Efficient Synthesis of 4-Amino-4-deoxy-l-arabinose and Spacer-Equipped 4-Amino-4-deoxy-l-arabinopyranosides by Transglycosylation Reactions

Abstract

Methyl 4-azido-4-deoxy-β-l-arabinopyranoside has been synthesized in five steps starting from methyl β-d-xylopyranoside in a multigram scale without chromatographic purification in 78% overall yield. The transformation relied on selective tosylation/nosylation at O-4 followed by acylation, S_N2 displacement with sodium azide, and subsequent deprotection. The methyl 4-azido-4-deoxy-arabinoside was then converted into allyl, propenyl, ω-bromohexyl, and chloroethoxyethyl spacer glycosides by transglycosylation with the respective alcohols in good yields and fair anomeric selectivity. Reduction of the azido group and further transformations of the aglycone afforded ω-thiol-containing spacer derivatives. Coupling to maleimide-activated BSA provided a potent immunogen, which was used to generate murine and rabbit polyclonal sera binding to LPS-core epitopes containing 4-amino-4-deoxy-arabinose residues.

4-Amino-4-deoxy-l-arabinose (Ara4N) is an important sugar constituent of complex glycolipids constituting the outer leaflet of the cell wall of Gram-negative bacteria. Specifically, Ara4N has been detected in substoichiometric up to stoichiometric amounts ester-linked to the 1- and 4′-phosphate groups of the glucosamine disaccharide backbone of lipid A, which inserts the lipopolysaccharide (LPS) chain into the outer membrane. [¹-³] In addition, Ara4N residues have been found glycosidically linked to position 8 of the higher carbon sugar 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) as in the core region of Proteus strains [4] [5] and also linked to d-glycero-d-talo-oct-2-ulosonic acid (Ko) detected in the core region of Burkholderia, [6-9] as well as in a Serratia marcescens strain. [¹0] The incorporation of Ara4N has been implicated in the onset and maintenance of antibiotic resistance in bacteria by masking the anionic charges of the phosphate and carboxylic acid groups. [¹¹] In order to study the antigenic properties of Ara4N-substituted LPS domains and develop monoclonal antibodies for diagnostic applications, a high-yielding approach to generate sufficient amounts of Ara4N building blocks was needed. Furthermore, to produce neoglycoconjugates carrying covalently linked Ara4N ligands, suitable spacer groups should be introduced allowing for a selective attachment onto the protein carrier. The conjugation chemistry has to be compatible with the presence of both amino and carboxylic acid groups in order to retain the antigenic properties of LPS core sugars. The selection of conjugation conditions would also have to take into account the labile phospho­diester linkages of Ara4N as well as the acid-labile glycosidic linkages of Kdo residues.

In this communication, we describe the preparation of Ara4N building blocks in multigram amounts as well as their conversion into a thiol-equipped spacer derivative, which was coupled to bovine serum albumin (BSA).

Previously, Ara4N has been prepared from methyl β-d-xylopyranoside via isopropylidene protection of O-2 and O-3 followed by triflation of O-4 and subsequent inversion. [¹²] [¹³] Since in our hands the formation of the acetonide turned out to be unsuitable for large-scale preparation of the compound, we resorted to regioselective tosylation of O-4 via the intermediate stannylene acetal. [¹4-¹8] Thus, reaction of the commercially available methyl xyloside 1 with dibutyltin oxide and p-toluenesulfonyl chloride in 1,4-dioxane afforded the 4-O-tosylate 2 in 99% yield, followed by benzoylation with benzoyl chloride in pyridine, which gave the known dibenzoate 3 in 92% yield. [¹9] (Scheme  [¹] ). As an alternative option, the 4-O-nosylate 4 was prepared from 1 in 99% yield. [²0] The subsequent conversion of nosylate 4 into the dibenzoate 5, however, suffered from reduced yields, whereas the acetylation was smoothly effected in 93% yield to give the 2,3-di-O-acetyl-4-O-nosyl derivative 6. The introduction of the 4-azido group was achieved uneventfully by treatment of 3 with sodium azide in DMSO at 110 ˚C to furnish methyl α-l-arabino-pyranoside 7 in 93% yield. Transesterification of the benzoate groups under Zemplén conditions then afforded the 4-azido derivative 8 in 93% yield.

In addition, direct conversion of the unprotected nosylate 4 into compound 8 could be achieved in 85% yield. This three-step approach required a chromatography purification thereby limiting a large-scale application.

Reduction of the azide-group of 8 was accomplished by hydrogenation to furnish methyl 4-amino-4-deoxy-α-l-arabino-pyranoside 9 in 99% yield.

The synthesis of 8 could thus be achieved in multigram amounts via 7 without the need of chromatography and more than twice overall yield to previous reports. [¹²] The methyl glycoside 8 constitutes a suitable precursor for further transformations into glycosyl donors as well as spacer glycosides. Whereas acid hydrolysis of the methyl glycoside 8 under various conditions suffered from reduced yields and side reactions, transglycosylation has turned out as a versatile approach for direct conversion into glycosides.

Thus, an anomeric mixture of allyl arabinosides 10 and 11 was generated from the methyl glycoside precursor 8 by reaction with excess allyl alcohol and 1 M ethereal HCl at 60 ˚C (Table  [¹] , entry 1, Scheme  [²] ). The glycosides were obtained in a combined yield of 55% with a higher proportion of the axially oriented β-glycoside 10 (ratio of 10:11 ∼2:1).

Reaction of the methyl glycoside 8 with acetyl chloride and excess of allyl alcohol proceeded smoothly at room temperature and gave a higher product yield (Table  [¹] , entry 2) due to less by-product formation. Also, these conditions resulted in slightly higher enrichment of the respective β-anomers (entries 3 and 4). Similarly, the pent-4-enyl glycosides 14, 15, the (2-chloroethoxy)ethyl derivatives 16, 17, and the ω-bromohexyl glycosides 18 and 19 were obtained in satisfactory yields (Table  [¹] , entries 3-6). The anomeric mixtures of 10/11 and 18/19 could be partially resolved by HPLC-separation, whereas the acetylated allyl glycosides 12 and 13 were amenable to separation on a standard silica gel phase. The pent-4-enyl derivatives 14/15 and the (2-chloroethoxy)ethyl derivatives 16/17 were also isolated as the 2,3-di-O-acetyl derivatives in order to allow for a facile separation of the excess alcohol components via their respective acetates. The assignment of the anomeric configuration of the β-glycosides 10, 12, 14, 16, and 18 was based on the low-field shifted ^¹H NMR signals of the anomeric proton (in the range of δ = 5.19-4.85) and the small value of the homonuclear coupling constant (J _1,2 = ∼3.6 Hz). In addition, the values of the optical rotation showed a higher dextrorotation for the axial glycosides 10, 12, 14, and 18 in comparison to their equatorial counterparts.

In order to introduce reactive end groups for subsequent conjugation, the terminal bromide group of the glycoside derivative 18 was exchanged for a thioacetyl group by reaction with KSAc in DMF to afford the ω-thioacetyl derivative 20 in 83% yield. Finally, the S-acetyl group was hydrolyzed under alkaline conditions and with exclusion of oxygen to furnish the ω-thiol compound 21 with minimal dimer formation (Scheme  [²] ).

Notably, the allyl aglycone in compound 10 could also be diversified with respect to spacer elongation (Scheme  [³] ).

Reaction of 10 with propane-1,3-dithiol under UV irradiation led to chain elongation and provided the thioether bridged ω-thiol derivative 22 in 65% yield (Scheme  [³] ). A small portion of the respective dimer was also present in the product mixture. This strategy thus broadens the scope of using allyl glycosides as precursor of spacer glycosides, which have previously been obtained by reaction with cysteamine, cysteine, by epoxide formation, or via oxidative cleavage of the double bond. Alternatively, selective reduction of the azido group leaving the allyl group unaffected could be accomplished by reaction of 10 with propane-1,3-dithiol in the absence of UV irradiation in aqueous pyridine to give compound 23 in 91% yield. [²¹]

As additional haptenic ligands to be used in inhibition studies, the 4-amino-4-deoxy-derivative 24 and the N-acetylated derivative 25 were synthesized. Thus, the propyl 4-amino-4-deoxy-glycoside 24 was prepared by reduction of the 4-azido group with concomitant saturation of the allyl group by hydrogenation on palladium/charcoal in 97% yield. The ^¹³C NMR data of the glycoside 24 compare favorably with those reported for arabinoside units in LPS core and lipid A oligosaccharides, confirming the configurational assignments. [7] The N-acetyl derivative 25 was produced in 95% yield via peracetylation of 24 followed by Zemplén de-O-acetylation.

For the synthesis of the neoglycoconjugate 27, the 4-azido-4-deoxy spacer derivative 21 was converted into the corresponding 4-amino compound 26 by reaction with propane-1,3-dithiol in 81% yield (Scheme  [4] ). The material contained ca. 15% of the corresponding disulfide and was directly used for the conjugation step. Coupling of the thiol-containing ligand to maleimide-activated BSA at pH 7.4 afforded the neoglycoprotein 27. MALDI MS analysis of the Ara4N-BSA product indicated a high ligand density of ∼34 mol ligand/mol BSA.

