Design and assessment of dimeric trehalose glycolipids as a strategy to enhance Mincle-mediated vaccine adjuvanticity

Abstract

It has been suggested that the clustering of the macrophage inducible C-type lectin (Mincle), either with itself or with the related macrophage C-type lectin (MCL), can lead to improved Mincle-mediated signalling and adjuvant activity. To this end, we synthesised dimeric ligands that contained Mincle agonists trehalose dibehenate (TDB) or C18-brartemicin (C18Brar), and which were linked by C5, C10 or C14 acyl chains. Using mMincle NFAT-GFP reporter cells and murine WT and Mincle^−/− bone marrow derived macrophages (BMDMs), we demonstrated that the dimeric ligands activate Mincle signalling, with the C10-linked TDB dimer 3b leading to high levels of IL-1β, IL-6 and, in particular, TNF-α in vitro. Dimer 3b was then tested for its adjuvant properties in vivo using OVA as a model antigen and was found to induce significantly more germinal centre B cells compared to OVA alone and TDB + OVA (unconjugated); however neither TDB nor dimer 3b led to an increase in T cell numbers relative to OVA alone. Taken together, these data provide proof of concept that dimeric Mincle ligands are a new class of potential Mincle-mediated vaccine adjuvants.

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Introduction

The macrophage inducible C-type lectin (Mincle, Clec4e, or Clecsf9) is a pattern-recognition receptor (PRR) that has shown much promise as a molecular target for the development of T helper (T_H)-1/T_H-17-skewing vaccine adjuvants.¹ CAF (Cationic Adjuvant Formulation) liposomal adjuvant systems, which contain trehalose dibehenate (TDB, 1, Fig. 1) in combination with dimethyldioctadecylammonium bromide (DDA) and, at times, other immunostimulants, have shown exceptional potential as clinically relevant vaccine adjuvants for both subcutaneous and mucosally-delivered vaccines.^1–3 Other 6,6′-acylated trehalose glycolipids, including branched trehalose glycolipids^1,4,5 and lipidated brartemicin derivatives^6–10 such as C18Brar (2),⁶ have also demonstrated promising adjuvant activity in several studies, including in combination with TB antigens^11,12 and in the veterinary health space.^13,14 To elicit their immunomodulatory effects, Mincle ligands, such as TDB (1), bind to the receptor and initiate the FcRγ-Syk-Card9-Bcl10-Malt1 signalling axis.^15–18 This ultimately leads to the NFκB-mediated transcription of cytokines, chemokines, and small molecule mediators, such as NO. More recently, a C18Brar-muramyl dipeptide (MDP)-conjugate that was developed to target two PRRs, Mincle and NOD2, respectively, was found to lead to a synergistic improvement in the immune response and a reduction in adjuvant loading.¹⁹ Mincle and the related C-Type lectin receptor (CLR) macrophage C-type lectin (MCL) are mutually regulated and act as heterodimers for ligand binding.^20–22 Mincle has also been found to exist as a pre-formed disulfide-linked dimer.²³ While much remains to be elucidated about the role of Mincle clustering in immune responses, it has been suggested that Mincle clustering enhances the immune response via a variety of mechanisms that include enhanced phagocytosis and inflammatory gene responses,^20,21,24 as well as inflammasome activation.²⁵ These responses may in turn augment Mincle-mediated adjuvant activity.¹ Clustering may be achieved via the 3D aggregation of individual Mincle ligands using a delivery vehicle^1,26 or via a single Mincle agonist that can bridge multiple receptors, as evidenced by crystallographic data which indicated that the highly immunostimulatory Mincle agonist phenolic glycolipid III (PGL-III) bridged two hMincle proteins.²⁷

Fig. 1 Representative Mincle-signalling trehalose glycolipids, TDB (1) and C18Brar (2).

The Mincle binding site can accommodate a trehalose glycolipid acyl chain of approximately eight carbon atoms from the trehalose to the edge of the carbohydrate recognition domain (CRD).^28–30 With this in mind, we envisioned preparing two series of dimeric Mincle ligands: those based on TDB (3a–c) and those based on C18Brar (4a–c) (Fig. 2). The individual monomeric units within each dimer would be joined by acyl chains of varying lengths (n = 1, 6, 10). We proposed that these dimers could bind to Mincle heterodimers or Mincle/MCL dimers, with the non-conjugated acyl chain on each trehalose monomer being accommodated by the major hydrophobic groove in Mincle.^28,29 If successful, these ligands would be the first examples of dimeric Mincle agonists. For comparison, we were also interested in determining whether the constrained ring-closed product 5 would activate Mincle. Potentially, such a compound may be a Mincle antagonist; however, to date, Mincle-binding DNA aptamers, rather than glycolipids,¹ have shown the most promise as Mincle-mediated antagonists.³¹

Fig. 2 Target dimeric Mincle ligands, 3a–c and 4a–c, and cyclic derivative 5.

Results and discussion

To prepare the target compounds, a retrosynthetic strategy was proposed whereby dimers 3a–c and 4a–c could be accessed via a metathesis reaction of protected asymmetric diesters (6a–c and 7a–c) with alkene functionalities at the end of one lipid tail, followed by desilylation using Dowex-H⁺ (Scheme 1). The synthesis of the TMS-protected asymmetric diesters 6a–c and 7a–c could in turn be achieved via an EDCI/DMAP mediated coupling between the commercially available alkene functionalised carboxylic acids 8a–c and the corresponding monoesters 9 or 10. Behenic acid monoester 9 was prepared from readily accessible TMS-protected trehalose 11 by controlling the reaction conditions to obtain the mono-esterified product, as previously described.³² A similar methodology was proposed for the synthesis of benzoyl monoester 10. The preparation of the constrained glycolipid 5 was envisaged to proceed under RCM conditions employing the TMS-protected diester of undecenoic acid 12 followed by global deprotection, with undecenoic acid diester 12 being prepared via an esterification reaction between TMS-protected trehalose 11 and undecenoic acid (8b).

Scheme 1 Retrosynthetic analysis for the synthesis of dimeric glycolipids, 3a–c, and 4a–c, and ring-closed product 5.

With the retrosynthetic strategy in place, α,α′-D-trehalose (13) was persilyated using bis(trimethylsilyl)acetamide (BSA) and catalytic tetrabutylammonium fluoride (TBAF), with the more labile primary TMS groups subsequently being removed by the addition of K₂CO₃ to afford the required TMS-product 11 according to literature procedures³² (Scheme 2). By carefully controlling the ratio of behenic acid (14), TMS-trehalose 11, EDCI and DMAP (1 : 1.8 : 2 : 4.4, respectively), and by performing the reaction under dilute conditions (30–40 mL dry toluene per mmol of 11), monoester 9 was synthesised in 60% yield, which was an improvement on the previously reported literature yield.³² In an analogous manner, benzoyl monoester 10 was synthesised in 47% yield from TMS-protected trehalose 11 and p-(octadecyloxy)benzoic acid (15), whereby the latter was prepared according to our previously published protocols.¹⁴

Scheme 2 Synthesis of monoester intermediates 9 and 10.

With the target monoesters 9 and 10 in hand, these were then conjugated with the alkene-functionalised carboxylic acids 8a–c (where n = 1, 6, and 10) under the mediation of EDCI and DMAP to give the asymmetric diesters 6a–c and 7a–c in 60–80% yield (Scheme 3). The asymmetric diesters were treated with Grubb's second-generation catalyst in CH₂Cl₂ under reflux to form the cross-coupled dimeric ligands. This led to TMS-protected dimers 16a–c and 17a–c in 46–72% yield. Evidence for the desired dimerisation was observed in the ¹H NMR spectra by the presence of vinylic proton resonances at ca. δ 5.30–5.40 ppm and the absence of proton resonances at ca. δ 5.80 and 5.03–4.89 ppm, which were assigned to terminal alkene protons in the starting diesters. In addition, HRMS data corresponding to the dimeric products (e.g., m/z 1327.8482 for 16c) matched the calculated values (m/z 1327.8489 for [C₁₃₂H₂₇₂O₂₆Si₁₂ + 2Na]²⁺). The protected dimers 16a–c and 17a–c were then subjected to desilylation reactions using Dowex-H⁺ to generate the final target glycolipids 3a–c and 4a–c in good to excellent yields.

Scheme 3 Synthesis of dimeric targets 3a–c and 4a–c.

To synthesise the constrained trehalose glycolipid 5, TMS-protected trehalose 11 was reacted with undecenoic acid 8b in toluene in the presence of EDCI and DMAP to produce diester 18 in 86% yield (Scheme 4). Diester 18 was then subjected to RCM using Grubbs’ second-generation catalyst. This led to the cyclic glycolipid 19 in 28% yield. Use of Hoveyda–Grubbs’ second generation catalyst did not improve the outcome, with the desired ring-closed product being isolated in only 11%. This low yield observed for the olefin RCM was proposed to be, in part, due to the loss of TMS-protecting groups from the starting material/product, as observed by TLC, whereby multiple products with lower R_f values were observed, and by HRMS data consistent with partially deprotected by-products. We also speculated that intermolecular reactions between two or more diester units occurred, which additionally reduced the yield of the target product. Nevertheless, as protected glycolipid 19 was obtained in sufficient yield, it was subjected to desilylation using Dowex-H⁺ to generate the constrained glycolipid 5 in quantitative yield.

Scheme 4 Synthesis of the constrained glycolipid 5.

To establish whether the target ligands could signal via Mincle, nuclear factor activated T cells (NFAT)–green fluorescent protein (GFP) reporter cells expressing mMincle and coupled to FcRγ were stimulated with the synthesised dimers (3a–c, 4a–c), cyclic ligand (5), as well as TDB (1) and C18Brar (2), and the production of GFP was measured via flow cytometry (Fig. 3). All dimeric compounds (3a–c, 4a–c) activated mMincle at both 0.1 and 1 nmol per well concentrations, while the cyclic TDB ligand 5 showed a modest ability to signal through Mincle at lower ligand concentrations and exhibited similar activity to TDB (2) when tested at 1 nm per well. For the dimeric ligands, the effect of concentration on reporter cell activation was modest, although a slight trend towards increasing GFP production with increasing linker length was observed.

Fig. 3 Mincle reporter cell assay. NFAT-GFP-2B4 reporter cells expressing mMincle + FcRγ or FcRγ only were stimulated with ligands coated on plates at 0.1 and 1 nmol per well. GFP expression by the harvested cells after 18 h was measured using flow cytometry. Data represent the mean of three independent experiments performed in duplicate (mean ± SEM).

Having demonstrated that the ligands can signal through mMincle, we then tested the ability of compounds to activate both wild-type and Mincle^−/− bone marrow-derived macrophages (BMDMs), as determined by measuring the release of the proinflammatory cytokines IL-1β, IL-6 and TNF-α (Fig. 4). From these studies, it was observed that all dimers induced the Mincle-dependent production of IL-1β, except dimer 4b for which Mincle-independent IL-1β production was also observed (Fig. 4A and B). We previously observed a Mincle-independent increase in IL-1β for a C18Brar derivative with a C18 alkyl chain at the ortho-, rather than the para-position, and determined that this was due to caspase-1 dependent NLRP3 inflammasome-mediated cell death.^7,33 It remains to be seen whether a similar Mincle-independent mechanism of action occurs for 4b. All dimeric ligands also led to a significant increase in IL-6 and TNF-α production by the BMDMs in a Mincle-dependent manner (Fig. 4A), with dimer 3b (n = 6) leading to TNF-α production by the BMDMs that was significantly greater than that elicited by the ‘gold-standard’ Mincle agonist, TDB. Apart from very modest levels of IL-6, the cyclic compound 5 did not lead to cytokine production in these assays. As with the NFAT-GFP assays, there was no consistent effect of concentration on the cellular responses. This phenomenon has been observed for several promising Mincle-mediated adjuvants^6,7,10,11,34 and is poorly understood, although it may be due to immunological feedback mechanisms^25,35 or, perhaps more likely, the complex way in which the ligands aggregate in solution.^1,26 In the BMDM assays, there was no correlation between linker length and cytokine production.

Fig. 4 BMDM activation assay. BMDMs, either from wild type c57 mice (A) or from c57 Mincle^−/− mice (B), were stimulated with TDB, C18Brar or trehalose glycolipids 3a–c/4a–c coated on plates (0.1 or 1 nmol per well), or LPS (100 ng mL⁻¹). IL-1β, IL-6 and TNF-α production was measured by ELISA from the supernatants collected after 24 hours of incubation. Data represent three independent experiments performed in triplicate (mean ± SEM). Statistical significance was determined using one-way ANOVA with Dunnett's multiple comparisons test compared to iPrOH control, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Statistical significance was determined using one-way ANOVA, with Dunnett's multiple comparisons test compared to TDB, ^####P < 0.0001.

The ability of the C10-linked TDB dimer 3b to induce good cytokine production in vitro was promising, particularly since IL-6 and IL-1β are thought to be important for the activation of Th-1 immunity,^36–38 while TNF-α has been shown to play a critical role in dendritic cell (DC) maturation^36,37,39 and in host protection against a variety of infectious diseases, including those caused by viruses (e.g., foot-and-mouth disease³⁷) and intracellular (e.g., Mycobacterium tuberculosis)⁴⁰ and extracellular (e.g., Klebsiella pneumoniae)⁴¹ bacteria, and can activate macrophages to phagocytose and kill mycobacteria and other pathogens.⁴² To this end, we were interested in exploring the adjuvant potential of the C10-linked TDB dimer 3b using OVA (which contains the immunogenic peptide sequence SIINFEKL as a model antigen).

