Solid-Phase Synthesis of α-Glucosamine Sulfoforms With Fragmentation Analysis by Tandem Mass Spectrometry

Abstract

Sulfated epitopes of α-glucosamine (GlcN sulfoforms) were prepared by solid-phase synthesis as models of internal glucosamines within heparan sulfate. An orthogonally protected 2’-hydroxyethyl GlcN derivative was immobilized on a trityl resin support and subjected to regioselective deprotection and sulfonation conditions, which were optimized with the aid of on-resin infrared or Raman analysis. The sulfoforms were cleaved from the resin under mild Lewis acid conditions without affecting the O- or N-sulfate groups, and purified by reverse-phase reverse-phase high performance liquid chromatography (HPLC). The α-GlcN sulfoforms and their 4-O-benzyl ethers were examined by electrospray ionization tandem mass spectrometry (ESI-MS/MS), with product ion spectra produced by collision-induced dissociation (CID). ESI-MS/MS revealed significant differences in parent ion stabilities and fragmentation rates as a function of sulfate position. Ion fragmentation by CID resulted in characteristic mass losses with strong correlation to the positions of both free hydroxyl groups and sulfate ions. Most of these fragmentation patterns are consonant with elimination pathways, and suggest possible strategies for elucidating the structures of glucosamine-derived sulfoforms with identical m/z ratios. In particular, fragmentation analysis can easily distinguish GlcN sulfoforms bearing the relatively rare 3-O-sulfate from isomers with the more common 6-O-sulfate.

Introduction

The biological functions of cell-surface carbohydrates and related glycoconjugates are frequently regulated by postsynthetic modifications, particularly via their derivatization as sulfate esters.^1,2 In many cases, protein—carbohydrate recognition is determined by the presentation of specific sulfate epitopes, more recently referred to as sulfoforms.^2,3,4,5 A famous example of sulfoform diversity is provided by the glycosaminoglycan heparan sulfate (HS), a linear polysaccharide comprised of α-d-glucosamine (GlcN) linked in alternating 1→4 fashion to either β-d-glucuronic acid (GlcA) or α-l-iduronic acid (IdoA).⁶ At least 21 disaccharide sulfoforms have been identified within HS, assembled from 6 out of 8 possible sulfated glucosamine derivatives (GlcNAc, GlcNAc-6S, GlcNS, GlcNS-3S, GlcNS-6S, and GlcNS-3S,6S) and 4 different uronic acid derivatives (GlcA, GlcA-2S, IdoA, and IdoA-2S);^7,8 another 3 sulfoforms containing a free amine (GlcNH₃⁺, GlcNH₃⁺-3S, GlcNH₃⁺-3S,6S) have also been observed.⁹ These are already sufficient to produce an enormous number of low molecular-weight HS fragments: for example, HS octasaccharides generated randomly from naturally occurring disaccharide epitopes can result in well over 10⁵ possible sulfoforms. Even greater structural diversity can be attained by the incorporation of unnatural (or yet to be identified) sulfoforms.

HS and other sulfated glycosaminoglycans have attracted great interest because of their important and potentially therapeutic roles in vascular diseases, inflammation, viral infections, and cancer.^6,10,11,12 However only a handful of high-affinity HS ligands have been characterized due to the challenges of purification and structural determination, ^{13, 14} presenting a serious bottleneck for target-oriented synthetic efforts. The structural elucidation of HS sulfoforms with specific biological activities may be particularly challenged by their low natural abundance and their temporal or transient expression on cell-surface proteoglycans. For example, 3-O-sulfate esters are generated during a late stage of HS modification and considered to be relatively rare, yet are found in high-affinity ligands for extracellular proteins such as antithrombin and the herpes simplex virus glycoprotein gD.^15,16 Several recent studies have shown that the upregulation of glucosamine 3-O-sulfotransferases (3-OSTs) are associated with fetal and neural development^8,17 and also with several forms of human cancer.¹⁸ The presence of 3-OSTs implies that the biosynthesis of HS ligands containing GlcN-3S are critical in many important biological pathways; nevertheless, the isolation and characterization of such ligands remain elusive.

Mass spectrometry (MS) has become a leading method for characterizing sulfated oligosaccharides, many of which are available only in trace quantities.¹⁹ Three strategies have emerged in recent years. In one approach, molecular ion signals are generated by matrix-assisted laser desorption/ionization and correlated with fragments produced by enzymatic digestion.^{14b, 20 , 21} A second one is based on electrospray ionization tandem MS (ESI-MS/MS) for characterizing fragments generated in situ by collision-induced dissociation (CID),²² with the expectation that isomers having identical m/z ratios can be distinguished by unique ion peaks or by signature patterns of mass fragments.^{19,23,24,25,26} A third strategy employs MS/MS and electron detachment dissociation to produce signature mass fragment peaks, which are likely generated via radical processes and can also be used to differentiate isomeric sulfoforms.²⁷ These approaches are complementary and may even be performed in sequence, as the latter two are applicable toward the compositional analysis of mono- and disaccharide fragments generated from longer oligosaccharide sequences. Regardless of the method, the ionization conditions for producing mass spectra must be optimized for a given molecular structure. Organic salts such as sulfated carbohydrates are particularly sensitive to sampling conditions, as their stabilities and volatilities may be affected by counterions as well as by the parent ion structure.²⁸

ESI-MS/MS with CID can be applied toward the quantitative analysis of sulfoforms within HS. Saad et al. demonstrated that the population distribution of isomeric disaccharides could be determined to within 5% by a ratiometric analysis of fragment ion peaks, and validated their method by the compositional profiling of several HS oligosaccharides.²⁴ However, the sample set was limited to commercially available HS disaccharides, and did not include rare sulfoforms containing GlcNS-3S or GlcNS-3S,6S. Subsequent studies on sulfated monosaccharides bearing a 3-O-sulfate ester indicate that these compounds may produce MS fragmentation patterns and abundance ratios distinct from those of isomeric sulfoforms.^26,29 The growing significance of 3-O-sulfate esters in biological signaling supports the value of including such derivatives in the compositional profiling of HS oligosaccharides.

In this article we present the comprehensive synthesis and ESI-MS/MS analysis of sulfoforms based on glucosamine, a primary source of structural diversity within HS (Figure 1). GlcN sulfoforms are also found in mucin-like glycoproteins, and have been shown to mediate binding to the cell adhesion protein L-selectin.³⁰ We elected to use a solid-phase approach to generate α-GlcNAc 1 and sulfoforms 2−8 with up to 3 sulfate esters at the C2, C3, and C6 positions, in overall yields ranging from 35−88%. Solid-phase and polymeric supports have been applied toward the linear synthesis of protected oligosaccharides,³¹ but there are surprisingly few reports involving the multistep deprotection of oligosaccharides,³² and no verifiable demonstrations involving sulfated carbohydrate derivatives.³³ Here we examine some practical issues enabling the solid-phase synthesis of GlcN sulfoforms from a common precursor, and provide a systematic analysis of molecular ion stability and fragmentation behavior as a function of sulfonation pattern. The synthetic GlcN sulfoforms reveal important differences between positional isomers, and offer new insights for structural analysis by MS/MS techniques.

FIGURE 1.

2’-Hydroxyethyl α-N-acetylglucosaminoside 1 and GlcN sulfoforms 2−8 (a: R=Bn; b: R=H).

Results and Discussion

Synthesis

Sulfoform diversity can be generated from combinations of carbohydrate building blocks having protecting groups at predesignated sulfate sites, or by developing synthetic precursors with orthogonal protecting group systems.^3,34,35,36 Both approaches have their merits, but the latter offers the benefit of using a universal intermediate to generate an entire set of sulfoforms, unnatural as well as natural. The challenges of developing a unified sulfation strategy increase geometrically with the number of possible sulfate positions, as each additional protecting group reduces the chemical space for further orthogonality. We have recently addressed the issue of generating diverse sulfoforms from a common intermediate by introducing a heparan disaccharide (GlcN(α1→4)GlcA) with 6 orthogonal protecting groups.³⁵ A high density of sulfate groups also raises some concerns for the handling of highly charged intermediates in a multistep synthetic sequence. Here we address this important practical issue by using a solid-phase approach to produce sulfated carbohydrates in deprotected forms.

α-Thioethyl 2-azidoglucoside 9 was prepared in three steps and 57% overall yield from glucosamine hydrochloride by Cu-mediated diazo transfer,³⁷ peracetylation, and TMSOTf-mediated glycosylation using TMSSEt,³⁸ added in two portions (Scheme 1). Triacetate 9 was converted into 4,6-benzylidene acetal 10 in 85% yield over three steps, followed by reductive cleavage to the 4-O-benzyl ether using BH₃/Bu₂BOTf and 2,6-lutidine as a proton scavenger.³⁹ The C6 alcohol was protected as a t-butyldiphenylsilyl (TBDPS) ether to afford compound 11 in 65% overall yield from 10. A glycol linker was installed by NIS/TfOH-promoted coupling of α-thioethyl glycoside 11 with 2’-hydroxyethyl 2-bromoisobutyrate, followed by treatment with DBU to yield a 6:1 α:β mixture of glycol-linked GlcN derivatives.⁴⁰ Careful separation by silica gel chromatography afforded 2’-hydroxyethyl α-GlcN derivative 12 in 66% isolated yield from 11. It is worth mentioning that the coupling of β-thioglycosides with monoprotected glycol linkers generally favored the formation of β-O-glycosides,⁴⁰ contrary to expectations of glycosyl donors with non-participatory C2 groups.^41,42,43

SCHEME 1.

Synthesis and Loading of 2’-Hydroxyethyl α-GlcN Derivatives Onto a Trityl-PS Resin ^a

^a Select abbreviations: DBU = 1,8-diazabicyclo[5.4.0]undec-7-ene; HE-BriB = 2’-hydroxyethyl 2-bromoisobutyrate; TBDPS = t-butyldiphenylsilyl.

Orthogonally protected α-GlcN 12 was tethered onto a trityl-PS resin in dry pyridine at 65 °C, assisted by 4-dimethylaminopyridine (DMAP) to produce α-GlcN-loaded resin 13. A loading ratio of 0.8−0.9 mmol/g was attained using stoichiometric amounts of 12; a higher ratio of 1.3 mmol/g could be obtained by using 1.5 equivalent of monosaccharide relative to the maximum loading density. The loading ratios of 13 were determined by the amount of recovered 12 after cleavage from the resin.

Orthogonal cleavage conditions for the TBDPS, Ac, and N₃ groups were first optimized for homogeneous (solution) conditions, in accordance with our earlier studies of an orthogonally protected heparan disaccharide.³⁵ These deprotection conditions could also be employed in solid-phase reactions, but the order of functional group transformations and the responsivity of the supporting PS resin had some influence on reaction efficiency. In some cases, reaction speed was reduced by changes in local chemical environment; in others, a loss of chemoselectivity was observed. For example, deprotection of the TBDPS ether with unneutralized TBAF (pH ≥ 10) did not affect the C3 acetate in azidoglucose 13, but performing the same reaction on the corresponding N-acetyl derivative resulted in partial C3 deacetylation. We thus employed a dual set of deprotection conditions, one optimized for speed but which could be slightly aggressive toward other functionalities, and a slower but milder condition which could be used without any concern for chemoselectivity (Table 1).

TABLE 1.

