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. Author manuscript; available in PMC: 2022 Dec 17.
Published in final edited form as: J Org Chem. 2021 Oct 7;86(24):18257–18264. doi: 10.1021/acs.joc.1c01781

Synthesis of Cell-Permeable N-Acetylhexosamine 1-Phosphates

Badrinath N Kakde 1, Emanuela Capota 1, Jennifer J Kohler 1, Uttam K Tambar 1
PMCID: PMC9071850  NIHMSID: NIHMS1801404  PMID: 34618463

Abstract

We recently reported the incorporation of diazirine photo-cross-linkers onto the O-GlcNAc posttranslational modification in mammalian cells, enabling the identification of binding partners of O-GlcNAcylated proteins. Unfortunately, the syntheses of the diazirine-functionalized substrates have exhibited inconsistent yields. We report a robust and stereoselective synthesis of cell-permeable GlcNAc-1-phosphate esters based on the use of commercially available bis(diisopropylamino)-chlorophosphine. We demonstrate this approach for two diazirine-containing GlcNAc analogues, and we report the cellular incorporation of these compounds into glycoconjugates to support photo-cross-linking applications.

Graphical Abstract

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Metabolic oligosaccharide engineering (MOE) is a commonly used method in glycoscience research.1 In MOE, researchers prepare monosaccharide analogues to which unnatural functional group modifications have been appended. These monosaccharide analogues are metabolized by cells, leading to the production of complex glycoconjugates displaying the functional group modifications at specific sites. Introduction of azides, alkynes, or other bioorthogonal functional groups enables selective labeling of specific glycoconjugates, while addition of diazirines or aryl azides allows for cross-linking of glycoconjugates to neighboring molecules. MOE exploits the natural biosynthetic pathways used by cells to produce nucleotide sugar donors; however, the enzymes in these pathways vary in their tolerance of unnatural substituents. Enzymes in the sialic acid biosynthetic pathway accept a wide variety of functional group modifications, but enzymes in the hexosamine biosynthetic and salvage pathways have a more restricted substrate scope, limiting the functional group modifications that can be introduced onto N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) residues.

In 2012, we reported a metabolic engineering strategy for incorporating the diazirine photo-cross-linker onto the O-GlcNAc posttranslational modification in mammalian cells (Figure 1A).2 We envisioned exploiting the hexosamine salvage pathway, in which GlcNAc is initially phosphorylated at position 6 and then isomerized to GlcNAc-1-P through the sequential actions of N-acetylglucosamine kinase (NAGK) and phosphoacetylglucosamine mutase 1 (AGM1). UDP-N-acetylglucosamine pyrophosphorylase 1 (UAP1) converts GlcNAc-1-P to UDP-GlcNAc, which serves as a substrate for the O-GlcNAc transferase (OGT). Initially, we prepared a cell-permeable GlcNAc analogue that included a diazirine modification attached to the N-acyl position [Ac4GlcNDAz-(2me)]. Unfortunately, we found that HeLa cells were unable to transform this compound to the corresponding diazirine-modified UDP-GlcNAc analogue [UDP-GlcNDAz(2me)]. To bypass this metabolic bottleneck, we prepared a cell-permeable, diazirine-modified GlcNAc-1-phosphate analogue [Ac3GlcNDAz(2me)-1-P(Ac-SATE)2]. To make the compound less polar and improve cellular uptake, hydroxyl groups were modified with acetyl protecting groups that can be removed by intracellular esterases.3 To mask the phosphate, we used S-acetyl-2-thioethyl (Ac-SATE) protecting groups, which have been widely used to improve delivery of nucleoside phosphate and phosphonate prodrugs.4,5 Intracellular esterases act on Ac-SATE groups to produce an unstable O-2-mercaptoethyl substituent, which has been proposed to further cyclize to form a thiirane and reveal the unprotected phosphate (Figure 1B).4 Using this protecting group strategy, we successfully delivered diazirine-modified GlcNAc-1-phosphate [GlcNDAz(2me)-1-P] to a variety of mammalian cell lines.2,6,7 This compound can be transformed to the corresponding UDP-GlcNDAz(2me) by the action of the F383G mutant of human UAP1. UDP-GlcNDAz(2me) is a substrate for the O-GlcNAc transferase (OGT), allowing for intracellular production of diazirine-modified O-GlcNAc modifications (O-GlcNDAz).8 Irradiation of cells at 365 nm activates the diazirine, leading to covalent cross-linking of O-GlcNAcylated proteins to neighboring molecules. Note that our prior work did not include the “2me” modifier in these compound names;8a it is included here to distinguish these compounds from the extended linker “4me” compounds introduced later in this work.

Figure 1.

Figure 1.

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(A) MOE approach for the production of diazirine-modified O-GlcNAcylated proteins in a mammalian cell culture. (B) Esterase-mediated removal of Ac-SATE protecting groups.

To support ongoing cell culture experiments, we require a robust synthetic route to Ac3GlcNDAz(2me)-1-P(Ac-SATE)2. Additionally, we have been interested in preparing additional diazirine-modified GlcNAc-1-phosphate esters, and indeed, Ac-SATE protecting groups have been used to facilitate delivery of other GlcNAc-1-phosphate and GalNAc-1-phosphate analogues.9,10 Unfortunately, the synthesis of GlcNAc-1-phosphate esters is often plagued by inconsistent yields. Thus, we were motivated to develop a more robust synthesis of cell-permeable GlcNAc-1-phosphate esters. Herein, we describe a synthetic strategy that can be more reliably employed by nonspecialists. We demonstrate this approach in the context of two GlcNDAz analogues. We also report that these compounds can be metabolized by mammalian cells to support photo-cross-linking applications.

To highlight the challenges in synthesizing diazirine-modified GlcNAc-1-phosphate esters through the previously reported strategies, we attempted to synthesize model phosphate ester Ac3GlcNAc-1-P(Ac-SATE)2 6 from N,N-diisopropylphosphoramidite 4 (Scheme 1). d-(+)-Glucosamine hydrochloride 1 was peracetylated to form Ac4GlcNAc 2. Selective deacetylation at the anomeric carbon in the presence of piperidine produced Ac3GlcNAc 3. We explored several conditions for the coupling of monosaccharide 3 with N,N-diisopropylphosphoramidite 4. Unfortunately, we did not observe the desired phosphite product 5, presumably because of the chemical instability of N,N-diisopropylphosphoramidite 4. Our inability to successfully form model phosphate ester Ac3GlcNAc-1-P(Ac-SATE)2 6 through this N,N-diisopropylphosphoramidite-mediated strategy motivated us to explore an alternate approach.

Scheme 1.

Scheme 1.

