Synthesis of Disaccharide and Trisaccharide Building Blocks.

With the aforementioned requirements in mind, monosaccharide units 10 - 17 were prepared according to the standard reaction conditions (see Supporting Information 2.1). Having electrophilic donors 10 – 14 and nucleophilic acceptors 15 – 17 in hand, we next focused on the parallel synthesis of disaccharide building blocks incorporating 1,2-trans relative stereochemistry (Scheme 1). A combination of N-iodosuccinimide and silver triflate (NIS/AgOTf)-mediated glycosylation of each of the acceptors 15-17 with thioglycoside donor 14 afforded three different disaccharides (18, 19, and 24) bearing an aminoethyl spacer at the reducing end. In each coupling, only a β−1,2-trans glycoside was observed due to neighboring group participation by the C2-acetyl of the donor 14, affording the corresponding disaccharides in excellent yields (85% to 96%). Next, the coupling products 18, 19, and 24 were transformed into disaccharide acceptors 22, 23, and 26 bearing a −1 GlcA derivative and a +1 GlcN derivative, utilizing a standard set of reaction conditions (Scheme 1). Accordingly, the benzylidene acetals of 18, 19, and 24 were removed by treatment with 80% acetic acid (AcOH) at 80 °C to generate the desired diols 20, 21, and 25, respectively, whose C6-hydroxyls were selectively oxidized with substoichiometric amount of 2,2,6,6-teteramethyl-1-piperidinyloxy (TEMPO) in the presence of excess iodobenzene diacetate (BAIB) as a co-oxidant.¹⁹ The resulting carboxylic acids were then protected as methyl esters by treatment with methyl iodide (MeI) to afford disaccharide acceptors 22, 23, and 26 ranging from 78% to 83% over two steps.

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Scheme 1.

Synthesis of disaccharides with GlcA-GlcN backbone^a

^a Reagents and conditions: (a) NIS, AgOTf, DCM, 0 °C (18, 85%; 19, 96%; 24, 87%); (b) 80% AcOH, 80 °C (20, 72%; 21, 82%; 25, 75%); (c) BAIB, TEMPO, DCM/H₂O; then MeI, KHCO₃, DMF (22, 78%; 23, 80%; 26, 83%).

To illustrate that the spacer-containing disaccharide acceptors 22, 23, and 26 (Scheme 1) and −2 GlcN-derived donors 10 – 13 (Figure 3) can be employed for the stereoselective construction of α−1,4-linkages of trisaccharides 27 – 33, we first investigated a NIS/AgOTf-mediated coupling of disaccharide acceptor 23 with thioglycoside donor 12 (Table 1). The desired trisaccharide 27 was not observed at 0 °C or room temperature (entries 1 and 2) due to relatively low reactivity of C4-hydroxyl of the GlcA unit. Increasing the reaction temperature (25°C → 35°C) and longer reaction time (2 h → 12 h) afforded 27 in 56% yield (entry 4) as a mixture of anomers (α:β = 3:1). Following the seminal work of Mong and co-workers,²⁰ we investigated N,N-dimethylforamide (DMF) as an α-modulating additive to direct the stereochemical course of glycosylation. As illustrated in entry 5, the stereoselectivity of the reaction with DMF (6 equiv.) was significantly much better (α:β >20:1). This optimal set of glycosylation conditions were then applied to the coupling of disaccharide acceptors 22 and 26 with thioglycoside donors 10, 11 and 12 to furnish the trisaccharides 28 – 32 (57% to 65%) with exclusive α-anomeric configuration at the newly-formed glycosidic bond (Scheme 2).

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Scheme 2.

Stereoselective construction of trisaccharides 28-32^a

^a Reagents and conditions: (a) NIS, AgOTf, DMF (6 equiv.), DCM, 35 °C, 5 h (28, 63%; 29, 65%; 30, 62%; 31, 57%; 32, 60%; α:β >20:1).

Table 1.

Stereoselective glycosylation of disaccharide acceptor 23with C2-azido thioglycoside donor 12^a

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entry	AgOTf (equiv)	additive	Temp.	time (h)	yield (%)	α:β
1	0.1	none	0 °C	1	NR
2	0.1	none	25 °C	1	NR
3	0.1	none	35 °C	2	45	3:1
4	0.1	none	35 °C	12	56	3:1
5	0.1	DMF (6 equiv)	35 °C	12	NR
6	1	DMF (6 equiv)	35 °C	12	51	>20:1

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^aThe glycosylation was conducted with NIS (2.0 equiv.) and AgOTf (0.1 or 1.0 equiv.). Yields were reported as isolated yields. The (α/β) ratios were determined by ¹H NMR analysis.

After establishing the effectiveness of glycosylation conditions for the synthesis of trisaccharides 27 – 32, we examined the coupling of disaccharide acceptor 22 with C2-N-benzylidene donor 13 (Scheme 3). The N-benzylidene group can be selectively removed under acidic conditions in the presence of the azido moiety. The N-benzylidene and azido offer a set of orthogonal amino protecting groups that allows selective functionalization of each function, further increasing the structural diversity of trisaccharides. We have recently discovered that the release of triflic acid from nickel triflate in combination with the unique stereoelectronic nature of the C(2)-N-benzylidene group on glycosyl N-phenyl trifluoroacetimidates donor provided the highly favorable formation of 1,2-cis-amino- glycosides.^21–24 We found that the reaction of donor 13 with disaccharide acceptor 22 (Scheme 2) using triflic acid (TfOH) provided trisaccharide 33 with moderate α-selectivity (α:β = 5:1). Addition of DMF as an α-modulating additive²⁰ significantly improved α-selectivity (α:β > 20:1) of 33 (Scheme 3). In this reaction, it is worth noting that stoichiometric amount of triflic acid was required because the DMF additive can quench the acid. In addition, room temperature and long reaction time (8 h) were neccesaary for the coupling to proceed to completion while degradation was observed at 35 °C.

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Scheme 3.

Preparation of trisaccharide 33 containing C2-N-benzylidene^a

^a Reagents and conditions: (a) TfOH (1 equiv.), DMF (6 equiv.), 25 °C, 8 h, DCM, 57%, α:β > 20:1.

We next focused on the synthesis of trisaccharides having the β-anomeric configuration at the reducing end so that we can compare their ability to modulate heparanase activity with their α-counterparts. Accordingly, we embarked on the synthesis of α- and β-adducts 37 and 38 (Scheme 4) starting from commercially available trisaccharide 34.¹⁸ The hemiacetal 34 was modified at the reducing end to furnish trichloroacetimidate 35, ready for coupling to the aminoethyl spacer 36. Since the C2-azido moiety of the reducing end glucosamine unit of 35 lacks traditional neighboring participation, we anticipated that a mixture of α-and β-anomers would be generated. The stereochemical outcome of this reaction is necessary for us to access both α- and β-trisaccharides for subsequent studies of heparanase activity. Accordingly, glycosylation of the acceptor 36 with trisaccharide trichloroacetimidate 35 promoted by trimethylsilyl trifluoromethanesulfonate (TMSOTf) afforded α-adduct 37 (51%) and β-adduct 38 (30%), which could be separated by traditional silica column chromatography (Scheme 4).

Synthesis of Sulfated Trisaccharides.

After constructing the trisaccharide frameworks, functional group transformations were next conducted to achieve the desired products incorporating different sulfation patterns at 6-O, 3-O and 2-N of glucosamine unit at −2 and +1 binding subsites as well as 2-O of glucuronic acid unit at the −1 binding subsite. First, selective deacetylation at C6 of trisaccharide 37 were investigated (Table 2) using sodium methoxide (NaOMe) in a 1:1 mixture of methanol (MeOH) and dichloromethane (DCM). Employing substoichiometric amount (0.2 equiv.) of NaOMe at low reaction concentration (0.02 M) for 5 h (entry 3) afforded a separable mixture of 39 (removal of −2 C6-acetyl group of GlcN unit) and 40 (removal of +1 C6-acetyl group of GlcN unit), further increasing the structural diversity of synthetic targets. On the other hand, using equimolar sodium methoxide at high concentration (0.1 M) predominantly yielded 40 (entry 6). In all cases, an inseparable mixture of the byproducts 41 and 42 which were tentatively identified by Mass spectrometry was also detected. A higher amount of 41 and 42 was observed at higher concentration and longer reaction time. The optimal conditions developed for the selective hydrolysis were then exploited with trisaccharide 38 incorporating the reducing end β-linkage (Scheme 5) to deliver the separable products 43 (41%) and 44 (35%).

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Scheme 5.

Selective C6-deacetylation of −2 and +1 glucosamine units of β-linked trisaccharide 38^a

^a Reagents and conditions: (a) CH₃ONa (0.2 equiv.), 1:1 mixture of MeOH and DCM (0.02 M), 25 °C, 5 h (43, 41%; 44, 35%).

Table 2.

Selective C6-deacetylation of −2 and +1 glucosamine units of α-linked trisaccharide 37^a

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entry	CH3ONa (equiv.)	concentration (M)	time (h)	39: yield (%)	40: yield (%)	41+42: yield (%)
1	0.1	0.02	8	23	8	none
2	0.2	0.02	8	35	40	17
3	0.2	0.02	5	42	33	11
4	0.5	0.02	5	24	48	22
5	1.0	0.1	3	7	57	21
6	1.0	0.1	5	5	51	32

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^aThe reaction was conducted using trisaccharide 37 (0.19 mmol). Yields were reported as isolated yields. Nuclear magnetic resonance and mass spectrometry analyses confirmed the product structures.

