Synthesis and Evaluation of 1,5-Dithialaminaribiose and - triose Tetravalent Constructs

Abstract

We report the synthesis of novel tetravalent glucoclusters containing 1,5-dithia mimetics of laminaribiose and triose. The new constructs were evaluated for their ability to inhibit anti-CR3 fluorescent staining of human neutrophils, for which they showed moderate affinity. Evaluation of the synthesized glycoclusters for their ability to inhibit anti-Dectin-1 fluorescent staining of mouse macrophages revealed little to no affinity for Dectin-1.

Graphical Abstract

1. Introduction

The β-(1→3)-glucans (Figure 1) are widely occurring natural immunomodulating agents found in yeasts, seaweeds, and fungi [1–4]. They are known to potentiate tumor-specific antibodies, modulate the effects of radiation and photodynamic therapy [5–7], mitigate allergic rhinitis [8], regulate stress [9], afford protection of the liver [10], and protect from symptoms of inflammatory bowel disease [11–14].

Figure 1.

Structure of β-(1→3)-glucans and synthetic analogs.

Despite widespread availability in nature, the isolation of pure homogenous glycoforms of β-(1→3)-glucans is complicated. Extensive aqueous extractions and chromatographic purification are required to isolate oligomeric glycans. To date many efforts have been devoted to the synthesis of various β-(1→3)-glucan oligomers and evaluation of their immunomodulating properties [15–18]. The immunostimulating properties of the β-(1→3)-glucans are considered to arise from their affinity for the lectin regions of complement receptor 3 (CR3) [9, 19, 20] and Dectin-1 [21–23], binding to which triggers a cascade of effects including phagocytosis. Studies [24] of homogenous β-(1→3)-glucans demonstrated that the shortest oligomer capable of detectable binding to recombinant murine Dectin-1 in a microarray format is the 10- or 11-mer. However, surface plasmon resonance assays later revealed a heptasaccharide unit to be capable of binding to Dectin-1 [25]. Additionally, it was subsequently shown [26, 27] that short synthetic β-(1→3)-glucans (penta- and hexamers) modified at the reducing end by the replacement of the terminal glucopyranose residue by its manno-stereoisomer or by gluco- and manno-configured glycitols retain the ability to promote phagocytosis. X-ray crystallographic studies of recombinant Dectin-1 revealed the presence of a hydrophobic pocket lined by the Trp 221 and His 223 residues in the carbohydrate binding region, but to date no structures exist of Dectin-1 with a β-(1→3)-glucan located in the carbohydrate binding domain [28]. Likewise, although multiple X-ray crystallographic structures of CR3 have been described, none as yet include a β-(1→3)-glucan [29–31]. For Dectin-1, NMR spectroscopic experiments showed that the α-face of terminal pyranose rings in natural β-(1→3)-glucan are in close contact with the aromatic rings of the amino acid residues in the lectin domain (Figure 2) [32]. More recent STD NMR studies revealed that a synthetic β-(1→3)-glucan hexadecamer, but not a hexamer, bound to the lectin domain of Dectin-1 [33]. This may be the result of enhancement of relatively weak carbohydrate-protein [34–36] interactions by a multivalent effect arising from the repeated presence of the epitope in the polymeric glycan. Surface plasmon resonance (SPR) experiments demonstrated binding of multiple Dectin-1 molecules to a single polymeric β-(1→3)-glucan backbone with the affinity increasing in an additive fashion [25].

Figure 2.

Schematic representation of the hydrophobic α-face of a disaccharide unit of a β-(1→3)-glucan in complex with hydrophobic residues in the binding pocket of the Dectin-1–lectin domain.

The increasing evidence that hydrophobic binding pockets in CR3 and Dectin-1 can accept small carbohydrate epitopes suggests that the ligand–receptor interaction may be enhanced by modification of the carbohydrate epitope. Following this idea several short synthetic β-(1→3)-glucans and their 1-thia analogs were described [37]. Pursuing a glycomimetic approach, as opposed to a multivalent glycoconjugate approach, for the synthesis of improved β-(1→3)-glucan analogues we designed and evaluated hydroxylamine-based glycan analogs 2–3, which showed significant affinity for CR3 and Dectin-1 [38]. We hypothesized that the enhanced binding of the mimetics 2–3 was caused by replacement of the C2-OH group with a C-H bond and of the ring oxygen with methylene group (Figure 1) resulting in increased hydrophobicity. Extrapolating from this hypothesis, we most recently synthesized and evaluated short thioether linked carbosugars 4–6 [39], and 1,5-dithia analogs 7–8 of laminaribiose, triose, and tetraose (Figure 1) [40]: both mimetics showed affinity for the carbohydrate binding domains of the lectin, which was reflected by a comparable ability to stimulate phagocytosis and pinocytosis. The improved affinity of the dithia mimetics in comparison to simple glycosides possibly arises from the weak sulfur/π-interactions occurring between ring and/or glycosidic sulfur and aromatic side chains in the binding domain, a well-recognized effect [41, 42]. Inspired by the affinity of the dithia analogs, and by previous work on the development of multivalent constructs of short β-(1→3)-glucans and related sugars and their Dectin-1 binding properties [43–45], we hypothesized that their incorporation into a multivalent construct could improve the overall activity as a result of multiple ligand-protein interactions occurring between the glycocluster and the lectin. To date much effort has been made in designing multivalent cores [46] and developing glycoclusters based on those cores [35, 47–50]. Building on these observations we designed two multivalent glycoclusters based on a conformationally unrestricted tetravalent pentaerythritol core linked to our short 1,5-dithia β-(1→3)-glucan mimetics via amide bonds, 11–12, and report on their synthesis and preliminary evaluation (Figure 3) together with that of appropriate controls 9–10, and 13.

Figure 3.

Structure of synthetic dithiaglycoclusters and comparators

2. Results and Discussion

2.1. Synthesis

We chose to employ a conformationally mobile pentaerythritol-based tetravalent core, of a similar size to other small molecule cores used in other recent studies of glycan interactions with Dectin-1 [45, 51], linked to the carbohydrate units by amide bonds. Therefore, we sought to synthesize a core functionalized with remote carboxyl groups, which would provide sufficient distance from the core, and a carbohydrate moiety bearing a terminal amino group as a well reported building block [52–54]. Synthetic efforts began with preparation of the tetravalent core 17.

Adopting a reported protocol [55], pentaerythritol was alkylated with allyl bromide affording tri- 15a and tetraallylated 15b derivatives in 66% and 25% yield, respectively (Scheme 1). Triallylated 15a derivative then was converted into the corresponding tetraallylated derivative 15b by alkylation with allyl bromide in the presence of NaH in 93% yield. Irradiation of a DMF solution of tetraallyl ether 15b, methyl thioglycolate and 2,2-dimethoxy-2-phenylacetophenone (DMPAP) with UV light (λ = 260 nm) gave tetraester 16 in 69% yield, admixed with a small amount of regioisomers arising from anti-Markovnikoff addition [56]. Saponification of the esters with an excess of aqueous KOH then gave the tetracarboxylic acid core 17 quantitatively.

Scheme 1.

Synthesis of tetravalent core 17

To prepare the carbohydrate epitopes, we started with glucose thioacetate 18 [57]. Adopting a reported protocol [58], thioacetate 18 was alkylated with tert-butyl (2-(2-iodoethoxy)ethyl)carbamate [59] in the presence of diethylamine to give carbamate 19 in 73% yield. Subsequent Boc group removal with aqueous trifluoroacetic acid then gave 20 as the trifluoroacetate salt quantitatively (Scheme 2). Coupling of trifluoroacetate 20 with tetracarboxylic acid 17 in presence of Hünig’s base and PyBOP gave glucocluster 21 in 80% yield. Finally, Zemplen deesterification of 21 afforded tetraamide 9 in 57% yield. We then applied an analogous route to the preparation of the thiaglucose derivatives. Thus, 1,5-dithiaglucose pentaacetate 22 [40] was alkylated with tert-butyl (2-(2-iodoethoxy)ethyl)carbamate to give carbamate 23 in 91% yield, followed by carbamate deprotection with aqueous TFA to give ammonium salt 24 in 96% yield. Coupling of trifluoroacetate 24 with multivalent core 17 in presence of PyBOP and DIPEA afforded tetraamide 25 in 78% yield, which upon deacetylation gave tetraamide 10 in 70% yield.

Scheme 2.

Synthesis of monosaccharyl clusters 9–10

To prepare clusters of higher oligomers (Scheme 3) disaccharyl thioacetate [40] 26 was alkylated with tert-butyl (2-(2-iodoethoxy)ethyl)carbamate to give carbamate 27 in 90% yield. Hydrolysis of the Boc protecting group with aqueous TFA then gave a 98% yield of the trifluoroacetate ammonium salt 28, which was coupled with tetracarboxylic acid 17 in presence of PyBOP and DIPEA to afford tetraamide 29 in 66% yield. Final deacetylation was performed with excess NaOMe. In this manner the peracetylated tetraamide 29 was converted into the final glycocluster 11 in 76% yield. The trisaccharide derivatives were found to be less reactive, as evidenced by reduced yields in the majority of transformations. Thus, alkylation of thioacetate 30 [40] with tert-butyl (2-(2-iodoethoxy)ethyl)carbamate in presence of Et₂NH gave only 57% of the carbamate derivative 31, which on hydrolysis with aqueous TFA gave trifluoroacetate salt 32 quantitatively. Coupling of trifluoroacetate salt 32 with tetravalent core 17 under the optimized conditions gave tetraamide 33 in 57% yield. Deacetylation of 33 with either catalytic or stoichiometric amounts of NaOMe did not go to completion and led to complications during purification. Therefore, we devised an alternative deacetylation method using NEt₃ in aqueous MeOH under microwave irradiations. In this way peracetylated trisaccharide derivative 33 was successfully deacetylated to give final tetraamide 12 in 50% yield.

Scheme 3.

Synthesis of targeted dithiaglycoclusters 11–12

A monovalent comparator 13 was synthesized by analogous methods by coupling ammonium salt 32 with propionic acid in presence of PyBOP and DIPEA to give 90% of the trisaccharide derivative 34, which was deacetylated with NaOMe to afford propionyl amide 13 in 86% yield (Scheme 4).

Scheme 4.

Synthesis of monovalent amide 13.

2.2. Evaluation of Binding to CR3 and Dectin-1

Multivalent glucoclusters 9, 10, 11, 12 and the monovalent comparator 13 were screened for their ability to inhibit anti-CR3 or anti-Dectin-1 fluorescein isothiocyanate (FITC)-conjugated antibody staining of human neutrophils and mouse macrophages, which is indicative of their affinity for CR3 and Dectin-1 (Table 1) [60]. For comparison purposes, the previously reported performances of the di- and trimeric hydroxylamines 2 and 3, as well as 1,5-dithia di- and trisaccharides 7 and 8 in the same assay are also presented in Table 1. The glycolusters 9, 10, 11, 12, 13 were also examined for their ability to stimulate phagocytosis of fluorescein isothiocyanate (FITC)-labeled E. coli particles [61] by human macrophage-like U937 cells, but no such activity was detected. All the synthesized tetravalent glucans 9–12 and monovalent comparator 13 showed inhibition of anti-CR3 staining of human neutrophils comparable to that of the monovalent parent 1,5-dithia mimetics 7–8, suggesting that the multivalent presentation tested is not effective at enhancing binding. No activity was found when 9-13 were submitted to the anti-Dectin-1 staining of mouse macrophages, possibly suggesting that the aglycon is incompatible with binding domain of Dectin-1.

