Crystal and molecular structure of methyl L-glycero-α-D-manno-heptopyranoside, and synthesis of 1→7 linked L-glycero-D-manno-heptobiose and its methyl α-glycoside.

Methyl l-glycero-α-d-manno-heptopyranoside was synthesized in good yield by a Fischer-type glycosylation of the heptopyranose with methanol in the presence of cation-exchange resin under reflux and microwave conditions, respectively. The compound crystallized from 2-propanol in an orthorhombic lattice of space group P2(1)2(1)2 showing a comparatively porous structure with a 2-dimensional O-H⋯O hydrogen bond network. As model compounds for the side chain domains of the inner core structure of bacterial lipopolysaccharide, l-glycero-α-d-manno-heptopyranosyl-(1→7)-l-glycero-d-manno-heptopyranose and the corresponding disaccharide methyl α-glycoside were prepared. The former compound was generated via glycosylation of a benzyl 5,6-dideoxy-hept-5-enofuranoside intermediate followed by catalytic osmylation and deprotection. The latter disaccharide was efficiently synthesized in good yield by a straightforward coupling of an acetylated N-phenyltrifluoroacetimidate heptopyranosyl donor to a methyl 2,3,4,6-tetra-O-acetyl heptopyranoside acceptor derivative followed by Zemplén deacetylation.

Introduction

Heptoses of the l- and d-glycero-d-manno configuration are important carbohydrate constituents of the lipopolysaccharide (LPS) of many Gram-negative bacteria. As an example, l,d-heptose is found in a common branched trisaccharide [l-α-d-Hepp-(1→7)]-l-α-d-Hepp-(1→3)-l-α-d-Hepp linked to position 5 of the lipid A-connected Kdo residue in the inner core region of LPS.

The heptosyl domain contributes to fundamental binding interactions of LPS with components of the innate and adaptive immune system. Recently, lectins, such as the human lung surfactant protein (SP-D) as well as the Burkholderia cenocepacia BC2L-C lectin have been found to bind to heptoses either via the side chain diol system or via the trans-diol part of the pyranose ring.2, 3 In order to generate simple model glycosides for crystallographic as well as STD-NMR and lectin binding studies we have set out to develop a straightforward preparation of mono- and disaccharide methyl heptopyranosides.

Results and discussion

Preparation of methyl glycosides of l-glycero-d-manno-heptose

Previously, the l,d-Hepp methyl α-glycoside 4 had been obtained by chain elongation at position 6 of methyl α-d-mannopyranoside or via reaction of related l,d-heptosyl thioglycoside, chloride as well as trichloroacetimidate donors with methanol. A central synthetic intermediate within the Brimacombe approach toward heptoses may be considered as a protected precursor for Fischer-type heptoside synthesis and could also serve as a suitable glycosyl acceptor for the synthesis of (1→7)-linked heptobiosides. Thus, benzyl (E)-5,6-dideoxy-2,3-O-isopropylidene-α-d-lyxo-hept-5-enofuranoside 1 was converted via catalytic osmylation into the triol derivative 2 according to the literature.5, 6 While the reported two-step procedure, comprising hydrogenolysis of the anomeric benzyl group followed by acidic cleavage of the isopropylidene group with H2SO4, had been applied for the preparation of the reducing l-glycero-d-manno heptose 3, monitoring of the hydrogenolysis reaction by thin layer chromatography (TLC) revealed a major highly polar fraction which had lost the isopropylidene group. Hence, a direct conversion of 2 into the methyl glycoside was envisaged. In a model study, reducing heptose 3 was subjected to treatment with Dowex 50 H+ cation-exchange resin and methanol at 60 °C, which in the course of several hours revealed the formation of four less polar products by TLC. Eventually, the composition of the mixture converged into a main component and a minor byproduct in 81% combined yield. 1H NMR analysis of the mixture identified the presence of methyl l-glycero-α-d-manno-heptopyranoside 4 as the main isomer (H-1: δ 4.565) and the corresponding β-isomer 6 (H-1: δ 4.32) as the minor constituent (α to β ratio 7:1). Reaction of 3 under microwave irradiation at 100 °C for 25 min furnished a 10:1 mixture of 4 and 6 containing 4% of the anomeric methyl heptofuranosides (H-1: δ 5.27 and δ 5.10) in a combined yield of 94%.

For ease of purification, the mixture was acetylated with pyridine–acetic anhydride–4-N,N-dimethylaminopyridine (DMAP) to furnish a separable mixture of the penta-O-acetyl derivatives 5 and 7 in 81% yield (Scheme 1). Following separation of the isomers, Zemplén deacetylation of 5 afforded known methyl l-glycero-α-d-manno-heptopyranoside 4 in 73% yield, whereas deacetylation of 7 gave methyl l-glycero-β-d-manno-heptopyranoside 6 in 91% yield, respectively. 1H NMR and 13C NMR spectroscopic data as well as the optical rotation values of 4 and 6 were in full agreement with the assigned anomeric configuration.

Scheme 1

Reagents and conditions: (a) H2, 10%Pd–C, MeOH, 20 h, rt; (b) MeOH, Dowex 50 H+, 60 °C, 12 h, 84% or MW, 100 °C, 25 min 94% for 4 and 6; (c) Ac2O, pyr., DMAP, 4 h, rt 81%; (d) 0.1 M NaOMe, MeOH, 3.5–5.5 h, rt, 73% for 4, 91% for 6, 99% for 11; (e) TMSOTf, CH2Cl2, molecular sieves 4 Å, 2 h, rt, then Ac2O, pyr, DMAP, 30 h, rt, 50% for 9; (f) NMO, OsO4, CH2Cl2, 3.5 h rt; 95% (46% for isolated 10); (g) H2, 10%Pd–C, MeOH, 28 h, rt, 38% for 13.

Compound 4 could also be directly produced from intermediate 2 as follows. Hydrogenolysis of 2 was performed in methanol with 10% Pd–C at atmospheric pressure for 20 h at room temperature. The catalyst was removed by filtration, cation-exchange resin (H+) was then added and the filtrate was refluxed for 15 h. Filtration of the suspension and concentration furnished the anomeric mixture of methyl glycosides in 84% yield, which crystallized from hot 2-propanol to give compound 2 containing traces of β-isomer.

Preparation of α-(1→7)-linked heptobiose and its methyl α-glycoside

Previously, the α-(1→7)-linked heptobiose has been synthesized by employing a benzyl 2,3,5,6-tetra-O-benzyl heptofuranoside acceptor. In addition, 7-silane derivatives as well as 7-O-allyl, 7-O-tBDPSi, and 7-O-tBDMSi heptopyranoside derivatives have served as suitable heptosyl precursors.10, 11, 12, 13, 14 The allylic alcohol derivative 1, however, may also be regarded as an implicitly protected heptose glycosyl acceptor. Previously a 7-O-phosphate derivative of d-glycero-d-manno-heptose has been synthesized via phosphorylation of 1 followed by osmylation and deprotection. Compound 1 was subjected to a glycosylation reaction with the known trichloroacetimidate donor 8 promoted by TMSO-triflate in dichloromethane. In order to avoid orthoester formation, the promoter had to be added in larger proportions (∼0.3 equiv), leading to partially deacetylated products. Subsequent reacetylation afforded the α-(1→7)-linked disaccharide 9 in 50% isolated yield (Scheme 1).