Prior to examining the conditions for selective removal of the Nap and PMB groups, the C2-acetyl group of −1 glucuronic acid (GlcA) derivative of trisaccharide 29 was first hydrolyzed under mild conditions to reveal secondary alcohol 45 (Scheme 6). Next, the C2-N-benzylidene of −2 GlcN moiety of trisaccharide 33 was removed in less than 20 min under acidic conditions. Subsequent acetylation of the resulting amine salt intermediate 46 afforded trisaccharide 47 (Scheme 6) having a −2 GlcNAc moiety. The stage was set for chemoselective cleavage of the Nap and PMB groups from trisaccharides 27, 28, 30-32, 45, and 47.

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Scheme 6.

Removal of C2-acetyl group of −1 GlcA unit and C2-N-benzylidene group of −2 GlcN unit^a

^a Reagents and conditions: (a) CH₃ONa (4.0 equiv.), CH₃OH/DCM (45, 85%); (b) 6N HCl, acetone, 25 °C, 20 min (46, 78%); (c) Ac₂O, Et₃N, DCM/CH₃OH (47, 81%).

Proper Nap and PMB deprotection procedure is necessary to reveal key hydroxyls for subsequent O-sulfonations. As such, we first examined the conditions with the most available trisaccharide 32 as it contains the C6-Nap group at −2 GlcN unit as well as the C6-PMB and C3-Nap groups at +1 GlcN unit (Table 3). To this end, we studied the reaction of 32 under acidic conditions using a 8:1 mixture of TFA and DCM (entries 1 and 2), affording the corresponding triol 48 in poor yield (26% to 35%). Reasoning that the acidic conditions were causing product decomposition, we next examined the reaction of 32 with DDQ in the presence of DCM and water.²⁵ In the absence of an acid scavenger of the acidic 2,3-dichloro-5,6-dicyanohydroquinone byproduct resulting from the DDQ deprotection, the desired adduct 48 was isolated in moderate yield (entries 3–4). Following the seminal work of Bennett and co-workers,²⁶ we investigated the strained olefin, β-pinene, as an acid scavenger in combination with DDQ, leading to the formation of the desired product 48 in a significantly higher yield (73%, entry 6). The optimal conditions were applied to other trisaccharides to give compounds 49 – 54 in an overall yield ranging from 65% to 82% (Scheme 7).

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Scheme 7.

Removal of the Nap and PMB protecting groups.

^a Reagents and conditions: (a) DDQ (9 equiv.), β-pinene (9 equiv.), 25 °C, DCM/H₂O (10/1) (49, 82%; 50, 75%; 51, 71%; 52, 76%; 53, 71%; 54, 65%).

Table 3.

Chemoselective cleavage of the Nap and PMB protecting groups of trisaccharide 32^a

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entry	solvent	DDQ	β-pinene	temperature	time (h)	yield (%)
1	TFA/toluene (8/1)	none	none	0 °C	3	26
2	TFA/toluene (8/1)	none	none	0 °C→25 °C	3	35
3	DCM/H2O (10/1)	9 equiv.	none	25 °C	4	36
4	DCM/H2O (10/1)	9 equiv.	none	25 °C	16	43
5	DCM/H2O (10/1)	9 equiv.	9 equiv.	25 °C	4	65
6	DCM/H2O (10/1)	9 equiv.	9 equiv.	25 °C	8	73

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^aThe reaction was conducted using trisaccharide 32 (0.05 mmol). Yields were reported as isolated yields. Nuclear magnetic resonance and mass spectrometry analyses confirmed the product structure.

With the eleven differently protected trisaccharides 39, 40, 43, 44, and 48 - 54 in hand, they could now individually undergo selective azide reduction followed by N- and O-sulfonations. A key issue of this approach is to identify the conditions that orthogonally reduce the azido moiety as well as chemoselectively sulfate the hydroxyl and amine groups (Scheme 8). We found that utilizing trimethyl phosphine (PMe₃) in THF/H₂O in the absence of NaOH to avoid deacetylation of C3-acetyl of +1 GlcN unit of 40 resulted in no conversion.²⁷ On the other hand, using Zn/AcOH conditions resulted in a complex mixture.²⁸ Ultimately, we discovered that the smooth azido-to-amino conversion was successfully implemented using propane-1,3-dithiol in MeOH in the presence of trimethylamine at 50 °C for 10 h.²⁹ The resulting amine 55 was immediately subjected to simultaneous N- and O-microwave sulfonation developed by the Yu group.³⁰ Interestingly, the over-sulfated byproduct 57 was observed along with the desired product 56. No such issue was found in the previous study while methyl glycosides were utilized as substrates.³⁰ We subsequently discovered that the amount of sulfur trioxide trimethylamine complex and reaction time play a key role in controlling the ratio of the desired polysulfated product 56 and the oversulfated product 57. The search revealed that sulfation of compound 55 using 15 equivalents of sulfur trioxide trimethylamine complex for 15 min gave almost exclusively the desired sulfated product 56 (83% yield over 2 steps) with a trace of 57. For the subsequent hydrogenolysis process to proceed smoothly, it was essential for the N-sulfate counterions to be sodium cation (Na⁺) as opposed to the triethylammonium (Et₃NH⁺). Accordingly, treatment of 56 with Amberlite IR120 Na+ resin afforded the sodium sulfated product 58. Finally, 58 was subjected to hydrogenolysis over Pd(OH)₂ in a mixture of MeOH and pH 7 buffer to remove the benzyl ethers followed by deacetylation³¹ to provide trisaccharide 1α in 88% yield over 3 steps (Scheme 8).

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Scheme 8.

Azide reduction followed by sulfations and deprotection to form HS trisaccharide 1α^a

^a Reagents and conditions: (a) propane-1,3-dithiol, DIPEA, CH₃OH, 50 °C, 10 h; (b) SO₃^.Et₃N, pyridine, 100 °C, 15 min, microwave (58, 83% over 2 steps); (c) Amberlite IR120 Na+ resin, 25 °C, 24 h; (d) 100 psi H₂, Pd(OH)₂, CH₃OH/pH 7 buffer, 25 °C, 24 h; (e) LiOH, H₂O, 25 °C 24 h; then Amberlite IR120 Na+ resin, 25 °C, 24 h (1α, 88% over 3 steps).

The optimal azido-amine conversion and sulfation conditions developed in Scheme 8 were then applied to give the polysulfated trisaccharides 59 – 68 in an overall yield ranging from 73% to 85% (Scheme 9). Finally, these compounds were subjected to hydrogenolysis and deacetylation to supply the desired HS trisaccharides 1β, 2α, 2β, and 3α−9α in overall good yields. The structures of HS-liked compounds were confirmed by 1D and 2D NMR and mass spectrometry analysis (see Supporting Information). The α−1,2-cis anomeric configuration of 2-aminoglucosides was confirmed by J_1,2 coupling constants. While O-sulfonation was observed by ¹H NMR downfield shifts from 3.5–3.8 ppm to 4.0–4.4 ppm, N-sulfonation was observed by ¹³C NMR upfield shifts from 62–65 ppm to 55–58 ppm.³¹

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Scheme 9.

Synthesis of HS trisaccharides 1β, 2α, 2β, and 3α−9α^a

^a Reagents and conditions: (a) propane-1,3-dithiol, DIPEA, CH₃OH, 50 °C, 10 h; (b) SO₃.Et₃N, pyridine, 100 °C, 15 min, microwave (59, 80%; 60, 76%; 61, 78%; 62, 81%; 63, 82%; 64, 85%; 65, 82%; 66, 84%; 67, 76%; 68, 73%; 2 steps); (c) Amberlite IR120 Na+ resin, 25 °C; (d) 100 psi H₂, Pd(OH)₂, CH₃OH/pH 7 buffer, 25 °C, 24 h; (b) LiOH, H₂O, 25 °C 24 h; then Amberlite IR120 Na+ resin, 25 °C (1β, 84%; 2α, 87%; 2β, 85%; 3α, 86%; 4α, 83%; 5α, 86%; 6α, 91%; 7α, 80%; 8α, 88%; 9α, 82%; 3 steps).