Table 1.

Percentage Inhibition of Anti-CR3 Antibody Staining of Neutrophils by 0.1 μg mL⁻¹ Substrate

compound	Oligomer unit no.	Type of epitope	Valency	% Inhibition of anti-CR3-FITC staining of human neutrophilsa
2	dimer	isofagamine	monovalent	20.2 ± 1.7
3	trimer	isofagamine	monovalent	37.3 ± 3.2
7	dimer	1,5-dithiaglucose	monovalent	26.4 ± 2.7
8	trimer	1,5-dithiaglucose	monovalent	34.2 ± 3.3
9	monomer	glucose	tetravalent	39.7b
10	monomer	1,5-dithiaglucose	tetravalent	33b
11	dimer	1,5-dithiaglucose	tetravalent	46.3b
12	trimer	1,5-dithiaglucose	tetravalent	36.8b
13	trimer	1,5-dithiaglucose	monovalent	34.3b

^aMean ± SD.

^bMean calculated from two experiments

3. Conclusions

In summary, we designed and synthesized novel tetravalent glucoclusters 9–12 and monovalent propionylamide comparator 13. All the synthesized compounds demonstrated some activity in inhibition of staining of human neutrophils by fluorescent anti-CR3 antibodies comparable to the parent 1,5-dithiaglucose mimetics 7–8. On the other hand, the same glycoclusters showed little to no activity in inhibition of staining of mouse macrophages by fluorescent anti-Dectin-1 antibodies.

4. Experimental section

4.1. General methods

All reactions were conducted in flame/oven dried glassware capped with rubber septa under an atmosphere of argon unless otherwise stated. Commercially available starting materials were used without purification, unless otherwise stated. All organic solutions were concentrated under reduced pressure on a rotary evaporator and water bath. Flash-column chromatography was performed using a COMBIFLASH^® NextGen system, unless otherwise stated. Thin-layer chromatography (TLC) was carried out with 250 μm glass backed silica (XHL) plates with fluorescent indicator (254 nm) or 250 μm glass backed C18 silica plates with fluorescent indicator (254 nm). TLC plates were visualized by submersion in ceric ammonium molybdate solution (CAM), or aqueous potassium permanganate solution (KMnO₄), or 10% sulfuric acid in ethanol followed by heating on a hot plate (120 °C). Nuclear magnetic resonance (NMR) spectra of all compounds were obtained in CDCl3 (δ 7.27 and 77.0 ppm, respectively), CD3OD (δ 3.31 and 49.00 ppm, respectively), DMSO-d6 (δ 2.5 and 39,5 ppm, respectively), or D2O (δ 4.67 ppm), or CD3CN (δ 1.94 and 1.32 ppm, respectively) at 500 MHz or 900 MHz. The chemical shifts (δ) are calculated with respect to residual solvent peak and are given in ppm. Multiplicities are abbreviated as follows: s (singlet), m (multiplet), br (broad), d (doublet), t (triplet), q (quartet), p (quintet), and comp (complex). Assignments were made with the help of COSY, HMBC, HSQC HMQC, and DEPT-135 spectra. Specific rotations were recorded in CHCl₃ or DMSO, at 589 nm and 20–22 °C on a digital polarimeter with a path length of 10 cm. High resolution mass spectra were obtained on a ThermoFisher Orbitrap Q-Exactive instrument using electrospray ionization (ESI). Reactions under microwave irradiation were performed using a BIOTAGE Initiator microwave. Photochemical reactions were performed in a Rayonet photochemical reactor.

4.1.1. Cell lines

U937 cells, a human monocytic cell line (ATCC CRL-1593.2) and P388D1(IL-1), a murine monocytic cell line (ATCC, TIB-63), were obtained from the ATCC and maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 IU/mL), streptomycin (100 mg/mL) and L-glutamine (2 mM) in an incubator at 37 °C and a humidified atmosphere containing 5% CO₂.

4.1.2. Antibodies

Mouse anti-human Dectin-1 (MCA4661F), rat anti-mouse Dectin-1 (MCA2289F) and a rat anti-mouse CD11b antibody (also detects human CD11b (MCA74F)), all fluorescein labeled (FITC), were obtained from Bio-Rad and used in the staining inhibition experiments at 1:200 dilution.

4.2.1. Tri-O-allylpentaerythritol (15a) and tetra-O-allylpentaerythritol (15b)

Pentaerythritol (3 g, 22.0 mmol) was dissolved in DMSO (40 mL) and NaOH (40% aq solution) (1.5 mL) was added followed by addition of allyl bromide (14.3 mL, 165 mmol, 7.5 equiv). The reaction mixture then was stirred for 16 h at 20 °C before it was diluted with H₂O (40 mL) and extracted with Et₂O (3×50 mL). The combined organic layers were washed with brine (100 mL), dried over MgSO₄, and concentrated to dryness. The crude reaction mixture was purified by a flash column chromatography on a silica gel eluting with hexanes:EtOAc (0 → 30% EtOAc (v/v)) to give 15a (3.74 g, 66%) and 15b (1.6 g, 25%) ethers as a colorless liquids. The triallylated derivative 15a (3.7g, 14.43 mmol) was then dissolved in anhydrous THF (48 mL) and the reaction mixture was cooled down to 0 °C before NaH (60% dispersion in mineral oil, 630 mg, 17.32 mmol, 1.2 equiv) was added. The reaction mixture was then stirred for 0.25 h at 0 °C before addition of allyl bromide (1.5 mL, 17.3 mmol, 1.2 equiv). The reaction mixture then heated to reflux with stirring for 12 h before further allyl bromide (1.5 mL, 17.3 mmol, 1.2 equiv) was added. After 1 h the reaction mixture was cooled to room temperature, quenched with MeOH (10 mL), concentrated under vacuo, redissolved in EtOAc (70 mL), washed with H₂O (40 mL), brine (40 mL), dried over MgSO₄ and concentrated to dryness. The crude product was purified by flash column chromatography on silica gel eluting with hexanes:EtOAc (0 → 20% EtOAc (v/v)) to afford the tetraallylated derivative 15b as a colorless liquid (4.0 g, 93%).

Tri-O-allylpentaerythritol (15a): a colorless liquid with R_f 0.47 (hexanes:EtOAc 4:1, (KMnO₄)) had spectral data identical to the literature values [55]. ¹H NMR (500 MHz, CDCl₃) δ 5.87 (ddt, J = 17.2, 10.7, 5.4 Hz, 3H, OCH₂CH=CH₂), 5.31– 5.07 (m, 6H, OCH₂CH=CH₂), 3.95 (dt, J = 5.4, 1.4 Hz, 6H, OCH₂CH=CH₂), 3.72 (s, 2H, CCH₂OH), 3.49 (s, 6H, C(CH₂OCH₂CH=CH₂)₃), 2.82 (br, 1H, OH); ¹³C NMR (126 MHz, CDCl₃) δ 134.7 (OCH₂CH=CH₂), 116.5 (OCH₂CH=CH₂), 72.4 (OCH₂CH=CH₂), 70.8 (C(CH₂OCH₂CH=CH₂)₃), 66.0 (CCH₂OH), 44.8 (C(CH₂O)₄); ESI-HRMS [M+K]⁺ calcd. for C₁₄H₂₄O₄K⁺ 295.1306, found 295.1297.

Tetra-O-allylpentaerythritol (15b): a colorless liquid with R_f 0.88 (hexanes:EtOAc 4:1, (KMn-O₄)); ¹H NMR (500 MHz, CDCl₃) δ 5.89 (ddt, J = 17.3, 10.6, 5.4 Hz, 4H, OCH₂CH=CH₂), 5.26 (dq, J = 17.2, 1.8 Hz, 4H, OCH₂CH=CH₂), 5.14 (dq, J = 10.5, 1.6 Hz, 4H, OCH₂CH=CH₂), 3.96 (dt, J = 5.3, 1.6 Hz, 8H, OCH₂CH=CH₂), 3.47 (s, 8H, CCH₂O); ¹³C NMR (126 MHz, CDCl₃) δ 135.1 (OCH₂CH=CH₂), 115.9 (OCH₂CH=CH₂), 72.1 (OCH₂CH=CH₂), 69.2 (C(CH₂OCH₂CH=CH₂)₄), 45.3 (C(CH₂O)₄); ESI-HRMS [M+K]⁺ calcd. for C₁₇H₂₈O₄K⁺ 335.1619, found 335.1610.

4.2.2. Tetraester core (16)

Tetra-O-allyl-pentaerythritol 15b (2.1 g, 7.08 mmol) was dissolved in anhydrous DMF (30 mL) and 2,2-dimethoxy-2-phenylacetophenone (DMPAP) (0.544 g, 2.13 mmol, 0.3 equiv) was added followed by methyl thioglycolate (6.3 mL, 70.8 mmol, 10 equiv). The reaction vessel then was flushed with Ar, sealed, and irradiated with UV light (λ = 254 nm) for 2.5 h. After such time the reaction mixture was allowed to cool down to the room temperature (20 °C), quenched with H₂O (40 mL), and extracted with Et₂O (3×50 mL). The combined organic layers were dried over Na₂SO₄, concentrated to dryness and co-evaporated with toluene (3×15 mL). The crude material was purified by flash column chromatography on a silica gel eluting with hexanes:EtOAc (0 → 30% EtOAc (v/v)) to give product 16 as a colorless viscous liquid (3.55 g, 69%) containing ~5% of anti-Markovnikoff regioisomers^a: R_f 0.2 (hexanes:EtOAc 7:3 (CAM)); ¹H NMR (500 MHz, CDCl₃) δ 3,72 (s, 12H, CO₂Me), 3.43 (t, J = 6.0 Hz, 8H, OCH₂CH₂CH₂S), 3.33 (s, 8H, CCH₂O ), 3.21 (s, 8H, SCH₂CO₂Me), 2.68 (t, J = 7.3 Hz, 8H, OCH₂CH₂CH₂S), 1.88 – 1.74 (m, 8H, OCH₂CH₂CH₂S), 1.24 (t, J = 6.9 Hz)¹; ¹³C NMR (126 MHz, CDCl₃) δ 171.1 (CO), 69.6 (CCH₂O/OCH₂CH₂CH₂S), 52.4 (CO₂Me), 45.5 (C(CH₂O)₄), 33.5 (SCH₂CO₂Me), 29.6 (OCH₂CH₂CH₂S), 29.2 (OCH₂CH₂CH₂S); ESI-HRMS [M+Na]⁺ calcd. for C₂₉H₅₂NaO₁₂S₄⁺ 743.2234, found 743.2209.

