Synthesis of C-Glycoside Analogues of β-Galactosamine-(1–>4)-3-O-Methyl-D-Chiro-Inositol and Assay as Activator of Protein Phosphatases PDHP and PP2Cα

doi:10.1016/j.bmc.2009.12.056

Bioorg Med Chem. Author manuscript; available in PMC 2012 July 30.

Published in final edited form as:

Bioorg Med Chem. 2010 February; 18(3): 1103–1110.

Published online 2010 January 4. doi: 10.1016/j.bmc.2009.12.056

PMCID: PMC3408221

NIHMSID: NIHMS171319

Synthesis of C-Glycoside Analogues of β-Galactosamine-(1–>4)-3-O-Methyl-D-Chiro-Inositol and Assay as Activator of Protein Phosphatases PDHP and PP2Cα

Sunej K. Hans,^a Fatoumata Camara,^a Ahmad Altiti,^a Alejandro Martin-Montalvo Sanchez,^c David L. Brautigan,^d Douglas Heimark,^b Joseph Larner,^b Scott Grindrod,^e Milton L. Brown,^e and David R. Mootoo^a^*

^aDepartment of Chemistry, Hunter College, 695 Park Avenue New York, NY 10065 and The Graduate Center, CUNY, 365 5th Avenue, New York, NY 10016

^bAllomed Pharmaceuticals, Charlottesville Virginia 22908, Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, Virginia 22908-0735

^cCentro Andaluz de Biologia del Desarrollo, Universidad Pablo de Olavide, 41013 Seville, Spain

^dCenter for Cell Signaling and Department of Microbiology, University of Virginia School of Medicine, Charlottesville, Virginia 22908

^eGeorgetown University Medical Center, Department of Neuroscience, Research Building EP07, 3970 Reservoir Road, Washington, D.C. 20057

^*Corresponding author: Tel: +1-2-772-4356; fax: +1-2-772-5332; Email: dmootoo/at/hunter.cuny.edu

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Abstract

The glycan β-galactosamine-(1–4)-3-O-methyl-D-chiro-inositol, called INS-2, was previously isolated from liver as a putative second messenger-modulator for insulin. Synthetic INS-2 injected intravenously in rats is both insulin-mimetic and insulin-sensitizing. This bioactivity is attributed to allosteric activation of pyruvate dehydrogenase phosphatase (PDHP) and protein phosphatase 2Cα (PP2Cα). Towards identification of potentially metabolically stable analogues of INS-2 and illumination of the mechanism of enzymatic activation, C-INS-2, the exact C-glycoside of INS-2, and C-INS-2-OH the deaminated analog of C-INS-2, were synthesized and their activity against these two enzymes evaluated. C-INS-2 activates PDHP comparable to INS-2, but failed to activate PP2Cα. C-INS-2-OH was inactive against both phosphatases. These results and modeling of INS-2, C-INS-2 and C-INS-2-OH into the 3D structure of PDHP and PP2Cα, suggest that INS-2 binds to distinctive sites on the two different phosphatases to activate insulin signaling. Thus the carbon analog could selectively favor glucose disposal via oxidative pathways.

Keywords: C-glycoside, insulin mediator-modulator, pyruvate dehydrogenase, protein phosphatase

1. Introduction

Soluble inositol-glycans have been proposed to operate as insulin messengers-modulators in concert with the insulin receptor tyrosine kinase signaling system to control intracellular oxidative and non-oxidative glucose disposal.¹ By analogy with the PIP3 signaling pathway, the insulin receptor is thought to regulate, via heterotrimeric G proteins, specifically G_q,² the GPI-phospholipase cleavage of a glycolipid to generate diacylglycerol and inositol glycans as messengers-modulators. In earlier studies, we isolated INS-2, 1 from beef liver as a novel inositol glycan pseudo-disaccharide (Figure 1). The INS-2 structure was determined by 2D NMR and confirmed by chemical synthesis.³ Synthetic INS-2 was insulin-mimetic and insulin-sensitizing when injected in vivo, and when added to living cells.³ In biochemical assays, INS-2 activated two related Mg²⁺ dependent protein phosphatases, mitochondrial pyruvate dehydrogenase phosphatase (PDHP) and a Ser/Thr protein phosphatase, PP2Cα.^3,4 These protein phosphatases respectively dephosphorylate the rate limiting enzymes of intracellular oxidative glucose disposal (i.e. PDH), and non-oxidative glucose disposal (glycogen synthase, GS).⁵ Both PDH and GS are classical targets of activation by insulin via dephosphorylation.^6,7 When docked into the X-ray crystal structure of PP2Cα, INS-2 bound to an allosteric pocket adjoining the catalytic pocket that was occupied by a phosphopeptide substrate.⁴ INS-2 enhanced reaction with the phosphopeptide substrate, but not with the small molecule substrate p-nitrophenyl phosphate. A point mutation D163A in PP2Cα completely eliminated allosteric activation by INS-2, with no loss of catalytic activity.⁴ Thus a mechanism of allosteric activation of PP2Cα was proposed.⁴

Figure 1

1. INS-2, 2. C-INS-2 and 3. C-INS-2-OH

To further define the structure-activity relationship of INS-2, we synthesized C-INS-2, 2, the C-glycoside of 1 in which the glycosidic oxygen is replaced with a methylene and C-INS-2-OH, 3, the corresponding C-glycoside in which the 2-amino substituent is replaced with a hydroxyl group. These analogues were compared to INS-2 for activation of the two protein phosphatases, PP2Cα and PDHP. C-glycoside probes like 2 and 3⁸ may provide insight on the chemical and conformational requirements for activity,⁹ and are of special interest as possible therapeutic agents that are stable to hydrolysis.^10,11,12

2. Synthesis

C-glycoside 3 was prepared using our protocol for β-C-galactosides and the aminated analog 2 was obtained from an intermediate en route to 3.¹³ Accordingly, di-O-isopropylidene pinitol 4 was transformed to the thiocarbonate 5, which when subjected to the Keck radical allylation procedure gave the equatorial C-propenyl derivative 6 in greater than 90% stereoselectivity as determined by ¹H NMR.¹⁴ (Scheme 1). Oxidative processing of 6 led to the C-branched-ethanoic acid 7. Acid 7 and known 1-thio-1,2-O-isopropylidene acetal 8 were next subjected to the C-glycosidation sequence (Scheme 2). Thus, DCC mediated esterification on 7 and 8, followed by Tebbe olefination of the derived ester 9 afforded enol ether 10. In the key step, treatment of 10 with methyl triflate in the presence of 2,6-di-tert-butyl-4-methylpyridine (DTBMP), provided glycal 11 in 77% yield. Hydroboration of 11 afforded C-pseudodisaccharide 12 as a single diastereomer. Removal of the isopropylidene protecting groups provided C-INS-2-OH 3. For the synthesis of 2, alcohol 12 was first oxidized to ketone 13 using PCC. Treatment of 13 with O-methylhydroxylamine afforded a mixture of oximes 14. Attempted reduction of 14 with standard hydride reducing agents produced complex mixtures with low yield of the desired hydroxylamine ether 15.^15,16 The optimal conditions were found to be Bu₃SnH in the presence of BF₃.OEt₂, which afforded 15 in 82% isolated yield, and a minor compound (less than 5%) for which the physical data supported the axial isomer.¹⁷ Cleavage of the silylether in 15 followed by methoxylamine reduction and cleavage of acetal protecting groups provided C-INS-2 in 73% overall yield from 15. The structures of 2 and 3 were confirmed by 2D NMR and HRMS analysis (Supporting Information).

Scheme 1

Synthesis of the C-inositol segment

Scheme 2

Assembly of C-INS-2 and C-INS-2-OH

3. Enzyme assays

PP2Cα was assayed using two separate substrates with and without added INS-2 analogues. p-Nitrophenyl phosphate (pNPP) as substrate assayed the activity of the enzyme catalytic center and this activity is unaffected by the allosteric site for INS-2. PP2Cα activity with an octapeptide-phosphate substrate (RRRRPp-TPA) was sensitive to the allosteric site regulating the active site.⁴ As seen in Fig 2A, with the octapeptide substrate and Malachite green, as previously shown, Mn²⁺ alone and INS-2 showed phosphate release assayed by dose-dependent increase in PP2Cα activity, while the C-glycosides 2 and 3 were inactive. As shown in Fig 2B, with the pNPP substrate, as previously shown,⁴ only Mn²⁺ increased the catalytic activity of PP2Cα, not INS-2 or its analogues.