Immunization of rabbits and mice with Ara4N-BSA resulted in a specific response in all animals except one mouse against the immunizing antigen as measured by ELISA. The titers reached values of up to 1 million whereby no reactivity (<1000) was seen with BSA alone (Table  [²] ). We also tested a panel of bacterial LPS known to contain Ara4N in a terminal position. No reactivity was observed with Re-mutant LPS of E. coli, which is not substituted with Ara4N, and LPS of S. enterica serovar Minnesota, which carries Ara4N on the 4′-phosphate of the lipid A backbone in nonstoichiometric amounts. However, both rabbits developed antibodies against the LPS of P. mirabilis and B. cepacia whereby the titers against the latter antigen were significantly higher. From 10 mice only two (animal M6 and M8) had developed antibodies against LPS of P. mirabilis and B. cepacia. Interestingly, animal M8 had a higher titer against P. mirabilis LPS than against B. cepacia LPS of 3200 and 800, respectively.

Maleimide-activated BSA was purchased from Sigma Aldrich. Known compounds were identified by comparison with reported melting points as well as ^¹H and ^¹³C NMR data. Melting points were determined with a Kofler hot stage microscope and are uncorrected. Optical rotations were measured with a Perkin-Elmer 243 B polarimeter. [α]_D ^²0 Values are given in units of 10^-¹ degcm^²g^-¹. ^¹H NMR spectra were recorded at 297 K with a Bruker DPX instrument operating at 300 MHz or 400 MHz for ^¹H using CDCl₃ as solvent and TMS as standard, unless otherwise stated. ^¹³C NMR spectra were measured at 75.47 or 100.62 MHz and referenced to 1,4-dioxane (δ = 67.40). Homo- and heteronuclear 2D NMR spectroscopy was performed with Bruker standard software. MALDI-TOF-MS ionization spectra were recorded in the positive ion mode, with sinapic acid as matrix. TLC was performed on Merck precoated plates (5 × 10 cm, layer thickness 0.25 mm, silica gel 60 F₂₅₄); spots were detected by spraying with anisaldehyde/H₂SO₄. For column chromatography, silica gel (0.040-0.063 mm) was used. Concentration of solutions was performed at reduced pressure at temperatures <40 ˚C. Elemental analyses were provided by Dr. J. Theiner, Mikroanalytisches Laboratorium, Institut für Physikalische Chemie, Universität Wien. LPS of Re mutants of E. coli (strain F576), P. mirabilis (strain R45) and S. enterica serovar Minnesota (strain R595) and LPS from B. cepacia (strain Ko2b) were obtained by extraction of bacteria with phenol-chloroform-light petroleum ether as described. [²²]

Methyl 4- O - p -Toluenesulfonyl-β- d -xylopyranoside (2); Large-Scale Preparation

Commercially available methyl β-d-xylopyranoside (1; 25.00 g, 152.3 mmol) and Bu₂SnO (37.92 g, 152.3 mmol) were suspended in anhyd toluene (500 mL) and heated to reflux with a Dean-Stark trap. Full conversion was accomplished when 2.74 mL of H₂O had been collected after 5 h. The reaction mixture was cooled to 40 ˚C and concentrated. The remaining, slightly yellow solid was dried under reduced pressure overnight and used without further purification. A solution of TsCl (28.95 g, 151.85 mmol) in anhyd 1,4-dioxane (110 mL) was added dropwise with cooling to a suspension of the stannylidene derivative in anhyd 1,4-dioxane (400 mL). The reaction mixture was stirred for 48 h at r.t. SiO₂ (25 g) was added and the solvent was removed. The remaining, slightly yellowish solid was purified by flash chromatography first with toluene (1.2 L) followed by elution with EtOAc (1.2 L). The EtOAc fraction was concentrated until a slightly yellow oil remained, which crystallized overnight. Crystallization from n-hexane-EtOAc gave 2 (48.16 g, 99%) as colorless needles; mp 128-130 ˚C (Lit. [¹8] mp 135-136 ˚C).

^¹H NMR (400 MHz, CDCl₃): δ = 7.82 (d, J = 12.0 Hz, 2 H_arom), 7.36 (d, J = 12.0 Hz, 2 H_arom), 4.36 (dt, J = 4.5, 8.0 Hz, 1 H, H-4), 4.23 (d, J = 6.5 Hz, 1 H, H-1), 4.05 (dd, J = 4.8, 12.2 Hz, 1 H, H-5 _eq ), 3.68 (dt, J = 4.5, 8.0 Hz, 1 H, H-3), 3.49 (s, 3 H, OCH₃), 3.35-3.44 (m, 2 H, H-2, H-5 _ax ), 3.04-3.12 (br s, 1 H, 3-OH), 2.81-2.92 (br s, 1 H, 2-OH), 2.45 (s, 3 H, ArCH ₃).

^¹³C NMR (100 MHz, CDCl₃): δ = 145.44 (C1-Ar), 132.84 (C4-Ar), 130.01 (C3-, C5-Ar), 128.05 (C2-, C6-Ar), 103.27 (C-1), 77.58 (C-4), 72.39 (C-2, C-3), 62.05 (C-5), 56.97 (OCH₃), 21.70 (ArCH₃).

Methyl 2,3-Di- O -benzoyl-4- O - p -toluenesulfonyl-β- d -xylopyranoside (3)

Benzoyl chloride (2.30 mL, 9.71 mmol) was added dropwise to a stirred solution of 2 (3.00 g, 9.4 mmol) in anhyd pyridine (15 mL) at 0 ˚C. The colorless solution turned pink and a colorless solid precipitated. The ice-bath was removed and the reaction mixture was stirred overnight at r.t. The mixture was dissolved in CHCl₃ (50 mL) and washed with H₂O (50 mL). The aqueous layer was extracted with CHCl₃ (40 mL), the organic were layers combined, washed with aq 0.1 M HCl (50 mL) and sat. aq NaHCO₃ (50 mL), and dried (MgSO₄). The solution was concentrated and the solid residue was crystallized from n-hexane-EtOAc to give 3; yield: 4.34 g (92%); colorless solid; mp 138-140 ˚C (Lit. [¹9] mp 139-141 ˚C); [α]_D ^²0 +39.5 (c 0.31, CHCl₃).

^¹H NMR (400 MHz, CDCl₃): δ = 7.93-7.87 (m, 2 H_arom), 7.68 (d, J = 8.3 Hz, 2 H_arom), 7.61 (d, J = 8.3 Hz, 2 H_arom), 7.53-7.45 (m, 2 H_arom), 7.37-7.28 (m, 4 H_arom), 6.95 (d, J = 8.3 Hz, 2 H_arom), 5.57 (t, J = 8.7 Hz, 1 H, H-3), 5.24 (dd, J = 6.8, 8.8 Hz, 1 H, H-2), 4.70-4.60 (m, 1 H, H-4), 4.58 (d, J = 6.8 Hz, 1 H, H-1), 4.38 (dd, J = 5.1, J = 12.0 Hz, 1 H, H-5 _eq ), 3.69 (dd, J = 12.0, 8.8 Hz, 1 H, H-5 _ax ), 3.47 (s, 3 H, OCH₃), 2.17 (s, 3 H, ArCH ₃).

^¹³C NMR (100 MHz, CDCl₃): δ = 165.00, 164.74 (C=O), 144.92, 133.23, 133.17, 132.45, 129.73, 129.71, 128.91, 128.60, 128.26, 128.11, 127.57 (C-Ar), 101.59 (C-1), 74.93 (C-4), 70.73 (C-2, C-3), 62.86 (C-5), 56.91 (OCH₃), 21.50 (ArCH₃).

Methyl 4- O -4-Nitrobenzenesulfonyl-β- d -xylopyranoside (4)

Compound 4 was prepared according to the procedure described for 2 from the stannylidene intermediate (4.00 g, 10.1 mmol) and nosyl chloride (2.20 g, 9.9 mmol) in dioxane (30 mL) with stirring for 20 h at r.t. and worked up as described; yield: 3.46 g (99%); colorless solid; mp 64-67 ˚C (Lit. [²0] mp 62-65 ˚C).

^¹H NMR (400 MHz, CDCl₃): δ = 8.40 (d, J = 9.1 Hz, 2 H_arom), 8.17 (d, J = 9.1 Hz, 2 H_arom), 4.39 (dt, J = 5.4, 9.3 Hz, 1 H, H-4), 4.14 (dd, J = 5.2, 11.5 Hz, 1 H, H-5 _eq ), 4.13 (d, J = 7.4 Hz, 1 H, H-1), 3.57 (app. t, J = 8.9 Hz, 1 H, H-3), 3.51 (s, 3 H, OCH₃), 3.43 (dd, J = 9.8, 11.8 Hz, 1 H, H-5 _ax ), 3.36 (br s, 2 H, 2-OH, 3-OH), 3.22 (dd, J = 7.4, 9.0 Hz, 1 H, H-2).

^¹³C NMR (100 MHz, CDCl₃): δ = 150.65 (C4-Ar), 141.71 (C1-Ar), 129.40 (C3-, C5-Ar), 124.09 (C2-, C6-Ar), 103.88 (C-1), 79.11 (C-4), 73.24 (C-2), 72.90 (C-3), 62.88 (C-5), 56.95 (OCH₃).

Methyl 2,3-Di- O -benzoyl-4- O -4-nitrobenzenesulfonyl-β- d -xylopyranoside (5)

Compound 5 was prepared from 4 (1.71 g, 3.89 mmol) and benzoyl chloride (1.16 mL, 9.71 mmol) in anhyd pyridine (12 mL) with stirring for 4 h at r.t. according to the procedure described for 3. Workup as described for 3 gave compound 5; yield: 1.29 g (47%); colorless crystals; mp 151-152 ˚C (n-hexane); [α]_D ^²0 +6.7 (c 1.0, CHCl₃).

