Determination of substitution positions in hyaluronic acid hydrogels using NMR and MS based methods

Article abstract of DOI:10.1016/j.carbpol.2015.09.112

In hydrogels of cross-linked polysaccharides, the total amount of cross-linker and the degree of cross-linking influence the properties of the hydrogel. The substitution position of the cross-linker on the polysaccharide is another parameter that can influence hydrogel properties; hence methods for detailed structural analysis of the substitution pattern are required. NMR and LC-MS methods were developed to determine the positions and amounts of substitution of 1,4-butanediol diglycidyl ether (BDDE) on hyaluronic acid (HA), and for the first time it is shown that BDDE can react with any of the four available hydroxyl groups of the HA disaccharide repeating unit. This was achieved by studying di-, tetra-, and hexasaccharides obtained from degradation of BDDE cross-linked HA hydrogel by chondroitinase. Furthermore, amount of linker substitution at each position was shown to be dependent on the size of the oligosaccharides. For the disaccharide, substitutions were predominantly at ΔGlcA-OH2 and GlcNAc-OH6 while in the tetra- and hexasaccharides, it was mainly at the reducing end GlcNAc-OH4. In the disaccharide there was no substitution at this position. Since chondroitinase is able to completely hydrolyse non-substituted HA into unsaturated disaccharides, these results indicate that the enzyme is prevented to cleave on the non-reducing side of an oligosaccharide substituted at the reducing end GlcNAc-OH4. The procedure can be adopted for the determination of substitution positions in other types of polymers.

