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. Author manuscript; available in PMC: 2022 Dec 14.
Published in final edited form as: J Org Chem. 2022 Jun 29;87(14):9419–9425. doi: 10.1021/acs.joc.2c00901

Design and Synthesis of a Doubly Functionalized Core Structure of Glycosylphosphatidylinositol Anchor Containing a Photoreactive and a Clickable Functional Groups

Venkanna Babu Mullapudi 1, Kendall C Craig 1, Zhongwu Guo 1,*
PMCID: PMC9749488  NIHMSID: NIHMS1856402  PMID: 35766889

Abstract

A bifunctional derivative of the core structure of glycosylphosphatidylinositol (GPI) anchors having a clickable alkynyl group and a photoreactive diazirine group attached to the GPI glucosamine and lipid moieties, respectively, was synthesized from myo-inositol, D-glucosamine, and (R)-1,2-O-acetonized glycerol. The target molecule should be useful for the investigation of GPI-interacting components in the cell membrane that play a key role in the signal transduction and other biological functions of GPI-anchored proteins.

Graphical Abstract

graphic file with name nihms-1856402-f0001.jpg

Glycosylphosphatidylinositols (GPIs) are a subclass of glycolipids. Their attachment to the protein C-terminus is ubiquitous in eukaryotic organisms and represents one of the most common and important posttranslational modifications of proteins.1 GPIs serve as the membrane anchor of many cell-surface proteins/glycoproteins, and GPI-anchored proteins/glycoproteins are involved in various biological and pathological processes.2–4

Since the first structural elucidation of an intact GPI anchor by Ferguson and coworkers more than three decades ago,5 numerous GPIs and GPI-anchored proteins have been discovered and characterized.6 Investigations of these molecules suggest the biological functions of GPIs, such as signal transduction,7–8 beyond providing a stable anchoring system for proteins.9 However, among all GPI anchors identified thus far, the length of their lipid chains rarely exceeds twenty carbon atoms, which is not long enough to span the whole cell membrane. Consequently, to accomplish transmembrane signal transduction and other functions, GPI anchors need to interact with other components in the cell membrane. For example, a study using fluorescence-labeled analogs of the GPI core structure have shown that GPIs may selectively interact with specific phosphatidylserine (PS) molecules in the cell membrane.10 Despite this and other progresses in the field, currently, there is still limited information about the exact molecules that interact with GPI anchors and thus a poor understanding of the functional mechanisms of GPIs and GPI-anchored proteins. In the present research, we intend to address this issue through designing and developing new molecular tools to explore GPI-cell membrane interactions.

To meet the above demand, the molecular tool should be bifunctional as illustrated in Figure 1A. On the one hand, it should contain a functional group that is reactive under defined conditions to help catch the membrane components interacting with GPIs. On the other hand, it should contain a functional group that can selectively couple with solid materials, such as beads, for the efficient isolation of caught GPI-interacting molecules for proteomics and lipidomics analysis. Accordingly, we have designed a GPI analog 1 (Figure 1B), which has a photoreactive diazirine moiety and a clickable alkynyl group linked to the lipid and glycan, respectively, of the core structure of GPI anchors. It has been established that diazirines can be effectively activated by UV light at 350 nm to form reactive carbenes, which react with molecules in close proximity to form stable covalent linkages.11–13 The alkynyl group can be used to selectively couple with azides by a click reaction under biocompatible conditions for the attachment to azide-modified materials.14–15

Figure 1.

Figure 1.

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(A) A strategy to identify cell membrane components that interact with GPIs to enable their transmembrane functions. (B) The designed molecular probe for investigating GPI-cell membrane interactions. The probe contains a photoreactive diazirine group on the lipid to enable UV-activated cross-reaction with cell membrane components and a clickable alkynyl group to facilitate the isolation of labeled cell membrane components for structural analysis.

In specific, the diazirine group in 1 is incorporated at the C-12-position in the O-2-steric group of the phosphatidyl moiety. This design will facilitate the cross-reaction of 1 with molecules that interact with the lipid moiety of GPI anchors. In the meantime, its pseudodisaccharide motif, α-D-glucosaminyl-(1→6)-inositol, represents the core structure shared by all GPIs. Furthermore, its glucosamine 4’-O-position, where the GPI glycan should elongate, bears the alkynyl group, which by design can block the participation of 1 in GPI-anchored protein biosynthesis. Comparing this GPI analog with glycolipids without the inositol residue will provide insights into the functional role of inositol in the organization and recognition of GPIs in the cell membrane.

Scheme 1 shows our overall synthetic plan. A notable challenge of this synthesis is that the target molecule contains several base-sensitive O-acyl groups and hydrogenation-vulnerable diazirine and alkynyl motifs. To address this problem, we intended to use the para-methoxybenzyl (PMB) group for permanent protection of hydroxyl groups during the synthesis. PMB ethers can be readily and selectively deprotected under mild conditions by oxidation using ceric ammonium nitrate (CAN) or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ)16–17 or by acids, e.g., 10% trifluoroacetic acid (TFA),18 which conditions would not affect the phosphatidyl moiety or the alkynyl group. As a result, finally, 2 could be deprotected by a three-step protocol, including selective reduction of the azide, removal of the cyanoethyl group under mildly basic conditions, and global deprotection of PMB ethers by oxidation or with acids. Disconnecting the phosphoryl and pentynoyl linkages in fully protected 2 led to pseudodisaccharide 3 and functionalized lipid-containing phosphoramidite 4 as key intermediates.

Scheme 1.

Scheme 1.

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Retrosynthesis of the target molecule 1.

Our synthesis commenced with the preparation of phosphoramidite 4 as depicted in Scheme 2. First, diazirine-modified fatty acid 6 was synthesized from keto acid 5 in a yield similar to that of the literature19 (Scheme 2A). Next, 6 was coupled with alcohol 9, derived from acetonized glycerol 7 as reported,20 in the presence of dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP). Finally, the tert-butyldimethylsilyl (TBS) group in 10 was removed with triethylamine trihydrofluoride (Et3N•3HF), followed by reaction of the resultant 11 with 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite (12) to provide 4 (Scheme 2B). All the reactions afforded good yields (75–85%). Because 4 was moisture-sensitive, it was directly subjected to the next reaction after brief purification by column chromatography.

Scheme 2.

Scheme 2.

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Synthesis of phosphoramidite intermediate 4.