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Unsuccessful Synthesis of Model Ac3GlcNAc-1-P(Ac-SATE)2 6 from N,N-Diisopropylphosphoramidite 4

A seminal study by Boons and co-workers employed a different protocol for converting N-acetylglucosamine phosphoramidites into proteophosphoglycans.11 A key component of this approach was the use of commercially available bis(diisopropylamino)chlorophosphine as the phosphorus(III) reagent. Notably, this approach also led to selective formation of the α-phosphate linkage, unlike other anomeric phosphorylations that yield a mixture of α and β anomers. We surmised that a similar strategy could be successfully implemented for the stereoselective synthesis of the α anomers of more complex N-acylated glucosamine phosphate esters, such as our desired S-acetyl-2-thioethanol-containing, diazirine-modified GlcNAc-1-phosphate esters.

To assay the viability of the bis(diisopropylamino)-chlorophosphine-mediated approach for the synthesis of GlcNAc-1-phosphate esters, our initial target molecule was dibenzyl phosphate 11 (Scheme 2). Ac3GlcNAc 3 was coupled with bis(diisopropylamino)chlorophosphine 7 in the presence of Hünig’s base, as reported by Boons, to furnish phosphordiamide 8 in 36% yield after 2 days. A more efficient reaction was observed in the presence of 1,8-diazabicyclo-[5.4.0]undec-7-ene as a base, which resulted in the formation of phosphordiamide 8 in 89% yield after 1 h. Subsequent coupling of the phosphordiamide with benzyl alcohol 9 in the presence of 1H-tetrazole resulted in the formation of dibenzyl Ac3GlcNAc-1-phosphite 10, which was oxidized to dibenzyl phosphate 11. Gratifyingly, the GlcNAc-1-phosphate ester was formed exclusively as the α anomer, as confirmed by NMR spectroscopy.12 The stereoselective formation of this anomer is presumably due to its stability via the anomeric effect between the ring oxygen’s lone pair of electrons and the C1 axial phosphate substituent in the α anomer.

Scheme 2. New Bis(diisopropylamino)chlorophosphine-Mediated Synthesis of Model Dibenzyl Ac3GlcNAc-1-phosphate 11a.

Scheme 2.

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aAbbreviations: DIPEA, N,N-diisopropylethylamine; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; m-CPBA, m-chloroperbenzoic acid.

We next employed the bis(diisopropylamino)chlorophosphine-mediated strategy for the synthesis of model Ac3GlcNAc-1-P(Ac-SATE)2 14 (Scheme 3). To our delight, phosphordiamide 8 was coupled with S-acetyl-2-thioethanol 12 in the presence of 1H-tetrazole to yield AcGlcNAc-1-phosphite 13. Subsequent oxidation of phosphorus with m-chloroperbenzoic acid resulted in the formation of desired Ac3GlcNAc-1-P(Ac-SATE)2 14. Once again, the GlcNAc-1-phosphate ester was formed exclusively as the α anomer. This stereochemistry is required for metabolism of the S-acetyl-2-thioethanol-containing GlcNAc-1-phosphate ester by cells for the production of complex glycoconjugates (vide infra).

Scheme 3. New Bis(diisopropylamino)chlorophosphine-Mediated Synthesis of Model Ac3GlcNAc-1-P(Ac-SATE)214a.

Scheme 3.

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aAbbreviation: m-CPBA, m-chloroperbenzoic acid.

With a robust synthesis of cell-permeable GlcNAc-1-phosphate in hand, we examined the bis(diisopropylamino)-chlorophosphine-mediated strategy for the synthesis of diazirine-modified GlcNDAz-1-P(Ac-SATE)2 phosphate esters. We first explored the synthesis of Ac3GlcNDAz-1-P(Ac-SATE)2(2me) 21 (Scheme 4A). Coupling of d-(+)-glucosamine hydrochloride 1 with diazirine-containing carboxylic acid 1513 resulted in the formation of N-acylglucosamine 16. Peracetylation with acetic anhydride followed by selective deacetylation at the anomeric carbon with ammonium carbonate yielded Ac3GlcNDAz(2me)-1-OH 18. Next, we treated this diazirine-modified N-acylglucosamine with bis(diisopropylamino)chlorophosphine 7 in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene, which furnished phosphordiamide 19. Treatment with S-acetyl-2-thioethanol 12 and 1H-tetrazole resulted in the formation of phosphite 20, which was oxidized in the presence of m-chloroperbenzoic acid to provide Ac3GlcNDAz(2me)-1-P(Ac-SATE)2 21 exclusively as the α anomer.

Scheme 4. (A) New Bis(diisopropylamino)chlorophosphine-Mediated Synthesis of Ac3GlcNDAz-1-P(Ac-SATE)2(2me) 21 and (B) New Bis(diisopropylamino)chlorophosphine-Mediated Synthesis of Ac3GlcNDAz-1-P(Ac-SATE)2(4me) 28a.

Scheme 4.

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aAbbreviations: EDC, N-[3-(dimethylamino)propyl]-N′-ethylcarbodiimide; HOBt, 1-hydroxybenzotriazole; DIPEA, N,N-diisopropylethylamine; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; m-CPBA, m-chloroperbenzoic acid.

Through a similar reaction sequence, we synthesized the longer Ac3GlcNDAz-1-P(Ac-SATE)2(4me) 28 (Scheme 4B). Carboxylic acid 2214 was coupled to d-(+)-glucosamine hydrochloride 1 to form N-acylglucosamine 23. Manipulation of the acetylation pattern of the monosaccharide resulted in Ac3GlcNDAz-1-OH(4me) 25, which was treated with bis(diisopropylamino)chlorophosphine 7 and 1,8-diazabicyclo-[5.4.0]undec-7-ene to yield phosphordiamide 26. Formation of bis-S-acetyl-2-thioethanol phosphite 27 followed by oxidation of the phosphorus resulted in desired diazirine-modified Ac3GlcNDAz-1-P(Ac-SATE)2(4me) 28.

With two cell-permeable, diazirine-modified GlcNAc-1-phosphate esters in hand (21 and 28), we first evaluated whether they were toxic to mammalian cells. Neither compound showed overt toxicity as evaluated by trypan blue staining (data not shown), although both caused modest growth inhibition (Figure S1A). Next, we evaluated metabolism of 21 and 28 to the corresponding UDP-GlcNAc analogues. We used Colo205 cells that stably express the F383G mutant of UAP1, which is required to convert GlcNDAz-1-P to UDP-GlcNDAz.2 As a control, we also used wild-type (wt) Colo205 cells. The wt or UAP1 F383G cells were cultured with 21 or 28. Nucleotide sugars were extracted from cells and analyzed by high-performance anion exchange chromatography (HPAEC) with UV detection. UAP1 F383G cells cultured with 21 yielded a peak at a retention time of ~60 min that was absent from UAP1 F383G cells cultured without any compound and from wt cells cultured with 21 (Figure S1B and S1C). The retention time of this peak matched that of a synthetic UDP-GlcNDAz(2me) standard. Similarly, UAP1 F383G cells cultured with 28 yielded a peak at a retention time of ~66 min that was absent from wt cells cultured with 28 and from UAP1 F383G cells cultured without any compound. This peak was postulated to be UDP-GlcNDAz(4me), although no standard compound was available for comparison.