^¹H NMR (400 MHz, CDCl₃): δ = 8.20-7.23 (m, 14 H_arom), 5.57 (t, J = 8.6 Hz, 1 H, H-3), 5.26 (dd, J = 6.8, 8.8 Hz, 1 H, H-2), 4.79 (dt, J = 5.1 Hz, 1 H, H-4), 4.61 (d, J = 6.8 Hz, 1 H, H-1), 4.17 (dd, J = 5.1, 12.1 Hz, 1 H, H-5 _eq ), 3.75 (dd, J = 12.1, 8.8 Hz, 1 H, H-5 _ax ), 3.50 (s, 3 H, OCH₃).

^¹³C NMR (100 MHz, CDCl₃): δ = 164.48, 164.92 (C=O), 150.17, 141.30, 133.88, 133.37, 129.71, 129.44, 128.73, 128.33, 128.28, 128.03, 124.21 (C2-, C6-Ar), 101.67 (C-1), 76.57 (C-4), 70.72 (C-3), 70.60 (C-2), 62.90 (C-5), 57.00 (OCH₃).

Anal. Calcd for C₂₆H₂₃NO₁₁S: C, 55.75; H, 3.95; N, 2.51. Found: C, 56.01; H, 4.16; N, 2.51.

Methyl 2,3-Di- O -acetyl 4- O -4-nitrobenzenesulfonyl-β- d -xylopyranoside (6)

Compound 6 was prepared from 4 (3.00 g, 8.6 mmol) and Ac₂O (15 mL, 132 mmol) in anhyd pyridine (15 mL) with stirring for 2 h at r.t. The formed precipitate was filtered off and the filtrate was concentrated to 1/3 volume, which led to additional precipitation. Solids were combined and dried at 40 ˚C under reduced pressure to afford 6; yield: 3.4 g (93%); colorless crystals; mp 157 ˚C (n-hexane); [α]_D ^²0 -65.7 (c 0.4, CHCl₃).

^¹H NMR (400 MHz, CDCl₃): δ = 8.42 (d, J = 8.8 Hz, 2 H_arom), 8.11 (d, J = 8.8 Hz, 2 H_arom), 5.15 (app. t, J = 8.5 Hz, 1 H, H-3), 4.84 (dd, J = 7.0, 8.5 Hz, 1 H, H-2), 4.67 (dt, J = 5.1, 8.6 Hz, 1 H, H-4), 4.38 (d, J = 6.8 Hz, 1 H, H-1), 4.17 (dd, J = 5.1, 11.9 Hz, 1 H, H-5 _eq ), 3.51 (dd, J = 12.1, 9.1 Hz, 1 H, H-5 _ax ), 3.46 (s, 3 H, OCH₃), 2.03 (s, 3 H, 2-OAc), 1.85 (s, 3 H, 3-OAc).

^¹³C NMR (100 MHz, CDCl₃): δ = 169.50, 169.38 (C=O), 150.95 (ArNO₂), 141.75 (C1-Ar), 129.24 (C3-, C5-Ar), 124.52 (C2-, C6-Ar), 101.47 (C-1), 75.68 (C-4), 70.82 (C-3), 70.64 (C-2), 62.36 (C-5), 56.90 (OCH₃), 20.61 (COCH₃), 20.46 (COCH₃).

Anal. Calcd for C₁₆H₁₉NO₁₁S: C, 44.34; H, 4.42; N, 3.23. Found: C, 44.00; H, 4.23; N, 3.22.

Methyl 4-Azido-2,3-di- O -benzoyl-4-deoxy-α- l -arabinopyranoside (7)

Compound 3 (69.15 g, 131 mmol) and NaN₃ (13.7 g, 210.6 mmol) were suspended in anhyd DMSO (280 mL) and stirred at 90 ˚C overnight. The reaction mixture was dissolved in CHCl₃ (0.8 L) and washed with H₂O (3 × 400 mL). The combined organic layers were washed with sat. aq NaHCO₃ (400 mL) and brine (400 mL), dried (Na₂SO₄), and concentrated. The residual oil was dissolved in EtOAc (20 mL) and n-hexane was added until a colorless solid precipitated. The crystals were collected and the filtrate was evaporated to dryness and to afford an additional crop. Crystallization from 2:1 n-hexane-EtOAc afforded 7; yield: 48.64 g (93%); mp 104-105 ˚C (EtOH); [α]_D ^²0 +18.6 (c 0.3, CHCl₃).

^¹H NMR (300 MHz, CDCl₃): δ = 8.06-7.97 (m, 4 H_arom), 7.58-7.50 (m, 2 H_arom), 7.45-7.38 (m, 4 H_arom), 5.58 (dd, J = 5.4, 7.9 Hz, 1 H, H-2), 5.51 (dd, J = 3.2, 7.9 Hz, 1 H, H-3), 4.60 (d, J = 5.6 Hz, 1 H, H-1), 4.22-4.14 (m, 2 H, H-4, H-5 _eq ), 3.84-3.75 (m, J = 4.3, 13.8 Hz, 1 H, H-5 _ax ), 3.50 (s, 3 H, OCH₃).

^¹³C NMR (75 MHz, CDCl₃): δ = 165.63, 165.03 (C=O), 133.57, 133.32, 130.01, 129.80, 129.30, 128.78, 128.48, 128.39, (C-Ar), 101.05 (C-1), 71.47 (C-3), 69.18 (C-2), 61.66 (C-5), 57.56 (C-4), 56.45 (OCH₃).

Anal. Calcd for C₂₀H₁₉N₃O₆: C, 60.45; H, 4.82; N, 10.57. Found: C, 60.58; H, 4.78; N, 10.36.

Synthesis of 7 under Microwave Irradiation: Compound 3 (3.70 g, 7.03 mmol) and NaN₃ (0.69 g, 10.6 mmol) were suspended in anhyd DMSO (10 mL) and heated in a MSL Microwave to 110 ˚C (max. 250 W). After 30 min, a second portion of NaN₃ (200 mg) was added and heating was continued for 15 h. Cooling to r.t and workup as described above afforded 7 (2.26 g, 81%) as colorless needles.

Methyl 4-Azido-4-deoxy-α- l -arabinopyranoside (8)

Method A, from 7: Azide 7 (47.42 g, 119.3 mmol) was suspended in anhyd MeOH (300 mL). A 1 M solution of methanolic NaOMe (12 mL) was added and the mixture was stirred for 1 h at r.t. The solution was made neutral by adding DOWEX 50 H⁺ resin. The resin was removed by filtration and the filtrate was concentrated. n-Hexane (200 mL) was added, the suspension was stirred overnight and the solid product 8 was collected by filtration and dried; yield: 20.97 g (93%); colorless solid; mp 94-95 ˚C {Lit. [¹²]  mp 94-95 ˚C); [α]_D ^²0 -22.6 (c 0.3, CHCl₃); Lit.⁸ -26.1 (c 0.5, CHCl₃)}.

^¹H NMR (300 MHz, CDCl₃): δ = 5.48-5.43 (m, 1 H, 3-OH), 5.25-5.20 (m, 1 H, 2-OH), 4.04 (d, J = 7.2 Hz, 1 H, H-1), 3.83-3.71 (m, 2 H, H-4, H-5 _eq ), 3.70-3.60 (m, 1 H, H-3), 3.56-3.48 (m, 1 H, H-5 _ax ), 3.38 (s, 3 H, OCH₃), 3.34-3.24 (m, 1 H, H-2).

^¹³C NMR (75 MHz, CDCl₃): δ = 104.33 (C-1), 72.54 (C-3), 70.66 (C-2), 63.03 (C-5), 61.13 (C-4), 55.84 (OCH₃).

Method B, from 4: Nosylate 4 (0.200 g, 0.57 mmol) and NaN₃ (0.40 g, 6.15 mmol) were suspended in anhyd DMSO (1 mL) and stirred at 45 ˚C for 5 h. The reaction mixture was dissolved in CHCl₃ (30 mL) and washed with H₂O (20 mL), sat. aq NaHCO₃ (2 20 mL), and brine (15 mL). The organic layer was dried (MgSO₄) and concentrated. Purification of the residue by column chromatography (n-hexane-EtOAc, 3:1) afforded 8; yield: 0.092 g (85%); colorless solid.

Methyl 4-Amino-4-deoxy-α- l -arabinopyranoside (9)

Pd/C (10 mol%) was added to a solution of azide 8 (308 mg, 1.63 mmol) in MeOH (25 mL) and the suspension was stirred at r.t. overnight under H₂ at atmospheric pressure. The catalyst was removed by filtration over Celite and the filtrate was concentrated and dried to give 9; yield: 264 mg (99%); colorless syrup; [α]_D ^²0 -36.4 (c 0.31, MeOH).

^¹H NMR (300 MHz, CDCl₃): δ = 4.33 (d, J = 6.9 Hz, 1 H, H-1), 3.86 (dd, J = 3.2, 12.4 Hz, 1 H, H-5 _eq ), 3.76 (dd, J = 4.2, 8.8 Hz, 1 H, H-3), 3.71 (dd, J = 2.4, 12.4 Hz, 1 H, H-5 _ax ), 3.56 (s, 3 H, OCH₃), 3.52-3.59 (m, 1 H, H-2), 3.08-3.14 (m, 1 H, H-4).

^¹³C NMR (75 MHz, CDCl₃): δ = 103.84 (C-1), 72.07 (C-3), 70.19 (C-2), 65.29 (C-5), 56.88 (OCH₃), 49.72 (C-4).