Deprotection Conditions and Characteristic Vibrational Bands of Protecting Groups

group	deprotection conditions	IR, Raman bands (cm−1) a
N3 (to -NH2)	(i) Bu3P (5 eq), CH2Cl2, 5 h; (ii) 95% aq DMF, 10 h, rt b	IR: 2107 (−)
N3 (to -NHAc)	as above, then 1:1 pyridine:Ac2O, 1 day, rt	IR: 1687 (+)
6-O-TBDPS c	I: 0.9 M TBAF in THF (pH ≥10), 1 day, rt	Raman: 1407, 1462, 1568 (−)
	II: 0.9 M TBAF in THF (pH 7) 1−2 days, rt
3-O-Ac	NaOMe or Bu4NOH (5 eq), 1:1 MeOH:CH2Cl2, 5 h, rt	IR: 1753 (−)

^aVibrational bands used to monitor the disappearance (−) or appearance (+) of functional groups.

^bRef. ³⁵.

^cCondition I preferred for efficient cleavage; condition II preferred for chemoselectivity.

The efficacy of each deprotection step could be monitored on-resin by Fourier transform infrared (IR) or Raman spectroscopy.³¹ Each protecting group exhibited one or more characteristic vibrational bands, whose presence could be correlated with the endpoint of the deprotection reaction (Table 1). Typical IR and Raman spectra following a deprotection reaction starting from immobilized α-GlcN derivative 13 are shown in Figure 2.⁴⁴ It is interesting to note that the IR frequency corresponding to the amide carbonyl stretch of NHAc appears at 1687 cm⁻¹ while immobilized on resin, but is restored to a more typical value of 1652 cm⁻¹ after cleavage from the resin support.

FIGURE 2.

Raman and IR spectra of α-GlcN derivatives on trityl-PS resin. (a,b) IR and Raman spectra of fully protected α-GlcN (13).⁴⁴ (c) IR spectrum following N₃ reduction to free amine. (d) Raman spectrum following 6-O-TBDPS deprotection. (e) IR spectrum following conversion of N₃ to −NHAc. (f) IR spectrum following 3-O-Ac deprotection.

Partially deprotected α-GlcN derivatives were directly converted on-resin into sulfate esters using SO₃·pyridine, then treated with a neutral Bu₄NHSO₄ solution (30−50 mM) in 1:1 MeOH:DMF to produce the Bu₄N salt. This procedure minimized any negative effects of the sulfate esters on the swelling properties of the resin, which is essential for maintaining access to the reaction medium. Mono-O-sulfates were readily formed at room temperature in DMF, whereas N-sulfate formation required basic conditions and was best achieved using 5:1 pyridine:Et₃N.⁴⁵ Multiple sulfates could be installed in a single step in some instances but more often required prolonged reaction times at 55 °C using pyridine:Et₃N, or an intermediate ion-exchange step with Bu₄NHSO₄ to restore resin swelling followed by a second round of SO₃·pyridine to achieve saturation. We note that while direct sulfonation is suitably efficient, one might also consider the use of protected sulfate diesters for installing multiple sulfate groups while maintaining charge neutrality.⁴⁶ However, this approach introduces an additional deprotection step, which imposes another condition for orthogonality.

The 2’-hydroxyethyl α-GlcN derivatives were cleaved from the resin as their 4-O-benzyl ethers for characterization and to determine overall yield (Table 2). We first established the efficiency of the loading and deprotection steps by cleaving the unsulfated glucosamine using 3% CF₃COOH in CH₂Cl₂, and obtained α-GlcNAc 1a in 88% yield over 6 operations after purification by reverse-phase HPLC. We note that this acidic condition did not cause the decomposition of α-GlcNAc-6S 2a, α-GlcNAc-3S 3a, or α-GlcNAc-3S,6S 5a, which were recovered intact by adding acetic acid to the filtrate prior to concentration under reduced pressure. However, N-sulfated α-GlcN derivatives were more labile to acid and typically experienced partial or complete desulfation during recovery.

TABLE 2.

Generation of 4-O-Benzyl GlcN 1a and Sulfoforms 2a-8a (Bu₄N salts) From 13 ^a

product	yield, no. operations	react cond
	88%, 6 ops	a, b, c, d, e, f
	58%, 8 ops	a, b, c, g, h, i, j, k
	53%, 8 ops	a, b, c, e, h, i, d, k
	63%, 7 ops	a, b, l, i, d, j, k
	42%, 10 ops	a, b, c, d, e, m, i, m, i, k
	46%, 7 ops	e, a, b, m, i, d, k
	35%, 9 ops	d, a, b, h, i, l, i, j, k
	38%, 9 ops	e, a, b, d, m, i, m, i, k

^aReagents and conditions: (a) Bu₃P, CH₂Cl₂, rt; (b) 95% aq DMF, rt; (c) Ac₂O, pyridine, rt; (d) TBAF (pH ≥ 10), THF, rt; (e) NaOMe, MeOH/CH₂Cl₂, rt; (f) TFA, CH₂O₂, rt; (g) TBAF (pH 7), THF, rt; (h) SO₃-py, DMF, rt; (i) BU₄NHSO₄ (pH 7), DMF/MeOH, rt; (j) BU₄NOH, MeOH/CH₂Cl₂, rt; (k) BF₃-Et₂O, CH₂Cl₂, −20 °C; (l) SO₃·py, pyridine/Et₃N, rt; (m) SO₃·py, pyridine/Et₃N, 55 °C.

Prior experience suggested that Lewis acid conditions might be used to cleave the GlcN sulfoforms from the resin without sulfate ester decomposition.³⁵ After evaluating several possibilities we were pleased to find that both O- and N-sulfates were stable in the presence of 0.3 M BF₃·Et₂O at −20 °C. This condition was also efficient at cleaving the glycol linker from the trityl-PS resin, and proved to be of general utility for obtaining 4-O-benzyl GlcN sulfoforms 2a–8a as their Bu₄N salts (Table 2). The C4 benzyl ether facilitated HPLC purification by reducing the sulfoforms’ polarity and provided a convenient UV-active chromophore for detection during elution. The benzyl ethers were then cleaved by catalytic hydrogenation, followed by treatment with an ion-exchange resin in aqueous methanol to produce the fully deprotected GlcN 1b and sulfoforms 2b–8b as Na salts (Scheme 2). The efficient sorption of Bu₄N ions is noteworthy as it can be used to purify polar organic compounds at the final stage of synthesis,⁴⁷ if care is taken to remove residual polyelectrolyte from the resin beforehand.

SCHEME 2.

Synthesis of 2’-Hydroxyethyl α-GlcN 1b and Sulfoforms 2b–8b ^a

^a R₁,R₂ = H or SO₃⁻; R₃ = Ac or SO₃⁻

Fragmentation analysis

ESI-MS/MS analyses were performed in negative ion mode on 2’-hydroxyethyl α-GlcN sulfoforms with 4-O-benzyl ethers (2a–8a) as well as on fully deprotected sulfoforms (2b–8b), with each set respectively serving as models of internal and terminal α-GlcN residues. Preliminary analyses were performed on sulfoforms 2a–8a using an ion trap mass analyzer with the CID energy set to 40% of the resonance excitation rf voltage (5 V_pp), in order to identify differences in mass losses or fragment patterns as a function of the parent ion (Table 3). Among monosulfate derivatives 2a–4a, the loss of SO₃ [M–H–80]⁻ was responsible for the major fragment produced from GlcNAc-3S, but was a minor fragmentation pathway for GlcNS or GlcNAc-6S. Among monoprotonated disulfate derivatives 5a–7a, the loss of SO₃ was produced by all sulfoforms but the spectrum of GlcNS-6S also featured a significant loss of glycol [M–H–62; B ion]⁻. The parent ion generated from diprotonated trisulfate 8a [M–H]⁻ was too weak for MS/MS analysis under these conditions, although standard ESI-MS revealed a substantial loss of one or two SO₃ groups. These losses are well known to be produced from sulfonic acids, and indicate the importance of performing electrospray under basic conditions.²¹ Overall, the results suggest that sulfoforms bearing a 3-O-sulfate are significantly more prone to fragmentation, relative to their positional isomers.

TABLE 3.

ESI-MS² Fragments of 4-O-Benzyl GlcN Sulfoforms 2a-8a

compd a	M (amu) b	MS2 fragment mass loss (% intensity) c
2a (GlcNAc-6S)	435	M-H (12)
		M-H-62 (100)
		M-H-80 (3)
3a (GlcNAc-3S)	435	M-H (100)
		M-H-62 (15)
		M-H-80 (67)
4a (GlcNS)	393	M-H (100)
		M-H-62 (2)
		M-H-80 (<1)
		M-H-108 (5)
5a (GlcNAc-3S,6S)	515	M-H (5)
		M-H-62 (1.4)
		M-H-80 (100)
6a (GlcNS-3S)	473	M-H-62 (<1)
		M-H-80 (100)
7a (GlcNS-6S)	473	M-H-62 (14)
		M-H-80 (100)
8a (GlcNS-3S,6S) d	553	M-H (3)
		M-H-80 (36)
		M-H-160 (11)
		M-3H+Na, Bu4N (100)

^aBu₄N counterions.

^bMass of neutral (acidic) form.

^cNegative ions.

^dESI-MS data; no MS² was obtained from the parent ion [M-H]⁻.

Several 4-O-benzyl GlcN sulfoforms were also examined by ESI-MS/MS using a dueling-ion trap at lower CID voltages (100 mV_pp) to measure the rates of parent ion dissociation and fragment ion formation (Table 4). First-order dissociation rate constants (k_P) were obtained from the exponential decay of normalized parent ion density P during activated collision:

$$k_{p}t=-\ln \left[\frac{P\left(t\right)}{\sum F+P\left(t\right)}\right]$$ 1

where ΣF is the sum of the normalized fragment peak intensities. A typical semilog plot of parent ion dissociation versus collision time t is shown in Figure 3. Differences in dissociation rates among monosulfated GlcN derivatives were modest: the k_P values of O-sulfated GlcNAc isomers 2a and 3a were greater than that of GlcNS 4a by 1.5 to 2 times, respectively. On the other hand, a threefold rate difference was observed between isomeric disulfates 6a and 7a (GlcNS-3S and GlcNS-6S, respectively). The faster dissociation rate of 6a can be attributed to the destabilizing effect of electrostatic repulsion between neighboring N- and O-sulfates at C2 and C3.

TABLE 4.

Dissociation Rate Constants of 4-O-Benzyl GlcN Sulfoforms

compd a	kP, s−1b
2a (GlcNAc-6S) c	151±12
3a (GlcNAc-3S) c	189±11
4a (GlcNS) d	100±9
6a (GlcNS-3S) e	741±24
7a (GlcNS-6S) e	239±11

^aBU₄N counterions.

^b95% confidence limits. CID conditions:

^c100 mV_pp, 49.016 kHz

^d100 mV_pp, 54.273 kHz

^e100 mV_pp, 90.806 kHz. Bath gas temperature = 298 K; gas pressure = 1.4 × 10⁻⁴ Torr.

FIGURE 3.

Parent ion dissociation rate data of 6a, with each data point based on the average of 50 individual mass analysis scans. CID conditions: 100 mV_pp, 90.806 kHz; bath gas temperature = 298 K; total gas pressure = 1.4 × 10⁻⁴ Torr (uncorrected for ion gauge detector response to helium).