4.2.3. Tetracarboxylic acid core (17)

Tetraester 16 (3.5 g, 4.85 mmol) was dissolved in THF:H₂O (1:1) (140 mL) and KOH (1.36 g, 24.3 mmol, 5 equiv) was added, and the reaction mixture was stirred for 3 h. After such time the reaction mixture was acidified with 1M aq H₂SO₄ (30 mL), and extracted with EtOAc (3×40 mL). The combined organic layers were washed with brine (100 mL), dried over Na₂SO_4, and concentrated to dryness to give tetracarboxylic acid 17 as a syrup (3.2 g, quant.) containing ~4% of the anti-Markovnikoff regioisomers, which were retained throughout the synthesis of glucoclusters^b: ¹H NMR (500 MHz, DMSO-d₆) δ 3.38 (t, J = 6.0 Hz, 8H, OCH₂CH₂CH₂S), 3.26 (s, 8H, C(CH₂O)₄), 3.19 (s, 8H, SCH₂CO₂H), 2.60 (t, J = 7.3 Hz, 8H, OCH₂CH₂CH₂S), 1.73 (p, J = 6.5 Hz, 8H, OCH₂CH₂CH₂S), 1.18 (d, J = 6.9 Hz)^b; ¹³C NMR (126 MHz, DMSO-d₆) δ 171.7 (CO₂H), 69.2 (CCH₂O), 69.0 (OCH₂CH₂CH₂S), 45.2 (C(CH₂O)₄), 33.4 (SCH₂CO₂H), 28.8 (OCH₂CH₂CH₂S), 28.7 (OCH₂CH₂CH₂S); ESI-HRMS [M-2H]²⁻/2 calcd. for C₂₅H₄₂O₁₂S₄²⁻ 331.0785, found 331.0792

4.2.4. ((2-(tert-Butylcarbamoyl)ethoxy)ethyl) 2,3,4,6-Tetra-O-acetyl-1-thio-β-D-glucopyranoside (19)

1-S-Acetyl-2,3,4,6-tetra-O-acetyl-1-thio-β-D-glucopyranose 18 (250 mg, 0.615 mmol) and tert-butyl (2-(2-iodoethoxy)ethyl)carbamate (932.5 mg, 2.96 mmol, 4.8 equiv) were dissolved in anhydrous DMF (12 mL), treated with HNEt₂ (153 μL, 1.48 mmol, 2.4 equiv) and stirred for 4 h at 20 °C. After such time the reaction mixture was diluted with EtOAc (60 mL), washed with H₂O (50 mL), brine (50 mL), dried over Na₂SO₄, concentrated to dryness and co-evaporated with toluene (3×15 mL). The crude product was purified by flash column chromatography on silica gel eluting with hexanes:EtOAc (0 → 50% EtOAc (v/v)) to give compound 19 as a colorless syrup (247 mg, 73%): R_f 0.14 (hexanes:EtOAc 1:1 (H₂SO₄/EtOH)); [α]_D²⁰ + 14.7 (c 0.01, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 5.23 (t, J = 9.4 Hz, 1H, H-3), 5.08 (t, J = 9.7 Hz, 1H, H-4), 5.03 (t, J = 9.7 Hz, 1H, H-2), 4.93 (br, 1H, NH), 4.57 (d, J = 10.0 Hz, 1H, H-1), 4.25 (dd, J = 12.4, 4.9 Hz, 1H, H-6a), 4.20 – 4.07 (m, 1H, H-6b), 3.72 (ddd, J = 10.0, 5.0, 2.4 Hz, 1H, H-5), 3.65 (qt, J = 10.0, 6.5 Hz, 2H, SCH₂CH₂O), 3.51 (t, J = 5.2 Hz, 2H, OCH₂CH₂NHBoc), 3.31 (q, J = 5.5 Hz, 2H, OCH₂CH₂NHBoc), 2.93 (dt, J = 13.4, 6.7 Hz, 1H, SCH₂CH₂O), 2.78 (dt, J = 13.2, 6.4 Hz, 1H, SCH₂CH₂O), 2.09 (s, 3H, CH₃CO), 2.07 (s, 3H, CH₃CO), 2.03 (s, 3H, CH₃CO), 2.01 (s, 3H, CH₃CO), 1.61 (s, 2H, C(CH₃)₃^c), 1.45 (s, 9H, C(CH₃)₃); ¹³C NMR (126 MHz, CDCl₃) δ 170.6, 170.2, 169.4, 169.4 (4×CO), 83.5 (C-1), 76.0 (C-5), 73.8 (C-3), 70.6 (SCH₂CH₂O), 70.0 (OCH₂CH₂NHBoc), 70.0 (C-2), 68.3 (C-4), 62.1 (C-6), 60.4 (C(CH₃)₃), 40.4 (OCH₂CH₂NHBoc), 29.4 (SCH₂CH₂O), 28.4 (C(CH₃)₃), 20.8, 20.7, 20.6, 20.6 (4×CH₃CO); ESI-HRMS [M+Na]⁺ calcd. for C₂₃H₃₇NNaO₁₂S⁺ 574.1929, found 574.1919.

4.2.5. (2-(2-Aminoethoxy)ethyl) 2,3,4,6-Tetra-O-acetyl-1-thio-β-D-glucopyranoside trifluoroacetate salt (20)

Carbamate 19 (219 mg, 0.40 mmol) was dissolved in CH₂Cl₂ (20 mL), 90% aq TFA (4 mL) was added, and the reaction mixture was stirred for 40 min at 20 °C. After such time the reaction mixture was concentrated to dryness, co-evaporated with toluene (3×10 mL) and dried under high vacuum for 12 h to give compound 20 as a colorless syrup (223 mg, quant): [α]_D²⁰ + 5.2 (c 0.021, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.77 (br, 3H, NH₃), 5.23 (t, J = 9.3 Hz, 1H, H-3), 5.07 (t, J = 9.8 Hz, 1H, H-4), 5.00 (t, J = 9.6 Hz, 1H, H-2), 4.55 (d, J = 10.0 Hz, 1H, H-1), 4.26 – 4.14 (m, 2H, H-6a, H-6b), 3.81 – 3.66 (m, 5H, H-5/SCH₂CH₂O/OCH₂CH₂NH₃),3.25 (s, 2H, OCH₂CH₂NH₃), 2.93 (dt, J = 12.7, 6.2 Hz, 1H, SCH₂CH₂O), 2.80 (dt, J = 13.5, 6.1 Hz, 1H, SCH₂CH₂O), 2.08 (s, 3H, CH₃CO), 2.05 (s, 3H, CH₃CO), 2.03 (s, 3H, CH₃CO), 2.01 (s, 3H, CH₃CO); ¹³C NMR (126 MHz, CDCl₃) δ 171.0, 170.2, 169.8, 169.5 (4×CO), 83.3 (C-1), 76.0 (C-5), 73.6 (C-3), 70.1 (OCH₂CH₂NH₃), 70.0 (C-2), 68.2 (C-4), 66.0 (SCH₂CH₂O), 61.9 (C-6), 39.8 (OCH₂CH₂NH₃), 29.4 (SCH₂CH₂O), 20.6, 20.6, 20.5, 20.4 (4×CH₃CO); ¹⁹F NMR (470 MHz, CDCl₃) δ –75.70; ESI-HRMS [M+H]⁺ calcd. for C₁₈H₃₀NO₁₀S⁺ 452.1585, found 452.1572.

4.2.6. Peracetylated tetraamide (21)

Tetracarboxylic acid 17 (10 mg, 15.0 μmol) and trifluoroacetate 20 (42.5 mg, 75.2 μmol, 5 equiv) were dissolved in anhydrous DMF (6 mL), DIPEA (34 μL, 195.5 μmol, 13 equiv) was added followed by PyBOP (47 mg, 90.24 μmol, 6 equiv), and the reaction mixture was stirred for 16 h at 20 °C. After such time H₂O (10 mL) was added and the reaction mixture was extracted with EtOAc (4×10 mL). The combined organic layers were washed with H₂O (30 mL), brine (30 mL), dried over Na₂SO₄, concentrated to dryness and co-evaporated with toluene (3×10 mL). The crude product was purified by a flash column chromatography on a silica gel eluting with hexanes:acetone (10 → 100% acetone (v/v)) to give compound 21 as a colorless syrup (29 mg, 80%) containing ~4% of the anti-Markovnikoff regioisomers^d: R_f 0.21 (Acetone (H₂SO₄/EtOH)); ¹H NMR (500 MHz, CDCl₃) δ 7.17 (br, 4H, NH), 5.23 (t, J = 9.4 Hz, 4H, H-3), 5.08 (t, J = 9.8 Hz, 4H, H-4), 5.03 (t, J = 9.7 Hz, 4H, H-2), 4.57 (d, J = 10.0 Hz, 4H, H-1), 4.25 (dd, J = 12.4, 4.9 Hz, 4H, H-6a), 4.15 (dd, J = 12.4, 2.5 Hz, 4H, H-6b), 3.74 (ddd, J = 10.2, 4.9, 2.4 Hz, 4H, H-5), 3.69 (d, J = 7.1 Hz, 8H, SCH₂CH₂O), 3.55 (t, J = 5.3 Hz, 8H, OCH₂CH₂NH), 3.50 – 3.40 (m, 16H, OCH₂CH₂CH₂S/OCH₂CH₂NH), 3.33 (s, 8H, SCH₂CONH), 3.23 (s, 8H, C(CH₂O)₄), 2.93 (dt, J = 13.4, 6.6 Hz, 4H, SCH₂CH₂O), 2.80 (dt, J = 13.3, 6.5 Hz, 4H, SCH₂CH₂O), 2.62 (t, J = 7.4 Hz, 8H, OCH₂CH₂CH₂S), 2.09 (s, 12H, CH₃CO), 2.06 (s, 12H, CH₃CO), 2.03 (s, 12H, CH₃CO), 2.01 (s, 12H, CH₃CO), 1.82 (p, J = 6.4 Hz, 8H, OCH₂CH₂CH₂S), 1.26 (s)^d; ¹³C NMR (126 MHz, CDCl3) δ 170.6, 170.2, 169.5, 169.5, 169.2 (CO), 83.5 (C-1), 76.0 (C-5), 73.9 (C-3), 70.5 (SCH₂CH₂O), 69.9 (C-2), 69.7 (C(CH₂O)₄), 69.6 (OCH₂CH₂CH₂S), 69.5 (OCH₂CH₂NH), 68.4 (C-4), 62.2 (C-6), 39.5 (OCH₂CH₂NH), 36.1 (SCH₂CONH), 29.9 (OCH₂CH₂CH₂S), 29.5 (SCH₂CH₂O), 29.3 (OCH₂CH₂CH₂S), 20.8, 20.8, 20.7, 20.7 (CH₃CO); ESI-HRMS [M+2Na]²⁺/2 calcd. for C₉₇H₁₅₂N₄Na₂O₄₈S₈²⁺ 1221.3563, found 1221.3525.