Figure 2

Protein phosphatase 2Cα activity in response to added INS-2, C-INS-2-OH and C-INS-2. The recombinant GST-PP2C fusion protein was assayed as described in the experimental section, using either phosphopeptide (A) or p-nitrophenyl phosphate (B) as (more ...)

As shown in Figure 3, INS-2, C-INS-2, C-INS-2-OH were assayed ± Mn²⁺ for their activation of PDHP. Only INS-2 and C-INS-2 activated PDHP significantly, and activation was seen only in the presence of Mn²⁺, relative to control. The C-INS-2-OH analog was inactive in the presence or absence of Mn²⁺. The results were combined from two separate sets of experiments with two separate chemically synthesized samples. These data show equal activation of PDHP by INS-2 and its carbon analog, C-INS-2. Thus while C-INS-2-OH was inactive with both phosphatases, there was a marked dichotomy with C-INS-2 which activated PDHP, but not PP2Cα.

Figure 3

PDHP assay of INS-2, C-INS-2 and C-INS-2-OH ± 5 mM Mn²⁺. Activity is expressed as change in absorbance at 340 nm. Mean + s.e.m. (n=6) * p < 0.016; ** p < 0.004 vs. Control.

4. Computer Modeling

Docking of INS-2 and its analogues was accomplished using the FlexX flexible docking suite in the SYBYL shell.^4,18 The crystal structure for PP2Cα (1ASQ) and PDHP1 (2PNQ) were acquired from the Protein Data Bank. The allosteric sites for the two proteins were detailed through a residue selection followed by an 8 Å radius selection. Compound structures were then prepared in the SYBYL shell and minimized using the conjugate gradient method with an endpoint of 0.01 kcal. The top 30 conformations of each molecule were then analyzed using G-score, D-score, PMF-score as previously described.⁴ Multiple scoring functions were then combined for analysis by the C-score function to rate the conformations.¹⁹ In each case the top scoring two to three conformations of the molecule overlap well, but then the scores of the conformations drop off quickly and the position of the molecule is less reproducible (Supporting Information). Putative hydrogen bonds were analyzed through the SYBYL shell by the addition of hydrogen atoms to the protein structure with the Biopolymer function.

Docking of INS-2 and C-INS-2 into the X-ray crystal structure of PP2Cα revealed very different orientations and conformations (with respect to the intersaccharide torsions) for the two glycans (Figure 4A and 4B).¹¹ The intersaccharide torsions Φ,Ψ for INS-2 and C-INS-2 are (270, 64) and (300, 210) respectively.²⁰ In particular, Asp 243, which is proximal to the catalytic site (vide infra), interacts with different residues on the two ligands, the sugar 2-amino and 3-hydroxyl on INS-2, and the sugar 4-hydroxyl and the inositol 2- and 3- hydroxyls on C-INS-2 (Figures 4A, 4B and Supporting Information). The orientation of INS-2 suggests that Asp 243 is moved toward the allosteric pocket and away from the catalytic site. In contrast, bound C-INS-2 does not appear to have a significant effect on the position of Asp 243. However, both INS-2 and C-INS-2 dock very similarly into the crystal structure for PDHP1. The respective intersaccharide torsions are (244, 60) and (240, 60) and both ligands appear to pull Glu 351 (the comparable acidic residue to Asp 243 in PPC2α) from the active site (Figure 4B and Supporting Information).

Figure 4

A. INS-2 and C-INS-2 docked in silico into the allosteric site of PP2Cα. H-bonds to the amino group indicated in pink. For the enzyme to be fully active Asp 243 moves to open up the catalytic site. Note the significant difference in position of (more ...)

When C-INS-2-OH was docked into the X-ray crystal structures of PP2Cα (Fig 5A) and PDHP1 (Fig 5B), it bound to both enzymes but not in a fashion that would be expected to pull the acidic residue (either Asp 243 or Glu 351) away from the catalytic pocket.

Figure 5

A: C-INS-2-OH docked into the allosteric site of PP2Cα. Note that C-INS-2-OH still binds but is blocked from pulling Asp 243 toward the allosteric pocket. B: C-INS-2-OH docked in silico into the allosteric site of PDHP1. Note again that C-INS-2-OH (more ...)

5. Discussion

Carbon in place of oxygen bridges in carbohydrates are commonly synthesized to produce analogues that are resistant to intestinal hydrolytic breakdown.^8,10 A similar strategy was imagined for INS-2, a natural inositol glycan pseudo-disaccharide, initially isolated from liver and then chemically synthesized and shown to be an insulin-mimetic and sensitizing agent.³

INS-2, in vitro activates two phosphoprotein phosphatases, mitochondrial PDHP and cytosolic PP2Cα,^3,4 both members of the same PPM family and with ~ 20% amino acid sequence identity.²¹ Both have been crystallized and X-ray structures determined.^21,22 By computer modeling, we have previously reported that INS-2 docked into an allosteric site on PP2Cα adjacent to the catalytic site. With a point mutant D163A in the allosteric site, we showed a specific loss of allosteric activation of the enzyme with INS-2, but with full retention of catalytic activity measured with a non-peptide substrate.⁴ Thus we were able to propose a mechanism of allosteric action. By occupying the allosteric site, INS-2 would hydrogen bond to Asp 243 and thus prevent Asp 243 from interfering with positioning of the phosphopeptide substrate at the catalytic site.⁴

In the present study, C-INS-2, the C-glycoside analog of INS-2 was found to be inactive on PP2Cα (Fig 3). To get insight into the molecular basis for the difference in activity of INS-2 and C-INS-2 on PP2Cα, the binding of the two ligands to the X-ray crystal structure of PP2Cα was modeled (Fig 4A). Significant differences in their binding suggest that INS-2 but not C-INS-2 pulls Asp 243 into the allosteric site and away from the catalytic pocket, thereby providing a possible explanation for the inactivity of C-INS-2. In contrast to their activity on PP2Cα, INS-2 and C-INS-2 showed similar activity on PDHP1. This result was somewhat surprising because PDHP1 and PP2Cα are related enzymes. We therefore modeled the binding of INS-2 and C-INS-2 to the recently published 3D structure of PDHP1 (Fig 4B) and compared the results with those for PP2Cα (Fig 4A). INS-2 and C-INS-2 bound similarly to PDHP1 and in a way that suggests they both pull Glu-351 (the comparable residue to Asp 243 on PP2Cα), away from the catalytic pocket. Interestingly, the bound conformations of both ligands on PDHP1 are particularly high energy rotamers with respect to the intersaccharide torsions, whereas PP2Cα selects relatively low energy conformations. The selection of high energy conformers by PDHP1 may be a reflection of an intrinsically higher binding affinity with this enzyme. Such conformational variations in bound glycans are influenced by both glycan structure and receptor architecture and have previously been noted.^11,23,24 C-INS-2-OH was inactive with both phosphatases and its inactivity is consistent with the absence of an appropriately placed amino group that contributes to pulling Asp 243 or Glu 351 toward the allosteric site. (Fig 5A and B). However, it is also possible that the 2’-amino group may function to chelate Mg²⁺ or Mn²⁺ in conjunction with an OH on the pinitol. This provides an alternative possible mechanism for the inactivity of C-INS-2-OH.