High-accuracy ion-trap CID experiments performed on a quadrupole/time-of-flight (QqTOF) tandem mass spectrometer generated characteristic daughter ions and fragmentation patterns from 4-O-benzyl GlcN sulfoforms under basic electrospray conditions (Figure 4 and Table 5).⁴⁸ Most fragments can be derived from their parent ions via elimination pathways and assigned according to Domon–Costello nomenclature.⁴⁹ The 3-O-sulfate ester again plays a defining role in fragment production: hydrogensulfate (HOSO₃⁻, m/z 97) is generated as the dominant fragment from GlcNAc-3S 3a, whereas the mass spectra of GlcNS-6S 2a and GlcNS 4a also feature ion peaks generated by ring fragmentation pathways leading to A and X ions. In these cases the C3 hydroxyl is likely to promote the formation of ^3,5A and ^0,2X fragments,²⁶ whereas the C2 sulfamate may contribute toward the formation of ^1,3A and ^1,5A fragments. Sulfamate (H₂NSO₃⁻, m/z 96) is also produced as a major fragment ion in the spectrum of GlcNS 4a but in lower relative abundance from N,O-disulfates 6a and 7a. The appearance of a small HOSO₃⁻ peak in the GlcNS spectrum indicates that N-to-O-sulfate migration is possible, but minor compared with other pathways. GlcNS-6S 7a produces fragments with m/z 374 (M–2H–97) and m/z 375 (M–2H–96), corresponding to the monovalent parent anion with loss of HOSO₃⁻ and H₂NSO₃⁻ respectively, whereas GlcNS-3S 6a does not produce the latter.

FIGURE 4.

ESI-MS/MS spectra with CID of 4-O-benzyl GlcN sulfoforms. (a) GlcNAc-6S (2a); (b) GlcNAc-3S (3a); (c) GlcNS (4a); (d) GlcNS-6S (7a); (e) GlcNS-3S (6a). Parent ion [M–H]⁻ for 2a–4a; [M–2H]²⁻ for 7a and 6a. See Table 5 for CID conditions.

TABLE 5.

Major Fragments of 4-O-Benzyl GlcN Sulfoforms by ESI-MS/MS With CID

compd	mass loss or ions formed (m/z)a	rel abund (%)b	cleavage patterns
2a c	SO3− (80)	4.3
	HOSO3− (97)	22.3
	3,5A (243)	8.9
	B-H2O (354)	1.8
	B (372)	44.1
3a d	SO3− (80)	11.6
	HOSO3− (97)	100
	B-H2O (354)	3.7
	B (372)	2.1
4a e	SO3− (80)	16.0
	HSO3− (81)	9.5
	H2NSO3− (96)	22.3
	HOSO3− (97)	2.9
	1,3A (137, 138)	14.5, 4.5
	0,2X (182)	14.7
	Z (284)	10.8
	B (330)	6.4
7a f	SO3− (80)	14.1
	H2NSO3− (96)	3.6
	HOSO3− (97)	24.8
	0,2X (182)	1.6
	B2- (204.5)	2.6
	3,5A (243)	3.1
	[M-2H-97]− (374)	5.0
	[M-2H-96]− (375)	2.3
6a f	SO3− (80)	37.6
	H2NSO3− (96)	6.2
	HOSO3− (97)	100.0
	Z & 1,5A (174, 176) g	5.9, 3.9
	Z & extrusion (191) g	6.2
	Z & C (221) g	5.0
	Z & extrusion (253) g	5.2
	[M-2H-97]− (374)	43.7

^aDerived from parent ion [M-H]⁻ or [M-2H]^2- as described in Figure 4.

^bRelative abundance based on parent ion peak, except 3a and 6a.

^cCID at 380 mV, 80.39 KHz (10 ms).

^dCID at 330 mV, 80.39 KHz (10 ms).

^eCID at 350 mV, 89.04 KHz (10 ms).

^fCID at 220 mV, 149.22 KHz (10 ms).

^gSee Scheme 3 for mechanistic details.

The sulfation patterns have considerable influence on the generation of neutral mass losses, i.e. glycosidic and aglyconic cleavage pathways leading to B and Z ions, respectively. B ion formation (mass loss of 62) is especially pronounced in GlcNAc-6S 2a, whereas GlcNS derivative 4a has a capacity to form Z ions (mass loss of 108). In the case of GlcNS-3S 6a, aglycone (C4) elimination may operate in conjunction with other fragmentation processes, not easily described by conventional nomenclature. Mechanistically speaking, “Z elimination” may be combined with “^1,5A fragmentation” and various eliminations to produce conjugated fragments with m/z 174 and 176 (Scheme 3a), with elimination of H₂NSO₃ and “C elimination” (loss of ethanol) to produce an α-pyrone with m/z 221 (Scheme 3b), or with loss of HSO₃⁻ and ring contraction with extrusion of HCN and loss of H₂ to produce furans with m/z 191 and 253 (Scheme 3c).

SCHEME 3.

Proposed Fragmentation Mechanisms in the ESI-MS/MS of GlcNS-3S 6a ^a

^a Domon–Costello nomenclature has been applied purely for descriptive purposes; fragmentation pathways may be acting in concert.

ESI-MS/MS of fully deprotected sulfoforms 2b–4b, 6b, and 7b using similar CID conditions also produces characteristic fragmentation patterns, but with somewhat greater complexity (Figure 5 and Table 6).⁵⁰ The C4 hydroxyl adds considerable diversity to the possible types of fragmentation pathways. It significantly alters the ring cleavage pathways for monosulfates 2b and 4b with a higher production of ^0,nAA and ^0,nX fragments, sometimes at the expense of other ring fragments such as the ^3,5A ion. The C4 hydroxyl also appears to promote the generation of SO₃⁻, most likely by serving as a hydrogen bond donor to the adjacent O-sulfate. In the case of GlcNS 4b, the relative intensity of the HOSO₃⁻ peak (16.8%) is much higher than that produced from 4a (2.9%), which suggests that hydrogen bonding can also promote N-to-O-sulfate migration to GlcN-4-O-sulfate (Scheme 4).

FIGURE 5.

ESI-MS/MS spectra with CID of fully deprotected GlcN sulfoforms. (a) GlcNAc-6S (2b); (b) GlcNAc-3S (3b); (c) GlcNS (4b); (d) GlcNS-6S (7b); (e) GlcNS-3S (6b). Parent ion [M–H]⁻ for 2b–4b; [M–2H]²⁻ for 7b and 6b.⁵¹ See Table 6 for CID conditions.

TABLE 6.

Major Fragments of Fully Deprotected GlcN Sulfoforms by ESI–MS/MS With CID

compd	mass loss or ions formed (m/z)a	rel abund (%)b	cleavage patterns
2b c	SO3− (80)	8.1
	HOSO3− (97)	83.2
	0,4A (139)	19.4
	0,3A (169)	3.5
	0,2A–H2O (181)	18.2
	0,2A (199)	9.8
	B–SO3 (202)	5.3
	B (282)	100
3bc	SO3− (80)	12.3
	HOSO3− (97)	100
	B–SO3 (202)	6.6
	B–H2O or [M– H– SO3]− (264)	6.5
	B (282)	7.4
4b d	SO3− (80)	42.4
	HSO3− (81)	34.8
	H2NSO3− (96)	100
	HOSO3− (97)	16.8
	H2CNSO3− (108)	8.5
	0,2X–C2H5OH (136)	63.6
	1,3A (137, 138)	25.1, 65.1
	B’ & 3,5X (150) e	56.4
	0,4X–SO3 (162)	6.8
	B & 3,5X (166) e	6.4
	0,2X (182)	31.9
	B’–CH2O (192) e	5.3
	B’ (222) e	5.0
	B (240)	57.9
	0,4X (242)	12.5
	[M–H–H2O]− (284)	8.3
7b f	SO3− (80)	27.4
	H2NSO3− (96)	4.4
	HOSO3− (97)	45.0
	[M–3H–156]2- (112) g	6.8
	B’ & 3,5A (137) e	13.2
	0,4A (139)	6.4
	[M–3H–96]2- (142) g	6.0
	0,4A +H2O (157)	18.9
	B2- (159.5)	100
	0,2A–H2O (181)	6.1
	0,2A (199)	4.6
	[M–2H–97]− (284)	4.1
	[M–2H–96]− (285)	1.7
6b f	SO3− (80)	17.5
	H2NSO3− (96)	37.9
	HOSO3− (97)	100.0
	B2- (159.5)	74.0
	C2- (168.5)	11.2
	[M–2H–145]− (236)	5.6
	0,4X–HSO3 (240)	10.3
	[M–2H–127]− (254)	8.5
	[M–2H–97]− (284)	17.8

^aDerived from parent ion [M–H]⁻ or [M–2H]^2- as described in Figure 5.

^bRelative abundance based on ions with highest intensity.

^cCID at 350 mV, 101.51 KHz (10 ms).

^dCID at 400 mV, 115.60 KHz (10 ms).

^eSee Scheme 5a for mechanistic details.

^fCID at 250 mV, 184.88 KHz (10 ms).

^gSee Scheme 5b for mechanistic details.

SCHEME 4.

Elimination and Migration Pathways in the ESI-MS/MS of GlcNS 4b

The 3,4-diols in 4b and 7b can participate in [1,2]-hydride shifts (semipinacol rearrangements), a mechanism previously reported for vicinal diols in carbohydrate rings.⁵² The rearrangement of B ions (B’) can lead to fragmentation by retroaldol or retro-Diels-Alder reaction mechanisms (Scheme 5a). In addition, vicinal diols and especially enediols are easily deprotonated, due to electronic stabilization of the corresponding planar anion.⁵³ This can explain the observation of additional dianion fragments from GlcNS-6S 7b, which are not observed in the spectra of the corresponding 4-O-benzyl sulfoform 7a (Scheme 5b).

SCHEME 5.

Proposed Fragmentation Pathways in the ESI-MS/MS of GlcNS 4b and GlcNS-6S 7b

Conclusions

α-GlcN sulfoforms can be synthesized by orthogonal deprotection and sulfonation of a common intermediate tethered on a solid support, enabling their systematic analysis by tandem mass spectrometry. The sulfoforms were efficiently cleaved from trityl-PS resins as 4-O-benzyl ethers using Lewis acid conditions at low temperature, with overall yields ranging from 88% (6 operations) to 35% (9 operations), and were readily converted to the fully deprotected sulfoforms by catalytic hydrogenation. ESI-MS/MS analysis with CID revealed that GlcN derivatives with a 3-O-sulfate ester eliminate hydrogensulfate ion more readily than other sulfoforms, whereas derivatives with a C3 hydroxyl are more prone to ring fragmentation. GlcNS derivatives with a 4-O-benzyl ether tend to form Z ions, whereas those with a C4 hydroxyl produce additional ^0,nA and ^0,nX fragments. The increased number of fragmentation pathways introduced by the C4 hydroxyl suggests that GlcN sulfoforms with O4 protecting groups are better suited for modeling the MS fragmentation of internal GlcN units within HS derivatives. Lastly, the unique ESI-MS/MS spectra produced by GlcN derivatives with identical m/z ratios support the use of fragmentation analysis to distinguish HS sulfoforms with 3-O-sulfate esters from those with 6-O-sulfate esters.

Experimental Section

General experimental methods

See Supporting Information.

Solid-phase generation of α-GlcN sulfoforms on trityl-PS resin

Trityl-PS resins were obtained from Novabiochem and used directly without activation. Standard workup conditions for solid-phase reactions involved (i) filtering and washing the resin (50 mg) three times alternatively with DMF (3 × 3 mL) and CH₂Cl₂ (3 × 3 mL), then with additional CH₂Cl₂ (2 × 3 mL), and (ii) drying the resin under reduced pressure in the presence of KOH pellets prior to the next operation. The dry loaded resin (50 mg) was swollen in CH₂Cl₂ (3 mL) for 5 min under an inert atmosphere, then cooled to −20 °C and treated dropwise with BF₃·Et₂O (114 μL, 0.9 mmol). The mixture was stirred for 1 h, then quenched with pyridine (3 mL) and slowly warmed to rt. The resin was filtered and washed with MeOH (4 × 3 mL), and the extracts were concentrated under reduced pressure at rt. Compound 1a and sulfoforms 2a–8a were purified by reverse-phase HPLC (Hydro-RP C18). The 4-O-benzyl group was removed using standard hydrogenation conditions in MeOH (see below); reaction mixtures were filtered then passed through an ion-exchange column (Dowex Marathon MSC, Na form) to yield fully deprotected sulfoforms 2b–8b as Na salts. These were purified by reverse-phase HPLC to remove residual polystyrenesulfonate.