Interaction of the HS Trisaccharides with Heparanase:

With the library of trisaccharides 1α, 1β, 2α, 2β, and 3α – 9α in hand, we evaluated how their varied sulfation patterns and anomeric configuration at the reducing end modulated heparanase activity employing a time-resolved fluorescence resonance energy transfer (TR-FRET) assay against fluorescent labeled-HS.³² The assay is based on time-resolved fluorescence energy transfer (TR-FRET) between two fluorophores, a donor (europium cryptate) and an acceptor (XL665, allophycocyanin). When the donor and acceptor apart, there is no FRET signal. Once they are brought in proximity, the FRET signals are generated (for example, the emission spectrum at 665 nm). Heparan sulfate substrate labeled with both biotin and europium cryptate, then incubated with streptavidin-XL665 will give maximum emission. Cleavage of biotin-HS-cryptate substrate by heparanase results in decrease in FRET signal. If the inhibitor is present, biotin-HS-cryptate substrate remains intact resulting in high FRET signal. The FRET emission signal observed will depend on the inhibitory potency of the compound at a given concentration. As can be seen in Table 4, HS trisaccharide 1α bearing an α-anomeric linkage at the reducing end was able to inhibit heparanase with relatively high potency (entry 1, IC₅₀ = 0.393 ± 0.010 μM). To compare, compound 1β which is composed of a β-anomeric linkage at the reducing end, displayed modest inhibitory activity (entry 2, IC₅₀ = 42.06 ± 1.287 μM). These results could be attributed to differences in their interactions with the active site of heparanase (vide infra).¹¹ Furthermore, these results provide the first example that the anomeric configuration at the reducing end of HS trisaccharide can modulate heparanase activity and underscore the importance of the saccharide framework for recognition by heparanase. Next, we examined whether site-defined modification to the sulfation pattern of 1α would alter its affinity for heparanase. Removal of the 6-O-sulfate group at +1 GlcN (2α, entry 3) and at −2 GlcN (3α, entry 4) decreased binding to heparanase (IC₅₀ = 8.225 ± 0.102 μM and 3.012 ± 0.058 μM, respectively). The observation, that a) removal of 6-O-sulfation reduces potency, and b) removal of 6-O-sulfate group at +1 GlcN has a greater effect than removal of 6-O-sulfate group at −2 GlcN, suggests the importance of precise positioning of the sulfate groups on the trisaccharides for heparanase recognition. This result is also consistent with Davies’ the co-crystalized HS-heparanase structures,¹¹ which indicate 6-O-SO₃⁻ at the +1 subsite forms a hydrogen bonding network with Gln270.¹⁵ On the other hand, compounds 5α and 6α, which have N-acetyl groups instead of N-sulfates, have 30-fold reduced potency (entries 7 and 8) compared to 1α, highlighting the importance of the −2 and +1 N-sulfates for heparanase recognition.

Table 4.

Modulation of heparanase activity by HS-like trisaccharides using a TR-FRET assay

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Previous studies have demonstrated that heparanase can recognize GlcN unit (GlcN) carrying either C6- and/or C3-O-sulfate.^14–15 As can be seen in Table 4, trisaccharides bearing 3-O-sulfate at −2 GlcN (8α, entry 10) and at +1 GlcN (9α, entry 11) were significantly less active (IC₅₀ = 12.39 ± 0.362 μM and 8.118 ± 0.153 μM, respectively) than trisaccharide 1α (entry 1). The addition of a third sulfate to the C3 position of both GlcNS6S units, forming trisaccharide 7α (entry 9, IC₅₀ = 9.961 ± 0.261 μM), did not prove to be advantageous. Furthermore, addition of a sulfate ester at C2 of −1 glucuronic acid (4α, entry 6) unit led to a large reduction in heparanase recognition (IC₅₀ = 11.961 ± 0.341 μM), validating that the presence of GlcA(2S) at the −1 subsite cannot be tolerated by heparanase.¹¹ Collectively, our results illustrate that controlling the sulfation pattern and the reducing end anomeric configuration within the trisaccharides enable to modulate their heparanase-inhibitory activity.

It has been previously demonstrated that HS oligosaccharides are less active than full-length HS polysaccharides.³¹ As such, we questioned whether relatively larger oligosaccharides exhibit higher activity than a well-defined sulfated trisaccharide 1α having an α-anomeric linkage at the reducing end linker. Surprisingly, both HS heptasaccharide 69 (IC₅₀ = 41.68 ± 3.64 μM, Scheme 10) and octasaccharide 70 (IC₅₀ = 3.94 ± 0.20 μM, Scheme 10) exhibited significantly decreased binding to heparanase compared to 1α.³³ The low potency compound 69 is similar in activity to trisaccharide 1β (Table 4, entry 2) bearing the β-configured reducing end. Although octasaccharide 70 is more potent than 69, its potency is approximately 10-fold lower than that of trisaccharide 1α (Table 4, entry 1). The observation that trisaccharide 1α is more potent than larger oligosaccharides 69 and 70 suggests that saccharide framework, precise positioning of the sulfate groups, overall charge, and the reducing-end anomeric configuration play critical roles in determining the affinity of HS oligosaccharides for heparanase and the preferential cleavage recognized by heparanase.³⁴

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Scheme 10.

Heparanase activities of large HS oligosaccharides using a TR-FRET assay

Computational Study of HS Trisaccharides 1α and 1β.

It has never been established how the anomeric configuration at the reducing end of HS oligosaccharides could have a great effect on their binding affinity for heparanase. As can be clearly demonstrated in Table 4, trisaccharide 1α with the α-configured reducing end is by far more potent than trisaccharide 1β with the β-configured reducing end. Accordingly, an in silico docking study with 1α and 1β into the apo crystal structure of heparanase (PDB code: 5E8M) was performed.^{11, 35–36} We initiated our study by docking the natural HS substrate, GlcNS(6S)α(1,4)GlcAβ(1,4)GlcNS(6S) trisaccharide, into heparanase to obtain a benchmark for comparison with our compounds (see Figure S6 for actual docked structures). Next, trisaccharide 1α (Figure 4A) and trisaccharide 1β (Figure 4B) were constructed in silico to be docked in the apo heparanase crystal structure. Upon examination of the docked structures for the trisaccharide-heparanase complexes, both 1α and 1β placed the GlcNS(6S)α(1,4)GlcAβ(1,4)GlcNS(6S) into subsites −2/−1/+1 (Figure 4) in a similar fashion to Davies’ the published co-crystalized HS-heparanase structures.¹¹ The trisaccharide was oriented in the same direction as the co-crystalized structures with the non-reducing end towards HBD-1 and the reducing end closer to HBD-2. When looking at specific interactions for both trisaccharides, we found that while the N-sulfate of the aminoethyl spacer of trisaccharide 1α formed a hydrogen bond with Lys231, the spacer of trisaccharide 1β formed a hydrogen bond with Arg272. Both the +1 C6-O-sulfate of 1α and 1β formed a strong divalent hydrogen bonding network with Gln270 while both the −2 N-sulfate of 1α and 1β formed a hydrogen bond with Gly389 (Figure 4A and ​and4B).4B). Interestingly, while −2 C6-O-sulfate of trisaccharide 1α potentially formed a salt bridge to Lys159 (Figure 4A) in a similar fashion to Davies’ the co-crystalized HS-heparanase structures;¹¹ −2 C6-O-sulfate of trisaccharide 1β formed a hydrogen bond with Thr97 (Figure 4B).

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Figure 4.

Trisaccharides docked into the apo crystal structure of heparanase (Protein Data Bank (PDB) code: 5E8M): (A) trisaccharide 1α and (B) trisaccharide 1β¸ using the Autodock Vina suite in the YASARA program. Hydrogen bonding is shown in magenta. HBD-2 is on the left and HBD-1 is on the right.

The ligand-heparanase complexes illustrated in Figure 4 were then subjected to 25000 ps molecular dynamic (MD) simulations (see Figures S7–S9) and established the complex’s stability and the distance between the scissile GlcA–GlcNS(6S) bond of trisaccharides 1α (Figure 5A) and 1β (Figure 5B) to the catalytic residues Glu225 and Glu343.¹¹ As shown in Figure 5A, the distance of both catalytic residues to the scissile bond for 1α complex tends to be steady. However, the distance increases between Glu343 and C1 of the GlcA unit at the −1 position toward the end of the simulations (around 24000 ps). As a result, the simulation for 1α complex was further extended to 30000 ps, but no change in distance was observed (see Figure S8). The sudden increase in distance between Glu343 and the scissile bond around 24000 ps was due to a shift in hydrogen bonding interaction between Glu343 and C2/C3-hydroxyl group of the −1 GlcA unit (see superimposed snapshots in Figure 5A). During the duration of the simulations, there is a hydrogen bond interaction between Glu343 and C2-OH of the −1 GlcA unit. Near the end of the simulations, Glu343 participates in a hydrogen bond with C3-OH group of the −1 GlcA unit. Meanwhile, Glu225 maintained at approximately 5 Å away from the scissile bond as it participates in a hydrogen bond with 6-O-sulfate of +1 GlcNS6S. Collectively, these docking data illustrate that a nucleophilic residue, Glu343, moved away from the scissile bond and a proton donor, Glu225, did not participate in a hydrogen bond with C1-oxygen of the scissile bond. As a result, hydrolysis is unlikely to occur for compound 1α. On the other hand, the distance between Glu225 and the scissile GlcA–GlcNS(6S) bond of 1β fluctuated during the simulations, while Glu343 was maintained at approximately 4 Å away from the scissile bond (Figure 5B). As demonstrated in the superimposed snapshots in Figure 5B, Glu225 shifted its participation in a hydrogen bond with 6-O-sulfate of +1 GlcNS6S to C1-oxygen of the scissile bond during the fluctuation. Overall, a proton donor, Glu225, formed a hydrogen bond to C1-oyxgen of the scissile bond, while nucleophile Glu343 maintained a close distance to C1 of the scissile bond, suggesting that hydrolysis is likely to occur for compound 1β.

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Figure 5.

Distance of scissile bond between −1 GlcA and +1 GlcNS6S of trisaccharides 1α (A) and trisaccharide 1β (B) to the catalytic residues (Glu343 and Glu225).

Since the difference between trisaccharide 1α and 1β was the orientation of the aminoethyl linker at +1 GlcNS6S, we took a close look at the +1 region in the ligand-enzyme complex (Figure 6). Seemingly, the hydrogen bonding patterns on the aminoethyl linker are different between compound 1α and 1β. While the axial oriented linker (1α) interacts with Asn227, Ser 228, and Lys 231 (Figure 6A), the equatorial oriented linker (1β) was hydrogen-bonded to Arg272 (Figure 6B). The major difference between the two complexes is the hydrogen bonding pattern of Arg272. In 1α complex, Arg272 formed a hydrogen bond with 6-O-sulfate of the +1 subset, which allowed the catalytic residue Glu225 to interact with that 6-O-sulfate and stay away from the scissile bond (Figure 6A). On the other hand, in 1β complex, Arg272 formed a hydrogen bond with the N-sulfate of the linker, consequently shifting the position of the ligand in the binding pocket. The shifting position further leads to loose interaction of Glu225 with 6-O-sulfate of the +1 subset and allows hydrogen bonding to C1-oxygen of the scissile bond (Figure 5A).