It is of interest that INS-2 alone activates PP2Cα, but requires addition of Mn²⁺ for activating PDHP1. PP2Cα is purified and crystallized in the presence of Mn²⁺.²² Two binding sites in a bimetallic center are well established. In contrast, PDHP1catalytic subunit is isolated in the presence of Mg²⁺.²¹ A Mg²⁺ bimetallic center has been established. However, the PDHP1 catalytic subunit is known to interact with its regulatory subunit, which also has a low affinity Mg²⁺ binding site that is inhibitory. We propose that in the case of PP2Cα, sufficient Mn²⁺ is present bound to the bimetallic center of the enzyme to chelate to added INS-2 to activate the enzyme. In contrast, INS-2 and C-INS-2 need addition of Mn²⁺ to activate PDHP1 and the effect is possibly to displace loosely bound Mg²⁺ in the regulatory subunit.²¹

6. Conclusion

The relative activity of the inositol glycan INS-2 and its C-glycoside analog C-INS-2 on PP2Cα and PDHP, two enzymes that are implicated in insulin modulation was evaluated. Both glycans activated PDHP similarly, but PP2Cα was only activated by INS-2. This result suggests that the inositol-glycan binding sites on the two enzymes are distinct. Preliminary modeling experiments appear to support this conclusion. What possible benefit may be derived from these results? Since PDHP is a selective activator of mitochondrial pyruvate oxidation, C-INS-2, in contrast to INS-2, would selectively activate one of two pathways of glucose disposal, glucose oxidation, bypassing the other pathway, glycogen synthesis. Such a selective tool would be useful in investigating insulin and insulin-mimetic agents for partitioning of intracellular carbon disposal between oxidation and glycogen synthesis. Presently this may be done with inhibitors of oxidation or of glycogen synthesis, which tend to be nonspecific. In terms of a potential therapeutic agent, selective glucose disposal via oxidation without glycogen storage may be beneficial against insulin resistance and diabetes for disposal of carbon overload. For future studies, in the context of the advancement of analogues of INS-2 as potential clinical agents, it will be of interest to determine whether C-INS-2 does escape intestinal breakdown and manifests insulin-mimetic effects in vivo.

7. Experimental

7.1 General – Synthesis

Unless otherwise stated, all reactions were carried out under a nitrogen atmosphere in oven-dried glassware using standard syringe and septa technique. ¹H and ¹³C NMR spectra were obtained on a Varian Unity Plus 500 (500 MHz) spectrometer. Chemical shifts are relative to the deuterated solvent peak or the tetramethylsilane (TMS) peak at (δ 0.00) and are in parts per million (ppm). Assignments for selected nuclei were determined from ¹H COSY experiments. Unless otherwise stated, thin layer chromatography (TLC) was done on 0.25 mm thick precoated silica gel HF₂₅₄ aluminum sheets. Chromatograms were observed under UV (short and long wavelength) light, and were visualized by heating plates that were dipped in a solution of ammonium (VI) molybdate tetrahydrate (12.5 g) and cerium (IV) sulfate tetrahydrate (5.0 g) in 10% aqueous sulphuric acid (500 mL). Flash column chromatography (FCC) was performed using silica gel 60 (230–400 mesh) and employed a stepwise solvent polarity gradient, correlated with TLC mobility.

7.2. 1,2,5,6-di-O-isopropylidene-3-O-methyl-4-O-phenylcarbonothioyl-D-chiro-inositol 5

Phenyl chlorothionoformate (8.75 mL, 64.7 mmol) was added dropwise to a suspension of alcohol 4 (8.05 g, 29.4 mmol) and DMAP (0.72 g, 5.87 mmol), in dry toluene (120 mL) at rt. Pyridine (12.0 mL, 147 mmol) was then added and the resulting suspension was stirred for 2h at rt. The reaction mixture was then washed with HCl (0.1M) and saturated NaHCO₃, and the organic extracts were dried (Na₂SO₄) and concentrated in vacuo. FCC of the residue gave 5 (11.5 g, 95%). R_f = 0.43 (10% ethyl acetate: petroleum ether). ¹H NMR (CDCl₃) ™ 2.40 (s, 6H, 2 × CH₃-C), 2.60 (two s, 6H, 2 × CH₃-C), 3.40 (dd, 1H, J = 7.7, 10.9 Hz, H-3), 3.58 (s, 3H, CH₃O), 4.39 (t, 1H, J = 7.7 Hz, H-2), 4.48–4.51 (m, 3H. H-1, H-5, H-6), 5.60 (dd, 1H, J = 7.1, 10.9 Hz, H-4), 7.16 (d, 2H, J = 8.4 Hz, Ar-H), 7.31 (t, 1H, J = 7.4 Hz, Ar-H), 7.44 (t, 2H, J = 7.9 Hz, Ar-H); ¹³C NMR (CDCl₃) ™ 25.4 (CH₃-C), 25.5 (CH₃-C), 27.5 (CH₃-C), 27.8 (CH₃-C), 60.2 (CH₃O), 75.4, 75.5, 76.0, 78.4, 80.4, 82.5, 109.5 (OCO), 110.0(OCO), 122.0 (Ar), 126.6 (Ar), 129.5 (Ar), 153.6 (Ar), 195.0 (C=S). HRMS (ESI) m/z calcd for C₂₀H₂₇O₇S (M + H)⁺ 411.1472, found 411.1471.

7.3 4-Deoxy-4-C-(3’-propenyl)-1,2,5,6-di-O-isopropylidene-3-O-methyl-D-chiro-inositol 6

A solution of 5 (4.17 g, 10.2 mmol), allyltributyltin (9.35 mL, 30.6 mmol), AIBN (0.33 g, 2.04 mmol) and toluene (18 mL) was heated at reflux for 4 h at which point TLC showed complete consumption of the starting material. The solution was concentrated in vacuo. FCC of the residue afforded 6 (1.70 g, 56%). R_f = 0.64 (10% ethyl acetate: petroleum ether). ¹H NMR (5% CDCl₃/C₆D₆) ™ 1.40 (two s, 6H, 2 × CH₃-C), 1.51 (two s, 6H, 2 × CH₃-C), 1.79–1.87 (m, 1H, H-4), 2.54–6.67 (m, 2H, CH₂-3’), 3.02 (dd, 1H, J = 6.8, J = 12.4 Hz, H-3), 3.54 (s, 3H, CH₃O), 4.06 (dd, 1H, J = 6.9, 9.2 Hz, H-5), 4.15 (t, 1H, J = 6.3 Hz, H-6), 4.21 (t, 1H, J = 7.1 Hz, H-2), 4.30 (t, 1H, J = 6.6 Hz, H-1), 5.20 (d, 1H, J = 10.1 Hz, ½ × =CH₂), 5.27 (d, 1H, J = 17.1 Hz, ½ × =CH₂); 6.10 (m, 1H, -CH=) ¹³C NMR (5% CDCl₃/C₆D₆) ™ 25.1 (CH₃-C), 25.2 (CH₃-C), 27.8 (CH₃-C), 27.8 (CH₃-C), 32.7 (C-3’), 41.5 (C-4), 58.4 (CH₃O), 75.9, 77.2, 77.8, 79.2, 81.1, 108.7 (OCO), 108.9 (OCO), 117.3 (=CH₂), 135.2 (-CH=).

7.4 4-Deoxy-4-C-(2’-ethanoic acid)-1,2,5,6-di-O-isopropylidene-3-O-methyl-D-chiro-inositol 7

Alkene 6 (1.35 g, 4.55 mmol) was dissolved in a 5/1 mixture of CH₂Cl₂/MeOH (24 mL). The solution was cooled to −78 °C and treated with a stream of O₃ in O₂ until TLC indicated complete disappearance of the starting material. The reaction was then purged with nitrogen, and Ph₃P (2.64 g, 9.09 mmol) was added. The mixture was warmed to rt, stirred for 1 h at this temperature, and concentrated under reduced pressure. FCC of the residue gave the derived aldehyde (1.15 g, 85%). R_f = 0.16 (10 % ethyl acetate: petroleum ether). ¹H NMR (CDCl₃) ™ 1.30 (two s, 6H, 2 × CH₃-C), 1.51 (two s, 6H, 2 × CH₃-C), 2.09–2.18 (m, 1H, H-4), 2.48 (m, 1H, H-2’), 2.23 (dd, 1H, J = 4.7, 16.2 Hz, H-2’), 3.15 (dd, 1H, J = 6.7, 12.1 Hz, H-3), 3.45 (s, 3H, CH₃O), 4.04 (dd, 1H, J = 6.5, 9.7 Hz, H-5), 4.21 (m, 2H, H- 2, 6), 4.36 (dd, 1H, J = 4.7, 6.7 Hz, H-1), 9.65 (d, 1H, J = 2.1 Hz, CH=O); ¹³C NMR (CDCl₃) ™ 25.3 (CH₃-C), 25.4 (CH₃-C), 27.8 (2 × CH₃-C), 39.1 (CH₂), 43.9 (C-4), 59.1 (CH₃O), 76.4, 76.8, 76.9, 80.2, 80.5, 109.3 (2 × OCO), 200.9 (C=O). HRMS (ESI) m/z calcd for C₁₅H₂₄NaO₆ (M + Na)⁺ 323.1468, found 323.1468.

