Formation of substituted dioxanes in the oxidation of gum arabic with periodate

Renewable polysaccharide feedstocks are of interest in bio-based food packaging, coatings and hydrogels. Their physical properties often need to be tuned by chemical modification, e.g. by oxidation using periodate, to introduce carboxylic acid, ketone or aldehyde functional groups. The reproducibility required for application on an industrial scale, however, is challenged by uncertainty about the composition of product mixtures obtained and of the precise structural changes that the reaction with periodate induces. Here, we show that despite the structural diversity of gum arabic, primarily rhamnose and arabinose subunits undergo oxidation, whereas (in-chain) galacturonic acids are unreactive towards periodate. Using model sugars, we show that periodate preferentially oxidises the anti 1,2-diols in the rhamnopyranoside monosaccharides present as terminal groups in the biopolymer. While formally oxidation of vicinal diols results in the formation of two aldehyde groups, only traces of aldehydes are observed in solution, with the main final products obtained being substituted dioxanes, both in solution and in the solid state. The substituted dioxanes form most likely by the intramolecular reaction of one aldehyde with a nearby hydroxyl group, followed by hydration of the remaining aldehyde to form a geminal diol. The absence of significant amounts of aldehyde functional groups in the modified polymer impacts crosslinking strategies currently attempted in the preparation of renewable polysaccharide-based materials.


Reaction monitoring with Raman spectroscopy
The reaction times reported for the oxidation by periodate are up to hours, [2][3][4][5] and are reported to depend on temperature and pH, 6 the presence of metal salts, 7 and the concentration of periodate. 4,8,9 As reaction times reported for the periodate oxidations of 1,2-diols vary substantially, in the present study in-line monitoring of the conversion of periodate to iodate was carried out using Raman spectroscopy as described earlier. 10 The Raman spectrum of periodate (IO 4 -) shows two bands at 789 and 851 cm −1 . Its reduction product, iodate (IO 3 -), has a characteristic broad Raman band at 798 cm −1 (Fig. S2-A). As gum arabic has a low Raman scattering cross-section with a broad distribution of functional group types, significant changes in its Raman spectrum are not apparent over the course of the reaction. Several of the model sugars show more distinct bands, but for comparison, all rates were determined by adding solutions of (poly)saccharide to solutions of periodate under rapid stirring and following solely the intensity of the periodate bands over time.

Gum arabic (GA1)
The oxidation of gum arabic with periodate, followed by Raman spectroscopy, shows full conversion of IO 4 to IO 3 within 3.5 min (Fig. S2-B).

Model sugars
Compared to the polymer, the oxidation of model sugars proceeds on a similar timescale and not over several hours as reported elsewhere for mono-and dissaccharides. 4 Nevertheless, the reaction of periodate with m-Rhap takes 300 s ( Fig. S2-C), in contrast to the oxidation of GA1, which proceeds mostly during the initial mixing time.
Importantly, the addition of protected m 5 -Galp does not result in reduction of IO 4 -, indicating also that loss of periodate due to light or heating from the laser can be disregarded (Fig. S2-D and Fig. S3).