For the synthesis of pseudodisaccharide intermediate 3 (Scheme 3), we first prepared 1320 and inositol derivative 1421 from D-glucosamine and myo-inositol, respectively, according to reported methods. Glycosylation of 14 with 13 under the promotion of trimethylsilyl triflate (TMSOTf)22 went smoothly to give a good yield (75%) of 15 as an anomeric mixture (α:β = 1:1) with essentially no stereoselectivity. The two anomers were inseparable. Therefore, the mixture was directly subjected to the next reaction, i.e., deprotection of the allyl-protected inositol 1-O-position using Ir-complex-catalyzed allyl rearrangement and then HgO-promoted vinyl ether hydrolysis.23 At this stage, the products were readily separated by silica gel column chromatography to provide pure 3. Its α-anomeric configuration was confirmed by 1H NMR data, which had a small coupling constant (J = 3.7 Hz) between H-1 and H-2 for the glucosamine residue, whereas the other product (i.e., the β-anomer) had a large coupling constant (J = 7.7 Hz).

Scheme 3.

Scheme 3.

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Synthesis of key intermediate 3 and its phosphorylation reaction.

After 3 and 4 were obtained, we tried the phospholipidation reaction by a two-step method24 using 1H-tetrazole as the catalyst in the first step and oxidation of the resultant phosphite ester by tert-butyl peroxide (tBuO2H) in the second step (Scheme 3). Although both steps were smooth and completed quickly, the purification of 16 was tedious because of the side products generated from a large excess (~4 equiv) of the phosphorylating reagent 4 used in the reaction. Thus, 16 was only briefly purified by column chromatography before being applied to desilylation. However, tetra-n-butylammonium fluoride (TBAF) was not suitable for the desilylation of 16 due to phosphate deprotection under the condition. Removing the silyl group with Et3N•3HF was successful to give easily separable 17, but this reaction was extremely slow, taking 6 days to complete, which might account for the low overall yield (40%). It should be noted that the phosphorylation reaction gave a 1:1 mixture of two stereoisomers originated from the stereogenic phosphorus atom. Thereafter, 17 was condensed with 4-pentynoic acid in the presence of DCC and DMAP to afford 2. However, this reaction was also slow and low-yielding (52%), thus upon 72 h of reaction, there was still a substantial amount of 17 remaining. Clearly, after installing the phosphatidyl moiety, all reactions started to become sluggish and inefficient.

To overcome this problem, we attempted another synthetic strategy, as outlined in Scheme 4, to introduce the 4-pentynoic group before the phosphatidyl moiety. Accordingly, the silyl ether at the 4’-O-position of 3 was deprotected with TBAF. Compared to the desilylation reaction above, this one was fast and clean (finished in 3 h), affording diol 18 in an 87% isolated yield. Similarly, the condensation reaction between 18 and 4-pentynoic acid in the presence of DCC and DMAP was also smooth and fast (finished in 10 h) to afford the desired 19 in a significantly improved yield (68%) along with 9% of the diacylated byproduct 20. The regiochemistry of both products was confirmed by 1H NMR data, e.g., significant increases of the chemical shifts of glucosamine H-4 proton for 19 and of glucosamine H-4 and inositol H-1 protons for 20. These results proved that the phosphatidyl moiety at the inositol 1-O-position in 16 and 17 had a drastically negative impact on the reactivity of the glucosamine 4-O-position. Subsequently, 19 was phospholipidated by the above-described method to give 2 (58%) as a 1:1 diastereomeric mixture of the stereogenic phosphate. Global deprotection of 2 was furnished by the designed three-step protocol, including selective reduction of the azide with PMe3,25 deprotection of the cyanoethoxyl phosphate using 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), and removal of all PMB groups using 10% TFA in CH2Cl2. These reactions went smoothly to afford the final synthetic target 1 in a 58% overall yield, which contained a small quantity of impurities such as PMB alcohol and solvent residues. It should be pointed out that Zinc and acetic acid could not be used to reduce the azido group in 17 due to concomitant reduction of the diazirine group26 and elongated exposure of 1 to TFA would result in the hydrolysis of the diazirine group as shown by MS data.

Scheme 4.

Scheme 4.

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Optimized synthesis of target molecule 1.

In summary, we have designed and synthesized a new, bifunctional GPI anchor analog 1. After exploring different options, we have established the optimal synthetic route for the target molecule, and this synthetic route may also be applicable to other GPI analogs. An interesting observation is that the phosphatidyl moiety at the inositol 1-O-position can have a huge impact on the reactivity of the intermediates, thus the point in time to introduce this moiety should be carefully evaluated during the design of a synthesis. The core pseudodisaccharide of GPIs is proximal to the plasma membrane and should play a pivotal role in their biological functions, such as GPI distribution in the lipid rafts of cell membrane. Therefore, studies using molecular probe 1 should provide useful information about GPI interaction with other membrane components and functional mechanisms of GPI anchors, which is still poorly understood. Currently, our laboratory is pursuing protein pull-down and proteomics studies using the new molecular tool.

Experimental Section

Synthesis of 11-(3-hexyl-3H-diazirin-3-yl)undecanoic acid (6).

In a flame-dried flask under an N2 atmosphere was added 5 (0.47 g, 1.57 mmol), followed by NH3 in MeOH (7 M, 8.5 mL, 59.5 mmol). The mixture was stirred at 0 °C for 5 h. NH2OSO3H (0.28 g, 2.44 mmol) dissolved in MeOH (1.0 mL) was added at 0 °C. The reaction was warmed to rt and stirred overnight (16 h). N2 gas was bubbled through the solution to remove NH3. The precipitates were filtered off and washed with MeOH. The organic layers were combined and condensed to dryness in vacuum. CH2Cl2 (3.0 mL) was added, followed by Et3N (0.20 mL, 1.29 mmol). The flask was cooled to 0 °C, and I2 was added until the color remained. After 2 h, the mixture was diluted with H2O and extracted with CH2Cl2 (100 mL × 3). The organic layers were combined, washed with brine, dried with MgSO4, filtered, and then condensed in vacuum. The product was purified by column chromatography with 20% EtOAc in hexane as the eluent to give 6 (0.15 g, 30%) as a white solid. Rf = 0.33 (20% EtOAc in Hex). 1H NMR (600 MHz, CDCl3): δ 2.35 (t, J = 7.5 Hz, 2 H), 1.63 (quin, J = 7.5 Hz, 2 H), 1.37−1.17 (m, 22 H), 1.11−1.02 (m, 4 H, α-CH2 of diazirine), 0.87 (t, J = 7.2 Hz, 3 H, CH3). 13C{1H} NMR (150 MHz, CDCl3): δ 180.3 (C=O), 34.2, 33.1 (2 C), 31.8, 29.5 (3 C), 29.4 (2 C), 29.2 (2 C, C-diazirine), 29.1, 24.8, 24.0 (2 C), 22.7, 14.2. HR ESI-TOF MS: calcd for m/z C18H33N2O2 [M − H]−, 309.2548; found, 309.2559.