Mass spectrometry

Preliminary ESI-MS/MS analyses were carried out on a mass spectrometer with an ion trap mass analyzer. Electrospray experiments were typically performed with the needle voltage at 4.0 kV and the capillary voltage at 10 V with the capillary heated to 207 °C, the background source pressure at 1.2 × 10⁻² mTorr as read by an ion gauge, and a sample flow rate of approximately 8 μL/min with N₂ as the drying gas. The sample was administered as an unbuffered aqueous solution, and scanned to 1000 amu. CID experiments were conducted using helium as the bath gas, at approximately 1 mTorr for optimal trapping efficiency. The collision energy was set to 40% of the maximum resonance excitation voltage (5 V_pp).

High-accuracy ion-trap CID experiments were performed on parent ions using a quadrupole/time-of-flight (QqTOF) tandem mass spectrometer coupled with a nanoelectronspray ionization source.⁵⁴ Nanospray capillaries were prepared from borosilicate glass (1.5 mm o.d., 0.86 mm i.d.) using a micropipette puller. The sample was administered as a 0.5 mM solution in 98% aqueous CH₃CN containing 1% NH₄OH, with parent ions isolated by the ion-trap filter in mass-resolving mode, followed by injection into a collision chamber at 5 mTorr with nitrogen as the bath gas. The ions were cooled prior to collisional activation (10 ms) followed by QqTOF mass analysis; the final mass spectra are averages of 50 individual scans.

Parent ion dissociation rates were measured on a dueling ion-trap MS with nanoelectrospray ionization.⁵⁵ Ions were accumulated over several hundred milliseconds, followed by isolation of the parent ion and collisional activation for periods ranging from 2 to 50 ms. CID was performed by applying resonance excitation voltages with an amplitude of 100 mV_pp, and optimized for each substrate based on the signal intensities in the product ion spectra.

Thioethyl 3,4,6-O-acetyl-2-azido-2-deoxy-α-d-glucopyranoside (9)

Procedure for synthesis of TfN₃(caution: potentially explosive!)

A solution of NaN₃ (37.6 g, 580 mmol) in water (48 mL) and CH₂Cl₂ (80 mL) at 0 °C was treated with Tf₂O (19.6 mL, 116 mmol). The mixture was stirred for 2 h at 0 °C, extracted with CH₂Cl₂ (2 × 35 mL), washed with saturated aqueous Na₂CO₃ solution (75 mL), then dried over Na₂SO₄ and used immediately.

A solution of glucosamine hydrochloride (10 g, 46.4 mmol) in 66% aqueous MeOH (231 mL) was treated with K₂CO₃ (9.6 g, 70 mmol), CuSO₄·5 H₂O (1.2 g, 4.6 mmol) and fresh prepared TfN₃ (70 mL, 1.66 M solution in CH₂Cl₂). An additional 30 mL of MeOH was added to homogenize the mixture. The mixture was stirred for 12 h at rt, concentrated and azeotroped with toluene, then dried under reduced pressure. The crude azide was dissolved in pyridine followed by addition of Ac₂O at 0 °C. The mixture was warmed to rt and stirred for another 12 h, concentrated, azotroped with toluene and dried under reduced pressure. The product was purified by recrystallization in 25% EtOAc in hexanes to afford the desired tetraacetate as a white solid and a mixture of anomers (14.3 g; 2.4:1 α:β). These were dissolved in CH₂Cl₂ (60 mL) and treated with TMSSEt (18.6 mL, 15 mmol, added in two portions) and TMSOTf (2.76 mL, 15.3 mmol). The mixture was heated to reflux for 20 h under Ar, quenched with saturated NaHCO₃ solution (50 mL), extracted with CH₂Cl₂ (3 × 35 mL), washed with brine (50 mL), then dried over Na₂SO₄ and concentrated. The crude product was purified by recrystallization in 95% EtOH to afford α-thioethyl glycoside 9 as an amorphous white solid (9.92 g, 57% over 3 steps). ¹H NMR (300 MHz, CDCl₃): δ 5.43 (d, 1 H, J = 6 Hz), 5.27 (t, 1 H, J = 9.6 Hz), 4.99 (t, 1 H, J = 9.8 Hz), 4.44 (ddd, 1 H, J = 1.8, 4.5, 10.2 Hz), 4.29 (dd, 1 H, J = 4.8, 12.3 Hz), 4.05 (dd, 1 H, J = 2.1,12.3 Hz), 3.99 (dd, 1 H, J = 6, 10.5 Hz), 2.6 (m, 2 H), 2.06 (s, 6 H), 2.02 (s, 3 H), 1.30 (t, 3 H, J = 7.2 Hz); ¹³C NMR (75 MHz, CDCl₃): δ 170.3, 168.5, 82.8, 71.9, 68.5, 67.7, 61.7, 61.3, 24.5, 20.4, 14.4; IR (KBr): 2965, 2112, 1749, 1367, 1225, 1054, 1016 cm⁻¹; [α]²⁰_D = +180.2 (c 1.04, CH₂Cl₂); HRESI–MS: m/z calcd for C₁₄H₂₁N₃O₇SNa [M + Na]⁺ : 398.0998; found: 398.1000.

Thioethyl 3-O-acetyl-2-azido-4,6-O-benzylidene-2-deoxy-α-d-glucopyranoside (10)

A solution of triacetate 9 (7.7 g, 20.5 mmol) in MeOH (60 mL) was treated with 1 M NaOMe solution in MeOH (8.2 mL, 8.2 mmol). The mixture was stirred at rt for 3 h, neutralized with activated Dowex 50X-W-H⁺ ion-exchange resin, filtered, concentrated, and dried under reduced pressure. A solution of the crude triol in THF (48 mL) was treated with benzaldehyde dimethyl acetal (9.3 mL, 61.5 mmol) and TsOH·H₂O (390 mg, 2.05 mmol). The mixture was refluxed for 10 h, quenched with saturated NaHCO₃ solution (30 mL), extracted with CH₂Cl₂ (3 × 50 mL), dried over Na₂SO₄ and concentrated under reduced pressure. The crude acetal was dissolved in pyridine (64 mL) followed by addition of Ac₂O (32 mL) at 0 °C. The mixture was stirred for 10 h at rt, then concentrated under reduced pressure. Purification by recrystallization in 95% EtOH yielded 4,6-O-benzylidene acetal 10 as an amorphous white solid (6.6 g, 85% over 3 steps). ¹H NMR (300 MHz, CDCl₃): δ 7.45 (m, 1 H), 7.35 (m, 1 H), 5.50 (s, 1 H), 5.46 (d, 2 H, J = 5.7 Hz), 5.46 (t, 1 H, J = 9.9 Hz), 4.36 (m, 1 H), 4.25 (dd, 1 H, J = 4.5, 9.9 Hz), 3.97 (dd, 1 H, J = 5.7, 9.9 Hz), 3.78 (t, 1 H, J = 9.9 Hz), 3.62 (t, 1 H, J = 10.2 Hz), 2.63 (m, 2 H), 2.12 (s, 3 H), 1.31 (t, 3 H, J = 7.6 Hz); ¹³C NMR (75 MHz, CDCl₃): δ 169.6, 136.9, 129.2, 128.3, 126.2, 101.7, 84.1, 79.8, 70.6, 68.7, 63.3, 62.4, 24.9, 20.9, 14.9; IR (KBr): 2966, 2933, 2901, 2108, 1750, 1370, 1218, 1098, 969 cm⁻¹; [α]²⁰_D = +126.7 (c 1.03, CH₂Cl₂); HRESI–MS: m/z calcd for C₁₇H₂₁N₃O₅SNa [M + Na]⁺: 402.1100; found: 402.1103.

Thioethyl 3-O-acetyl-2-azido-4-O-benzyl-6-O-(tert-butyldiphenylsilyl)-2-deoxy-α-d-glucopyranoside (11)

Compound 10 (2.49 g, 6.55 mmol) was dissolved in a 1 M BH₃ solution in THF (66 mL), then treated with a 1 M Bu₂BOTf solution in THF (6.88 mL) at 0 °C in the presence of 2,6-lutidine (2.29 mL, 19.66 mmol). The mixture was stirred for 10 h at 0 °C then cooled to −78 °C and quenched with Et₃N (1.82 mL, 13.11 mmol) followed by the slow addition of cold MeOH (30 mL). The mixture was slowly warmed to rt, concentrated, then purified by silica gel chromatography (20% EtOAc in hexanes) to yield the intermediate C6 alcohol (2.04 g, 5.34 mmol). This was dissolved in DMF (7 mL) and treated with imidazole (1.1 g, 16.02 mmol) and TBDPS-Cl (2.78 mL, 10.68 mmol). The mixture was stirred for 12 h at rt, quenched with saturated NaHCO₃ solution (40 mL), extracted with CH₂Cl₂ (2 × 50 mL), washed with brine (40 mL), dried over Na₂SO₄ then concentrated under reduced pressure. Purification by silica gel chromatography (6.25% EtOAc in hexanes) yielded C6 TBDPS ether 11 as an amorphous white solid (2.52 g, 62% over 2 steps). ¹H NMR (300 MHz, CDCl₃): δ 7.77 (m, 4 H), 7.45 (m, 6 H), 7.34 (m, 3 H), 7.25 (m, 2 H), 5.51 (d, 1 H, J = 6 Hz), 5.49 (t, 1 H, J = 9.75 Hz), 4.69 (m, 2 H), 4.26 (dd, 1 H, J = 1.2, 9.6 Hz), 4.35 (dd, 1 H, J = 3.6, 11.1 Hz), 3.94 (m, 2 H), 3.86 (t, 1 H, J = 9.3 Hz), 2.61 (m, 2 H), 2.08 (s, 3 H), 1.31 (t, 3 H, J = 7.7 Hz), 1.16 (s, 9 H); ¹³C NMR (75 MHz, CDCl₃): δ 169.7, 137.7, 135.8, 135.6, 133.4, 132.9, 129.8, 128.5, 127.9, 127.8, 127.6, 82.4, 76.5, 74.6, 73.6, 72.0, 62.5, 62.4, 26.9, 24.3, 20.9, 19.3, 14.6; IR (KBr): 3070, 2931, 2858, 2103, 1752, 1428, 1217, 1112 cm⁻¹; [α]²⁰_D = +77.6 (c 1.10, CH₂Cl₂); HRESI–MS: m/z calcd for C₃₃H₄₁N₃O₅SSiNa [M + Na]⁺: 642.2434; found: 642.2435.

2’-Hydroxyethyl 3-O-acetyl-2-azido-4-O-benzyl-6-O-(tert-butyldiphenylsilyl)-2-deoxy-α-d-glucopyranoside (12)

Synthesis of 2’-hydroxyethyl 2-bromoisobutyrate

A solution of 2-bromopropionic acid (500 mg, 3.3 mmol) and ethylene glycol (1.8 mL, 33 mmol) in CH₂Cl₂ (16 mL) was treated at 0 °C with dicyclohexylcarbodiimide (DCC; 1.0 g, 4.95 mmol) and DMAP (37 mg, 0.3 mmol). The mixture was stirred for 5 h at 0 °C, quenched with saturated NaHCO₃ solution (20 mL), extracted with EtOAc (3 × 30 mL), washed with water (30 mL), brine (30 mL), and dried over Na₂SO₄, then concentrated under reduced pressure. Purification by silica gel chromatography (20% EtOAc in hexanes) afforded 2’-hydroxyethyl 2-bromoisobutyrate in 67% yield as a light yellow oil.