We also evaluated the ability of compounds 21 and 28 to support photo-cross-linking of an O-GlcNAcylated protein, following our previously described protocol.7 The wt or UAP1 F383G Colo205 cells were cultured with 21 or 28 and then subjected to 365 nm irradiation to induce cross-linking. Cells were lysed, and lysates were analyzed by immunoblotting using an antibody that recognizes NUP153, a heavily O-GlcNAcy-lated protein. Additional bands of NUP153 reactivity and reduced mobility were observed when UAP1 F383G cells were cultured with 21 or 28 and subjected to UV irradiation (Figure 2). These cross-linked species were absent when UV irradiation was not applied, and their levels were dramatically reduced when compound 21 or 28 was omitted or when wt cells were used. The intensity of cross-linking observed for 28 was lower than that observed for 21. We speculate that the diminished cross-linking may reflect less efficient metabolism of compound 28, consistent with the less intense HPAEC-UV peak detected for putative UDP-GlcNDAz(4me) compared to that for UDP-GlcNDAz(2me) (Figure S1C). Additionally, a small amount of diazirine-independent but UV-dependent cross-linking was observed, which may be due to UV-induced formation of free radicals.15

Figure 2.

Figure 2.

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Diazirine-modified GlcNAc 1-phosphate esters are metabolized to enable cross-linking of NUP153. Wild-type Colo205 cells or Colo205 cells stably expressing UAP1 F383G were cultured in the presence of 21, 28, or no compound for 48 h and then UV irradiated to induce cross-linking. Cells were lysed, and the lysates were analyzed by immunoblotting using an anti-NUP153 antibody. The blot is representative of three biological replicates.

EXPERIMENTAL SECTION

General Information.

All reactions were carried out in capped reaction vials or round-bottom flasks with magnetic stirring unless otherwise indicated. Commercially obtained reagents were used as received. Solvents were dried by being passed through an activated alumina column under argon. Liquids and solutions were transferred via syringe. All reactions were monitored by thin-layer chromatography with E. Merck silica gel 60 F254 precoated plates (0.25 mm). Silica gel (particle sizes of 0.032–0.063 mm) purchased from SiliCycle was used for flash chromatography. 1H NMR spectra were recorded on Varian Inova-500, Agilent 400-MR DD2, and Bruker AVANCE NEO Nano 400 spectrometers. 13C{1H} NMR spectra were recorded on Agilent 400-MR DD2, Bruker AVANCE NEO Nano 400, and Bruker AVANCE NEO 600 spectrometers. 19F NMR spectra were recorded on Varian Inova-500 and Agilent 400-MR DD2 spectrometers. Data for 1H NMR spectra are reported relative to chloroform or benzene as an internal standard (7.26 or 7.16 ppm, respectively) and are reported as follows: chemical shift (δ), multiplicity, coupling constant (hertz), and integration. Data for 13C NMR spectra are reported relative to chloroform or benzene as an internal standard (77.2 or 128.1 ppm, respectively) and are reported in terms of chemical shift (δ). A FTIR Mettler Toledo ReactIR 15 instrument with a DST Series 6.3 mm AgX FiberCoduit Au/Silicon Probe was used to record infrared spectra. HRMS (ESI) data were obtained at the Shimadzu Center for Advanced Analytical Chemistry at The University of Texas at Arlington on the Shimadzu LCMS-IT-TOF instrument.

Synthesis and Characterization of Compounds.

Phosphoramidite 8.

On the basis of a procedure reported in the literature,10 a solution of acylated glucosamine 3 (105 mg, 0.302 mmol) in methylene chloride (3 mL) was cooled to 0 °C and treated with bis(diisopropylamine) phosphine chloride 7 (81 mg, 0.302 mmol). The solution was then treated dropwise with DBU (64 μL, 0.453 mmol) using a microliter syringe. The ice bath was allowed to warm to 23 °C, and the mixture was stirred for 1 h at the same temperature. After completion of the reaction (monitored by TLC), the solution was concentrated under vacuum. The product was purified by flash column chromatography on silica gel (5:20:75 Et3N/ethyl acetate/hexanes) to afford phosphordiamide 8 as a white solid (155 mg, 89% yield): TLC Rf = 0.36 [3:2 (v/v) hexanes/EtOAc]; 1H NMR (400 MHz, CDCl3) δ 5.59 (d, J = 9.3 Hz, 1H), 5.25 (dd, J = 9.5, 9.4 Hz, 1H), 5.18–5.13 (m, 2H), 4.35–4.29 (m, 1H), 4.27–4.22 (m, 1H), 4.10–4.04 (m, 2H), 3.59–3.47 (m, 4H), 2.08 (s, 2H), 2.02 (s, 3H), 2.01 (s, 3H), 1.92 (s, 3H), 1.21–1.14 (m, 24H); 13C{1H} NMR (100 MHz, CDCl3) δ 171.9, 170.9, 169.9, 169.5, 92.8, 92.6, 71.6, 68.6, 68.2, 62.2, 53.3, 53.2, 45.5, 45.4, 45.3, 45.3, 24.7, 24.6, 24.5, 24.4, 24.4, 23.3, 20.9, 20.8; 31P NMR (243 MHz, CDCl3) δ 113.8; FT-IR (CH2Cl2) 2536, 2371, 1745, 1685, 1511, 1375, 1234, 1189, 1044, 953 cm−1; ESI-HRMS calcd for [C26H49N3O9P, M + H]+ 578.3201, found 578.3196.

Phosphordiamidite intermediates 8, 19, and 26 are not stable under certain acidic conditions. For example, during column chromatography, we neutralized the silica gel with 1% triethylamine before purification. Although the phosphordiamidites are stable at room temperature, they were stored for long periods of time in a −20 °C freezer. The phosphordiamidites are also stable to light exposure.

Phosphite 10.