4.2.7. Tetraamide (9)

Peracetylated tetraamide 21 (33 mg, 13.7 μmol) was suspended in anhydrous MeOH (1 mL) and NaOMe (freshly prepared 1M solution in MeOH) (5.5 μL, 5.5 μmol, 0.4 eq) was added. The reaction mixture then was stirred until full consumption of the starting material was detected by LCMS. The reaction mixture was quenched with Amberlite IR-120 H⁺ (washed with MeOH, CH₂Cl₂) until pH 6. The reaction mixture was filtered, and the filtrate was concentrated to dryness and the crude product was purified by flash column chromatography on a C18 silica gel eluting with H₂O:MeOH (20 → 50% MeOH (v/v)) to give the product as a colorless foam (13.4 mg, 57%): R_f 0.54 (C18 plate, H₂O:MeOH 1:1 (v/v) (H₂SO₄/EtOH)); ¹H NMR (900 MHz, CD₃OD) δ 4.43 (d, J = 9.8 Hz, 4H, H-1), 3.87 (dd, J = 12.0, 1.6 Hz, 4H, H-6), 3.73 (dt, J = 10.0, 6.4 Hz, 4H, SCH₂CH₂O), 3.69 (dt, J = 10.0, 6.6 Hz, 4H, SCH₂CH₂O), 3.67 – 3.64 (m, 4H, H-6), 3.58 (tt, J = 5.6, 3.1 Hz, 8H, OCH₂CH₂NH), 3.49 (t, J = 5.9 Hz, 8H, OCH₂CH₂CH₂S), 3.41 (t, J = 5.4 Hz, 8H, OCH₂CH₂NH), 3.39 – 3.34 (m, 12H, H-3, C(CH₂O)₄), 3.31 – 3.28 (m, 8H, H-4, H-5)^e, 3.23 (s, 8H, SCH₂CONH), 3.21 (dd, J = 8.8 Hz, 4H, H-2), 2.96 (dt, J = 13.4, 6.6 Hz, 4H, SCH₂CH₂O), 2.84 (dt, J = 13.4, 6.5 Hz, 4H, SCH₂CH₂O), 2.68 (t, J = 7.2 Hz, 8H, OCH₂CH₂CH₂S), 1.85 (p, J = 6.4 Hz, 8H, OCH₂CH₂CH₂S), 1.28 (d, J = 6.8 Hz)^f; ¹³C NMR (226 MHz, CD₃OD) δ 172.7 (CONH), 87.3 (C-1), 82.1 (C-4), 79.6 (C-3), 74.5 (C-2), 71.9 (SCH₂CH₂O), 71.6 (C-5), 70.8 (OCH₂CH₂CH₂S), 70.7 (C(CH₂O)₄), 70.2 (OCH₂CH₂NH), 63.0 (C-6), 46.8 (q-C), 40.7 (OCH₂CH₂NH), 36.4 (SCH₂CONH), 30.5 (SCH₂CH₂O), 30.5 (OCH₂CH₂CH₂S), 30.5 (OCH₂CH₂CH₂S); ESI-HRMS [M+2Na]²⁺/2 calcd. for C₆₅H₁₂₀N₄Na₂O₃₂S₈²⁺ 885.2718, found 885.2710.

4.2.8. ((2-(tert-Butylcarbamoyl)ethoxy)ethyl) 2,3,4,6,-Tetra-O-acetyl-1,5-dithio-β-D-glucopyranoside (23)

1-S-Acetyl-2,3,4,6-tetra-O-acetyl-1,5-dithio-β-D-glucopyranose 22 (100 mg, 0.236 mmol) and tert-butyl (2-(2-iodoethoxy)ethyl)carbamate [59] (387 mg, 1.2 mmol, 5.2 equiv) were dissolved in anhydrous DMF (12 mL) treated with HNEt₂ (65 μL, 0.619 mmol, 2.6 equiv) and stirred for 4 h at 20 °C. After such time the reaction mixture was diluted with EtOAc (50 mL), washed with H₂O (30 mL), brine (30 mL), dried over Na₂SO₄, concentrated to dryness and co-evaporated with toluene (3×15 mL). The crude product was purified by flash column chromatography on a silica gel eluting with hexanes:EtOAc (10 → 60% EtOAc (v/v)) to give compound 23 as a colorless syrup (122 mg, 91%): R_f 0.43 (hexanes:EtOAc 1:1 (H₂SO₄/EtOH)); [α]_D²² – 3.06 (c 0.047, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 5.26 (dd, J = 10.7, 9.6 Hz, 1H, H-4), 5.15 (dd, J = 10.7, 9.4 Hz, 1H, H-2), 5.06 (t, J = 9.5 Hz, 1H, H-3), 4.97 (br, 1H, NH), 4.26 (dd, J = 12.0, 5.7 Hz, 1H, H-6a), 4.15 – 4.09 (m, 1H, H-6b), 3.96 (d, J = 10.7 Hz, 1H, H-1), 3.69 – 3.58 (m, 2H, SCH₂CH₂O), 3.51 (t, J = 5.2 Hz, 2H, OCH₂CH₂NHBoc), 3.35 – 3.26 (m, 3H, H-5/OCH₂CH₂NHBoc), 2.92 (dt, J = 12.8, 6.3 Hz, 1H, SCH₂CH₂O), 2.83 (dt, J = 13.2, 6.3 Hz, 1H, SCH₂CH₂O), 2.08 (s, 3H, CH₃CO), 2.07 (s, 3H, CH₃CO), 2.02 (s, 3H, CH₃CO), 2.00 (s, 3H, CH₃CO), 1.45 (s, 9H, C(CH₃)₃); ¹³C NMR (126 MHz, CDCl3) δ 170.4, 169.6, 169.4, 169.2 (4×CO), 155.9 (CONH), 79.2 (C(CH₃)₃), 74.4 (C-3), 73.2 (C-2), 71.7 (C-4), 70.4 (SCH₂CH₂O), 70.0 (OCH₂CH₂NHBoc), 61.2 (C-6), 47.9 (C-1), 44.3 (C-5), 40.3 (OCH₂CH₂NHBoc), 30.3 (SCH₂CH₂O), 28.3 (C(CH₃)₃), 20.5, 20.5, 20.40, 20.35 (4×CH₃CO); ESI-HRMS [M+Na]⁺ calcd. for C₂₃H₃₇NNaO₁₁S₂⁺ 590.1700, found 590.1690.

4.2.9. (2-(2-Aminoethoxy)ethyl) 2,3,4,6,-Tetra-O-acetyl-1,5-dithio-β-D-glucopyranoside trifluoroacetate salt (24)

Carbamate 23 (122 mg, 0.215 mmol) was dissolved in CH₂Cl₂ (40 mL) and 90% aq TFA (10 mL) was added, the reaction mixture then was stirred for 40 min at 20 °C. After such time the reaction mixture was concentrated to dryness, co-evaporated with toluene (3×10 mL) and dried on a high vacuum for 12 h to give product 24 as a syrup (120 mg, 96%): [α]_D²⁰ – 1.75 (c 0.048, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.79 (br, 3H, NH₃), 5.25 (t, J = 10.0 Hz, 1H, H-4), 5.16 – 5.03 (m, 2H, H-2/H-3), 4.31 – 4.21 (m, 1H, H-6a), 4.13 (dd, J = 12.1, 3.2 Hz, 1H, H-6b), 3.95 (d, J = 10.2 Hz, 1H, H-1), 3.74 – 3.65 (m, 4H, OCH₂CH₂NH₃/SCH₂CH₂O), 3.33 (dt, J = 9.5, 4.3 Hz, 1H, H-5), 3.25 (s, 2H, OCH₂CH₂NH₃), 2.97 – 2.90 (m, 1H, SCH₂CH₂O), 2.88 – 2.82 (m, 1H, SCH₂CH₂O), 2.07 (s, 3H, CH₃CO), 2.06 (s, 3H, CH₃CO), 2.03 (s, 3H, CH₃CO), 2.00 (s, 3H, CH₃CO); ¹³C NMR (126 MHz, CDCl₃) δ 170.9, 170.1, 169.9, 169.5 (4×CO), 74.3 (C-3), 73.4 (C-2), 71.8 (C-4), 70.3 (SCH₂CH₂O), 66.1 (OCH₂CH₂NH₃), 61.3 (C-6), 47.8 (C-1), 44.4 (C-5), 40.0 (OCH₂CH₂NH₃), 30.2, 20.6, 20.5, 20.4 (4×CH₃CO); ¹⁹F NMR (470 MHz, CDCl₃) δ –75.72; ESI-HRMS [M+H]⁺ calcd. for C₁₈H₃₀NO₉S₂⁺ 468.1357, found 468.1346.

4.2.10. Peracetylated tetraamide (25)

Tetracarboxylic acid 17 (10 mg, 15.04 μmol) and trifluoroacetate 24 (43.7 mg, 75.2 μmol, 5 equiv) were dissolved in anhydrous DMF (7 mL) followed by addition of DIPEA (34 μL, 195.5 μmol, 13 equiv) and PyBOP (47 mg, 90.24 μmol, 6 equiv). The reaction mixture then was stirred for 16 h at 20 °C. After such time H₂O (10 mL) was added and the reaction mixture was extracted with EtOAc (4×10 mL). The combined organic layers were washed with H₂O (30 mL), brine (30 mL), dried over Na₂SO₄, concentrated to dryness and co-evaporated with toluene (3×10 mL). The crude product was purified by a flash column chromatography on a silica gel eluting with hexanes:acetone (10 → 60% acetone (v/v)) to give compound 25 as a white solid (29 mg, 78%): R_f 0.08 (Acetone (H₂SO₄/EtOH)); ¹H NMR (500 MHz, CDCl₃) δ 7.17 (t, J = 5.6 Hz, 4H, NH), 5.25 (t, J = 10.2 Hz, 4H, H-4), 5.14 (t, J = 10.0 Hz, 4H, H-2), 5.06 (t, J = 9.5 Hz, 4H, H-3), 4.26 (dd, J = 12.0, 5.7 Hz, 4H, H-6a), 4.11 (dd, J = 12.0, 3.4 Hz, 4H. H-6b), 3.94 (d, J = 10.6 Hz, 4H, H-1), 3.70 – 3.59 (m, 8H, SCH₂CH₂O), 3.54 (t, J = 5.1 Hz, 8H, OCH₂CH₂NH), 3.51 – 3.40 (m, 16H, OCH₂CH₂CH₂S/OCH₂CH₂NH), 3.32 (s, 12H, H-5/SCH₂CONH), 3.23 (s, 8H, (C(CH₂O)₄)), 2.93 (dt, J = 12.9, 6.3 Hz, 4H, SCH₂CH₂O), 2.83 (dt, J = 12.9, 6.3 Hz, 4H, SCH₂CH₂O), 2.61 (t, J = 7.3 Hz, 8H, OCH₂CH₂CH₂S), 2.07 (s, 12H, CH₃CO), 2.06 (s, 12H, CH₃CO), 2.02 (s, 12H, CH₃CO), 1.99 (s, 12H, CH₃CO), 1.83 (p, J = 6.2 Hz, 8H, OCH₂CH₂CH₂S), 1.25 (d, J = 3.8 Hz)^g; ¹³C NMR (126 MHz, CDCl3) δ 170.5, 169.7, 169.5, 169.4, 169.2 (5×CO), 74.4 (C-3), 73.2 (C-2), 71.8 (C-4), 70.3 (SCH₂CH₂O), 69.6 ((C(CH₂O))₄), 69.5 (OCH₂CH₂CH₂S/OCH₂CH₂NH), 61.3 (C-6), 47.9 (C-1), 44.4 (C-5), 39.5 (OCH₂CH₂NH), 36.1 (SCH₂CONH), 30.3 (OCH₂CH₂CH₂S), 29.8 (SCH₂CH₂O), 29.3 (OCH₂CH₂CH₂S), 20.7, 20.6, 20.5, 20.5 (4×CH₃CO); ESI-HRMS [M+2Na]²⁺/2 calcd. for C₉₇H₁₅₂N₄Na₂O₄₄S₁₂²⁺ 1253.3106, found 1253.3070.