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Figure 6.

Hydrogen bonding pattern at the aminoethyl spacer region in the ligand-enzyme complex. (A) trisaccharide 1α and (B) trisaccharide 1β.

As can be seen in Figure 5B, The scissile GlcA–GlcNS(6S) bond of trisaccharide 1β is in proximity to the catalytic residues, Glu225 and Glu343 and aligned in the proper anti-orientation to these residues for cleavage by heparanase. In stark contrast, trisaccharide 1α preferred a distance further away from Glu225 and Glu343 and its GlcA–GlcNS(6S) bond is not aligned in the anti-orientation for cleavage by heparanase (Figure 5A). To confirm if cleavage of the scissile bond between −1 GlcA and +1 GlcNS6S of trisaccharides 1α and 1β is possible, a colorimetric assay was then performed (Figure 7).^37–38 This assay measures the appearance of the saccharide hemiacetal product of heparanase-catalyzed cleavage utilizing a tetrazolium sodium salt (WST-1), which reacts with a newly formed anomeric hemiacetal.³⁷ Since the ultralow molecular weight heparin, fondaparinux, is known to cleaved by heparanase,³⁷ it was used as a control. Monitoring the degradation over 24 h showed almost no change in absorbance for the sample containing a mixture of 1α and heparanase relative to the control, suggesting that 1α is not hydrolyzed by heparanase. In stark contrast, use of 1β in this type of experiment resulted in a drastic change in absorbance over the same amount of time, suggesting that compound 1β is readily cleaved by heparanase (Figure 7). Interestingly, for trisaccharide 2β that lacks 6-O-sulfate at the +1 position, there was a small change in absorbance starting around the 11 h time point, which could be attributed to the close distance between the scissile bond and the catalytic residues (Figure 7). To compare, trisaccharide 2α undergoes hydrolysis at a faster rate than does its counterpart 2β. The results obtained with both 2α and 2β are in accordance with a docking study (see Figure S6), wherein the scissile GlcA–GlcNS(6S) bond of 2α is in closer proximity to the catalytic residues than that bond of 2β. The rate of hydrolysis of trisaccharide 3α (that lacks 6-O-sulfate at the −2 position) is similar to that of trisaccharide 2α. Among all compounds tested, sulfated trisaccharide 6α incorporating with the N-acetyl group at the +1 position had the fastest change in absorbance over the same amount of time (Figure 7), suggesting that 6α is readily cleaved by heparanase. This hydrolysis result is consistent with the docking data. We found that replacement of N-sulfate with N-acetyl at the +1 position shifts the scissile GlcA–GlcNS(6S) bond closer to the catalytic residues, Glu225 and Glu343, for cleavage by heparanase (see Figure S6). Furthermore, to determine whether any acidic autohydrolysis took place during the assay due to the acidic media, each experiment were conducted concurrently with a control of trisaccharide without heparanase. From these control experiments, acid-catalyzed hydrolysis did not occur and any change in absorbance was a result of glycosidic cleavage by heparanase. Overall, these aforementioned results suggest that heparanase preferentially cleaves a HS trisaccharide with GlcNS6S residues at the −2 and +1 position and with the β-anomeric configuration at the reducing end. Furthermore, these results suggest that changes in the sulfation pattern and the reducing end anomeric configuration disrupt the positioning of the saccharide, reducing the number of ionic salt bridges and hydrogen bonding interactions, as well as altering the distance between the scissile bond and the catalytic residues.

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Figure 7.

Hydrolytic potential overtime of heparanase on trisaccharides 1α, 1β, 2α, 2β, 3α, 6α, and fondaprinux.

Cross-Bioactivity Studies:

After discovering that the GlcNS(6S)α(1,4)GlcAβ(1,4)GlcNS(6S) bearing an α-configured aminoethyl linker at the reducing end (1α) is the most effective trisaccharide to inhibit heparanase activity, we next sought to assess its potential off-target anticoagulant activity in comparison to heparin. It has been reported that heparin binds to antithrombin (AT) III to induce a conformational change that enables ATIII to inhibit the serine proteases factor Xa (FXa) and FIIa.³⁹ As such, protease inhibition of heparin and trisaccharide 1α was assessed using chromogenic substrate assays.⁴⁰ In a manner consistent with previous studies,⁴⁰ heparin exhibited a significant high activity against both FXa (IC₅₀ = 15.13 ± 0.6511 nM) and FIIa (IC₅₀ = 5.554 ± 0.2448 nM) (Figure 8). Since trisaccharide 1α lacks an iduronic acid (IdoA) which is key in biding to ATIII,⁴¹ it is anticipated that it would bind to FXa and FIIa with low affinity. As expected, compound 1α (IC₅₀ >2000 nM) did not inhibit either FXa or FIIa (Figure 8). To compare, a heparin pentasaccharide (fondaparinux) bearing an IdoA moiety, exhibited significantly higher activity against FXa (IC₅₀ = 11.0 ± 0.1 nM) and limited activity against FIIa.⁴⁰

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Figure 8.

Trisaccharides 1α (orange) displayed no measurable activity against serine proteases FIIa (A) and FXa (B) in comparison to heparin (blue).

Next, we screened the ability of trisaccharide 1α to bind to the HS-binding protein, platelet factor-4 (PF4), which is responsible for causing thrombocytopenia,^42–43 the main reason why clinical trials for other carbohydrate-based heparanase inhibitors were halted.^44–45 To achieve this goal, a solution-based biolayer interferometry (BLI) assay was utilized to determine the apparent K_d of compound 1α to PF4 in comparison to biotinylated-heparin (18 kDa) attached to the BLI streptavidin-probe.⁴⁶ Trisaccharide 1α exhibited very low affinity to PF4 (K_d = 16.01 ± 5.480 μM, Figure 9), which was significantly weaker than that of heparin (K_d = 0.3513 ± 0.0.2687 nM, see Figure S5) and that of PI-88 (K_d =16.0 ± 1.9 nM), a known oligosaccharide heparanase inhibitor.⁴⁷

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Figure 9.

Binding affinity of trisaccharide 1α to PF4.

Materials.

The procedure for the preparation of monosaccharide building blocks (10-17), disaccharides (22, 23, and 26), 11 partially protected trisaccharides (58-68), as well as their NMR spectral data can be found in the Supporting Information.

Monosaccharide building blocks preparation.

(see pS4 in the Suppporting Information for the Synthetic Scheme).

p-Tolyl 2-azido-2-deoxy-4,6-O-(p-methoxybenzylidene)-1-thio-D-glucopyranoside (11b):

To a solution of 2-azido-2-deoxy-thioglucoside⁵⁶ 11a (3.7 g, 11.86 mmol) in CH₃CN (100 mL) were added p-anisaldehyde dimethyl acetal (3.03 mL, 17.79 mmol) and p-TSA (225 mg, 1.186 mmol). After the reaction mixture had been stirring overnight at room temperature, it was quenched with Et₃N and concentrated in vacuo. The resulting residue was subjected to silica gel column chromatography (1:6, EtOAc/hexanes) to give 11b (4.28 g, 84%) as a pale yellow syrup.

α-isomer:

R_f 0.30 (1:4, EtOAc/hexanes); ¹H NMR (600 MHz, CDCl₃) δ 7.43 (d, J = 8.7 Hz, 2H), 7.38 (d, J = 8.1 Hz, 2H), 7.13 (d, J = 7.9 Hz, 2H), 6.91 (d, J = 8.8 Hz, 2H), 5.49 (s, 1H, Ph-CH-), 5.47 (d, J = 5.6 Hz, 1H, H-1α), 4.37 (td, J = 9.9, 5.0 Hz, 1H), 4.20 (dd, J = 10.3, 5.0 Hz, 1H), 4.01 (t, J = 9.6 Hz, 1H), 3.87 – 3.84 (m, 1H), 3.80 (s, 3H, -OCH₃), 3.71 (t, J = 10.3 Hz, 1H), 3.52 (t, J = 9.4 Hz, 1H), 3.00 (s, 1H, -OH), 2.33 (s, 3H, Ph-CH₃). ¹³C NMR (150 MHz, CDCl₃) δ 160.35, 138.37, 133.09, 133.07, 129.98, 129.96, 129.27, 129.13, 127.65, 113.78, 102.10, 88.08, 81.66, 70.67, 68.48, 63.87, 63.34, 55.31, 21.14. HR ESI-TOF MS (m/z): calcd for C₂₁H₂₃N₃O₅SNa [M + Na]⁺, 452.1251; found, 452.1260.