A solution of phosphoramidite 8 (48 mg, 0.083 mmol) in methylene chloride (0.5 mL) was cooled to 0 °C and treated with benzyl alcohol (21.6 μL, 0.208 mmol) followed by dropwise addition of 1H-tetrazole (554 μL, 0.249 mmol, 0.45 M in MeCN) using a microliter syringe. The reaction mixture was allowed to warm to 23 °C and stirred for 12 h. After completion of the reaction (monitored by TLC), the solution was concentrated under vacuum. The product was purified by flash column chromatography on silica gel (50:50 ethyl acetate/hexanes) to afford phosphite 10 as an oil (45 mg, 92% yield): TLC Rf = 0.33 [1:1 (v/v) hexanes/EtOAc]; 1H NMR (600 MHz, C6D6) δ 7.28–7.26 (m, 2H), 7.24–7.22 (m, 2H), 7.19–7.17 (m, 2H), 7.15–7.14 (m, 2H), 7.08–7.04 (m, 2H), 5.64 (dd, J = 7.7, 3.3 Hz, 1H), 5.51 (dd, J = 10.8, 9.5 Hz, 1H), 5.40–5.37 (m, 2H), 4.82 (dd, J = 12.3, 8.5 Hz, 1H), 4.74 (d, J = 8.2 Hz, 2H), 4.71–4.63 (m, 2H), 4.22 (dd, J = 12.2, 4.4 Hz, 1H), 4.19–4.16 (m, 1H), 4.06 (dd, J = 12.3, 2.3 Hz, 1H), 1.70 (s, 3H), 1.67 (s, 3H), 1.66 (s, 3H), 1.37 (s, 3H); 13C{1H} NMR (150 MHz, C6D6) δ 171.1, 170.1, 169.1, 169.0, 138.4, 138.4, 138.3, 138.2, 129.0, 128.8, 128.3, 128.3, 127.7, 93.2, 93.1, 71.5, 69.5, 68.6, 65.0, 64.9, 64.9, 64.8, 61.9, 52.8, 52.7, 22.5, 20.4, 20.3, 20.2; 31P NMR (243 MHz, C6D6) δ 140.5; FT-IR (CH2Cl2) 2537, 2370, 2338, 1747, 1685, 1374, 1233, 1155 cm−1; ESI-HRMS calcd for [C28H34NO11PNa, M + Na]+ 614.1762, found 614.1784.

Phosphate 11.

A solution of phosphite 10 (9 mg, 0.0152 mmol) in methylene chloride (0.15 mL) was cooled to −40 °C and treated with m-CPBA (4.1 mg, 0.018 mmol) in one portion. The mixture was stirred at −40 °C for 30 min and 23 °C for 2.5 h. After completion of the reaction (monitored by TLC), the solution was concentrated under vacuum. The product was purified by flash column chromatography on silica gel (90:10 ethyl acetate/hexanes) to afford phosphate 11 as an oil (6.5 mg, 70% yield): TLC Rf = 0.40 [1:4 (v/v) hexanes/EtOAc]; 1H NMR (600 MHz, CDCl3) δ 7.40–7.33 (m, 10H), 5.77 (d, J = 9.1 Hz, 1H), 5.66 (dd, J = 6.0, 3.3 Hz, 1H), 5.17–5.03 (m, 6H), 4.39–4.34 (m, 1H), 4.12 (dd, J = 12.6, 4.0 Hz, 1H), 4.00–3.97 (m, 1H), 3.90 (dd, J = 12.5, 2.3 Hz, 1H), 2.02 (s, 3H), 2.01 (s, 6H), 1.71 (s, 3H); 13C{1H} NMR (150 MHz, CDCl3) δ 171.3, 170.7, 170.5, 169.3, 135.4, 135.3, 135.2, 135.2, 129.1, 129.0, 128.9, 128.3, 128.2, 96.4, 96.3, 70.2, 70.2, 70.1, 70.1, 69.7, 67.3, 61.3, 51.9, 51.9, 22.9, 20.8, 20.7, 20.7; 31P NMR (243 MHz, CDCl3) δ 2.5; FT-IR (CH2Cl2) 2535, 2371, 1749, 1687, 1513, 1376, 1230, 955 cm−1; ESI-HRMS calcd for [C28H34NO11PNa, M + Na]+ 630.1711, found 630.1734. The anomeric configuration of 11 was assigned as α on the basis of the coupling constants of H1: JH1,H2 = 3.3 Hz, and JH1,P = 6.0 Hz.11

Phosphite 13.

A solution of phosphoramidite 8 (59 mg, 0.102 mmol) in methylene chloride (0.62 mL) was cooled to 0 °C. The resulting solution was treated with S-(2-hydroxyethyl) ethanethioate (31.0 mg, 0.255 mmol) followed by dropwise addition of 1H-tetrazole (680 μL, 0.306 mmol, 0.45 M in MeCN) using a microliter syringe. The reaction mixture was allowed to warm to 23 °C and stirred for 12 h. After completion of the reaction (monitored by TLC), the solution was concentrated under vacuum. The product was purified by flash column chromatography on silica gel (60:40 ethyl acetate/hexanes) to afford phosphite 13 as an oil (49 mg, 78% yield): TLC Rf = 0.23 [1:1 (v/v) hexanes/EtOAc]; 1H NMR (600 MHz, C6D6) δ 6.40 (d, J = 9.1 Hz, 1H), 5.70 (dd, J = 7.9, 3.4 Hz, 1H), 5.55 (dd, J = 10.9, 9.3 Hz, 1H), 5.41 (dd, J = 10.1, 9.4 Hz, 1H), 4.76–4.72 (m, 1H), 4.35 (dd, J = 12.3, 4.4 Hz, 1H), 4.30–4.28 (m, 1H), 4.20 (dd, J = 12.3, 3.2 Hz, 1H), 3.85–3.70 (m, 4H), 2.98–2.82 (m, 4H), 1.90 (s, 3H), 1.86 (s, 3H), 1.83 (s, 3H), 1.76 (s, 3H), 1.70 (s, 3H), 1.65 (s, 3H); 13C{1H} NMR (150 MHz, C6D6) δ 195.0, 194.4, 170.9, 170.2, 169.8, 169.1, 93.1, 93.0, 71.1, 69.6, 68.8, 62.1, 61.6, 61.5, 61.5, 52.8, 52.8, 30.3, 30.3, 30.2, 30.1, 30.1, 30.1, 22.8, 20.4, 20.2; 31P NMR (243 MHz, C6D6) δ 138.9; FT-IR (CH2Cl2) 2531, 2369, 2340, 1747, 1691, 1516, 1371, 1232, 1135, 1046, 953 cm−1; ESI-HRMS calcd for [C22H34NO13PS2Na, M + Na]+ 638.1101, found 638.1124.

Phosphate 14.

A solution of phosphite 13 (18 mg, 0.0293 mmol) in methylene chloride (0.3 mL) was cooled to −40 °C and treated with m-CPBA (8 mg, 0.035 mmol) in one portion. The mixture was stirred at −40 °C for 30 min and 23 °C for 2.5 h. After completion of the reaction (monitored by TLC), the solution was concentrated under vacuum. The product was purified by flash column chromatography on silica gel (100% ethyl acetate) to afford phosphate 14 as an oil (12.2 mg, 66% yield): TLC Rf = 0.35 (100% EtOAc); 1H NMR (600 MHz, CDCl3) δ 6.23 (d, J = 9.1 Hz, 1H), 5.71 (dd, J = 5.8, 3.2 Hz, 1H), 5.25–5.18 (m, 2H), 4.47–4.42 (m, 1H), 4.26 (dd, J = 12.5, 3.8 Hz, 1H), 4.23–4.13 (m, 5H), 4.11 (dd, J = 12.6, 2.3 Hz, 1H), 3.21–3.16 (m, 4H), 2.38 (s, 3H), 2.37 (s, 3H), 2.09 (s, 3H), 2.03 (s, 6H), 1.99 (s, 3H); 13C{1H} NMR (150 MHz, CDCl3) δ 195.3, 164.9, 171.3, 170.8, 170.6, 169.3, 96.6, 96.6, 70.0, 69.9, 67.5, 66.7, 66.7, 66.6, 66.6, 61.5, 52.0, 51.9, 30.8, 30.7, 29.3, 29.3, 29.2, 29.2, 23.1, 20.9, 20.8, 20.7; 31P NMR (243 MHz, CDCl3) δ 3.4; FT-IR (CH2Cl2) 2542, 2368, 2341, 1748, 1694, 1369, 1279, 1229, 1017, 956 cm−1; ESI-HRMS calcd for [C22H34NO14PS2Na, M + Na]+ 654.1051, found 654.1065. The anomeric configuration of 14 was assigned as α on the basis of the coupling constants of H1: JH1,H2 = 3.2 Hz, and JH1,P = 5.8 Hz.11

Monodeacylated GlcNDAz Sugar 18.