4.2.11. Tetraamide (10)

Peracetylated tetraamide 25 (8 mg, 3.3 μmol) was suspended in anhydrous MeOH (1 mL) and NaOMe (freshly prepared 1M solution in MeOH) (6.6 μL, 6.6 μmol, 2 eq) was added. The reaction mixture then was stirred until full consumption of the starting material was detected by LCMS. After that the reaction mixture was quenched with Amberlite IR-120 H⁺ (washed with MeOH, CH₂Cl₂) until pH 6. The reaction mixture then was filtered, and the filtrate was concentrated to dryness. The crude product was purified by column chromatography on a C18 silica gel eluting with H₂O:MeCN (H₂O:MeCN 1:1 (v/v)) to give product as a white foam (4.1 mg, 70%): R_f 0.68 (H₂O:MeCN 1:1, (H₂SO₄/EtOH)); ¹H NMR (900 MHz, D₂O) δ 3.88 – 3.80 (m, 8H, H-1, H-6a), 3.71 (dd, J = 11.9, 6.2 Hz, 4H, H-6b), 3.67 (t, J = 6.2 Hz, 8H, SCH₂CH₂O), 3.56 (t, J = 5.4 Hz, 8H, OCH₂CH₂NH), 3.52 – 3.45 (m, 12H, OCH₂CH₂CH₂S, H-4), 3.38 (t, J = 9.6 Hz, 4H, H-2), 3.36 – 3.30 (m, 16H, C(CH₂O)₄, OCH₂CH₂NH), 3.23 – 3.17 (m, 12H, H-3, SCH₂CONH), 2.94 (ddd, J = 9.8, 6.1, 3.2 Hz, 4H, H-5), 2.90 (dt, J = 12.6, 6.1 Hz, 4H, SCH₂CH₂O), 2.85 (dt, J = 13.2, 6.1 Hz, 4H, SCH₂CH₂O), 2.57 (t, J = 7.1 Hz, 8H, OCH₂CH₂CH₂S), 1.83 – 1.72 (m, 8H, OCH₂CH₂CH₂S); ¹³C NMR (226 MHz, D₂O) δ 172.7 (CO), 78.1 (C-3), 75.5 (C-2), 73.1 (C-4), 69.9 (SCH₂CH₂O), 69.6 (OCH₂CH₂CH₂S), 69.2 (C(CH₂O)₄), 68.6 (OCH₂CH₂NH), 60.2 (C-6), 49.1 (C-1), 48.7 (C-5), 45.1 (q-C), 39.3 (OCH₂CH₂NH), 35.2 (SCH₂CONH), 30.4 (SCH₂CH₂O), 29.0 (OCH₂CH₂CH₂S), 28.4 (OCH₂CH₂CH₂S); ESI-HRMS [M+2Na]²⁺/2 calcd. for C₆₅H₁₂₀N₄Na₂O₂₈S₁₂²⁺ 917.2261, found 917.2259.

4.2.12. ((2-(tert-Butylcarbamoyl)ethoxy)ethyl) 2,3,4,6-Tetra-O-acetyl-5-thio-β-D-glucopyranosyl-(1→3)-2,4,6-tri-O-acetyl-1,3,5-trideoxy-1,3,5-trithio-β-D-glucopyranoside (27)

Thioacetate 26 (100 mg, 0.135 mmol) and tert-butyl (2-(2-iodoethoxy)ethyl)carbamate[59] (212 mg, 0.67 mmol, 5 equiv) were dissolved in anhydrous DMF (1 mL) followed by addition of HNEt₂ (35 μL, 0.336 mmol, 2.5 equiv) and the reaction mixture then was stirred for 50 min at 20 °C. After such time the reaction mixture was diluted with EtOAc (50 mL), washed with H₂O (2×10 mL), brine (2×10 mL), dried over Na₂SO₄, concentrated to dryness and co-evaporated with toluene (3×15 mL). The crude product was purified by flash column chromatography on a silica gel eluting with hexanes:EtOAc (10 → 60%(EtOAc (v/v)) to give compound 27 as a colorless syrup (108 mg, 90%): R_f 0.44 (hexanes:EtOAc 2:3 (H₂SO₄/EtOH)); [α]_D²² – 0.94 (c 0.0053, CHCl₃); ¹H NMR (900 MHz, CDCl₃) δ 5.29 (t, J = 9.9 Hz, 1H, H-4a), 5.19 (t, J = 10.5 Hz, 1H, H-2b), 5.10 (t, J = 10.4 Hz, 1H, H-2a), 5.03 – 4.95 (m, 2H, H-4b, H-3a), 4.93 (br, 1H, NH), 4.27 (dt, J = 12.1, 3.2 Hz, 1H, H-6a), 4.20 (dd, J = 12.1, 5.7 Hz, 1H, H-6b), 4.17 – 4.11 (m, 2H, H-6b, H-6a), 4.05 (d, J = 11.1 Hz, 1H, H-1a), 3.79 (d, J = 10.4 Hz, 1H, H-1b), 3.70 – 3.57 (m, 2H, SCH₂CH₂O), 3.51 (s, 2H, OCH₂CH₂NHBoc), 3.32 (s, 2H, OCH₂CH₂NHBoc), 3.28 (s, 1H, H-5b), 3.21 (d, J = 10.9 Hz, 1H, H-5a), 3.00 – 2.89 (m, 2H, H-3b, SCH₂CH₂O), 2.83 (dd, J = 13.9, 7.5 Hz, 1H, SCH₂CH₂O), 2.22 (s, 3H, CH₃CO), 2.15 (s, 3H, CH₃CO), 2.09 (s, 3H, CH₃CO), 2.07 (s 3H, CH₃CO), 2.03 (s, 6H, 2×CH₃CO), 1.99 (s, 3H, CH₃CO), 1.60 (s, 4H, C(CH₃)₃^h), 1.46 (s, 9H, C(CH₃)₃); ¹³C NMR (226 MHz, CDCl3) δ 170.6, 170.5, 169.7, 169.4, 169.2, 169.2, 169.1 (7×CH3CO), 155.9 (CONH), 79.4 (C(CH₃)₃), 75.8 (C-2b), 74.4 (C-3a), 72.8 (C-2a), 71.8 (C-4a), 70.4 (SCH₂CH₂O), 70.3 (OCH₂CH₂NHBoc), 70.0 (C-4b), 61.9 (C-6a), 61.1 (C-6b), 55.2 (C-3b), 50.6 (C-1a), 49.7 (C-1b), 46.8 (C-5b), 44.5 (C-5a), 40.3 (OCH₂CH₂NHBoc), 30.0 (SCH₂CH₂O), 28.4 (C(CH₃)₃), 21.0, 20.9, 20.7, 20.6, 20.5, 20.4, 20.2 (7×CH₃CO); ESI-HRMS [M+Na]⁺ calcd. for C₃₅H₅₃NNaO₁₇S₄⁺ 910.2089, found 910.2050.

4.2.13. (2-(2-Aminoethoxy)ethyl) 2,3,4,6-Tetra-O-acetyl-5-thio-β-D-glucopyranosyl-(1→3)-2,4,6-tri-O-acetyl-1,3,5-trideoxy-1,3,5-trithio-β-D-glucopyranoside trifluoroacetate salt (28).

Carbamate 27 (99 mg, 0.11 mmol) was dissolved in CH₂Cl₂ (16 mL) and 90% aq TFA (4 mL) was added. The reaction mixture then was stirred for 40 min at 20 °C. After such time the reaction mixture was concentrated to dryness, co-evaporated with toluene (3×10 mL) and dried on a high vacuum for 12 h to give compound 28 as a colorless syrup (98 mg, 98%): [α]_D²⁰ – 6.5 (c 0.0023, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 8.08 (br, 2H, NH), 5.29 (t, J = 10.0 Hz, 1H, H-4a), 5.17 (t, J = 10.1 Hz, 1H, H-2b), 5.10 (t, J = 10.2 Hz, 1H, H-2a), 5.05 – 4.93 (m, 2H, H-3a, H-4b), 4.27 (dd, J = 12.0, 4.7 Hz, 1H, H-6a), 4.23 – 4.11 (m, 3H, 2×H-6b, H-6a), 4.05 (d, J = 10.9 Hz, 1H, H-1a), 3.80 (d, J = 10.3 Hz, 1H, H-1b), 3.77 – 3.63 (m, 4H, SCH₂CH₂O, OCH₂CH₂NH₃), 3.34 – 3.17 (m, 4H, H-5b, H-5a, OCH₂CH₂NH₃), 3.02 – 2.91 (m, 2H, H-3b, SCH₂CH₂O), 2.89 – 2.80 (m, 1H, SCH₂CH₂O), 2.22 (s, 3H, CH₃CO), 2.15 (s, 3H, CH₃CO), 2.09 (s, 3H, CH₃CO), 2.07 (s, 3H, CH₃CO), 2.02 (s, 6H, 2×CH₃CO), 2.00 (s, 3H, CH₃CO); ¹³C NMR (126 MHz, CDCl₃) δ 170.8, 170.6, 170.4, 169.8, 169.6, 169.4, 169.2, 169.2 (7×CH₃CO), 75.8 (C-2b), 74.4 (C-3a), 72.8 (C-2a), 71.8 (C-4a), 70.4 (C-4b), 70.0 (SCH₂CH₂O), 66.1 (OCH₂CH₂NH₃), 61.9 (C-6a), 61.1 (C-6b), 55.1 (C-3b), 50.6 (C-1a), 49.4 (C-1b), 46.8 (C-5b), 44.4 (C-5a), 39.8 (OCH₂CH₂NH₃), 30.0 (SCH₂CH₂O), 20.9, 20.9, 20.6, 20.5, 20.4, 20.4, 20.2 (7×CH₃CO); ¹⁹F NMR (470 MHz, CDCl₃) δ –75.44; ESI-HRMS [M+H]⁺ calcd. for C₃₀H₄₆NO₁₅S₄⁺ 788.1745, found 788.1726.