Synthesis of (S)-(2,2-dimethyl-1,3-dioxolan-4-yl)methyl stearate (8).

Optically pure 7 (3.00 g, 22.7 mmol) and stearic acid (7.08 g, 24.9 mmol) were dissolved in CH2Cl2 (6.0 mL). To the solution were added DCC (6.08 g, 29.5 mmol) and DMAP (0.28 g, 2.27 mmol) at rt with stirring. About 16 h later, the precipitates were filtered off and washed with CH2Cl2. The organic layers were combined and condensed in vacuum. The product was purified via column chromatography with 1–2% EtOAc in hexane as the eluent to give 8 (8.30 g, 92%) as a white solid. Rf = 0.32 (2% EtOAc in Hex). 1H NMR (600 MHz, CDCl3): δ 4.35−4.32 (m, 1 H), 4.16 (dd, J = 11.5, 4.7 Hz, 1 H), 4.11−4.06 (m, 2 H), 3.77−3.74 (m, 1 H), 2.34 (t, J = 7.6 Hz, 2 H), 1.62 (quin, J = 7.5 Hz, 2 H), 1.43 (s, 3 H), 1.37 (s, 3 H), 1.32−1.21 (m, 28 H), 0.88 (t, J = 7.0 Hz, 3 H, CH3). 1H NMR data match with those reported in the literature.20

Synthesis of (R)-3-[(tert-butyldimethylsilyl)oxy]-2-hydroxypropyl stearate (9).

Compound 8 (8.22 g, 20.6 mmol) was dissolved in MeOH (100 mL). HCl (1.0 M, 10.0 mL, 10.0 mmol) was added slowly, and the solution was stirred at 50 °C for 1.5 h. After the starting material disappeared as indicated by TLC, the mixture was added to aqueous NaHCO3 and extracted with ethyl acetate (100 mL × 3). The organic layers were combined, washed with brine, dried with MgSO4, filtered, and condensed in vacuum. To the resulting crude product dissolved in CH2Cl2 (166 mL) was added Et3N (2.8 mL, 20.1 mmol), and the solution was stirred at rt for 5 min. TBSCl (3.05 g, 20.2 mmol) and DMAP (0.20 g, 1.64 mmol) were added, and the solution was stirred at rt for 16 h. The solid was filtered off and washed with CH2Cl2. The organic layers were combined and condensed in vacuum. The product was purified via column chromatography with 5% EtOAc in hexane as the eluent to give 9 (6.77 g, 70% for two steps) as colorless syrup. Rf = 0.48 (10% EtOAc in Hex). 1H NMR (600 MHz, CDCl3): δ 4.16 (dd, J = 11.4, 4.8 Hz, 1 H), 4.12 (dd, J = 11.4, 6.3 Hz, 1 H), 3.90−3.84 (m, 1 H), 3.67 (dd, J = 10.1, 4.6 Hz, 1 H), 3.61 (dd, J = 10.1, 5.6 Hz, 1 H), 2.52 (bs, 1 H, OH), 2.33 (t, J = 7.6 Hz, 2 H), 1.64 (quin, J = 7.5 Hz, 2 H), 1.34−1.21 (m, 28 H), 0.90 (s, 9 H, tBu), 0.88 (t, J = 7.3 Hz, 3 H, CH3), 0.08 (s, 6 H, SiMe2). 1H NMR data match with those reported in the literature.21

Synthesis of (R)-3-(tert-butyldimethylsilyl)oxy-2-[11-(3-hexyl-3H-diazirine-3-yl)undecano yl]oxypropyl stearate (10).

Compound 9 (0.34 g, 0.72 mmol) and 6 (0.20 g, 0.65 mmol) were dissolved in CH2Cl2 (6.0 mL) under an N2 atmosphere. DCC (0.18 g, 0.85 mmol) and DMAP (7.0 mg, 0.06 mmol) were added, and the solution was stirred at rt for 16 h. The precipitates were filtered off and washed with CH2Cl2. The organic layers were combined and condensed in vacuum. The product was purified via column chromatography with 2% EtOAc in hexane as the eluent to give 10 (0.44 g, 79%) as colorless syrup. Rf = 0.23 (2% EtOAc in Hex). 1H NMR (600 MHz, CDCl3): δ 5.08−5.04 (m, 1 H, Gly-2), 4.34 (dd, J = 11.8, 3.7 Hz, 1 H, Gly-1), 4.16 (dd, J = 11.9, 6.3 Hz, 1 H, Gly-1), 3.72−3.69 (m, 2 H, Gly-3), 2.34−2.27 (m, 4 H), 1.64−1.57 (m, 4 H), 1.37−1.19 (m, 50 H), 1.10−1.03 (m, 4 H, α-CH2 of diazirine), 0.92−0.83 (m, 15 H, tBu and 2 CH3), 0.05 (s, 6 H, SiMe2). 13C{1H} NMR (150 MHz, CDCl3): δ 173.6 (C=O), 173.3 (C=O), 71.8, 62.6, 61.6, 34.5, 34.3, 33.1, 32.1, 31.8, 29.9 (multiple C), 29.8 (3 C), 29.6 (3 C), 29.5 (2 C), 29.4 (2 C), 29.3 (2 C), 29.1, 25.9 (3 C), 25.1 (2 C), 24.0 (2 C), 22.9, 22.7, 18.4, 14.3, 14.2, 1.2, −5.3 (2 C). HR ESI-TOF MS: calcd for m/z C45H88N2O5NaSi [M + Na]+, 787.6355; found, 787.6345.

Synthesis of (R)-2-[11-(3-hexyl-3H-diazirin-3-yl)undecanoyl]oxy-3-hydroxypropyl stearate (11).

Compound 10 (0.43 g, 0.57 mmol) was dissolved in CH3CN:THF (1:1, 9.0 mL). Et3N·3HF (1.1 mL, 6.79 mmol) was added slowly at 0 °C. The solution was stirred at 0 °C for 16 h. The mixture was condensed under vacuum, and the product was purified via column chromatography with 10–15% EtOAc in hexane as the eluent to give 11 (0.28 g, 75%) as a white solid. Rf = 0.43 (20% EtOAc in Hex). 1H NMR (600 MHz, CDCl3): δ 5.13−5.08 (m, 1 H, Gly-2), 4.32 (dd, J = 11.9, 4.6 Hz, 1 H, Gly-1), 4.24 (dd, J = 12.0, 5.6 Hz, 1 H, Gly-1), 3.75−3.72 (m, 2 H, Gly-3), 2.36−2.30 (m, 4 H), 1.89 (t, J = 6.6 Hz, 1 H, OH), 1.65−1.58 (m, 4 H), 1.36−1.18 (m, 50 H), 1.09–1.02 (m, 4 H, α-CH2 of diazirine), 0.82−0.85 (2 q, J = 6.9 Hz, 6 H, 2 CH3). 13C{1H} NMR (150 MHz, CDCl3): δ 173.9 (C=O), 173.6 (C=O), 72.3, 62.1, 61.7, 34.4, 34.3, 33.1 (2 C), 32.1, 31.8, 29.9 (multiple C), 29.8 (3 C), 29.6 (2 C), 29.5 (3 C), 29.4 (2 C), 29.3, 29.2, 29.1, 25.1 (2 C), 24.0 (2 C), 22.9, 22.7, 14.3, 14.2. HR ESI-TOF MS: calcd for m/z C39H74N2O5Na [M + Na]+, 673.5490; found, 673.5481.