Full text of DOI:10.1016/j.carbpol.2015.09.112

Accepted Manuscript
Title: Determination of substitution positions in hyaluronic
acid hydrogels using NMR and MS based methods
Author: Frida J. Wende Suresh Gohil Hotan Mojarradi
Thibaud Gerfaud Lars I. Nord Anders Karlsson Jean-Guy
Boiteau Anne Helander Kenne Corine Sandstr o¨ m
PII:
S0144-8617(15)00977-7
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.carbpol.2015.09.112
CARP 10414
To appear in:
Received date:
Revised date:
Accepted date:
10-5-2015
25-9-2015
30-9-2015
Please cite this article as: Wende, F. J., Gohil, S., Mojarradi, H., Gerfaud, T., Nord, L.
I., Karlsson, A., Boiteau, J.-G., Kenne, A. H., and Sandstrddotom, C.,Determination of
substitution positions in hyaluronic acid hydrogels using NMR and MS based methods,
Carbohydrate Polymers (2015), http://dx.doi.org/10.1016/j.carbpol.2015.09.112
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Determination of substitution positions in hyaluronic
acid hydrogels using NMR and MS based methods.
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Frida J.Wende , Suresh Gohil , HotanMojarradi , Thibaud Gerfaud , Lars I. Nord , Anders
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Karlsson , Jean-Guy Boiteau , Anne HelanderKenne , Corine Sandström *
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Department of Chemistry and Biotechnology, Uppsala BioCenter, Swedish University of
Agricultural Sciences, P.O. Box 7015, SE-750 07 Uppsala, Sweden. Frida.Wende@slu.se;
Suresh.Gohil@slu.se; Corine.sandstrom@slu.se
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ResearchA&CGalderma, Seminariegatan 21, SE-752 28 Uppsala, Sweden.
Hotan.Mojarradi@galderma.com; Lars.Nord@galderma.com; Anders.Karlsson@galderma.com;
Anne.HelanderKenne@galderma.com.
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Galderma R&D, Les Templiers, 2400 route des Colles BP 87, 06410 Biot Cedex, France.
Thibaud.Gerfaud@galderma.com; Jean-Guy.Boiteau@galderma.com
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*Corresponding author:
Prof Corine Sandström
Dept of Chemistry and Biotechnology
Uppsala BioCenter, Swedish University of Agricultural Sciences
PO Box 7015, SE-750 07 Uppsala, Sweden
e-mail: corine.sandstrom@slu.se
Tel : +46 671504
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Highlights:
• NMR and MS methods to determinelinker position in HA hydrogels were developed.
• Oligosaccharides of different sizes were obtained by enzymatic degradation
• Substitution occur at all four available hydroxyl groups of the HA repeating unit.
• Linker distribution among the four positions was obtained by NMR and MS.
• Distribution of substitution positions depend on the size of the oligosaccharides.
Graphical abstract
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Keywords
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Hyaluronic acid, Hydrogel, Cross-linked, Substitution, NMR, MS
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Abstract
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In hydrogels of cross-linked polysaccharides, thetotal amount of cross-linkerandthe degree of
cross-linking influence the properties of the hydrogel. The substitution position of the cross-
linker on the polysaccharideis another parameter that can influence hydrogel properties; hence
methods for detailed structural analysis of the substitution pattern are required.
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NMR and LC-MS methods were developed to determine the positions and amounts of
substitution of 1,4-butanediol diglycidyl ether (BDDE)on hyaluronic acid (HA), andfor the first
timeit is shown that BDDE can react with any of the four available hydroxyl groups of the HA
disaccharide repeating unit.This was achieved by studying di-, tetra-, and hexasaccharides
obtained from degradation of BDDE cross-linked HA hydrogel by chondroitinase. Furthermore,
amount of linkersubstitution at each position was shown to be dependent on the size of the
oligosaccharides.For the disaccharide, substitutions werepredominantlyat ∆GlcA-OH2 and
GlcNAc-OH6 while in the tetra- and hexasaccharides,it was mainly atthe reducing end GlcNAc-
OH4.In the disaccharide there was no substitution at this position. Since chondroitinase is able to
completely hydrolyse non-substituted HA into unsaturated disaccharides, these results indicate
that the enzyme is prevented to cleave on the non-reducing side of an oligosaccharide substituted
atthe reducing endGlcNAc-OH4. The procedure can be adopted for the determination of
substitution positions in other types ofpolymers.
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1.Introduction
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Hydrogels formed by cross-linking of polysaccharides such as hyaluronic acid (HA, also known
as hyaluronan) are materials that can be used for numerous applications, both medical and
aesthetic(Burdick & Prestwich, 2011; Fakhari & Berkland, 2013; Gutowska, Jeong &
Jasionowski, 2001; Kogan, Šoltés, Stern & Gemeiner, 2007; Schanté, Zuber, Herlin &
Vandamme, 2011).The hydrogels will have different physical properties depending on the
starting material and the manufacturing process(Edsman, Nord, Öhrlund, Lärkner & Kenne,
2012; Lapčík, Lapčík, De Smedt, Demeester & Chabreček, 1998; Xu, Jha, Harrington, Farach-
Carson & Jia, 2012)and characterization of the chemical structure of the cross-linked network is
essential for understanding and differentiating the different materials.
HA is a linear non-sulfated glycosaminoglycan built of the repeating unit disaccharide β-
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glucuronic acid-(1→3)-β-
D
-N-acetyl- -glucosamine-(1→4). HA is present in all vertebrates and
D
the primary structure is preserved through all species, the variation lying only in the molecular
weight and polydispersity index of the polymer. In humans, HA is abundant in the skin, eyes and
extracellular matrix(Laurent, 1987; Laurent & Fraser, 1992).Due to its numerous negative
charges, HA can retain large amounts of water and acts therefore as a space filler, lubricant and
osmotic buffer. However, the poor mechanical properties, rapid degradation and clearance in
vivo of soluble hyaluronic acid limit its use as a biomaterial. To improve the mechanical
properties andto increase the resistance to degradation by hyaluronidases, HA is chemically
modified or cross-linked to form hydrogels(Lapčík, Lapčík, De Smedt, Demeester & Chabreček,
1998; Segura, Anderson, Chung, Webber, Shull & Shea, 2005; Volpi, Schiller, Stern & Šoltés,
2009).
A common procedure for cross-linking HA into hydrogels is the reaction with 1,4-butanediol
diglycidyl ether (BDDE) under alkaline condition which yields a stable covalent ether linkage
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between HA and the cross-linker(Ågerup, Berg & Åkermark, 2005). During the cross-linking
process, the epoxide groups of BDDE react with nucleophiles forming derivatives of 1,4-
butanediol di-(propan-2,3-diolyl)ether (BDPE). Some of the cross-linker molecules form
truecross-links that are connected to HA atboth ends while other BDDE molecules only bind at
one end.Definitions of cross-linking parameters to describe the resulting HA hydrogels have
been described in detail earlier (Kenne et al., 2013) as: (a) the degree of modification (MoD),
which is the stoichiometric ratio between the sum of mono- and double-linked BDPE residues
and HA disaccharide units; (b) the degree of substitution (DS) which is the proportion of the HA
disaccharides that are substituted; (c) The degree of cross-linking (CrD), which is the
stoichiometric ratio between BDPE residues that are double-linked and HA disaccharide units;
(d) the effective cross-linker ratio (CrR), which is the fraction of cross-linker residues that are
double-linked (forming cross-linkages in the polysaccharide network), compared to all linked
cross-linkers.
The physical properties of the hydrogels can vary vastly depending on e.g. the molecular weight
of HA, the MoD andthe cross-linking efficiency.Thus methods to differentiate HA gels on the
molecular level are important to explain the different properties of the gels and also to
understand the effect of different manufacturing processes used. Detailed structural analysis of
intact HA hydrogels are however difficult due to the viscosity of the polymer and the complex
cross-linked polymer network(Barbucci et al., 2006; Guarise, Pavan, Pirrone & Renier, 2012;
Pouyani, Harbison & Prestwich, 1994).Recently, an analytical method that allows quantifying
the MoD and CrD in cross-linked HA hydrogels wasreported(Kenne et al., 2013).Using
chondroitinase, a lyase which by an elimination reaction cleaves all 1,4-linkages between N-
acetylglucosamine and glucuronic acid,HA is completely degraded into disaccharides with an
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unsaturated uronic acid at the non-reducing end (∆HA , Fig. 1). When HA is substituted with
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BDPE, the cleavage is not complete and longer BDPE-substituted oligosaccharide fragments are
generated. These oligosaccharidescan be separated from the main non-substituted
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disaccharide(∆HA ) products by size-exclusion chromatography (SEC) and analyzed by
electrospray ionization mass spectrometry (ESI-MS), allowing the determination of CrR(Kenne
et al., 2013).The total amount of BDPE linked to HA,expressed as the MoD, can be determined
1
by H NMR spectroscopy. From these two parameters which give information about the polymer
network,the DS and the CrD can be obtained(Kenne et al., 2013).
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Figure 1.∆HA formed by degradation of HA with chondroitinase ABC.
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Besides MoD and CrD, the substitution positions for the BDPE-molecules on HA couldalso
influence the properties of the hydrogel. In the reaction, nucleophilic groups of HA react with the
epoxide groups of BDDE. In theory, six positions in each HA-disaccharide building blocks are
available for reaction with BDDE, the hydroxyl groups, the carboxylate group and the amide
group. The hydroxyl groups are the most likely binding positions as any esters formed with the
carboxylate group of HA will probably be cleaved under the alkaline conditions used in the
synthesis and the amide nitrogen has the lowest reactivity. The relative tendency of binding to
the four different hydroxyl groups may vary due to steric differences between primary and
secondary alcohols, conformational effects of the polymer due to hydrogen bonding and
hydration(Nestor, Kenne & Sandström, 2010), differences in pKa of the hydroxyl groups and
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differences in reaction conditions during the manufacturing process.In this work, we have
established NMR and LC-MSbased methods to determine the position of substitution of mono-
linked BDPE in oligosaccharides of different lengths obtained by enzymatic hydrolysis of cross-
linked HA hydrogels.
2.Experimental
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2.1 Materials
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HA hydrogel cross-linked with BDDE was prepared under alkaline conditions and the HA
concentration was adjusted to 20 mg/ml after neutralization.The MoD and CrR determined using
the method by Kenne et al. (Kenne et al., 2013)were 8 and 0.1% respectively. Chondroitinase
ABC from Proteus Vulgaris (C2905) was purchased from Sigma Aldrich.
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2.2 Degradation and separation
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The hydrogel (1 g in 100 ml 1 mM sodium phosphate buffer pH 7.0) was treated with
chondroitinase ABC (10 UN) in a sealed flask at 37°Cfor 90 h. The degradation was followed by
analysing the sample on a Superdex Peptide 10/300 GL column (GE Healthcare, Uppsala,
Sweden) using analytical HPLC with a diode-array detector (LC10vp LC system, Shimadzu).
The degradation was considered complete when the viscosity of the sample was low, no
remaining gel particles were observed and the sample contained mostly low-molecular weight
oligosaccharides as demonstrated by HPLC-MS analysis (response for ∆HA
more than 70% out
2
of 21 detected modified and unmodified oligosaccharides with size up to 16 monosaccharides).
The enzyme digest was separated using preparative HPLC (ÄKTA purifier with UV-detector
UV-900, pump P-900 and sample pump P-960, GE Healthcare, Uppsala, Sweden) in two steps.