To this end, C57BL/6 mice (n = 5/group) were vaccinated using a routine vaccination schedule.⁴³ This involved intramuscular (i.m.) vaccination on day 0 with an oil-in-water emulsion containing the C10-linked TDB dimer 3b + OVA, TDB + OVA, PBS (negative control) or OVA only (negative control) and an i.m. boost 3 weeks later and, after a further 7 days, the mice were sacrificed and the inguinal lymph node (iLN) was harvested and B and T cell populations were analysed by flow cytometry (Fig. 5A–F) (see the SI for the flow gating strategies). Following this vaccination protocol, it was observed that only 3b induced significantly more SIINFEKL⁺CD8⁺T cells compared to PBS (Fig. 5D), and significantly more OVA⁺ germinal centre B cells compared to OVA alone and TDB + OVA (Fig. 5F). This illustrates the potential of 3b to expand B cell populations and augment the immune response. However, no significant increase in the T follicular CD4⁺ helper cells was observed in response to either TDB or 3b (Fig. 3B), which is an immune response that is essential for supporting germinal centre (GC) B cells activation, and survival within GC, as well as for the production of high-affinity antigen-specific antibodies and isotype switching.⁴⁴ Although TDB is a potent Mincle agonist,^1,2,4 it exhibited only modest adjuvant activity in this assay. The adjuvant activity of 3b could perhaps be improved by ensuring better co-delivery of the antigen and multiple copies of the adjuvant to the same antigen presenting cell (APC) either through adsorption of both antigen and adjuvant to a particulate delivery vehicle or via covalent conjugation of the antigen to the adjuvant.^1,12 Such studies will be explored in due course.

Fig. 5 Intramuscular vaccination with 3b + OVA induces SIINFEKL⁺ CD8⁺ T cells and OVA-specific germinal centre B cell populations in the inguinal lymph node. C57BL/6 mice (n = 5 per group) were immunised through the intramuscular route on day 0 and day 21 with 50 µL volumes per leg of PBS, OVA (50 µg), TDB (0.3 µmol) + OVA (50 µg), or 3b (0.3 µmol) + OVA (50 µg). Mice were sacrificed on day 28 and the inguinal lymph nodes were harvested for analysis by flow cytometry. (A) Frequency of activated CD4⁺ T cells as measured by CD44⁺ expression. (B) Frequency of activated Tfh cells as measured by PD-1⁺ and intracellular Bcl6⁺ co-expression. (C) Frequency of activated CD8⁺ T cells as measured by CD44⁺ expression. (D) Frequency of activated OVA-specific CD8⁺ T cells as measured by PD-1⁺ and MHC-I:SIINFEKL tetramer⁺ co-expression. (E) Frequency of activated germinal centre B cells gated on IgD⁻ B cells expressing Bcl6 transcription factor. (F) Frequency of OVA-specific germinal centre B cells as measured using OVA-biotin and detection with Sav-PECF594. Data are expressed as mean ± SEM. Statistical significance was calculated using one-way ANOVA with Tukey's multiple comparison test. (*) P ≤ 0.05; (**) P ≤ 0.01; (***) P ≤ 0.001; (****) P ≤ 0.0001.

Conclusions

In this study, the first dimeric Mincle ligands were synthesised and shown to signal and activate immune cells via Mincle. Notably, the dimeric ligand 3b, which contains two units of TDB linked by a C10 acyl group, led to IL-1β, IL-6 and TNF-α production, with the level of TNF-α being significantly greater than that shown by TDB alone. The potential of dimer 3b as a vaccine adjuvant was further demonstrated in an in vivo i.m. vaccination protocol using OVA as the model antigen. Here, 3b led to a significant increase in OVA-specific CD8⁺ T cells and GC B cells, with the B cell response to 3b being greater than that shown by TDB. Unfortunately, neither TDB nor dimer 3b led to increased CD4⁺ T cell numbers when formulated as the o.i.w. emulsion. This may be due to poor co-delivery of antigen and adjuvant to the same APC, which could be explored in future vaccination studies by using particulate carrier systems that can adsorb both antigen and adjuvant or via the chemical conjugation of antigen to adjuvant. In summary, we provide the first example to illustrate how two Mincle ligands can be conjugated to provide Mincle-mediated agonist activity, thereby highlighting a potential new approach for the development of improved Mincle-mediated adjuvants.

Experimental

General methods

Unless otherwise stated, all reactions were performed under an argon atmosphere. Methanol, EtOAc, and light petroleum ether (pet. ether) were distilled prior to use. Dichloromethane was distilled over phosphorous pentoxide, under an Ar atmosphere. All other chemicals were purchased from commercial suppliers and used as received. All solvents were evaporated under reduced pressure. Reactions were monitored by TLC-analysis using Macherey-Nagel silica gel pre-coated plastic sheets (0.20 mm, fluorescent indicator UV-254) and visualised under UV light (254 nm) or by dipping in 10% H₂SO₄ in EtOH followed by charring, dipping in ceric ammonium molybdate solution or dipping in KMnO₄ solution (2% in H₂O). Column chromatography was performed using silica gel (40–63 µm). High resolution mass spectroscopy data were obtained on an Agilent 6530 Q-TOF mass spectrometer with a JetStream™ electrospray ionisation source in positive or negative mode. Optical rotations were recorded on an Autopol II polarimeter (Rudolph Research Analytical) at 589 nm (sodium D-line). Infrared spectra were obtained for the compounds (as a thin film) using a Bruker Platinum ATR instrument and are reported in wave numbers (cm⁻¹). Nuclear magnetic resonance spectra were recorded at 20 °C in CDCl₃, C₅D₅N or D₂O as indicated, using JEOL JNM-ECZ spectrometers operating at 500 or 600 MHz. Chemical shifts (δ) are reported in ppm relative to residual solvent peaks. Peak assignments in NMR spectra were made using 2D-NMR experiments (COSY, HSQC, and HMBC). TDB (1),⁴⁵ C18Brar (2),⁴⁶ 2,2′,3,3′,4,4′-hexa-O-trimethylsislyl-α,α′-D-trehalose (11)⁴⁵ and p-(octadecyloxy)benzoic acid (15)⁴⁶ were prepared according to previously published procedures.

6-O-Docosanoyl-2,2′,3,3′,4,4′-hexa-O-trimethylsilyl-α,α′-D-trehalose (9). To a solution of TMS protected trehalose 11 (502 mg, 0.647 mmol) and behenic acid (14, 397 mg, 1.17 mmol) in dry toluene (25 mL) were added DMAP (34.8 mg, 2.85 mmol) and EDCI (24.8 mg, 1.30 mmol). After stirring for 22 h at 70 °C, the reaction was deemed complete, as confirmed by TLC. The reaction mixture was cooled to room temperature, diluted with EtOAc (150 mL) and washed with water (150 mL). The aqueous layer was extracted with EtOAc (150 mL) and the combined organic layers were washed successively with sat. aq. NaHCO₃ (250 mL) and brine (300 mL), dried over MgSO₄, filtered, and concentrated under reduced pressure. The resulting residue was then purified using gradient silica gel flash chromatography (eluting with pet. ether : EtOAc, 1 : 0 → 19 : 1, v/v) to obtain behenic acid monoester 9 as a colourless viscous oil (620 mg, 0.320 mmol, 60%). The data obtained for this compound matched literature values.⁴⁷

6-O-(p-(Octadecyloxy)benzoyl)-2,2′,3,3′,4,4′-hexa-O-trimethylsilyl-α,α′-D-trehalose (10). To a solution of TMS protected trehalose (11, 503 mg, 0.645 mmol) and 4-(octadecyloxy)benzoic acid (15, 455 mg, 1.16 mmol) in dry toluene (25 mL) were added DMAP (35.0 mg, 2.85 mmol) and EDCI (25.0 mg, 1.30 mmol). After stirring for 22 h at 70 °C, the reaction was deemed complete, as confirmed by TLC. The reaction mixture was cooled to room temperature, diluted with EtOAc (150 mL) and washed with water (150 mL). The aqueous layer was extracted with EtOAc (150 mL) and the combined organic layer was washed with a saturated solution of NaHCO₃ (150 mL) and brine (300 mL), dried over MgSO₄, filtered, and concentrated under reduced pressure. The resulting residue was then purified using gradient silica gel flash chromatography (eluting with pet. ether : EtOAc, 1 : 0 → 47 : 3, v/v) to obtain benzoic acid monoester 10 as a colourless viscous oil (347 mg, 0.302 mmol, 47%). R_f = 0.63 (pet. ether : EtOAc, 17 : 3, v/v); [α]^22.9_D = +80 (c = 0.1, CH₂Cl₂); ¹H NMR (500 MHz, CDCl₃) δ 7.98 (d, J_3″,4″ = 9.0 Hz, 2H, H-3″), 6.90 (d, J_3″,4″ = 9.0 Hz, 2H, H-4″), 4.93 (d, J_{1/1′,2/2′} = 3.1 Hz, 2H, H-1 and H-1′), 4.54 (dd, J_6a,b = 12.0 Hz, J_5,6a 2.4 Hz, 1H, H-6a), 4.24 (dd, J_6a,b = 12.0 Hz, J_5,6b 2.4 Hz, 1H H-6b), 4.13–4.07 (m, 1H, H-5), 3.99 (t, J_6″,7″ = 6.6 Hz, 2H, H-6″), 3.96–3.87 (m, 2H, H-3 and H-3′), 3.83 (dt, J_4′,5′ = 3.4 Hz, J_5′,6′a/b = 9.5 Hz, 1H, H-5′), 3.73–3.65 (m, 2H, H-6′), 3.63 (t, J_3,4 = J_4,5 = 9.1 Hz, 1H, H-4), 3.49–3.40 (m, 3H, H-2 and H-2′ and H-4′), 1.82–1.70 (m, 2H, H-7″), 1.48–1.39 (m, 2H, H-8″), 1.38–1.18 (m, 26H, H-9″–H-22″), 0.86 (t, J_22″,23″ = 7.0 Hz, H-23″), 0.16, 0.15, 0.14, 0.13, 0.12, 0.11 (6s, 54H, TMS); ¹³C NMR (125 MHz, CDCl₃) δ 166.4 (C-1″), 163.2 (C-5″), 131.8 (C-3″), 122.2 (C-2″), 114.2 (C-4″), 94.7, 94.6 (C-1 and C-1′), 73.8 (C-3), 73.4 (C-3′), 73.0 (C-5′), 72.9, 72.8 (C-2 and C-2′), 72.0 (C-4′), 71.5 (C-4), 71.0 (C-5), 68.3 (C-6″), 63.4 (C-6), 61.7 (C-6′), 32.0, 29.78, 29.75, 29.69, 29.66, 29.47, 29.45, 29.2 (C-9″–C-21″), 29.1 (C-7″), 26.1 (C-8″), 22.7 (C-22″), 14.3 (C-23′), 1.2, 1.1, 1.0, 0.9, 0.3, 0.2 (TMS); IR (film): 2955, 2924, 2854, 2091, 2052, 1988, 1719, 1607, 1581, 1510, 1458, 1387, 1313, 1251, 1167, 1109, 1076, 1009, 965, 944, 899, 972, 843, 749, 694, 646, 573, 514, 449, 421, 412 cm⁻¹; HRMS (ESI) m/z calcd for [C₅₅H₁₁₀O₁₃Si₆ + K]⁺: 1185.6194; obsd.: 1185.6191.

6-O-Docosanoyl-6′-O-(hex-5-enoyl)-2,2′,3,3′,4,4′-hexa-O-trimethylsilyl-α,α′-D-trehalose (6a). Monoester 9 (101 mg, 0.092 mmol) was co-evaporated twice with dry toluene (2 × 5–6 mL) and then a third time with toluene (6 mL), which was then concentrated to half the volume of toluene (3 mL). Hexenoic acid (8a) (0.010 mL, 0.111 mmol), DMAP (22.5 mg, 0.184 mmol) and EDCI (35.0 mg, 0.184 mmol) were then added to the reaction mixture, and the mixture was stirred at 60–70 °C for 24 hours. The reaction mixture was cooled to room temperature, diluted, and extracted with EtOAc (2 × 50 mL). The combined organic layers were washed with water (100 mL), sat. aq. NaHCO₃ (2 × 70 mL) and brine (80 mL), dried over MgSO₄, filtered, and concentrated under reduced pressure. The crude product was purified using gradient silica gel flash column chromatography (eluting with pet. ether : EtOAc, 1 : 0 → 24 : 1, v/v) to obtain the title compound 6a as a colourless waxy oil (67.4 mg, 0.056 mmol, 61%). R_f = 0.66 (pet. ether : EtOAc, 9 : 1, v/v); [α]^22.7_D = +100 (c = 0.1, CH₂Cl₂); ¹H NMR (500 MHz, CDCl₃) δ 5.77 (ddt, J_5‴,6‴a = 17.0 Hz, J_5‴,6‴b = 10.2 Hz, J_4‴,5‴ = 6.7 Hz, 1H, H-5‴), 5.02 (dq, J_5‴,6‴a = 17.0 Hz, J_6‴a,6‴b = J_6‴a,4‴ = 1.6 Hz, 1H, H-6‴a), 4.98 (ddt, J_5‴,6‴b = 10.2 Hz, J_6‴a,6‴b = 1.6 Hz, J_6‴b,4‴ = 1.1 Hz, 2H, H-6‴b), 4.91 (d, J_{1/1′,2/2′} = 3.0 Hz, 2H, H-1 and H-1′), 4.29–4.26 (m, 2H, H-6a and H-6′a), 4.08–4.04 (m, 2H, H-6b and H-6′b), 3.99 (ddd, J_{4/4′,5/5′} = 9.1 Hz, J_{5/5′,6/6′b} = 4.4 Hz, J_{5/5′,6/6′a} = 2.3 Hz, 2H, H-5 and H-5′), 3.90 (t, J_{3/3′,4/4′} = J_{2/2′,3/3′} = 9.1 Hz, 2H, H-3 and H-3′), 3.48 (t, J_{3/3′,4/4′} = J_{4/4′,5/5′} = 9.1 Hz, 2H, H-4 and H-4′), 3.43 (dd, J_{2/2′,3/3′} = 9.1 Hz, J_{1/1′,2/2′} = 3.0 Hz, 2H, H-2 and H-2′), 2.41–2.28 (m, 4H, H-2″ and H-2‴), 2.12–2.07 (m, 2H, H-4‴), 1.77–1.69 (m, 2H, H-3‴), 1.65–1.58 (m, 2H, H-3″), 1.33–1.22 (m, 36H, H-4″–H-21″), 0.88 (t, J_21″,22″ = 7.0 Hz, 3H, H-22″), 0.15, 0.13, 0.13 (3s, 54H, TMS); ¹³C NMR (125 MHz, CDCl₃) δ 173.9 (C-1″/C-1‴), 173.7 (C-1″/C-1‴), 137.8 (C-5‴), 115.5 (C-6‴), 94.5 (C-1 and C-1′), 73.62 (C-3/C-3′), 73.60 (C-3/C-3′), 72.8 (C-2 and C-2′), 72.0 (C-4 and C-4′), 70.9 (C-5 and C-5′), 63.5 (C-6/C-6′), 63.4 (C-6/C-6′), 34.3 (C-2″/C-2‴), 33.5 (C-2″/C-2‴), 33.1 (C-4‴), 32.1, 29.85, 29.81, 29.78, 29.63, 29.51, 29.46, 29.31, 22.8 (C-4″–C-21″), 24.9 (C-3″), 24.0 (C-3‴), 14.3 (C-22″), 1.2, 1.0, 0.3 (TMS); IR (film): 2955, 2924, 2854, 1743, 1458, 1251, 1164, 1111, 1100, 1077, 1045, 1010, 965, 898, 873 cm⁻¹; HRMS (ESI) m/z calcd for [C₅₈H₁₂₀O₁₃Si₆ + NH₄]⁺: 1210.7683; obsd.: 1210.7685.