To a solution of aldehyde from the previous step (1.4 g, 4.67 mmol) in a 5:1 mixture of CH₃CN:H₂O (85:17mL) at 0 °C, was added NaH₂PO₄ 3.H₂O (6.46 g, 46.8 mmol), and a solution of NaClO₂ (0.51 g, 5.62 mmol) in H₂O (85 mL) and H₂O₂ (0.55 mL). The mixture was stirred at 0 °C until TLC indicated the disappearance of the starting material. Solid Na₂SO₃ was then added to the reaction. The reaction mixture was diluted with water and the organic phase was extracted with ether. The combined extract was dried (Na₂SO₄), the solvent removed under reduced pressure, and the residue purified by FCC to give 7 (1.10 g, 75%). R_f = 0.16 (25% ethyl acetate: petroleum ether). ¹H NMR (CDCl₃) ™ 1.35 (s, 3H, CH₃-C), 1.38, (s, 3H, CH₃-C), 1.45 (s, 3H, CH₃-C), 1.53 (s, 3H, CH₃-C), 2.05 (m, 1H, H-4), 2.52 (dd, 1H, J = 7.0, 15.7 Hz, H-2’), 2.71 (dd, 1H, J = 4.3, 15.7 Hz, H-2’), 3.16 (dd, 1H, J = 7.3, 12.2 Hz, H-3), 3.54 (s, 3H, CH₃O), 4.14 (dd, 1H, J = 6.4, 9.9 Hz, H-5), 4.25 (m, 2H, H- 2, 6), 4.38 (dd, 1H, J = 4.7, 6.7 Hz, H-1); ¹³C NMR (CDCl₃) ™ 25.4 (2 × CH₃-C), 27.7 (CH₃-C), 27.8 (CH₃-C), 33.5 (CH₂CO), 39.7 (C-4), 59.5 (CH₃O), 76.4, 76.5, 76.7, 80.3, 80.4, 109.3 (2 × OCO), 176.9 (C=O).

7.5 Monothioacetal Ester 9

DCC (0.50 g, 2.42 mmol) was added at 0 °C to a mixture of acid 7 (0.64 g, 2.02 mmol), alcohol 8 (1.23 g, 2.42 mmol), and DMAP (74.0 mg, 0.61 mmol) in dry dichloromethane (10 mL). The reaction mixture was warmed to rt and stirred for 6 h. The mixture was then diluted with ether and filtered. The filtrate was successively washed with 0.1 N aqueous HCl and brine, dried (Na₂SO₄), filtered, and evaporated in vacuo. FCC of the residue gave 9 (1.45 g, 89%) as a colorless oil. R_f = 0.38 (10% ethyl acetate: petroleum ether). ¹H NMR (CDCl₃) ™ 1.05 (s, 9H, (CH₃)₃C), 1.26 (s, 3H, CH₃-C), 1.27 (s, 3H, CH₃-C), 1.28 (s, 3H, CH₃-C), 1.56 (two s, 6H, 2 × CH₃-C), 2.13 (m, 1H, H-4), 2.34 (dd, 1H, J = 7.9, 16.5 Hz, ½ × CH₂C=O), 2.69 (dd, 1H, J = 3.8, 15.4 Hz, ½ × CH₂C=O), 3.06 (dd, 1H, J = 7.1, 12.2 Hz, H-3), 3.20 (s, 3H, CH₃O), 3.80 (dd, 1H, J = 7.3, 10.0 Hz, ½ × CH₂O), 3.85 (dd, 1H, J = 6.0, 9.9 Hz, ½ × CH₂O), 4.17 (dd, 1H, J = 6.6, 9.8 Hz, H-5), 4.19 (m, 2H, H-2, 6), 4.29 (t, J = 7.0 Hz, H-1), 4.42 (dd, 1H, J = 2.4, 7.7 Hz, H-2’), 5.25 (m, H, H-3’), 5.50 (d, 1H, J = 6.8 Hz, H-1’), 7.24–7.34 (m, 3H, ArH), 7.37–7.49 (m, 6H, ArH), 7.56 (m, 2H, ArH), 7.67–7.74 (m, 4H, ArH); ¹³C NMR (CDCl₃) ™ 20.0 (Me₃C-Si), 25.3 (CH₃-C), 25.5 (CH₃-C), 26.2 (CH₃-C), 26.7 ((CH₃)₃C), 27.3 (CH₃-C), 27.7 (CH₃-C), 27.9 (CH₃-C), 34.5 (CH₂CO), 39.9 (C-4), 59.0 (CH₃O), 62.0 (CH₂O), 70.8, 76.5, 76.6, 78.9, 80.4, 80.5, 84.4 (OCS), 109.3 (2 × OCO), 111.5 (OCO), 127.5, 127.8, 127.9, 128.9, 129.7 (two signals), 132.3, 133.1 (two signals), 133.7, 135.6, 135.7, 171.4 (C=O). HRMS (ESI) m/z calcd for C₄₄H₅₈O₁₀SSiNa (M + Na)⁺ 829.3420, found 829.3423.

7.6 Monothioacetal enol ether 10

To a mixture of ester 9 (0.592 g, 0.735 mmol), and pyridine (0.3 mL) in anhydrous 3:1 toluene:THF (10:5 mL), was added under an argon atmosphere at −78 °C, the Tebbe reagent (3.7 mL, 0.5M in THF). The reaction mixture was warmed to rt and stirred at this temperature for 1 h. The mixture was then slowly poured into 1N aqueous NaOH at 0 °C, and the resulting suspension extracted with ether. The combined organic phase was washed with brine, dried (Na₂SO₄), filtered and concentrated in vacuo. FCC of the residue on basic alumina provided 10 (0.44g, 75%) as light yellow oil. R_f = 0.49 (basic alumina, 10% ethyl acetate: petroleum ether). ¹H NMR (CDCl₃) ™ 1.07 (s, 9H, (CH₃)₃C), 1.29 (s, 3H, CH₃-C), 1.35 (s, 6H, 2 × CH₃-C), 1.48 (s, 3H, 2 × CH₃-C), 1.50 (s, 3H, CH₃-C), 1.51 (s, 3H, CH₃-C), 1.86 (m, 1H, H-4), 2.31 (dd, 1H, J = 5.4, 14.4 Hz, ½ × CH₂C=), 2.45 (dd, 1H, J = 5.4, 14.4 Hz, ½ × CH₂C=), 3.14 (dd, 1H, J = 6.9, 12.5 Hz, H-3), 3.34 (s, 3H, CH₃O), 3.81 (d, 1H, J = 2.2 Hz, ½ × =CH₂), 3.85–3.93 (m, 3H, CH₂O, ½ × CH₂C=), 3.97 (t, 1H, J = 6.3 Hz, H-6), 4.10 (dd, 1H, J = 7.9, 8.9 Hz, H-5), 4.16 (m, 2H, H-1, 2), 4.25 (m, 1H, H-3’), 4.49 (dd, 1H, J = 3.2, 8.0 Hz, H-2’), 5.49 (d, 1H, J = 7.0 Hz, H-1’), 7.25–7.34 (m, 4H, ArH), 7.37–7.48 (m, 4H, ArH), 7.51–7.58 (m, 2H, ArH), 7.67–7.73 (m, 5H, ArH); ¹³C NMR (CDCl₃) ™ 19.1 (Me₃C-Si), 25.2 (CH₃-C), 25.3 (CH₃-C), 26.1 (CH₃-C), 26.7 ((CH₃)₃C), 27.4 (CH₃-C), 27.8 (2 × CH₃-C), 34.3 (CH₂C=), 39.3 (C-4), 58.6 (CH₃O), 61.1 (CH₂O), 74.2, 76.0, 76.9, 79.6, 79.7, 81.0, 83.7, 84.3, 109.3 (OCO), 111.3 (OCO), 112.0 (OCO), 127.3, 127.7, 127.8, 129.0, 129.7, 129.8, 131.8 (two signals), 133.0, 133.3, 134.1, 135.6, 159.4 (OC=CH₂). HRMS (ESI) m/z calcd for C₄₅H₆₀O₉NaSSi (M + Na)⁺ 827.3625, found 827.3625.