A mixture of thioglycoside 11 (1.24 g, 2 mmol) and 2’-hydroxyethyl 2-bromoisobutyrate (465 mg, 2.2 mmol) in 1:1 (CH₂Cl)₂:Et₂O (14 mL) was stirred with activated 4A molecular sieves for 1 h at rt under Ar. Then the mixture was cooled to 0 °C and treated with N-iodosuccinimide (495 mg, 2.2 mmol) and TfOH (53 μL, 0.6 mmol). The mixture was stirred continuously for another 5 h at 0 °C, quenched with Et₃N (167 μL, 1.2 mmol), warmed to rt, filtered through Celite, extracted with CH₂Cl₂ (3 × 40 mL), washed with brine (40 mL), dried over Na₂SO₄, then concentrated under reduced pressure to yield the corresponding O-glycoside as a 6:1 mixture of anomers. These were separated by silica gel chromatography (10% EtOAc in hexane) to afford the major α-glycoside as a pure compound.

A solution of α-glycoside (1.20 g, 1.56 mmol) in 1:1 MeOH:THF (10 mL) was treated at −5 °C with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (233 μL, 1.56 mmol). The mixture was stirred for 15 h at −5 °C, quenched with saturated NH₄Cl solution (20 mL), extracted with CH₂Cl₂ (3 × 40 mL), washed with brine (40 mL), dried over Na₂SO₄, then concentrated under reduced pressure. Purification by silica gel chromatography (20% EtOAc in hexanes) yielded α-glycol GlcN derivative 12 as a colorless oil (0.82 g, 66% over 2 steps). ¹H NMR (300 MHz, CDCl₃): δ 7.77 (m, 4 H), 7.46 (m, 6 H), 7.32 (m, 3 H), 7.22 (m, 2 H), 5.67 (dd, 1 H, J = 8.7, 10.8 Hz), 5.09 (d, 1 H, J = 3.6 Hz), 4.69 (s, 2 H), 3.89 (m, 7 H), 3.68 (m, 1 H), 3.26 (dd, 1 H, J = 3.3, 11.1 Hz), 2.73 (t, 1 H, J = 6 Hz), 2.09 (s, 3 H), 1.17 (s, 9 H); ¹³C NMR (75 MHz, CDCl₃): δ 169.8, 137.5, 135.6, 135.4, 133.2, 132.7, 129.6, 128.3, 127.7, 127.6, 127.5, 76.0, 74.6, 72.1, 71.6, 70.1, 62.4, 61.5, 61.4, 26.7, 20.7, 19.1; IR (KBr): 2931, 2858, 2109, 1752, 1428, 1225, 1113. 1037 cm⁻¹; [α]²⁰_D = +81.5 (c 0.69, CH₂Cl₂); HRESI–MS: m/z calcd for C₃₃H₄₁N₃O₇SiNa [M + Na]⁺: 642.2612; found: 642.2607.

2’-Hydroxyethyl 2-azido-3-O-acetyl-4-O-benzyl-6-O-(tert-butyldiphenylsilyl)-2-deoxy-α-d-glucopyranoside, bound to trityl PS resin (13)

Trityl-PS resin beads (1.07 g, 1.61 mmol) were suspended in dry pyridine for 5 min followed by addition of compound 12 (1.0 g, 1.61 mmol) and DMAP (79 mg, 0.645 mmol). The mixture was shaken for 3 days at 65 °C. The resin was filtered, washed with 5% diisopropylethylamine (DIPEA) in 8:1 CH₂Cl₂:MeOH (2 × 4 mL), then alternatively with DMF (2 × 4 mL) and CH₂Cl₂ (2 × 4 mL), then finally with additional CH₂Cl₂ (3 × 4 mL). The resin was dried under reduced pressure in the presence of KOH pellets to afford fully resin-bound α-GlcN derivative 13, with a loading ratio ranging from 0.83 mmol/g to 0.9 mmol/g. IR (KBr): 3026, 2929, 2107, 1753, 1600, 1493, 1448, 1224, 1040, 701 cm⁻¹. To determine the loading ratio, 50 mg of 13 was treated with a solution of 3% trifluoroacetic acid (TFA) in CH₂Cl₂ (3 mL). The mixture was stirred for 1 h at rt, then the resin was filtered and washed with CH₂Cl₂ (3 × 3 mL). AcOH (3 mL) was added to the combined washings, which was then concentrated under reduced pressure to give compound 12.

2’-Hydroxyethyl 2-acetamido-4-O-benzyl-2-deoxy-α-d-glucopyranoside (1a)

Op. 1: Resin-bound α-GlcN derivative 13 (40 mg, 0.022 mmol sugar) was swollen in CH₂Cl₂ (3 mL) for 5 min, then treated with Bu₃P (34 μL, 0.137 mmol). The mixture was shaken for 5 h at rt, filtered, then subjected to standard workup conditions to give the intermediate N-ylide. Op. 2: The resin was treated with 95:5 DMF:H₂O (4 mL), shaken for 1 day at rt, washed with THF (2 × 3 mL), then subjected to standard workup conditions to give the resin-bound primary amine. Op. 3: The resin was swollen in pyridine (3 mL) for 5 min, treated with Ac₂O (1.5 mL) and shaken for 1 day at rt. The resin was filtered, washed with 4:1 DMF:pyridine (2 × 3 mL), then subjected to standard workup conditions to give the N-acetylated intermediate. Op. 4: The resin was swollen in THF (0.3 mL) for 5 min, treated with 1 M TBAF in THF (2.7 mL), and shaken for 1 day at rt. The resin was filtered, washed with THF (2 × 3 mL), then subjected to standard workup conditions to give the C6 alcohol. Op. 5: The resin was swollen in CH₂Cl₂ (2 mL) for 5 min, treated with 1 M NaOMe in MeOH (66 μL) and shaken for 5 h at rt. The resin was filtered, washed with 1:1 MeOH:CH₂Cl₂ (2 × 3 mL), then subjected to standard workup conditions to give the fully deprotected resin-bound α-GlcN. Op. 6: The resin was treated with 3% TFA in CH₂Cl₂ (3 mL) for 1 h at rt, then filtered and washed with CH₂Cl₂ (3 × 2 mL). AcOH (3 mL) was added to the combined washings, which was then concentrated under reduced pressure at rt. Purification by reverse-phase HPLC yielded glycol-linked α-GlcN 1a as an amorphous white solid (7 mg, 88% over 6 operations). ¹H NMR (300 MHz, CD₃OD): δ 7.32 (m, 5 H), 4.96 (d, 1 H, J = 11.1 Hz), 4.76 (d, 1 H, J = 3.9 Hz), 4.66 (d, 1 H, J = 11.1 Hz), 4.00 (dd, 1 H, J = 3.3, 10.8 Hz), 3.76 (m, 7 H), 3.47 (m, 2 H), 2.00 (s, 3 H); ¹³C NMR (100 MHz, CD₃OD): δ 173.7, 140.0, 129.3, 129.0, 128.7, 98.9, 79.9, 76.0, 73.7, 73.0, 70.3, 62.3, 62.0, 55.5, 22.6; IR (neat): 3307, 2923, 1656, 1637, 1558, 1130, 1029 cm⁻¹; [α]²⁰_D = +73.4 (c 0.63, MeOH); HRESI–MS: m/z calcd for C₁₇H₂₅NO₇Na [M + Na]⁺ : 378.1529; found: 378.1532.

2’-Hydroxyethyl 2-acetamido-4-O-benzyl-2-deoxy-6-O-sulfonato-α-d-glucopyranoside tetrabutylammonium salt (2a)

Op. 1: Resin-bound α-GlcN derivative 13 (40 mg, 0.022 mmol sugar) was swollen in CH₂Cl₂ (3 mL) for 5 min, then treated with Bu₃P (34 μL, 0.137 mmol). The mixture was shaken for 5 h at rt, filtered, then subjected to standard workup conditions to give the intermediate N-ylide. Op. 2: The resin was treated with 95:5 DMF:H₂O (4 mL), shaken for 1 day at rt, washed with THF (2 × 3 mL) then subjected to standard workup conditions to give the resin-bound primary amine. Op. 3: The resin was swollen in pyridine (3 mL) for 5 min, treated with Ac₂O (1.5 mL) and shaken for 1 day at rt. The resin was filtered, washed with 4:1 DMF:Pyridine (2 × 3 mL), then subjected to standard workup conditions to give the N-acetylated intermediate. Op. 4: The resin was swollen in THF (0.3 mL) for 5 min, treated with 1 M TBAF in THF adjusted to pH 7 with AcOH (2.7 mL), and shaken for 1 day at rt. The resin was filtered, washed with 5:1 THF:pyridine (2 × 3 mL), then subjected to standard workup conditions to give the C6 alcohol. Op. 5: The resin was swollen in dry DMF (3 mL) for 5 min, then treated with SO₃·Py (22 mg, 0.137 mmol) and shaken for 10 h at rt. Op. 6: The resin was filtered, washed with DMF (2 × 3 mL) then treated twice with Bu₄NHSO₄ (47 mg, 0.137 mmol) in 1:1 DMF:MeOH (2 × 2 mL, adjusted to pH 7 with 1 M Bu₄NOH in MeOH). The mixture was shaken for 3 h each time at rt. The resin was filtered, washed with 8:1 MeOH:pyridine (2 × 3 mL) then subjected to standard workup conditions to give the resin-bound 6-O-sulfate. Op. 7: The resin was swollen in CH₂Cl₂ (2 mL) for 5 min, treated with 1 M Bu₄NOH in MeOH (137 μL, 0.137 mmol) and shaken for 5 h at rt. The resin was filtered, washed with 1:1 MeOH:CH₂Cl₂ (2 × 3 mL), then subjected to standard workup conditions to give the fully deprotected resin-bound 6-O-sulfate. Op. 8: The resin was treated with 0.3 M BF₃·OEt₂ in CH₂Cl₂ under standard cleavage conditions. Purification by reverse-phase HPLC yielded glycol-linked 6-O-sulfate 2a as an amorphous white solid (10.5 mg, 58% over 8 operations). ¹H NMR (300 MHz, CD₃OD): δ 7.43 (dd, 2 H, J = 1.8, 8.4 Hz), 7.29 (m, 3 H), 4.92 (d, 2 H, J = 10.5 Hz), 4.76 (d, 2 H, J = 10.5 Hz), 4.75 (d, 1 H, J = 3.3 Hz), 4.25 (m, 2 H), 4.02 (dd, 1 H, J = 3, 10.5 Hz), 3.86 (m, 2 H), 3.78 (dt, 1 H, J = 3, 9.3 Hz), 3.7 (m, 2 H), 3.49 (m, 2 H), 3.24 (m, 8 H), 2.00 (s, 3 H), 1.66 (m, 8 H), 1.42 (m, 8 H), 1.03 (t, 12 H, J = 6.9 Hz); ¹³C NMR (100 MHz, CD₃OD): δ 173.7, 139.9, 129.4, 129.2, 128.6, 98.9, 79.8, 76.1, 73.8, 70.9, 70.3, 67.8, 62.0, 59.5, 55.4, 24.8, 22.6, 20.7, 13.9; IR (neat): 3401, 2962, 2876, 1656, 1630, 1251, 1223, 1016 cm⁻¹; [α]²⁰_D = +34.4 (c 0.43, MeOH); HRESI–MS: m/z calcd for C₁₇H₂₄NO₁₀S [M–NBu₄]⁻ : 434.1121; found: 434.1124.