Allyl 4-Azido-4-deoxy-β- l -arabinopyranoside (10) and Allyl 4-Azido-4-deoxy-α- l -arabinopyranoside (11)

Method A: Azide 8 (1.03 g, 5.44 mmol) was dissolved in anhyd allyl alcohol (3 mL) and a 1 M solution of HCl in Et₂O (2.0 mL) was added. The solution was stirred for 4 h at 60 ˚C and for further 18 h at r.t. The brownish reaction mixture was diluted with EtOAc (30 mL), neutralized with sat. aq NaHCO₃ (30 mL), and washed with brine (20 mL). The solvent was removed and the remaining yellowish oil was purified by column chromatography (100 g SiO₂, 10 µm, toluene-EtOAc, 3.5:1). The product-containing fractions were pooled and concentrated to afford 10.

10

Yield: 320 mg (27%); R _f  = 0.66 (EtOAc); mp 32-33 ˚C; [α]_D ^²0 +192.4 (c 0.4, CHCl₃).

^¹H NMR (400 MHz, CD₃OD): δ = 5.93-6.04 (m, 1 H, =CH), 5.35 (qd, J = 1.7, 17.2 Hz, 1 H, CH=CH _{2 trans} ), 5.21 (qd, J = 1.5, 10.4 Hz, 1 H, CH=CH _{2 cis} ), 4.85 (d, J = 3.6 Hz, 1 H, H-1), 4.20 (tdd, J = 1.5, 5.2, 13.0 Hz, 1 H, OCH₂), 4.04 (dd, J = 3.6, 9.6 Hz, 1 H, H-3), 4.01-4.08 (m, 1 H, OCH₂), 3.86-3.93 (m, 2 H, H-5 _eq , H-4), 3.75 (dd, J = 3.6, 9.7 Hz, 1 H, H-2), 3.62 (dd, J = 2.6, 12.8 Hz, 1 H, H-5 _ax ).

^¹³C NMR (100 MHz, CD₃OD): δ = 133.37 (=CH), 118.02 (=CH₂), 97.70 (C-1), 70.41 (C-3), 69.75 (C-2), 68.74 (OCH₂), 61.42 (C-4), 60.85 (C-5).

Anal. Calcd for C₈H₁₃N₃O₄: C, 44.65; H, 6.09; N, 19.53. Found: C, 44.68; H, 5.79; N, 18.79.

Further elution provided a mixture of 10 and 11; yield: 233 mg (20%), followed by 11.

11

Yield: 98 mg (8%); R _f  = 0.55 (EtOAc); mp 39-42 ˚C; [α]_D ^²0 +61.8 (c 0.51, CHCl₃).

^¹H NMR (400 MHz, CD₃OD): δ = 5.89-6.00 (m, 1 H, =CH), 5.32 (qd, J = 1.7, 17.3 Hz, 1 H, CH=CH _{2 trans} ), 5.16 (qd, J = 1.5, 10.4 Hz, 1 H, CH=CH _{2 cis} ), 4.29 (tdd, J = 1.5, 5.2, 12.9 Hz, 1 H, OCH₂), 4.22 (d, J = 7.2 Hz, 1 H, H-1), 4.09 (tq, J = 1.4, 6.3 Hz, 1 H, OCH₂), 3.89 (dd, J = 2.7, 12.7 Hz, 1 H, H-5 _eq ), 3.74 (dd, J _3,4 = 3.9 Hz, J = 8.8 Hz, 1 H, H-3), 3.76-3.80 (m, 1 H, H-4), 3.58 (dd, J = 1.8, 12.7 Hz, 1 H, H-5 _ax ), 3.50 (dd, 1 H, J = 7.2, 8.9 Hz, H-2).

^¹³C NMR (75 MHz, CDCl₃): δ = 133.48 (=CH), 118.21 (=CH₂), 101.70 (C-1), 72.60 (C-3), 71.23 (C-2), 69.95 (OCH₂), 62.98 (C-5), 60.06 (C-4).

Anal. Calcd for C₈H₁₃N₃O₄: C, 44.65; H, 6.09; N, 19.53. Found: C, 44.51; H, 6.03; N, 19.22.

Method B: Freshly distilled AcCl (0.5 mL) was added at r.t. to a solution of 8 (400 mg; 2.11 mmol) in anhyd allyl alcohol (2.5 mL). Upon addition a strong exothermic reaction occurred. After cooling to r.t., the solution was stirred for 18 h. The slightly yellowish solution was diluted with EtOAc (30 mL), treated with sat. aq NaHCO₃ (2 20 mL), washed with brine (20 mL), and dried (MgSO₄). The solvent was removed and the remaining oil was purified by column chromatography (60 g, 63 µm SiO₂, n-hexane-EtOAc, 4:1). The product-containing fractions were pooled and concentrated to afford 522 mg (76%) 10 and 11 as a 2.3:1 anomeric mixture.

Allyl 2,3-Di- O -acetyl-4-azido-4-deoxy-β- l -arabinopyranoside (12) and Allyl 2,3-Di- O -acetyl-4-azido-4-deoxy-α- l -arabinopyranoside (13)

A solution of the anomeric mixture 10,11 (100 mg, 0.46 mmol) in anhyd pyridine (5 mL) and Ac₂O (500 µL, 3.9 mmol) was stirred at r.t. overnight. The solution was co-evaporated with toluene (2 20 mL) and concentrated. Purification of the residue on silica gel (toluene-EtOAc, 8:1) afforded first 12 as a colorless oil.

12

Yield: 25 mg (18%); colorless syrup; R _f  = 0.55 (n-hexane-EtOAc, 3:1); [α]_D ^²0 +187.8 (c 0.4, CHCl₃).

^¹H NMR (300 MHz, CDCl₃): δ = 5.92-5.79 (m, 1 H, =CH), 5.40 (dd, J = 3.8, 10.5 Hz, 1 H, H-3), 5.30 (dq, 1 H, J = 1.6, 17.2 Hz, CH=CH _{2 trans} ), 5.21 (dq, 1 H, J = 1.3, 10.5 Hz, CH=CH _{2 cis} ), 5.20 (dd, J = 3.6, 10.5 Hz, 1 H, H-2), 5.09 (d, J = 3.6 Hz, 1 H, H-1), 4.19 (ddt, 1 H, J = 1.5, 5.1, 13.1 Hz, OCH₂), 4.11 (m, 1 H, H-4), 4.03-3.94 (m, 2 H, OCH₂, H-5 _eq ), 3.67 (dd, J = 2.0, 12.6 Hz, 1 H, H-5 _ax ), 2.12 (s, 3 H, COCH₃), 2.09 (s, 3 H, COCH₃).

^¹³C NMR (75 MHz, CDCl₃): δ = 170.22 and 170.17 (C=O), 133.23 (=CH), 117.84 (=CH₂), 95.51 (C-1), 69.28 (C-3), 68.56 (OCH₂), 68.20 (C-2), 60.01 (C-5), 59.86 (C-4), 20.78 and 20.59 (CH₃C=O).

Further elution of the column afforded 13.

13

Yield: 10 mg (8%); R _f  = 0.45 (n-hexane-EtOAc, 3:1); [α]_D ^²0 +26.3 (c 0.2 CHCl₃).

^¹H NMR (300 MHz, CDCl₃): δ = 5.92-5.78 (m, 1 H, =CH), 5.28 (dq, J = 1.7, 17.2 Hz, 1 H, CH=CH_{2 trans} ), 5.19 (dq, J = 1.4, 10.4 Hz, 1 H, CH=CH_{2 cis} ), 5.17 (dd, J = 5.4, 7.9 Hz, 1 H, H-2), 5.07 (dd, J = 3.6, 7.7 Hz, 1 H, H-3), 4.49 (d, J = 5.4 Hz, 1 H, H-1), 4.28 (ddt, J = 1.6, 4.8, 13.2 Hz, 1 H, OCH₂), 4.05 (dd, J = 5.2, 12.0 Hz, 1 H, H-5 _eq ), 4.04 (ddt, J = 5.2, 12.0 Hz, 1 H, OCH₂), 3.96-3.90 (m, 1 H, H-4), 3.61 (dd, J = 2.6, 12.1 Hz, 1 H, H-5 _ax ), 2.12 (s, 3 H, COCH₃), 2.08 (s, 3 H, COCH₃).

^¹³C NMR (75 MHz, CDCl₃): δ = 170.59 and 169.50 (C=O), 133.43 (=CH), 117.23 (CH=CH₂), 98.65 (C-1), 71.06 (C-3), 69.11 (OCH₂), 68.63 (C-2), 61.36 (C-5), 56.98 (C-4), 20.78 and 20.63 (COCH₃).

ESI-TOF: m/z calcd for C₁₂H₁₇O₆N₃ + Na [M + Na]⁺: 322.1010; found: 322.1024.

Pent-4-enyl 2,3-Di- O -acetyl-4-azido-4-deoxy-β- l -arabinopyranoside (14) and Pent-4-enyl 2,3-Di- O -acetyl-4-azido-4-deoxy-α- l -arabinopyranoside (15)

Compound 8 (400 mg, 2.11 mmol) and anhyd pent-4-en-1-ol (2.5 mL) was treated with freshly distilled AcCl (0.50 mL) and processed as described for the allyl derivatives 10 and 11 (Method B). Purification of the crude material by column chromatography (n-hexane-EtOAc, 4:1) afforded first a pure fraction of 14.

14

Yield: 250 mg (34%); colorless oil; [α]_D ^²0 +171.9 (c 0.36, CHCl₃).