6-O-Docosanoyl-2,2′,3,3′,4,4′-hexa-O-trimethylsilyl-6′-O-(undec-10-enoyl)-α,α′-D-trehalose (6b). Behenic acid monoester 9 (94.2 mg, 0.086 mmol) was co-evaporated twice with dry toluene (2 × 2–3 mL) and then a third time with toluene (5 mL), which was then concentrated to half the volume of toluene (2.5 mL). Carboxylic acid 8b (0.02 mL, 0.10 mmol), DMAP (21.0 mg, 0.172 mmol) and EDCI (32.9 mg, 0.172 mmol) were then added to the reaction mixture. The mixture was stirred at 70 °C for 24 hours, by which time the reaction was deemed complete, as gauged by TLC analysis. The reaction mixture was cooled to room temperature, diluted with EtOAc (80 mL), washed with water (80 mL) and brine (80 mL), dried over MgSO₄, filtered, and concentrated under reduced pressure. The crude product was subjected to gradient silica gel flash column chromatography (eluting with pet. ether : EtOAc, 1 : 0 → 97 : 3, v/v) to obtain the title compound 6b as a colourless waxy oil (64.8 mg, 0.051 mmol, 60%). R_f = 0.65 (pet. ether : EtOAc, 9 : 1, v/v); [α]^22.8_D = +80 (c = 0.1, CH₂Cl₂); ¹H NMR (600 MHz, CDCl₃) δ 5.80 (ddt, J_10‴,11‴a = 16.9 Hz, J_10‴,11‴b = 10.1 Hz, J_10‴,9‴ = 6.7 Hz, 1H, H-10‴), 5.01–4.91 (m, 4H, H-1, H-1′, H-11‴a and H-11‴b), 4.27 (dd, J_6/6′a,b = 11.9 Hz, J_{5/5′,6/6′a} = 2.3 Hz, 2H, H-6a and H-6′a), 4.05 (dd, J_6/6′a,b = 11.9 Hz, J_{5/5′,6/6′b} = 4.4 Hz, 2H, H-6b and H-6′b), 4.00 (ddd, J_{4/4′,5/5′} = 9.2 Hz, J_{5/5′,6/6′b} = 4.4 Hz, J_{5/5′,6/6′a} = 2.3 Hz, 2H, H-5 and H-5′), 3.90 (t, J_{2/2′,3/3′} = J_{3/3′,4/4′} = 9.2 Hz, 2H, H-3 and H-3′), 3.48 (t, J_{3/3′,4/4′} = J_{4/4′,5/5′} = 9.2 Hz, 2H, H-4 and H-4′), 3.43 (dd, J_{2/2′,3/3′} = 9.2 Hz, J_{1/1′,2/2′} = 3.1 Hz, 2H, H-2 and H-2′), 2.38–2.29 (m, 4H, H-2″ and H-2‴), 2.05–2.01 (m, 2H, H-9‴), 1.65–1.59 (m, 4H, H-3″ and H-3‴), 1.38–1.25 (m, 46H, H-4″–H-21″ and H-4‴–H-8‴), 0.88 (t, J_21″,22″ = 7.0 Hz, 3H, H-22″), 0.15, 0.13, 0.13 (3s, 54H, TMS); ¹³C NMR (150 MHz, CDCl₃) δ 173.9 (C-1″ and C-1‴), 139.3 (C-10‴), 114.3 (C-11‴), 94.5 (C-1 and C-1′), 73.6 (C-3 and C-3′), 72.8 (C-2 and C-2′), 72.1 (C-4 and C-4′), 70.9 (C-5 and C-5′), 63.5 (C-6 and C-6′), 34.3 (C-2″ and C-2‴), 33.9, 32.1, 29.9, 29.6, 29.51, 29.45, 29.38, 29.31, 29.27, 29.21, 29.0, 22.8 (C-4″–C21″ and C-4‴–C-8‴), 24.9 (C-3″ and C-3‴), 14.3 (C-22″), 1.2, 1.0, 0.3 (TMS); IR (film): 2970, 2924, 2854, 1742, 1630, 1459, 1369, 1250, 1164, 1110, 1100, 1076, 1009, 898, 871, 840 cm⁻¹; HRMS (ESI) m/z calcd for [C₆₃H₁₃₀O₁₃Si₆ + NH₄]⁺: 1280.8465; obsd.: 1280.8473.

6-O-Docosanoyl-6′-O-(pentadec-14-enoyl)-2,2′,3,3′,4,4′-hexa-O-trimethylsilyl-α,α′-D-trehalose (6c). Monoester 9 (24.9 mg, 0.023 mmol) was co-evaporated twice with dry toluene (2 × 2–3 mL) and then a third time with excess toluene, which was then concentrated to 1.5 mL. Carboxylic acid 8c (6.5 mg, 0.027 mmol), DMAP (5.5 mg, 0.045 mmol) and EDCI (8.7 mg, 0.045 mmol) were then added to the reaction mixture. The mixture was stirred at 70 °C for 26 h, by which time the reaction was deemed complete, as gauged by TLC analysis. The reaction mixture was cooled to room temperature, diluted with EtOAc (60 mL), washed with water (60 mL) and a saturated solution of brine (60 mL), dried over MgSO₄, filtered, and concentrated under reduced pressure. The crude product was subjected to gradient silica gel flash column chromatography (eluting with pet. ether : EtOAc, 1 : 0 → 97 : 3, v/v) to obtain the title compound 6c as a colourless waxy oil (17.5 mg, 0.013 mmol, 58%). R_f = 0.47 (pet. ether : EtOAc, 9 : 1, v/v); [α]^23.4_D = +60 (c = 0.1, CH₂Cl₂); ¹H NMR (500 MHz, CDCl₃) δ 5.80 (ddt, J_14‴,15‴a = 16.9, J_14‴,15‴b = 10.2, J_13‴,14‴ = 6.7 Hz, 1H, H-14‴), 5.01–4.89 (m, 4H, H-1 and H-1′ and H-15‴a and H-15‴), 4.26 (dd, J_6/6′a,b = 11.8, J_{5/5′,6/6′a} = 2.3 Hz, 2H, H-6a and H-6′a), 4.04 (dd, J_6/6′a,b = 11.9, J_{5/5′,6/6′b} = 4.4 Hz, 2H, H-6b and H-6′b), 4.01–3.96 (m, 2H, H-5 and H-5′), 3.89 (t, J_{2/2′,3/3′} = J_{3/3′,4/4′} = 8.9 Hz, 2H, H-3 and H-3′), 3.47 (t, J_{3/3′,4/4′} = J_{4/4′,5/5′} = 9.0 Hz, 2H, H-4 and H-4′), 3.43 (dd, J_{2/2′,3/3′} = 9.3, J_{3/3′,4/4′} = 3.1 Hz, 2H, H-2 and H-2′), 2.39–2.26 (m, 4H, H-2″ and 2‴), 2.02 (q, J_12‴,13‴ = J_13‴,14‴ = 6.9 Hz, 2H, H-13‴), 1.68–1.57 (m, 4H, H-3″ and H-3‴), 1.43–1.33 (m, 2H, H-12‴), 1.33–1.15 (m, 52H, H-4″–H-21″ and H-4‴–H-11‴), 0.87 (t, J_21″,22″ = 6.8 Hz, 3H, H-22″), 0.14, 0.12, 0.12 (3s, TMS, 54H); ¹³C NMR (125 MHz, CDCl₃) δ 173.9 (C-1″ and C-1‴), 139.4 (C-14‴), 114.2 (C-15‴), 94.5 (C-1 and C-1′), 73.6 (C-3 and C-3′), 72.7 (C-2 and C-2′), 72.0 (C-4 and C-4′), 70.8 (H-5 and H-5′), 63.4 (C-6 and C-6′), 34.2 (C-2″ and C-2‴), 33.9 (H-13‴), 32.0 (C-20″), 29.8, 29.8, 29.7, 29.7, 29.6, 29.6, 29.5, 29.4, 29.3, 29.0, 22.8, (C-4″–C-19″ and C-4‴–C-12‴), 24.9 (C-3″ and C-3‴), 14.2 (C-22″), 1.2, 1.0, 0.3 (TMS); IR (film): 2924, 2854, 1743, 1459, 1251, 1165, 1111, 1077, 1046, 1011, 966, 931, 898, 873, 843, 749 cm⁻¹; HRMS (ESI) m/z calcd for [C₆₇H₁₃₈O₁₃Si₆ + NH₄]⁺: 1336.9091; obsd.: 1336.9094.

6-O-(Hex-5-enoyl)-6′-O-(p-(octadecyloxy)benzoyl)-2,2′,3,3′,4,4′-hexa-O-trimethylsilyl-α,α′-D-trehalose (7a). Monoester 10 (188 mg, 0.163 mmol) was co-evaporated twice with dry toluene (2 × 6–8 mL) and then a third time with excess toluene, which was then concentrated to 5 mL. Carboxylic acid 8a (0.02 mL, 0.196 mmol), DMAP (39.9 mg, 0.326 mmol) and EDCI (70.0 mg, 0.365 mmol) were then added to the reaction mixture. The mixture was stirred at 70 °C for 24 hours, by which time the reaction was deemed complete, as gauged by TLC analysis. The reaction mixture was cooled to room temperature, diluted with EtOAc (150 mL), washed with water (150 mL) and brine (80 mL), dried over MgSO₄, filtered, and concentrated under reduced pressure. The crude product was subjected to gradient silica gel flash column chromatography (eluting with pet. ether : EtOAc, 1 : 0 → 97 : 3, v/v) to obtain the title compound 7a as a colourless waxy oil (113.3 mg, 0.091 mmol, 56%). R_f = 0.68 (pet. ether : EtOAc, 10 : 1, v/v); [α]^23.5_D = +60 (c = 0.1, CH₂Cl₂); ¹H NMR (500 MHz, CDCl₃) δ 7.98 (d, J_3″,4″ = 8.9 Hz, 2H, H-3″), 6.90 (d, J_3″,4″ = 8.9 Hz, 2H, H-4″), 5.76 (ddt, J_5‴,6‴a/b = 16.9, J_5‴,6‴a/b = 10.2, J_4‴,5‴ = 6.7 Hz, 1H, H-5‴), 5.04–4.91 (m, 4H, H-6‴a/b and H-1 and H-1′), 4.52 (dd, J_6a,b = 12.0, J_5,6a = 2.4 Hz, 1H, H-6a), 4.27 (dd, J_6′a,b = 11.9, J_5′,6′a = 2.3 Hz, 1H, H-6′a), 4.23 (dd, J_6a,b = 12.0, J_5,6b = 3.6 Hz, 1H, H-6b), 4.09 (dt, J_4′,5′ = 9.6, J_5′,6′a/b = 3.0 Hz, 1H), 4.06 (dd, J_6′a,b = 11.9, J_5′,6′b = 4.5 Hz, 1H, H-6′b), 4.02–3.97 (m, 3H, H-6′ and H-5), 3.94 (t, J_{3/3′,4/4′} = J_{2/2′,3/3′} = 8.9 Hz, 1H, H-3 or H-3′), 3.91 (t, J_{3/3′,4/4′} = J_{2/2′,3/3′} = 8.9 Hz, 1H, H-3 or H-3′), 3.63 (t, J_4′,5′ = J_3′,4′ = 8.9 Hz, 1H, H-4′), 3.51–3.41 (m, 3H, H-2 and H-2′ and H-4), 2.42–2.25 (m, 2H, H-2‴), 2.13–2.03 (m, 2H, H-4‴), 1.83–1.75 (m, 2H, H-7″), 1.75–1.67 (m, 2H, H-3‴), 1.49–1.40 (m, 2H, H-8″), 1.31–1.20 (m, 26H, H-9″–H-22″), 0.86 (t, J_22″,23″ = 6.9 Hz, 3H, H-23″), 0.16, 0.15, 0.14, 0.12, 0.12, 0.11 (6s, 54H, CH₃, TMS); ¹³C NMR (125 MHz, CDCl₃) δ 173.6 (C-1‴), 166.4 (C-1″), 163.2 (C-5″), 137.8 (C-5‴), 131.8 (C-3″), 122.2 (C-2″), 115.5 (C-6‴), 114.2 (C-4″), 94.6 (C-1 and C-1′), 73.8, 73.6 (C-3 and C-3′), 72.79, 72.75 (C-2 and C-2′), 72.0 (C-4 and C-4′), 71.0, 70.8 (C-5 and C-5′), 68.3 (C-6″), 63.5 (C-6 and C-6′), 33.5 (C-2‴), 33.1 (C-4‴), 32.0 (C-21″ or C-22″), 29.8, 29.7, 29.7, 29.5, 29.5 (C-9″–C-20″), 29.2 (C-7″) 26.1 (C-8″), 24.0 (C-3‴), 22.8 (C-21″ or C-22″), 14.2 (C-23″), 1.2, 1.2, 1.0, 1.0, 0.3, 0.3 (TMS); IR (film): 2924, 2853, 1741, 1719, 1606, 1510, 1458, 1389, 1313, 1250, 1165, 1109, 1099, 1076, 1044, 1008, 965, 934, 898, 871, 842, 766, 748, 694, 645, 577, 536, 518, 473, 455, 423, 414 cm⁻¹; HRMS (ESI) calcd for [C₆₁H₁₁₈O₁₄Si₆ + NH₄]⁺: 1260.7475; obsd.: 1260.7472.