Catalytic osmylation in the presence of NMO produced a mixture of the diastereoisomeric diols in 97% yield, which was resolved by repeated HPLC-separation to eventually furnish pure l-glycero-d-manno-isomer 10 in 46% yield. Deprotection of disaccharide 10 was achieved by Zemplén deacetylation to afford 11 in 99% yield. Removal of the benzylic aglycon from 11 was accomplished by hydrogenolysis on 10% Pd/C which also led to partial cleavage of the isopropylidene ketal to produce a 3:2 mixture of the reducing heptobioses 12 and 13. Reductive removal of ketal groups in the presence of Pd(OH)2 has previously been reported. Attempted acid-catalyzed hydrolysis of the isopropylidene group from 12 was tested under various conditions. Treatment of 12 with 10% aq TFA in CH2Cl2 or aq THF and 15% TFA in toluene consistently resulted in concomitant hydrolysis of the interglycosidic linkage. Treatment of 12 or 2 with Lewis acids did not lead to cleavage of the acetonide (ZnCl2 in dichloromethane, FeCl3 in HOAc).18, 19 The isopropylidene group of 12 could also not be removed by microwave treatment under aqueous conditions. Thus target compound 13 was eventually isolated from the mixture obtained in the hydrogenolysis step. Precipitation of 13 from a methanolic solution containing the mixture of 12 and 13 with EtOAc followed by purification on BioGel P-2 afforded 13 in 38% yield. 1H and 13C NMR spectral data of 13 were in full agreement with published values of l-glycero-α-d-manno-heptopyranosyl-(1→7)-l-glycero-α-d-manno-heptopyranose. The values of the heteronuclear coupling constants were in agreement with the assigned anomeric configurations (JC-1′/H-1′ 174.2, JC-1/H-1α 174.1, JC-1/H-1β 167.8 Hz).

Finally, the formation of the α-(1→7)-linked methyl heptobiosides 14 under conditions as described for the monosaccharide 4 was evaluated in a trial experiment. Thus treatment of 12 with Dowex H+ resin in methanol at 50 °C for an extended period of 8 days indicated the presence of several components. Acetylation of the mixture and flash chromatography afforded the corresponding acetylated product amenable to NMR analysis. The 1H NMR spectrum of the mixture revealed the peracetylated methyl α-heptobioside as the main component (∼50%). In conclusion, the recalcitrant removal of the isopropylidene group at the disaccharide stage disfavors the approach.

As second approach a straightforward strategy toward the synthesis of methyl α-disaccharide was then elaborated starting from methyl glycoside 4, which was converted into the 7-O-tertbutyldimethylsilyl ether derivative 15 by reaction with tBDMSiCl/diazabicyclo[2.2.2]octane in MeCN in 86% yield (Scheme 2). Subsequent acetylation with acetic anhydride–pyridine–DMAP gave the tetra-O-acetyl derivative 16 (∼98%). Removal of the silyl group by the action of 2% HF in MeCN afforded the glycosyl acceptor 17 which was immediately subjected to the coupling reaction in order to prevent extensive acetyl migration from the neighboring 6-position. As glycosyl donors, the known trichloroacetimidate donor 8 and the novel N-phenyltrifluoroacetimidate heptosyl donor 19 were employed. The latter donor was obtained in near quantitative yield by the reaction of hemiacetal 18 with N-phenyltrifluoroacetimidoyl chloride/K2CO3 in acetone. The donor was isolated by silica gel chromatography and was present as an anomeric mixture (α/β ratio 8:1). Glycosylation of 17 with donor 19 proceeded at room temperature within 1.25 h and furnished the α-(1→7)-linked disaccharide 20, which was isolated in 51% yield following extensive purification by column chromatography. A comparative experiment utilizing the trichloroacetimidate donor 8 under analogous conditions led to a substantial amount of 6-linked disaccharide which was rather difficult to separate thus leading to very low yields of isolated pure disaccharide 20. Small amounts of (1→6) linked disaccharide were isolated by preparative HPLC to confirm its structure by 2D-NMR experiments.

Scheme 2

Reagents and conditions: (a) MeCN, DABCO, tBDMSiCl, 20 h, rt 86% for 15; (b) Ac2O, pyr., DMAP, 3 h, rt ∼quant.; (c) 2% HF, MeCN; (d) PhN = CCl(CF3), acetone, K2CO3, 3 h, rt, ∼quant. for 19; (e) TMSOTf, CH2Cl2, molecular sieves 4Å, 75 min, -10→0 °C, 65% (51% for isolated 20); (f) 0.1 M NaOMe, MeOH, 2 h, rt, 85% for 21.

Transesterification of 20 with sodium methoxide eventually gave the known disaccharide glycoside 21 in 85% yield after final purification on Bio-Gel P2.

Crystal structure of 4

Previously only the crystal structure of the β-anomeric hexa-O-acetyl derivative of l-glycero-d-manno-heptose has been reported. Compound 4 was crystallized from 2-propanol to give prismatic crystals. Using this material, the crystal structure was determined at T = 150 K as described in the experimental section. A view of the molecular structure is shown in Figure 1. Selected bond lengths and angles are given in Table 1. In the solid state, 4 adopts the usual chair conformation for the pyran ring with ring bond lengths of 1.522–1.530 Å for C–C, and 1.408, and 1.438 Å for C–O(5). The alternating positive and negative values of the ring torsion angles vary between absolute values of 53.6° and 56.7°. The methoxy oxygen O(1) and the hydroxyl oxygen O(2) are in axial positions to the ring and mutually trans-configured, O(1)–C(1)–C(2)–O(2) = 169.5°. The remaining ring substituents O(3), O(4), and C(6) adopt equatorial positions. The dihydroxyethyl side chain HO(6)–C(6)H–C(7)H2–O(7)H features an antiperiplanar conformation for the carbon chain moiety C(4)–C(5)–C(6)–C(7) (torsion angle −169.3°) and the internal torsion angle O(6)–C(6)–C(7)–O(7) is −64.2°. All five hydroxyl groups are involved in short intermolecular O–H⋯O hydrogen bonds with O⋯O distances of 2.658–2.789 Å and O–H⋯O angles between 149° and 175°. As visualized in Figure 2, these hydrogen bonds link the heptose molecules into double layers of molecules parallel to (0 0 1) with crystallographic C2-symmetry perpendicular to (0 0 1). These double layers are centered at z ≈ 0, 1, 2, etc., and are mutually held together only via Van der Waals forces in the region of z ≈ 1/2, 3/2, etc., where continuous open channels parallel to the a-axis do exist. The channels, which are centered at y,z = 0,0.56, ½,0.44, 1,0.56, etc., (Fig. 2), have cross sections of ca. 1.5 Å too narrow to incorporate solvent molecules, but they are responsible for a comparatively low X-ray density of the compound, 1.370 g cm−3 at T = 297 K. This becomes clearly evident by comparison with the isochemical methyl d-glycero-β-d-gulo-heptopyranoside, C8H16O7, which crystallizes in the monoclinic space group P21 with a 3-dimensional hydrogen bond network and has an X-ray density of 1.464 g cm−3, by 6.8% larger than for 4.