2’-Hydroxyethyl 2-acetamido-4-O-benzyl-2-deoxy-3-O-sulfonato-α-d-glucopyranoside tetrabutylammonium salt (3a)

Op. 1: Resin-bound α-GlcN derivative 13 (40 mg, 0.022 mmol sugar) was swollen in CH₂Cl₂ (3 mL) for 5 min, then treated with Bu₃P (34 μL, 0.137 mmol). The mixture was shaken for 5 h at rt, filtered, then subjected to standard workup conditions to give the intermediate N-ylide. Op. 2: The resin was treated with 95:5 DMF:H₂O (4 mL), shaken for 1 day at rt, washed with THF (2 × 3 mL) then subjected to standard workup conditions to give the resin-bound primary amine. Op. 3: The resin was swollen in pyridine (3 mL) for 5 min, treated with Ac₂O (1.5 mL) and shaken for 1 day at rt. The resin was filtered, washed with 4:1 DMF:pyridine (2 × 3 mL), then subjected to standard workup conditions to give the N-acetylated intermediate. Op. 4: The resin was swollen in CH₂Cl₂ (2 mL) for 5 min, treated with 1 M NaOMe in MeOH (82 μL) and shaken for 5 h at rt. The resin was filtered, washed with 1:1 MeOH:CH₂Cl₂ (2 × 3 mL), then subjected to standard workup conditions to give the C3 alcohol. Op. 5: The resin was swollen in dry DMF (3 mL) for 5 min, then treated with SO₃·Py (22 mg, 0.137 mmol) and shaken for 10 h at rt. Op. 6: The resin was filtered, washed with DMF (2 × 3 mL) then treated twice with Bu₄NHSO₄ (47 mg, 0.137 mmol) in 1:1 DMF:MeOH (2 × 2 mL, adjusted to pH 7 with 1 M Bu₄NOH in MeOH). The mixture was shaken for 3 h each time at rt. The resin was filtered, washed with 8:1 MeOH:pyridine (2 × 3 mL) then subjected to standard workup conditions to give the resin-bound 3-O-sulfate. Op. 7: The resin was swollen in THF (0.3 mL) for 5 min, treated with 1 M TBAF in THF (2.7 mL) and shaken for 1 day at rt. The resin was filtered, washed with THF (2 × 3 mL), then subjected to standard workup conditions to give the fully deprotected resin-bound 3-O-sulfate. Op. 8: The resin was treated with 0.3 M BF₃·OEt₂ in CH₂Cl₂ under standard cleavage conditions. Purification by reverse-phase HPLC yielded glycol-linked 3-O-sulfate 3a as an amorphous white solid (9.5 mg, 53% over 8 operations). ¹H NMR (300 MHz, CD₃OD): δ 7.44 (dd, 2 H, J = 1.5, 7.8 Hz), 7.29 (m, 3 H), 5.19 (d, 1 H, J = 10.2 Hz), 4.95 (d, 1 H, J = 3.3 Hz), 4.76 (dd, 1 H, J = 8.7, 11.1 Hz), 4.54 (d, 1 H, J = 10.5 Hz), 4.98 (dd, 1 H, J = 3.3, 11.1 Hz), 3.71 (m, 6 H), 3.56 (t, 1 H, J = 9 Hz), 3.47 (m, 1 H), 3.23 (m, 8 H), 1.99 (m, 8 H), 1.66 (m, 8 H), 1.42 (m, 8 H), 1.03 (t, 12 H, J = 7.2 Hz); ¹³C NMR (100 MHz, CD₃OD): δ 173.7, 139.9, 129.9, 129.1, 128.6, 98.6, 79.5, 78.0, 76.0, 73.3, 70.6, 62.2, 62.0, 59.4, 55.1, 24.8, 22.9, 20.7, 13.9; IR (neat): 3413, 2962, 2876, 1662, 1630, 1263, 1217, 1128, 1038 cm⁻¹; [α]²⁰_D = +57.1 (c 0.62, MeOH); HRESI–MS: m/z calcd for C₁₇H₂₄NO₁₀S [M–NBu₄]⁻: 434.1121; found: 434.1118.

2’-Hydroxyethyl 2-amino-4-O-benzyl-2-deoxy-2-N-sulfonato-α-d-glucopyranoside tetrabutylammonium salt (4a)

Op. 1: Resin-bound α-GlcN derivative 13 (40 mg, 0.022 mmol sugar) was swollen in CH₂Cl₂ (3 mL) for 5 min, then treated with Bu₃P (34 μL, 0.137 mmol). The mixture was shaken for 5 h at rt, filtered, then subjected to standard workup conditions to give the intermediate N-ylide. Op. 2: The resin was treated with 95:5 DMF:H₂O (4 mL), shaken for 1 day at rt, washed with THF (2 × 3 mL) then subjected to standard workup conditions to give the resin-bound primary amine. Op. 3: The resin was swollen in dry pyridine (3 mL) for 5 min, then treated with Et₃N (0.6 mL), SO₃·Py (87 mg, 0.548 mmol) and shaken for 10 h at rt. Op. 4: The resin was filtered, washed with 1:2 MeOH:pyridine (2 × 3 mL) then treated twice with Bu₄NHSO₄ (47 mg, 0.137 mmol) in 1:1 DMF:MeOH (2 × 2 mL, adjusted to pH 7 with 1 M Bu₄NOH in MeOH). The mixture was shaken for 3 h each time at rt. The resin was filtered, washed with 8:1 MeOH:pyridine (2 × 3 mL) then subjected to standard workup conditions to give the resin-bound 2-N-sulfate. Op. 5: The resin was swollen in THF (0.3 mL) for 5 min, treated with 1 M TBAF in THF (2.7 mL) and shaken for 1 day at rt. The resin was filtered, washed with THF (2 × 3 mL), then subjected to standard workup conditions to give the C6 alcohol. Op. 6: The resin was swollen in CH₂Cl₂ (2 mL) for 5 min, treated with 1 M Bu₄NOH in MeOH (137 μL) and shaken for 5 h at rt. The resin was filtered, washed with 1:1 MeOH:CH₂Cl₂ (2 × 3 mL), then subjected to standard workup conditions to give the fully deprotected resin-bound N-sulfate. Op. 7: The resin was treated with 0.3 M BF₃·OEt₂ in CH₂Cl₂ under standard cleavage conditions. Purification by reverse-phase HPLC yielded glycol-linked N-sulfate 4a as an amorphous white solid (11 mg, 63% over 7 operations). ¹H NMR (300 MHz, CD₃OD): δ 7.25−7.39 (m, 5 H), 5.12 (d, 1 H, J = 3.6 Hz), 4.98 (d, 1 H, J = 11.1 Hz), 4.64 (d, 1 H, J = 11.1 Hz), 3.63−3.84 (m, 8 H), 3.55 (m, 1 H), 3.42 (t, 1 H, J = 9 Hz), 3.24 (m, 8 H), 1.66 (m, 8 H), 1.42 (m, 8 H), 1.03 (t, 12 H, J = 7.5 Hz); ¹³C NMR (100 MHz, CD₃OD): δ 140.1, 129.2, 129.1, 128.6, 99.1, 79.8, 75.6, 74.5, 72.5, 70.4, 62.5, 62.0, 60.0, 59.4, 24.8, 20.7, 13.9; IR (neat): 3383, 2962, 2876, 1630, 1455, 1382, 1151, 1029 cm⁻¹; [α]²⁰_D = +31.5 (c 0.41, MeOH); HRESI–MS: m/z calcd for C₁₅H₂₂NO₉S [M–NBu₄]⁻ : 392.1015; found: 392.1010.

2’-Hydroxyethyl 2-acetamido-4-O-benzyl-2-deoxy-3,6-di-O-sulfonato-α-d-glucopyranoside tetrabutylammonium salt (5a)

Op. 1: Resin-bound α-GlcN derivative 13 (40 mg, 0.022 mmol sugar) was swollen in CH₂Cl₂ (3 mL) for 5 min, then treated with Bu₃P (34 μL, 0.137 mmol). The mixture was shaken for 5 h at rt, filtered, then subjected to standard workup conditions to give the intermediate N-ylide. Op. 2: The resin was treated with 95:5 DMF:H₂O (4 mL), shaken for 1 day at rt, washed with THF (2 × 3 mL) then subjected to standard workup conditions to give the resin-bound primary amine. Op. 3: The resin was swollen in pyridine (3 mL) for 5 min, treated with Ac₂O (1.5 mL) and shaken for 1 day at rt. The resin was filtered, washed with 4:1 DMF:pyridine (2 × 3 mL), then subjected to standard workup conditions to give the N-acetylated intermediate. Op. 4: This resin was swollen in THF (0.3 mL) for 5 min, treated with 1 M TBAF in THF (2.7 mL) and shaken for 1 day at rt. The resin was filtered, washed with THF (2 × 3 mL), then subjected to standard workup conditions to give the C6 alcohol. Op. 5: The resin was swollen in CH₂Cl₂ (2 mL) for 5 min, treated with 1 M NaOMe in MeOH (82 μL) and shaken for 5 h at rt. The resin was filtered, washed with 1:1 MeOH:CH₂Cl₂ (2 × 3 mL), then subjected to standard workup conditions to give the corresponding 3,6-diol. Op. 6: The resin was swollen in dry pyridine (3 mL) for 5 min, then treated with Et₃N (0.6 mL), SO₃·Py (218 mg, 1.37 mmol) and shaken for 2 day at 55 °C. Op. 7: The resin was filtered, washed with 1:2 MeOH:pyridine (2 × 3 mL) then treated twice with Bu₄NHSO₄ (94 mg, 0.274 mmol) in 1:1 DMF:MeOH (2 × 2 mL, adjusted to pH 7 with 1 M Bu₄NOH in MeOH). The mixture was shaken for 3 h each time at rt. The resin was filtered, washed with 8:1 MeOH:pyridine (2 × 3 mL) then subjected to standard workup conditions. Op. 8,9: The sulfation and cation exchange steps were repeated one more time to give the resin-bound 3,6-di-O-sulfate. Op. 10: The resin was cleaved with 0.3 M BF₃·OEt₂ in CH₂Cl₂ under standard conditions. Purification by reverse-phase HPLC yielded 3,6-di-O-sulfate 5a as an amorphous white solid (11.5 mg, 42% over 10 operations). ¹H NMR (300 MHz, CD₃OD): δ 7.54 (dd, 2 H, J = 1.8, 8.1 Hz), 7.26 (m, 3 H), 5.12 (d, 1 H, J = 9.3 Hz), 4.94 (d, 1 H, J = 3.6 Hz), 4.76 (dd, 1 H, J = 9.3, 11.1 Hz), 4.64 (d, 1 H, J = 9.6 Hz), 4.26 (dd, 1 H, J = 4.2, 10.8 Hz), 4.22 (dd, 1 H, J = 2.4, 10.5 Hz), 3.98 (dd, 1 H, J = 3.6, 11.1 Hz), 3.90 (ddd, 1 H, J = 2.4, 4.4, 9.9 Hz), 3.59−3.80 (m, 4 H), 3.46 (m, 1 H), 3.24 (m, 16 H), 1.98 (s, 3 H), 1.66 (m, 16 H), 1.42 (m, 16 H), 1.03 (t, 24 H, J = 7.4 Hz); ¹³C NMR (100 MHz, CD₃OD): δ 173.6, 139.7, 130.5, 129.0, 128.6, 98.6, 79.4, 77.7, 76.0, 71.2, 70.6, 67.5, 62.0, 59.4, 50.0, 24.8, 22.9, 20.7, 13.9; IR (neat): 3410, 2962, 2876, 1671, 1629, 1459, 1258, 1223, 1024 cm⁻¹; [α]²⁰_D = +20.8 (c 0.52, MeOH); HRESI–MS: m/z calcd for C₁₇H₂₃NO₁₃S₂Na [M–2NBu₄+Na]⁻ : 536.0509; found: 536.0505.