The first separation was performed by injecting 5 ml of the enzyme digest (sample pump, 5
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ml/min)on to anin-house-packed QSepharoseHigh Performance 16/170 column (GE Healthcare,
Uppsala, Sweden) using agradient of1 mM sodium phosphate buffer pH 7.0 and 1 mM sodium
phosphate buffer pH 7.0 with 200 mM sodium chloride (0-40% 43 min, 40% 6.5 min, 100% 9.5
min) at 5 ml/min. UV detection (232 nm) was used to monitor the separation and fractions were
collected automatically according to predetermined limits of the UV-signal. The content of the
collected fractions were analysed with the Superdex Peptide 10/300 GL on the analytical HPLC.
Fractions containing equal oligosaccharides were pooled and lyophilized. The obtained powder
2 4 6
from the fractions containing ∆HA -B, ∆HA -B, ∆HA -B were dissolved in 10 ml of 100 mM
ammonium acetate pH 7.8 and were then further purified from salt and other impurities on the
ÄKTA purifier by injecting 1 ml with a sample loop on a self-packed Superdex Peptide Prep
Grade 16/750 column (GE Healthcare, Uppsala, Sweden). Isocratic elution with 100 mM
ammoniumacetate pH 7.8 at 1.0 ml/min was used. UV detection (232nm) was used to monitor
the separation and fractions were collected automatically based on the UV-signal. The pure
2 4 6
fractions containing ∆HA -B, ∆HA -B, ∆HA -B were analysed with Superdex Peptide 10/300
GL on the analytical HPLC and thenlyophilized. The fractions containing HA oligosaccharides
at both ends of BDPE were also collected but not analysed further.
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2.3 NMR-spectroscopy
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The freeze dried di-, tetra- and hexasaccharidesamples were dissolved in D
2
O and transferred
into either 5 or 3mm NMR sample tubes. The NMR spectra were recorded either on a Bruker
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AVANCE™ III 600 MHz spectrometer using a 5mm PABBO BB/ F- H/D Z-GRD probe or a
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5mm H/ C/ N/ P cryoprobe, both equipped with a z-gradient. All experiments were
performed using D O as solvent and the signals were referenced by adding a small portion of
acetone-d to the sample and setting the acetone-d
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signal to δ
H
2.204. The temperatures were set
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between 280 and 300K in order to get as little interference from the residual HDO peak as
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possible. The assignments of H and C resonances were obtained from homonuclear H- H
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COSY, TOCSY and NOESY and heteronuclear H- C HSQC, HSQC-TOCSY and HMBC
experiments from the Bruker pulse sequence library. Mixing times from 10 to 120 ms were used
for TOCSY spectra. Suppression of the water signal was achieved using the noesypr1d pulse
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sequence. For quantification of amount of substitution by integration of NMR signals, the 1D H
1
NMR spectra were recorded with a recycle delay of 100s. The H NMR spectra of
enantiomericallyenriched (2'R,2''R)-BDDE were recorded on a Bruker Avance 400 MHz.
2.4 Mass spectrometry
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All LC-MS analyses were performed on a Bruker Maxis Impact Mass spectrometer connected to
TM
an Agilent 1100LC system equipped with a diode-array UV detector. A Hypercarb column
ofdimension 100 x 4.6 mm and with particle size 3 µm (Thermo Scientific) was used and kept at
60 °C.The mobile phases were water and acetonitrile with 0.05 or 0.1% trifluoroacetic acid and
the flow rate was 0.5 ml/min. A gradient of acetonitrile from 20-40% enabled a reasonable
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separation of isomers of ∆HA -B. UV absorbance was measured at 235 nm and the peak areas in
the chromatogram were used to estimate the relative amounts of each isomer assuming that all
isomers had the same response factors. For MS/MS, the technique of multiple reactions
monitoring (MRM) was utilized. Whenever possible, direct infusion or flow injection analyses
were used to optimize the collision energy to be used in CID. A collision energy of 45-50 eV
was sufficient to give rise to cross-ring cleavages.
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Reduction of HA oligomers: Since the column separates the α and β isomers, the hemiacetal
form of the oligosaccharides were reduced to alditol usingsodium borohydride in order to
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simplify the chromatographic peak profiles. The oligosaccharide fragments were dissolved in
200 µl of freshly prepared stock solution of 1 M NH OH containing 4 mg/ml of NaBH and
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incubated at 40 °C for 30 minutes in a heating block. The reaction was quenched by adding 0.5
ml of 10% acetic acid in methanol followed by evaporation to dryness at 40 °C using a rotary
evaporator. Addition of 10% acetic acid and evaporation was repeated 2 more times followed by
100% methanol. After the final evaporation, the residue was dissolved in H
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O: acetonitrile
(80:20) before analysis.
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2.5 Hydrolytic kinetic resolution of BDDE
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To
a
stirred
solution
of
(1S,2S)-(-)-1,2-cyclohexanediamino-N,N'-bis(3,5-di-t-
butylsalicylidene)cobalt(II) (0.60 g; 0.99 mmol; 0.01 eq.) in 10 ml dichloromethane (DCM) at
23°C acetic acidwas added (566 µl; 9.89 mmol; 0.10 eq.) and the reaction mixture stirred at 23°C
under air for 30 min. Once the color changed from red to dark brown (Co(II) to Co(III)
oxidation) the reaction mixture was evaporated to dryness under vacuum. The catalyst was then
dissolved in THF (20.0 ml) and racemic BDDE (18.2 ml; 98.9 mmol; 1.00 eq.) was added. The
mixture was cooled down to 0 °C and water (1.43 ml; 79.1 mmol; 0.80 eq.) was added .The
reaction mixture was allowed to warm up to 23°C and stirred at this temperature for 24h. After
this time, the reaction mixture was evaporated to dryness. The dark red oil was purified two
times by column chromatography on silica gel eluting with DCM and then with
DCM/diisopropyl ether 85/15 to yield enantiomerically enriched (2'R,2''R)-BDDE (3.60 g; 18%
1
yield) as a pale brown oil. H NMR (400 MHz, DMSO-d
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):δ 3.67 (dd, J = 11.6, 2.8 Hz, 2H), 3.49
– 3.38 (m, 4H), 3.23 (dd, J = 11.5, 6.3 Hz, 2H), 3.09 (ddt, J = 6.9, 4.3, 2.8 Hz, 2H), 2.72 (dd, J =
5.2, 4.2 Hz, 2H), 2.54 (dd, J = 5.2, 2.7 Hz, 2H), 1.58 – 1.51 (m, 4H).
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2.6 Derivatization of enantiomerically enriched BDDE for enantiopurity measurement
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To a stirred solution of enantiomerically enriched (2'R,2''R)-BDDE (300mg; 1.48 mmol; 1.00
eq.) in methanol (3.00 ml) at 0°C was added 2-naphthalenethiol (0.48 g; 2.97 mmol; 2.00 eq.)
followed by triethylamine (411 µl; 2.97 mmol; 2.00 eq.). The reaction mixture was stirred at 0°C
for 2h and then allowed to warm up to 23°C and stirred at this temperature for 18h. The reaction
mixture was filtered and the filtrate partitioned between DCM (50 mL) and water (50 mL).
Phases were separated and the organic layer was washed with 1 M aqueous HCl (50 mL), dried
over MgSO
4
and evaporated to dryness to yield the desired product (700 mg; 90% yield) as a
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colorless oil. H NMR (400 MHz, DMSO-d
6
):δ 7.88 – 7.75 (m, 8H), 7.53 – 7.38 (m, 6H), 5.18
(d, J = 5.3 Hz, 2H), 3.80 (h, J = 5.4 Hz, 2H), 3.46 – 3.32 (m, 8H), 3.21 (dd, J = 13.3, 5.2 Hz,
+
+
2H), 3.05 (dd, J = 13.3, 6.6 Hz, 2H), 1.57 – 1.40 (m, 4H).MS (m/z, ES ): 545.2 ([M+Na] )
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HPLC with a chiral column was recorded on an Agilent 1100 Series system using the following
parameters:Column: Chiralpak ID 5µm, 250 x 4.6mm; Flow rate= 1.0 mL/min; 30.0 min runs;
solvent system: heptane 65%, iPrOH 35%; isocratic flow. Chiral HPLC showed the following
R R R
ratio: enantiomer 1 (t = 19.9 min): 5%; meso isomer (t = 22.8 min): 7%; enantiomer 2 (t = 26.2
min): 86%.
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2.7 Determination of absolute configuration
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A
FTIR spectrometer (Vertex 70, Bruker) equipped with a vibrational circular dichroism
(VCD)module (PMA 50, Bruker) for VCD measurements was used.
2.8 Computational methods
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The DFT calculations were carried out at 298 Kin gas phase with Gaussian 09.
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3.Results and discussion
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3.1 Isolation of HA-BDPE fragments
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The HA hydrogel formed by reaction with BDDE was degraded as previously described(Kenne
et al., 2013), but using chondroitinase ABC instead of chondroitinase AC. The product mixture
obtained after the enzymatic degradation was separated by anion exchange chromatography (Fig.
1 Supplementary Material). The fractionscorresponding to the monosubstituted di-, tetra- and
hexasaccharides were collected for further analysis of the position of substitution by BDPE.
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3.2 Nomenclature
The fragments obtained from enzymatic digestion with chondroitinase ABC are unsaturated HA
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oligosaccharides which are abbreviated ∆HA
The fragments with mono-linked BDPE which are investigated in this work are abbreviated as
∆HA -B. The monosaccharide residues within the chain are named ∆GlcA (unsaturated
glucuronic acid), GlcA (glucuronic acid), and GlcNAc (N-acetyl glucosamine) where “y”
x
where x refers to the length of the oligosaccharide.
x
y
y
indicates the position of the residue starting from the reducing end. The substitution positions are
referred to as the monosaccharide residue together with the suffix –OHZ where “Z” is the
position on the ring which is substituted.
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3.3 NMR analysis of fractions ∆HA -B, ∆HA -B and ∆HA -B
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The H and C resonances were assigned from a combination of 1D and 2D NMR experiments
including TOCSY, HSQC, HSQC-TOCSY, NOESY and HMBC and by comparison with the
NMR spectra of HAoligosaccharides(Blundell, Reed & Almond, 2006; Nestor, Kenne &
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Sandström, 2010).The substitution positions were determined based on changes in H and C
chemical shifts (∆δ) in the substituted oligosaccharides when compared to those in the
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unsubstituted oligosaccharides. Substitutions by BDPE cause an upfield shift of the proton signal
attached to the carbon whose hydroxyl group is substituted by BDPE while the carbon
experience a large downfield shift. Adjacent proton and carbon signals are also affected but in
the opposite direction and to lesser extent.
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3.3.1 Substitution positions in fraction ∆HA -B
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73
74
75
76
In the fraction ∆HA
2
-B, three types of disaccharides (named A-C, Fig. 2) with substitution by
-OH6 (C)
-B with those in the
showed large ∆δ for H2/C2 and H3/C3 of ∆GlcA and for
BDPE at three different positions, ∆GlcA-OH3 (A), ∆GlcA-OH2 (B) and GlcNAc
1
1
13
were identified. Comparison of the H and C chemical shifts in ∆HA
2
unsubstituted disaccharide ∆HA
2
H6/C6 of GlcNAc(Fig. 2, Table 1). For example, the upfield shifts of H2 by ~ 0.17 ppm together
with the downfield shift of C2 by ~ 8.5 ppm proved substitution by BDPE at this position. The
neighbouring C1/H1 and C3/H3 showed opposite behaviour with C1 and C3 being slightly
shielded while H1 and H3 were deshielded in good agreement with what is expected upon
substitution on sugar rings. The same trends in chemical shift changes were observed for
substitution at ∆GlcA-OH3 and at GlcNAc
GlcNAc
-OH4was not identified in the ∆HA
compared to the unsubstituted disaccharide are shown in Fig.2.
1
-OH6. Adisaccharide with substitution by BDPE at
2
2
77
78
1
2
-B fraction. The largest changes in chemical shifts
13
Page 13 of 31