4.2.14. Peracetylated tetraamide (29)

Tetracarboxylic acid 17 (11 mg, 16.5 μmol) and trifluoroacetate 28 (75 mg, 82.5 μmol, 5 equiv) were dissolved in anhydrous DMF (7 mL) followed by addition of DIPEA (37 μL, 214.5 μmol, 13 equiv) and PyBOP (52 mg, 99 μmol, 6 equiv). The reaction mixture then was stirred for 16 h at 20 °C. After such time H₂O (10 mL) was added and the reaction mixture was extracted with EtOAc (4×20 mL). The combined organic layers were washed with H₂O (2×20 mL), brine (2×20 mL), dried over Na₂SO₄, concentrated to dryness and co-evaporated with toluene (3×10 mL). The crude product was purified by a flash column chromatography on a silica gel eluting with CH₂Cl₂:MeOH (0 → 5% MeOH (v/v)) to give compound 29 as a yellowish syrup (41 mg, 66%): R_f 0.52 (CH₂Cl₂:MeOH (95:5) (v/v) (H₂SO₄/EtOH)); ¹H NMR (500 MHz, CDCl₃) δ 7.15 (t, J = 5.7 Hz, 4H, NH), 5.28 (dd, J = 10.7, 9.5 Hz, 4H, H-4a), 5.17 (t, J = 10.4 Hz, 4H, H-2b), 5.08 (dd, J = 11.0, 9.5 Hz, 4H, H-2a), 5.04 – 4.89 (m, 8H, H-3a, H-4b), 4.26 (dd, J = 12.0, 5.0 Hz, 4H, H-6a), 4.20 (dd, J = 12.0, 5.7 Hz, 4H, H-6a), 4.16 – 4.09 (m, 8H, H-6b), 4.05 (d, J = 11.0 Hz, 4H, H-1a), 3.77 (d, J = 10.5 Hz, 4H, H-1b), 3.63 (qt, J = 10.1, 6.3 Hz, 8H, SCH₂CH₂O), 3.54 (t, J = 5.2 Hz, 8H, OCH₂CH₂NH), 3.47 (t, J = 5.3 Hz, 8H, OCH₂CH₂NH), 3.44 (q, J = 6.0, 5.2 Hz, 8H, OCH₂CH₂CH₂S), 3.32 (s, 8H, (C(CH₂O)₄), 3.28 (td, J = 6.0, 2.9 Hz, 4H, H-5b), 3.22 (s, 8H, SCH₂CONH), 3.20 (dd, J = 5.0, 3.7 Hz, 4H, H-5a), 2.96 – 2.89 (m, 8H, SCH₂CH₂O, H-3a), 2.82 (dt, J = 12.8, 6.3 Hz, 4H, SCH₂CH₂O), 2.61 (t, J = 7.3 Hz, 8H, OCH₂CH₂CH₂S), 2.21 (s, 12H, CH₃CO), 2.14 (s, 12H, CH₃CO), 2.08 (s, 12H, CH₃CO), 2.06 (s, 12H, CH₃CO), 2.02 (s, 24H, CH₃CO), 1.98 (s, 12H, CH₃CO), 1.85 – 1.79 (m, 8H, OCH₂CH₂CH₂S), 1.25 (d, J = 3.9 Hz)ⁱ; ¹³C NMR (126 MHz, CDCl₃) δ 170.5, 170.5, 169.7, 169.4, 169.2, 169.1, 169.1, 169.1 (CO), 75.6 (C-2b), 74.4 (C-3a), 72.8 (C-2a), 71.8 (C-4a), 70.4 (C-4b), 70.2 (SCH₂CH₂O), 69.6 ((C(CH₂O))₄), 69.5 (OCH₂CH₂CH₂S), 69.3 (OCH₂CH₂NH), 61.9 (C-6a), 61.1 (C-6b), 55.2 (C-3b), 50.6 (C-1a), 49.6 (C-1b), 46.8 (C-5b), 45.4 (q-C), 44.4 (C-5a), 39.4 (OCH₂CH₂NH), 36.0 (SCH₂CONH), 29.9 (SCH₂CH₂O), 29.8 (OCH₂CH₂CH₂S), 29.2 (OCH₂CH₂CH₂S), 21.0, 20.9, 20.6, 20.6, 20.4, 20.4, 20.2 (7×CH₃CO); ESI-HRMS [M+2Na]²⁺/2 calcd. for C₁₄₅H₂₁₆N₄Na₂O₆₈S₂₀²⁺ 1893.8900, found 1893.8904.

4.2.15. Tetraamide (11)

Peracetylated teraamide 29 (5 mg, 1.33 μmol) was suspended in anhydrous MeOH (0.3 mL) and NaOMe (1.0 mg, 19.95 μmol, 15 equiv) was added, and the reaction mixture was stirred for 24 h at 20 °C. After such time the reaction mixture was quenched with amberlite IR-120 H⁺ washed with MeOH, CH₂Cl₂) until pH 6, filtered and the filter cake was additionally washed with MeOH (10 mL). The filtrate was concentrated to dryness, redissolved in H₂O (10 mL) and washed with EtOAc (2×10 mL). The aqueous layer was concentrated to dryness and co-evaporated with EtOH. The crude product was purified by column chromatography on a C18 silica gel eluting with MeCN:H₂O (1:1) to give tetraamide 11 as a colorless syrup (2.6 mg, 76%): R_f 0.68 (C18, MeCN:H₂O (1:1), (H₂SO₄/EtOH)); ¹H NMR (900 MHz, D₂O) δ 4.14 (d, J = 10.4 Hz, 4H, H-1a), 4.02 – 3.90 (m, 12H, H-6a, H-6b, H-1b), 3.90 – 3.81 (m, 8H, H-6a, H-6b), 3.80 – 3.76 (m, 8H, SCH₂CH₂O), 3.70 – 3.55 (m, 28H, OCH₂CH₂NH, OCH₂CH₂CH₂S, H-4a, H-4b, H-2b), 3.51 (t, J = 9.8 Hz, 8H, H-2a), 3.48 – 3.41 (m, 16H, C(CH₂O)₄, OCH₂CH₂NH), 3.34 (d, J = 9.6 Hz, 8H, SCH₂CONH), 3.15 – 3.08 (m, 4H, H-5a), 3.06 – 2.95 (m, 12H, SCH₂CH₂O, H-5b), 2.89 (t, J = 10.3 Hz, 4H, H-3b), 2.69 (t, J = 7.3 Hz, 8H, OCH₂CH₂CH₂S), 1.94 – 1.82 (m, 9H, OCH₂CH₂CH₂S)^j; ¹³C NMR (226 MHz, D₂O) δ 172.6 (CONH), 78.0 (C-3a), 76.3 (C-2a), 75.6 (C-4a), 72.9 (C-2b), 71.1 (C-4b), 69.9 (SCH₂CH₂O), 69.6 (OCH₂CH₂CH₂S), 69.2 (C(CH₂O)), 68.7 (OCH₂CH₂NH), 62.5 (q-C), 60.7 (C-6a), 60.1 (C-6b), 59.6 (C-3b), 50.8 (C-1b, C-5a,), 49.2 (C-1a, C-5b), 48.8 (OCH₂CH₂NH), 39.4 (OCH₂CH2NH2), 35.3 (SCH₂CONH), 30.6 (SCH₂CH₂O), 29.0 (OCH₂CH₂CH₂S), 28.5 (OCH₂CH₂CH₂S), 23.2^j; ESI-HRMS [M+2Na]²⁺/2 calcd. for C₈₉H₁₆₀N₄Na₂O₄₀S₂₀²⁺ 1305.2404, found 1305.2424.

4.2.16. ((2-(tert-Butylcarbamoyl)ethoxy)ethyl) 2,3,4,6-tetra-O-acetyl-5-thio-β-D-glucopyranosyl-(1→3)-2,4,6-tri-O-acetyl-1,3,5-trideoxy-1,3,5-trithio-β-D-(1→3)-2,4,6-tri-O-acetyl-1,3,5-trideoxy-1,3,5-trithio-β-D-glucopyranoside (31)

Thioacetate 30 (128 mg, 0.12 mmol) and tert-butyl (2-(2-iodoethoxy)ethyl)carbamate[59] (190 mg, 0.6 mmol, 5 equiv) were dissolved in anhydrous DMF (0.9 mL) followed by addition of HNEt₂ (31 μL, 0.300 mmol, 2.5 equiv) and the reaction mixture then was stirred for 1.5 h at 20 °C. After such time the reaction mixture was diluted with EtOAc (60 mL), washed with H₂O (2×10 mL), brine (2×10 mL), dried over Na₂SO₄, concentrated to dryness and co-evaporated with toluene (3× mL). The crude product was purified by flash column chromatography on a silica gel eluting with CH₂Cl₂:Et₂O (0 → 40% Et₂O (v/v)) to give compound 31 as a colorless syrup (83 mg, 57%): R_f 0.46 (CH₂Cl₂:Et₂O 7:3 (H₂SO₄/EtOH)); [α]_D²² –2.7 (c 0.00073, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 5.31 (dd, J = 10.3, 5.4 Hz, 1H, H-4a), 5.18 (t, J = 10.5 Hz, 1H, H-2c), 5.18 (t, J = 10.5 Hz, 1H, H-3a), 5.15 – 5.01 (m, 2H, H-2a, H-2b), 4.96 (t, J = 10.6 Hz, 2H, H-4b, H-4c), 4.74 (t, J = 10.2 Hz, 1H, H-6a), 4.27 – 4.09 (m, 6H, H-6a, H-6b, H-6c, H-1a), 4.00 (d, J = 9.7 Hz, 1H, H-1b), 3.80 (d, J = 9.7 Hz, 1H, H-1c), 3.63 (d, J = 6.3 Hz, 2H, SCH₂CH₂O), 3.52 (t, J = 5.0 Hz, 2H, OCH₂CH₂NHBoc), 3.35 – 3.18 (m, 4H, OCH₂CH₂NHBoc, H-5a, H-5b), 3.10 (s, 1H, H-5c), 3.03 – 2.88 (m, 3H, H-3b, H-3c, SCH₂CH₂O), 2.87 – 2.77 (m, 1H, SCH₂CH₂O), 2.25 (s, 3H, CH₃CO), 2.18 (s, 3H, CH₃CO), 2.17 (s, 3H, CH₃CO), 2.13 (s, 6H, 2×CH₃CO), 2.07 (s, 2H, CH₃CO), 2.06 (s, 3H, CH₃CO), 2.05 (s, 2H, CH₃CO), 2.02 (s, 3H, CH₃CO), 2.00 (s, 2H, CH₃CO), 1.64 (s, 1H, C(CH₃)₃^k), 1.46 (s, 9H, C(CH₃)₃); ¹³C NMR (126 MHz, CDCl₃) δ 170.6, 169.5, 169.3, 169.3, 169.3, 169.3, 169.3, 169.2, 169.2, 169.1 (10×CH₃CO), 155.9 (CONH), 79.3 (C(CH₃)₃), 75.8 (C-2c), 75.5 (C-2b), 72.6 (C-2a), 72.6 (C-4a), 70.6 (C-3a), 70.5 (C-4b, C-4c), 70.3 (SCH₂CH₂O), 70.0 (OCH₂CH₂NHBoc), 62.0 (C-6b), 61.9 (C-6c), 59.5 (C-6a), 54.9 (C-3b), 54.7 (C-3c), 52.3 (C-1b), 49.6 (C-1c), 47.8 (C-1a), 46.9 (C-5a), 43.3 (C-5b), 40.3 (C-5c), 30.3 (OCH₂CH₂NHBoc), 29.9 (SCH₂CH₂O), 28.4 (C(CH₃)), 21.1, 20.9, 20.9, 20.8, 20.7, 20.6, 20.6, 20.4, 20.2 (10×CH₃CO); ESI-HRMS [M+Na]⁺ calcd. for C₄₇H₆₉NNaO₂₃S₆⁺ 1230.2477, found 1230.2463.