Figure 1

Molecular structure of 4 showing 50% ellipsoids.

Table 1

Selected bond lengths and angles for 4

Bond lengths (Å)
C(1)–C(2)	1.523(2)
C(2)–C(3)	1.526(2)
C(3)–C(4)	1.522(2)
C(4)–C(5)	1.530(2)
C(5)–C(6)	1.526(2)
C(6)–C(7)	1.522(2)
C(1)–O(1)	1.410(2)
C(1)–O(5)	1.408(2)
C(2)–O(2)	1.419(2)
C(3)–O(3)	1.429(2)
C(4)–O(4)	1.427(2)
C(5)–O(5)	1.438(2)
C(6)–O(6)	1.429(2)
C(7)–O(7)	1.424(2)
C(8)–O(1)	1.421(2)
Bond angles (°)
C(5)–O(5)–C(1)	114.63(11)
O(5)–C(1)–C(2)	111.80(12)
C(1)–C(2)–C(3)	110.46(12)
C(2)–C(3)–C(4)	110.30(12)
C(3)–C(4)–C(5)	109.99(12)
C(4)–C(5)–O(5)	111.47(11)
O(1)–C(1)–O(5)	112.58(13)
O(1)–C(1)–C(2)	106.58(13)
O(2)–C(2)–C(1)	106.28(12)
O(2)–C(2)–C(3)	111.82(12)
O(3)–C(3)–C(2)	108.54(11)
O(3)–C(3)–C(4)	110.59(13)
O(4)–C(4)–C(3)	110.41(12)
O(4)–C(4)–C(5)	106.66(11)
C(6)–C(5)–C(4)	112.65(12)
C(6)–C(5)–O(5)	105.48(11)
C(5)–C(6)–C(7)	110.88(13)
O(6)–C(6)–C(5)	109.72(12)
O(6)–C(6)–C(7)	111.49(13)
C(6)–C(7)–O(7)	112.99(15)
C(1)–O(1)–C(8)	112.80(15)
Torsion angles (°)
O(5)–C(1)–C(2)–C(3)	54.34(17)
C(1)–C(2)–C(3)–C(4)	−53.59(16)
C(2)–C(3)–C(4)–C(5)	53.58(16)
C(3)–C(4)–C(5)–O(5)	−54.14(15)
C(4)–C(5)–O(5)–C(1)	56.72(16)
C(5)–O(5)–C(1)–C(2)	−56.65(17)
O(1)–C(1)–C(2)–O(2)	169.45(12)
O(5)–C(1)–C(2)–O(2)	−67.14(16)
O(2)–C(2)–C(3)–O(3)	−56.74(16)
O(3)–C(3)–C(4)–O(4)	−68.91(15)
O(4)–C(4)–C(5)–O(5)	−173.88(11)
O(5)–C(5)–C(6)–O(6)	−54.70(15)
O(6)–C(6)–C(7)–O(7)	−64.23(16)
O(4)–C(4)–C(5)–C(6)	67.77(15)
C(4)–C(5)–C(6)–C(7)	−169.31(12)
C(4)–C(5)–C(6)–O(6)	67.11(16)
C(5)–C(6)–C(7)–O(7)	173.20(12)
Hydrogen bonds (Å) with symmetry codes of the acceptor atoms
O(2)→O(3) [−x+1,−y,z]	2.754(2)
O(3)→O(7) [x+1,y,z]	2.704(2)
O(4)→O(6) [x+½,−y+½,−z]	2.658(2)
O(6)→O(2) [−x,−y,z]	2.789(2)
O(7)→O(4) [−x,−y+1,z]	2.746(2)

Figure 2

Packing diagram of 4 viewed along the a-axis and showing two double layers of 2-dimensionally hydrogen bonded molecules extending parallel to (0 0 1) at z ≈ 0 and 1. Hydrogen bonds are indicated by red dashed lines, the channels parallel to a by green rectangles. Crystallographic C2 axes extend parallel to c at y = 0, ½, and 1.

Experimental

General

Concentration of solutions was performed at reduced pressure at temperatures <40 °C. Dichloromethane and dry pyridine were dried by refluxing with CaH2 (5 g per L) for 16 h, then distilled and stored under argon over molecular sieves 0.4 nm. Column chromatography was performed on Silica Gel 60 (230–400 mesh, Merck). Analytical TLC was performed using Silica Gel 60 F254 HPTLC plates with 2.5 cm concentration zone (Merck). Spots were detected by treatment with anisaldehyde—H2SO4. Size-exclusion chromatography was performed on Bio-Gel® P-2 Gel, Extra fine <45 μm (wet) from Bio-Rad Laboratories. Ion exchange treatment was performed with Dowex 50 W X 8 resin, H+ form, 50–100 mesh. Melting points were determined on a Kofler hot stage microscope and are uncorrected. Optical rotations were measured with a Perkin–Elmer 243 B polarimeter. NMR spectra were recorded at 297 K in D2O, CD3OD, or CDCl3 with Bruker DPX 300, Avance II 400 and Avance III 600 spectrometers (1H at 300.13 MHz, 13C at 75.47 MHz or 1H at 400.13 MHz, 13C at 100.61 MHz, 1H at 600.13 MHz, 13C at 150.9 MHz), respectively, using standard Bruker NMR software. 1H NMR spectra were referenced to tetramethylsilane (in CDCl3) or 2,2-dimethyl-2-silapentane-5-sulfonic acid (in D2O). 13C NMR spectra were referenced to chloroform for solutions in CDCl3 (δ 77.00) or dioxane (δ 67.40) for solutions in D2O. HRMS analysis was carried out from H2O/MeCN solutions (concentration 1 mg/L) using an HTC PAL system autosampler (CTC Analytics AG), an Agilent 1100/1200 HPLC with binary pumps, degasser, and column thermostat (Agilent Technologies, Waldbronn, Germany) and Agilent 6210 ESI-TOF mass spectrometer (Agilent Technologies, Palo Alto, US). The mass spectrometer was previously tuned with Agilent tune mix and further reference masses were added to the method to provide a mass accuracy below 2 ppm. Data analysis was performed with Mass Hunter software (Agilent Technologies). Elemental analyses were provided by Dr. J. Theiner, Mikroanalytisches Laboratorium, Institut für Physikalische Chemie, Universität Wien.