2
79
2
2
2
2
80
81
82
83
Figure 2.Structures for the six oligosaccharides (A-F) identified from fractions ∆HA
-B, ∆HA
-
2 4
B and ∆HA -B.∆δ values (ppm) show chemical shift differences compared to the unsubstituted
6
1
13
oligosaccharides for the atoms closest to the substitution position. See Tables 1-3 for H and C
chemical shifts.
14
Page 14 of 31

2
84
In solution, the GlcNAc sugar at the reducing end exists in equilibrium between the α- and β-
anomeric forms in a 65/35% ratio. Thus, in the NMR spectra of ∆HA -B, two signals should be
2
2
2
2
2
2
2
2
2
2
2
85
86
87
88
89
90
91
92
93
94
95
2
observed for all the protons on the reducing sugaras well as for all the protons on the non-
reducing sugar due to the two anomeric forms of the reducing rings. However, because the
anomeric configuration usually has a negligible influence on the conformation, the proton
chemical shifts of the non-reducing units are very similar, and due to limited spectral resolution,
only the protons on the reducing residue as well as the anomeric protons on the neighbouring
sugars are usually clearly differentiated. Besides the observation of anomeric pairs of isomers,
the oligosaccharides present in fraction ∆HA
-Bwere also shown to exist as a mixture of
2
diastereoisomers as demonstrated by the doubling of the signals in the NMR spectra (Fig. 3) and
of the peaks in the LC-MS chromatograms (vide infra) originating from the use of racemic
BDDE in the synthesis of the HA cross-linked gel.
2
96
1
2
2
97
98
Figure 3.Portion of the H NMR spectra of (top) ∆HA and (bottom) ∆HA -B showing the H4,
2
2
H1 of ∆GlcA and the H1 of GlcNAc(α).
15
Page 15 of 31