2’-Hydroxyethyl 2-amino-4-O-benzyl-2-deoxy-2,3-di-N,O-sulfonato-α-d-glucopyranoside tetrabutylammonium salt (6a)

Op. 1: Resin-bound α-GlcN derivative 13 (50 mg, 0.0274 mmol sugar) was swollen in CH₂Cl₂ (2 mL) for 5 min, treated with 1 M NaOMe in MeOH (82 μL) and shaken for 5 h at rt. The resin was filtered, washed with 1:1 MeOH:CH₂Cl₂ (2 × 3 mL), then subjected to standard workup conditions to give the C3 alcohol. Op. 2: The resin was swollen in CH₂Cl₂ (3 mL) for 5 min, then treated with Bu₃P (34 μL, 0.137 mmol). The mixture was shaken for 5 h at rt, filtered, then subjected to standard workup conditions to give the intermediate N-ylide. Op. 3: The resin was treated with 95:5 DMF:H₂O (4 mL), shaken for 1 day at rt, washed with THF (2 × 3 mL) then subjected to standard workup conditions to give the resin-bound 2,3-amino alcohol. Op. 4: The resin was swollen in dry pyridine (3 mL) for 5 min, then treated with Et₃N (0.6 mL), SO₃·Py (218 mg, 1.37 mmol) and shaken for 2 days at 55 °C. Op. 5: The resin was filtered, washed with 1:2 MeOH:pyridine (2 × 3 mL), then treated twice with Bu₄NHSO₄ (94 mg, 0.274 mmol) in 1:1 DMF:MeOH (2 × 2 mL, adjusted to pH 7 with 1 M Bu₄NOH in MeOH). The mixture was shaken for 3 h each time at rt. The resin was filtered, washed with 8:1 MeOH:pyridine (2 × 3 mL) then subjected to standard workup conditions to give the resin-bound 2,3-di-N,O-sulfate. Op. 6: The resin was swollen in THF (0.3 mL) for 5 min, treated with 1 M TBAF in THF (2.7 mL) and shaken for 2 days at rt. The resin was filtered, washed with THF (2 × 3 mL), then subjected to standard workup conditions to give the deprotected, resin-bound 2,3-di-N,O-sulfate. Op. 7: The resin was treated with 0.3 M BF₃·OEt₂ in CH₂Cl₂ under standard cleavage conditions. Purification by reverse-phase HPLC yielded glycol-linked 2,3-di-N,O-sulfate 6a as an amorphous white solid (12 mg, 46% over 7 operations). ¹H NMR (300 MHz, CD₃OD): δ 7.44 (dd, 2 H, J = 1.8, 8.1 Hz), 7.28 (m, 3 H), 5.30 (d, 1 H, J = 3.3 Hz), 5.19 (d, 1 H, J = 10.8 Hz), 4.71 (dd, 1 H, J = 9, 11.1 Hz), 4.40 (d, 1 H, J = 10.2 Hz), 3.52−3.76 (m, 8 H), 3.47 (dd, 1 H, J = 3, 11.1 Hz), 3.24 (m, 16 H), 1.66 (m, 16 H), 1.42 (m, 16 H), 1.03 (t, 24 H, J = 7.5 Hz); ¹³C NMR (100 MHz, CD₃OD): δ 139.9, 129.9, 129.1, 128.6, 98.9, 79.7, 78.3, 76.0, 72.9, 70.4, 62.3, 62.0, 59.4, 59.1, 24.8, 20.7, 13.9; IR (neat): 3402, 2961, 2875, 1630, 1460, 1382, 1225, 1035 cm⁻¹; [α]²⁰_D = +30.2 (c 0.58, MeOH); HRESI–MS: m/z calcd for C₁₅H₂₂NO₁₂S₂ [M–2NBu₄+H]⁻ : 472.0583; found: 472.0585.

2’-Hydroxyethyl 2-amino-4-O-benzyl-2-deoxy-2,6-di-N,O-sulfonato-α-d-glucopyranoside tetrabutylammonium salt (7a)

Op. 1: Resin-bound α-GlcN derivative 13 (50 mg, 0.0274 mmol sugar) was swollen in THF (3 mL) for 5 min, treated with 1 M TBAF in THF (164 μL) and shaken for 10 h at rt. The resin was filtered, washed with THF (2 × 3 mL), then subjected to standard workup conditions to give the C6 alcohol. Op. 2: The resin was swollen in CH₂Cl₂ (3 mL) for 5 min, then treated with Bu₃P (34 μL, 0.137 mmol). The mixture was shaken for 30 h at rt, filtered, then subjected to standard workup conditions to give the intermediate N-ylide. Op. 3: The resin was treated with 95:5 DMF:H₂O (4 mL), shaken for 1 day at rt, washed with THF (2 × 3 mL), then subjected to standard workup conditions to give the resin-bound 2,6-amino alcohol. Op. 4: The resin was swollen in dry DMF (3 mL) for 5 min, then treated with SO₃·Py (21.8 mg, 0.137 mmol) and shaken for 10 h at rt. Op. 5: The resin was filtered, washed with DMF (2 × 3 mL) then treated twice with Bu₄NHSO₄ (49 mg, 0.137 mmol) in 1:1 DMF:MeOH (2 × 2 mL, adjusted to pH 7 with 1 M Bu₄NOH in MeOH). The mixture was shaken for 3 h each time at rt. The resin was filtered, washed with 8:1 MeOH:pyridine (2 × 3 mL), then subjected to standard workup conditions to give the resin-bound 6-O-sulfate. Op. 6: The resin was swollen in dry pyridine (3 mL) for 5 min, treated with Et₃N (0.6 mL) and SO₃·Py (88 mg, 0.548 mmol), then shaken for 10 h at rt. Op. 7: The resin was filtered, washed with 1:2 MeOH:pyridine (2 × 3 mL), then treated twice with Bu₄NHSO₄ (94 mg, 0.274 mmol) in 1:1 DMF:MeOH (2 × 2 mL, adjusted to pH 7 with 1 M Bu₄NOH in MeOH). The mixture was shaken for 3 h each time at rt. The resin was filtered, washed with 8:1 MeOH:pyridine (2 × 3 mL), then subjected to standard workup conditions to give the resin-bound 2,6-di-N,O-sulfate. Op. 8: The resin was swollen in CH₂Cl₂ (2 mL) for 5 min, treated with 1 M Bu₄NOH in MeOH (137 μL), and shaken for 5 h at rt. The resin was filtered, washed with 1:1 MeOH:CH₂Cl₂ (2 × 3 mL), then subjected to standard workup conditions to give the deprotected, resin-bound 2,6-di-N,O-sulfate. Op. 9: The resin was treated with 0.3 M BF₃·OEt₂ in CH₂Cl₂ under standard cleavage conditions. Purification by reverse-phase HPLC yielded glycol-linked 2,6-di-N,O-sulfate 7a as an amorphous white solid (9.2 mg, 35% over 9 operations). ¹H NMR (300 MHz, CD₃OD): δ 7.50 (d, 2 H, J = 8.1 Hz), 7.39 (m, 3 H), 5.10 (d, 1 H, J = 4.2 Hz), 4.94 (d, 1 H, J = 10.5 Hz), 4.74 (d, 1 H, J = 10.5 Hz), 4.27 (dd, 1 H, J = 2.7, 10.5 Hz), 4.20 (dd, 1 H, J = 4.8, 10.5 Hz), 3.69−3.88 (m, 5 H), 3.43−3.57 (m, 3 H), 3.24 (m, 16 H), 1.67 (m, 16 H), 1.42 (m, 16 H), 1.03 (t, 24 H, J = 7.5 Hz); ¹³C NMR (100 MHz, CD₃OD): δ 140.0, 129.5, 129.2, 128.5, 99.1, 79.7, 75.8, 74.5, 70.5, 67.9, 62.0, 59.9, 59.5, 24.8, 20.7, 13.9; IR (neat): 3401, 2962, 2876, 1631, 1459, 1251, 1226, 1055, 1027 cm⁻¹; [α]²⁰_D = +26.9 (c 0.52, MeOH); HRESI–MS: m/z calcd for C₁₅H₂₁NO₁₂S₂Na [M–2NBu₄+Na]⁻: 494.0403; found: 494.0406.

2’-Hydroxyethyl 2-amino-4-O-benzyl-2-deoxy-2,3,6-tri-N,O-sulfonato-α-d-glucopyranoside tetrabutylammonium salt (8a)

Op. 1: Resin-bound α-GlcN derivative 13 (50 mg, 0.0274 mmol sugar) was swollen in CH₂Cl₂ (2 mL) for 5 min, treated with 1 M NaOMe in MeOH (82 μL) and shaken for 5 h at rt. The resin was filtered, washed with 1:1 MeOH:CH₂Cl₂ (2 × 3 mL), then subjected to standard workup conditions to give the C3 alcohol. Op. 2: The resin was swollen in CH₂Cl₂ (3 mL) for 5 min, then treated with Bu₃P (34 μL, 0.137 mmol). The mixture was shaken for 5 h at rt, filtered, then subjected to standard workup conditions to give the intermediate N-ylide. Op. 3: The resin was treated with 95:5 DMF:H₂O (4 mL), shaken for 1 day at rt, washed with THF (2 × 3 mL) then subjected to standard workup conditions to give the resin-bound 2,3-amino alcohol. Op. 4: The resin was swollen in THF (0.3 mL) for 5 min, then treated with 1 M TBAF in THF (2.7 mL) and shaken for 10 h at rt. The resin was filtered, washed with THF (2 × 3 mL), then subjected to standard workup conditions to give the resin-bound, fully deprotected α-GlcN. Op. 5: The resin was swollen in dry pyridine (3 mL) for 5 min, then treated with Et₃N (0.6 mL), and SO₃·Py (218 mg, 1.37 mmol) and shaken for 2 days at 55 °C. Op. 6: The resin was filtered, washed with 1:2 MeOH:pyridine (2 × 3 mL), then treated twice with Bu₄NHSO₄ (140 mg, 0.411 mmol) in 1:1 DMF:MeOH (2 × 2 mL, adjusted to pH 7 with 1 M Bu₄NOH in MeOH). The mixture was shaken for 3 h each time at rt. The resin was filtered, washed with 8:1 MeOH:pyridine (2 × 3 mL), then subjected to standard workup conditions. Op. 7,8: The sulfation and cation exchange steps were repeated one more time to yield the resin-bound, 2,3,6-tri-N,O-sulfate. Op. 9: The resin was treated with 0.3 M BF₃·OEt₂ in CH₂Cl₂ under standard conditions. Purification by reverse-phase HPLC yielded glycol-linked 2,3,6-tri-N,O-sulfate 8a as an amorphous white solid (13.3 mg, 38% over 9 operations). ¹H NMR (300 MHz, CD₃OD): δ 7.54 (dd, 2 H, J = 1.2, 8.1 Hz), 7.26 (m, 3 H), 5.27 (d, 1 H, J = 3.3 Hz), 5.16 (d, 1 H, J = 9.9 Hz), 4.71 (d, 1 H, J = 10.8 Hz), 4.66 (t, 1 H, J = 9.3 Hz), 4.26 (dd, 1 H, J = 3.9, 10.5 Hz), 4.20 (dd, 1 H, J = 2.1, 10.2 Hz), 3.84 (ddd, 1 H, J = 2.4, 3.9, 9.6 Hz), 3.53−3.80 (m, 5 H), 3.47 (dd, 1 H, J = 3.3, 10.8 Hz), 3.24 (m, 24 H), 1.66 (m, 24 H), 1.42 (m, 24 H), 1.03 (t, 36 H, J = 7.5 Hz); ¹³C NMR (100 MHz, CD₃OD): δ 139.8, 130.5, 129.0, 128.5, 98.9, 79.6, 78.0, 76.0, 70.9, 70.4, 67.6, 61.9, 59.5, 59.1, 24.8, 20.7, 13.9; IR (neat): 3401, 2962, 2875, 1489, 1460, 1257, 1227, 1035 cm⁻¹; [α]²⁰_D = +22.0 (c 0.65, MeOH); HRESI–MS: m/z calcd for C₁₅H₂₀NO₁₅S₃Na₂ [M–3NBu₄+2Na]⁻: 595.9791; found: 595.9795.