6-O-(p-(Octadecyloxy)benzoyl)-2,2′,3,3′,4,4′-hexa-O-trimethylsilyl-6′-O-(undec-10-enoyl)-α,α′-D-trehalose (7b). Monobenzoate 10 (188 mg, 0.164 mmol) and carboxylic acid 8b (0.04 mL, 0.20 mmol) were co-evaporated twice with dry toluene (2 × 10 mL) and then a third time with excess toluene, which was then reduced to half the volume (8 mL). To this mixture, DMAP (40.0 mg, 0.328 mmol) and DCC (62.9 mg, 0.328 mmol) were added, and the reaction mixture was then stirred at 70 °C overnight. Once the reaction was deemed complete (24 h), as gauged by TLC analysis, the reaction mixture was cooled to room temperature, diluted with EtOAc (150 mL), and washed with water (150 mL). The aqueous layer was extracted with EtOAc (80 mL), and the combined organic layers were washed with sat. aq. NaHCO₃ (200 mL) and brine (200 mL), dried over MgSO₄, filtered, and concentrated under reduced pressure. The resulting residue was purified using gradient silica gel flash chromatography (eluting with pet. ether : EtOAc, 1 : 0 → 49 : 1, v/v), which gave the title compound 7b as a colourless viscous oil (172 mg, 0.130 mmol, 80%). R_f = 0.68 (pet. ether : EtOAc, 3 : 17, v/v); [α]^22.0_D = +60 (c = 0.1, CH₂Cl₂); ¹H NMR (600 MHz, CDCl₃) δ 7.99 (d, J_3″,4″ = 8.9 Hz, 2H, H-3″), 6.91 (d, J_3″,4″ = 8.9 Hz, 2H, H-4″), 5.80 (ddt, J_10‴,11‴a = 16.9 Hz, J_10‴,11‴b = 10.2 Hz, J_10‴,9‴ = 6.7 Hz, 1H, H-10‴), 5.03–4.89 (m, 4H, H-1, H-1′, H-11‴a and b), 4.53 (dd, J_6a,b = 12.0 Hz, J_5,6a = 2.4 Hz, 1H, H-6a), 4.27 (dd, J_6′a,b = 11.9 Hz, J_5,6′a = 2.4 Hz, 1H, H-6′a), 4.24 (dd, J_6a,b = 12.0 Hz, J_5,6b = 3.6 Hz, 1H, H-6b), 4.11–4.09 (m, 1H, H-5), 4.06 (dd, J_6′a,b = 11.9 Hz, J_5,6′b = 4.5 Hz, 1H, H-6′b), 4.02–3.99 (m, 3H, H-5′ and H-6″), 3.96–3.91 (m, 2H, H-3 and 3′), 3.64 (dd, J_4,5 = 9.5 Hz, J_3,4 = 8.5 Hz, 1H, H-4), 3.50–3.44 (m, 3H, H-2, H-2′ and H-4′), 2.38–2.29 (m, 2H, H-2‴), 2.04–2.00 (m, 2H, H-9‴), 1.84–1.76 (m, 2H, H-7″), 1.63–1.59 (m, 2H, H-3‴), 1.48–1.43 (m, 2H, H-8″), 1.37–1.24 (m, 36H, H-9″–H-22″ and H-4‴–H-8‴), 0.88 (t, J_22″,23″ = 7.0 Hz, 3H, H-23″), 0.17, 0.16, 0.15, 0.13, 0.13, 0.12 (6s, 54H, CH₃-TMS); ¹³C NMR (150 MHz, CDCl₃) δ 173.9 (C-1‴), 166.4 (C-1″), 163.2 (C-5″), 139.3 (C-10‴), 131.9 (C-3″), 122.3 (C-2″), 114.29, 114.24 (C-4″ and C-11‴), 94.7 (C-1 and C-1′), 73.8, 73.6 (C-3 and C-3′), 72.86, 72.84 (C-2 and C-2′), 72.1 (C-4 and C-4′), 71.0 (C-5), 70.9 (C-5′), 68.4 (C-6″), 63.5 (C-6 and C-6′), 34.3 (C-2‴), 33.9 (C-9‴), 32.1, 29.84, 29.75, 29.72, 29.5, 29.43, 29.38, 29.27, 29.20, 29.0, 26.2, 24.9, 22.8 (C-3‴–C-8‴ and C-7″–C-22″), 14.3 (C-23″), 1.25, 1.22, 1.04, 0.38 (TMS); IR (film): 2970, 2924, 2854, 1741, 1719, 1607, 1510, 1458, 1250, 1165, 1109, 1099, 1075, 1008, 899, 870, 838, 748 cm⁻¹; HRMS (ESI) m/z calcd for [C₆₆H₁₂₈O₁₄Si₆ + NH₄]⁺: 1330.8258; obsd.: 1330.8251.

6-O-(p-(Octadecyloxy)benzoyl)-2,2′,3,3′,4,4′-hexa-O-trimethylsilyl-6′-O-(pentadec-14-enoyl)-α,α′-D-trehalose (7c). Monobenzoate 10 (94.3 mg, 0.082 mmol) and carboxylic acid 8c (23.7 mg, 0.099 mmol) were co-evaporated twice with dry toluene (2 × 4 mL) and then a third time with excess toluene, which was then reduced to half the volume (ca. 4 mL). To this mixture, DMAP (20.0 mg, 0.164 mmol) and EDCI (31.0 mg, 0.164 mmol) were added, and the reaction mixture was stirred at 70 °C overnight. After 24 h, another equivalent of EDCI (15.0 mg, 0.078 mmol) was added and the reaction mixture was stirred overnight. The reaction was then cooled to room temperature, diluted with EtOAc (100 mL), and washed with water (100 mL). The aqueous layer was extracted with EtOAc (50 mL) and the combined organic layers were washed with sat. aq. NaHCO₃ (150 mL) and brine (150 mL), dried over MgSO₄, filtered, and concentrated under reduced pressure. The resulting residue was then purified using gradient silica gel flash chromatography (eluting with pet. ether : EtOAc, 1 : 0 → 97 : 3, v/v). The title compound 7c was obtained as a colourless viscous oil (78.7 mg, 0.057 mmol, 70%). R_f = 0.48 (pet. ether : EtOAc, 9 : 1, v/v); [α]^27.4_D = +51.7 (c = 1.0, CH₂Cl₂); ¹H NMR (500 MHz, CDCl₃) δ 7.97 (d, J_3″,4″ = 8.8 Hz, 2H, H-3″), 6.90 (d, J_3″,4″ = 8.8 Hz, 2H, H-4″), 5.84–5.80 (m, 1H, H-14‴), 5.00–4.89 (m, 4H, H-1 and H-1′ and H-15‴), 4.52 (dd, J_6a,b = 12.1 Hz, J_5,6a = 2.1 Hz, 1H, H-6a), 4.30–4.20 (m, 2H, H-6′a and H-6b), 4.1 (dt, J_4,5 = 9.5 Hz, J_5,6a/b = 2.7 Hz, 1H, H-5), 4.10 (dd, J_6′a,b = 11.9 Hz, J_5′,6′b = 4.4 Hz, 1H, H-6′b), 4.02–4.00 (m, 3H, H-5′ and H-6″), 3.94 (t, J_{2/2′,3/3′} = J_{3/3′,4/4′} = 8.9 Hz, 1H, H-3 or H-3′), 3.91 (t, J_{2/2′,3/3′} = J_{3/3′,4/4′} = 8.9 Hz, 1H, H-3 or H-3′), 3.63 (t, J_3,4 = J_4,5 = 9.0 Hz, 1H, H-4), 3.50–3.41 (m, 3H, H-2 and H-2′ and H-4′), 2.38–2.26 (m, 2H, H-2‴), 2.06–1.99 (m, 2H, H-13‴), 1.83–1.75 (m, 2H, H-7″), 1.65–1.57 (m, 2H, H-3‴), 1.49–1.40 (m, 2H, H-8″), 1.39–1.32 (m, 2H, H-9″), 1.32–1.17 (m, 44H, H-4‴-H–12‴ and H-10″–H-22″), 0.87 (t, J_22″,23″ = 6.9 Hz), 0.16, 0.15, 0.14, 0.12, 0.11 (6s, 54H, CH₃, TMS); ¹³C NMR (125 MHz, CDCl₃) δ 173.9 (C-1‴), 166.4 (C-1″), 163.2 (C-5″), 139.4 (C-14‴), 131.8 (C-3″), 122.2 (C-2″), 114.2 (C-4″ and C-15‴), 94.6 (C-1 and C-1′), 73.8 (C-3 or C-3′), 73.6 (C-3 or C-3′), 72.8, 72.8 (C-2 and C-2′ and C-4′), 72.0 (C-4), 71.0 (C-5), 70.8 (C-5′), 68.3 (C-6″), 63.5, 63.4 (C-6 and C-6′), 34.2 (C-2‴), 33.9 (C-13‴), 32.0, 29.8, 29.8, 29.7, 29.7, 29.6, 29.6, 29.5, 29.5, 29.4, 29.2, 29.2, 29.0, 22.8 (C-7″ and C-9″–C-22″ and C-5‴–C-12‴), 26.1 (C-8″), 24.9 (C-3‴), 14.2 (C-23″), 1.2, 1.2, 1.0, 0.3, 0.3 (TMS); IR (film): 2924, 2854, 1742, 1720, 1607, 1511, 1458, 1250, 1165, 1109, 1099, 1075, 1044, 1008, 965, 934, 898, 870, 838, 748, 695, 536, 519 cm⁻¹; HRMS (ESI) m/z calcd for [C₇₀H₁₃₆O₁₄Si₆ + NH₄]⁺: 1386.8884; obsd.: 1386.8844.

General procedure for olefin metathesis

The olefin starting material was co-evaporated with dry toluene (2 × 40 mL mmol⁻¹) and re-dissolved in dry CH₂Cl₂ (20 mL mmol⁻¹). To this was added Grubbs’ catalyst (2^nd generation, 1 mol%) and the mixture heated under reflux for 24 hours. At this point, the reaction was deemed complete, as confirmed by TLC and the reaction mixture was concentrated under reduced pressure.

Bis(6-O-docosanoyl-2,2′,3,3′,4,4′-hexa-O-trimethylsilyl-α,α′-D-trehalose-6′-yl) (E/Z)-dec-5-enedioate (16a). Diester 6a (52.0 mg, 0.044 mmol) was subjected to the general metathesis procedure and the resulting crude residue was purified by gradient silica gel flash column chromatography (gradient elution with pet. ether : EtOAc, 1 : 0 → 93 : 7, v/v) to obtain the title compound 16a as a colourless viscous oil (32.4 mg, 0.014 mmol, 64%). R_f = 0.68 (pet. ether : EtOAc, 9 : 1, v/v); [α]^29.5_D = +80 (c = 0.1, CH₂Cl₂); ¹H NMR (500 MHz, CDCl₃) δ 5.45–5.34 (m, 2H, H-5‴), 4.90 (d, J_{1/1′,2/2′} = 3.0 Hz, 4H, H-1 and H-1′), 4.26 (dd, J_6/6′a,b = 11.9 Hz, J_{5/5′,6/6′a} = 2.3 Hz, 4H, H-6a and H-6′a), 4.04 (dd, J_6/6′a,b = 11.9 Hz, J_{5/5′,6/6′b} = 4.4 Hz, 4H, H-6b and H-6′b), 3.98 (ddd, J_{4/4′,5/5′} = 9.2 Hz, J_{5/5′,6/6′b} = 4.4 Hz, J_{5/5′,6/6′a} = 2.3 Hz, 4H, H-5 and H-5′), 3.88 (t, J_{2/2′,3/3′} = J_{3/3′,4/4′} = 9.2 Hz, 4H, H-3 and H-3′), 3.47 (t, J_{3/3′,4/4′} = J_{4/4′,5/5′} = 9.2 Hz, 4H, H-4 and H-4′), 3.42 (dd, J_{2/2′,3/3′} = 9.2 Hz, J_{1/1′,2/2′} = 3.1 Hz, 4H, H-2 and H-2′), 2.41–2.27 (m, 8H, H-2″ and H-2‴), 2.07–2.00 (m, 4H, H-4‴), 1.70–1.57 (m, 8H, H-3″ and H-3‴), 1.31–1.23 (m, 72H, H-4″–H-21″), 0.86 (t, J_21″,22″ = 6.9 Hz, 6H, H-22), 0.13, 0.12, 0.11, 0.11 (4s, 108H, TMS); ¹³C NMR (125 MHz, CDCl₃) δ 173.9 (C-1″/C-1‴), 173.6 (C-1″/C-1‴), 130.2 (C-5‴), 94.6 (C-1 and C-1′), 73.6 (C-3 and C-3′), 72.7 (C-2 and C-2′), 72.0 (C-4 and C-4′), 70.8 (C-5 and C-5′), 63.42 (C-6/C-6′), 63.38 (C-6/C-6′), 34.2 (C-2″/C-2‴), 33.6 (C-2″/C-2‴), 32.01(C-4‴, Z/E), 31.97 (C-4‴, Z/E), 29.79, 29.74, 29.58, 29.45, 29.41, 29.26, 22.8 (C-4″–C-21″), 24.9 (C-3″), 24.6 (C-3‴), 14.2 (C-22″), 1.1, 1.0, 0.3 (TMS); IR (film): 2956, 2923, 2853, 1741, 1720, 1659, 1633, 1607, 1511, 1467, 1420, 1314, 1251, 1166, 1110, 1099, 1076, 1044, 1009, 966, 935, 899, 871, 841, 767, 748 cm⁻¹; HRMS (ESI) m/z calcd for [C₁₁₄H₂₃₆O₂₆Si₁₂ + Na]⁺: 2381.4302; obsd.: 2381.4366.