2
99
3
3
3
3
3
3
3
3
00
01
02
03
04
05
06
07
2
The origin of the different types of isomers observed for ∆HA -B is clarified in Fig. 4. In fact,
four diastereoisomersare expected to be formed after the reaction of HA with racemic BDDE:
HA-(2'R,9'R)-BDPE, HA-(2'S,9'S)-BDPE, HA-(2'S,9'R)-BDPE and HA-(2'R,9'S)-BDPE (see
Fig. 2for BDPE atom numbering). The asymmetric carbon in position 9', resulting from
hydrolysis of the epoxide, is on a very flexible chain having minimal impact on column
separation and NMR properties. Only the asymmetric carbon in position 2', resulting from the
sugar-reacted epoxide, is close enough to permit separation of the corresponding
diastereoisomerson column chromatography and modify the NMR spectra.
BDPE-
position
2´-BDPE
configuration configuration
Anomeric
Oligosaccharide
3
08
000
3
3
09
10
Figure 4.Schematic representation of the different types of isomers observed in the NMR and
LC-MS spectra of ∆HA -B
2
3
3
11
12
1
13
Once the H and Cchemical shifts of the different types of substituted disaccharides have been
3
3
13
14
assigned from a combination of 2D NMR experiments,the position and amount of substitution at
1
each position can be determined directly from the 1D H NMR spectra.Indeed, it can be seen in
16
Page 16 of 31

3
3
3
3
3
3
15
16
17
18
19
20
Fig. 3 that the H1 and H4 signals of ∆GlcAexperience a downfield shift upon substitution at the
2- and 3-position respectively and can be used as chemical shift reporters of substitution at these
positions. Integration of these signals gave ~ 52% substitution at ∆GlcA-OH2 and ~ 13% at
∆GlcA-OH3. Since there is no substitution at GlcNAc
integrates for the disaccharides with BDPE at ∆GlcA-OH2, ∆GlcA-OH3 and ∆GlcNAc
Thus, integration of this signal gave~ 35% of BDPE substitution at GlcNAc -OH6.
1
-OH4, the anomeric signal of GlcNAc
1
1
-OH6.
1
1
13
3
3
21
22
Table 1. H and C chemical shifts of the resonances for the three substituted disaccharides (A-
C) in ∆HA
-B.Values in bold face indicate the position of substitution.
2
1
13
Sugar residue
H/ C
1
5.18
1
2
3
4
5.94
5
-
6
-
6´
-
-
Me
-
-
β-D-∆GlcA-(1→
3.88 4.05
67.4 74.6
4.05 4.05
53.3 78.9
3.83 3.85
55.9 81.1
00.4
104.7 144.3 169.1
→
3)-α-GlcNAc
1
5.16
3.53
68.2
3.51
68.2
3.90
71.4
3.51
75.6
3.83
60.4
3.91
60.6
3.83 2.13
60.4 22.1
3.76 2.13
60.6 22.1
A
B
9
0.8
4.76
4.4
→3)-β-GlcNAc
1
9
β-D-∆GlcA-(1→
5.34
8.9
5.16
0.8
4.76
4.4
3.60 4.24
78.3 64.1
4.05 4.05
53.3 78.9
3.83 3.85
55.9 81.1
5.89
-
-
-
-
-
-
9
107.1 144.3 169.1
→3)-α-GlcNAc
1
3.53
68.2
3.51
68.2
3.90
71.4
3.51
75.6
3.83
60.4
3.91
60.6
3.83 2.13
60.4 22.1
3.76 2.13
60.6 22.1
9
→
3)-β-GlcNAc
1
9
β-D-∆GlcA-(1→
5.20
3.77 4.16
69.7 66.0
4.07 4.01
53.1 79.3
3.83 3.84
55.8 81.5
5.87
-
-
-
-
-
-
1
00.5
107.1 144.3 169.1
→
3)-α-GlcNAc
1
5.16
3.58
68.4
3.55
68.3
4.02
70.2
3.63
74.3
3.80
69.7
nd
3.80 2.13
C
9
0.8
4.76
4.4
69.7 22.1
a
a
→
3)-β-GlcNAc
1
nd
2.13
9
69.9
69.9 22.1
a
Could not be determined due to overlapping signals.
3
23
17
Page 17 of 31

3
3
23
24
4
3.3.2 Substitution positions in fraction HA -B
∆
3
3
3
3
3
3
3
3
3
25
26
27
28
29
30
31
32
33
In the ∆HA
abundant are presented in Table 2, Fig. 2). The signals at δ
H4 and H1 of ∆GlcAshowed, by comparison with the assignments for ∆HA
4
-B fraction, four different types of tetrasaccharides were identified (the two most
5.94 and δ 5.34corresponding to
-B, that small
H
H
2
amounts of tetrasaccharides substituted in positions ∆GlcA-OH2 and ∆GlcA-OH3 were present.
1
The two other substituted tetrasaccharides (D-E) were present in larger amount and the H and
1
3
C chemical shift values revealed that in both compounds, substitutionoccurred on the GlcNAc
residueat the reducing end, one on GlcNAc -OH4 (D) and the other one on GlcNAc -OH6
(E)(Fig. 2).Tetrasaccharides with substitution by BDPE on the internal GlcNAcorGlcA sugars
were not identified in the ∆HA
-B fraction.
1
1
4
3
3
3
3
34
35
36
37
2 4
As for ∆HA -B, the positionsof substitution by BDPE in ∆HA -B can be determined from the 1D
1
H NMR spectra and the amount quantifiedby integration of characteristic signals (Fig. 5): The
H1 and H4 signals of ∆GlcAare used for determination of amount of substitution at ∆GlcA-OH2
and -OH3. The H1signals of GlcNAc
substitution at GlcNAc -OH4 while the H1signals of GlcNAc
sumof substitution at the GlcNAc -OH4 and -OH6 positions. Integration of these signals gave
72% substitution by BDPE at GlcNAc -OH4 and 21% at GlcNAc -OH6. The remaining two
1
in the α-anomeric form allow obtaining the amount of
3
3
3
3
3
38
39
40
41
42
1
1
in the β-anomeric form give the
1
1
1
substitution positions contributed with less than 1% for ∆GlcA-OH3 and ca. 5-6% for ∆GlcA-
OH2.
18
Page 18 of 31

3
43
1
3
3
3
3
44
45
46
47
Figure 5.Anomeric and H4 (∆GlcA) region of the H NMR spectra of (a) ∆HA
2 4
-B, (b) ∆HA
-B
and (c) ∆HA -B. The NMR signals that can be used as reporters for identification and
quantification of the position of substitution of BDPE on the sugars are indicated. * Residual
HDO signal after water suppression.
6
19
Page 19 of 31