X-ray crystallographic study

Compound 4 was crystallized from 2-propanol to give excellent prismatic colorless crystals. X-ray data were collected with an oval of 0.51 × 0.43 × 0.35 mm on a Bruker Smart APEX CCD diffractometer using graphite-monochromated Mo-Kα radiation (λ = 0.71073 Å) and ω-scan frames covering a 3/4 sphere of the reciprocal space. After data integration with program SAINT, the structure was solved by direct methods and refined on F2 with the program package SHELX97. All non-hydrogen atoms were refined anisotropically. Hydrogen atoms were inserted in idealized positions and were treated as riding using the AFIX 83 restraint for OH hydrogen atoms. Crystal data are: C8H16O7, Mr = 224.21, orthorhombic, space group P21212 (no. 18), T = 150 K, a = 8.9740(3) Å, b = 9.9424(4) Å, c = 11.9632(4) Å, α = β = γ = 90°, V = 1067.39(7) Å3, Z = 4, δcalcd = 1.395 g/cm3, μ = 0.123 mm−1. Of 9379 reflections collected up to θmax = 30.0°, 1791 were independent, Rint = 0.0271, and 1636 were observed (I > 2σ(I)); final R indices: R1 = Σ||Fo| − |Fc||/Σ|Fo| = 0.044 (all data), wR2 = Σ(w(Fo2 − Fc2)2/Σ(w(Fo2)2) = 0.100 (all data). Unit cell dimensions and density of 4 at room temperature (297 K) are: a = 8.9929(1) Å, b = 9.9970(1) Å, c = 12.0940(2) Å, V = 1087.28 Å3, and δcalcd = 1.370 g cm−3. Atomic parameters and further details of the structure determination have been deposited. CCDC 881898 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

Methyl glycoside formation of l-glycero-d-manno-heptopyranose

Method A: A solution of 2 (3.31 g, 9.72 mmol) in dry methanol (80 mL) was stirred with 10% Pd–carbon (400 mg) under H2 at atmospheric pressure at room temperature for 20 h. The suspension was filtered over a bed of Celite and washed with methanol. The solution was concentrated to give a syrupy residue (2.27 g). A 1/3 aliquot (754 mg) of the debenzylated material was dissolved in dry methanol (50 mL), Dowex 50 H+ resin (4 g) was added and the suspension was stirred at 60 °C for 15 h. The resin was filtered off and the filtrate was concentrated to give a 10:1 mixture of 4 and 6 (603 mg, 84% based on 2).

Method B: A suspension of 3 (88 mg, 0.42 mmol) and DOWEX H+ resin (∼1 g) in dry methanol (5 mL) was stirred under reflux for 12 h. After this time, TLC (10:10:3 MeOH–CHCl3–H2O) indicated complete conversion of the starting material to a main product along with the presence of one by-product. Work-up as described for method A afforded a mixture of 4 and 6 (76 mg, 81%) as syrup.

Method C: A suspension of 3 (20 mg, 0.095 mmol) and DOWEX H+ resin (∼200 mg) in dry methanol (2 mL) was stirred in the MW-oven at 100 °C for 25 min. After this time, TLC (3:7:0.5 MeOH–CHCl3–H2O) indicated almost complete conversion of the starting material to a main product along with the presence of two minor by-products and no further progress toward target compound within the last 5 min. Work-up as described for method A afforded a 10:1 mixture of 4 and 6 (21 mg, 94%) as a white solid.

Acetylation of the anomeric heptopyranosides

A solution of 4 and 6 (76 mg) and a catalytic amount of DMAP in dry pyridine (5 mL) was stirred with Ac2O (0.5 mL) for 4 h at room temperature. Methanol (0.1 mL) was added and the solution was coevaporated with toluene (3 × 20 mL) and concentrated. Purification of the residue by column chromatography (3:2 toluene/EtOAc) afforded 5 as the major component (103 mg, 70%) followed by a fraction containing mainly compound 7 (8.5 mg, 6%) and pure compound 7 (7.5 mg, 5%). Data for 5: colorless syrup, +10 (c 0.75, CHCl3); lit.: +22 (c 1.0, CHCl3). 1H NMR (400 MHz, CDCl3): δ = 5.33–5.24 (m, 4H, H-2, H-3, H-4, H-6), 4.745 (d, 1H, J1,2 1.6 Hz, H-1), 4.31 (dd, 1H, J7a,6 5.8, J7a,7b 11.2 Hz, H-7a), 4.24 (dd, 1H, J6,7b 7.5 Hz, H-7b), 4.07–4.04 (m, 1H, H-5), 3.39 (s, 3H, OMe), 2.17 (s, 3H), 2.14 (s, 3H), 2.05 (s, 3H), 2.01 (s, 3H) and 1.98 (s, 3H, 5 × Ac). Data for 7: colorless syrup, −75 (c 0.75, CHCl3). 1H NMR (400 MHz, CDCl3): δ = 5.51 (dd, 1H, J1,2 1.1, J3,2 3.4 Hz, H-2), 5.31 (app. t, 1H, J3,4 10.2, J4,5 10.1 Hz, H-4), 5.31 (ddd, 1H, H-6), 5.06 (dd, 1H, H-3), 4.55 (d, 1H, H-1), 4.49 (dd, 1H, J7a,6 5.4, J7a,7b 11.4 Hz, H-7a), 4.20 (dd, 1H, J6,7b 7.6 Hz, H-7b), 3.69 (dd, 1H, J5,6 2.4 Hz, H-5), 3.54 (s, 3H, OMe), 2.22 (s, 3H), 2.14 (s, 3H), 2.07 (s, 3H), 2.03 (s, 3H) and 1.99 (s, 3H, 5 × Ac).

Methyl l-glycero-α-d-manno-heptopyranoside (4)

A solution of 5 (99.8 mg, 0.27 mmol) in dry MeOH (10 mL) was stirred with 0.1 M NaOMe solution (0.3 mL) at room temperature for 5.5 h. DOWEX H+ resin was added until pH 6–7 was achieved. The resin was removed and the filtrate was concentrated to afford 4. Crystallization of the residue from hot 2-propanol afforded 37.5 mg (73%) of 4, colorless plates, mp 133–134 °C (2-PrOH); +79.5 (c 0.5, MeOH), lit.: +70 (c 0.5, MeOH). 1H NMR (400 MHz, CD3OD): δ = 4.565 (d, 1H, J1,2 1.5 Hz, H-1), 3.88 (ddd, 1H, J5,6 1.2, J7b,6 6.5, J7a,6 7.4 Hz, H-6), 3.75 (app. t, 1H, J3,4 = J4,5 9.7 Hz, H-4), 3.68 (dd, 1H, J2,3 3.4 Hz, H-2), 3.595 (dd, 1H, J7a,7b 10.8 Hz, H-7a), 3.58 (dd, 1H, H-3), 3.54 (dd, 1H, H-7b), 3.44 (dd, 1H, H-5), 3.26 (s, 3H, OMe). 13C NMR (100 MHz, CD3OD): δ = 101.42 (C-1), 71.39 (C-3), 71.03 (C-5), 70.68 (C-2), 69.30 (C-6), 66.33 (C-4), 63.03 (C-7) and 53.91 (CH3O). Anal. Calcd for C8H16O7: C, 42.86; H, 7.19. Found: C, 42.84; H, 7.01.