Synthesis of 2-azido-4-O-(tert-butyldimethylsilyl)-2-deoxy-3,6-di-O-(para-methoxybenzyl)-α-D-glucosyl-(1→6)-2,3,4,5-tetra-O-(para-methoxybenzyl)-myo-inositol (3).

To a stirred mixture of 14 (0.56 g, 0.79 mmol), 13 (0.70 g, 0.99 mmol), and freshly activated MS 4A (0.60 g) in dry Et2O (8.0 mL) was added TMSOTf (18 μL, 0.10 mmol) at −10 °C under an N2 atmosphere. After being stirred for 30 min, the mixture was neutralized with Et3N, filtered, and concentrated under vacuum. The residue was subjected to silica gel column chromatography with 10% EtOAc in hexane as the eluent to afford the anomeric mixture 15 as colorless syrup. [Ir(COD)(PMePh2)2]PF6 (62.0 mg, 0.07 mmol) dissolved in THF (3.0 mL) was stirred under a H2 atmosphere at rt until the red color turned to pale yellow (in ca. 10 min), at which time point the H2 was exchanged with argon for three times. To this solution was slowly added 15 (0.92 g, 0.74 mmol) in anhydrous THF (4.0 mL). The solution was stirred at rt for 30 min until TLC showed the completion of reaction and then concentrated in vacuum. The residue was dissolved in acetone and water (10.0 mL, 9:1, v/v), and the solution was treated with HgCl2 (1.01 g, 3.70 mmol) and HgO (16.0 mg, 0.07 mmol). Ten minutes later, the solution was concentrated in vacuum, and the residue was purified by silica gel column chromatography with 13% EtOAc in hexane as the eluent to afford 3 (0.40 mg, 45%; as well as 45% for β-anomer; over two steps) as a white foamy solid. Rf = 0.30 (40% EtOAc in Hex). 1H NMR (600 MHz, CDCl3): δ 7.27–7.32 (m, 8 H), 7.16 (m, 4 H), 6.87–6.89 (m, 6 H), 6.98−6.84 (m, 6 H), 5.50 (d, J = 3.6 Hz, 1 H, GlcN-1), 4.92 (d, J = 11.2 Hz, 1 H), 4.9 (d, J = 10.6 Hz, 1 H), 4.8 (d, J = 10.2 Hz, 1 H), 4.79 (d, J = 10.9 Hz, 1 H), 4.70–4.75 (m, 3 H), 4.66 (d, J = 11.4 Hz, 1 H), 4.60–4.65 (m, 2 H), 4.27 (d, J = 11.6, Hz, 1 H), 4.25 (d, J = 11.6 Hz, 1 H), 4.01 (t, J = 9.6 Hz, 1 H), 3.96 (d, J = 9.4 Hz, 1 H), 3.95 (t, J = 2.3 Hz, 1 H), 3.88 (dt, J = 2.8, 9.4 Hz, 1 H), 3.83 (s, 3 H), 3.82 (s, 3 H), 3.81 (s, 3 H), 3.80 (s, 3 H), 3.80 (s, 3 H), 3.80 (s, 3 H), 3.70–3.76 (m, 3 H), 3.58 (ddd, J = 2.7, 6.8, 9.4 Hz, 1 H), 3.42–3.44 (m, 2 H), 3.39 (t, J = 4.0 Hz, 1 H), 3.37 (dd, J = 1.9, 5.4 Hz, 1 H), 3.35 (d, J = 9.2 Hz, 1 H), 3.22 (dd, J = 2.0, 10.7 Hz, 1 H), 3.11 (d, J = 6.8 Hz, 1 H), 0.84 (s, 9 H, tBu), 0.0 (3 H, SiMe), −0.01 (s, 3 H, SiMe). 1H NMR data match with those reported in the literature.21

Synthesis of 2-azido-2-deoxy-3,6-di-O-(para-methoxybenzyl)-α-D-glucosyl-(1→6)-2,3,4,5-tetra-O-(para-methoxybenzyl)-myo-inositol 2-cynoethanol (R)-2-O-[11-(3-hexyl-3H-diazirin-3-yl)undecanoyl]-3-O-stearoyl-glycerol phosphate (17).