7.7. 4-Deoxy-4-C-(2,6-anhydro-7-O-tertbutyldiphenylsilyl-4,5-O-isopropylidene-1,3-dideoxy-D-galacto-hept-2-enit-1-C-yl)-1,2,5,6-di-O-isopropylidene-3-O-methyl-D-chiro-inositol 11

A mixture of enol ether 10 (5.22 g, 6.49 mmol), 2,6-di-tert-butyl-4-methylpyridine (20.0 g, 97.3 mmol), and freshly activated, powdered 4 Å molecular sieves (15.3 g) in anhydrous CH₂Cl₂ (200 mL), was stirred for 15 min at rt, under an atmosphere of argon, then cooled to 0 °C. Methyl triflate (9.50 mL, 84.4 mmol) was then introduced, and the mixture warmed to rt, and stirred for an additional 18 h, at which time, triethylamine (15 mL) was added. The mixture was diluted with ether, washed with saturated aqueous NaHCO₃ and brine, dried (Na₂SO₄), filtered and evaporated in vacuo. FCC of the residue provided recovered 10 (1.22 g) and 11 (2.66 g, 77% based on recovered 10) as a light yellow oil. R_f = 0.58 (basic alumina, 10 % ethyl acetate: petroleum ether). ¹H NMR (C₆D₆) ™ 1.29 (s, 9H, (CH₃)₃C), 1.30 (s, 3H, CH₃-C), 1.34 (s, 3H, CH₃-C), 1.50 (s, 3H, CH₃-C), 1.56 (s, 3H, CH₃-C), 1.66 (s, 3H, CH₃-C), 2.16 (m, 2H, H-4, ½ × CH₂C=), 2.89 (bd, 1H, J = 10.2 Hz, ½ × CH₂C=), 3.07 (dd, 1H, J = 6.9, 12.0 Hz, H-3), 3.56 (s, 3H, CH₃O), 4.10 (t, 1H, J = 6.9 Hz, H-5), 4.25, 4.33, 4.36 (m, m, t, J = 6.4 Hz, 3H, 2H, 1H resp. H-1, H-2, H-6, H-6’, CH₂-7’), 4.47 (d, 1H, J = 6.4 Hz, H-5’), 4.73 (dd, 1H, J = 2.5, 6.4 Hz, H-4’), 4.94 (d, 1H, J = 2.5 Hz, H-3’), 7.31–7.41 (m, 6H), 7.87 (m, 4H); ¹³C NMR (C₆D₆) ™ 19.3 (Me₃C-Si), 25.2 (CH₃-C), 25.4 (CH₃-C), 26.8 ((CH₃)₃C), 27.0 (CH₃-C), 27.8 (CH₃-C), 28.0 (CH₃-C), 28.4 (CH₃-C), 34.1 (CH₂C=), 39.8 (C-4), 58.5 (CH₃O), 63.8 (CH₂O), 70.1, 72.0, 75.9, 80.0, 81.1, 100.5 (C-3’), 108.5 (OCO), 108.8 (OCO), 110.0 (OCO), 128.5, 129.8, 133.6 (two signals), 135.8 (two signals), 157.1. HRMS (ESI) m/z calcd for C₃₉H₅₄O₉NaSi (M + Na)⁺ 717.3429, found 717.3422.

7.8 4-Deoxy-4-C-(2,6-anhydro-7-O-tertbutyldiphenylsilyl-4,5-O-isopropylidene-1-deoxy-D-galacto-D-glycero-heptit-1-C-yl)-1,2,5,6-di-O-isopropylidene-3-O-methyl-D-chiro-inositol 12

BH₃.Me₂S (1.1 mL, 1M solution, mmol) was added at 0 °C to a solution of glycal 11 (0.191 g, 0.275 mmol) in anhydrous THF (8 mL) under an atmosphere of argon. The mixture was warmed to rt and stirred for an additional 1 h. At that time the solution was recooled to 0 °C and treated with a mixture of 3N NaOH (2 mL) and 30% aqueous H₂O₂ (2 mL) for 30 min. The solution was then diluted with ether and washed with saturated aqueous NaHCO₃ and brine, dried (Na₂SO₄), filtered and evaporated under reduced pressure. FCC of the residue provided 12 (0.149 g, 76 %). R_f = 0.51 (25 % ethyl acetate: petroleum ether). ¹H NMR (CDCl₃) ™ 1.06 (s, 9H, (CH₃)₃C)), 1.32 (s, 3H, CH₃-C), 1.34 (s, 3H, CH₃-C), 1.40 (s, 3H, CH₃-C), 1.44 (s, 3H, CH₃-C), 1.51 (s, 3H, CH₃-C), 1.54 (s, 3H, CH₃-C), 1.75 (m, 1H, ½ × CH₂), 2.01 (m, 2H, H-4, ½ × CH₂), 2.42 (d, 1H, J = 3.4 Hz, OH), 3.01 (dd, 1H, J = 6.3, 12.2 Hz, H-3), 3.45 (m, 2H), 3.49 (s, 3H, CH₃O), 3.86 (m, 3H), 4.03 (t, 1H, J = 5.4 Hz, H-3’), 4.07 (m, 2H), 4.20 (m, 2H), 4.39 (dd, 1H, J = 1.8, 5.4 Hz, H-4’), 7.35–7.46 (m, 6H), 7.67–7.75 (m, 4H); ¹³C NMR (CDCl₃) ™ 19.2 (Me₃C-Si), 25.3 (CH₃-C), 25.4 (CH₃-C), 26.4 (CH₃-C), 26.7 ((CH₃)₃C),, 27.7 (CH₃-C), 27.8 (CH₃-C), 28.4 (CH₃-C), 32.7 (CH₂-1’), 37.5 (C-4), 58.7 (CH₃O), 62.7 (CH₂O), 73.8, 74.8, 75.5, 76.1, 77.2, 79.1, 80.1, 80.3 (two signals), 109.2 (OCO), 109.4 (OCO), 109.6 (OCO), 127.6, 127.7, 129.6, 133.2, 133.4, 135.6 (two signals). HRMS (ESI) m/z calcd for C₃₉H₅₆O₁₀NaSi (M + Na)⁺ 735.3534, found 735.3537.