Methyl l-glycero-β-d-manno-heptopyranoside (6)

A solution of 7 (10.1 mg, 0.027 mmol) in MeOH (7 mL) was treated with 0.1 M methanolic NaOMe solution (0.08 mL) and processed as described for the deacetylation of 5. Yield: 4.8 mg (91%), colorless syrup, −49 (c 0.5, CHCl3). 1H NMR (400 MHz, CD3OD): δ = 4.32 (d, 1H, J1,2 0.9 Hz, H-1), 3.84 (ddd, 1H, J5,6 1.6, J 6.9, J 8.5 Hz, H-6), 3.73 (dd, 1H, J2,3 3.2 Hz, H-2), 3.70 (app. t, 1H, J3,4 = J4,5 9.6 Hz, H-4), 3.56 (app. d, 2H, H-7a, H-7b), 3.40 (s, 3H, OMe), 3.35 (dd, 1H, H-3), 3.16 (dd, 1H, H-5), 13C NMR (100 MHz, CD3OD): δ = 101.43 (C-1), 74.69 (C-5), 74.08 (C-3), 71.01 (C-2), 69.33 (C-6), 66.20 (C-4), 62.77 (C-7) and 55.57 (CH3O). HR MS: m/z = 223.0834 [M−H]−; calcd 223.0739.

Benzyl 2,3,4,6,7-penta-O-acetyl-l-glycero-α-d-manno-heptopyranosyl-(1→7)-(E)-5,6-dideoxy-2,3-O-isopropylidene-α-d-lyxo-hept-5-enofuranoside (9)

A suspension of the glycosyl acceptor 1 (181 mg, 0.59 mmol) and trichloroacetimidate donor 8 (128 mg, 0.22 mmol) in freshly distilled dichloromethane (5 mL) was stirred with powdered mol sieves 4 Å at 0 °C for 3 h under Ar. TMSO-triflate (0.012 mL, 0.066 mmol) was added in three portions and the suspension was stirred for 2 h at rt. Et3N (60 μL) was added, the suspension was diluted with dichloromethane (20 mL), filtered over a bed of Celite®, and washed with dichloromethane (100 mL). The filtrate was concentrated and the residue was purified by column chromatography (toluene/EtOAc 5:1) to give 9 as colorless syrup (39.5 mg, 24.6%) as major compound and a partially deacetylated product (49.4 mg) as by-product. The partially deacetylated glycoside was taken up in pyridine (2 mL) and stirred with Ac2O (1 mL) for 30 h at rt. The solution was cooled to −10 °C and MeOH (3 mL) was added and the mixture was stirred for 15 min. The solution was co-evaporated with toluene (3 × 50 mL) and concentrated. Flash chromatography of the residue (toluene/EtOAc 5:1) gave a second crop of 9 which crystallized from n-hexane/EtOAc as colorless needles. Combined yield: 80 mg (50%); mp 114–115°; +35.6 (c 0.5, CHCl3). 1H NMR (300 MHz, CDCl3): δ = 7.34 (m, 5H, Ph), 5.91 (m, 2H, H-5, H-6), 5.36 (dd, 1H, J2′,3′ 3.2, J3′,4′ 9.7 Hz, H-3′), 5.34–5.26 (m, 3H, H-2′, H-4′, H-6′), 5.10 (s, 1H, H-1), 4.92 (d, 1H, J1′,2′ 1.5 Hz, H-1′), 4.70 and 4.50 (AB system, 2H, JAB 11.7 Hz, CHPh), 4.70 (dd, 1H, J2,3 3.2, J3,4 5.9 Hz, H-3), 4.50 (dd, 1H, J4,5 6.0, H-4), 4.30 (dd, 1H, J6′,7a′ 6.0, J7a′,7b′ 11.3 Hz, H-7a′), 4.25 (dd, J6′,7b′ 7.5 Hz, H-7b′), 4.23 (m, 1H, J7a,7b 12.7 Hz, H-7a), 4.13 (dd, J4′,5′ 9.3, J5′,6′ 2.0 Hz, H-5′), 4.06 (m, 1H, H-7b), 2.18 (s, 3H), 2.15 (s, 3H), 2.06 (s, 3H), 2.03 (s, 3H) and 1.99 (s, 3H, 5 × COCH), 1.46 (s. 3H) and 1.30 [s, 3H, C(CH3)2]. 13C NMR (75 MHz, CDCl3): δ = 170.43, 170.25, 170.07, 169.87, 169.65 (COCH3), 137.27 (Cq, Ph), 129.35 (CPh), 128.50 (CPh), 128.41 (C-5), 128.01 (CPh), 127.88 (C-6), 112.63 [C(CH3)2], 105.25 (C-1), 97.19 (C-1′), 85.38 (C-2), 81.27 (C-3), 80.00 (C-4), 69.58 (C-2′), 69.26 (C-3′), 68.99 (CH2Ph), 68.57 (C-5′), 67.97 (C-7), 66.96 (C-6′), 64.90 (C-4′), 61.82 (C-7′), 26.06, 24.84 [C(CH3)2], 21.00, 20.78, 20.76, 20.69 and 20.66 (COCH3). Anal. Calcd for C34H44O16: C, 57.62; H, 6.26. Found: C, 57.56; H, 5.79.

Benzyl 2,3,4,6,7-penta-O-acetyl-l-glycero-α-d-manno-heptopyranosyl-(1→7)-2,3-O-isopropylidene-l-glycero-α-d-manno-heptofuranoside (10)