To a solution of 11 (130.0 mg, 0.20 mmol) and commercially available 12 (78.2 mg, 0.26 mmol) in anhydrous CH2Cl2/acetonitrile (2:1, 4.0 mL) was added diisopropylammonium tetrazolide (37.6 mg, 0.22 mmol). After being stirred at rt under an Ar atmosphere for 1 h, the reaction mixture was diluted with CH2Cl2 and then poured into saturated aqueous NaHCO3. The aqueous layer was extracted with CH2Cl2 three times. The combined organic layers were dried with Na2SO4 and concentrated in vacuum. The product was briefly purified by flush column chromatography using triethylamine-neutralized silica gel with 8% EtOAc in hexane as the eluent to give phosphoramidite 4 (145.0 mg, 85%) as colorless syrup. Rf = 0.33 (20% EtOAc in Hex). 1H NMR (600 MHz, CDCl3): δ 5.15–5.21 (m, 1 H), 4.30 (ddd, J = 3.8, 11.8, 15.4 Hz, 1 H), 4.12–4.20 (m, 1 H), 3.56–3.87 (m, 6 H), 2.61 (t, J = 6.4 Hz, 2 H), 2.28–2.32 (m, 4 H), 1.58–1.65 (m, 4 H), 1.24–1.34 (m, 26 H), 1.17 (t, J = 8.0 Hz, 12 H), 1.04–1.07 (m, 4 H), 0.85–0.87 (m, 6 H). 31P{1H} NMR (243 MHz, CDCl3): δ 8.3, 8.0. The freshly prepared 4 (141.6 mg, 0.17 mmol) was dissolved in dry CH2Cl2 (1.0 mL) and slowly added to a mixture of 3 (50.0 mg, 0.042 mmol) and tetrazole (0.45 M solution in acetonitrile, 0.92 mL, 0.42 mmol) in anhydrous CH2Cl2-CH3CN (3:1, 8.0 mL) and molecular sieves (MS) 4 A under an Ar atmosphere. The mixture was stirred at rt for 30 min and then cooled to 0 °C, which was followed by addition of tBuO2H (5.5 M in decane, 151 μL, 0.83 mmol). The solution was stirred at 0 °C for 1 h, warmed to rt, and filtered through a celite pad. The solution was poured into saturated aqueous NaHCO3, and the mixture was extracted with CH2Cl2 three times. The combined organic layers were dried with Na2SO4 and concentrated in vacuum. The product was purified by silica gel column chromatography with 30% EtOAc in hexane as the eluent to give 16 as an epimeric mixture (56.0 mg), which was directly applied to the next step. After 16 was dissolved in anhydrous THF-CH3CN (1:1, 4.0 mL), Et3N•3HF (1.0 mL) was added at rt under an Ar atmosphere. After stirring at rt for 6 days, saturated aqueous NaHCO3 was added to quench the reaction. The mixture was extracted with CH2Cl2 three times, and the combined organic layers were dried with Na2SO4 and concentrated in vacuum. The product was purified by silica gel column chromatography with 40% EtOAc in hexane as the eluent to give 17 (31.0 mg, 40%, 2 steps; a ~1:1 mixture of two epimers) as a white solid. Rf = 0.20 (40% EtOAc in Hex). 1H NMR (400 MHz, CDCl3): δ 7.36−7.28 (m, 12 H), 7.22−7.21 (m, 4 H), 7.16−7.14 (m, 8 H), 6.90−6.84 (m, 16 H), 6.80−6.77 (m, 8 H), 5.41 (d, J = 3.7 Hz, 1 H, GlcN-1), 5.40 (d, J = 3.7 Hz, 1 H, GlcN-1), 5.26−5.22 (m, 2 H), 4.95−4.90 (m, 4 H), 4.87 (d, J = 10.4 Hz, 2 H), 4.81−4.80 (m, 4 H), 4.71−4.66 (m, 6 H), 4.64−4.60 (m, 4 H), 4.45−4.40 (m, 4 H), 4.38−4.31 (m, 8 H), 4.30−4.20 (m, 10 H), 4.12−4.08 (m, 2 H), 4.04−4.01 (m, 4 H), 3.83 (s, 6 H), 3.81 (s, 6 H), 3.80 (m, 12 H), 3.79 (s, 6 H), 3.76 (s, 6 H), 3.70 (td, J = 3.7, 9.7 Hz, 2 H), 3.53 (dd, J = 2.0, 9.8 Hz, 2 H), 3.42 (m, 2 H), 3.32−3.26 (m, 4 H), 3.19 (dd, J = 3.6, 10.4 Hz, 2 H), 2.83−2.75 (m, 4 H, CH2-CN), 2.32−2.25 (m, 8 H, CH2-C=O), 2.11 (d, J = 3.6 Hz, 1 H, OH), 2.10 (d, J = 3.6 Hz, 1 H, OH), 1.65−1.58 (m, 8 H), 1.37−1.33 (m, 12 H), 1.26−1.20 (m, 88 H), 1.10−1.07 (m, 8 H, α-CH2 of diazirine), 0.91−0.87 (m, 12 H, CH3). 13C{1H} NMR (150 MHz, CDCl3): δ 173.2 (2 C, C=O), 173.1 (C=O), 172.8 (C=O), 159.4 (2 C), 159.2 (4 C), 159.1 (4 C), 159.0 (2 C), 130.9 (2 C), 130.8 (2 C), 130.7 (2 C), 130.3 (2 C), 130.2 (2 C), 130.0 (2 C), 129.9 (2 C), 129.8 (4 C), 129.4 (2 C), 129.3 (6 C), 129.2 (6 C), 129.0 (2 C), 128.9 (2 C), 113.9 (4 C), 113.8 (4 C), 113.7 (8 C), 113.6 (4 C), 113.6 (4 C), 97.6 (GlcN-1), 97.5 (GlcN-1), 81.2 (2 C), 80.6 (2 C), 80.5 (2 C), 80.3, 78.5, 78.3, 76.9 (2 C), 76.4, 76.3, 75.4 (2 C), 75.3 (2 C), 74.9, 74.8, 74.3 (2 C), 73.0 (4 C), 72.6, 72.5, 72.1, 72.0, 69.8, 69.7, 69.3 (4 C), 69.2, 68.7 (2 C), 66.3 (4 C), 66.1, 62.5 (2 C), 62.4, 62.3, 62.2, 61.5, 61.4, 55.2 (12 C), 35.9, 34.1 (2 C), 34.0, 33.9, 32.9 (2 C), 31.9 (2 C), 31.6, 29.8 (20 C), 29.7, 29.6, 29.5, 29.4 (2 C), 29.3 (2 C), 29.2 (2 C), 29.1 (4 C), 28.9, 27.2, 25.5, 24.8 (4 C), 23.8 (2 C), 22.7 (2 C), 22.5, 19.6 (2 C), 19.5 (2 C), 14.1 (2 C), 14.0 (2 C). 31P{1H} NMR (243 MHz, CDCl3): δ −2.5, −2.8. HR ESI-TOF MS: calcd for m/z C102H145N6O23NaP [M + Na]+, 1875.9991; found, 1875.9977.

Synthesis of 2-azido-2-deoxy-3,6-di-O-(para-methoxybenzyl)-4-O-(pent-4-ynoyl)-α-D-glucosyl-(1→6)-2,3,4,5-tetra-O-(para-methoxybenzyl)-myo-inositol 2-cynoethanol (R)-2-O-[11-(3-hexyl-3H-diazirin-3-yl)undecanoyl]-3-O-stearoyl-glycerol phosphate (2) from 17.