^¹H NMR (300 MHz, CDCl₃): δ = 5.87-5.72 (m, 1 H, =CH), 5.37 (dd, J = 3.8, 10.5 Hz, 1 H, H-3), 5.16 (dd, J = 3.6, 10.5 Hz, 1 H, H-2), 5.04 (d, J = 3.6 Hz, 1 H, H-1), 5.07-4.95 (m, 2 H, CH=CH ₂), 4.12-4.07 (m, 1 H, H-4), 3.94 (dd, J = 1.9, 12.6 Hz, 1 H, H-5 _eq ), 3.62-3.73 (m, 2 H, CH₂O and H-5 _ax ), 3.45-3.35 (m, 1 H, CH₂O), 2.18-2.06 (m, 2 H, CH₂), 2.12 (s, 3 H, COCH₃), 2.07 (s, 3 H, COCH₃), 1.74-1.63 (m, 2 H, CH₂).

^¹³C NMR (75 MHz, CDCl₃): δ = 170.18 and 170.28 (C=O), 137.76 (=CH), 115.21 (CH=CH₂), 96.41 (C-1), 69.39 (C-3), 68.42 (C-2), 67.75 (OCH₂), 59.92 (C-5), 59.86 (C-4), 30.09 and 28.49 (CH₂), 20.73 (COCH₃), 20.59 (COCH₃).

ESI-TOF: m/z calcd for C₁₄H₂₁N₃O₆ [M + Na]⁺: 350.1323; found: 350.1328.

Further elution gave a fraction containing 14 and 15 (208 mg, 28%) and pure 15.

15

Yield: 70 mg (9%); colorless oil; [α]_D ^²0 -14.3 (c 0.21, CHCl₃).

^¹H NMR (300 MHz, CDCl₃): δ = 5.87-5.72 (m, 1 H, CH=), 5.10-4.93 (m, 3 H, CH=CH ₂, H-3), 5.37 (dd, J = 3.8, 10.5 Hz, 1 H, H-3), 5.14 (dd, J = 5.4, 7.7 Hz, 1 H, H-2), 4.44 (d, J = 5.3 Hz, 1 H, H-1), 4.03 (dd, J = 5.3, 12.1 Hz, 1 H, H-5 _eq ), 3.95-3.89 (m, 1 H, H-4), 3.81 (dt, J = 6.3, 9.5 Hz, 1 H, CH₂O), 3.62 (dd, J = 2.6, 12.1 Hz, 1 H, H-5 _ax ), 3.44 (dt, J = 6.4, 9.5 Hz, 1 H, CH₂O), 2.17-2.06 (m, 2 H, CH₂), 2.12 (s, 3 H, COCH₃), 2.07 (s, 3 H, COCH₃), 1.72-1.62 (m, 2 H, CH₂).

^¹³C NMR (75 MHz, CDCl₃): δ = 170.10 and 169.15 (C=O), 137.84 (=CH), 114.98 (CH=CH₂), 99.78 (C-1), 71.11 (C-3), 68.70 (C-2), 68.33 (OCH₂), 61.30 (C-5), 57.02 (C-4), 29.94 and 28.61 (CH₂), 20.71 (COCH₃), 20.56 (COCH₃).

ESI-TOF: m/z calcd for C₁₄H₂₁N₃O₆ [M + Na]⁺: 350.1323; found: 350.1329.

2-(2-Chloroethoxy)ethyl 2,3-Di- O -acetyl-4-azido-4-deoxy-β- l -arabinopyranoside (16) and 2-(2-Chloroethoxy)ethyl 2,3-Di- O -acetyl-4-azido-4-deoxy-α- l -arabinopyranoside (17)

Compound 8 (400 mg, 2.11 mmol) and anhyd (2-chloroethoxy)ethanol (2.5 mL) were treated with freshly distilled AcCl (0.50 mL) and processed as described for the allyl derivatives 10 and 11 (Method B). The product-containing fractions were pooled, and concentrated to afford 430 mg (72%) 16 and 17 as a ∼2.9:1 anomeric mixture of colorless oil.

β-Anomer 16

NMR-data were extracted from anomeric mixture.

^¹H NMR (400 MHz, CDCl₃): δ = 5.41 (dd, J = 3.7, 10.5 Hz, 1 H, H-3), 5.19 (dd, J = 3.6, 10.5 Hz, 1 H, H-2), 5.11 (d, J = 3.6 Hz, 1 H, H-1), 4.12-4.09 (m, 1 H, H-4), 4.05 (ddd, J = 1.8, 12.6 Hz, 1 H, H-5 _eq ), 3.85-3.79 (m, 1 H, CH₂O), 3.78-3.74 (m, 2 H, CH₂O), 3.71-3.60 (m, 6 H, CH₂, H-5 _ax ), 2.12 (s, 3 H, COCH₃), 2.09 (s, 3 H, COCH₃).

^¹³C NMR (100 MHz, CHCl₃): δ = 170.11 (C=O), 96.48 (C-1), 71.36, 71.34, 70.12, 69.25 (C-3), 68.25 (C-2), 67.37, 59.97 (C-5), 59.89 (C-4), 42.81 (CH₂Cl), 20.59 (COCH₃), 20.79 (COCH₃).

α-Anomer 17

NMR-data were extracted from anomeric mixture.

^¹H NMR (300 MHz, CDCl₃): δ = 5.16 (dd, J = 5.5, 8.0 Hz, 1 H, H-2), 5.07 (dd, J = 3.6, 8 Hz, 1 H, H-3), 4.53 (d, J = 5.6 Hz, 1 H, H-1), 4.09-4.12 (m, 1 H, H-4), 4.05 (dd, J = 5.1, 12.2 Hz, 1 H, H-5 _eq ), 3.91-3.95 (m, 1 H, H-4), 3.88-3.91 (m, 1 H, CH₂O), 3.58-3.77 (m, 6 H), 2.12 (s, 3 H, COCH₃), 2.08 (s, 3 H, COCH₃).

^¹³C NMR (75 MHz, CDCl₃): δ = 169.22 (C=O), 99.90 (C-1), 71.37, 71.20, 70.33, 68.61 (C-3), 68.25 (C-2), 68.15, 61.60 (C-5), 57.13 (C-4), 42.78 (CH₂Cl), 20.74 (COCH₃), 20.56 (COCH₃).

ESI-TOF: m/z calcd for C₁₃H₂₀ClN₃O₇ [M + Na]⁺: 388.0882; found: 388.0887.

6-Bromohexyl 4-Azido-4-deoxy-β- l -arabinopyranoside (18) and 6-Bromohexyl 4-azido-4-deoxy-α- l -arabinopyranoside (19)

Method A: 6-Bromohexanol (1.6 mL) was added to a suspension of compound 8 (700 mg, 3.7 mmol) in 1 M ethereal HCl (1.0 mL) and heated for 4 h at 70 ˚C in a 25 mL two-necked flask. The resulting solution was then stirred overnight at r.t. EtOAc (80 mL) and solid Na₂CO₃ (2.5 g) were added and the suspension was filtered. The filtrate was washed with brine (100 mL), dried (Na₂SO₄), and concentrated. The residue was purified by column chromatography (120 g SiO₂, n-hexane-EtOAc, 7:3 → 0:1) yielding the pure β-anomer 18.

18

Yield: 542 mg (44%); colorless crystals; mp 33-34 ˚C; [α]_D ^²0 +132.8 (c 1.3, CHCl₃).

^¹H NMR (400 MHz, CDCl₃): δ = 4.87 (d, J = 3.7 Hz, 1 H, H-1), 3.97 (dd, J = 3.8, 9.2 Hz, 1 H, H-3), 3.96-3.92 (m, 1 H, H-4), 3.83 (dd, J = 1.7, 12.5 Hz, 1 H, H-5 _eq ), 3.79 (dd, J = 3.7, 9.2 Hz, 1 H, H-2), 3.72 (dt, J = 6.8, 9.7 Hz, 1 H, OCH₂), 3.69 (dd, J = 2.2, 12.5 Hz, 1 H, H-5 _ax ), 3.46 (dt, J = 6.3, 9.5 Hz, 1 H, OCH₂), 3.35 (t, J = 6.7 Hz, 2 H, BrCH₂), 2.34 (br s, 2 H, OH), 1.84-1.76 (m, 2 H, CH ₂CH₂Br), 1.61-1.51 (m, 2 H, CH₂), 1.45-1.26 (m, 4 H, CH₂).

^¹³C NMR (100 MHz, CDCl₃): δ = 98.44 (C-1), 70.74 (C-2), 69.93 (C-3), 68.39 (OCH₂), 61.19 (C-4), 60.53 (C-5), 33.75 (BrCH₂), 32.54, 29.24, 27.83, 25.30 (CH₂).

Anal. Calcd for C₁₁H₂₀BrN₃O₄: C, 39.07; H, 5.96; N, 12.42. Found: C, 39.44; H, 5.71; N, 12.32.

Further elution gave a fraction containing 18 and 19 (240 mg, 19%) and pure 19.

19

Yield: 108 mg (9%); colorless crystals; mp 47-49 ˚C; [α]_D ^²0 -11 (c 0.8, CHCl₃).