β-isomer:

R_f 0.40 (1:4, EtOAc–hexane); ¹H NMR matches with the literature report.⁵⁷

p-Tolyl 2-azido-2-deoxy-3-O-(2-naphthylmethyl)-4,6-O-(p-methoxybenzylidene)-1-thio-α-D-glucopyranoside (11c):

A solution of 11b (1.8 g, 4.19 mmol) in 45 mL of anhydrous DMF was mixted with NaH (201 mg, 8.37 mmol) at 0 °C under a N₂ atmosphere. After stirring for 15 min, 2-naphtylmethyl bromide (1.39 g, 6.28 mmol) was added and the reaction was monitored by TLC. After 3 h of stirring at room temperature, the excessive NaH was quenched with saturated aq. NH₄Cl solution, and the mixture was diluted with ethyl acetate. The organic layer, after being washed with saturated aq. NaCl solution, was dried with Na₂SO₄ and concentrated in vacuo. The residue was purified by silica gel column chromatography (1:8, EtOAc/hexanes) to give compound 11c (2.1 g, 88%) as syrup.

R_f 0.50 (1:4, EtOAc/hexanes); ¹H NMR (600 MHz, CDCl₃) δ 7.89 – 7.74 (m, 4H), 7.51 (dd, J = 8.5, 1.5 Hz, 1H), 7.48 – 7.44 (m, 2H), 7.44 – 7.41 (m, 2H), 7.41 – 7.37 (m, 2H), 7.14 (d, J = 7.9 Hz, 2H), 6.94 – 6.88 (m, 2H), 5.56 (s, 1H, Ph-CH-), 5.50 (d, J = 5.2 Hz, 1H, H-1α), 5.11 (d, J = 11.2 Hz, 1H), 4.99 (d, J = 11.2 Hz, 1H), 4.44 (td, J = 10.0, 4.9 Hz, 1H), 4.21 (dd, J = 10.4, 4.9 Hz, 1H), 4.04 – 3.95 (m, 2H), 3.83 (s, 3H, -OCH₃), 3.78 – 3.72 (m, 2H), 2.34 (s, 3H, Ph-CH₃). ¹³C NMR (150 MHz, CDCl₃) δ: 160.13, 138.33, 135.22, 133.27, 133.10, 133.07, 129.97, 129.60, 129.05, 128.16, 128.00, 127.66, 127.37, 126.97, 126.09, 125.99, 125.90, 113.66, 101.53, 88.13, 82.65, 77.95, 75.16, 68.55, 63.73, 63.69, 55.31, 21.15. HR ESI-TOF MS (m/z): calcd for C₃₂H₃₁N₃O₅SNa [M + Na]⁺, 592.1877; found, 592.1883.

p-Tolyl 2-azido-2-deoxy-3-O-(2-naphthylmethyl)-6-O-(p-methoxybenzyl)-1-thio-α-D-glucopyranoside (11d):

To a solution of 11c (1.1 g, 1.93 mmol) in THF (30 mL) was added NaBH₃CN (1.72 g, 19.3 mmol) at 0 °C. A 2N HCl/Et₂O solution was then added dropwise to maintain the reaction mixture at pH 1. After the reaction mixture had been stirring at room temperature for 2 h, it was diluted with ethyl acetate, washed with aqueous NaHCO₃ and brine, dried over anhydrous Na₂SO₄, and concentrated. The residue was subjected to flush silica gel column chromatography (1:5, EtOAc/hexanes) to give 11d (0.87 g, 79%) as syrup.

Rf 0.55 (1:3, EtOAc/hexanes); ¹H NMR (600 MHz, CDCl₃) δ 7.84 (ddd, J = 8.4, 6.8, 4.3 Hz, 4H), 7.54 (dd, J = 8.5, 1.5 Hz, 1H), 7.52 – 7.45 (m, 2H), 7.42 – 7.35 (m, 2H), 7.23 (d, J = 8.7 Hz, 2H), 7.08 (d, J = 7.9 Hz, 2H), 6.91 – 6.81 (m, 2H), 5.50 (d, J = 5.4 Hz, 1H, H-1α), 5.09 (d, J = 11.3 Hz, 1H), 5.02 (d, J = 11.3 Hz, 1H), 4.51 (d, J = 11.5 Hz, 1H), 4.43 (d, J = 11.5 Hz, 1H), 4.34 (dt, J = 9.1, 4.5 Hz, 1H), 3.90 (dd, J = 9.9, 5.4 Hz, 1H), 3.79 (s, 3H, -OCH₃), 3.78 – 3.68 (m, 3H), 3.64 (dd, J = 10.4, 4.4 Hz, 1H), 2.64 (s, 1H, -OH), 2.32 (s, 3H, Ph-CH₃). ¹³C NMR (150 MHz, CDCl₃) δ 159.33, 137.95, 135.40, 133.33, 133.10, 132.71, 129.85, 129.73, 129.53, 129.38, 128.43, 128.02, 127.71, 126.96, 126.16, 126.03, 125.93, 113.82, 87.57, 81.30, 75.43, 73.28, 72.71, 70.78, 69.49, 63.58, 55.26, 21.12. HR ESI-TOF MS (m/z): calcd for C₃₂H₃₃N₃O₅SNa [M + Na]⁺, 594.2033; found, 594.2028.

p-Tolyl 2-azido-2-deoxy-3-O-(2-naphthylmethyl)-4-O-benzyl-6-O-(p-methoxybenzyl)-1-thio-α-D-glucopyranoside (11):

To a solution of 11d (1.0 g, 1.75 mmol) in 18 mL of anhydrous DMF was added NaH (84 mg, 3.5 mmol) at 0 °C under a N₂ atmosphere. After the mixture had been stirring for15 min, benzyl bromide (311 μL, 2.62 mmol) was added. The resulting mixture was kept at room temperature for 3 h, quenched with saturated aq. NaCl solution, and diluted with ethyl acetate. The organic layer, after washing with water and brine, was dried with Na2SO4 and concentrated in vacuo. The residue was subjected to silica gel column chromatography (1:8, EtOAc/hexanes) to give 11 (1.05 g, 91%) as a yellow solid.

R_f 0.60 (1:4, EtOAc/hexanes); ¹H NMR (600 MHz, CDCl₃) δ: 7.92 – 7.72 (m, 4H), 7.55 – 7.37 (m, 5H), 7.34 – 7.28 (m, 3H), 7.28 – 7.21 (m, 2H), 7.16 (dd, J = 6.8, 2.5 Hz, 2H), 7.10 (d, J = 8.2 Hz, 2H), 6.88 – 6.78 (m, 2H), 5.58 (d, J = 5.3 Hz, 1H, H-1α), 5.09 (d, J = 10.8 Hz, 1H), 5.03 (d, J = 10.8 Hz, 1H), 4.82 (d, J = 10.9 Hz, 1H), 4.58 (d, J = 11.7 Hz, 1H), 4.52 (d, J = 10.9 Hz, 1H), 4.45 – 4.31 (m, 1H), 3.99 (dd, J = 10.3, 5.3 Hz, 1H), 3.94 – 3.86 (m, 2H), 3.83 – 3.77 (m, 2H), 3.76 (s, 3H, -OCH₃), 3.63 (dd, J = 10.9, 1.8 Hz, 1H), 2.34 (s, 3H, Ph-CH₃). ¹³C NMR (150 MHz, CDCl₃) δ: 159.30, 137.93, 137.87, 135.26, 133.32, 133.07, 132.63, 129.86, 129.83, 129.66, 129.65, 128.42, 128.23, 128.01, 127.80, 127.69, 127.65, 126.85, 126.07, 125.99, 125.96, 113.77, 87.62, 81.89, 78.33, 75.74, 75.03, 73.10, 71.75, 67.83, 64.18, 55.21, 21.14. HR ESI-TOF MS (m/z): calcd for C₃₉H₃₉N₃O₅SNa [M + Na]⁺, 684.2503; found, 684.2507.

Phenyl 2-azido-2-deoxy-3,4-di-O-benzyl-6-O-(2-naphthylmethyl)-1-thio-D-glucopyranoside (12):

To a solution of 2-azido-2-deoxy-3-O-benzyl-6-O-(2-naphthylmethyl)-thioglucoside⁵⁸ 12a (3.0 g, 5.69 mmol) in 60 mL of anhydrous DMF was added NaH (273.36 mg, 11.39 mmol) at 0 °C under a N₂ atmosphere. After the mixture had been stirring for 15 min, benzyl bromide (1.01 mL, 8.54 mmol) was added. The resulting mixture was kept at room temperature for 3 h, quenched with saturated aq. NaCl solution and diluted with ethyl acetate. The organic layer, after washing with water and brine, was dried with Na2SO4 and concentrated in vacuo. The residue was subjected to silica gel column chromatography (1:15, EtOAc/hexanes) to give 12 (3.27 g, 93%) as α/β (4/3) mixture.

R_f 0.50 (1:8, EtOAc/hexanes); ¹H NMR (600 MHz, CDCl₃) of α/β mixture δ: 7.92 – 7.71 (m, 5.5H), 7.68 – 7.59 (m, 1H), 7.58 – 7.42 (m, 5.8H), 7.42 – 7.11 (m, 14.6H), 7.06 (dd, J = 16.8, 6.6 Hz, 1H), 5.64 (d, J = 5.4 Hz, 1 H, H-1α), 4.97 – 4.75 (m, 6H), 4.71 (d, J = 12.2 Hz, 0.75H), 4.64 – 4.56 (m, 1.5H), 4.52 (d, J = 10.8 Hz, 1H), 4.44 (d, J = 10.1 Hz, 0.7H, H-1β), 4.42 – 4.36 (m, 1H), 3.98 (dd, J = 10.1, 5.4 Hz, 1H), 3.89 – 3.74 (m, 4H), 3.68 (dd, J = 10.9, 1.8 Hz, 1H), 3.63 (t, J = 9.4 Hz, 0.73H), 3.51 (ddd, J = 9.6, 6.0, 5.5 Hz, 1.3H), 3.38 (t, J = 9.7 Hz, 0.74H). ¹³C NMR (150 MHz, CDCl₃) of α/β mixture δ: 137.76, 137.70, 137.64, 137.58, 135.62, 135.16, 133.60, 133.48, 133.26, 133.20, 133.02, 132.99, 132.04, 131.20, 129.06, 129.00, 128.51, 128.43, 128.39, 128.35, 128.23, 128.21, 128.17, 128.14, 128.02, 127.97, 127.88, 127.86, 127.80, 127.79, 127.71, 127.66, 127.64, 126.73, 126.32, 126.16, 126.13, 125.95, 125.90, 125.88, 125.69, 87.27, 85.97, 85.08, 81.86, 79.35, 78.24, 77.54, 75.90, 75.76, 75.08, 75.05, 73.56, 73.54, 71.79, 68.68, 68.21, 65.06, 64.11. HR ESI-TOF MS (m/z): calcd for C₃₇H₃₅N₃O₄SNa [M + Na]⁺, 640.2240; found, 640.2238.