The solution of DCC (5.2 mg, 25.2 μmol), 4-pentynoic acid (1.8 mg, 18.2 μmol), and DMAP (0.24 mg, 2.0 μmol) in CH2Cl2:THF (1:1) was cooled to 0 °C with stirring for 30 min. Then, 17 (26.0 mg, 14.0 μmol) was added, and the reaction mixture was stirred at rt for 3 days. The mixture was filtered to remove solid urea byproduct, diluted with CH2Cl2, and washed with NaHCO3 and brine. The organic layer was dried with Na2SO4 and concentrated in vacuum. The product was purified by silica gel column chromatography with 25% EtOAc in hexane as the eluent to give 2 (14.0 mg, 52%) as a white solid. Rf = 0.36 (40% EtOAc in Hex). 1H NMR (600 MHz, CDCl3): δ 7.34−7.32 (m, 4 H), 7.33−7.29 (m, 6 H), 7.22−7.07 (m, 14 H), 6.90−6.83 (m, 16 H), 6.79−6.75 (m, 8 H), 5.46 (d, J = 3.7 Hz, 1 H, GlcN-1), 5.43 (d, J = 3.7 Hz, 1 H, GlcN-1), 5.23–5.32 (m, 2 H), 5.14 (ddd, J = 6.5, 9.5, 16.0 Hz, 2 H), 4.92–5.01 (m, 4 H), 4.82 (dd, J = 7.5, 10.2 Hz, 2 H), 4.62–4.71 (m, 12 H), 4.50 (dd, J = 5.2, 10.8 Hz, 2 H), 4.41–4.44 (m, 4 H), 4.37–439 (m, 2 H), 4.31–4.36 (m, 6 H), 4.24–4.30 (m, 6 H), 4.12–4.20 (m, 10 H), 4.02–4.05 (m, 2 H), 3.88 (td, J = 3.4, 9.8 Hz, 2 H), 3.84 (d, J = 6.4 Hz, 2 H), 3.83 (s, 6 H), 3.82 (s, 6 H), 3.80 (s, 12 H), 3.78 (m, 12 H), 3.52 (dt, J = 2.0, 9.9, 19.6 Hz, 2 H), 3.38 (dt, J = 3.5, 9.4, 18.8 Hz, 2 H), 3.32 (ddd, J = 3.6, 6.0, 9.8 Hz, 2 H), 3.20 (bd, J = 10.7 Hz, 2 H), 2.88 (dt, J = 2.9. 11.0 Hz, 2 H), 2.77–2.80 (m, 2 H), 2.62–2.70 (m, 2 H), 2.24–2.37 (m, 12 H), 2.13–2.18 (m, 2 H), 2.02–2.08 (m, 2 H), 1.91 (t, J = 2.6 Hz, 2 H, acetylene C-H), 1.62–1.66 (m, 8 H), 1.33–1.37 (m, 12 H), 1.22–1.24 (m, 88 H), 1.07–1.08 (m, 8 H, α-CH2 of diazirine), 0.87–0.91 (m, 12 H, CH3). 13C{1H} NMR (150 MHz, CDCl3): δ 173.2 (C=O), 173.1 (C=O), 172.8 (2 C, C=O), 169.8 (2 C, C=O), 159.4 (2 C), 159.2 (2 C), 159.1 (6 C), 158.8 (2 C), 130.9 (2 C), 130.8 (2 C), 130.6 (2 C), 130.4 (2 C), 130.2 (4 C), 129.8 (4 C), 129.7 (4 C), 129.6 (4 C), 129.3 (4 C), 129.1 (2 C), 129.0 (2 C), 128.2 (2 C), 128.1 (2 C), 116.3 (2 C), 113.8 (8 C), 113.7 (8 C), 113.5 (8 C), 97.4 (GlcN-1), 97.3 (GlcN-1), 82.4 (2 C), 81.2 (2 C), 80.6 (2 C), 80.3, 80.2, 76.5 (2 C), 76.4 (2 C), 75.5 (2 C), 75.1 (2 C), 75.0, 74.8, 74.2 (2 C), 74.1 (2 C), 73.3, 73.2, 72.9 (2 C), 72.6, 72.5, 70.0, 69.4, 69.3 (2 C), 69.2 (2 C), 68.6 (2 C), 66.8 (2 C), 66.3 (2 C) 66.1 (2 C), 65.9 (2 C), 65.8 (2 C), 62.4 (2 C), 62.3, 62.2 (2 C), 62.0 (2 C), 61.5 (2 C), 61.4, 55.3 (4 C), 55.2 (8 C), 34.1 (3 C), 34.0 (2 C), 33.9, 33.1, 32.9 (2 C), 31.9 (2 C), 31.6 (2 C), 29.7 (16 C), 29.6 (4 C), 29.5 (2 C), 29.4 (2 C), 29.3 (2 C), 29.2 (2 C), 29.1 (2 C), 28.9 (2 C), 28.4 (4 C), 23.9 (2 C), 23.8 (2 C), 22.7 (2 C), 22.5 (2 C), 14.1(2 C), 14.0 (2 C). 31P{1H} NMR (243 MHz, CDCl3): δ −2.8, −2.7. HR ESI-TOF MS: calcd for m/z C107H153N7O24P [M + NH4]+, 1951.0699; found, 1951.0698.

Synthesis of 2-azido-2-deoxy-3,6-di-O-(para-methoxybenzyl)-α-D-glucosyl-(1→6)-2,3,4,5-tetra-O-(para-methoxybenzyl)-myo-inositol (18).

To a solution of 3 (100.0 mg, 83.0 μmol) in THF (4.0 mL) was added dropwise a solution of TBAF (1.0 M in THF, 0.13 mL, 0.13 mmol) at 0 °C, and the reaction was stirred at rt for 3 h. The reaction mixture was concentrated under vacuum. The product was purified by silica gel column chromatography with 30% EtOAc in hexane as the eluent to give 18 (79.0 mg, 87%) as colorless syrup. Rf = 0.2 (30% EtOAc in Hex). 1H NMR (600 MHz, CDCl3): δ 7.35−7.30 (m, 4 H), 7.26−7.24 (m, 4 H), 7.18−7.13 (d, J = 8.6 Hz, 4 H), 6.90−6.82 (m, 12 H), 5.45 (d, J = 3.7 Hz, 1 H, GlcN-1), 4.96 (d, J = 5.3 Hz, 1 H), 4.92 (d, J = 6.3 Hz, 1 H), 4.87 (d, J = 10.1 Hz, 1 H), 4.87 (d, J = 10.1 Hz, 1 H), 4.79 (dd, J = 10.8, 2.9 Hz, 2 H), 4.71 (d, J = 10.3 Hz, 1 H), 4.66 (d, J = 11.2 Hz, 1 H), 4.61–4.66 (m, 3 H), 4.31 (d, J = 11.3 Hz, 1 H), 4.18 (d, J = 11.3 Hz, 1 H), 4.01 (t, J = 9.5 Hz, 1 H), 3.97 (t, J = 2.4 Hz, 1 H), 3.90–3.94 (m, 2 H), 3.84 (s, 3 H), 3.83 (s, 3 H), 3.81 (m, 3 H), 3.80 (s, 6 H), 3.78 (s, 3 H), 3.67 (dt, J = 3.0, 9.5 Hz, 1 H), 3.60 (ddd, J = 2.7, 6.8, 9.5 Hz, 1 H), 3.43 (dd, J = 2.3, 10.0 Hz, 1 H), 3.39 (dd, J = 3.6, 10.0 Hz, 1 H), 3.35 (t, J = 9.2 Hz, 1 H), 3.30 (dd, J = 3.6, 10.2 Hz, 1 H), 3.24 (dd, J = 5.6, 10.2 Hz, 1 H), 3.15 (d, J = 6.8 Hz, 1 H), 2.38 (d, J = 3.0 Hz, 1 H, OH). 13C{1H} NMR (150 MHz, CDCl3): δ 159.4, 159.3 (2 C), 159.2, 159.1, 158.9, 130.9, 130.8, 130.7, 130.3 (2 C), 129.9, 129.8 (2 C), 129.6 (4 C), 129.3 (4 C), 128.9 (2 C), 114.0 (2 C), 113.9 (2 C), 113.8 (2 C), 113.7 (4 C), 113.6 (2 C), 98.0 (GlcN-1), 81.6, 81.0, 80.7, 80.2, 79.7, 76.7, 75.4, 74.8, 74.7, 74.4, 73.5, 73.1, 72.7, 72.6, 69.8, 69.2, 63.5, 55.3 (2 C), 55.2 (4 C). HR ESI-TOF MS: calcd for m/z C60H73N4O16 [M + NH4]+, 1105.5016; found, 1105.5050.