^¹H NMR (300 MHz, CDCl₃): δ = 4.26 (d, J = 6.4 Hz, 1 H, H-1), 3.90-3.81 (m, 3 H, H-3, H-4, OCH₂), 3.77 (dd, J = 3.8, 12.6 Hz, 1 H, H-5 _eq ), 3.70 (dd, J = 6.4, 7.9 Hz, 1 H, H-2), 3.59 (dd, J = 2.5, 12.6 Hz, 1 H, H-5 _ax ), 3.49 (td, J = 6.6, 9.6 Hz, 1 H, OCH₂), 3.41 (t, J = 6.7 Hz, 2 H, BrCH₂), 2.94 (br s, 1 H, OH), 2.54 (br s, 1 H, OH), 1.84-1.76 (m, 2 H, CH ₂CH₂Br), 1.61-1.51 (m, 2 H), 1.45-1.26 (m, 4 H, CH₂).

^¹³C NMR (75 MHz, CDCl₃): δ = 102.45 (C-1), 72.61 (C-2), 71.38 (C-3), 69.55 (OCH₂), 62.54 (C-4), 59.33 (C-5), 33.78 (BrCH₂), 32.55, 29.30, 27.82, 25.15 (CH₂).

Anal. Calcd for C₁₁H₂₀BrN₃O₄: C, 39.07; H, 5.96; N, 12.42. Found: C, 39.36; H, 5.78; N, 12.28.

Method B: Freshly distilled AcCl (0.25 mL) was added at r.t. to a solution of 8 (200 mg; 1.6 mmol) in 6-bromohexanol (1 mL, 6.6 mmol). Upon addition a strong exothermic reaction occurred. After cooling to r.t., the solution was stirred for 18 h. The slightly yellowish solution was diluted with EtOAc (50 mL), treated with sat. aq NaHCO₃ (60 mL), washed with brine (40 mL), and dried (Na₂SO₄). The solvent was removed and the remaining oil was purified by column chromatography (50 g, 63 µm SiO₂, n-hexane-EtOAc, 4:1). The product-containing fractions were pooled and concentrated to afford 238 mg (65%) 18 and 19 as a 3:1 anomeric mixture.

6- S -Thioacetylhexyl 4-Azido-4-deoxy-β- l -arabinopyranoside (20)

A solution of KSAc (17 mg, 0.15 mmol) in anhyd DMF (1mL) was added to a solution of bromide 18 (98 mg, 0.29 mmol) in anhyd DMF (2 mL) and the solution was stirred at r.t. overnight whereupon a colorless solid precipitated.The reaction mixture was diluted with EtOAc (20 mL) and poured onto H₂O (20 mL). The aqueous layer was extracted with EtOAc (2 × 15 mL). The combined organic layers were washed with sat. NaHCO₃ (40 mL), brine (30 mL), and dried (MgSO₄). Concentration of the solution afforded a slightly yellow oil, which was further purified by column chromatography (15.0 g SiO₂; n-hexane-EtOAc, 3:2) to furnish 20; yield: 82 mg (83%); colorless syrup; [α]_D ^²0 +133 (c 0.5 CHCl₃).

^¹H NMR (300 MHz, CDCl₃): δ = 4.86 (d, J = 3.7 Hz, 1 H, H-1), 4.00-3.91 (m, 2 H, H-4, H-5 _eq ), 3.88-3.65 (m, 4 H, H-2, H-3, H-5 _ax , OCH₂), 3.49-3.39 (m, 1 H, OCH₂), 2.86 (t, J = 7.2 Hz, 2 H, SCH₂), 2.32 (s, 3 H, CH₃COS), 2.17 (br s, 2 H, OH), 1.68-1.51 (m, 4 H, CH₂), 1.46-1.29 (m, 4 H, CH₂).

^¹³C NMR (75 MHz, CDCl₃): δ = 196.11 (C=O), 98.46 (C-1), 70.82 (C-2), 69.97 (C-3), 68.46 (OCH₂), 61.17 (C-4), 60.57 (C-5), 30.66 (CH₃), 29.37, 29.23, 28.88, 28.35, 25.60 (CH₂).

Anal. Calcd for C₁₁H₂₀BrN₃O₄: C, 46.83; H, 6.95; N, 12.60. Found: C, 46.94; H, 6.96; N, 12.35.

6-Mercaptohexyl 4-Azido-4-deoxy-β- l -arabinopyranoside (21)

Thioacetate 20 (210 mg, 0.629 mmol) was stirred with aq 0.2 M NaOH (50 mL) under argon at r.t. for 1.5 h. The reaction mixture was extracted with EtOAc (3 × 40 mL), and the combined organic layers were dried (MgSO₄) and concentrated to give 21 as a colorless oil, which solidified at r.t.; yield: 169 mg (87%); R _f = 0.14 (n-hexane-EtOAc, 1:1); [α]_D ^²0 +146 (c 0.5, MeOH).

^¹H NMR (300 MHz, CD₃OD): δ = 4.77 (d, J = 3.7 Hz, 1 H, H-1), 4.00 (dd, J = 3.8, 9.7 Hz, 1 H, H-3), 3.91-3.84 (m, 2 H, H-4, H-5 _eq ), 3.74 (dd, J = 3.7 Hz, 1 H, H-2), 3.69 (dt, J = 7.2, 9.7 Hz, 1 H, OCH₂), 3.61 (dd, J = 2.6, 12.8 Hz, 1 H, H-5 _ax ), 3.46 (dt, J = 6.3, 9.7 Hz, 1 H, OCH₂), 2.52 (t, J = 7.1 Hz, 2 H, SCH₂), 1.72-1.55 (m, 4 H, CH₂), 1.50-1.36 (m, 4 H, CH₂).

^¹³C NMR (75 MHz, CD₃OD): δ = 100.72 (C-1), 70.90 (C-3), 70.55 (C-2), 69.41 (OCH₂), 64.07 (C-4), 61.63 (C-5), 35.11 (SCH₂), 30.46, 29.16, 26.76, 24.88 (CH₂).

ESI-TOF: m/z calcd as the disulfide C₂₂H₄₀O₈N₆S₂ [M - H]^-: 579.2276; found: 579.2275.

3-Mercaptopropyl-3-thiopropyl 4-Azido-4-deoxy-β- l -arabino­pyranoside (22)

Propane-1,3-dithiol (0.2 mL) was added to a solution of azide 10 (42 mg, 0.19 mmol) in MeOH (1.0 mL) in a quartz flask. The reaction mixture was stirred under UV radiation (254 nm) for 4 h at r.t. to give full conversion. The solvent was removed and the residue was purified by chromatography (n-hexane-EtOAc-MeOH, 1:1:0.1) to afford 22 as a colorless oil; yield: 40 mg (65%).

^¹H NMR (300 MHz, CDCl₃): δ = 4.88 (d, J = 3.7 Hz, 1 H, H-1), 4.02-3.75 (m, 5 H, H-2, H-3, H-4, H-5 _eq , OCH₂), 3.69 (dd, J = 2.2, 12.6 Hz, 1 H, H-5 _ax ), 3.53 (dt, J = 6.1, 9.9 Hz, 1 H, OCH₂), 2.70-2.57 (m, 6 H, CH₂), 1.95-1.81 (m, 4 H, CH₂), 1.38 (t, J = 8.0 Hz, 1 H, SH).

^¹³C NMR (75 MHz, CDCl₃): δ = 98.65 (C-1), 70.68 (C-3), 69.89 (C-2), 67.24 (OCH₂), 61.17 (C-4), 60.63 (C-5), 33.16, 30.51, 29.09, 29.03, 23.34 (CH₂). 

ESI-TOF: m/z calcd for C₁₁H₂₀N₃O₄S₂ [M - H]^-: 322.0901; found: 322.0905.

Allyl 4-Amino-4-deoxy-β- l -arabinopyranoside (23)

A solution of azide 10 (50 mg, 0.23 mmol) in pyridine-H₂O (10:1, 2.2 mL) was purged with argon. Et₃N (0.48 mL) and propane-1,3-dithiol (0.49 mL, 4.65 mmol) were added at r.t. and the mixture was stirred overnight. The mixture was co-evaporated with toluene (2 10 mL) and the residue was purified on an Isolute Si column (500 mg) using EtOAc as eluent. The first eluate (15 mL) contained residual propane-1,3-dithiol. Further elution with MeOH and pooling of the fractions afforded product 23; yield: 40 mg (91%); colorless syrup; R _f = 0.18 (CHCl₃-MeOH-AcOH, 4:1:0.1); [α]_D ^²0 +183.6 (c 0.4, MeOH).

^¹H NMR (300 MHz, CD₃OD): δ = 6.03-5.88 (CH=), 5.32 (dq, J = 1.7, 17.3 Hz, 1 H, CH=CH _{2 trans} ), 5.16 (dq, J = 1.5, 10.4 Hz, 1 H, CH=CH _{2 cis} ), 4.75 (d, J = 3.5 Hz, 1 H, H-1), 4.19 (m, 1 H, OCH₂), 4.02 (m, 1 H, OCH₂), 3.87 (dd, J = 2.5, 11.9 Hz, 1 H, H-5 _eq ), 3.82 (dd, J = 4.1, 9.2 Hz, 1 H, H-3), 3.68 (dd, J = 3.4, 9.2 Hz, 1 H, H-2), 3.48 (dd, J = 3.1, 11.9 Hz, 1 H, H-5 _ax ), 3.06-3.01 (m, 1 H, H-4).

^¹³C NMR (75 MHz, CD₃OD): δ = 134.23 (=CH), 116.07 (CH=CH₂), 98.33 (C-1), 69.32 (C-3), 68.84 (C-2), 68.27 (OCH₂), 62.29 (C-5), 50.96 (C-4).

ESI-TOF: m/z calcd for C₈H₁₅NO₄ [M + Na]⁺: 212.0893; found: 212.0898.