7.9 C-INS-2-OH 3

To a solution of TBDPS protected alcohol 12 (0.065 g, 0.09 mmol) in dry THF (1 mL), was added TBAF (0.18 mL, 0.18 mmol) at rt. The reaction was stirred for 5 h at rt. The mixture was then diluted with saturated aqueous NaHCO₃ and extracted with ether. The combined organic phase was dried (Na₂SO₄) and concentrated in vacuo. FCC of the residue afforded the derived primary alcohol (0.041 g, 95%) as a colorless oil. The product (0.040 g, 0.09 mmol) was dissolved in dry methanol (3 mL). The pH of the solution was adjusted to 2–3 by addition of a 2M solution of HCl in anhydrous ether, and the mixture stirred for 18 h. The solution was then diluted with methanol and evaporated under reduced pressure. The dilution-evaporation procedure was repeated three times and the residue dried in vacuo to afford 3 (0.029 g, 97%) as a clear gum. R_f = 0.45 (50% methanol:ethyl acetate). ¹H NMR (D₂O) ™ 1.68–1.78 (m, 1H, H-1’a), 1.98 (m, 1H, H-4), 2.05 (m, 1H, H-1’b), 3.23 (t, 1H, J = 9.6 Hz, H-3), 3.33 (t, 1H, J = 9.5 Hz, H-3’), 3.43 (s, 3H, CH₃O), 3.45 (m, 1H, H-2’), 3.49–3.67 (m, 4H, H-6’, 4’, CH₂-7’), 3.75 (dd, 1H, J = 3.1, 9.6 Hz, H-2), 3.89–3.79 (m, 3H, H-1, 5, 5’), 3.89 (t, 1H, J = 3.3 Hz, H-6); ¹³C NMR (CDCl₃) δ 28.8 (C-1’), 37.9 (C-4), 58.5 (CH₃O), 61.4 (C-7’), 69.1 (C-1/5/5’), 69.9 (C-1/5/5’), 71.2 (C-2), 71.3 (C-3’), 71.8 (C-6), 72.0 (C-1/5/5’), 73.9 (C-4’), 77.8 (C-2’), 78.2 (C-6’), 79.9 (C-3). HRMS (ESI) m/z calcd for C₁₄H₂₆NaO₁₀ (M + Na)⁺ 377.1425, found 377.1425.

7.10 C-pseudodisaccharide ketone 13

To a mixture of PCC (0.401 g, 1.87 mmol), florisil (0.996 g), sodium acetate (0.153 g, 1.87 mmol), freshly activated, powdered 4Å molecular sieves (0.996 g) and Celite (0.996 g) in dry dichloromethane (15 mL), was added dropwise a solution of alcohol 12 (0.322 g, 0.466 mmol) in dry dichloromethane (5 mL). The reaction mixture was stirred at rt until TLC indicated total consumption of the starting material. The mixture was then diluted with ether and filtered through a column of florisil. The filtrate was evaporated in vacuo and the residue purified by FCC to afford ketone 13 (0.315 g, 95%) as colorless oil. R_f = 0.51 (25% ethyl acetate: petroleum ether). ¹H NMR (CDCl₃) ™ 1.07 (s, 9H, (CH₃)₃C)), 1.30 (s, 3H, CH₃-C), 1.35 (s, 3H, CH₃-C), 1.42 (s, 3H, CH₃-C), 1.45 (2s, 6H, 2 × CH₃-C), 1.51 (s, 3H, CH₃-C), 1.80 (ddd, 1H, J = 3.8, 10.5, 14.4 Hz, H-1a’), 1.90 (m, 1H, H-4), 2.21(ddd, 1H, J = 2.5, 9.2, 14.4 Hz, H-1’b), 3.01 (dd, 1H, J = 6.3, 12.1 Hz, H-3), 3.49 (s, 3H, CH₃O), 3.93 (m, 2H, CH₂-7’), 4.12 (m, 3H, H- 1, 5, 6’), 4.20 (m, 2H, H-2, 6), 4.26 (dd, 1H, J = 3.8, 9.2 Hz, H-2’), 4.47 (d, 1H, J = 5.8 Hz, H-4’), 4.77 (dd, 1H, J = 1.7, 5.8 Hz, H-5’), 7.32–7.56 (m, 6H, ArH), 7.67–7.99 (m, 4H, ArH); ¹³C NMR (CDCl₃) ™ 19.2 (Me₃C-Si), 25.3 (CH₃-C), 25.4 (CH₃-C), 26.1 (CH₃-C),, 26.8 ((CH₃)₃C), 27.2 (CH₃-C), 27.7 (CH₃-C), 27.9 (CH₃-C), 29.8 (C-1’), 38.3 (C-4), 58.9 (CH₃O), 62.5 (C-7’), 76.7, 77.1, 77.2, 77.5, 77.8, 79.0, 79.1, 80.0, 80.3, 109.2 (OCO), 109.4 (OCO), 110.8 (OCO), 127.7 (two signals), 128.3, 129.7, 133.2, 133.3, 135.5, 135.6, 204.6 (C=O). HRMS (ESI) m/z calcd for C₃₉H₅₄O₁₀SiNa (M + Na)⁺ 733.3393, found 733.3393.

7.11 Pseudodisaccharide oxime 14

To a solution of ketone 13 (0.312 g, 0.439 mmol) in a 1:1 mixture of THF/MeOH (5.5 mL), was added a mixture of O-methyl hydroxylamine hydrochloride (0.367 g, 4.39 mmol) and NaOAc (0.397 g, 4.82 mmol) in water (2.75 mL), which was adjusted to pH = 4.5 with a few drops of glacial acetic acid. The reaction mixture was stirred at rt for 4 h then diluted with EtOAc and washed with saturated aqueous NaHCO₃ and water, dried (Na₂SO₄), and filtered. The solvent was removed in vacuo to give 14 (0.280 g, 86%, 1.5:1 mixture) as a colorless oil. R_f = 0.60 (20 % ethyl acetate: petroleum ether). For major isomer: ¹H NMR (CDCl₃) ™ 1.07 (s, 9H, (CH₃)₃C), 1.32 (s, 3H, CH₃-C), 1.34 (s, 3H, CH₃-C), 1.36 (s, 3H, CH₃-C), 1.42 (s, 3H, CH₃-C), 1.49 (s, 3H, CH₃-C), 1.50 (s, 3H, CH₃-C), 1.83 (dd, 1H, J = 9.5, 14.0 Hz, H-1’a), 2.05 (m, 1H, H-4), 2.40 (ddd, J = 1.9, 10.6, 14.0 Hz, H-1’b), 3.12 (dd, 1H, J = 7.0, 12.5 Hz, H-3), 3.45 (dt, 1H, J = 1.7, 6.4 Hz, H-6’), 3.48 (s, 3H, CH₃O), 3.82 (m, 2H, CH₂-7’), 3.93 (s, 3H, CH₃O), 4.03 (t, 1H, J = 7.0 Hz, H-6), 4.15 (t, 1H, J = 7.0 Hz, H-1), 4.23 (t, 1H, J = 7.0 Hz, H-2), 4.35 (t, 1H, J =7.0 Hz, H-5), 4.53 (dd, 1H, J = 1.7, J = 7.8 Hz, H-5’), 4.78 (d, 1H, 7.8 Hz, H-4’), 5.09 (d, 1H, J = 9.5 Hz, H-2’), 7.36–7.48 (m, 6H), 7.68–7.75 (m, 4H); ¹³C NMR (CDCl₃) ™ 19.2 (Me₃C-Si), 25.3 (2 × CH₃-C), 25.4 (CH₃-C), 26.0 (CH₃-C), 26.8 ((CH₃)₃C), 27.8 (CH₃-C), 27.9 (CH₃-C), 34.0 (C-1’), 38.6 (C-4), 58.4 (CH₃O), 62.3 (CH₃ON), 62.5 (CH₂-7’), 72.5, 74.2, 75.7, 76.7, 77.1, 77.5, 78.9, 79.5, 109.2 (OCO), 109.5 (OCO), 111.2 (OCO), 127.6, 127.7, 133.4, 133.7, 135.7, 135.8, 156.1 (C=N). HRMS (ESI) m/z calcd for C₄₀H₅₇NO₁₀NaSi (M + Na) + 762.3643, found 762.3643.