Synthesis of 2-azido-2-deoxy-3,6-di-O-(para-methoxybenzyl)-4-O-(pent-4-ynoyl)-α-D-glucosyl-(1→6)-2,3,4,5-tetra-O-(para-methoxybenzyl)-myo-inositol (19).

A solution of DCC (22.8 mg, 110 μmol), 4-pentynoic acid (7.9 mg, 89.0 μmol), and DMAP (1.4 mg, 11.4 μmol) in CH2Cl2:THF (1:1) was stirred at 0 °C for 30 min, and then 18 (80.0 mg, 73.0 μmol) was added. The reaction mixture was stirred at rt for 10 h, filtered to remove the solid urea byproduct, diluted with CH2Cl2, and washed with NaHCO3 and brine. The organic layer was dried with Na2SO4 and concentrated in vacuum, and the product was purified by silica gel column chromatography with 20% EtOAc in hexane as the eluent to give 19 (58.5 mg, 68%) and 20 (8.3 mg, 9%) as colorless syrup. 19: Rf = 0.33 (30% EtOAc in Hex). 19: 1H NMR (600 MHz, CDCl3): δ 7.32−7.12 (m, 12 H), 6.91−6.78 (m, 12 H), 5.58 (d, J = 3.6 Hz, 1 H, GlcN-1), 5.15 (t, J = 9.8 Hz, 1 H, GlcN-4), 4.95 (m, 2 H), 4.87 (d, J = 10.3 Hz, 1 H), 4.70 (dd, J = 4.6, 10.3 Hz, 2 H), 4.63–4.69 (m, 4 H), 4.53 (d, J = 10.8 Hz, 1 H), 4.31 (d, J = 11.6 Hz, 1 H), 4.13 (d, J = 11.6 Hz, 1 H), 4.01–4.06 (m, 2 H), 3.93–3.97 (m, 2 H, Ino-6), 3.88 (t, J = 9.7 Hz, 1 H), 3.84 (s, 3 H), 3.83 (s, 3 H), 3.81 (s, 3 H), 3.80 (s, 3 H), 3.79 (s, 6 H), 3.59 (ddd, J = 2.9, 7.9, 10.5 Hz, 1 H, Ino-1), 3.47 (dd, J = 3.6, 10.3 Hz, 1 H), 3.43 (dd, J = 2.3, 9.9 Hz, 1 H), 3.35 (t, J = 9.4 Hz, 1 H), 3.15 (dd, J = 2.8, 10.9 Hz, 1 H), 2.96 (dd, J = 3.3, 10.9 Hz, 1 H), 2.85 (d, J = 7.8 Hz, 1 H), 2.26–2.31 (m, 2 H), 2.12–2.23 (m, 2 H), 1.90 (t, J = 2.6 Hz, 1 H, acetylene C-H). 13C{1H} NMR (150 MHz, CDCl3): δ 169.9 (C=O), 159.4, 159.3 (2 C), 159.2 (2 C), 158.8, 130.8 (2 C), 130.6, 130.2, 129.8 (2 C), 129.7 (4 C), 129.6 (4 C), 129.3 (2 C), 128.4 (2 C), 113.9 (4 C), 113.8 (2 C), 113.7 (2 C), 113.5 (4 C), 97.4 (GlcN-1), 82.4, 81.5, 81.0, 80.7, 79.1, 77.6, 76.9 (Ino-6), 75.4, 74.7, 74.5, 74.0, 73.4 (Ino-1), 73.0, 72.8, 70.5 (GlcN-4), 69.2, 68.8, 67.3, 63.4, 55.3 (2 C), 55.2 (4 C), 33.1, 14.1. HR ESI-TOF MS: calcd for m/z C65H77N4O17 [M + NH4]+, 1185.5278; found, 1185.5270. 20: Rf = 0.4 (30% EtOAc in Hex). 1H NMR (600 MHz, CDCl3): δ 7.28−7.11 (m, 12 H), 6.89−6.83 (m, 8 H), 6.79−6.75 (m, 4 H), 5.32 (d, J = 3.6 Hz, 1 H, GlcN-1), 5.19 (dd, J = 9.4, 10.2 Hz, 1 H, GlcN-4), 5.03 (d, J = 11.1 Hz, 1 H), 4.87 (d, J = 10.2 Hz, 1 H), 4.84 (dd, J = 2.3, 10.2 Hz, 1 H, Ino-1), 4.76 (d, J = 11.5 Hz, 1 H), 4.70 (d, J = 10.2 Hz, 1 H), 4.69 (d, J = 10.2 Hz, 1 H), 4.63−4.59 (m, 2 H), 4.57 (t, J = 10.8 Hz, 2 H), 4.51 (d, J = 10.9 Hz, 1 H), 4.37 (d, J = 11.6 Hz, 1 H, Ino-6), 4.27 (t, J = 9.9 Hz, 1 H), 4.15 (d, J = 11.6 Hz, 1 H), 4.08−4.06 (m, 2 H), 4.02 (dt, J = 2.5, 10.4 Hz, 1 H), 3.86 (dd, J = 9.4, 10.3 Hz, 1 H), 3.83 (s, 3 H), 3.82 (s, 3 H), 3.80 (s, 3 H), 3.79 (s, 3 H), 3.78 (s, 3 H), 3.77 (s, 3 H), 3.57 (dd, J = 2.3, 9.8 Hz, 1 H), 3.41 (t, J = 9.4 Hz, 1 H), 3.32 (dd, J = 3.7, 10.4 Hz, 1 H), 3.11 (dd, J = 2.6, 11.1 Hz, 1 H), 2.74 (dd, J = 2.7, 11.1 Hz, 1 H), 2.57−2.52 (m, 1 H), 2.47−2.40 (m, 3 H), 2.30−2.18 (m, 3 H), 2.14−2.09 (m, 1 H), 1.98 (t, J = 2.5 Hz, 1 H, acetylene C-H), 1.90 (t, J = 2.5 Hz, 1 H, acetylene C-H). 13C{1H} NMR (150 MHz, CDCl3): δ 171.0 (C=O), 170.0 (C=O), 159.3 (2 C), 159.1 (3 C), 158.8, 131.0, 130.6, 130.5, 130.4, 130.1, 129.8 (2 C), 129.7, 129.6 (2 C), 129.6 (4 C), 129.2 (2 C), 128.2 (2 C), 113.8 (2 C), 112.8 (2 C), 113.7 (2 C), 113.7 (2 C), 113.5 (4 C), 97.7 (GlcN-1), 82.3, 82.2, 81.5, 80.7, 80.6, 77.1, 76.9, 75.4, 75.3 (Ino-1), 74.8, 74.5, 74.4, 74.3, 73.4 (Ino-6), 73.0, 72.6, 70.0 (GlcN-4), 69.3, 69.2, 68.6, 66.5, 62.3, 55.3 (3 C), 55.2 (3 C), 33.2, 33.1, 14.0 (2 C). HR ESI-TOF MS: calcd for m/z C70H81N4O18 [M + NH4]+, 1265.5568; found, 1265.5575.