Propyl 4-Amino-4-deoxy-β- l -arabinopyranoside (24)

A suspension of azide 10 (37.0 mg, 0.53 mmol) and 10% Pd/C (15 mg) in anhyd MeOH (4 mL) was stirred at r.t. under H₂ at atmospheric pressure for 18 h. The suspension was filtered over a bed of Celite and the filtrate was concentrated. The remaining oil was further dried under high vacuum yielding the pure product 24; yield: 33 mg (97%); colorless oil; R _f = 0.17 (CHCl₃-MeOH-AcOH, 2:1:0.1); [α]_D ^²0 +197.5 (c 0.28, MeOH).

^¹H NMR (300 MHz, CD₃OD): δ = 4.75 (d, J = 3.6 Hz, 1 H, H-1), 3.87 (dd, J = 2.6, 11.9 Hz, 1 H, H-5 _eq ), 3.81 (dd, J = 4.2, 9.1 Hz, 1 H, H-3), 3.66 (dd, J = 3.6, 9.0 Hz, 1 H, H-2), 3.69-3.60 (m, 1 H, OCH₂), 3.47 (dd, J = 3.1, 11.9 Hz, 1 H, H-5 _ax ), 3.45-3.36 (m, 1 H, OCH₂), 3.07-3.01 (m, 1 H, H-4), 1.66-1.61 (m, 2 H, CH₂), 0.95 (t, J = 7.4 Hz, 3 H, CH₃).

^¹³C NMR (75 MHz, CD₃OD): δ = 100.5 (C-1), 71.1 (OCH₂), 70.6 (C-3), 70.2 (C-2), 66.99 (C-3), 63.4 (C-5), 52.3 (C-4), 23.7 (CH₂), 10.9 (CH₃).

MS: ESI-TOF: m/z calcd for C₈H₁₇NO₄ [M + H]⁺: 192.123; found: 192.1231.

Propyl 4-Acetamido-4-deoxy-β- l -arabinopyranoside (25)

A solution of amine 24 (40 mg, 0.209 mmol) in anhyd pyridine (2 mL) and Ac₂O (0.5 mL) was stirred for 36 h at r.t. The reaction mixture was diluted with EtOAc (15 mL) and stirred for 15 min with EtOH (0.5 mL). The organic phase was washed with aq 2 M HCl (8 mL), H₂O (10 mL), and sat. aq NaHCO₃ (10 mL), and dried (MgSO₄). Concentration afforded a colorless residue (63 mg, 95%). A portion of the residue (56 mg, 0.176 mmol) was dissolved in anhyd MeOH (2 mL) and treated with 0.1 M methanolic NaOMe (1.4 mL) for 15 h at r.t. The solution was neutralized with Dowex 50 H⁺ resin. The resin was filtered off, washed with MeOH (10 mL), and the filtrate was concentrated to give 25; yield: 30 mg (95%); colorless oil; R _f = 0.48 (CHCl₃-MeOH, 4:1); [α]_D ^²0 +138 (c 0.3, MeOH).

^¹H NMR (300 MHz, CD₃OD): δ = 5.97 (d, J = 8.7 Hz, 1 H, NHCOCH₃), 4.78 (d, J = 3.3 Hz, 1 H, H-1), 4.20-4.26 (m, 1 H, H-4), 3.92 (dd, J = 4.4, 9.0 Hz, 1 H, H-3), 3.83 (dd, J = 2.7, 11.8 Hz, 1 H, H-5 _eq ), 3.68 (dd, J = 3.3, 9.2 Hz, 1 H, H-2), 3.59-3.69 (m, 1 H, OCH₂), 3.50 (dd, J = 3.6 Hz, 11.8 Hz, 1 H, H-5 _ax ), 3.37-3.46 (m, 1 H, OCH₂), 1.99 (s, 3 H, COCH₃), 1.63 (m, J = 7.2 Hz, 2 H, CH₂), 0.96 (t, J = 7.4 Hz, 3 H, CH₃).

^¹³C NMR (75 MHz, CD₃OD): δ = 100.43 (C-1), 71.28 (C-1′), 70.82 (C-3), 69.71 (C-2), 62.51 (C-5), 51.21 (C-4), 23.79 (NHCOCH₃), 22.62 (C-2′), 10.96 (C-3′).

ESI-TOF: m/z calcd for C₁₀H₁₉NO₅ + Na [M + Na]⁺: 256.1155; found: 256.1151.

6-Mercaptohexyl 4-Amino-4-deoxy-β- l -arabinopyranoside (26)

Azide 21 (150 mg, 0.51 mmol) was dissolved in pyridine (5 mL), deionized H₂O (0.6 mL), and Et₃N (0.6 mL). The solution was purged with argon for 5 min and propane-1,3-dithiol (0.6 mL) was added. Stirring at r.t. overnight gave full conversion. The reaction mixture was co-evaporated with toluene (2 20 mL), and purified by flash chromatography over SiO₂ (6 g) using EtOAc and MeOH as eluents. The methanolic product fractions were pooled and evaporated to dryness to give 26 as a syrup, which was stored under argon; yield: 110 mg (80%, containing 15% disulfide according to NMR spectroscopy).

^¹H NMR (300 MHz, CD₃OD): δ = 4.76 (d, J = 3.7 Hz, 1 H, H-1), 3.86 (dd, J = 2.7, 11.8 Hz, 1 H, H-5 _eq ), 3.81 (dd, J = 4.4, 9.5 Hz, 1 H, H-3), 3.74-3.66 (m, 1 H, OCH₂), 3.67 (dd, J = 3.3, 9.5 Hz, 1 H, H-2), 3.52-3.39 (m, 2 H, H-5 _ax , OCH₂), 3.06-3.01 (m, 1 H, H-4), 2.52 (t, 2 H, SCH₂), 1.78-1.54 (m, 4 H, CH₂), 1.53-1.33 (m, 4 H, CH₂).

^¹³C NMR (75 MHz, CD₃OD): δ = 100.56 (C1), 70.69 (C3), 70.24 (C2), 69.34 (OCH₂), 63.56 (C5), 52.32 (C4), 35.08 (CH₂S), 30.46 (CH₂), 29.14 (CH₂), 26.73 (CH₂), 24.92 (CH₂).

BSA-Conjugate 27

According to the general procedure of the commercially available ‘Sigma-Aldrich Maleimide Activated BSA, KLH Conjugatio Kit- Stock No. MBK-1’: Thiol 26 (5 mg) was dissolved in buffer (0.5 mL; 20 mM sodium phosphate buffer, 100 mM EDTA, and 80 mM sucrose, pH 6.6) and immediately added to a solution of 5 mg/mL maleimide-activated BSA (20 mM sodium phosphate buffer, 230 mM NaCl, 2 mM EDTA, and 80 mM sucrose, pH 6.6). The reaction mixture was purged with argon for 2 min, and stirred at 4 ˚C overnight. The reaction mixture was purified over a Sephadex G-25M column (part of the BSA-Kit), and as eluent 0.01 M PBS-buffer in double distilled H₂O was used. The protein containing fractions were pooled and lyophilized to afford 1.5 mg of 27 as colorless solid.

Immunization of Mice and Rabbits with Ara4N-BSA (27)

Ten mice (Balb/c) were injected on day 0 subcutaneously into four sites of the back skin with Ara4N-BSA conjugate (50 µg in 200 µL of PBS) emulsified with an equal volume of Freund’s complete adjuvant. The animals received a booster injection intraperitoneally on day 28 (50 µg in 200 µL of PBS emulsified with an equal volume of Freund’s incomplete adjuvant) and bled on day 35. From two rabbits (chinchilla bastards) preimmune sera were collected on day -2. On day 0 they were injected with Ara4N-BSA conjugate (100 µg in 1 mL of PBS) emulsified with an equal volume of Freund’s complete adjuvant into the popliteal and elbow lymph nodes. The animals received a booster injection subcutaneously on day 47 (150 µg in 1 mL of PBS emulsified with an equal volume of Freund’s incomplete adjuvant) into 6 sites of the back skin and were bled on day 56.

ELISA Using Neoglycoconjugate and LPS Antigens

i) Neoglycoconjugates in carbonate buffer (50 mM, pH 9.2) were coated onto MaxiSorp microtiter plates (96-well, U-bottom, NUNC) at 4 C overnight. Antigen solutions were adjusted to equimolar concentrations based on the amount of ligand present in the respective glycoconjugate. If not stated otherwise, a volume of 50 µL was used. Plates were washed twice in PBS supplemented with Tween 20 (0.05%, Bio-Rad) and thimerosal (0.01%, PBS-T) and were then blocked with PBS-T supplemented with casein (2.5%, PBS-TC) for 1 h at 37 ˚C on a rocker platform followed by two washings. Appropriate antibody dilutions in PBS-TC supplemented with 5% BSA (PBS-TCB) were added and incubated for 1 h at 37 ˚C. After two washings, peroxidase-conjugated goat anti-mouse IgG or goat-antirabbit IgG (both heavy and light chain specific; Dianova; diluted 1:1.000 in PBS-TCB) was added and incubation was continued for 1 h at 37 ˚C. The plates were washed three times with BPS-T. Substrate solution was freshly prepared and was composed of 2,2′-azinobis(3-ethylbenzthiazolinsulfonic acid) diammonium salt (1 mg) dissolved in substrate buffer (0.1 M sodium citrate, pH 4.5; 1 mL) followed by the addition of H₂O₂ (25 µL of a 0.1% solution). After 30 min at 37 ˚C, the reaction was stopped by the addition of aq oxalic acid (2%) and the plates were read by a microplate reader (Tecan Sunrise) at 405 nm. Tests were run twice in quadruplicates with confidence values not exceeding 10%.

ii) When LPS was used as an antigen in ELISA, microtiter polyvinyl plates (96-well, Falcon 3911; Becton Dickinson) were coated with LPS of varying concentration (2 to 250 ng/well) diluted in PBS (pH 7.2) and were incubated overnight at 4 ˚C. PBS and PBS-containing solutions were supplemented with thimerosal (0.01%). Further incubation steps were performed at 37 ˚C under gentle agitation. The coated plates were washed four times with PBS and were blocked for 1 h with PBS supplemented with casein (2.5%, Sigma; PBS-C; 200 µL per well). Serial serum dilutions in PBS-C were subsequently added, and the mixture was incubated for 1 h at 37 ˚C. After washing as described above, secondary antibodies diluted in PBS-C (same source and dilution as above) were added. After four washings in PBS, the following steps were done as described for ELISA using neoglycoconjugate antigens.