3
48
1
13
3
3
49
50
Table 2. H and C chemical shifts of the resonances for the two most abundant substituted
4
tetrasaccharides (D-E) in ∆HA -B.Values in bold face indicate the position of substitution.
1
/13
Sugar residue
H C
1
5.17
2
3.76
3
4
5.86
5
-
6
-
6´
-
-
Me
-
-
β-∆GlcA-(1→
4.16
66.0
3.84
81.7
3.59
73.4
3.59
73.5
4.09
77.5
3.91
79.7
100.6 69.6
107.1 144.3 169.1
→
→
→
→
→
3)-β-GlcNAc
3
-(1→
4.62
00.5 54.5
4.56 3.34
103.4 72.5
4.52 3.35
03.4 72.4
3.86
3.53
68.1
3.77
80.4
3.77
80.4
3.49
76.9
3.46
76.8
3.53
75.3
3.66
76.5
3.66
76.5
3.92
70.5
3.51
74.6
3.80
60.3
-
173.7
-
173.7
3.84
60.1
3.79
60.1
3.93 2.07
60.3 22.3
-
-
-
-
3.84 2.03
60.1 21.8
3.89 2.03
60.1 22.2
1
a
4)-β-GlcA
4)-β-GlcA
2
-(1→
-(1→
-
-
-
-
D
b
2
1
3)-α-GlcNAc
1
5.11
0.8
4.68
4.5
4.06
53.5
3.86
56.0
9
3)-β-GlcNAc
1
9
β-∆GlcA-(1→
5.17
3.75
4.16
66.0
3.84
81.9
3.59
73.4
3.59
73.4
3.92
79.5
3.73
82.0
5.86
-
-
-
-
-
-
1
00.6 69.6
4.59 3.86
00.2 54.5
4.52 3.38
102.9 72.4
4.48 3.38
02.9 72.2
107.1 144.3 169.1
→
→
→
→
→
3)-β-GlcNAc
3
-(1→
3.52
68.1
3.76
79.8
3.75
79.8
3.60
72.1
3.56
68.2
3.52
75.4
3.72
76.2
3.72
76.2
4.01
68.4
3.61
73.2
3.80
60.3
-
174.0
-
174.0
3.79
69.7
3.93 2.07
60.3 22.3
-
-
-
-
1
a
4)-β-GlcA
2
2
-(1→
-
-
-
-
E
b
4)-β-GlcA
-(1→
1
3)-α-GlcNAc
1
5.16
0.8
4.72
4.6
4.06
52.8
3.84
55.3
3.79 2.03
69.7 21.8
9
c
c
3)-β-GlcNAc
1
nd
69.7
nd
2.03
9
69.7 22.2
a
3
3
3
3
51
52
53
54
Connecting to reducing end GlcNAc in α configuration
Connecting to reducing end GlcNAc in β configuration
Could not be determined due to overlapping signals.
b
c
20
Page 20 of 31

3
55
6
3.3.3 Substitution positions in the fraction HA -B
∆
3
3
3
3
3
56
57
58
59
60
Analysis of the NMR spectra of the ∆HA
almost exclusively atGlcNAc -OH4on the reducing end (Table 3, Fig. 2).Hexasaccharides with
BDPEsubstitution on ∆GlcA-OH2, ∆GlcA-OH3 and GlcNAc -OH6 were present in very small
amounts (Table 4). Hexasaccharides with BDPE substitution on the internal sugars werenot
present in the∆HA
-B fraction.
6
-B fraction showed that substitution by BDPE occurred
1
1
6
3
3
3
3
3
61
62
63
64
65
4
As for ∆HA -B, reporter signals that can be used for determination of amounts of substitution in
∆HA -Bat the different positions are H1 and H4 of ∆GlcA for C2 and C3 substitution
6
respectively, H1 of GlcNAc(α) for C4 substitution and H1 of GlcNAc(β) for C4 and C6
1
substitution (Fig. 5).Integration of the 1D H NMR spectrum showed that more than 90% of the
1
substitution occurs at GlcNAc -OH4.
1
13
3
3
66
67
Table 3. H and C chemical shifts of the resonances for the most abundant substituted
hexasacharide (F) in ∆HA
-B.Values in bold face indicate the position of substitution.
6
1
13
Sugar residue
H/ C
1
2
3
4
5
-
6
-
6´
-
Me
-
-
β-∆GlcA-(1→
5.17 3.76
00.5 69.6
4.58 3.86
00.2 54.5
4.47 3.35
03.0 72.2
4.58 3.85
00.7 54.1
4.56 3.34
03.3 72.5
4.51 3.33
03.4 72.4
5.11 4.05
0.8 53.5
4.67 3.84
4.5 55.9
Connecting to reducing end GlcNAc in α configuration
4.15 5.86
65.8 107.0 144.3 169.2 -
1
→
→
→
3)-β-GlcNAc
4)-β-GlcA
3)-β-GlcNAc
5
-(1→
3.83 3.51 3.51
81.8 68.0 75.3
3.59 3.74 3.72
73.3 79.7 76.0
3.72 3.54 3.49
82.1 68.1 75.0
3.58 3.76 3.65
73.4 80.3 76.5
3.58 3.75 3.64
73.4 80.3 76.4
4.08 3.48 3.92
77.5 76.9 70.3
3.89 3.46 3.51
79.6 76.8 74.6
3.79 3.93 2.07
60.2 60.2 22.2
-
174.2 -
3.79 3.92 2.03
60.2 60.2 22.2
1
4
-(1→
-
-
-
1
3
-(1→
1
a
F →4)-β-GlcA
2
-(1→
-(1→
-
-
-
-
-
-
1
173.7 -
-
173.7 -
3.83 3.83 2.03
60.0 60.0 21.8
3.78 3.92 2.03
60.1 60.1 22.2
b
→
→
→
4)-β-GlcA
2
-
1
3)-α-GlcNAc
1
9
3)-β-GlcNAc
1
9
a
3
68
21
Page 21 of 31

b
3
3
3
69
70
71
Connecting to reducing end GlcNAc in β configuration
3.3.4 Total distribution of BDPE in the oligosaccharides
3
3
3
72
73
74
Size-exclusion chromatography and NMR analysis revealed the distribution and position of
BDPE in the oligosaccharide fractions obtained by enzymatic hydrolysis of the HA hydrogel. In
the present example, ∆HA
-Bwas the most abundant fraction (59%) collected by SEC followed
-B(15%) fractions (Table 5). In total in these three fractions, ca.
6
3
3
3
75
76
77
by the∆HA -B (26%) and ∆HA
4
2
90% of substitution occured on the GlcNAcresidue at the reducing end and only 10% on the
unsaturated non-reducing end ∆GlcA.
3
78
3
3
79
80
Table 4.Amount (%) of substitution at the different positions in ∆HA -B, ∆HA -B and ∆HA
-B
2 4 6
obtained from integration of H signals in NMR spectra and from areas of UV chromatograms.
1
Substitution
position
∆
HA -B
∆
HA -B
∆
HA -B
2
4
6
a
b
a
b
a
∆
∆
GlcA-OH2
GlcA-OH3
52 /55
6 /-
~ 1
a
b
a
b
a
13 /11
< 1 /-
~ 1
a
b
a
b
a
GlcNAc -OH4
-a/-
35 /34
72 /78b
21 /22
92a
~ 6
1
b
a
GlcNAc -OH6
1
a
b
3
3
81
82
NMR. LC-UV, -: not observed
3
3
3
83
84
85
Table 5.Total distribution (%) of monosubstitutedBDPE calculated from SEC and NMR data.
The proportion of monosubstituted oligosaccharides (fraction of fragment) was obtained from
SEC.
∆
GlcA
GlcNAc1
Fragment
Fraction of fragment (%)
-
OH2
-OH3
-OH4
-OH6
∆
∆
∆
HA -B
15
26
59
52
6
1
13
1
1
0
35
21
6
2
HA -B
72
92
73
4
HA -B
6
Total distribution of monosubstitutedBDPE (%)
10
3
14
3
86
22
Page 22 of 31