N-Methylmorpholine-N-oxide (22 mg, 0.188 mmol) was added to a solution of 9 (46 mg, 0.065 mmol) in 8:1 acetone/water (20 mL). Upon complete dissolution a catalytic amount of OsO4 was added, the mixture turned slightly yellow and was stirred for 3.5 h at rt. The mixture was diluted with CHCl3 (80 mL) and was washed with 5 M HCl (10 mL). The organic phase was treated vigorously with 45% aq Na2S2O5 and dried (Na2SO4). Concentration gave a residue which was purified by column chromatography (toluene/EtOAc 1:1) to afford an 8:1 isomeric mixture of the l-glycero-d-manno- and d-glycero-l-gulo-diastereomer. Yield: 54 mg (95%). Separation of the isomers was achieved by repeated HPLC chromatography (toluene/EtOAc 1:1→1:2) to give 10 (28 mg, 46%) as the less mobile isomer (Rf 0.31; toluene/EtOAc 1:1) as a syrup; +40.5 (c 0.4, CHCl3). 1H NMR (400 MHz, CDCl3): δ = 7.38–7.15 (m, 5H, Ph), 5.33–5.27 (m, 4H, H-2′, H-3′, H-4′, H-6′), 5.11 (s, 1H, H-1), 4.93 (d, 1H, J1′,2′ ∼1.0 Hz, H-1′), 4.88 (dd, 1H, J2,3 6.0, J3,4 3.8 Hz, H-3), 4.67 (d, 1H, H-2), 4.65 and 4.53 (AB system, 2H, JAB 11.9 Hz, CHPh), 4.38 (dd, 1H, J6′,7a′ 5.4, J7a′,7b′ 11.4 Hz, H-7a′), 4.24 (dd, 1H, J6′,7b′ 7.6 Hz, H-7b′), 4.14 (m, 1H, H-5′), 4.11 (dd, 1H, J4,5 8.1, H-4), 3.96 (m, 2H, H-5, H-6), 3.80 (dd, 1H, J7a,6 5.0, J7a,7b 10.0 Hz, H-7a), 3.61 (dd, 1H, J7b,6 7.0 Hz, H-7b), 2.75 (br. signal, 1H, OH), 2.18 (s, 3H), 2.14 (s, 3H), 2.04 (s, 3H), 2.02 (s, 3H) and 1.98 (s, 3H, 5 × COCH), 1.47 (s, 3H) and 1.33 [s, 3H, C(CH3)2]. 13C NMR (100 MHz, CDCl3): δ = 170.54, 170.25, 170.01, 169.85 and 169.57 (COCH3), 137.48 (Cq-Ph), 128.48, 128.41, 127.89 (CPh), 112.68 [C(CH3)2], 105.55 (C-1), 98.36 (C-1′), 84.96 (C-2), 80.14 (C-3), 78.75 (C-4), 69.84 (C-7), 69.71 (C-2′), 69.40 (C-5, C-6), 69.36 (CH2Ph), 69.26 (C-3′), 68.76 (C-5′), 67.10 (C-6′), 64.82 (C-4′), 62.03 (C-7′), 25.95, 24.59 [C(CH3)2], 20.34, 20.72 (d.i.) and 20.64 (d.i., total 5C, COCH3). Anal. Calcd for C34H46O18: C, 54.98; H, 6.24. Found: C, 54.40; H, 6.08.

l-Glycero-α-d-manno-heptopyranosyl-(1→7)-benzyl 2,3-O-isopropylidene-l-glycero-α-d-manno-heptofuranoside (11)

A solution of 10 (36 mg, 0.049 mmol) in dry MeOH (1.5 mL) was stirred with 1 M methanolic NaOMe (100 μL) for 3.5 h at rt. Dowex 50 H+ ion exchange resin was then added to adjust the pH ∼6. The resin was filtered off and the filtrate was concentrated under reduced pressure giving 11 as colorless syrup. Yield: 26 mg (99%); +37.4 (c 0.5, MeOH). 1H NMR (400 MHz, CD3OD): δ = 7.39–7.26 (m, 5H, Ph), 5.02 (s, 1H, H-1), 4.89–4.85 (m, 2H, H-3, H-1′), 4.73 and 4.48 (AB system, 2H, JAB 11.8 Hz, CH2Ph), 4.64 (d, 1H, J2,3 5.8 Hz, H-2), 4.19 (dd, 1H, J4,5 9.2, J4,3 3.2 Hz, H-4), 4.04–3.95 (m, 3H, H-5, H-6, H-6′), 3.90–3.82 (m, 3H, H-4′, H-2′, H-7a), 3.74 (dd, 1H, J5′,6′ 3.4, J5′,4′ 9.8 Hz, H-5′), 3.71–3.66 (m, 3H, H-3′, H-7′a, H-7′b), 3.57 (dd, 1H, J7a,7b 9.5, J7b,6 6.8 Hz, H-7b), 1.44 (s, 3H) and 1.33 [s, 3H, C(CH)2]. 13C NMR (100 MHz, CD3OD): δ = 138.91 (Cq-Ph), 129.31, 129.26, 129.70 (CPh), 113.49 [C(CH3)2], 106.51 (C-1), 102.25 (C-1′), 86.28 (C-2), 81.26 (C-3), 79.92 (C-4), 72.84 (C-5′), 72.53 (C-3′), 72.13 (C-2′), 70.59 and 70.51 (C-5, C-6), 70.26 (C-7), 69.57 (CH2Ph), 69.14 (C-6′), 67.70 (C-4′), 64.19 (C-7′), 26.52 and 25.09 [C(CH3)2].

l-Glycero-α-d-manno-heptopyranosyl-(1→7)-l-glycero-d-manno-heptopyranose (13)

A suspension of heptobiose 11 (22 mg, 0.042 mmol) in dry MeOH (3 mL) was stirred with 10% Pd–C (∼50 mg) under hydrogen at atmospheric pressure for 28 h at room temperature. The catalyst was removed by filtration over a bed of Celite® and the filtrate was concentrated under reduced pressure. NMR analysis of the residue indicated the presence of 12 and 13 (3:2 ratio). The residue was dissolved in the minimal amount of MeOH and compound 13 was selectively precipitated by addition of EtOAc. The precipitate was separated by centrifugation, dissolved in water and lyophilized. The residue was purified over Bio-Rad Bio-Gel® P2 column (water, 5% EtOH) and lyophilized to give 13 as an amorphous solid. Yield: 6.4 mg (38%); +24 (c 0.3, H2O, equilibrium after 15 h); lit: +45.4 (c 1.0, H2O). 1H NMR (300 MHz, D2O): δ = 5.19 (d, 0.7H, J1α,2α 1.6 Hz, H-1α), 4.93 (m, 1H, H-1′α, H-1′β), 4.90 (d, 0.3H, J1β,2β 1.0 Hz, H-1β), 4.22–4.19 (m, 1H, H-6α, H-6β), 4.02–4.06 (m, 1H, H-6′α, H-6′β), 4.00 (br m, 1H, H-2′α, H-2′β), 3.96–3.92 (br m, 1H, H-2α, H-2β), 3.90–3.59 (m, 9.7H, H-3α, H-3′α, H-4α, H-4β, H-4′α, H-4′β, H-3′β, H-5α, H-7aα, H-7aβ, H-7′bα, H-7′bβ, H-3β, H-5′α, H-5′β, H-7bα, H-7bβ) and 3.35 (dd, 0.3H, J5β,6β 1.8, J4β,5β 8.3 Hz, H-5β). 13C NMR (100 MHz, D2O): δ = 101.04 (C-1′α), 100.94 (C-1′β), 94.81 (C-1α), 94.59 (C-1β), 75.35 (C-5β), 73.89 (C-3β), 72.11 (C-5′β), 71.93 (2C, C-2β, C-5′α), 71.73 (C-5α), 71.43 (2C, C-3α, C-2α), 71.16 (C-3′α), 70.59 (2C, C-2′α, C-2′β), 69.57 (C-6′β), 69.47 (C-6′α), 69.32 (C-7α), 68.97 (C-7β), 67.87 (C-6α), 67.70 (C-6β), 66.74 (2C, C-4α, C-4′α), 66.42 (2C, C-4β, C-4′β), 63.71 (C-7′β) and 63.57 (C-7′α). ESI-TOF MS: m/z = 403.1339 [M+H]; calcd 403.1452.