2-azido-2-deoxy-3,4-di-O-benzyl-6-O-(2-naphthylmethyl)-D-glucopyranoside (13a):

To a solution of 12 (2.18 g, 3.53 mmol) in a mixture of acetone/H₂O (v/v, 50/1, 30 mL) were added NIS (1.47 g, 7.05 mmol) and AgOTf (168.6 mg, 0.705 mmol). After the reaction mixture had been stirring at room temperature for 20 min, it was quenched with Et₃N, and concentrated. The residue was subjected to silica gel column chromatography (1:2, EtOAc/hexanes) to give 13a (1.52 g, 82%) as α/β (1/0.8) mixture.

R_f 0.40 (3:7, EtOAc/hexanes); ¹H NMR (600 MHz, CDCl₃) of α/β mixture δ: 7.80 (ddd, J = 30.8, 16.5, 12.7 Hz, 6H), 7.48 (dt, J = 15.9, 6.2 Hz, 5H), 7.42 – 7.27 (m, 7H), 7.25 – 7.11 (m, 5H), 7.04 (dd, J = 17.3, 7.2 Hz, 3H), 5.34 (t, J = 3.4 Hz, 1H, H-1α), 4.92 – 4.62 (m, 8H), 4.55 (dd, J = 7.3, 5.5 Hz, 0.78H, H-1β), 4.51 – 4.38 (m, 2H), 4.18 – 4.09 (m, 1H), 4.07 – 3.99 (m, 1H), 3.86 (d, J = 2.8 Hz, 1H), 3.73 – 3.56 (m, 4H), 3.55 – 3.31 (m, 4H). ¹³C NMR (150 MHz, CDCl₃) of α/β mixture δ: 137.82, 137.68, 137.54, 135.00, 134.94, 133.21, 133.09, 133.08, 128.48, 128.47, 128.36, 128.34, 128.33, 128.12, 128.06, 127.91, 127.90, 127.84, 127.83, 127.78, 127.72, 126.95, 126.19, 126.18, 126.01, 126.00, 125.97, 125.96, 96.15, 92.02, 83.09, 80.12, 78.58, 77.71, 75.53, 75.52, 74.99, 74.74, 73.60, 73.57, 70.50, 68.58, 67.32, 64.00. HR ESI-TOF MS (m/z): calcd for C₃₁H₃₁N₃O₅Na [M + Na]⁺, 548.2156; found, 548.2166.

2-N-[(2-trifluoromethyl)benzylidene]-2-deoxy-3,4-di-O-benzyl-6-O-(2-naphthylmethyl)-D-glucopyranoside (13c):

To a solution of 13a (1.4 g, 2.67 mmol) in CH₃OH (30 mL) was added 1,3-propanedithiol (6 mL) and DIPEA (2 mL). After the reaction mixture had been stirring at 50 °C for overnight, it was concentrated in vacuo and the residue was purified by silica gel column chromatography (1:15, CH₃OH/CH₂Cl₂) to afford 13b as colorless syrup. The resulting amine intermediate was then dissolved in pyridine/CH₂Cl₂ (1:10, 30 mL), and 2-(trifluoromethyl) benzaldehyde (421.82 μL, 3.2 mmol) was then added. After the reaction mixture had been stirring under reflux for overnight, it was concentrated. The resulting residue was subjected to silica gel column chromatography (1:2, EtOAc/hexanes) to give 13c (1.08 g, 62% for 2 steps) as α/β (0.28/1) mixture.

R_f 0.20 (1:2, EtOAc/hexanes); ¹H NMR (600 MHz, CDCl₃) δ: 8.75 (d, J = 2.0 Hz, 0.26H), 8.71 (d, J = 2.1 Hz, 1H), 8.21 (d, J = 7.6 Hz, 0.28H), 8.17 (dd, J = 5.2, 3.8 Hz, 1H), 7.86 – 7.64 (m, 6H), 7.54 – 7.41 (m, 5H), 7.23 – 7.01 (m, 10H), 5.24 (d, J = 3.4 Hz, 0.29H, H-1α), 5.04 (d, J = 7.7 Hz, 1H, H-1β), 4.84 – 4.76 (m, 1.3H), 4.75 – 4.61 (m, 3.2H), 4.57 – 4.45 (m, 2.3H), 3.91 (t, J = 9.2 Hz, 1H), 3.85 – 3.57 (m, 5H), 3.36 (dd, J = 9.4, 7.8 Hz, 1H). ¹³C NMR (150 MHz, CDCl₃) δ: 160.69, 137.85, 135.28, 133.67, 133.20, 133.02, 131.89, 130.70, 130.38, 129.48, 128.43, 128.31, 128.29, 128.22, 127.96, 127.93, 127.89, 127.85, 127.75, 127.71, 127.69, 127.61, 127.57, 126.73, 126.08, 125.97, 125.91, 125.87, 125.62, 124.89, 123.08, 95.58 (C-1β), 93.76(C-1α), 83.23, 80.21, 78.28, 77.87, 75.38, 75.27, 75.17, 75.05, 74.95, 73.63, 73.57, 71.22, 68.98, 68.67, 60.39. HR ESI-TOF MS (m/z): calcd for C₃₉H₃₆F₃NO₅Na [M + Na]⁺, 678.2438; found, 678.2433.

2-N-[(2-trifluoromethyl)benzylidene]-2-deoxy-3,4-di-O-benzyl-6-O-(2-naphthylmethyl)-D-glucopyranosyl N-phenyl-trifluoroacetimidate (13):

To a solution of 13c (950 mg, 1.45 mmol) in acetone (15 mL) was added 2,2,2-trifluoro-N-phenyl-ethanimidoyl chloride (695 μL, 4.35 mmol) and K₂CO₃ (400 mg, 2.9 mmol) under a N₂ atmosphere After the reaction mixture had been stirring overnight at room temperature, it was filtered and concentrated in vacuum. The resulting residue was purified by silica gel column chromatography (1:10, EtOAc/hexanes + 1% triethylamine) to afford compound 13 (1.15 g, 96%) as α/β mixture.

For β anomer (majority): R_f 0.50 (1:5, EtOAc/hexanes);¹H NMR (600 MHz, CDCl₃) δ: 8.76 (s, 1H), 8.19 (d, J = 7.7 Hz, 1H), 7.87 – 7.77 (m, 3H), 7.73 (d, J = 7.6 Hz, 1H), 7.58 (dt, J = 29.1, 7.5 Hz, 2H), 7.53 – 7.44 (m, 3H), 7.28 – 7.00 (m, 14H), 6.75 (s, 2H), 6.03 (s, 1H), 4.87 – 4.78 (m, 2H), 4.78 – 4.65 (m, 2H), 4.59 (t, J = 11.1 Hz, 2H), 3.97 (s, 1H), 3.84 (dd, J = 21.4, 12.1 Hz, 3H), 3.67 (s, 2H). ¹³C NMR (150 MHz, CDCl₃) δ: 161.48, 143.45, 137.85, 137.80, 135.43, 133.62, 133.24, 133.03, 131.94, 130.63, 129.65, 129.44, 128.60, 128.36, 128.27, 128.19, 127.91, 127.89, 127.87, 127.78, 127.69, 127.68, 126.67, 126.10, 125.92, 125.87, 125.76, 125.72, 124.84, 124.17, 123.02, 119.36, 83.14, 76.23, 76.10, 75.20, 75.07, 73.54, 68.14. HR ESI-TOF MS (m/z): calcd for C₄₇H₄₀F₆N₂O₅Na [M + Na]⁺, 849.2734; found, 849.2736.

2-Chloroethyl 2-azido-2-deoxy-4,6-O-(2-naphthylmethylbenzylidene)-α-D-glucopyranoside (15c):

To a solution of 2-azido-2-deoxy-glucopyranoside⁵⁹ 15a (5.4 g, 26.21 mmol) in 2-chloroethanol (120 mL) was added acetyl chloride (2.79 mL, 39.32 mmol). After the reaction mixture had been stirring at 70 °C for 4 h, it was concentrated in vacuo and the residue was purified by silica gel column chromatography (1:15, CH₃OH/CH₂Cl₂) to afford 2-chloroehyl 2-azido-2-deoxy-α-glucopyranoside 15b as colorless syrup. The intermediate 15b was dissolved in CH₃CN (160 mL), and 2-naphtylaldehyde dimethyl acetal (7.94 g, 39.32 mmol) and p-TSA (497.8 mg, 2.62 mmol) were added. After the reaction mixture had been stirring overnight at 50 °C, it was quenched with Et₃N and concentrated. The residue was subjected to silica gel column chromatography (1:5, EtOAc/hexanes) to give 15c (6.18 g, 58% for 2 steps) as syrup.