4.2.17. (2-(2-Aminoethoxy)ethyl) 2,3,4,6-tetra-O-acetyl-5-thio-β-D-glucopyranosyl-(1→3)-2,4,6-tri-O-acetyl-1,3,5-trideoxy-1,3,5-trithio-β-D-(1→3)-2,4,6-tri-O-acetyl-1,3,5-trideoxy-1,3,5-trithio-β-D-glucopyranoside trifluoroacetate salt (32)

Carbamate 31 (60 mg, 49.65 μmol) was dissolved in CH₂Cl₂ (16 mL) and 90% aq TFA (4 mL) and the reaction mixture was stirred for 40 min at 20 °C. After such time the reaction mixture was concentrated to dryness, co-evaporated with toluene (3×10 mL) and dried on a high vacuum for 12 h to give compound 32 as a colorless syrup (60.5 mg, quant): [α]_D²⁰ –6.0 (c 0.002, CHCl₃); ¹H NMR (500 MHz, CD₃CN) δ 7.08 (br, 3H, OCH₂CH₂NH₃⁺), 5.34 – 5.21 (m, 2H, H-3a, H-4a), 5.06 (t, J = 10.5 Hz, 1H, H-2c), 5.01 – 4.87 (m, 4H, H-2a, H-4b, H-2b, H-4c), 4.59 (dd, J = 11.9, 8.1 Hz, 1H, H-6a), 4.29 – 4.20 (m, 2H, H-1a, H-6c), 4.19 – 4.10 (m, 5H, H-6a, H-6c, H-1b, H-6b), 4.01 (d, J = 10.5 Hz, 1H, H-1c), 3.68 – 3.61 (m, 4H, SCH₂CH₂O, OCH₂CH₂NH₃), 3.46 – 3.38 (m, 1H, H-5b), 3.35 – 3.25 (m, 2H, H-5a, H-5c), 3.17 – 3.03 (m, 4H, H-3b, H-3c, OCH₂CH₂NH₃), 2.91 (dt, J = 13.0, 6.3 Hz, 1H, SCH₂CH₂O), 2.83 (dt, J = 13.3, 6.5 Hz, 1H, SCH₂CH₂O), 2.23 (s, 3H, CH₃CO), 2.17 (s, 3H, CH₃CO), 2.12 (s, 3H, CH₃CO), 2.11 (s, 3H, CH₃CO), 2.09 (s, 3H, CH₃CO), 2.00 (s, 3H, CH₃CO), 1.98 (s, 6H, 2×CH₃CO), 1.98 (s, 3H, CH₃CO), 1.97 (s, 3H, CH₃CO)^l; ¹³C NMR (126 MHz, CD3CN) δ 171.6, 171.3, 170.9, 170.6, 170.5 (CO), 77.1 (C-2a), 76.5 (C-2c), 74.0 (C-2b), 73.4 (C-3a), 72.0 (C-4c), 71.6 (C-4b), 71.3 (C-4a), 71.1 (OCH₂CH₂NH₃), 66.8 (SCH₂CH₂O), 62.9 (C-6c), 62.6 (C-6b), 62.2 (C-6a), 55.5 (C-3b, C-3c), 52.3 (C-1a), 50.1 (C-1c), 48.9 (C-1b), 47.0 (C-5b), 46.9 (C-5b), 43.4 9 (C-5c), 40.7 (OCH₂CH₂NH₃), 31.3 (SCH₂CH₂O), 21.5, 21.4, 21.4, 20.9, 20.8, 20.8, 20.7 (CH₃CO); ¹⁹F NMR (470 MHz, CD₃CN) δ −76.2; ESI-HRMS [M+H]⁺ calcd. for C₄₂H₆₂NO₂₁S₆⁺ 1108.2133, found 1108.2136.

4.2.18. Peracetylated tetraamide (33)

Tetracarboxylic acid 17 (7 mg, 10.5 μmol) and trifluoroacetate 32 (64 mg, 52.5 μmol, 5 equiv) were dissolved in anhydrous DMF (4 mL) followed by addition of DIPEA (24 μL, 136.5 μmol, 13 equiv) and PyBOP (33 mg, 63 μmol, 6 equiv). The reaction mixture then was stirred for 16 h at 20 °C. After such time H₂O (10 mL) was added and the reaction mixture was extracted with EtOAc (4×20 mL). The combined organic layers were washed with H₂O (2×20 mL), brine (2×20 mL), dried over Na₂SO₄, concentrated to dryness and co-evaporated with toluene (3×10 mL). Crude product was purified by a flash column chromatography on a silica gel eluting with CH₂Cl₂:MeOH (0 → 5% MeOH (v/v)) to give compound 33 as colorless syrup (30 mg, 57%): R_f 0.4 (CH₂Cl₂:MeOH (95:5) (v/v) (H₂SO₄/EtOH)); ¹H NMR (500 MHz, CDCl₃) δ 7.15 (t, J = 5.7 Hz, 4H, CONH), 5.31 (dd, J = 10.3, 5.5 Hz, 4H, H-4a), 5.23 – 5.13 (m, 8H, H-2c, H-3a), 5.12 – 5.03 (m, 8H, H-2a, H-2b), 4.95 (t, J = 10.6 Hz, 8H, H-4b, H-4c), 4.75 (dd, J = 11.7, 9.8 Hz, 4H, H-6a), 4.26 – 4.11 (m, 20H, H-6b, H-6c, H-1a), 4.06 (dd, J = 11.6, 4.4 Hz, 4H, H-6a), 3.99 (d, J = 10.6 Hz, 4H, H-1b), 3.78 (d, J = 10.3 Hz, 4H, H-1c), 3.67 – 3.60 (m, 8H, SCH₂CH₂O), 3.55 (t, J = 5.2 Hz, 8H, OCH₂CH₂NH), 3.46 (dt, J = 23.0, 5.6 Hz, 16H, OCH₂CH₂NH, OCH₂CH₂CH₂S), 3.32 (s, 8H, C(CH₂O)), 3.28 (ddd, J = 9.7, 5.6, 3.6 Hz, 4H, H-5c ), 3.25 – 3.20 (m, 12H, SCH₂CONH, H-5a), 3.09 (dd, J = 10.6, 4.9 Hz, 4H, H-5b), 3.01 – 2.89 (m, 12H, H-3b, H-3c, SCH₂CH₂O), 2.83 (dt, J = 12.9, 6.3 Hz, 4H, SCH₂CH₂O), 2.62 (t, J = 7.3 Hz, 8H, OCH₂CH₂CH₂S), 2.24 (s, 12H, CH₃CO), 2.18 (s, 12H, CH₃CO), 2.17 (s, 12H, CH₃CO), 2.13 (s, 24H, 2×CH₃CO), 2.06 (s, 12H, CH₃CO), 2.06 (s, 12H, CH₃CO), 2.04 (s, 12H, CH₃CO), 2.02 (s, 12H, CH₃CO), 2.00 (s, 12H, CH₃CO), 1.83 (p, J = 6.5 Hz, 8H, OCH₂CH₂CH₂S); ¹³C NMR (126 MHz, CDCl₃) δ 170.6, 170.5, 170.5, 169.5, 169.3, 169.2, 169.2, 169.1, 169.1 (CO), 75.8 (C-2c), 75.4 (C-2b), 72.6 (C-2a), 72.5 (C-4a), 70.6 (C-3a), 70.5 (C-4b, C-4c), 70.2 (SCH₂CH₂O), 69.6 ((C(CH₂O))₄), 69.5 (OCH₂CH₂CH₂S), 69.3 (OCH₂CH₂NH), 62.0 (C-6b), 61.9 (C-6c), 59.5 (C-6a), 54.9 (C-3b), 54.7 (C-3c), 52.3 (C-1b), 49.5 (C-1c), 47.6 (C-1a), 46.9 (C-5a), 46.6 (C-5b), 45.4 (q-C), 43.2 (C-5c), 39.4 (OCH₂CH₂NH), 36.0 (SCH₂CONH), 29.8 (SCH₂CH₂O), 29.3 (OCH₂CH₂CH₂S /OCH₂CH₂CH₂S), 21.1, 20.9, 20.9, 20.7, 20.7, 20.6, 20.6, 20.4, 20.2 (11×CH₃CO); ESI-HRMS [M+3Na]³⁺/3 calcd. For C₁₉₃H₂₈₀N₄Na₃O₉₂S₂₈³⁺ 1697.9745, found 1697.9778.