Methyl 7-O-tertbutyldimethylsilyl-l-glycero-α-d-manno-heptopyranoside (15)

Diazabicyclo[2.2.2]octane (86 mg, 0.77 mmol) was added at room temperature to a solution of 4 (0.15 g, 0.669 mmol) in dry acetonitrile (2.7 mL), followed by addition of tBDMS-chloride (111 mg, 0.736 mmol). After 2 h additional MeCN (2.5 mL) was added followed by addition of DABCO (53 mg, 0.468 mmol) and tBDMSCl (71 mg, 0.468 mmol) in two portions after 5 h and 7 h, respectively. The suspension was stirred overnight at room temperature. MeOH (5 mL) was added and stirring was continued for 1 h. The suspension was concentrated and the residue was applied on a column of silica gel and eluted with EtOAc/MeOH 100:15→100:25→MeOH. Appropriate fractions were pooled and concentrated to give 15 as a crystalline solid. Yield: 194 mg (86%); mp 166–167 °C (EtOAc), +59 (c 0.3, CHCl3). 1H NMR (400 MHz, CD3OD): δ = 4.65 (d, 1H, J1,2 1.6 Hz, H-1), 3.94 (ddd, 1H, J5,6 1.3, J7b,6 6.5, J7a,6 7.9 Hz, H-6), 3.84 (app. t, 1H, J3,4 = J4,5 9.7 Hz, H-4), 3.76 (dd, 1H, J2,3 3.4 Hz, H-2), 3.74 (dd, 1H, J7a,7b 9.7 Hz, H-7a), 3.69 (dd, 1H, H-7b), 3.68 (dd, 1H, H-3), 3.63 (dd, 1H, H-5), 3.36 (s, 3H, OCH), 0.92 (s, 9H, tBu), 0.10 (s, 3H) and 0.08 (s, 3H, 2 × SiMe). 13C NMR (100 MHz, CD3OD): δ = 102.83 (C-1), 72.86 (C-3), 72.18 (C-2), 71.61 (C-5), 70.36 (C-6), 67.63 (C-4), 64.67 (C-7), 55.31 (CH3O), 26.40 [C(CH3)3], 19.16 [C(CH3)3], −5.14 and −5.29 (SiCH3). HR MS: m/z = 337.1713 [M−H]−; calcd 337.1682.

Methyl 2,3,4,6-tetra-O-acetyl-7-O-tertbutyldimethylsilyl-l-glycero-α-d-manno-heptopyranoside (16)

A solution of 15 (180 mg, 0.532 mmol) in dry pyridine (5 mL) was stirred with Ac2O (1 mL, 14 mmol) and a catalytic amount of 4-N,N-dimethylaminopyridine for 3 h at room temperature. The solution was cooled to 0 °C, MeOH (2 mL) was added and stirring was continued for 20 min. The solution was concentrated and coevaporated with toluene. The residue was purified by silica gel chromatography (n-hexane/EtOAc 2:1) to furnish 270 mg (∼quant.) of 16 as colorless solid, mp 86–88 °C (CH2Cl2); +31.5 (c 0.6, CHCl3). 1H NMR (400 MHz, CDCl3): δ = 5.34 (dd, 1H, J3,4 10.0, J2,3 3.4 Hz, H-3), 5.29 (app. t, 1H, J4,5 8.9 Hz, H-4), 5.26 (dd, 1H, J1,2 1.5 Hz, H-2), 4.95 (ddd, 1H, J5,6 1.8, J7b,6 6.0, J7a,6 9.0 Hz, H-6), 4.76 (d, 1H, H-1), 4.16 (dd, 1H, H-5), 3.81 (dd, 1H, J7a,7b 9.7 Hz, H-7a), 3.70 (dd, 1H, H-7b), 3.42 (s, 3H, OCH3), 2.20 (s, 3H), 2.12 (s, 3H), 2.03 (s, 3H), 1.98 (s, 3H, CH3CO), 0.89 (s, 9H, tBu), 0.08 (s, 3H) and 0.06 (s, 3H, 2 × SiMe). 13C NMR (100 MHz, CDCl3): δ = 170.30, 170.08, 169.91 and 169.66 (CO), 98.88 (C-1), 69.65 (C-2), 69.59 (C-3), 69.50 (C-6), 66.98 (C-5), 65.06 (C-4), 59.28 (C-7), 55.28 (CH3O), 25.73 [C(CH3)3], 20.93, 20.79, 20.65 and 20.59 (COCH3), 18.09 [C(CH3)3], −5.36 and −5.49 (SiCH3)2. HR MS: m/z = 507.2281 [M+H]+; calcd 507.2261.

Desilylation of 16

A solution of 16 (50 mg, 0.1 mmol) in dry MeCN was placed in a teflon vessel and stirred with 2% HF in MeCN (1.2 mL) for 3 h at room temperature. A spatula tip of solid NaHCO3 was added and stirring was continued for 20 min. Filtration, washing of solids with dry CH3CN (5 mL), and concentration of the filtrate gave a crude material which was dried and directly submitted to the ensuing glycosylation reaction. Material 17 was immediately used for the subsequent glycosylation step. 1H NMR (400 MHz, CDCl3): δ = 5.35–5.29 (m, 2H, H-3, H-4), 5.26 (dd, 1H, J2,3 3.1 J1,2 1.7 Hz, H-2), 5.02 (dt, 1H, J5,6 2.0, J7b,6 = J7a,6 8.1 Hz, H-6), 4.76 (d, 1H, H-1), 4.14–4.11 (m, 1H, H-5), 3.91–3.80 (m, 2H, H-7a, H-7b), 3.42 (s, 3H, OMe), 2.17 (s, 3H), 2.16 (s, 3H), 2.02 (s, 3H) and 1.98 (s, 3H, 4 × COCH). 13C NMR (100 MHz, CDCl3): δ = 171.15, 170.03, 169.85 and 169.62 (CO), 98.99 (C-1), 70.25 (C-6), 69.48 and 69.26 (C-3, C-2), 68.75 (C-5), 65.10 (C-4), 61.33 (C-7), 55.46 (CH3O), 20.90, 20.81, 20.62 and 20.60 (COCH3).

2,3,4,6,7-Penta-O-acetyl-l-glycero-d-manno-heptopyranosyl N-phenyltrifluoroacetimidate (19)

A solution of 18, prepared according to lit., (96 mg, 0.228 mmol) in acetone (1.8 mL) was stirred with K2CO3 (63 mg) and N-phenyltrifluoroacetimidoyl chloride (95 mg, 0.457 mol) for 3 h at room temperature. The solids were filtered over a bed of Celite® and the filtrate was concentrated. The residue was purified by silica gel chromatography (5:1 toluene/EtOAc) to furnish 135 mg (∼quant.) of 19 as a syrup. 1H NMR (400 MHz, CDCl3): δ = 7.32 (t, 2H, J 8.8 Hz, PhH-3, PhH-5), 7.13 (t, 1H, J 6.9 Hz, PhH-4), 6.81 (t, 2H, J 8.0 Hz, PhH-2, PhH-6), 6.26 (br. s, 1H, H-1), 5.49 (dd, 1H, J2,3 2.6, J2,1 1.6 Hz, H-2), 5.40–4.38 (m, 2H, H-3, H-4), 5.28 (dt, 1H, J6,5 2.0, J6,7a 6.8 Hz, H-6), 4.24 (dd, 1H, J7a,7b 11.0 Hz, H-7a), 4.22 (dd, 1H, J6,7b 7.0 Hz, H-7b), 4.19–4.16 (m, 1H, H-5), 2.20 (s, 3H), 2.14 (s, 3H), 2.06 (s, 3H), 2.03 (s, 3H) and 2.02 (s, 3H, 5 × CH3CO). 13C NMR (100 MHz, CDCl3): δ = 170.31, 170.07, 170.01, 169.76, 169.67 and 169.49 (COCH3), 142.74 (C1-Ph), 128.94 (C3-Ph, C-5Ph), 124.77 (C4-Ph), 119.14 (C2-Ph, C-6Ph), 93.49 (C-1), 70.56 (C-5), 68.93 (C-3), 68.02 (C-2), 66.45 (C-6), 64.23 (C-4), 61.08 (C-7), 20.76, 20.63, 20.58 and 20.48 (COCH3).