Bis(6-O-docosanoyl-2,2′,3,3′,4,4′-hexa-O-trimethylsilyl-α,α′-D-trehalose-6′-yl) (E/Z)-eicos-10-enedioate (16b). Diester 6b (48.9 mg, 0.039 mmol) was subjected to the general metathesis procedure and the resulting crude residue was purified by gradient silica gel flash column chromatography (gradient elution with pet. ether : EtOAc, 1 : 0 → 24 : 1, v/v) to obtain the title compound 16b as a colourless viscous oil (25.7 mg, 0.010 mmol, 51%). R_f = 0.55 (pet. ether : EtOAc, 9 : 1, v/v); [α]^23.3_D = +60 (c = 0.1, CH₂Cl₂); ¹H NMR (600 MHz, CDCl₃) δ 5.39–5.32 (m, 2H, H-10‴), 4.92 (d, J_{1/1′,2/2′} = 3.1 Hz, 4H, H-1 and H-1′), 4.27 (dd, J_6/6′a,b = 11.9 Hz, J_{5/5′,6/6′a} = 2.3 Hz, 4H, H-6a and H-6′a), 4.05 (dd, J_6/6′a,b = 11.9 Hz, J_{5/5′,6/6′b} = 4.4 Hz, 4H, H-6b and H-6′b), 4.00 (ddd, J_{4/4′,5/5′} = 9.2 Hz, J_{5/5′,6/6′b} = 4.4 Hz, J_{5/5′,6/6′a} = 2.3 Hz, 4H, H-5 and H-5′), 3.90 (t, J_{2/2′,3/3′} = J_{3/3′,4/4′} = 9.2 Hz, 4H, H-3 and H-3′), 3.48 (t, J_{3/3′,4/4′} = J_{4/4′,5/5′} = 9.2 Hz, 4H, H-4 and H-4′), 3.44 (dd, J_{2/2′,3/3′} = 9.2 Hz, J_{1/1′,2/2′} = 3.1 Hz, 4H, H-2 and H-2′), 2.38–2.29 (m, 8H, H-2″ and H-2‴), 2.02–1.93 (m, 4H, H-9‴), 1.64–1.60 (m, 8H, H-3″ and H-3‴), 1.33–1.22 (m, 92H, H-4″–H-21″ and H-4‴–H-8‴), 0.88 (t, J_21″,22″ = 7.0 Hz, 6H, H-22″), 0.15, 0.13, 0.13 (3s, 108H, TMS); ¹³C NMR (150 MHz, CDCl₃) δ 173.94, 173.92 (C-1″ and C-1‴), 130.5 (C-10‴), 94.6 (C-1 and C-1′), 73.6 (C-3 and C-3′), 72.8 (C-2 and C-2′), 72.1 (C-4 and C-4′), 70.9 (C-5 and C-5′), 63.4 (C-6 and C-6′), 34.3 (C-2″ and C-2‴), 32.8 (C-9‴), 32.1, 29.85, 29.81, 29.79, 29.64, 29.51, 29.46, 29.3, 22.8 (C-4″–C21″ and C-4‴–C-8‴), 24.9 (C-3″ and C-3‴), 14.3 (C-22″), 1.2, 1.0, 0.3 (TMS); IR (film): 2970, 2923, 2853, 1742, 1459, 1368, 1250, 1163, 1110, 1099, 1076, 1045, 1009, 871, 837, 747 cm⁻¹; HRMS (ESI) m/z calcd for [C₁₂₄H₂₅₆O₂₆Si₁₂ + 2NH₄]²⁺: 1267.3326; obsd.: 1267.3320.

Bis(6-O-docosanoyl-2,2′,3,3′,4,4′-hexa-O-trimethylsilyl-α,α′-D-trehalose-6′-yl) (E/Z)-octacos-14-enedioate (16c). Diester 6c (32.7 mg, 0.025 mmol) was subjected to the general metathesis procedure and the resulting crude residue was purified by gradient silica gel flash column chromatography (gradient elution with pet. ether : EtOAc, 1 : 0 → 24 : 1, v/v) to obtain the title compound 16a as a colourless viscous oil (18.4 mg, 0.007 mmol, 57%). R_f = 0.60 (pet. ether : EtOAc, 9 : 1, v/v); [α]^24.3_D = +60 (c = 0.1, CH₂Cl₂); ¹H NMR (500 MHz, CDCl₃) δ 5.41–5.31 (m, 2H, H-14‴), 4.91 (d, J_{1/1′,2/2′} = 2.8 Hz, 4H, H-1 and H-1′), 4.26 (dd, J_6/6′a,b = 11.8, J_{5/5′,6/6′a} = 2.1 Hz, 4H, H-6a and H-6′a), 4.04 (dd, J_6/6′a,b = 12.0, J_{5/5′,6/6′b} = 4.3 Hz, 4H, H-6b and H-6′b), 3.98 (ddd, J_{4/4′,5/5′} = 9.6, J_{5/5′,6/6′b} = 4.2, J_{5/5′,6/6′a} = 2.1 Hz, 4H, H-5 and H-5′), 3.89 (t, J_{2/2′,3/3′} = J_{3/3′,4/4′} = 9.0 Hz, 4H, H-3 and H-3′), 3.47 (t, J_{3/3′,4/4′} = J_{4/4′,5/5′} = 9.0 Hz, 4H, H-4 and H-4′), 3.43 (dd, J_{2/2′,3/3′} = 9.1, J_{1/1′,2/2′} = 2.9 Hz, 4H, H-2 and H-2′), 2.38–2.26 (m, 8H, H-2″ and H-2‴), 2.02–1.91 (m, 4H, H-13‴), 1.67–1.56 (m, 8H, H-3″ and H-3‴), 1.34–1.20 (m, 108H, H-4″–H-21″ and H-4‴–H-12‴), 0.87 (t, J_21″,22″ = 7.0 Hz, H-22″), 0.14, 0.12, 0.12 (3s, 108H, TMS); ¹³C NMR (125 MHz, CDCl₃) δ 173.9 (C-1″ and C-1‴), 130.4 (C-14‴), 94.5 (C-1 and C-1′), 73.6 (C-3 and C-3′), 72.7 (C-2 and C-2′), 72.0 (C-4 and C-4′), 70.8 (C-5 and C-5′), 63.4 (C-6 and C-6′), 34.2 (C-2″ and C-2‴), 32.7 (C-13″), 32.0, 29.8, 29.8, 29.6, 29.5, 29.4, 29.3, 29.3, 22.8 (C-4″–C-21″ and C-4‴–C-12‴), 24.9 (C-3″ and C-3‴), 14.2 (C-22″), 1.2, 1.0, 0.3 (TMS); IR (film): 2923, 2853, 1742, 1632, 1459, 1411, 1250, 1164, 1110, 1099, 1076, 1045, 1009, 965, 897, 871, 839, 747, 684, 624, 579, 536, 518 cm⁻¹; HRMS (ESI) m/z calcd for [C₁₃₂H₂₇₂O₂₆Si₁₂ + 2Na]²⁺: 1327.8489; obsd.: 1327.8482.

Bis(6-O-(p-(octadecyloxy)benzoyl)-2,2′,3,3′,4,4′-hexa-O-trimethylsilyl-α,α′-D-trehalose-6′-yl) (E/Z)-dec-5-enedioate (17a). Diester 7a (55.0 mg, 0.044 mmol) was subjected to the general metathesis procedure and the resulting crude residue was purified by silica gel flash chromatography (gradient elution with pet. ether : EtOAc, 1 : 0 → 93 : 7, v/v) to obtain the title compound 17a as a colourless viscous oil (40.5 mg, 0.016 mmol, 75%). R_f = 0.53 (pet. ether : EtOAc, 9 : 1, v/v); [α]^27.3_D = +40 (c = 0.1, CH₂Cl₂); ¹H NMR (500 MHz, CDCl₃) δ 7.97 (d, J_3′,4′ = 9.1 Hz, 4H, H-3″), 6.90 (d, J_3′,4′ = 9.0 Hz, 4H, H-4″), 5.42–5.34 (m, 2H, H-5‴), 4.93 (t, J_{1/1′,2/2′} = 3.5 Hz, 4H, H-1 and H-1′), 4.52 (dd, J_6a,b = 12.1, J_5,6a = 2.4 Hz, 2H, H-6a), 4.28–4.20 (m, 4H, H-6b and H-6a′), 4.12–4.07 (m, 2H, H-5′), 4.05 (dd, J_6′a,b = 11.8, J_5′,6′b = 4.4 Hz, 2H, H-6′b), 4.02–3.97 (m, 6H, H-5 and H-6″), 3.93 (t, J_{2/2′,3/3′} = J_{3/3′,4/4′} = 8.9 Hz, 2H, H-3 or H-3′), 3.90 (t, J_{2/2′,3/3′} = J_{3/3′,4/4′} = 9.0 Hz, 2H, H-3 or H-3′), 3.64 (t, J_3′,4′ = J_4′,5′ = 9.0 Hz, 2H, H-4′), 3.50–3.41 (m, 6H, H-2 and H-2′ and H-4), 2.47–2.22 (m, 5H, H-2‴), 2.12–1.93 (m, 4H, H-4‴), 1.84–1.74 (m, 5H, H-7″), 1.70–1.62 (m, 4H, H-3‴), 1.48–1.40 (m, 4H, H-8″), 1.37–1.17 (m, 52H, H-9″–H-22″), 0.86 (t, J_22″,23″ = 6.9 Hz, 6H, H-23″), 0.15, 0.14, 0.13, 0.11, 0.10 (6s, 108H, CH₃, TMS); ¹³C NMR (125 MHz, CDCl₃) δ 173.7 (C-1‴), 166.3 (C-1″), 163.2 (C-5″), 131.8 (C-3″), 130.2 (C-5‴), 122.2 (C-2″), 114.2 (C-4″), 94.7 (C-1 and C-1′), 73.8, 73.6 (C-3 and C-3′), 72.8, 72.7 (C-2 and C-2′), 72.0 (C-4 and C-4″), 70.9, 70.8 (C-5 and C-5′), 68.3 (C-6″), 63.5 (C-6 and C-6′), 33.6 (C-2‴), 32.0 (C-4‴), 29.8, 29.8, 29.7, 29.7, 29.5, 29.5, 22.8 (C-9″–C-22″), 29.2 (C-7″), 26.1 (C-8″), 24.6 (C-3‴), 14.2 (C-23″), 1.2, 1.2, 1.0, 0.3, 0.3 (TMS); IR (film): 2924, 2854, 1740, 1719, 1606, 1511, 1457, 1249, 1165, 1099, 1075, 1044, 1008, 966, 934, 898, 870, 835, 748, 695, 537, 518 cm⁻¹; HRMS (ESI) m/z calcd for [C₁₂₀H₂₃₂O₂₈Si₁₂ + 2NH₄]²⁺: 1247.2336; obsd.: 1247.2339.