R_f 0.50 (1:2, EtOAc–hexane); ¹H NMR (600 MHz, CDCl₃) δ: 7.96 (s, 1H), 7.89 – 7.80 (m, 3H), 7.59 (dd, J = 8.5, 1.5 Hz, 1H), 7.53 – 7.45 (m, 2H), 5.68 (s, 1H, Ph-CH-), 4.95 (d, J = 3.7 Hz, 1H, H-1α), 4.31 (dd, J = 10.3, 5.0 Hz, 1H), 4.26 (t, J = 9.6 Hz, 1H), 4.03 – 3.91 (m, 2H), 3.79 (ddd, J = 27.1, 15.9, 8.0 Hz, 2H), 3.68 (t, J = 5.7 Hz, 2H), 3.55 (t, J = 9.4 Hz, 1H), 3.28 (dd, J = 10.1, 3.7 Hz, 1H), 2.79 (s, 1H, -OH). ¹³C NMR (150 MHz, CDCl₃) δ: 134.11, 133.76, 132.85, 128.34, 128.31, 127.71, 126.58, 126.29, 125.83, 123.59, 102.17, 98.88, 81.75, 68.78, 68.74, 68.68, 62.89, 62.74, 42.36. HR ESI-TOF MS (m/z): calcd for C₁₉H₂₀ClN₃O₅Na [M + Na]⁺, 428.0984; found, 428.0989.

2-Azidoethyl 2-azido-2-deoxy-3-O-benzyl-4,6-O-(2-naphthylmethylbenzylidene)-α-D-glucopyranoside (15e):

To a solution of 15c (1.7 g, 4.20 mmol) in DMF (40 mL) was added NaN₃ (1.36 g, 20.99 mmol). After the reaction mixture had been stirring at 80 °C for 24 h, it was diluted with CH₂Cl₂ and washed with water and brine, dried over anhydrous Na₂SO₄, and concentrated. The resulting residue 15d was dissolved in 40 mL of anhydrous DMF and NaH (201 mg, 8.39 mmol) was added at 0 °C under a N₂ atmosphere. After the reaction mixture had been stirring for 15 min, benzyl bromide (747.68 μL, 6.30 mmol) was added. After the reaction mixture had been stirring at room temperature for 2 h, it was quenched with saturated aq. NaCl solution and diluted with ethyl acetate. The organic layer, after washing with water and brine, was dried with Na₂SO₄ and concentrated in vacuo. The residue was subjected to silica gel column chromatography (1:5, EtOAc/hexanes) to give 15e (1.81 g, 86% for 2 steps) as a yellow solid.

R_f 0.60 (1:3, EtOAc/hexanes); ¹H NMR (600 MHz, CDCl₃) δ: 7.99 (s, 1H), 7.91 – 7.84 (m, 3H), 7.61 (d, J = 8.5 Hz, 1H), 7.55 – 7.47 (m, 2H), 7.40 (d, J = 7.3 Hz, 2H), 7.31 (dt, J = 26.5, 7.3 Hz, 3H), 5.75 (s, 1H, Ph-CH-), 4.98 (d, J = 11.0 Hz, 1H, Ph-CH₂-), 4.95 (d, J = 3.6 Hz, 1H, H-1α), 4.84 (d, J = 11.0 Hz, Ph-CH₂-), 4.36 (dd, J = 10.3, 4.9 Hz, 1H), 4.15 (t, J = 9.5 Hz, 1H), 4.00 (td, J = 10.0, 4.9 Hz, 1H), 3.90 (ddd, J = 10.6, 7.1, 3.6 Hz, 1H), 3.80 (dt, J = 18.7, 9.8 Hz, 2H), 3.71 – 3.64 (m, 1H), 3.55 (ddd, J = 13.0, 7.1, 3.5 Hz, 1H), 3.44 (dt, J = 7.6, 4.6 Hz, 2H). ¹³C NMR (150 MHz, CDCl₃) δ: 137.78, 134.50, 133.64, 132.90, 128.43, 128.40, 128.20, 128.16, 127.89, 127.72, 126.50, 126.25, 125.52, 123.64, 101.66, 98.80, 82.79, 75.96, 75.09, 68.92, 67.16, 63.13, 62.86, 50.60. HR ESI-TOF MS (m/z): calcd for C₂₆H₂₆N₆O₅Na [M + Na]⁺, 525.1857; found, 525.1864.

2-Azidoethyl 2-azido-2-deoxy-3-O-benzyl-6-O-(2-naphthylmethyl)-α-D-glucopyranoside (15):

To a solution of 15e (1.5 g, 2.99 mmol) in THF (30 mL) was added NaBH₃CN (1.72 g, 29.88 mmol) at 0 °C. A 2N HCl/Et₂O solution was added dropwise to maintain the reaction mixture at pH 1. After the reaction mixture had been stirring at room temperature for 2 h, it was diluted with ethyl acetate, washed with aqueous NaHCO₃ and brine, dried over anhydrous Na₂SO₄, and concentrated. The resulting residue was subjected to silica gel column chromatography (1:5, EtOAc/hexanes) to give 15 (1.17 g, 78%) as syrup.

R_f 0.50 (1:3, EtOAc/hexanes); ¹H NMR (600 MHz, CDCl₃) δ: 7.82 (dd, J = 9.0, 6.0 Hz, 3H), 7.76 (s, 1H), 7.50 – 7.43 (m, 3H), 7.38 (ddd, J = 13.0, 8.0, 3.9 Hz, 4H), 7.33 – 7.28 (m, 1H), 4.94 (d, J = 3.5 Hz, 1H, H-1α), 4.92 (d, J = 11.2 Hz, 1H, Ph-CH₂-), 4.77 (dd, J = 14.2, 11.8 Hz, 2H, Ph-CH₂-), 4.70 (d, J = 12.2 Hz, 1H, Ph-CH₂-p), 3.90 – 3.81 (m, 3H), 3.79 – 3.69 (m, 3H), 3.65 (ddd, J = 10.6, 5.8, 3.5 Hz, 1H), 3.54 (ddd, J = 13.2, 7.5, 3.5 Hz, 1H), 3.41 – 3.32 (m, 2H), 2.43 (s, 1H, -OH). ¹³C NMR (150 MHz, CDCl₃) δ: 137.99, 135.14, 133.19, 133.01, 128.65, 128.29, 128.16, 128.08, 127.85, 127.69, 126.60, 126.18, 125.98, 125.60, 98.19, 79.60, 75.09, 73.79, 71.80, 70.57, 69.49, 66.99, 62.69, 50.59. HR ESI-TOF MS (m/z): calcd for C₂₆H₂₈N₆O₅Na [M + Na]⁺, 527.2013; found, 527.2020.

2-Azidoethyl 2-acetamido-2-deoxy-3-O-benzyl-4,6-O-(2-naphthylmethylbenzylidene)-α-D-glucopyranoside (16c):

To a solution of 2-azidoehyl 2-acetamido-2-deoxy-α-glucopyranoside⁶⁰ 16a (1.04 g, 3.66 mmol) in CH₃CN (30 mL) were added 2-naphtylaldehyde dimethyl acetal (1.11 g, 5.48 mmol) and p-TSA (69.5 mg, 0.366 mmol). After the reaction mixture had been stirring overnight at 50 °C, it was quenched with Et₃N and concentrated. The residue was purified by silica gel column chromatography (1:10, CH₃OH/CH₂Cl₂) to afford benzylidene 16b as white solid. The product 16b was then dissolved in DMF (30 mL), and NaN₃ (1.19 g, 18.28 mmol) was added. After the reaction mixture had been stirring overnight at 80 °C, it was diluted with CH₂Cl₂, washed with water and brine, dried over anhydrous Na₂SO₄, and concentrated. The resulting intermediate was then dissolved in 30 mL anhydrous DMF, and NaH (175.7 mg, 7.32 mmol) was added at 0 °C under a N₂ atmosphere. After the reaction mixture had been stirring for15 min, benzyl bromide (651.9 μL, 5.49 mmol) was added. The resulting mixture was kept at room temperature for 1 h, quenched with saturated aq. NaCl solution and diluted with ethyl acetate. The organic layer, after washing with water and brine, was dried with Na2SO4 and concentrated in vacuo. The residue was recrystallized in EtOAc/hexanes to give 16c (1.19 g, 63% for 2 steps) as a white solid.

R_f 0.50 (EtOAc); ¹H NMR (600 MHz, CDCl₃) δ: 7.98 (s, 1H), 7.91 – 7.77 (m, 3H), 7.60 (dd, J = 8.5, 1.4 Hz, 1H), 7.54 – 7.43 (m, 2H), 7.38 – 7.19 (m, 5H), 5.74 (s, 1H, Ph-CH-), 5.40 (d, J = 9.1 Hz, 1H, -NHAc), 4.91 (dd, J = 15.1, 8.0 Hz, 2H), 4.65 (d, J = 12.2 Hz, 1H), 4.32 (dt, J = 13.8, 4.4 Hz, 2H), 3.96 – 3.80 (m, 4H), 3.80 – 3.70 (m, 1H), 3.59 (ddd, J = 10.8, 8.2, 2.7 Hz, 1H), 3.48 (ddd, J = 13.3, 8.2, 2.8 Hz, 1H), 3.26 (ddd, J = 13.4, 5.1, 2.7 Hz, 1H), 1.90 (s, 3H, -Ac). ¹³C NMR (150 MHz, CDCl₃) δ: 170.03, 138.29, 134.60, 133.59, 132.88, 128.41, 128.38, 128.11, 128.00, 127.75, 127.68, 126.44, 126.20, 125.46, 123.63, 101.49, 98.44, 82.69, 75.55, 74.12, 68.96, 67.37, 63.18, 52.31, 50.43, 23.26. HR ESI-TOF MS (m/z): calcd for C₂₈H₃₀N₄O₆Na [M + Na]⁺, 541.2058; found, 541.2055.

