalents ADH using the two-pot reductive amination protocol.

Figure S9: 1

 1 Figure S9: 1 H-NMR characterisation of fraction I. from Figure S8.

Figure S10: 1

 1 Figure S10: 1 H-NMR characterisation of fraction II. from Figure S8. The structures identified by MS are included in the figure.

Figure S11: 1

 1 Figure S11: 1 H-NMR characterisation of fraction III. from Figure S8. The structures identified by MS are included in the figure.

Figure S12: 1

 1 Figure S12: 1 H-NMR characterisation of fraction IV. from Figure S8. The structure identified by MS is included in the figure.

Figure S13: 1

 1 Figure S13: 1 H-NMR characterisation of fraction V. from Figure S8. The structure identified by MS is included in the figure.

Figure S14: 1

 1 Figure S14: 1 H-NMR characterisation of fraction VI. from Figure S8. The structure identified by MS is included in the figure.

  Characterisation of A2M and ADH dissolved in HFIP is included for comparison (FigureS15b and c, respectively). Only resonances from the protons of ADH was observed in the spectrum obtained for the insoluble fraction, and hence, the sample only contained polymerized ADH.

Figure S15: 1

 1 Figure S15: 1 H-NMR characterisation of a) the insoluble fraction from the reaction of oxidised A2M oligomers and 10 equivalents ADH, b) A2M and c) ADH in HFIP.

Figure S16: 1 H

 1 Figure S16: 1 H-NMR characterisation of a) the reaction mixture formed with oxidised A2M oligomers and a sub-stochiometric amount of PDHA (0.5 equivalents), b) the reaction mixture formed with oxidised A2M an excess of PDHA (10 equivalents) and c) the purified A2M-PDHA conjugate with annotations of identified resonances

Figure S17 :

 S17 Figure S17: GFC fractionation of a) a standard mixture of AnM oligomers for calibration and b) the reaction mixture obtained for oxidised A2M oligomers reacted with 0.5 equivalents PDHA using the two-pot reductive amination protocol.

Figure S18: 1 H

 1 Figure S18: 1 H-NMR characterisation of fraction E from Figure S17.

Figure S19: 1

 1 Figure S19: 1 H-NMR characterisation of fraction F from Figure S17.

Figure S20: 1 H

 1 Figure S20: 1 H-NMR characterisation of fraction G from Figure S17. The structure identified by MS is included in the figure.

Figure S21: 1

 1 Figure S21: 1 H-NMR characterisation of fraction H from Figure S17. The structures identified by MS are included in the figure.

Figure S22: 1 H

 1 Figure S22: 1 H-NMR characterisation of fraction I from Figure S17. The structures identified by MS are included in the figure.

Figure S23 :

 S23 Figure S23: GFC fractionation of a) a standard mixture of AnM oligomers for calibration, b) the reaction mixture obtained for A2M oligomers, oxidised with 4 equivalents periodate, reacted with 0.5 equivalents PDHA and c) the reaction mixture obtained for A2M oligomers, oxidised with 2 equivalents periodate, reacted with 0.5 equivalents PDHA using the two-pot reductive amination protocol.

Figure S24 :

 S24 Figure S24: GFC fractionation of a) the reaction mixture obtained for oxidised A5M oligomers reacted with 0.5 equivalents PDHA using the two-pot reductive amination protocol and b) a standard mixture of AnM oligomers for calibration.

Figure S25: 1

 1 Figure S25: 1 H-NMR characterisation of the main fraction from Figure S24. The structures identified by MS are included in the figure.

Figure S26: 1 H

 1 Figure S26: 1 H-NMR characterisation of fractions from Figure S24.

  1 H-NMR characterisation after purification by dialysis is given in Figure S27, revealing complete disappearance of the resonances resulting from the secondary amines and a drastic change in the proton resonances resulting from the reducing end M residue. Hence, the results indicate that the secondary amine linkages of the diblock are destroyed during the periodate oxidation.

Table S2 :

 S2 Results of the MS characterisation of fractions obtained in the reaction with oxidised A2M oligomers and 10 equivalents ADH (fraction II.-.VI.). All structures identified were fully reduced. The m/z ions in bold represent ions identified in the fractions.

	Structure	Fraction	Theoretical mass (Da)	[M+H] +	m/z [M+Na] +	[M+K] +	[M+2H] 2+
		VI.	316.19	317.20	339.18	355.15
		V.	458.26	459.27	481.25	497.22
		IV.	726.33	727.34	749.32	765.29
		III.	866.43	867.44	889.42	905.39
		II.	1008.51	1009.52	1031.50	1047.47	505.26

S4 Characterisation of the water-insoluble fraction from the reaction with oxidised A2M oligomers and an excess ADH

Acknowledgements

This work was supported by a grant from the Norwegian University of Science and Technology to I. V.

Mo. Rangnhild Bardal Roness is thanked for running some preliminary experiments with periodate oxidised AnM oligomers during the work with her master thesis (2015-2017). Kåre Kristiansen is thanked for running the MS analysis.

Supporting information