7.12 C-Pseudodisaccharide methoxylamine 15

To a mixture of oxime 14 (0.092 g, 0.124 mmol), and Bu₃SnH (0.065 mL, 0.248 mmol) in CH₂Cl₂ (3 mL), was added at −20 °C, BF₃.OEt₂ (0.015 mL, 0.124 mmol). The mixture was maintained at this temperature for 2 h, then diluted with saturated aqueous NaHCO₃ and extracted with CH₂Cl₂. The combined organic phase was dried (Na₂SO₄) and concentrated in vacuo. FCC of the residue afforded 15 (0.075 g, 82%) as a colorless oil. R_f = 0.81 (25 % ethyl acetate: petroleum ether). ¹H NMR (CDCl₃) ™ 1.06 (s, 9H, (CH₃)₃C), 1.33 (s, 3H, CH₃-C), 1.35 (s, 3H, CH₃-C), 1.38 (s, 3H, CH₃-C), 1.46 (s, 3H, CH₃-C), 1.49 (s, 3H, CH₃-C), 1.51 (s, 3H, CH₃-C), 1.82 (ddd, 1H, J = 1.7, 10.0, 11.8 Hz, H-1’a), 2.05 (m, 2H, H-1’b, H-4), 2.59 (dd, 1H, J = 8.1, 9.7 Hz, H-3’), 3.05 (dd, 1H, J = 6.1, 12.3 Hz, H-3), 3.49 (s, 3H, CH₃O), 3.56 (s, 3H, CH₃O), 3.73 (dt, 1H, J = 1.7, 9.9 Hz, H-2’), 3.84 (m, 2H, CH₂-7’), 3.91 (dd, 1H, J = 7.5, 9.0 Hz, H-6’), 4.06 (t, 1H, J = 6.5 Hz, H-6), 4.13 (dd, 1H, J = 7.2, 8.6 Hz, H-5), 4.19 (m, 2H, H-1, 2), 4.40 (m, 2H, H-4’, 5’), 6.01 (bs, 1H, NH), 7.31–7.51 (m, 6H, ArH), 7.71–7.82 (m, 4H, ArH); ¹³C NMR (CDCl₃) ™ 19.2 (Me₃C-Si), 25.3 (2 × CH₃-C), 26.4 (CH₃-C), 26.8 ((CH₃)₃C),, 27.8 (CH₃-C), 27.9 (CH₃-C), 28.5 (CH₃-C),, 33.2 (C-1’), 37.8 (C-4), 58.5 (CH₃O), 62.3 (CH₃O), 63.0 (CH₂-O), 65.7 (CH-N), 72.9, 73.2, 73.7, 75.9, 77.4, 79.1, 79.9, 80.4, 109.2 (OCO), 109.3 (OCO), 109.4 (OCO), 127.6, 127.7, 129.6, 133.5, 133.6, 135.5, 135.6. HRMS (ESI) m/z calcd for C₄₀H₅₉NO₁₀SiNa (M + Na)+ 764.3688, found 764.3688.

7.13 C-INS-2-hydrochloride

To a solution of silylether 15 (0.033 g, 0.046 mmol) in dry THF (1 mL), was added TBAF (0.09 mL, 0.09 mmol) at rt. The reaction mixture was stirred for 5 h at rt then diluted with saturated aqueous NaHCO₃ and extracted with ether. The combined organic phase was dried (Na₂SO₄) and concentrated in vacuo. FCC of the residue afforded the primary alcohol derivative (0.022 g, 95%) as a colorless oil. R_f = 0.20 (50 % ethyl acetate: petroleum ether). ¹H NMR (CDCl₃) ™ 1.35 (s, 3H, CH₃-C), 1.36 (s, 3H, CH₃-C), 1.37 (s, 3H, CH₃-C), 1.50 (s, 3H, CH₃-C), 1.51 (s, 3H, CH₃-C), 1.53 (s, 3H, CH₃-C), 1.80 (dd, 1H, J = 3.9, 10.8, 13.9 Hz, H-1’a), 2.10 (m, 2H, H-1’b, H-4), 2.44 (dd, 1H, J = 1.9, 9.4 Hz, OH), 2.66 (t, 1H, J = 7.7 Hz, H-3’), 3.10 (dd, 1H, J = 6.5, 12.3 Hz, H-3), 4.06 (t, 1H, J = 7.1 Hz, H-6), 4.12 (t, 1H, J = 6.9 Hz, H-5), 4.17 (dd, 1H, J = 1.8, 5.5 Hz, H-1), 4.21 (t, 1H, J = 7.4 Hz, H-5’), 4.25 (t, 1H, J = 4.2 Hz, H-2), 4.39 (dd, 1H, J = 5.5, 8.2 Hz, H-4’), 5.97 (d, 1H, J = 1.3 Hz, NH); ¹³C NMR (CDCl₃) ™ 25.3 (CH₃-C), 25.5 (CH₃-C), 26.4 (CH₃-C), 27.8 (CH₃-C), 28.0 (CH₃-C),, 28.3 (CH₃-C), 33.4 (C-1’), 37.8 (C-4), 58.6 (CH₃O), 62.4 (CH₃O), 63.0 (CH₂-7’), 65.7 (CH-N), 69.8, 73.3 (two signals), 76.5, 77.4, 77.5, 78.3, 80.2, 80.5, 109.5 (OCO), 109.7 (OCO), 109.9 (OCO).

Liquid NH₃ was condensed into a solution of the product from the previous step (0.122 g, 0.242 mmol) in THF (4 mL) at −78 °C, under an atmosphere of argon. Sodium was then carefully added to the solution until a blue color persisted for several minutes. After slowly warming to rt, the reaction was quenched with solid NH₄Cl, and filtered. The filtrate was concentrated in vacuo. FCC of the residue afforded the amine derivative (0.090 g, 78%) as a colorless oil. R_f = 0.48 (25 % methanol:ethyl acetate). ¹H NMR (CDCl₃) ™ 1,30 (s, 3H), 1.35 (3, 6H), 1.47 (s, 3H), 1.50 (s, 6H), 2.05 (m, 2H), 2.70 (t, 1H, J = 8.8 Hz), 3.05 (dd, 1H, J = 6.4, 12.3 Hz), 3.23 (t, 1H, J = 9.2 Hz), 3.46 (s, 3H), 3.66 (m, 2H), 3.83 (m, 2H), 4.04 (t, 1H, J = 7.0 Hz), 4.05 (m, 2H), 4.15 (t, 1H, J = 7.4 Hz), 4.23 (t, 1H, J = 7.0 Hz); ¹³C NMR (CDCl₃) δ 25.2, 25.5, 26.5, 27.8, 28.0, 28.3, 32.9, 37.6, 56.7, 58.5, 62.9, 73.5, 76.1, 76.7, 76.8, 77.4, 77.5, 78.2, 80.0, 80.6, 80.7, 109.5, 109.7, 110.0.

To a solution of product from the previous step (52 mg, 0.11 mmol) in dry methanol (3 mL) was added a 2M solution of HCl in anhydrous ether to a pH of 2–3. The reaction mixture was stirred for 18 h. The mixture was then diluted with methanol and evaporated under reduced pressure. The dilution-evaporation procedure was repeated three times and the residue dried in vacuo to give 2 as the hydrochloride salt (38 mg, 98%). R_f = 0.67 (4:4:1:2 of 2-propanol: pyridine: acetic acid: water). ¹H NMR (500 MHz, D₂O) ™ 1.77 (ddd, 1H, J = 3.0, 7.5, 14.8 Hz, H-1’a), 1.85 (ddd, 1H, J = 1.8, 9.9, 14.8 Hz, H-1’b), 2.05 (m, 1H, H-4), 3.05 (t, 1H, J = 10.3 Hz, H-3’), 3.52 (t, 1H, J = 3.2 Hz, H-3), 3.40 (s, 3H, CH₃O), 3.57 (dd, 1H, J = 3.9, 8.1 Hz, H-6’), 3.61 (dd, 1H, J = 3.9, J =11.8 Hz, H-7’), 3.69 (dd, 1H, J = 8.1, 11.8 Hz, H-7’), 3.75 (dd, 1H, J = 3.0, 10.6 Hz, H-2’), 3.77 – 3.81 (m, 4H, H-1, 2, 5, 6), 3.87 (d, 1H, J = 3.5 Hz, H-5’), 3.90 (t, 1H, J = 3.5 Hz, H-4’); ¹³C NMR (CDCl₃) ™ 30.9 (C-1’), 36.6 (C-4), 54.0 (CH-N), 57.5 (CH₃O), 61.3 (CH₂-7’), 68.0 (C-5’), 70.3 (C-1/2/5/6), 70.4 (C-2’), 71.2 (C-1/2/5/6), 71.9 (C-4’), 72.1 (C-1/2/5/6), 75.2 (C-1/2/5/6), 78.5 (C-6’), 79.8 (C-3). HRMS (ESI) m/z calcd for C₁₄H₂₈NO₉ (RNH₃)⁺ 354.1759, found 354.1762.