2-Azidoethyl 2-acetamido-2-deoxy-3-O-benzyl-6-O-(2-naphthylmethyl)-α-D-glucopyranoside (16):

To a solution of 16c (580 mg, 1.12 mmol) in THF (15 mL) was added NaBH₃CN (705.4 mg, 11.2 mmol). A 2N HCl/Et₂O solution was added dropwise to keep the reaction mixture at pH 1. After the mixture had been stirring at room temperature for 5 h, it was diluted with ethyl acetate, washed with aqueous NaHCO₃ and brine, dried over anhydrous Na₂SO₄, and concentrated. The residue was subjected to silica gel column chromatography (2:1, EtOAc/hexanes) to give 16 (472 mg, 81%) as syrup.

R_f 0.50 (4:1, EtOAc/hexanes); ¹H NMR (600 MHz, CDCl₃) δ: 7.85 – 7.78 (m, 3H), 7.76 (s, 1H), 7.50 – 7.41 (m, 3H), 7.36 – 7.22 (m, 5H), 5.50 (d, J = 9.3 Hz, 1H, -NHAc), 4.84 (d, J = 3.6 Hz, 1H, H-1α), 4.79 – 4.64 (m, 4H), 4.27 (ddd, J = 10.5, 9.4, 3.6 Hz, 1H), 3.86 (ddd, J = 10.7, 5.3, 2.9 Hz, 1H), 3.82 – 3.69 (m, 4H), 3.64 – 3.51 (m, 2H), 3.42 (ddd, J = 13.4, 8.0, 2.9 Hz, 1H), 3.21 (ddd, J = 13.4, 5.3, 2.7 Hz, 1H), 3.03 (s, 1H, -OH), 1.86 (s, 3H, -Ac). ¹³C NMR (150 MHz, CDCl₃) δ: 170.10, 138.35, 135.31, 133.20, 132.98, 128.58, 128.24, 128.12, 127.90, 127.87, 127.69, 126.49, 126.15, 125.93, 125.61, 97.97, 79.58, 74.01, 73.77, 71.73, 70.84, 70.04, 67.17, 51.77, 50.45, 23.29. HR ESI-TOF MS (m/z): calcd for C₂₈H₃₂N₄O₆Na [M + Na]⁺, 543.2214; found, 543.2205.

2-Chloroethyl 2-azido-2-deoxy-4,6-O-(p-methoxybenzylidene)-α-D-glucopyranoside (17a):

To a solution of 15b (4.0 g, 14.9 mmol) in CH₃CN (100 mL) were added para-anisaldehyde dimethyl acetal (3.8 mL, 22.35 mmol) and p-TSA (283 mg, 1.49 mmol). After the reaction mixture had been stirring overnight at room temperature, it was quenched with Et₃N and concentrated. The resulting residue was subjected to silica gel column chromatography (1:4, EtOAc/hexanes) to give 17a (4.43 g, 77%).

R_f 0.40 (1:2, EtOAc/hexanes); ¹H NMR (600 MHz, CDCl₃) δ: 7.40 (d, J = 8.7 Hz, 2H), 6.89 (d, J = 8.7 Hz, 2H), 5.47 (s, 1H, Ph-CH-), 4.93 (d, J = 3.7 Hz, 1H, H-1α), 4.28 – 4.15 (m, 2H), 3.97 – 3.87 (m, 2H), 3.84 – 3.75 (m, 4H), 3.72 – 3.65 (m, 3H), 3.47 (t, J = 9.4 Hz, 1H), 3.25 (dd, J = 10.1, 3.7 Hz, 1H), 2.89 (s, 1H, -OH). ¹³C NMR (150 MHz, CDCl₃) δ: 160.31, 129.29, 127.60, 113.74, 102.02, 98.87, 81.63, 68.75, 68.62, 62.88, 62.73, 55.31, 42.35. HR ESI-TOF MS (m/z): calcd for C₁₆H₂₀ClN₃O₆Na [M + Na]⁺, 408.0933; found, 408.0925.

2- Azidoethyl 2-azido-2-deoxy-4,6-O-(p-methoxybenzylidene)-α-D-glucopyranoside (17b):

To a solution of 17a (4.3 g, 11.13 mmol) in DMF (100 mL) was added NaN₃ (3.62 g, 55.63 mmol). After the reaction mixture had been stirring overnight at 80 °C, it was diluted with CH₂Cl₂, washed with water and brine, dried over anhydrous Na₂SO₄, and concentrated. The residue was subjected to silica gel column chromatography (1:3, EtOAc/hexanes) to give 17b (4.11 g, 94%).

R_f 0.40 (1:2, EtOAc/hexanes); ¹H NMR (600 MHz, CDCl₃) δ: 7.40 (d, J = 8.8 Hz, 2H), 6.89 (d, J = 8.8 Hz, 2H), 5.47 (s, 1H, Ph-CH-), 4.91 (d, J = 3.6 Hz, 1H, H-1α), 4.25 (dd, J = 10.3, 4.9 Hz, 1H), 4.23 – 4.16 (m, 1H), 3.87 (tdd, J = 9.7, 6.2, 3.4 Hz, 2H), 3.79 (s, 3H, -OCH₃), 3.70 (t, J = 10.3 Hz, 1H), 3.64 (ddd, J = 10.5, 6.1, 3.5 Hz, 1H), 3.53 (ddd, J = 13.3, 7.2, 3.5 Hz, 1H), 3.47 (t, J = 9.3 Hz, 1H), 3.40 (ddd, J = 13.3, 6.0, 3.5 Hz, 1H), 3.27 (dd, J = 10.1, 3.7 Hz, 1H), 2.91 (s, 1H, -OH). ¹³C NMR (150 MHz, CDCl₃) δ: 160.31, 129.30, 127.62, 113.74, 102.04, 98.77, 81.64, 68.66, 68.61, 67.22, 62.85, 62.75, 55.30, 50.60. HR ESI-TOF MS (m/z): calcd for C₁₆H₂₀N₆O₆Na [M + Na]⁺, 415.1337; found, 415.1338.

2- Azidoethyl 2-azido-2-deoxy-3-O-(2-naphthylmethyl)-4,6-O-(p-methoxybenzylidene)-α-D-glucopyranoside (17c):

To a solution of 17b (3.4 g, 8.65 mmol) in 80 mL of anhydrous DMF was added NaH (415.27 mg, 17.3 mmol) at 0 °C under a N₂ atmosphere. After the mixture had been stirring for 15 min, NapBr (2.87 g, 12.97mmol) was added. After the resulting mixture had been stirring at room temperature for 1 h, it was quenched with saturated aq. NaCl solution and diluted with ethyl acetate. The organic layer, after washing with water and brine, was dried with Na2SO4 and concentrated in vacuo. The residue was subjected to silica gel column chromatography (1:4, EtOAc/hexanes) to give 17c (3.92 g, 85%).

R_f 0.40 (1:4, EtOAc/hexanes); ¹H NMR (600 MHz, CDCl₃) δ: 7.86 – 7.73 (m, 4H), 7.51 (dd, J = 8.5, 1.5 Hz, 1H), 7.49 – 7.36 (m, 4H), 6.95 – 6.85 (m, 2H), 5.55 (s, 1H, Ph-CH-), 5.09 (d, J = 11.3 Hz, 1H, Ph-CH₂-), 4.97 (d, J = 11.3 Hz, 1H, Ph-CH₂-), 4.93 (d, J = 3.6 Hz, 1H, H-1α), 4.28 (dd, J = 10.3, 4.9 Hz, 1H), 4.20 – 4.11 (m, 1H), 3.94 (td, J = 10.0, 4.9 Hz, 1H), 3.87 (ddd, J = 10.7, 7.1, 3.6 Hz, 1H), 3.82 (s, 3H, -OCH₃), 3.73 (dt, J = 17.4, 9.9 Hz, 2H), 3.65 (ddd, J = 10.6, 6.1, 3.6 Hz, 1H), 3.53 (ddd, J = 13.2, 7.1, 3.6 Hz, 1H), 3.47 – 3.36 (m, 2H). ¹³C NMR (150 MHz, CDCl₃) δ: 160.12, 135.34, 133.28, 133.07, 129.63, 128.16, 128.01, 127.64, 127.38, 127.01, 126.14, 125.95, 125.86, 113.64, 101.52, 98.77, 82.59, 76.04, 75.07, 68.78, 67.12, 63.11, 62.94, 55.30, 50.57. HR ESI-TOF MS (m/z): calcd for C₂₇H₂₈N₆O₆Na [M + Na]⁺, 555.1963; found, 555.1967.

This research was supported by the National Institutes of Health (R01 GM098285) and Wayne State University. The authors acknowledge the Wayne State Proteomic Core and Chemistry Lumigen Instrument Center for assistance. The authors would also like to thank Professor Jian Liu at the University of North Carolina at Chapel Hill for providing HS oligosaccharides 69 and 70.