A solution of acylated diazirine sugar 1716 (1.2 g, 2.62 mmol) in a 2:1 methanol/THF mixture (8.0 mL) was treated with ammonium carbonate (981 mg, 10.22 mmol) in one portion at 23 °C. The resulting reaction mixture was stirred for 18 h. After completion of the reaction (monitored by TLC), the solution was concentrated under vacuum. The resulting crude reaction mixture was resuspended in ethyl acetate and filtered through a small pad of Celite with washing with ethyl acetate (100 mL). The filtrate was concentrated under vacuum and purified by flash column chromatography on silica gel (1:1 ethyl acetate/hexanes) to afford monodeacylated GlcNDAz sugar 18 as a white solid (721 mg, 77% yield): TLC Rf = 0.42 [1:1 (v/v) hexanes/EtOAc]; 1H NMR (600 MHz, CDCl3) δ 5.85 (d, J = 9.2 Hz, 1H), 5.30–5.27 (m, 2H), 5.13 (t, J = 9.8 Hz, 1H), 4.31–4.27 (m, 1H), 4.22–4.18 (m, 2H), 4.15–4.12 (m, 1H), 3.67 (s, 1H), 2.10 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H), 2.01–1.93 (m, 2H), 1.75–1.72 (m, 2H), 1.01 (s, 3H); 13C{1H} NMR (150 MHz, CDCl3) δ 171.7, 171.6, 171.1, 169.6, 91.7, 70.9, 68.3, 67.8, 62.2, 52.4, 30.7, 29.7, 25.5, 21.0, 20.9, 20.8, 20.0; FT-IR (CH2Cl2) 2536, 2369, 1746, 1685, 1517, 1372, 1233, 1050 cm−1; ESI-HRMS calcd for [C17H26N3O9, M + H]+ 416.1664, found 416.1678.

Phosphoramidite 19.

A solution of monodeacylated GlcNDAz sugar 18 (150 mg, 0.361 mmol) in methylene chloride (3.6 mL) was cooled to 0 °C and treated with bis(diisopropyl amine) phosphine chloride 7 (97 mg, 0.361 mmol). The solution was then treated dropwise with DBU (77 μL, 0.541 mmol) using a microliter syringe. The ice bath was allowed to warm to 23 °C, and the mixture was stirred for 1 h at the same temperature. After completion of the reaction (monitored by TLC), the solution was concentrated under vacuum. The product was purified by flash column chromatography on silica gel (20:80 ethyl acetate/hexanes) to afford phosphordiamide 19 as a solid (210 mg, 89% yield). Note that the silica in the column was initially equilibrated with 5% Et3N/hexanes (100 mL): TLC Rf = 0.45 [2:1 (v/v) hexanes/EtOAc]; 1H NMR (600 MHz, C6D6) δ 5.74 (d, J = 8.9 Hz, 1H), 5.64 (dd, J = 10.8, 9.5 Hz, 1H), 5.47 (t, J = 9.9 Hz, 1H), 5.38 (dd, J = 10.3, 3.2 Hz, 1H), 4.68–4.64 (m, 1H), 4.39 (dd, J = 12.0, 4.5 Hz, 1H), 4.35–4.32 (m, 1H), 4.26 (dd, J = 12.0, 2.3 Hz, 1H), 3.49–3.39 (m, 4H), 1.84–1.81 (m, 1H), 1.77 (s, 3H), 1.76 (s, 3H), 1.75–1.71 (m, 2H), 1.63 (s, 3H), 1.55–1.52 (m, 1H), 1.23–1.21 (m, 12H), 1.14–1.13 (m, 12H), 0.66 (s, 3H); 13C{1H} NMR (150 MHz, C6D6) δ 171.8, 170.4, 170.1, 169.1, 93.1, 93.0, 71.8, 69.5, 68.7, 62.3, 54.0, 54.0, 45.5, 45.4, 30.5, 29.8, 25.4, 24.6, 24.5, 24.5, 24.5, 24.4, 24.4, 20.5, 20.4, 20.2, 19.7; 31P NMR (243 MHz, C6D6) δ 113.5; FT-IR (CH2Cl2) 2539, 2368, 2341, 1744, 1685, 1512, 1368, 1232, 1045, 956 cm−1; ESI-HRMS calcd for [C29H53N5O9P, M + H]+ 646.3575, found 646.3597.

Phosphite 20.

A solution of phosphoramidite 19 (130 mg, 0.201 mmol) in methylene chloride (1.22 mL) was cooled to 0 °C. The resulting solution was treated with S-(2-hydroxyethyl) ethanethioate (61.0 mg, 0.502 mmol) followed by dropwise addition of 1H-tetrazole (1.34 mL, 0.603 mmol, 0.45 M in MeCN) using a syringe. The reaction mixture was allowed to warm to 23 °C and stirred for 12 h. After completion of the reaction (monitored by TLC), the solution was concentrated under vacuum. The product was purified by flash column chromatography on silica gel (40:60 ethyl acetate/hexanes) to afford phosphite 20 as an oil (114 mg, 83% yield): TLC Rf = 0.40 [1:1 (v/v) hexanes/EtOAc]; 1H NMR (600 MHz, C6D6) δ 6.43 (d, J = 9.0 Hz, 1H), 5.67 (dd, J = 7.7, 3.3 Hz, 1H), 5.55 (dd, J = 11.0, 9.4 Hz, 1H), 5.40 (t, J = 9.9 Hz, 1H), 4.77–4.73 (m, 1H), 4.35 (dd, J = 12.1, 4.4 Hz, 1H), 4.31–4.28 (m, 1H), 4.20 (dd, J = 12.3, 2.3 Hz, 1H), 3.86–3.70 (m, 4H), 2.99–2.83 (m, 4H), 1.96 (s, 3H), 1.95–1.88 (m, 2H), 1.88 (s, 3H), 1.76 (s, 3H), 1.75 (s, 3H), 1.74–1.71 (m, 1H), 1.66 (s, 3H), 1.65–1.62 (m, 1H), 0.69 (s, 3H); 13C{1H} NMR (150 MHz, C6D6) δ 195.2, 194.4, 171.2, 170.9, 170.2, 169.1, 93.2, 93.1, 71.0, 69.6, 68.8, 62.1, 61.6, 61.5, 61.5, 61.5, 52.7, 52.7, 30.3, 30.3, 30.3, 30.2, 30.2, 30.1, 30.1, 29.9, 25.6, 20.5, 20.4, 20.2, 19.8; 31P NMR (243 MHz, C6D6) δ 139.1; FT-IR (CH2Cl2) 2537, 2369, 2340, 1747, 1691, 1516, 1370, 1232, 12138, 1046, 930 cm−1; ESI-HRMS calcd for [C25H38N3O13PS2Na, M + Na]+ 706.1476, found 706.1511.