3
3
3
3
3
87
88
89
90
91
The fact that substitution occurredpredominantly at GlcNAc
substitution was also found at GlcNAc -OH6 suggest that hydrolysis of the hexasaccharide by
the enzyme did not proceed to completion during degradation of the gel. Indeed, addition of
chondroitinase ABC in the NMR tube containing ∆HA
-B followed by incubation at 37 °C
resulted in further degradation of the hexasaccharide into the unsubstituted disaccharide∆HA
-B with BDPE at GlcNAc -B
and the tetrasaccharide∆HA -OH4. No further hydrolysis of ∆HA
was observed. Since the ∆HA -B fraction contains disaccharides substituted at ∆GlcA-OH2,
∆GlcA-OH3 and GlcNAc -OH6 but not at GlcNAc -OH4, it can be concluded that ∆HA -B with
1 6 4
-OH4in ∆HA -B, while in ∆HA
-B
1
6
2
3
3
92
93
4
1
4
2
3
3
3
94
95
96
1
1
4
BDPE at GlcNAc
1
-OH4 is not a substrate for the enzyme while the tetrasaccharides with BDPE
at other sugar positions can be further hydrolyzed by the enzyme.
3
97
2 4 6
3.4 LC-MS analysis of fractions ∆HA -B, ∆HA -B and ∆HA -B
3
3
4
4
4
4
4
4
4
4
98
99
00
01
02
03
04
05
06
07
3.4.1 Fraction
2
∆HA -B
A good separation of the disaccharides constituting the ∆HA
-B fraction was achieved within a
2
run time of 40 minutes using a porous graphite carbon based HPLC column.Analysis by LC-ESI-
MS/MS in the negative ion mode showed the presence of twelveisobaric peaks (Fig. 6a). Since
porous graphite carbon based columns are often able to resolve α- and β-anomers of
carbohydrates, these twelve peaks were fractionated on an analytical scale and the partially pure
fractions were, after lyophilization,injected once again into the LC-MS, one at a time. This
caused additional peaks to appear in the chromatograms (data not shown) due to mutarotation
occurring at the reducing-end GlcNAc
1
revealing the retention time of the other anomer. Hence
the twelve peaks in Fig. 6a were confirmed to belong to six different isomers each one resolved
23
Page 23 of 31

4
4
08
09
as α- and β- anomeric pairs, namelypeaks 1-2, 5-7, 3-6, 4-8, 9-9* and 10-10*, the anomers9*
and 10*being hidden under other peaks.
4
10
4
4
4
4
11
12
13
14
Figure 6.LC-ESI-MS/MS of ∆HA
the twelveisomers that could be partially separated; (b) and (c) extracted ion chromatograms
(EIC) of the glycosidic cleavage ions C , i.e. m/z 395 and m/z 175 indicating an attachment of
2
-B in the negative ion mode: (a) UV chromatogram showing
1
BDPE on the ∆GlcA, and GlcNAc, respectively.
4
4
15
16
The MS/MS spectra of peaks 1 and 2 were, as expected,identical since these two peaks
correspond to the α and β-anomeric pair. The MS/MS spectra of peaks 1 and 5 were also
identical(See Fig. 2, Supplementary Material)to the spectra of peaks 2 and 7 showing that
substitution by BDPE at one position on the disaccharide gave rise to four peaks in the HPLC
4
4
4
4
4
4
4
4
17
18
19
20
21
22
23
24
chromatogram, two of them originating from the α- and β-anomers. Reduction of fraction ∆HA
2
-
B using sodium borohydride in 0.1M NH OH (see Experimental) followed by LC-MS analysis
4
showed that the number of peaks decreased from twelve to six (See Fig. 3a, Supplementary
Material), further confirming the existence of six α-,β- anomeric pairs of isomers in the fraction
∆HA -B.
2
24
Page 24 of 31

4
4
4
4
4
4
4
4
4
4
25
26
27
28
29
30
31
32
33
34
1
The extracted ion chromatograms (EIC) of the glycosidicbond cleavage ion C i.e. m/z 395
indicated that peaks 1, 2, 5 and 7 as well as peaks 9, 9*, 10 and 10* correspond to disaccharides
with BDPE linked on ∆GlcAwhilem/z 175 indicated that peaks 3, 4, 6 and 8 represent a
disaccharide with BDPE linked on GlcNAc(Fig. 6b and 6c).
From the peaks 1, 3 and 9 shown in the UV chromatogram (Fig. 6a), the position of substitution
by BDPE was determined from MS/MS analysis in the positive ion mode of the precursor ion at
+
m/z 622 ([M+Na] )(Fig. 7). The glycosidic cleavage fragments B and Y indicated on which
sugars the BDPE molecule is attached while the cross-ring fragmentations, that involve rupturing
two bonds on the same sugar residue, provided information on the linkage position of BDPE.
∆GlcA-OH2 substitution was deduced from the diagnostic glycosidic cleavage ions B at m/z
1
4
4
4
4
35
36
37
38
401 and Y
cross-ring ion
OH3 substitution was deduced from the diagnostic glycosidic cleavage ions B
at m/z 244 indicating BDPE substitution on ∆GlcA and from the diagnostic cross-ring ions
1
at m/z 244,Y
1
–H
at m/z 506 indicating BDPE substitution at ∆GlcA-OH2 (Fig. 7a). ∆GlcA-
1
2
O at m/z 226 indicating BDPE substitution on ∆GlcA and from the
0
,2
X
1
at m/z 401 and Y
1
1
0
,2
X
0
,2
4
4
4
4
4
4
39
40
41
42
43
44
at m/z 286 and
A
1
at m/z 359 indicating BDPE attachment on ∆GlcA-OH3 (Fig. 7b).
2
–H O
∆GlcNAc-OH6 substitution was deduced from the diagnostic glycosidic cleavage ions Y
1
0
,4
at m/z 446 and from the diagnostic cross-ring ion
2
A at m/z 303 (Fig. 7c).
The relative amount of each isomer was obtained from the area under the peaks in the UV
chromatogram of the reduced isomers and the data were in very good agreement with the data
obtained by NMR (Table 4).
25
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4
45
+
4
4
4
4
46
47
48
49
Figure 7.ESI-MS/MS[M+Na] spectra of peaks 1, 9 and 3 (from chromatogram a, Fig. 6)
corresponding to BDDE at (a) ∆GlcA-OH2,(b) ∆GlcA-OH3 and (c) GlcNAc-OH6 respectively.
The fragments are described using the nomenclature ofDomon and Costello (1988). All the
fragments are sodiated but Na is purposely excluded in the annotations above and in the text.
4
4
50
51
3.4.2 Fraction
A total of eight peaks were detected for the ∆HA
could be resolved. As with ∆HA
-B, when LC-MS was run on the reduced sample, only half the
numbers of signals were present indicating the presence of two major diastereoisomers (Fig. 8b).
∆
4
HA -B
4
4
4
52
53
54
4
-B fraction (Fig.8a) although not all of them
2
26
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4
4
55
56
These peaks were assigned as tetrasaccharides with BDPE substitution at C4 and C6 of GlcNAc
at the reducing end.
1
4
57
4
4
4
4
4
4
58
59
60
61
62
63
Figure 8.(a)UV chromatogram (235nm) from the LC-MS analysis of ∆HA
chromatogram (235nm) from the LC-MS analysis of borohydride reduced ∆HA
major substitutions at GlcNAc -OH4 and GlcNAc -OH6 of the two diasteroisomers. X denote
4
-B.(b)UV
4
-Bshowing the
1
1
minor components that appear as weak peaks in the extracted ion chromatogram of m/z 981,
+
[M+H] , (data not shown).These peaksprobably correspond to minor isomers with substitutions
on ∆GlcA-OH2 and ∆GlcA-OH3 andrepresent approx. 5% of the total substitution.
4
4
64
65
Since fraction ∆HA
-B was shown by NMR to be further degraded by the enzymeinto ∆HA
and
2
6
4
4
4
4
4
4
4
66
67
68
69
70
71
72
∆HA -B, it was not investigated by LC-MS.
4
3.5 Preparation of HA gel using enantiomerically enriched BDDE
2 4
NMR and LC-MS showed that the oligosaccharides present in fractions ∆HA -B, ∆HA
-B and
∆HA -B exist as a mixture of diastereoisomers. To demonstrate that the diastereoisomers
6
originated from the use of racemic BDDE in the synthesis of the HA cross-linked
gel,enantiomerically enriched BDDE was prepared.
27
Page 27 of 31