Bis(6-O-(p-(octadecyloxy)benzoyl)-2,2′,3,3′,4,4′-hexa-O-trimethylsilyl-α,α′-D-trehalose-6′-yl) (E/Z)-icos-10-enedioate (17b). Diester 7b (70.5 mg, 0.054 mmol) was subjected to the general metathesis procedure and the resulting crude residue was purified by silica gel flash chromatography (gradient elution with pet. ether : EtOAc, 1 : 0 → 19 : 1, v/v) to obtain the title compound 17b as a colourless viscous oil (41.8 mg, 0.016 mmol, 60%) R_f = 0.75 (pet. ether : EtOAc, 3 : 22, v/v); [α]^21.0_D = +40 (c = 0.1, CH₂Cl₂); ¹H NMR (500 MHz, CDCl₃) δ 7.99 (d, J_3″,4″ = 8.9 Hz, 4H, H-3″), 6.91 (d, J_3″,4″ = 8.9 Hz, 4H, H-4″), 5.37–5.32 (m, 2H, Z- and E-H-10‴), 4.95 (d, J_{1/1′,2/2′} = 3.5 Hz, 2H, H-1/1′), 4.94 (d, J_{1/1′,2/2′} = 3.5 Hz, 2H, H-1/1′), 4.53 (dd, J_6a,b = 12.0 Hz, J_5,6a = 2.4 Hz, 2H, H-6a), 4.28–4.23 (m, 4H, H-6b and H-6′a), 4.11–4.05 (m, 4H, H-5 and H-6′b), 4.03–3.99 (m, 6H, H-5′ and H-6″), 3.97–3.90 (m, 4H, H-3 and 3′), 3.65 (t, J_3,4 = J_4,5 = 9.1 Hz, 2H, H-4), 3.50–3.43 (m, 6H, H-2, H-2′ and H-4′), 2.37–2.30 (m, 4H, H-2‴), 2.00–1.90 (m, 4H, H-9‴), 1.83–1.77 (m, 4H, H-7″), 1.63–1.59 (m, 4H, H-3‴), 1.46 (p, J_7″,8″ = J_8″,9″ = 7.2 Hz, 4H, H-8″), 1.36–1.21 (m, 76H, H-9″–H-22″ and H-4‴–H-8‴), 0.88 (t, J_22″,23″ = 7.1 Hz, 6H, H-23″), 0.17, 0.16, 0.15, 0.13, 0.12 (5s, 54H, TMS); ¹³C NMR (150 MHz, CDCl₃) δ 174.0 (C-1‴), 166.4 (C-1″), 163.2 (C-5″), 131.8 (C-3″), 130.5 (C-10‴), 122.3 (C-2″), 114.2 (C-4″), 94.7 (C-1 and C-1′), 73.8, 73.6 (C-3 and C-3′), 72.84, 72.79 (C-2 and C-2′), 72.0 (C-4 and C-4′), 70.98 (C-5), 70.86 (C-5′), 68.4 (C-6″), 63.51 (C-6′), 63.45 (C-6), 34.3 (C-2‴), 32.8(C-9‴), 32.1, 29.84, 29.81, 29.75, 29.72, 29.53, 29.51, 29.49, 29.45, 29.32, 29.26, 26.1, 24.9, 22.8 (C-3‴–C-8‴ and C-7″–C-22″), 14.3 (C-23″), 1.24, 1.21, 1.04, 1.03, 0.38, 0.36 (TMS); IR (film): 2970, 2925, 2854, 1741, 1720, 1607, 1511, 1459, 1251, 1166, 1110, 1100, 1076, 1045, 1009, 899, 872, 843 cm⁻¹; HRMS (ESI) m/z calcd for [C₁₃₀H₂₅₂O₂₈Si₁₂ + HCOO]⁻: 2642.5509; obsd.: 2642.5522.

Bis(6-O-(p-(octadecyloxy)benzoyl)-2,2′,3,3′,4,4′-hexa-O-trimethylsilyl-α,α′-D-trehalose-6′-yl) (E/Z)-hex-5-enedioate (17c). Diester 7c (48.2 mg, 0.035 mmol) was subjected to the general metathesis procedure and the resulting crude residue was purified by silica gel flash chromatography (gradient elution with pet. ether : EtOAc, 1 : 0 → 19 : 1, v/v) to obtain the title compound 17c as a colourless viscous oil (21.8 mg, 0.008 mmol, 46%). R_f = 0.67 (pet. ether : EtOAc, 9 : 1, v/v); [α]^23.2_D = +49.4 (c = 0.5, CH₂Cl₂); ¹H NMR (500 MHz, CDCl₃) δ 7.98 (d, J_3″,4″ = 8.7 Hz, 4H, H-3″), 6.90 (d, J_3″,4″ = 8.7 Hz, 4H, H-4″), 5.38–5.31 (m, 2H, H-14‴), 4.94 (d, J_{1/1′,2/2′} = 3.5 Hz, 2H, H-1/1′), 4.93 (d, J_{1/1′,2/2′} = 3.5 Hz, H-1/1′), 4.52 (dd, J_6a,b = 12.0, J_5,6a = 2.4 Hz, 2H, H-6a), 4.29–4.20 (m, 4H, H-6b and H-6′a), 4.09 (dt, J_4,5 = 9.5, J_5,6a/b = 2.8 Hz, 2H, H-5), 4.05 (dd, J_6′a,b = 11.9 Hz, J_5′,6′b = 4.4 Hz, 2H, H-6′b), 4.02–3.97 (m, 6H, H-5′ and H-6″), 3.94 (t, J_{2/2′,3/3′} = J_{3/3′,4/4′} = 8.9 Hz, 2H, H-3/3′), 3.91 (t, J_{2/2′,3/3′} = J_{3/3′,4/4′} = 8.9 Hz, 2H, H-3/3′), 3.63 (t, J_3,4 = J_4,5 = 9.0 Hz, 2H, H-4), 3.50–3.41 (m, 6H, H-2 and H-2′ and H-4′), 2.38–2.26 (m, 4H, H-2‴), 2.02–1.90 (m, 4H, H-13‴), 1.82–1.75 (m, 4H, H-7″), 1.65–1.56 (m, 4H, H-3‴), 1.48–1.40 (m, 4H, H-8″), 1.38–1.17 (m, 92H, H-9″–H-22″ and H-4‴–H-12‴), 0.87 (t, J_22″,23″ = 6.8H, 6H, H-23″), 0.16, 0.15, 0.14, 0.12, 0.11 (6s, 54H, TMS); ¹³C NMR (125 MHz, CDCl₃) δ 173.9 (C-1‴), 166.3 (C-1″), 163.2 (C-5″), 131.8 (C-3″), 130.4 (C-14‴), 122.2 (C-2″), 114.2 (C-4″), 94.6 (C-1 and C-1′), 73.8, 73.6 (C-3 and C-3′), 72.8 (C-2 and C-2′), 72.0 (C-4 and C-4′), 71.0, 70.8 (C-5 and C-5′), 68.3 (C-6″), 63.5, 63.4 (C-6 and C-6′), 34.2 (C-2‴), 32.7 (C-13‴), 32.0, 29.8, 29.8, 29.7, 29.7, 29.7, 29.6, 29.5, 29.5, 29.4, 29.3, 29.3, 29.2, 22.8 (C-7″ and C-9″-C-22″ and C-4‴-C-12‴), 26.1 (C-8″), 24.9 (C-3‴), 14.2 (C-23″), 1.2, 1.2, 1.0, 1.0, 0.3, 0.3 (TMS); IR (film): 2924, 2854, 1742, 1720, 1607, 1511, 1458, 1313, 1251, 1166, 1110, 1100, 1077, 1045, 1010, 966, 934, 899, 872, 843, 767, 748 cm⁻¹.

General procedure for the desilylation

To a solution of the TMS protected trehalose ester in CH₂Cl₂ : MeOH (20 mL mmol⁻¹, 1 : 1, v/v) was added Dowex-H⁺ until the pH of the reaction mixture reached 2–3. The mixture was then stirred at room temperature for 1.5–2.5 hours until the reaction was deemed complete, as confirmed by TLC. The reaction mixture was then diluted with CH₂Cl₂ : MeOH (1 : 1, v/v), filtered, washed with CH₂Cl₂ : MeOH (1 : 1, v/v), and the filtrate was concentrated under vacuum.

Bis(6-O-docosanoyl-α,α′-D-trehalose-6′-yl) (E/Z)-dec-5-enedioate (3a). TMS protected glycolipid 16a (18.5 mg, 0.0078 mmol) was subjected to the general procedure for desilylation, followed by purification using silica gel flash column chromatography (eluted with CH₂Cl₂ : MeOH, 1 : 0 → 41 : 9, v/v) to obtain the final glycolipid 3a as a thin white film (10.6 mg, 0.0071 mmol, 91%). R_f = 0.52 (CH₂Cl₂ : MeOH, 4 : 1, v/v); [α]^25.2_D= + 40 (c = 0.1, pyridine); ¹H NMR (500 MHz, C₅D₅N) δ 5.89 (2d, J_{1/1′,2/2′} = 3.8 Hz, 4H, H-1 and H-1′), 5.30–5.28 (m, 2H, H-5‴), 5.12–5.06 (m, 4H, H-5 and H-5′), 5.00–4.98 (m, 4H, H-6a and H-6′a), 4.86–4.81 (m, 4H, H-6b and H-6′b), 4.75 (t, J_{2/2′,3/3′} = J_{3/3′,4/4′} = 9.3 Hz, 4H, H-3 and H-3′), 4.32 (dd, J_{2/2′,3/3′} = 9.3 Hz, J_{1/1′,2/2′} = 3.8 Hz, 4H, H-2 and H-2′), 4.21–4.16 (m, 4H, H-4 and H-4′), 2.35–2.29 (m, 8H, H-2″ and H-2‴), 1.94–1.90 (m, 4H, H-4‴), 1.69–1.59 (m, 8H, H-3″ and H-3‴), 1.32–1.19 (m, 72H, H-4″–H-21″), 0.87 (t, 6H, H-22″); ¹³C NMR (125 MHz, C₅D₅N) δ 174.1 (C-1″/C-1‴), 173.9 (C-1″/C-1‴), 130.8 (C-5‴), 96.3 (C-1 and C-1′), 75.3 (C-3 and C-3′), 73.7 (C-2 and C-2′), 72.4 (C-4 and C-4′), 72.0 (C-5 and C-5′), 64.8 (C-6 and C-6′), 34.8 (C-2‴), 34.2 (C-2″), 32.5 (C-4‴), 30.4, 30.3, 30.2, 29.98, 29.76, 23.3 (C-4″–C21″), 25.7 (C-3″), 25.4 (C-3‴), 14.7 (C-22″). IR (film): 3358 (broad), 2917, 2850, 1716, 1717, 1467, 1240, 1174, 1148, 1103, 1076, 1050, 1020, 987 cm⁻¹; HRMS (ESI) m/z calcd for [C₇₈H₁₄₀O₂₆ + NH₄]⁺: 1510.9971; obsd.: 1510.9970.

Bis(6-O-docosanoyl-α,α′-D-trehalose-6′-yl) (E/Z)-icos-10-enedioate (3b). TMS-protected metathesis product 16b (18.7 mg, 0.0074 mmol) was subjected to the general procedure for desilylation, followed by purification using silica gel flash column chromatography (eluted with CH₂Cl₂ : MeOH, 1 : 0 → 41 : 9, v/v) to obtain glycolipid 3b as a thin white film (10.1 mg, 0.0062 mmol, 83%). R_f = 0.63 (CH₂Cl₂ : MeOH, 4 : 1, v/v); [α]^23.5_D = +60 (c = 0.1, pyridine); ¹H NMR (600 MHz, C₅D₅N) δ 5.90 (d, J_{1/1′,2/2′} = 3.7 Hz, 4H, H-1 and H-1′), 5.53–5.48 (m, 2H, H-10‴), 5.11–5.08 (m, 4H, H-5 and H-5′), 5.02–4.99 (m, 4H, H-6a and H-6′a), 4.84 (dd, J_6/6′a,b = 11.9 Hz, J_{5/5′,6/6′b} = 5.3 Hz, 4H, H-6b and H-6′b), 4.76 (t, J_{2/2′,3/3′} = J_{3/3′,4/4′} = 9.3 Hz, 4H, H-3 and H-3′), 4.32 (dd, J_{2/2′,3/3′} = 9.3 Hz, J_{1/1′,2/2′} = 3.7 Hz, 4H, H-2 and H-2′), 4.18 (t, J_{3/3′,4/4′} = J_{4/4′,5/5′} = 9.3 Hz, 4H, H-4 and H-4′), 2.36–2.29 (m, 8H, H-2″ and H-2‴), 2.12–2.03 (m, 4H, H-9‴), 1.65–1.59 (m, 8H, H-3″ and H-3‴), 1.38–1.15 (m, 92H, H-4″–H-21″ and H-4‴–H-8‴), 0.88 (t, J_21″,22″ = 6.9 Hz, 6H, H-22″); ¹³C NMR (150 MHz, C₅D₅N) δ 174.1 (C-1″ and C-1‴), 131.1 (C-10‴), 96.2 (C-1 and C-1′), 75.3 (C-3 and C-3′), 73.8 (C-2 and C-2′), 72.4 (C-4 and C-4′), 71.9 (C-5 and C-5′), 64.7 (C-6 and C-6′), 34.8 (C-2″ and C-2‴), 33.4 (C-9‴), 32.5, 30.40, 30.36, 30.30, 30.15, 29.98, 29.94, 29.84, 29.75, 23.3 (C-4″–C21″ and C-4‴–C-8‴), 25.6 (C-3″ and C-3‴), 14.7 (C-22″); IR (film): 3343 (broad), 2918, 2850, 1736, 1735, 1466, 1354, 1241, 1175, 1150, 1103, 1077, 1053, 1019, 989, 941 cm⁻¹; HRMS (ESI) m/z calcd for [C₈₈H₁₆₀O₂₆ + HCOO]⁻: 1679.1213; obsd.: 1679.1188.