Methyl 2,3,4,6,7-penta-O-acetyl-l-glycero-α-d-manno-heptopyranosyl-(1→7)-2,3,4,6-tetra-O-acetyl-l-glycero-α-d-manno-heptopyranoside (20)

Dry toluene was added to 17 (39 mg, 0.099 mmol) and donor 19 (88 mg, 0.148 mmol) and the solution was coevaporated several times with addition of toluene and then concentrated. The residue was dissolved in dry dichloromethane (1.6 mL) and a spatula tip of ground activated molecular sieves 4 Å was added. The suspension was stirred under Ar at 0 °C for 30 min and TMSO-triflate (10 μL, 0.049 mmol) was added via a syringe. The suspension was slowly warmed to room temperature and stirred for 75 min. Et3N (0.1 mL) was added and the suspension was filtered over a bed of Celite® and washed with dichloromethane (5 mL). The filtrate was washed with satd aq NaHCO3, dried (Na2SO4), and concentrated to give 135 mg of an oily residue. Purification by silica gel chromatography (toluene/EtOAc 2:1→1.5:1→1:1) afforded crude 20 (51 mg, 65%). In order to remove traces of a more polar by-product, the disaccharide was further purified by chromatography using 15:1 CH2Cl2/acetone as eluant, which furnished 40 mg (51%) of pure disaccharide 20 and additional fractions containing slightly contaminated material. +22 (c 0.5, CHCl3). 1H NMR (600 MHz, CDCl3): δ = 5.34 (dd, 1H, J2,3 3.2, J3,4 10.0 Hz, H-3), 5.31 (t, 1H, J4,5 9.4 Hz, H-4), 5.29 (t, 1H, J4′,5′ 9.9 Hz, H-4′), 5.29–5.27 (m, 2H, H-2, H-6′), 5.24 (dd, 1H, J2′,3′ 3.4, J4′,3′ 10.1 Hz, H-3′), 5.21 (dd, 1H, H-2′), 5.20 (ddd, 1H, H-6), 4.90 (d, 1H, J1′,2′ 0.7 Hz, H-1′), 4.79 (d, 1H, J1,2 1.8 Hz, H-1), 4.34 (dd, 1H, J7′a,7′b 11.4, J7′a,6′ 5.1 Hz, H-7′a), 4.19 (dd, 1H, J7′b,6′ 7.8 Hz, H-7′b), 4.10 (dd, 1H, J5,4 9.8, J5,6 2.2 Hz, H-5), 4.08 (dd, 1H, J5′,6′ 1.8 Hz, H5′), 3.85 (dd, 1H, J7a,7b 10.2, J7a,6 7.0 Hz, H-7a), 3.68 (dd, 1H, J6,7b 7.2 Hz, H-7b), 3.47 (s, 3H, OCH3), 2.18 (s, 3H), 2.17 (s, 3H), 2.16 (s, 3H), 2.14 (s, 3H), 2.053 (s, 3H), 2.050 (s, 3H), 2.01 (s, 3H), 1.988 (s, 3H), 1.986 (s, 3H, 9 × COCH). 13C NMR (150 MHz, CDCl3): δ = 170.39, 170.20 (d.i.), 170.01, 169.92, 169.81, 169.68 (d.i.) and 169.47 (COCH3), 98.98 (C-1), 98.24 (C-1′), 69.37 (C-2′), 69.27 (C-3, C-2), 68.98 (C-3′), 68.88 (C-5′), 67.88 (C-5), 67.28 (C-6), 66.92 (C-6′), 64.94 (C-7), 64.82 and 64.72 (C-4′, C-4), 62.10 (C-7′), 55.57 (OCH3), 20.94, 20.92, 20.72 (d.i.), 20.69 (triple intensity) and 20.61 (d.i., COCH3).

Methyl l-glycero-α-d-manno-heptopyranosyl-(1→7)-l-glycero-α-d-manno-heptopyranoside (21)

A solution of 20 (28 mg, 0.0352 mmol) in dry MeOH (1.5 mL) was stirred with 0.1 M methanolic NaOMe (0.4 mL) for 3 h at room temperature and processed as described for the deacetylation of 5. Final purification was achieved on a BioGel P-2 column (28 × 1 cm, H2O) to give 12.5 mg (85%) of 21 as an amorphous solid after lyophilization. +68 (c 0.3, H2O); Lit.: +65 (c 1.0, H2O). 1H NMR (600 MHz, MeOD): δ = 4.73 (d, 1H, J1′,2′ 1.3 Hz, H-1′), 4.55 (d, 1H, J1,2 1.5 Hz, H-1), 4.02 (ddd, 1H, J7b,6 7.6, J7a,6 5.4, J5,6 1.6 Hz, H-6), 3.85 (td, 1H, J6′,5′ 1.5 Hz, H-6′), 3.75–3.74 (m, 1H, H-2), 3.75, 3.74 (2 × app. t, 1H, J 9.7 Hz, H-4, H-4′), 3.68 (dd, 1H, J7a,7b 10.5 Hz, H-7a), 3.66 (dd, 1H, J3′,2′ 3.4 Hz, H-2′), 3.62 (dd, 1H, J3′,4′ 9.6 Hz, H-3′), 3.56 (dd, 1H, J7′a,7′b 10.9, J7′a,6′ 6.7 Hz, H-7′a), 3.56 (dd, 1H, J3,2 3.8 Hz, H-3), 3.54–3.50 (m, 3H, H-5′, H-7b, H-7′b), 3.38 (dd, 1H, J5,4 9.8 Hz, H-5), 3.26 (s, 3H, OCH3). 13C NMR (150 MHz, MeOD): δ = 102.97 (C-1), 102.50 (C-1′), 72.95 (C-5′), 72.81 (d.i.) and 72.75 (C-5, C-3′, C-3), 72.09 and 72.07 (C-2, C-2′), 70.79 (C-6′), 70.67 (C-7), 68.91 (C-6), 67.76 and 67.68 (C-4, C-4′), 64.47 (C-7′) and 55.51 (OCH3). HR MS: m/z = 415.1505 [M−H]−; calcd 415.1451.