4
4
4
4
4
4
4
4
73
74
75
76
77
78
79
80
Hydrolytic kinetic resolution was used for the preparation of enantiomerically enriched BDDE
from racemic BDDE (See Fig. 4 Supplementary Material)(Schaus et al., 2002). Enantiomeric
excess (ee) was determined using a chiral HPLC column after derivatization of enantiomerically
enriched BDDE (See Fig. 5 Supplementary Material) while the absolute configuration was
obtained using VCD associated with density functional theory (DFT) calculations. The absolute
configuration of this enantiomerically enriched BDDE was established to be (2'R,2''R).
(2'R,2''R)-BDDE was then used to produce a HA hydrogel which was degraded and separated
using the same experimental procedure as the one obtained with racemic BDDE.
4
4
4
4
4
4
4
4
4
81
82
83
84
85
86
87
88
89
LC-MS analysis of the ∆HA
2
-Bfraction obtained from the HA gel prepared with
enantiomerically enriched BDDEshowed that one of the two isomers of ∆GlcA-
OH2,∆GlcA-OH3 and GlcNAc-OH6 had decreased considerably in amount (see Fig. 3
1
supplementary Material). Similarly, the H NMR spectra of the∆HA
-B fraction obtained with
2
(2'R,2''R)- BDDE clearly showed that H1 of ∆GlcA-OH2now appeared as two doublets of
relative intensities 65/35 representing the α- and β-anomeric forms of the disaccharide, thus
consisting of only one HA-BDPE diastereoisomer(See Fig. 6 supplementary Material). The other
two doublets that were slightly upfield shifted in the ∆HA
-B fraction obtained from gel prepared
2
with racemic BDDE were not present.
4
4
4
4
4
90
91
92
93
94
These data thus confirm that the additional signals observed in the NMR (See Fig. 6a
Supplementary material)and LC-MS spectra (Fig. 6a) of∆HA
-B originating from HA-gel cross-
2
linked with racemic BDDE are due to the diastereomeric mixture.Peaks1 and 4 (Fig. 6) obtained
1
from LC-MS analysis of ∆HA
2
-B were collected and 1D H NMR spectra were obtained for
bothof them. Peak 1 was shown to contain one diastereoisomer of the disaccharide substituted at
28
Page 28 of 31

4
4
4
4
95
96
97
98
∆GlcA-OH2 (B) while peak 4 contained the other diastereoisomer (see Fig 6 Supplementary
material).As observed for the ∆HA -B fraction, the NMR spectrum of the ∆HA
-B fraction from
2
4
(2'R,2''R)- BDDE showed that only one of the isomer of the substituted tetrasaccharideswas
present (see Fig 7 Supplementary material).
4
99
4.Conclusion
5
5
5
5
5
5
5
00
01
02
03
04
05
06
In this study, the position and amount of BDPE substitution in di-, tetra- and
hexasaccharidesfromenzyme degraded hydrogel of cross-linked hyaluronic acid have been
determined by NMR and LC-MS. The data obtained from both methods are in excellent
agreement. In ∆HA
hydroxyl group at the terminal reducing or non-reducing sugar residue. Substitution is either on
GlcNAc -OH4/6 or on ∆GlcA-OH2/3 but does not occur on the internal sugar residues. In the
∆HA -B fraction, substitution in three of the four possible substitution positions is found.
-B, ∆HA -B and ∆HA
-B, the substitution with BDPE is located on the
2
4 6
1
2
5
5
5
5
5
5
5
5
5
5
07
08
09
10
11
12
13
14
15
16
Substitution occurs predominantly at ∆GlcA-OH2 (> 50%) and GlcNAc
disaccharide with substitution at GlcNAc
-OH4 was identified. In the ∆HA
fractions, substitution with BDPE was predominantly at GlcNAc -OH4 at the reducing end (>
70% in ∆HA -B and > 90% in ∆HA -B). The fact that the larger oligosaccharides are
predominantly substituted at GlcNAc -OH4 while such substitution is not observed in the
disaccharide indicates that tetrasaccharides with GlcNAc -OH4 substitution are not substrates for
chondroitinase ABC. Diagnosticglycosidic cleavage and cross-ring ions that established the
1
-OH6 (> 30%). No
-B and ∆HA
-B
1
4
6
1
4
6
1
1
1
13
2
position of substitution in ∆HA -B by LC-MS and H and C NMR signals that can be used as
chemical shifts reporters of the position of substitution of BDPE on the sugars of HA were also
identified. TheNMR and LC-MS methodologies presented in this work are not restricted to the
29
Page 29 of 31