Bis(6-O-docosanoyl-α,α′-D-trehalose-6′-yl) (E/Z)-octacos-14-enedioate (3c). TMS-protected metathesis product 16c (14.4 mg, 0.0055 mmol) was subjected to the general procedure for desilylation, followed by purification using silica gel flash column chromatography (eluted with CH₂Cl₂ : MeOH, 1 : 0 → 41 : 9, v/v) to obtain the final glycolipid 3c as a thin white film (9.6 mg, 0.0055 mmol, quant.). R_f = 0.26 (CH₂Cl₂ : MeOH, 4 : 1, v/v); [α]^24.9_D = +65.8 (c = 0.1, pyridine); ¹H NMR (500 MHz, C₅D₅N) δ 5.83 (d, J_{1/1′,2/2′} = 3.4 Hz, 4H, H-1 and H-1′), 5.50–5.43 (m, 2H, H-14‴), 5.06–5.00 (m, 4H, H-5 and H-5′), 4.97–4.91 (m, 4H, H-6a and H-6′a), 4.77 (dd, J_6/6′a,b = 11.7, J_{5/5′,6/6′b} = 5.3 Hz, 4H, H-6b and H-6′b), 4.69 (t, J_{2/2′,3/3′} = J_{3/3′,4/4′} = 9.1 Hz, 4H, H-3 and H-3′), 4.25 (dd, J_{2/2′,3/3′} = 9.7 Hz, J_{1/1′,2/2′} = 3.3 Hz, 4H, H-2 and H-2′), 4.11 (t, J_{3/3′,4/4′} = J_{4/4′,5/5′} = 9.4 Hz, 4H, H-4 and H-4′), 2.30–2.22 (m, 8H, H-2″ and H-2‴), 2.10–1.97 (m, 4H, H-13‴), 1.60–1.52 (m, 8H, H-3″ and H-3‴), 1.39–1.03 (m, 108H, H-4″–H-21″ and H-4‴–H-12‴), 0.81 (t, J_21″,22″ = 6.9 Hz, 6H, H-22″); ¹³C NMR (125 MHz, C₅D₅N) δ 173.5 (C-1″ and C-1‴), 130.5 (C-14‴), 95.7 (C-1 and C-1′), 74.7 (C-3 and C-3′), 73.2 (C-2 and C-2′), 71.9 (C-4 and C-4′), 71.4 (C-5 and C-5′), 64.2 (C-6 and C-6′), 34.3 (C-2″ and C-2‴), 32.9 (C-13‴), 32.0, 29.9, 29.8, 29.8, 29.7, 29.6, 29.5, 29.4, 29.4, 29.2, 22.8 (C-4″–C-21″ and C-4‴–C12‴), 25.1 (C-3″ and C-3‴), 14.1 (C-22″); IR (film): 3353 (broad), 2917, 2849, 2337, 2258, 2224, 2197, 2136, 2021, 1736, 1467, 1376, 1260, 12223, 1175, 1149, 1102, 1076, 1052, 1018, 988, 941, 805, 720, 661, 645, 578, 521, 457, 436 cm⁻¹; HRMS (ESI) m/z calcd for [C₉₈H₁₇₆O₂₆ + NH₄]⁺: 1764.2822; obsd.: 1764.2879.

Bis(6-O-(p-(octadecyloxy)benzoyl)-α,α′-D-trehalose-6′-yl) (E/Z)-dec-5-enedioate (4a). TMS-protected dimer 17a (19.0 mg, 0.0077 mmol) was subjected to the general procedure for desilylation, followed by purification using silica gel flash column chromatography (eluted with CH₂Cl₂ : MeOH, 1 : 0 → 41 : 9, v/v) to obtain the title compound 4a as a thin white film (10.1 mg, 0.00063 mmol, 82%). R_f = 0.59 (CH₂Cl₂ : MeOH, 4 : 1, v/v); [α]^26.2_D = +45.8 (c = 0.1, pyridine); ¹H NMR (500 MHz, C₅D₅N) δ 8.21 (d, J_3″,4″ = 8.9 Hz, 4H, H-3″), 6.90 (d, J_3″,4″ = 8.9 Hz, 4H, H-4″), 5.87 (d, J_{1/1′,2/2′} = 3.6 Hz, 2H, H-1/1′), 5.83 (d, J_{1/1′,2/2′} = 3.6 Hz, 2H, H-1/1′), 5.21–5.17 (m, 2H, H-5), 5.12 (dd, J_6a,b = 11.7, J_5,6a = 2.1 Hz, 2H, H-6a), 5.05–4.97 (m, 4H, H-5′ and H-6b), 4.90 (dd, J_6′a,b = 11.7, J_5′,6′a = 3.3 Hz, 2H, H-6′a), 4.77–4.66 (m, 6H, H-3 and H-3′ and H-6′b), 4.31–4.24 (m, 4H, H-2 and H-2′), 4.21 (t, J_3,4 = J_4,5 = 9.4 Hz, 2H, H-4), 4.10 (t, J_3′,4′ = J_4′,5′ = 9.4 Hz, 2H, H-4′), 3.84 (t, J_6″,7″ = 6.4 Hz, 4H, H-6″), 2.29–2.15 (m, 4H, H-2‴), 1.89–1.77 (m, 4H, H-4‴), 1.70–1.62 (m, 4H, H-7″), 1.60–1.51 (m, 4H, H-3‴), 1.38–1.30 (m, 4H, H-8″), 1.27–1.11 (m, 60H, H-9″–H-22″), 0.80 (t, J_22″,23″ = 7.0 Hz, 6H, H-23″); ¹³C NMR (125 MHz, C₅D₅N) δ 173.4 (C-1‴), 166.4 (C-1″), 163.2 (C-5″), 131.9 (C-3″), 130.2 (C-5‴), 114.4 (C-4″), 95.8 (C-1/1′), 74.9, 74.7 (C-3/3′), 73.2 (C-2/2′), 71.9, 71.8 (C-4/4′), 71.5, 71.4 (C-5/5′), 68.3 (C-6″), 64.8 (C-6), 64.2 (C-6′), 33.6 (C-2‴), 32.0 (C-4‴), 29.8, 29.8, 29.7, 29.5, 29.5, 22.8 (C-9″–C-22″), 29.2 (C-7″), 26.1 (C-8″), 24.8 (C-3‴), 14.1 (C-23″); IR (film): 3340 (broad), 2921, 2852, 1711, 1605, 1580, 1510, 1453, 1422, 1378, 1276, 1253, 1168, 1148, 1100, 1075, 1049, 1019, 983, 911, 846, 805, 769, 720, 696, 647, 578, 509, 444 cm⁻¹; HRMS (ESI) m/z calcd for [C₈₄H₁₃₆O₂₈ + NH₄]⁺: 1610.9556; obsd.: 1610.9544.

Bis(6-O-(p-(octadecyloxy)benzoyl)-α,α′-D-trehalose-6′-yl) (E/Z)-icos-10-enedioate (4b). TMS-protected dimer 17b (23.3 mg, 0.0090 mmol) was subjected to the general procedure for desilylation, followed by purification using silica gel flash column chromatography (eluted with CH₂Cl₂ : MeOH, 1 : 0 → 41 : 9, v/v) to obtain the title compound 4b as a thin white film (13.9 mg, 0.0080 mmol, 89%). R_f = 0.42 (CH₂Cl₂ : MeOH, 4 : 1, v/v); [α]^20.8_D = +60 (c = 0.1, pyridine); ¹H NMR (600 MHz, C₅D₅N) δ 8.30 (d, J_3″,4″ = 8.8 Hz, 4H, H-3″), 6.98 (d, J_3″,4″ = 8.8 Hz, 4H, H-4″), 5.96 (d, J_{1/1′,2/2′} = 3.6 Hz, 2H, H-1/1′), 5.93 (d, J_{1/1′,2/2′} = 3.6 Hz, 2H, H-1/1′), 5.57–5.46 (m, 2H, Z- and E-H-10‴), 5.27 (dd, J_{4/4′,5/5′} = 10.2 Hz, J_{5/5′,6/6′-a/b} = 5.3 Hz, 2H, H-5/5′), 5.21–5.00 (m, 6H, H-5/5′, H-6a, H-6b, and H-6′a), 4.88–4.76 (m, 6H, H-3, H-3′, and H-6′b), 4.39–4.35 (m, 4H, H-2 and H-2′), 4.29 (dd, J_{4/4′,5/5′} = 10.2 Hz, J_{3/3′,4/4′} = 9.3 Hz, 2H, H-4/4′), 4.20 (dd, J_{4/4′,5/5′} = 10.2 Hz, J_{3/3′,4/4′} = 9.3 Hz, 2H, H-4/4′), 3.93 (t, J_6″,7″ = 6.7 Hz, 4H, H-6″), 2.34–2.30 (m, 4H, H-2‴), 2.11–2.02 (m, 4H, H-9‴), 1.74 (p, J_6″,7″ = J_7″,8″ = 6.7 Hz, 4H, H-7″), 1.61 (p, J_2‴,3‴ = J_3‴,4‴ = 7.5 Hz, 4H, H-3‴), 1.44–1.17 (m, 80H, H-8″–H-22″ and H-4‴–H-8‴), 0.88 (t, J_22″,23″ = 7.0 Hz, 6H, H-23″); ¹³C NMR (150 MHz, C₅D₅N) δ 174.1 (C-1‴), 166.9 (C-1″), 163.7 (C-5″), 132.4 (C-3″), 131.1 (C-10‴), 123.6 (C-2″), 114.9 (C-4″), 96.3 (C-1 and C-1′), 75.4 (C-3/3′), 75.3 (C-3/3′), 73.8 (C-2 and C-2′), 72.5 (C-4/4′), 72.4 (C-4/4′), 72.1 (C-5/5′), 71.9 (C-5/5′), 68.8 (C-6″), 65.3 (C-6/6′), 64.7 (C-6/6′), 34.7 (C-2‴), 33.3 (C-9‴), 32.5, 30.4, 30.27, 30.26, 30.23, 30.0, 29.97, 29.89, 29.80, 29.74, 29.70, 26.6, 25.6, 23.3 (C-3‴–C-8‴ and C-7″–C-22″), 14.6 (C-23″); IR (film): 3343 (broad), 2919, 2851, 1712, 1606, 1279, 1254, 1170, 1149, 1101, 1076, 1050, 1019, 987, 769 cm⁻¹; HRMS (ESI) m/z calcd for [C₉₄H₁₅₆O₂₈ + NH₄]⁺: 1751.1121; obsd.: 1751.1198.

Bis(6-O-(p-(octadecyloxy)benzoyl)-α,α′-D-trehalose-6′-yl) (E/Z)-octacos-14-enedioate (4c). TMS-protected dimer 17c (12.9 mg, 0.0048 mmol) was subjected to the general procedure for desilylation, followed by purification using silica gel flash column chromatography (eluted with CH₂Cl₂ : MeOH, 1 : 0 → 41 : 9, v/v) to obtain the title compound 4c as a thin white film (6.2 mg, 0.0033 mmol, 71%). R_f = 0.23 (CH₂Cl₂ : MeOH, 4 : 1, v/v); [α]^25.2_D = +46.6 (c = 0.1, pyridine); ¹H NMR (500 MHz, CDCl₃) δ 8.22 (d, J_3″,4″ = 8.9 Hz, 4H, H-3″), 6.90 (d, J_3″,4″ = 8.9 Hz, 4H, H-4″), 5.89 (d, J_{1/1′,2/2′} = 3.7 Hz, 2H, H-1/H-1′), 5.85 (d, J_{1/1′,2/2′} = 3.7 Hz, 2H, H-1/1′), 5.50–5.43 (m, 2H, H-14″), 5.22–5.17 (m, 2H, H-5/5′), 5.13 (dd, J_6a,b = 11.7, J_5,6a = 2.0 Hz, 2H, H-6a), 5.07–4.81 (m, 6H, H-5/5′ and H-6b and H-6′a), 4.79–4.68 (m, 6H, H-6′, H-3 and H-3′), 4.32–4.25 (m, 4H, H-2 and H-2′), 4.21 (t, J_{3/3′,4/4′} = J_{4/4′,5/5′} = 9.1 Hz, 2H, H-4/4′), 4.12 (t, J_{3/3′,4/4′} = J_{4/4′,5/5′} = 9.3 Hz, 2H, H-4/4′), 3.85 (t, J_6″,7″ = 6.6 Hz, 4H, H-6″), 2.30–2.20 (m, 4H, H-2‴), 2.10–1.97 (m, 4H, H-13‴), 1.67 (p, J_6″,7″ = J_7″,8″ = 6.7 Hz, 4H, H-7″), 1.55 (p, J_2‴,3‴ = J_3‴,4‴ = 7.4 Hz, 4H, H-3‴), 1.40–1.05 (m, 96H, H-8″–H-22″ and H-4‴–H-12‴), 0.81 (t, J_22″,23″ = 7.0 Hz, 6H, H-23″); ¹³C NMR (125 MHz, CDCl₃) δ 173.5 (C-1‴), 166.4 (C-1″), 163.2 (C-5″), 131.9 (C-3″), 130.6 (C-14‴), 114.4 (C-4″), 95.7 (C-1/1′), 75.6, 75.4 (C-3/3′), 73.3 (C-2 and C-2′), 72.8, 72.6 (C-4/4′), 72.0, 71.4 (C-5/5′), 68.3 (C-6″), 64.2 (C-6 and C-6′), 34.2 (C-2‴), 32.9 (C-13‴), 32.0 (C-7″), 29.8, 29.8, 29.7, 29.5, 29.2, 26.1, 25.1, 22.8 (C-3‴–C-12‴ and C-7″–C-22″), 14.1 (C-23″); IR (film): 3354 (broad), 2948, 2919, 2838, 1647, 1450, 1407, 1253, 1168, 1109, 1078, 1016, 568, 475, 441, 421 cm⁻¹; HRMS: (ESI) m/z calcd for [C₁₀₂H₁₇₂O₂₈ + NH₄]⁺: 1864.2407, obsd. = 1864.23732.