Phosphate 21.

A solution of phosphite 20 (14 mg, 0.0209 mmol) in methylene chloride (0.21 mL) was cooled to −40 °C and treated with m-CPBA (5.5 mg, 0.0245 mmol) in one portion. The mixture was stirred at −40 °C for 30 min and 23 °C for 2.5 h. After completion of the reaction (monitored by TLC), the solution was concentrated under vacuum. The product was purified by flash column chromatography on silica gel (80:20 ethyl acetate/hexanes) to afford phosphate 21 as an oil (9.8 mg in 67% yield): TLC Rf = 0.36 [7:3 (v/v) EtOAc/hexane]; 1H NMR (600 MHz, CDCl3) δ 6.21 (d, J = 9.1 Hz, 1H), 5.71 (dd, J = 5.7, 3.3 Hz, 1H), 5.25–5.18 (m, 2H), 4.48–4.44 (m, 1H), 4.27 (dd, J = 12.5, 3.9 Hz, 1H), 4.24–4.10 (m, 6H), 3.21–3.16 (m, 4H), 2.40 (s, 3H), 2.38 (s, 3H), 2.10 (s, 3H), 2.07–2.06 (m, 1H), 2.04 (s, 6H), 1.98–1.93 (m, 1H), 1.80–1.71 (m, 2H), 1.02 (s, 3H); 13C{1H} NMR (150 MHz, CDCl3) δ 195.4, 195.0, 171.8, 171.3, 170.8, 169.3, 96.6, 96.5, 69.9, 67.6, 66.7, 66.7, 66.6, 66.6, 61.5, 51.9, 51.8, 30.8, 30.7, 30.4, 29.6, 29.3, 29.2, 29.2, 25.6, 20.9, 20.7, 20.1; 31P NMR (243 MHz, CDCl3) δ −3.2; FT-IR (CH2Cl2) 2869, 2710, 2533, 2375, 2337, 1745, 1692, 1516, 1464, 1376, 1241, 1043, 954 cm−1; ESI-HRMS calcd for [C25H38N3O14PS2Na, M + Na]+ 722.1425, found 722.1461. The anomeric configuration of 21 was assigned as α on the basis of the coupling constants of H1: JH1,H2 = 3.3 Hz, and JH1,P = 5.7 Hz.11

Synthesis of Amide 23.

On the basis of a procedure reported in the literature,17 a solution of d-(+)-glucosamine hydrochloride 1 (858 mg, 3.98 mmol) and diazirine acid 2214 (685 mg, 4.38 mmol) in methanol (4.0 mL) was cooled to 0 °C and treated with triethylamine (2.5 mL, 17.9 mmol). The reaction mixture was treated dropwise with a solution of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDCI) (680 mg, 4.38 mmol) and hydroxylbenzotriazole (HOBt) (592 mg, 4.38 mmol) in methanol (2.0 mL) using a syringe. The ice bath was allowed to warm to 23 °C, and the reaction mixture was then stirred for 18 h. After completion of the reaction (monitored by TLC), the solution was concentrated under vacuum. The product was purified by flash column chromatography on silica gel (eluent gradient from 9:1 to 7:1 methylene chloride/methanol) to afford amide 23 as a white solid (670 mg, 53% yield): TLC Rf = 0.36 [4:1 (v/v) CH2Cl2/MeOH]; 1H NMR (600 MHz, CD3OD) δ 5.10 (d, J = 3.5 Hz, 1H), 3.85–3.83 (m, 1H), 3.82–3.78 (m, 2H), 3.72–3.68 (m, 2H), 3.38–3.34 (m, 1H), 2.23 (d, J = 7.4 Hz, 1H), 1.62–1.57 (m, 2H), 1.38–1.34 (m, 2H), 1.26–1.22 (m, 2H), 0.98 (s, 3H); 13C{1H} NMR (150 MHz, CD3OD) δ 176.3, 92.6, 73.1, 72.6, 62.8, 55.8, 36.7, 35.1, 26.5, 24.7, 19.8, 18.7, 17.3; FT-IR (CH2Cl2) 2535, 2370, 2337, 1737, 1249, 1185 cm−1; ESI-HRMS calcd for [C13H24N3O6, M + H]+ 318.1660, found 318.1656.

Acylated GlcNDAz Sugar 24.

A solution of amide 23 (1.5 g, 4.72 mmol) in pyridine (18 mL, excess) was cooled to 0 °C and treated dropwise with acetic anhydride (4.5 mL, excess) via syringe. The resulting reaction mixture was stirred for 16 h at 23 °C. After completion of the reaction (monitored by TLC), the mixture was diluted with ethyl acetate, and the organic layer was washed with 1 M aqueous HCl and then brine. The organic layer was dried over sodium sulfate and concentrated in vacuo. The resulting crude reaction mixture was purified by flash column chromatography on silica gel (40:60 ethyl acetate/hexanes) to afford acylated GlcNDz sugar 24 as an oil (2.2 g, 96% yield): TLC Rf = 0.45 [1:1 (v/v) hexanes/EtOAc]; 1H NMR (600 MHz, CDCl3) δ 6.17 (d, J = 3.6 Hz, 1H), 5.54 (d, J = 8.9 Hz, 1H), 5.25–5.19 (m, 2H), 4.49–4.46 (m, 1H), 4.24 (dd, J = 4.1, 12.5 Hz, 1H), 4.05 (dd, J = 12.5, 2.3 Hz, 1H), 4.00–3.98 (m, 1H), 2.19 (s, 3H), 2.11–2.06 (m, 2H), 2.08 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H), 1.56–1.50 (m, 2H), 1.36–1.33 (m, 2H), 1.14–1.11 (m, 2H), 0.98 (s, 3H); 13C{1H} NMR (150 MHz, CDCl3) δ 172.6, 171.9, 170.8, 169.2, 168.7, 90.7, 70.7, 69.8, 67.5, 61.6, 51.1, 36.2, 34.0, 25.7, 24.9, 23.6, 21.0, 20.9, 20.8, 20.7, 19.9; FT-IR (CH2Cl2) 2540, 2368, 2341, 1752, 1685, 1510, 1371, 1218, 1013 cm−1; ESI-HRMS calcd for [C21H31N3O10Na, M + Na]+ 508.1902, found 508.1923.