Model peptide
The backbone of the gum arabic polymer consist of a polypeptide chain, the reactivity of which also has to be considered in the oxidation with periodate, especially since reactions have been reported between periodate and amino acids such as serine, threonine, tryptophan, methionine and cysteine. 11 Addition of albumin, a common model protein, to a solution of periodate has no effect on the Raman spectrum of periodate over the time interval typical for sugar oxidation (Fig. S4), indicating a low reactivity of periodate towards polypeptides under these conditions and allows us to assume the minor amount of peptide present in our polymer structure is not oxidised to a significant extent under the present conditions. Raman shift (cm −1 ) Counts Figure S4: Raman spectra (λ exc 785 nm) of NaIO 4 (0.234 M, aq) before (green) and after (green to blue) the addition of albumin (1:1, 2 w/v%, aq), A minor decrease in the periodate band at 792 cm −1 is observed, but the band at 854 cm −1 is unaffected over 4 min. The initial spectrum of NaIO 4 was corrected for dilution and all spectra were baseline corrected.        Table S1: Amounts used for the data obtained in Figure S15.   Figure Figure S24: Structure of product 1 with between brackets the J-coupling values of H1, H2, H3, H4, H5 and H6 as obtained from the 1 H-NMR spectrum (Fig. S26), which were used to determine the stereochemistry of product 1. DFT calculations on all four possible products obtained by changing the stereochemistry on the C2 and C4, show that for each of them the lowest energy state of the molecule is when it adopts a chair conformation. The large J-coupling of 8.2 Hz between H4 and H5 of product 1 indicates an axial-axial interaction, 13 and with H5 axial pointing up, this means that H4 has to be axial pointing down and the hydroxyl group on the C4 atom has to be pointing up in an equatorial position. The small J-coupling of 2.1 Hz between H1 and H2 indicates either an equatorial-equatorial or an axial-equatorial coupling. 13 Considering the way in which this product is formed (Fig.  S25), the only option for intramolecular ring-closing to happen is for the OH on the C2 to attack the C4. This results in in the C3 substituent on the C2 position pointing up, provided enolisation does not occur. With both the OMe on the C1 pointing up and the substituent on the C3 pointing up, whether they are axial or equatorial depends on whether the molecule adopts a boat or chair conformation. In the (most stable) chair conformation, the OMe on the C1 is axial and the substituent on the C2 is oriented equatorially. As a result, H1 and H2 are equatorial and axial, respectively. Hence, the small coupling constant of 2.1 Hz between H1 and H2 is due to an equatorial-axial interaction. Figure S25: Proposed mechanism for the formation of product 1. First, oxidation by periodate occurs on the anti diol of m-Rhap, forming an aldehyde on the C3 and C4 positions. Protonation of the aldehyde on the C4 position, followed by the attack of the C2 hydroxyl group and subsequent deprotonation, leads to ring-closing and the formation of a substituted dioxane, still containing one aldehyde on the C3. Hydration leads to the formation of product 1.  Figure 30  35  40  45  50  55  60  65  70  75  80  85  90  95  100 Chemical shift (ppm)  Figure Figure S31: (left) Structure of product 2 with between brackets the J-coupling values of H1, H2, H3, H4, H5 and H6 as obtained from the 1 H-NMR spectrum (Fig. S33), which were used to determine the stereochemistry of product 2. Similar to the reasoning for product 1 (Fig. S24), the small J-coupling of 2.1 Hz between H1 and H2 indicates axial-equatorial coupling. 13 With H1 being equatorial, this means that H2 has to be oriented axial, and pointing down, and the substituent on the C2 has to be pointing up. The additional small J-coupling of 2.0 Hz between H4 and H5 indicates another axial-equatorial interaction, 13 and with H5 in the axial position, this means that H4 has to be equatorial and pointing up, with the hydroxyl group on the C4 atom pointing down. The different J-coupling between H4 and H5 shows the different stereochemistry on the C4 position in products 1 and 2.    Figure Figure S38: Structure of product 3 with between brackets the J-coupling values of H1, H2, H4, H5 and H6 as obtained from the 1 H-NMR spectrum (Fig. S40), which were used to determine the stereochemistry of product 3. The not observable J-coupling between H1 and H2 indicates either equatorial-equatorial or axial-equatorial coupling. 13 With H1 pointing down, this means that H2 can point either up or down, and the same is the case for the hydroxyl group on the C2 position. The large J-coupling of 8.1 Hz between H4 and H5 indicates an axialequatorial interaction, 13 and with H5 pointing up, this means that H4 has to be pointing up and the hydroxyl group on the C4 atom has to be pointing down. H3 or C3 are not present in product 3, as they have formed formic acid, due to double oxidation (see Fig. S39). Figure S39: Proposed mechanism for the formation of product 3. After the initial oxidation of either the syn or the anti diol, periodate oxidises once again, forming an aldehyde on the C2 and C4 positions. The C3 carbon is converted to formic acid. Hydration on the C2 position, followed by protonation of the aldehyde on the C4 position, allows for ring-closing and deprotonation to formation of a substituted dioxane, product 3.  Figure 18  50  52  54  56  58  60  62  64  66  68  70  72  74  76  78  80  82  84  86  88  90  92  94  96  98 Chemical shift (ppm)  Figure Figure Figure Figure

DFT calculations
Computational details -NMR All density functional theory and NMR property calculations have been performed using ORCA 5.0.2. 14 Initial structures were drawn using Avogadro, 15 conformers were generated with meta-dynamics using CREST 16 with the GFN2-xTB method 17 and ALPB water solvation.
part0: b97-d3/def2-SV(P) // GFNn-xTB (Input geometry) (threshold = 4.0 kcal/mol) Briefly, the results from crest are taken and prescreened using b97-d3/def2-SV(P) and later r2scan-3c with a cheap GmRRHO free energy correction before they are optimized using r2scan-3c [SMD]. Between each step, conformers higher in energy than the threshold are discarded. Finally the NMR shifts are calculated using pbe0-d4/pcsseg-3 18,19 and coupling constants using pbe0-d4/pcJ-3, 20 both with SMD water solvation. From the NMR shifts and couplings the spectra are calculated and the conformers are weighted using ANMR. 21 For the reference, TMS was optimized and calculated using the same methods as above and its chemical shift was subtracted from the calculated values.

Computational details -energies
The Gibbs free energies were obtained for the conformers at the end of step 2 at the r2scan-3c + SMD[h2o] + GmRRHO(GFN2[alpb]-bhess) // r2scan-3c[SMD] level. Energies were weighted according to: With:  Table S2: Calculated energies for the various products that can be formed upon the oxidation of m-Rhap by periodate. Figure S45: Structures of possible products from the oxidation of m-Rhap by periodate used for the DFT calculations. Product 1, 2, and product 3 or its enantiomer 8 were found experimentally.     12. Solid state 13 C NMR spectroscopy Figure S50: The 13 C solid state NMR spectrum of oxidised GA1, showing the deconvolution of the signal for the methyl carbon of rhamnopyranoside units in the sample isolated by precipitation in ethanol. Figure S51: The 13 C solid state NMR spectrum of oxidised GA1, showing rhamnopyranoside methyl signal(s) in unoxidised GA1 (green), oxGA1 isolated by freeze drying (blue) and oxGA1 isolated by precipitation in ethanol (red). Figure S52: The 13 C solid state NMR spectra of oxidised GA1, isolated by freezedrying (blue) and precipitation in ethanol (orange).