Acknowledgment

The authors are grateful to Buko Lindner (Research Center Borstel) for recording the MALDI MS and Stephan Hann and Madeleine Dell’mour (BOKU Vienna) for recording the ESI-TOF MS. Financial support by Fonds zur Förderung der wissenschaftlichen Forschung (FWF grant P 19295) is gratefully acknowledged.

References

• For reviews, see:
• 1a Raetz C. Whitfield C. Annu. Rev. Biochem.  2002,  71:  635 
  Search in Google Scholar
• 1b Holst O. Molinaro A. In: Microbial Glycobiology   Moran AP. Holst O. Brennan PJ. von Itzstein M. Elsevier; Amsterdam: 2009.  p.29-55  ; and references cited therein
• 2 Gatzeva-Topalova PZ. May AP. Sousa MC. Biochem.  2005,  44:  5328 
  CrossrefSearch in Google Scholar
• 3 Breazeale SD. Ribeiro AA. Raetz CRH. J. Biol. Chem.  2003,  278:  24731 
  CrossrefSearch in Google Scholar
• 4 Sidorczyk Z. Kaca W. Brade H. Rieschel ET. Sinnwell V. Zähringer U. Eur. J. Biochem.  1987,  168:  269 
  CrossrefSearch in Google Scholar
• 5 Vinogradov EV. Sidorczyk Z. Knirel Y. Aust. J. Chem.  2002,  55:  61 
  CrossrefSearch in Google Scholar
• For reviews, see:
• 6a Vinion-Dubiel AD. Goldberg JB. J. Endotoxin Res.  2003,  9:  201 
  Search in Google Scholar
• 6b De Soyza A. Silipo A. Lanzetta R. Govan JR. Molinaro A. Innate Immun.  2008,  14:  127 
  Search in Google Scholar
• 7 Isshiki A. Kawahara K. Zähringer U. Carbohydr. Res.  1998,  313:  21 
  CrossrefSearch in Google Scholar
• 8 Gronow S. Xia G. Brade H. Eur. J. Cell Biol.  2010,  89:  3 
  CrossrefSearch in Google Scholar
• 9 Gronow S. Noah C. Blumenthal A. Lindner B. Brade H. J. Biol. Chem.  2003,  278:  1647 
  CrossrefSearch in Google Scholar
• 10 Vinogradov E. Lindner B. Seltmann G. Radziejewska-Lebrecht J. Holst O. Chem. Eur. J.  2006,  12:  6692 
  CrossrefSearch in Google Scholar
• 11 Moskowitz SM. Ernst RK. Miller SI. J. Bacteriol.  2004,  186:  575 
  CrossrefSearch in Google Scholar
• 12 Naleway JJ. Raetz CHR. Anderson L. Carbohydr. Res.  1988,  179:  199 
  CrossrefSearch in Google Scholar
• 13 Kline T. Trent SM. Stead CM. Lee MS. Sousa MC. Felise HB. Nguyen HV. Miller SI. Bioorg. Med. Chem. Lett.  2008,  18:  1507 
  CrossrefSearch in Google Scholar
• 14 Helm RF. Ralph J. Anderson L. J. Org. Chem.  1991,  56:  7015 
  CrossrefSearch in Google Scholar
• 15 Nilsson M. Westman J. Svahn C.-M. J. Carbohydr. Chem.  1993,  12:  23 
  CrossrefSearch in Google Scholar
• 16 Tadeo K. Ohguchi Y. Hasegawa R. Kitamura S. Carbohydr. Res.  1995,  278:  301 
  CrossrefSearch in Google Scholar
• 17 Ziser L. Withers SG. Carbohydr. Res.  1994,  265:  9 
  CrossrefSearch in Google Scholar
• 18 Tsuda Y. Nishimura M. Kobayashi T. Sato Y. Kanemitsu K. Chem. Pharm. Bull.  1991,  39:  2883 
  Search in Google Scholar
• 19 Kondo Y. Carbohydr. Res.  1982,  110:  339 
  CrossrefSearch in Google Scholar
• 20 Buchanan JG. Edgar AR. Large DG. J. Chem. Soc., Chem. Commun.  1969,  10:  558 
  Search in Google Scholar
• 21 Bayley H. Standring DN. Knowles JR. Tetrahedron Lett.  1978,  19:  3633 
  CrossrefSearch in Google Scholar
• 22 Westphal O. Jann K. Meth. Carbohydr. Chem.  1965,  5:  83 
  Search in Google Scholar

References

• For reviews, see:
• 1a Raetz C. Whitfield C. Annu. Rev. Biochem.  2002,  71:  635 
  Search in Google Scholar
• 1b Holst O. Molinaro A. In: Microbial Glycobiology   Moran AP. Holst O. Brennan PJ. von Itzstein M. Elsevier; Amsterdam: 2009.  p.29-55  ; and references cited therein
• 2 Gatzeva-Topalova PZ. May AP. Sousa MC. Biochem.  2005,  44:  5328 
  CrossrefSearch in Google Scholar
• 3 Breazeale SD. Ribeiro AA. Raetz CRH. J. Biol. Chem.  2003,  278:  24731 
  CrossrefSearch in Google Scholar
• 4 Sidorczyk Z. Kaca W. Brade H. Rieschel ET. Sinnwell V. Zähringer U. Eur. J. Biochem.  1987,  168:  269 
  CrossrefSearch in Google Scholar
• 5 Vinogradov EV. Sidorczyk Z. Knirel Y. Aust. J. Chem.  2002,  55:  61 
  CrossrefSearch in Google Scholar
• For reviews, see:
• 6a Vinion-Dubiel AD. Goldberg JB. J. Endotoxin Res.  2003,  9:  201 
  Search in Google Scholar
• 6b De Soyza A. Silipo A. Lanzetta R. Govan JR. Molinaro A. Innate Immun.  2008,  14:  127 
  Search in Google Scholar
• 7 Isshiki A. Kawahara K. Zähringer U. Carbohydr. Res.  1998,  313:  21 
  CrossrefSearch in Google Scholar
• 8 Gronow S. Xia G. Brade H. Eur. J. Cell Biol.  2010,  89:  3 
  CrossrefSearch in Google Scholar
• 9 Gronow S. Noah C. Blumenthal A. Lindner B. Brade H. J. Biol. Chem.  2003,  278:  1647 
  CrossrefSearch in Google Scholar
• 10 Vinogradov E. Lindner B. Seltmann G. Radziejewska-Lebrecht J. Holst O. Chem. Eur. J.  2006,  12:  6692 
  CrossrefSearch in Google Scholar
• 11 Moskowitz SM. Ernst RK. Miller SI. J. Bacteriol.  2004,  186:  575 
  CrossrefSearch in Google Scholar
• 12 Naleway JJ. Raetz CHR. Anderson L. Carbohydr. Res.  1988,  179:  199 
  CrossrefSearch in Google Scholar
• 13 Kline T. Trent SM. Stead CM. Lee MS. Sousa MC. Felise HB. Nguyen HV. Miller SI. Bioorg. Med. Chem. Lett.  2008,  18:  1507 
  CrossrefSearch in Google Scholar
• 14 Helm RF. Ralph J. Anderson L. J. Org. Chem.  1991,  56:  7015 
  CrossrefSearch in Google Scholar
• 15 Nilsson M. Westman J. Svahn C.-M. J. Carbohydr. Chem.  1993,  12:  23 
  CrossrefSearch in Google Scholar
• 16 Tadeo K. Ohguchi Y. Hasegawa R. Kitamura S. Carbohydr. Res.  1995,  278:  301 
  CrossrefSearch in Google Scholar
• 17 Ziser L. Withers SG. Carbohydr. Res.  1994,  265:  9 
  CrossrefSearch in Google Scholar
• 18 Tsuda Y. Nishimura M. Kobayashi T. Sato Y. Kanemitsu K. Chem. Pharm. Bull.  1991,  39:  2883 
  Search in Google Scholar
• 19 Kondo Y. Carbohydr. Res.  1982,  110:  339 
  CrossrefSearch in Google Scholar
• 20 Buchanan JG. Edgar AR. Large DG. J. Chem. Soc., Chem. Commun.  1969,  10:  558 
  Search in Google Scholar
• 21 Bayley H. Standring DN. Knowles JR. Tetrahedron Lett.  1978,  19:  3633 
  CrossrefSearch in Google Scholar
• 22 Westphal O. Jann K. Meth. Carbohydr. Chem.  1965,  5:  83 
  Search in Google Scholar