Monodeacylated GlcNDAz Sugar 25.

A solution of acylated diazirine sugar 24 (2.0 g, 4.12 mmol) in a 2:1 methanol/THF mixture (12.5 mL) was treated with ammonium carbonate (1.54 g, 16.7 mmol) in one portion at 23 °C. The resulting reaction mixture was stirred for 18 h. After completion of the reaction (monitored by TLC), the solution was concentrated under vacuum. The resulting crude reaction mixture was resuspended in ethyl acetate and filtered through a small pad of Celite with washing with ethyl acetate (150 mL). The filtrate was concentrated under vacuum and purified by flash column chromatography on silica gel (1:1 ethyl acetate/hexanes) to afford monodeacylated GlcNDAz sugar 25 as a colorless oil (1.33 g, 73% yield): TLC Rf = 0.33 [1:1 (v/v) hexanes/EtOAc]; 1H NMR (400 MHz, CDCl3) δ 5.89 (d, J = 9.2 Hz, 1H), 5.27 (dd, J = 10.8, 9.4 Hz, 1H), 5.23 (t, J = 3.7 Hz, 1H), 5.11 (t, J = 9.7 Hz, 1H), 4.30–4.24 (m, 2H), 4.22–4.17 (m, 2H), 4.13–4.09 (m, 1H), 2.13–2.07 (m, 2H), 2.08 (s, 1H), 2.02 (s, 3H), 2.01 (s, 3H), 1.57–1.49 (m, 2H), 1.37–1.33 (m, 2H), 1.16–1.08 (m, 2H), 0.98 (s, 3H); 13C{1H} NMR (100 MHz, CDCl3) δ 173.0, 171.5, 171.1, 169.6, 91.7, 71.0, 68.4, 67.6, 62.2, 52.3, 36.4, 34.0, 25.8, 25.1, 23.7, 20.9, 20.9, 20.7, 20.0; FT-IR (CH2Cl2) 2537, 2368, 2340, 1746, 1679, 1515, 1372, 1234, 1050, 909 cm−1; ESI-HRMS calcd for [C19H30N3O9, M + H]+ 444.1977, found 444.2002.

Phosphoramidite 26.

A solution of monodeacylated GlcNDAz sugar 25 (121 mg, 0.273 mmol) in methylene chloride (2.7 mL) was cooled to 0 °C and treated with bis(diisopropylamine) phosphine chloride 7 (73 mg, 0.273 mmol). The solution was then treated dropwise with DBU (58 μL, 0.410 mmol) using a microliter syringe. The ice bath was allowed to warm to 23 °C, and the mixture was stirred for 1 h at the same temperature. After completion of the reaction (monitored by TLC), the solution was concentrated under vacuum. The product was purified by flash column chromatography on silica gel (20:80 ethyl acetate/hexanes) to afford phosphordiamide 26 as an oil (139 mg, 76% yield). Note that the silica in the column was initially equilibrated with 5% Et3N/hexanes (100 mL): TLC Rf = 0.40 [2:1 (v/v) hexanes/EtOAc]; 1H NMR (600 MHz, C6D6) δ 5.73 (d, J = 9.0 Hz, 1H), 5.66 (dd, J = 10.8, 9.5 Hz, 1H), 5.49 (t, J = 9.8 Hz, 1H), 5.40 (dd, J = 10.3, 3.3 Hz, 1H), 4.72–4.68 (m, 1H), 4.39 (dd, J = 12.0, 4.6 Hz, 1H), 4.36–4.34 (m, 1H), 4.29 (dd, J = 12.0, 2.3 Hz, 1H), 3.49–3.42 (m, 4H), 2.00–1.88 (m, 2H), 1.77 (s, 3H), 1.76 (s, 3H), 1.63 (s, 3H), 1.56–1.48 (m, 2H), 1.25–1.23 (m, 12H), 1.16–1.14 (m, 12H), 1.04–1.01 (m, 2H), 0.98–0.94 (m, 2H), 0.63 (s, 3H); 13C{1H} NMR (150 MHz, C6D6) δ 171.6, 171.5, 170.1, 169.1, 93.2, 93.1, 72.0, 69.5, 68.7, 62.3, 53.8, 53.8, 45.6, 45.5, 45.5, 45.4, 36.3, 34.1, 25.5, 25.1, 24.6, 24.5, 24.5, 24.4, 24.0, 20.5, 20.4, 20.2, 19.6; 31P NMR (243 MHz, C6D6) δ 113.4; FT-IR (CH2Cl2) 2534, 2369, 2340, 1744, 1681, 1510, 1460, 1372, 1234, 1044 cm−1; ESI-HRMS calcd for [C31H57N5O9P, M + H]+ 674.3888, found 674.3912.

Phosphite 27.

A solution of phosphoramidite 26 (180 mg, 0.267 mmol) in methylene chloride (1.6 mL) was cooled to 0 °C. The resulting solution was treated with S-(2-hydroxyethyl) ethanethioate (81.0 mg, 0.668 mmol) followed by dropwise addition of 1H-tetrazole (1.78 mL, 0.800 mmol, 0.45 M in MeCN) using a syringe. The reaction mixture was allowed to warm to 23 °C and stirred for 12 h. After completion of the reaction (monitored by TLC), the solution was concentrated under vacuum. The product was purified by flash column chromatography on silica gel (40:60 ethyl acetate/hexanes) to afford phosphite 20 as an oil (165 mg, 87% yield): TLC Rf = 0.45 [1:1 (v/v) hexanes/EtOAc]; 1H NMR (600 MHz, C6D6) δ 6.30 (d, J = 9.1 Hz, 1H), 5.69 (dd, J = 7.8, 3.3 Hz, 1H), 5.56 (dd, J = 11.0, 9.4 Hz, 1H), 5.42 (t, J = 9.8 Hz, 1H), 4.77–4.73 (m, 1H), 4.35 (dd, J = 12.3, 4.4 Hz, 1H), 4.31–4.28 (m, 1H), 4.20 (dd, J = 12.3, 2.3 Hz, 1H), 3.88–3.73 (m, 4H), 2.99–2.84 (m, 4H), 2.09–2.07 (m, 2H), 1.94 (s, 3H), 1.86 (s, 3H), 1.77 (s, 3H), 1.75 (s, 3H), 1.65 (s, 3H), 1.59–1.50 (m, 2H), 1.06–1.00 (m, 4H), 0.64 (s, 3H); 13C{1H} NMR (150 MHz, C6D6) δ 195.0, 194.3, 172.2, 171.0, 170.2, 169.1, 93.2, 93.1, 71.2, 69.6, 68.8, 62.1, 61.6, 61.5, 61.5, 52.8, 52.7, 36.0, 34.2, 30.3, 30.3, 30.2, 30.2, 30.1, 25.3, 24.0, 20.5, 20.4, 20.2, 19.6; 31P NMR (243 MHz, C6D6) δ 139.2; FT-IR (CH2Cl2) 2535, 2370, 1747, 1690, 1514, 1372, 1233, 1147 cm−1; ESI-HRMS calcd for [C27H42N3O13PS2Na, M + Na]+ 734.1789, found 734.1830.

