HMF-MA and HMF-MM did not exhibit any absorption peaks in the UV region. Based on our previous studies, the polyethylene glycol (PEG) chain serves as the backbone of the biomacromolecule PDHPGA, with catechol moieties as regular substituents along the PEG backbone.1-8 Typically, catechol exhibits an absorbance peak at approximately 270–290 nm.17 The absence of such absorption peaks in HMF-MA and HMF-MM suggests that these fractions do not contain the caffeic acid-derived biopolymer PDHPGA.

FTIR of HMF-MA and HMF-MM
The FTIR spectra of HMF-MA and HMF-MM samples were interpreted according to the literature data, and the assignments of their infrared absorption bands are reported below.18-20 The strong and broad absorption peak at around 3400 cm-1 was attributed to O-H stretching vibration due to the galacturonic acid backbone’s intra- and intermolecular hydrogen bonding.  The band at 2925.5 cm-1 indicated a characteristic of C-H stretching vibration from -CH, -CH2, and -CH3, methyl esters of galacturonic acid in polysaccharide components. The strong band at 1732.7 cm-1 corresponded to the carbonyl (C=O) in the methyl-esterified group (-COOCH3). Meanwhile, the strong bands at 1611 cm-1 and 1365.4 cm-1 represented the asymmetrical and symmetric stretching vibration of the carboxylate ion (COO−), respectively. Thus, two peaks at 1732.7 cm-1 and 1365.4 cm-1 have been ascribed to the methyl esterified carbonyl groups (–COOCH3) and the ionic carboxyl groups (COO–) of galacturonic acid in the pectin, respectively. The intensities of the 1732.7 cm-1 and 1611 cm-1 absorption bands strongly suggested the high degree of esterification in pectin. In agreement with the above finding, an absorbance peak around 1200–1237 cm-1 has been assigned to the stretching and bending characteristic for -COOCH3 of galacturonic acid, and another peak at about 1421.3 cm−1 has been associated with the carboxylate groups. The characteristic bending asymmetric vibration of δas(CH3) methyl group appears in the region of 1438.6 cm-1, and the symmetric vibration of δs(CH3) acetyl group in the region of 1366 cm-1. A combination of peaks at 1231 cm-1 and 1086.7 cm-1 can be assigned to the presence of rhamnogalacturonan (RG-I). The absorption bands in the fingerprinted region, overlapped bands observed in the 1211-900 cm-1 characteristics for stretching the asymmetric vibration of pyranose C-O-C, are characteristic of the pectin backbone and side groups. The band at 1143.6 cm-1 is assigned to the C-O-C stretching vibrations of the α-1,4-D-glycosidic bonds in the homogalacturonan (HG) chains. The two bands, at 1086.7 and 1074 cm-1 in the IR spectra, result from neutral sugars in the side chains of RG-I and are assigned to the same stretching modes of L-arabinosyl and D-galactosyl units, respectively. The spectra observed between 1300 and 900 cm-1 corresponded to the ether R-O-R and cyclic C-C ring linkages of the pectin structure. Several 1143.6 to 1000 cm-1 peaks have been associated with the glycosidic bond vibrations involving C-O-C, C-C (C-O), and O=C-H bending in the pyranose ring in polysaccharides.  A peak at 829 cm-1 suggested the existence of α-glycosidic linkages, and a weak band near 745 cm-1 can be assigned to ring stretching and ring deformation of α-D-(1–4) linkages. In addition, a weak band near 665 cm-1 implied the presence of the galacturonan structures. The spectral region ranging from 800 to 1200 cm-1 has been generally recognized as the “fingerprint” region for different pectins.18-20

NMR of HMF-MA and HMF-MM

NMR spectroscopy is a powerful analytical technique for investigating synthetic and natural compounds in solution and solid state. It provides molecular-level insights by analyzing the behavior of the atomic nuclei in a magnetic field. Modern pulsed NMR methods facilitate the structural assignment and authentication of low-molecular-weight compounds while also enabling the interpretation of polymer spectra. This approach has significant potential for elucidating molecular fragments within complex mixtures. The present study's interpretation of NMR spectra and data was primarily based on previously published works.21,22

The chemical structures of the constituents in the HMF-MA and HMF-MM samples were identified by comparing their ¹H NMR and ¹³C NMR spectra with those literature data.23-27

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The ¹H and ¹³C NMR spectra of HMF-MA and HMF-MM did not display signals characteristic of aromatic nuclei in the regions 6-7 ppm (1H NMR) and 110-150 ppm (³C NMR), respectively. Consequently, based on the NMR spectral data, the presence of poly[3-(3,4-dihydroxyphenyl)glyceric acid] (PDHPGA) was ruled out in these fractions. The principal signals observed in the 1H NMR spectra of the main chemical component of HMF-MA and HMF-MM were 5.32, 5.20, 4.98, and 4.91 ppm, corresponding to the anomeric H1 centers of various sugars, including arabinose, rhamnose, galactose, and galacturonic acid fragments:

• →4)-α-GalpA-(1→ - (H1) 5.20 ppm;

• →2)-α-Rhap-(1→ - (H1) 5.32 ppm; 

• α-GalpA-(1→ (2) - (H1) 4.98 ppm;

• →2,4)-α-Rhap-(1→ - (H1) 5.20 ppm;

• →3)-β-Galp-(1→ - (H1) 4.91 ppm;

• Araf-(1→ - (H1) 5.32 ppm.

Additionally, the ¹H NMR spectra of HMF-MA and HMF-MM exhibited signals corresponding to O-methyl protons of carboxylic acid methyl ester (COOCH3) for D-galacturonic acid with a chemical shift of 3.8 ppm.  The signals at 2.5 ppm and 2.67 ppm were attributed to the methyl protons of the O-acetyl groups (OCCH3) attached to D-galacturonic acid. The former was assigned to 3-O-acetyl groups and the latter to 2-O-acetyl groups. The signals at 1.56 ppm and 1.77 ppm were respectively assigned to the methyl protons of rhamnose (Rha-CH3) linked only at O-2 and to the rhamnose (Rha-CH3) linked both at O-2 and O-4.

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The ¹³C NMR spectra of HMF-MA and HMF-MM revealed signals attributed to the anomeric C1 centers of various sugar fragments, including arabinose, rhamnose, galactose, and galacturonic acid, as well as the pyranoid rings of their derivatives (C2, C3, C4, C5) and C6:

• →4)-α-GalpA-(1→ - (C1) 101 ppm; (C 2,3,5) 71.6 ppm; (C4) 78 ppm;

• →3)-β-Galp-(1→ - (C1) 104.8 ppm; (C3) 82 ppm; (C6) 62 ppm;

• Araf-(1→ - (C1) 109 ppm; (C5) 62 ppm; 

• →2)-α-Rhap-(1→ - (C1) 95 ppm; (C2) 78 ppm;

• →2,4)-α-Rhap-(1→ - (C1) 95 ppm; (C2) 78 ppm; (C4) 82 ppm.

Furthermore, the ¹³C NMR spectra of HMF-MA and HMF-MM contained signals corresponding to the methyl carbon atoms of the O-acetyl groups (OCCH3) attached to D-galacturonic acid at 20 ppm and 24 ppm. The former was assigned to 3-O-acetyl groups and the latter to 2-O-acetyl groups. The signals at 17 ppm and 19 ppm were respectively assigned to the methyl carbon atoms of rhamnose (Rha-CH3) (C6) linked only at O-2 and to the methyl carbon atoms of rhamnose (Rha-CH3) (C6) linked both at O-2 and O-4.21-27