2’-Hydroxyethyl 2-acetamido-2-deoxy-α-d-glucopyranoside (1b)

Compound 1a (7 mg) was dissolved in MeOH (3 mL) and treated with Pd(OH)₂ on charcoal (5 mg), then stirred under positive H₂ pressure at rt for 20 h. The reaction mixture was filtered to remove catalyst, then concentrated and purified by reverse- phase HPLC to yield 1b as an amorphous white solid (5.5 mg, 90%). ¹H NMR (500 MHz, D₂O): δ 4.85 (d, 1 H, J = 3.6 Hz), 3.92 (dd, 1 H, J = 3.6, 10.6 Hz), 3.85 (dd, 1 H, J = 2.0, 12.0 Hz), 3.69−3.80 (m, 6H), 3.53 (m, 1H), 3.46 (t, 1H, J = 9.5 Hz); ¹³C NMR (125 MHz, D₂O): δ 174.4, 97.1, 71.8, 71.1, 69.9, 68.8, 60.5, 60.4, 53.5, 21.8; [α]²⁰_D = +75.7 (c 0.46, MeOH); HRESI–MS: m/z calcd for C₁₀H₁₉NO₇Na [M + Na]⁺: 288.1059; found: 288.1061.

2’-Hydroxyethyl 2-acetamido-2-deoxy-6-O-sulfonato-α-d-glucopyranoside sodium salt (2b)

Compound 2a (6.2 mg) was dissolved in MeOH and subjected to the hydrogenation conditions described above, then filtered and loaded onto a column packed with a cation-exchange resin (Dowex Marathon MSC, Na form) and eluted with MeOH. The fractions were concentrated and purified by reverse-phase HPLC to yield 2b as an amorphous white solid (2.6 mg, 82%). ¹H NMR (500 MHz, CD₃OD): δ 4.72 (d, 1 H, J = 3.5 Hz), 4.29 (dd, 1 H, J = 1.8, 10.8 Hz), 4.11 (dd, 1 H, J = 6.1, 10.9 Hz), 3.95 (dd, 1 H, J = 3.6, 10.6 Hz), 3.80 (m, 2H), 3.70 (m, 2H), 3.64 (dd, 1 H, J = 9.0, 10.4 Hz), 3.44 (ddd, 1 H, J = 3.7, 7.1, 10.8 Hz), 3.36 (t, 1H, J = 9.7 Hz); ¹³C NMR (125 MHz, CD₃OD): δ 172.1, 97.4, 71.6, 70.6, 70.4, 68.7, 66.9, 60.6, 53.6, 21.1; [α]²⁰_D = +81.9 (_c 0.17, MeOH); HRESI–MS: m/z calcd for C₁₀H₁₈NO₁₀S [M–Na]⁻: 344.0651; found: 344.0648.

2’-Hydroxyethyl 2-acetamido-2-deoxy-3-O-sulfonato-α-d-glucopyranoside sodium salt (3b)

Compound 3a (5.1 mg) was dissolved in MeOH and subjected to the hydrogenation conditions and ion-exchange chromatography described above, then concentrated and purified by reverse-phase HPLC to yield 3b as an amorphous white solid (2.1 mg, 81%). ¹H NMR (500 MHz, CD₃OD): δ 4.84 (d, 1 H, J = 3.5 Hz), 4.50 (dd, 1 H, J = 8.7, 10.7 Hz), 4.02 (dd, 1 H, J = 3.5, 10.7 Hz), 3.75−3.80 (m, 2H), 3.62−3.72 (m, 4H), 3.57 (t, 1H, J = 8.9 Hz), 3.47 (ddd, 1 H, J = 3.5, 6.8, 10.7 Hz); ¹³C NMR (125 MHz, CD₃OD): δ 172.1, 97.3, 78.4, 72.4, 69.6, 69.0, 60.9, 60.5, 52.1, 21.2; [α]²⁰_D = +96.4 (c 0.14, MeOH); HRESI– MS: m/z calcd for C₁₀H₁₈NO₁₀S [M–Na]⁻: 344.0651; found: 344.0652.

2’-Hydroxyethyl 2-amino-2-deoxy-2-N-sulfonato-α-d-glucopyranoside sodium salt (4b)

Compound 4a (3.8 mg) was dissolved in MeOH and subjected to the hydrogenation conditions and ion-exchange chromatography described above, then concentrated and purified by reverse-phase HPLC to yield 4b as an amorphous white solid (1.4 mg, 78%). ¹H NMR (500 MHz, CD₃OD): δ 3.78 (dd, 1 H, J = 2.3, 11.8 Hz), 3.75 (m, 1H), 3.68−3.72 (m, 1H), 3.65 (dd, 1 H, J = 5.8, 11.8 Hz), 3.58 (dd, 1 H, J = 8.8, 10.2 Hz), 3.51−3.57 (m, 2H), 3.33 (m, 2H), 3.22 (dd, 1 H, J = 3.7, 10.3 Hz); ¹³C NMR (125 MHz, CD₃OD): δ 97.6, 72.4, 71.9, 70.7, 68.9, 61.3, 60.6, 58.2; [α]²⁰_D = +131.8 (c 0.09, MeOH); HRESI–MS: m/z calcd for C₈H₁₆NO₉S [M–Na]⁻: 302.0546; found: 302.0548.

2’-Hydroxyethyl 2-acetamido-2-deoxy-3,6-di-O-sulfonato-α-d-glucopyranoside sodium salt (5b)

A mixture of 5a (5.7 mg) was dissolved in MeOH and subjected to the hydrogenation conditions and ion-exchange chromatography described above, then concentrated and purified by reverse-phase HPLC to yield 5b as an amorphous white solid (2.1 mg, 82%). ¹H NMR (500 MHz, CD₃OD): δ 4.83 (d, 1 H, J = 3.4 Hz), 4.50 (dd, 1 H, J = 8.8, 10.7 Hz), 4.34 (dd, 1 H, J = 1.9, 10.8 Hz), 4.10 (dd, 1 H, J = 6.5, 10.1 Hz), 4.03 (dd, 1 H, J = 3.5, 10.7 Hz), 3.91 (ddd, 1 H, J = 1.8, 6.5, 11.7 Hz), 3.81 (ddd, 1H, J = 3.1, 4.8, 10.6 Hz), 3.36−3.78 (m, 2H), 3.54 (dd, 1H, J = 9.0, 9.8 Hz), 3.47 (ddd, 1H, J = 3.4, 7.2, 8.8 Hz); ¹³C NMR (125 MHz, CD₃OD): δ 173.6, 98.7, 79.9, 71.9, 71.2, 70.5, 68.3, 62.1, 53.7, 22.8; [α]²⁰_D = +77.1 (c 0.14, MeOH); HRESI–MS: m/z calcd for C₁₀H₁₇NO₁₃S₂Na [M–Na]⁻ : 446.0039; found: 446.0042.

2’-Hydroxyethyl 2-amino-2-deoxy-2,3-di-N,O-sulfonato-α-d-glucopyranoside sodium salt (6b)

A mixture of 6a (3.1 mg) was dissolved in MeOH and subjected to the hydrogenation conditions and ion-exchange chromatography described above, then concentrated and purified by reverse-phase HPLC to yield 6b as an amorphous white solid (0.8 mg, 62%). ¹H NMR (500 MHz, CD₃OD): δ 5.27 (d, 1 H, J = 3.4 Hz), 4.43 (dd, 1 H, J = 8.6, 10.6 Hz), 3.78 (m, 2H), 3.65−3.72 (m, 4H), 3.58 (m, 2H), 3.39 (dd, 1 H, J = 3.4, 10.5 Hz); ¹³C NMR (125 MHz, CD₃OD): δ 97.3, 78.5, 72.1, 69.7, 68.9, 61.0, 60.5, 56.5; [α]²⁰_D = +65.6 (c 0.05, MeOH); HRESI–MS: m/z calcd for C₈H₁₅NO₁₂S₂Na [M–Na]⁻: 403.9933; found: 403.9931.

2’-Hydroxyethyl 2-amino-2-deoxy-2,6-di-N,O-sulfonato-α-d-glucopyranoside sodium salt (7b)

A mixture of 7a (6.9 mg) was dissolved in MeOH and subjected to the hydrogenation conditions and ion-exchange chromatography described above, then concentrated and purified by reverse-phase HPLC to yield 7b as an amorphous white solid (1.9 mg, 65%). ¹H NMR (500 MHz, CD₃OD): δ 5.07 (d, 1 H, J = 3.6 Hz), 4.27 (dd, 1 H, J = 2.0, 10.9 Hz), 4.12 (dd, 1 H, J = 5.9, 10.9 Hz), 3.79 (m, 2H), 3.71 (m, 2H), 3.59 (dd, 1 H, J = 9.1, 10.2 Hz), 3.52 (ddd, 1 H, J = 4.1, 6.2, 10.2 Hz), 3.25 (dd, 1 H, J = 3.5, 10.2 Hz); ¹³C NMR (125 MHz, D₂O): δ 95.8, 69.7, 68.1, 67.6, 65.5, 59.0, 56.1; [α]²⁰_D = +31.6 (c 0.13, MeOH); HRESI–MS: m/z calcd for C₈H₁₅NO₁₂S₂Na [M–Na]⁻: 403.9933; found: 403.9940.

2’-Hydroxyethyl 2-amino-2-deoxy-2,3,6-tri-N,O-sulfonato-α-d-glucopyranoside sodium salt (8b)

A mixture of 8a (9.8 mg) was dissolved in MeOH and subjected to the hydrogenation conditions and ion-exchange chromatography described above, then concentrated and purified by reverse-phase HPLC to yield 8b as an amorphous white solid (3 mg, 81%). ¹H NMR (500 MHz, CD₃OD): δ 5.23 (d, 1 H, J = 3.4 Hz), 4.42 (dd, 1 H, J = 8.8, 10.6 Hz), 4.33 (dd, 1 H, J = 1.9, 10.7 Hz), 4.10 (dd, 1 H, J = 6.3, 10.7 Hz), 3.88 (m, 1H), 3.80 (m, 1H), 3.72 (dd, 2 H, J = 3.8, 6.2 Hz), 3.54 (m, 1H), 3.41 (dd, 1 H, J = 3.4, 10.6 Hz; ¹³C NMR (125 MHz, CD₃OD): δ 97.2, 78.3, 70.1, 69.6, 68.9, 66.9, 60.4, 56.6; [α]²⁰_D = +23.5 (c 0.2, MeOH); HRESI–MS: m/z calcd for C₈H₁₆NO₁₅S₃ [M–3Na+2H]⁻: 461.9682; found: 461.9670.