Synthesis of compound 2 from 19.

To the solution of 19 (50.0 mg, 42.0 μmol) and tetrazole (0.45 M solution in acetonitrile, 0.95 mL, 0.43 mmol) in anhydrous CH2Cl2-CH3CN (3:1, 8.0 mL) was slowly added a solution of freshly prepared 4 (145.7 mg in 1.0 mL dry CH2Cl2, 0.17 mmol) with stirring at rt under an Ar atmosphere. After the reaction was stirred at rt for 30 min, it was cooled to −40 °C and then tBuO2H (5.5 M in decane, 156 μl, 0.86 mmol) was added. The solution was stirred at −40 °C for 1 h, and Me2S (127 μl, 1.71 mmol) was added. The reaction continued for another 1 h. The mixture was poured into saturated aqueous NaHCO3 followed by extraction with CH2Cl2 three times. The combined organic layers were dried with Na2SO4 and concentrated in vacuum. The product was purified by silica gel column chromatography with 25% EtOAc in hexane as the eluent to give 2 (48.0 mg, 58%, 1:1 mixture of two epimers) as colorless syrup. Spectroscopic data are described above.

Synthesis of 2-azido-2-deoxy-4-O-(pent-4-ynoyl)-α-D-glucosyl-(1→6)-myo-inositol (R)-2-O-[11-(3-hexyl-3H-diazirin-3-yl)undecanoyl]-3-O-stearoyl-glycerol phosphate (1).

To a solution of 2 (10.0 mg, 5.2 μmol) in THF:H2O (1:1, 2.0 mL) was added PMe3 (10 μL, 10.3 μmol), and the solution was stirred at rt for 12 h, followed by addition of saturated NH4Cl (1.0 mL) and stirring for another 12 h. The reaction mixture was diluted with CH2Cl2 and then washed with NaHCO3 and brine. The organic layers were combined, dried with Na2SO4, and concentrated in vacuum. The crude product was dissolved in CH2Cl2 (500 μL), and a drop of DBU was added and the solution was stirred at rt for 1 h. Then, 20% TFA in CH2Cl2 (500 μL) was added directly to give a final concentration of ~10% TFA. After stirring for 30 min, the reaction mixture was co-evaporated with toluene five times. The product was purified by silica gel column chromatography with 30% EtOAc in hexane as the eluent to gave 1 (4.0 mg, 68%) as an off-white solid. Rf = 0.33 (30% MeOH in CHCl3). 1H NMR (600 MHz, MeOD:CDCl3 1:1): δ 5.49 (bd, 1 H, GlcN-1), 5.24 (b, 1 H, Gly-2), 4.85 (dd, J = 9.6, 9.6 Hz, 1 H, GlcN-4), 4.44 (dd, J = 2.9, 12.2 Hz, 1 H, Ino-1), 4.29 (ddd, J = 2.1, 5.6, 12.0 Hz, 1 H), 4.20−4.10 (m, 3 H), 4.08−3.97 (m, 4 H), 3.93 (t, J = 9.4 Hz, 1 H), 3.80 (s, 1 H), 3.68−3.63 (m, 2 H), 3.60−3.57 (m, 1 H), 3.54 (t, J = 5.8 Hz, 2 H), 3.40−3.35 (m, 4 H), 3.18−3.15 (m, 1 H), 2.68−2.65 (m, 1 H), 2.63−2.60 (m, 2 H), 2.52−2.49 (m, 2 H), 2.41 (t, J = 7.4 Hz, 1 H), 2.35−2.29 (m, 5 H), 2.12 (t, J = 2.5 Hz, 1 H, acetylene C-H), 2.10−2.05 (m, 1 H), 1.78−1.73 (m, 2 H), 1.61−1.54 (m, 8 H), 1.36−1.26 (m, 32 H), 1.10−1.02 (m, 4 H, α-CH2 of diazirine), 0.88−0.84 (m, 6 H, CH3). 13C{1H} NMR (150 MHz, MeOD:CDCl3 1:1): δ 174.5 (C=O), 172.8 (C=O), 166.7 (C=O), 90.5 (GlcN-1), 78.4, 73.8, 73.3, 73.0, 71.2, 71.6, 70.9, 70.8, 70.7, 70.6, 69.6, 63.7, 63.0, 56.8, 55.0, 54.8, 49.0, 43.2, 38.6, 34.6, 34.5, 33.6, 33.3, 33.2, 32.3, 32.0, 30.1, 30.0, 29.9 (10 C), 29.8, 29.7, 29.7, 29.6, 29.5, 29.2, 26.9, 25.3, 24.2, 23.0, 22.8, 19.7, 14.5, 14.2. 31P{1H} NMR (243 MHz, MeOD:CDCl3 1:1): δ 0.2. HR ESI-TOF MS: calcd for m/z C56H101N3O18P [M + H]+, 1134.6818; found, 1134.6848.

Supplementary Material

Supporting Information

Acknoledgement

This work is supported by an NIH/NIGMS Grant (1R35 GM131686).. The MS instrument was funded by NIH (S10 OD021758). ZG is also grateful to Steven and Rebecca Scott for endowing our research.

Footnotes

Supporting Information

General experimental procedures and 1D, 2D NMR and HR MS spectra of all new compounds. Available free of charge from ACS website……..

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Supporting Information
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