7.14. PP2Cα Assays

The mouse wild type GST-PP2Cα fusion protein was expressed and purified as previously described.⁴ Phosphatase activity was assayed at rt in 0.1 M Tris-HCl (pH 8.0), 2 mM dithiothreitol, and various concentrations of added MnCl₂, INS-2, C-INS-2-OH or C-INS-2, in a 96-well plate in 50 µL reaction mixtures with 1 µg of GST-PP2Cα fusion protein and 5 mM p-nitrophenyl phosphate (PNPP). After 30 min, the reaction was stopped by addition of 200 µL of 0.5% SDS and the absorbance at 410 nm recorded with an Eldex microplate reader. Alternatively, activity of 2 µg of GST-PP2Cα was assayed in the same volume of the same buffer with a synthetic phosphopeptide (R-R-R-R-P-pT-P-A) at a final concentration of 5 µM. After 15 min, the reaction was stopped by addition of 100 µL of a malachite green solution (Millipore). After color development for 15 min, the absorbance at 650 nm was determined with a microplate reader. Values were corrected by subtracting the absorbance of blanks that were reactions without added enzyme.

7.15. PDHP Assay

PDHP was assayed in triplicate samples on a 96 well plate.³ An ATP inhibition curve was first established to determine the 50% inhibition concentration. 10 µL of assay buffer (1mM imidazole pH 7, 2 mM DTT, 10 mM MgCl₂, 0.1mM CaCl₂, 105 mg/mL BSA) was added to each well. 10 µL of 0.5 to 5 mM ATP is added and the volumes all adjusted with aliquots of distilled water (DW). 10 µL PDHP is added and the plate gently shaken. 10 µL PDH was added, the plate gently shaken and incubated at 37 °C for 30 minutes. 10 µL of 110 mM NaF was added and the plate gently shaken. 10 µL pyruvate-ADP (30 mM sodium pyruvate, 30 mM ADP) was added with gentle shaking. 45 µL NAD mix (83 mM imidazole pH 7, 83 mM ®-NAD, 1.65 mM cocarboxylase, 3.3 mM DTT, 3.3 mM CoA) was added with gentle shaking and incubated at rt for 2 min. The plate was read for change in O.D. at 340 nM. Activity was expressed as % of basal, e.g. ATP concentration for 50% inhibition.

Supplementary Material

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Acknowledgment

This investigation was supported by grants R01 GM57865 from the National Institute of General Medical Sciences of the National Institutes of Health (NIH). "Research Centers in Minority Institutions" award RR-03037 from the National Center for Research Resources of the NIH, which supports the infrastructure and instrumentation of the Chemistry Department at Hunter College, is also acknowledged.

Footnotes

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References

1. Larner J, Galasko G, Cheng K, DePaoli-Roach A, Huang L, Daggy P, Kellogg J. Science. 1979;206:1408–1410. [PubMed]

2. Sleight S, Wilson B, Heimark D, Larner J. Biochem Biophys Res Commun. 2002;295:561–569. [PubMed]

3. Larner J, Price JD, Heimark D, Smith L, Rule G, Piccariello T, Fonteles MC, Pontes C, Vale D, Huang L. J Med Chem. 2003;46:3283–3291. [PubMed]

4. Brautigan D, Brown M, Grindrod S, Chinigo G, Kruszewski A, Lukasik S, Bushweller J, Horal M, Keller S, Tamura S, Heimark D, Price J, Larner A, Larner J. Biochemistry. 2005;44:11067–11073. [PubMed]

5. Hiraga A, Kikuchi K, Tamura S, Tsuiki S. Eur J Biochem. 1981;119:503–510. [PubMed]

6. Villar-Palasi C, Larner J. Biochim Biophys Acta. 1960;39:171–173. [PubMed]

7. Linn T, Pettit F, Reed L. Proc Natl Acad Sci U S A. 1969;62:234–241. [PubMed]

8. (a) For C-glycoside reviews: Postema MHD C-Glycoside Synthesis. Boca Raton, Fl: CRC Press; 1995. Levy DE, Tang C The Synthesis of C-Glycosides. Oxford: Pergamon; 1995. Beau J-M, Gallagher T Top. Curr. Chem. 1997;187:1–54. Togo H, He W, Waki Y, Yokoyama M Synlett. 1998:700–717. Du Y, Linhardt RJ, Vlahov JR Tetrahedron. 1998;54:9913–9959. Yuan X, Linhardt RJ Curr. Top. Med. Chem. 2005;5:1393–1430. [PubMed] Levy DE In: The Organic Chemistry of Sugars. Levy DE, Fügedi P, editors. Boca Raton: CRC Press / Taylor & Francis; 2005. pp. 269–348.

9. Jiménez-Barbero J, Espinosa JF, Asensio JL, Cañada FJ, Poveda A. Adv. Carbohydr. Chem. Biochem. 2000;56:235–284. [PubMed]

10. For examples of relative activity of O-glycosides and their exact C-glycoside analogues: Acton EM, Ryan KJ, Tracy M, Arora SK Tetrahedron Letters. 1986;27:4245–4248. Wei A, Boy KM, Kishi Y J. Am. Chem. Soc. 1995;117:9432–9436. Uchiyama T, Vassilev VP, Kajimoto T, Wong W, Huang H, Lin C-C, Wong C-H J. Am. Chem. Soc. 1995;117:5395–5396. Xin Y-C, Zhang Y-M, Mallet J-M, Glaudemans CPJ, Sinaÿ P Eur. J. Org. Chem. 1999:471–476. Link JT, Sorensen BK Tetrahedron Lett. 2000;41:9213–9217. Yang G, Schmieg J, Tsuji M, Franck RW Angew. Chem. Int. Ed. 2004;43:3818–3822. [PubMed] Postema MH, Piper JL, Betts RL, Valeriote FA, Pietraszkewicz H J Org Chem. 2005;70:829–836. [PubMed]

11. For an example where O- and C- glycosides bind in different conformations with respect to the intersaccharide torsions: Espinosa JF, Montero E, Vian A, Garcia JL, Dietrich H, Schmidt RR, Martín-Lomas M, Imberty A, Cañada FJ, Jimenéz-Barbero J J. Am. Chem. Soc. 1998;120:1309–1318.

12. (a) Gabius HJ. Pharm Res. 1998;15:23–30. [PubMed](b) Dam TK, Brewer CF. Chem. Rev. 2002;102:387–429. [PubMed]

13. Khan N, Cheng X, Mootoo DR. J. Am. Chem. Soc. 1999;121:4918–4919.

14. Keck GE, Enholm EJ, Yates JB, Wiley MR. Tetrahedron. 1985;41:4079–4094.

15. Zunszain PA, Varela O. Tetrahedron: Asym. 1998;9:1269–1276.

16. Afarinkia K, Bahar A. Tetrahedron Asym. 2005;16:1239–1287.

17. Ueda M, Miyabe H, Namba M, Nakabayashi T, Naito T. Tetrahedron Lett. 2002;43:4369–4371.

18. Rarey M, Kramer B, Lengauer T, Klebe GA. J. Mol. Biol. 1996;261:470–489. [PubMed]

19. For definition of scoring functions see ref. ⁴.

20. For Φ: O5-C1-X-C4’, Ψ:C1-X-C4’-H4’ where X the glycosidic oxygen or “CH₂”

21. Vassylyev D, Symersky J. J Mol Biol. 2007;370:417–426. [PMC free article] [PubMed]

22. Das A, Helps N, Cohen P, Barford D. EMBO J. 1996;15:6798–6809. [PubMed]

23. Mikkelsen LM, Hernáiz MJ, Martín-Pastor M, Skrydstrup T, Jiménez-Barbero J. J. Am. Chem. Soc. 2002;124:14940–14951. [PubMed]

24. García-Herrero A, Montero E, Muñoz JL, Espinosa JF, Vián A, García JL, Asensio JL, Cañada FJ, Jiménez-Barbero J. J. Am. Chem. Soc. 2002;124:4804–4810. [PubMed]