4.2.19. Tetraamide (12)

A 200–500 μL microwave vial was charged with peracetylated tetraamide 33 (4 mg, 0.79 μmol) followed by addition of H₂O:MeOH:NEt₃ (5:4:1) mixture (200 μL). The reaction vessel then was sealed and subjected to microwave irradiation for 4 h at 71 °C. After such time the reaction mixture was diluted with H₂O (5 mL) and washed with EtOAc (2×5 mL). The aqueous layer was concentrated to dryness and co-evaporated with EtOH (2×5 mL). The crude product was purified by column chromatography on a C18 silica gel eluting with MeCN:H₂O (1:1) to give tetraamide 12 as a white foam (1.3 mg, 50%): R_f 0.57 (C18, H₂O:MeCN (3:2)); ¹H NMR (900 MHz, D₂O) δ 4.15 (d, J = 8.6 Hz, 4H, H-1a), 4.07 (d, J = 10.1 Hz, 4H, H-1b), 3.96 – 3.81 (m, 16H, H-6c, H-6c, H-1c, H-3a), 3.79 – 3.73 (m, 12H, H-4a, H-6a), 3.68 (s, 8H, SCH₂CH₂O), 3.61 – 3.47 (m, 28H, H-6b, H-6b, H-2c, H-4b, H-2b, OCH₂CH₂NH), 3.44 – 3.39 (m, 8H, H-2a, H-4c), 3.39 – 3.29 (m, 16H, C(CH₂O)₄, OCH₂CH₂NH), 3.24 (s, 8H, SCH₂CONH), 3.04 – 2.96 (m, 8H, H-5c, H-5b), 2.96 – 2.86 (m, 12H, SCH₂CH₂O, H-3b, H-5a), 2.81 (dt, J = 21.4, 10.3 Hz, 8H, SCH₂CH₂O), 2.59 (d, J = 7.4 Hz, 8H, OCH₂CH₂CH₂S), 1.84 – 1.73 (m, 8H, OCH₂CH₂CH₂S); ¹³C NMR (226 MHz, D₂O) δ 76.8 (C-4b), 76.2 (C-4c), 76.0 (C-3a), 73.8 (C-2a), 73.3 (C-4a), 71.1 (C-2b), 71.0 (C-2c), 69.9 (SCH₂CH₂O), 69.6 (OCH₂CH₂CH₂S), 69.2 (C(CH₂O)), 68.6 (OCH₂CH₂NH), 60.6 (C-6c), 60.5 (C-6b), 59.8 (C-6a), 59.6 (C-3c), 59.5 (C-3b), 58.4, 58.3 (C-6a), 51.0 (C-1b), 50.7 (C-1c), 48.0 (C-5a), 46.8 (C-5c), 46.7 (C-1a), 39.4 (OCH₂CH₂NH), 35.3 (SCH₂CONH), 30.5 (SCH₂CH₂O), 29.0 (OCH₂CH₂CH₂S), 28.5 (OCH₂CH₂CH₂S); ESI-HRMS [M+3Na]³⁺/3 calcd. For C₁₁₃H₂₀₀N₄Na₃O₅₂S₂₈⁺ 1137.4992, found 1137.4990.

4.2.20. Peracetylated propionylamide (34)

Trifluoroacetate 32 (21 mg, 17.3 μmol) and propionic acid (3 μL, 34.6 μmol, 2 equiv) were dissolved in anhydrous DMF (4 mL) followed by addition of DIPEA (12 μL, 69.2 μmol, 4 equiv) and PyBOP (18 mg, 34.6 μmol, 2 equiv). The reaction mixture then was stirred for 1 h at 20 °C. After such time H₂O (10 mL) was added and the reaction mixture was extracted with EtOAc (4×10 mL). The combined organic layers were washed with H₂O (30 mL), brine (30 mL), dried over Na₂SO₄, concentrated to dryness and co-evaporated with toluene (3×10 mL). The crude product was purified by a flash column chromatography on a silica gel eluting with CH₂Cl₂:MeOH (0 → 10% MeOH (v/v)) to give compound 34 as a colorless syrup (18 mg, 90%): R_f 0.38 (MeOH:CH₂Cl₂ 5:95 (H₂SO₄/EtOH)); [α]_D^20–22 +9.5 (c 0.004, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 5.95 (br, 1H, CONH), 5.32 (dd, J = 10.3, 5.5 Hz, 1H, H-4a), 5.23 – 5.14 (m, 2H, H-2c, H-3a), 5.13 – 5.02 (m, 2H, H-2b, H-2a), 4.96 (t, J = 10.6 Hz, 2H, H-4c, H-4b), 4.75 (t, J = 10.8 Hz, 1H, H-6a), 4.28 – 4.10 (m, 5H, H-6b, H-6c, H-1a), 4.05 (dd, J = 11.7, 4.5 Hz, 1H, H-6a), 4.00 (d, J = 10.7 Hz, 1H, H-1b), 3.78 (d, J = 10.4 Hz, 1H, H-1c), 3.70 – 3.58 (m, 1H, SCH₂CH₂O), 3.53 (t, J = 5.4 Hz, 2H, OCH₂CH₂NH), 3.46 (p, J = 5.2, 4.7 Hz, 2H, OCH₂CH₂NH), 3.29 (dt, J = 10.4, 4.8 Hz, 1H, H-5b), 3.23 (dt, J = 9.9, 5.0 Hz, 1H, H-5a), 3.09 (s, 1H, H-5c), 3.01 – 2.91 (m, 3H, SCH₂CH₂O, H-3c, H-3b), 2.86 (dt, J = 12.8, 6.2 Hz, 1H, SCH₂CH₂O), 2.27 – 2.22 (m, 7H, CH₃CH₂CO, CH₃CO), 2.18 (s, 3H, CH₃CO), 2.17 (s, 3H, CH₃CO), 2.13 (s, 6H, CH₃CO), 2.07 (s, 3H, CH₃CO), 2.07 (s, 3H, CH₃CO), 2.05 (s, 3H, CH₃CO), 2.02 (s, 3H, CH₃CO), 2.01 (s, 1H, CH₃CO), 1.18 (t, J = 7.6 Hz, 3H, CH3CH2CO); ¹³C NMR (126 MHz, CDCl3) δ 173.8, 170.6, 170.6, 170.6, 169.6, 169.4, 169.3, 169.3, 169.3 (12×CO), 75.7 (C-2b), 75.4 (C-2c), 72.6 (C-2a), 72.5 (C-4a), 70.5 (C-3a), 70.4 (C-4b, C-4c), 69.9 (SCH₂CH₂O), 69.5 (OCH₂CH₂NH), 61.9 (C-6b), 61.9 (C-6c), 59.4 (C-6a), 54.9 (C-3b), 54.7 (C-3c), 52.3 (C-1b), 49.4 (C-1c), 47.8 (C-1a), 46.9 (C-5a), 46.5 (C-5b), 43.3 (C-5c), 39.0 (OCH₂CH₂NH₂), 29.9 (SCH₂CH₂O), 29.7 (CH₃CH₂CO), 21.1, 20.9, 20.9, 20.8, 20.7, 20.7, 20.7, 20.6, 20.4, 20.2 (11×CH₃CO), 9.9 (CH₃CH₂CO); ESI-HRMS [M+Na]⁺ calcd. For C₄₅H₆₅NNaO₂₂S₆⁺ 1186.2215, found 1186.2207.

4.2.21. Propionylamide (13)

Peracetylated propionylamide 34 (9 mg, 7.7 μmol) was suspended in anhydrous MeOH (0.5 mL) and NaOMe (freshly prepared 1M solution in MeOH) (25 μL, 25 μmol, 3.25 equiv) was added. The reaction mixture then was stirred until full consumption of the starting material was detected by LCMS. The reaction mixture then was quenched with Amberlite IR-120 H⁺ (washed with MeOH, CH₂Cl₂) until pH 6, filtered, and the filtrate was concentrated to dryness. The crude product was purified by column chromatography on a C18 silica gel eluting with MeOH:H₂O (1:1) to give product as a colorless syrup (4.9 mg, 86%): R_f 0.59 (CH₂Cl₂:MeOH 4:1 (H₂SO₄/EtOH); [α]_D²⁰ –11.1 (c 0.00027, DMSO); ¹H NMR (500 MHz, D₂O) δ 4.16 (d, J = 10.1 Hz, 1H, H-1a), 4.08 (d, J = 10.2 Hz, 1H, H-1b), 3.98 – 3.90 (m, 2H, H-3a, H-6a), 3.89 – 3.81 (m, 4H, H-1c, H-6c), 3.80 – 3.72 (m, 3H, H-4a, H-6a, H-6b), 3.68 (t, J = 6.1 Hz, 2H, SCH₂CH₂O), 3.61 – 3.48 (m, 6H, H-2b, H-2c, H-4c, H-6b, OCH₂CH₂NH), 3.45 – 3.40 (m, 2H, H-2a, H-4b), 3.30 (t, J = 5.3 Hz, 3H, OCH₂CH₂NH), 2.96 (d, J = 25.6 Hz, 2H, H-5c, H-5b), 2.92 – 2.86 (m, 3H, H-3b, H-5a, SCH₂CH₂O), 2.80 (td, J = 10.2, 7.2 Hz, 2H, H-3c, SCH₂CH₂O), 2.18 (q, J = 7.7 Hz, 2H, CH₃CH₂CO), 1.02 (t, J = 7.7 Hz, 3H, CH₃CH₂O); ¹³C NMR (126 MHz, D₂O) δ 178.3 (CO), 76.9 (C-4b), 76.3 (C-4c), 75.6 (C-3a), 73.9 (C-2a), 73.4 (C-4a), 70.9 (C-2b), 70.8 (C-2c), 69.6 (SCH₂CH₂O), 68.7 (OCH₂CH₂NH), 60.6 (C-6c), 60.6 (C-6b), 59.6 (C-3c), 59.5 (C-3b), 58.5 (C-6a), 50.8 (C-1c, C-5c), 50.6 (C-1b, C-5b), 48.1 (C-5a), 46.8 (C-1a), 39.0 (OCH₂CH₂NH), 30.3 (SCH₂CH₂O), 29.3 (CH₃CH₂CO), 9.7 (CH₃CH₂CO); ESI-HRMS [M+Na]⁺ calcd. For C₂₅H₄₅NNaO₁₂S₆⁺ 766.1159, found 766.1158.

4.3. Inhibition of Anti-CR3-FITC Antibody Staining of Human Neutrophils and of Anti-Dectin 1-FITC Antibody Staining of Mouse Macrophages

After harvesting and counting, cells (5×10⁵/tube) were pre-incubated for 30 min on ice with medium alone or with medium containing the glucoclusters 9–13 (1 – 10 μM) in a final volume of 250 μL. Then, 250 μL of the antibodies diluted in staining buffer (PBS containing 1% heat-inactivated FBS and 1% sodium azide) were added and the tubes further incubated for 60 min on ice. The cells were then washed three times with ice-cold staining buffer, resuspended and fixed by incubation with 2% paraformaldehyde for at least 60 minutes. Before analysis, the cells were centrifuged and resuspended in 0.5 mL of staining buffer before analysis in a BD-FACSCanto instrument (Becton Dickinson).

4.4. Phagocytosis assay

U937 cells were used in this experiment. Phagocytosis was measured using a Phagocytosis Assay kit (Green E. coli) (Abcam, ab235900) following the instructions provided by the manufacturer. Briefly, cells were plated on 24-well plates at 1×10⁶ cells per well. After an overnight incubation at 37 °C, the cells were incubated with several concentrations of the glucoclusters 9–13 (0.5 – 5μM) for 2 hours at 37 °C. A suspension of FITC-labeled E. coli was added to the wells and further incubated for 3 hours at 37 °C. After washing by centrifugation and aspiration, a quenching solution of Trypan blue was added and incubated for 5 min at room temperature. Cells were then washed and resuspended in ice-cold phagocytosis buffer (provided by the kit) and fluorescence read using a plate reader at excitation/emission 490/520 nm respectively.

Highlights:

• Easily accessible tetracarboxyl multivalent core

• Novel dithiasugar-based epitope

• Novel multivalent glucoclusters

• Significant affinity of synthesized glucoclusters for CR3

• Synthesis of a 3,5-dithiogluose-based analog of laminaritriose

• Synthesis of a pentaerythritol-based tetravalent tetravalent construct

• Evaluation of a tetravalent tetravalent construct of 3,5-dithiolaminaritriose