2,2′,3,3′,4,4′-Hexa-O-trimethylsilyl-6,6′-di-O-(undec-10-enoyl)-α,α′-D-trehalose (18). Diol 11 (176 mg, 0.227 mmol) was co-evaporated twice with dry toluene (20 mL) and then a third time with excess toluene, which was reduced to a final volume of 10 mL. To this, undecenoic acid (8b) (0.18 mL, 0.91 mmol), DMAP (19.0 mg, 0.159 mmol), and EDCI (261 mg, 1.36 mmol) were added, and the reaction mixture was stirred at 70 °C for 22 hours, at which point the reaction was deemed complete, as gauged by TLC analysis. The reaction mixture was then cooled to room temperature, diluted with EtOAc (2 × 50 mL), washed with water (100 mL), a saturated solution of NaHCO₃ (100 mL), and brine (100 mL), dried over MgSO₄, filtered, and concentrated under reduced pressure. The resulting residue was then purified using a gradient silica gel flash column (eluting with pet. ether : EtOAc, 1 : 0 → 19 : 1, v/v) to obtain the title compound 18 as a colourless viscous oil (217 mg, 0.196 mmol, 86%). R_f = 0.69 (pet. ether : EtOAc, 9 : 1, v/v); [α]^21.7_D = +76 (c = 1, CH₂Cl₂); ¹H NMR (500 MHz, CDCl₃) δ 5.80 (ddt, J_16,17a = 16.9 Hz, J_16,17b = 10.2 Hz, J_15,16 = 6.7 Hz, 2H, H-16), 5.01–4.91 (m, 6H, H-17a, H-17b and H-1), 4.27 (dd, J_6a,b = 11.9 Hz, J_5,6a = 2.3 Hz, 2H, H-6a), 4.05 (dd, J_6a,b = 11.9 Hz, J_5,6b = 4.4 Hz, 2H, H-6b), 3.99 (ddd, J_4,5 = 9.4 Hz, J_5,6b = 4.4, J_5,6a = 2.3 Hz, 2H, H-5), 3.90 (t, J_2,3 = J_3,4 = 8.9 Hz, 2H, H-3), 3.49–3.42 (m, 4H, H-2 and H-4), 2.39–2.28 (m, 4H, H-8), 2.05–2.01 (m, 4H, H-15), 1.64–1.59 (m, 4H, H-9), 1.38–1.27 (m, 20H, H-10–H-14), 0.15, 0.13, 0.13 (3s, 54H, TMS); ¹³C NMR (125 MHz, CDCl₃) δ 173.9 (C-7), 139.3 (C-16), 114.3 (C-17), 94.5 (C-1), 73.6 (C-3), 72.8 (C-2), 72.0 (C-4), 70.9 (C-5), 63.4 (C-6), 34.3 (C-8), 33.9 (C-15), 29.43, 29.38, 29.26, 29.21, 29.0 (C-10–C-14), 24.9 (C-9), 1.2, 1.0, 0.3 (TMS); IR (film): 2971, 2926, 2855, 1741, 1640, 1457, 1415, 1250, 1075, 870, 836 cm⁻¹; HRMS (ESI) m/z calcd for [C₅₂H₁₀₆O₁₃Si₆ + NH₄]⁺: 1124.6587; obsd.: 1124.6597.

2,2′,3,3′,4,4′-Hexa-O-trimethylsilyl-α,α′-D-trehalose-6,6′-diyl (E/Z)-icos-10-enedioate (19). Diester 18 (86.0 mg, 0.078 mmol) was co-evaporated with dry toluene (2 × 4 mL) and re-dissolved in dry CH₂Cl₂ (2 mL). To this was added Grubbs’ catalyst (2^nd generation, 1 mol%) and the mixture was refluxed for 24 hours, after which point the reaction was deemed complete, as gauged by TLC. The reaction mixture was concentrated under vacuum and the crude residue was purified using silica gel flash column chromatography (eluted with pet. ether : EtOAc, 1 : 0 → 97 : 3, v/v) to obtain the title compound 19 as a colourless viscous oil (23.7 mg, 0.022 mmol, 28%). R_f = 0.56 (pet. ether : EtOAc, 9 : 1, v/v); [α]^21.9_D = +80 (c = 0.1, CH₂Cl₂); ¹H NMR (600 MHz, CDCl₃) δ 5.37–5.33 (m, 2H, Z/E-H-16), 4.95 and 4.91 (2d, J_1,2 = 3.1 Hz, 2H, H-1), 4.33–4.23 (m, 2H, H-6a), 4.07–4.03 (m, 2H, H-6b), 3.98–3.91 (m, 4H, H-3 and H-5), 3.48–3.38 (m, 4H, H-2 and H-4), 2.33–2.30 (m, 4H, H-8), 2.04–1.95 (m, 4H, H-15), 1.65–1.60 (m, 4H, H-9), 1.33–1.25 (m, 20H, H-10–H-14), 0.15 and 0.14 (2s, 54H, TMS); ¹³C NMR (150 MHz, CDCl₃) δ 174.0 (C-7), 130.5 (C-16), 93.5 (C-1), 73.4 (C-3/5), 72.8 (C-2), 72.5 (C-4), 70.9 (C-3/5), 63.8 (C-6), 34.2 (C-8), 32.5 (C-15), 29.36, 29.26, 28.97, 28.73 (C-10–C-14), 24.9 (C-9), 1.17, 1.03, 0.26 (TMS); IR (film): 2971, 2927, 2855, 1742, 1457, 1406, 1251, 1112, 1077, 873, 842 cm⁻¹; HRMS (ESI) m/z calcd for [C₅₀H₁₀₂O₁₃Si₆ + NH₄]⁺: 1096.6274; obsd.: 1096.6283.

α,α′-D-Trehalose-6,6′-diyl (E/Z)-icos-10-enedioate (5). TMS-protected RCM product 19 (15.7 mg, 0.015 mmol) was subjected to the general desilylation procedure, followed by purification using silica gel flash column chromatography (eluted with CH₂Cl₂ : MeOH, 1 : 0 → 17 : 3, v/v) to obtain the final glycolipid 5 as a thin white film (9.40 mg, 0.015 mmol, quantitative). R_f = 0.38 (CH₂Cl₂ : MeOH, 17 : 3, v/v); [α]^22.3_D = +80 (c = 0.1, pyridine); ¹H NMR (600 MHz, C₅D₅N) δ 5.90 and 5.91 (2d, 2H, Z/E-H-1), 5.51–5.46 (m, 2H, Z/E-H-16), 5.06–4.98 (m, 4H, H-5 and H-6a), 4.80–4.75 (m, 4H, H-3 and H-6b), 4.32–4.29 (m, 2H, H-2), 4.21–4.11 (m, 2H, H-4), 2.32–2.29 (m, 4H, H-8), 2.12–2.00 (m, 4H, H-15), 1.64 (p, J_8,9 = J_9,10 = 7.5 Hz, 4H, H-9), 1.38–1.16 (m, 20H, H-10–H-14); ¹³C NMR (150 MHz, C₅D₅N) δ 174.0 (C-7), 131.2 (Z/E-C-16), 130.7 (Z/E-C-16), 95.4 (Z/E-C-1), 95.3 (Z/E-C-1), 75.2(C-3), 73.8 (C-2), 72.5 (C-4), 71.9 (C-5), 64.7 (Z/E-C-6), 64.4 (Z/E-C-6), 34.84 (C-8), 33.0 (C-15), 29.8, 29.7, 29.5, 29.4, 29.1 (C-10–C-14), 25.6 (C-9). IR (film): 3346 (broad), 2924, 2853, 1736, 1552, 1442, 1349, 1262, 1183, 1150, 1105, 1077, 1052, 1020, 991, 807 cm⁻¹; HRMS (ESI) m/z calcd for [C₃₂H₅₄O₁₃ + NH₄]⁺: 664.3903; obsd.: 664.3907.

Biological methods

Endotoxin testing. Synthetic glycolipids 3a–c, 4a–c and 5 were confirmed to be endotoxin free at a sensitivity level of ≤0.25 EU per mL by using the ToxinSensor™ Gel Clot Endotoxin Assay Kit (Gen Script) prior to biological evaluation.

NFAT-GFP Mincle reporter assays. 2B4-reporter cells expressing NFAT-GFP along with mouse Mincle + FcRγ, human Mincle + FcRγ, or FcRγ only were maintained as previously described.⁴⁸ The 2B4 T cells transfected with NFAT-GFP were cultured in complete Roswell Park Memorial Institute medium [RPMI-1640 supplemented with 2 mM Glutamax (Gibco), 10% (v/v) fetal bovine serum (Gibco) and 1% (v/v) penicillin–streptomycin (Gibco)]. Cells (4 × 10⁵ cells per mL, 100 μL per well) were then incubated with ligands 3a–c, 4a–c or 5 coated on plates (0.1 or 1 nmol per well) for 18 h. The reporter cells were then harvested, stained with 4′,6-diamidino-2-phenylindole (DAPI), and NFAT-GFP expression was monitored by flow cytometry (FACS Canto II). Expression of GFP by reporter cells is given as a percentage of total live, single cells.

Plate-coated bone-marrow derived macrophage (BMDM) assay. Bone-marrow cells were collected from femurs and tibias of C57BL/6 wild type or Mincle^−/− mice and the cells were cultured as previously reported.⁴⁹ On day 8, all media together with non-adherent cells were removed by aspirating. BMDMs were harvested using Acutase (0.5 mL per well, 10–15 minutes). Cells were resuspended at 1 × 10⁶ cells per mL in cRPMI and were added (200 μL per well) to 96 well plates coated with glycolipids 3a–c, 4a–c and 5 (0.1 or 1 nmol per well). 100 ng mL⁻¹ LPS served as the positive control and iso-propanol was used as the negative control. Stimulated cells were incubated for 48 h at 37 °C before supernatants were collected and analysed for cytokine production.

Cytokine and chemokine analysis. Levels of mIL-1β (R&D systems), mIL-6 and mTNF-α (BD Biosciences) were determined using sandwich ELISA according to the manufacturer's instructions.

Mice. Mice were bred at the Biomedical Research Unit (BRU) at the Malaghan Institute of Medical Research (MIMR), Wellington, NZ. Mice were maintained under specific pathogen-free conditions with 12 hour light/dark cycles, provided with autoclaved meat-free dry chow and autoclaved acidified water ad libitum. Mice were sex and age matched at 6–8 weeks of age. Animal ethics approval was applied for and granted by the Victoria University Animal Ethics Committee Animal ethics (#31099). Experiments were conducted according to the VUW Code of Ethical Conduct for the use of live animals for research, and in accordance with the NZ Animal Welfare Act 1999. The inbred C57BL/6 WT and Mincle^−/− mice were used for experiments. Breeding pairs of WT mice were purchased from Jackson Laboratories (Bar Harbour, ME, USA) while Mincle^−/− were a gift from Professor Sho Yamasaki, and all mice were bred for use in the MIMR BRU facility.

Vaccine preparation. TDB and the dimeric ligand 3b were initially prepared as a 9 : 1 : 40 mineral oil : TWEEN 80 : PBS emulsion and then subsequently diluted with 1 : 1 PBS containing 50 µg EndoGrade OVA purchased from Lionex Diagnostics and Therapeutics.

In vivo experiment. Mice were anaesthetised with 100 mg kg⁻¹ ketamine and 3 mg kg⁻¹ xylazine through the intraperitoneal route followed by intramuscular vaccination, with a total volume of 50 µL being administered into each of the left and right quadricep muscles. This corresponded to doses of OVA alone (50 µg), TDB (0.3 µmol) + OVA (50 µg), or 3b (0.3 µmol) + OVA (50 µg).⁶ At the experimental endpoint, mice were euthanised by asphyxiation with CO₂ followed by close monitoring with heartbeat and reflex checks before any further procedures. The iLNs were collected from mice after euthanasia and placed in 1 mL IMDM in a 24-well plate maintained on ice. The iLNs were then mashed through a 70 µM cell strainer and washed with 10 mL IMDM into a 50 mL Falcon tube. The samples were then centrifuged for 10 minutes at 250g at 4 °C and the supernatant was discarded. The cell pellets were resuspended in 200 µL IMDM and transferred to a 96-well U-bottom plate for flow cytometry staining.

Cell surface, antigen, and intracellular transcription factor labelling. Cells were stained with Zombie NIR viability dye for 15 min at room temperature and then incubated with anti-mouse CD16/32 (clone 2.4G2) for 5 min at room temperature to block any non-specific antibody binding. Detection of OVA-specific B cells was achieved using biotin-conjugated OVA, prepared using the Sigma Aldrich Enzymatic Protein Biotinylation kit (CS0008) by incubation for 30 min at 4 °C followed by incubation with strepdavidin-PECF594 for 15 min at 4 °C. For labelling of OVA-specific CD8⁺ T cells, the cells were incubated with MHC-I tetramer loaded with the SIINFEKL peptide and labelled with PE (NIH Tetramer Core Facility) for 30 min at room temperature in the dark. Surface staining was performed at 4 °C for 15 min using fluorescent antibody cocktails prepared in PBS containing 0.01% sodium azide, 2 mM EDTA, and 2% FBS. Cells were then fixed and permeabilised using the eBioscience™ FoxP3/transcription factor buffer set (Invitrogen) before intracellular staining for 1 h for the detection of Bcl6 and identification of germinal centre B cells and T follicular helper CD4 cells.

Flow cytometry. Cell samples were run on an Aurora Spectral Flow Cytometer (Cytek, Fremont, USA). Unstained cell controls, FMOs, and single stained controls were used to set up the instrument for sample acquisition. The sample data were analysed using FlowJo software (version 10, Tree Star, USA), with appropriate controls used for cell gating. The presented flow cytometry plots were generated using the FlowJo software.

Conflicts of interest

There are no conflicts to declare.

Data availability

The data supporting this article have been included in the article and in the supplementary information (SI). Supplementary information: flow cytometry data and NMR data for all new compounds. See DOI: https://doi.org/10.1039/d5ob01835h.

Acknowledgements

We would like to thank the Health Research Council of New Zealand (HRC, Consolidator Grant, VUW20/928) for funding. We would also like to thank Professor Sho Yamasaki (Osaka University) for kindly providing the Mincle reporter cell lines and Mincle^−/− mice.

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Footnote

† These authors contributed equally.

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