Phosphate 28.

A solution of phosphite 20 (20 mg, 0.0289 mmol) in methylene chloride (0.3 mL) was cooled to −40 °C and treated with m-CPBA (6.4 mg, 0.037 mmol) in one portion. The mixture was stirred at −40 °C for 30 min and 23 °C for 2.5 h. After completion of the reaction (monitored by TLC), the solution was concentrated under vacuum. The product was purified by flash column chromatography on silica gel (80:20 ethyl acetate/hexanes) to afford phosphate 28 as an oil (17.8 mg, 72% yield): TLC Rf = 0.36 [7:3 (v/v) EtOAc/hexane]; 1H NMR (400 MHz, CDCl3) δ 6.11 (d, J = 9.1 Hz, 1H), 5.69 (dd, J = 5.8, 3.3 Hz, 1H), 5.26–5.17 (m, 2H), 4.48–4.42 (m, 1H), 4.29–4.09 (m, 7H), 3.22–3.16 (m, 4H), 2.39 (s, 3H), 2.37 (s, 3H), 2.22–2.12 (m, 2H), 2.10 (s, 3H), 2.04 (s, 6H), 1.60–1.52 (m, 2H), 1.38–1.34 (m, 2H), 1.19–1.11 (m, 2H), 0.99 (s, 3H); 13C{1H} NMR (100 MHz, CDCl3) δ 195.0, 194.7, 173.1, 171.1, 170.6, 169.2, 96.5, 96.5, 69.9, 69.8, 67.4, 66.6, 66.6, 66.6, 66.5, 61.4, 51.8, 51.7, 36.0, 33.9, 30.6, 30.6, 29.1, 29.1, 29.1, 29.0, 25.6, 24.9, 23.6, 20.7, 20.7, 20.6, 19.8; 31P NMR (161 MHz, CDCl3) δ 3.4; FT-IR (DCM) 2530, 2369, 2340, 1748, 1693, 1513, 1369, 1232, 1143, 1019, 956, 913 cm−1; ESI-HRMS calcd for [C27H42N3O14PS2Na, M + Na]+ 750.1738, found 750.1730. The anomeric configuration of 28 was assigned as α on the basis of the coupling constants of H1: JH1,H2 = 3.3 Hz, and JH1,P = 5.8 Hz.11

Evaluation of the Metabolic Production of UDP-GlcNAc Analogues.

Colo205 cells, wild-type or stably expressing mutant UAP1 F383G, were cultured with compound 21 or 28. Compounds were dissolved in DMSO and added to cell media to achieve a final concentration of 100 μM; control cells were treated with an equivalent volume of DMSO alone. After 8 h, cells were harvested and washed in PBS. Pelleted cells were stored at −80 °C. To extract UDP-sugars, cells were vortexed and incubated on ice in 50% acetonitrile followed by centrifugation. The soluble fraction was frozen in liquid nitrogen and dried with a SAVANT SC210A SpeedVac Concentrator (Thermo Fisher Scientific). Dried extracts were resuspended in 100 μL of 40 mM sodium phosphate buffer (pH 7.4) and centrifuged at 20000g and 4 °C for 1 min. Clarified samples were filtered using a 3K Amicon Ultra filter unit (Millipore Sigma, UFC5003BK). Twenty microliters of each sample was injected for analysis by HPAEC using a CarboPac PA1 column (Thermo Fisher Scientific, catalog no. 035391) with a guard column (Thermo Fisher Scientific, catalog no. 0343096), along with standards for UDP-GlcNAc (Promega, V7071) and UDP-GlcNDAz.2 Samples were eluted with buffer A (1 mM NaOH) and buffer B (1 M NaOAc, 1 mM NaOH) at a flow rate of 1 mL/min using the following gradient of buffer B in buffer A: 5% at 0 min, 30% at 40 min, 50% at 45 min, 60% at 75 min, 100% at 80 min, 100% at 90 min, 5% at 95 min, and 5% at 90 min. Elution of UDP-sugars was detected by UV absorption at 260 nm.

Photo-Cross-Linking of NUP153.

Colo205 cells stably expressing UAP1 F383G were prepared by lentiviral infection using pSin4 UAP1 F383G (Addgene ID 169891) as reported previously.7 Wild-type Colo205 cells, or Colo205 cells stably expressing UAP1 F383G, were cultured in RPMI-1640. Compound 21 or 28 was dissolved in DMSO and added to cell medium to achieve a final concentration of 100 μM; control cells were treated with an equivalent volume of DMSO alone. Twenty-four hours later, addition of the compounds was repeated. Twenty-four hours after the second addition of compounds, cells were counted to evaluate growth inhibition, using trypan blue (Invitrogen, T10282) staining to evaluate toxicity. Cells were UV irradiated for 15 min using an AnalitikJena UVP cross-linker and then lysed by resuspension in 100 μL of RIPA buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1% (w/v) SDS, 0.5% (w/v) sodium deoxycholate, and 1% (w/v) IGEPAL CA-630 in water] containing protease inhibitors (Millipore Sigma, 11836153001) followed by vortexing in eight bursts before incubation on ice for 1 h. After centrifugation of the lysate at 20000g for 15 min, the soluble fraction was boiled with SDS loading dye, subjected to 8% SDS–PAGE, and transferred to an Immobilon-P PVDF membrane (Sigma, IPVH00010). The membrane was immunoblotted using anti-NUP153 (1:2000, Abcam, ab96462), followed by probing with anti-β-actin (1:5000, Abcam, ab8227) as a loading control. Goat anti-mouse HRP (1:2000, Invitrogen, 62-6520) and goat anti-rabbit HRP (1:2000, Invitrogen, 65-6120) secondary antibodies were used with SuperSignal West Pico PLUS Chemiluminescent substrate (Thermo-Scientific, 34580).

Supplementary Material

SI

ACKNOWLEDGMENTS

The authors acknowledge support from the Welch Foundation (I-1686 and I-1748), the National Institutes of Health (U01CA242115 and R01GM102604), a W. W. Caruth, Jr. Endowed Scholarship, and a Teva Pharmaceuticals Marc A. Goshko Memorial Grant (60011-TEV). The authors thank Han Wu for assistance with HPAEC-PAD analysis. The authors acknowledge the Shimadzu Center for Advanced Analytical Chemistry (SCAAC) at The University of Texas at Arlington for HRMS data. The authors also thank our diverse collection of lab members for creating an environment that supported the success of this project.

Footnotes

Supporting Information

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.joc.1c01781.

NMR spectroscopic data for all new compounds and effects of compounds on cell growth and UDP-sugar production (PDF)

The authors declare no competing financial interest.

REFERENCES

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