Parasite glycoconjugates. Part 11. Preparation of phosphodisaccharide synthetic probes, substrate analogues for the elongating α-D-mannopyranosylphosphate transferase in the Leishmania

Abstract

A set of phosphodisaccharides, substrate analogues, which will be used to study acceptor–substrate specificity of the Leishmania biosynthetic enzymes, are synthesized using the Koenigs–Knorr and trichloroacetimidate methods for the glycosylation reactions, S_N2 nucleophilic displacement of a triflic ester for epimerization, and the glycosyl hydrogenphosphonate method for phosphorylation.

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Introduction

Throughout the tropics and subtropics Leishmania parasites cause a variety of diseases ranging from self-limiting skin lesions to the often fatal visceral leishmaniasis. The surface lipophosphoglycan (LPG) produced by the infectious promastigote stage of all species of the Leishmania contains a polymeric section consisting of (1→6)-linked β-D-galactosyl-(1→4)-α-D-mannosyl phosphate repeating units. The importance of the LPG for parasite infectivity and survival ² makes the enzymes responsible for the biosynthesis of this glycoconjugate of great interest. Phospho-oligosaccharide fragments of the LPG of L. donovani, L. mexicana and L. major were synthesized ^3–6 in our laboratory and tested as acceptor substrates (in vitro) for the Leishmania α-D-mannopyranosylphosphate transferase (MPT) responsible for the transfer of α-D-Manp phosphate from GDP-Man to the growing phosphoglycan chain. It was shown ⁷ that the phosphodisaccharide 1 ^4,8 (representing one repeating unit of the phosphoglycan) is the minimal structure exhibiting acceptor substrate activity for the MPT.

In Part 9 ⁸ of this series, we disclosed our interest in the design and synthesis of various structural analogues of compound 1 to test the fine acceptor substrate specificity of the MPT and to gain more information about enzyme–substrate recognition. Thus, phosphodisaccharides 2–5, which are epimers of the substrate 1 at C-1′, C-2′, C-3′ or C-4′, respectively, have been synthesized.

We now report the chemical synthesis of the disaccharide phosphates 6–10. Compounds 6 and 7 are epimers of the substrate 1 at C-2 and C-3, respectively, of the D-mannopyranose moiety. Compounds 8 and 9 are substrate analogues deoxygenated at positions C-6 and C-6′, respectively. In this context, the preparation of the analogue 10, which is an epimer of compound 9 at C-1′ and could be (as well as the analogue 9 itself ) a potential inhibitor of the enzyme, is also described. The information obtained from testing the acceptor activity of the substrate analogues 2–10 will be used to predict which sugar hydroxy groups of compound 1 are involved in enzyme–substrate recognition events and to design potential enzyme inhibitors.

Results and discussion

The synthetic schemes for the preparation of the phosphodisaccharides 6–10 consist of a few general steps (Scheme 1): 1) synthesis of fully protected disaccharide derivatives A; 2) anomeric de-O-protection ( → B); 3) H-phosphonylation at position O-1 ( → C); 4) coupling of the H-phosphonates C with dec-9-en-1–ol (using the glycosyl H-phosphonate method) ⁹ to furnish the protected glycosyl phosphodiesters D; 5) total de-O-protection.

Scheme 1 R = Ac, or Bz; R′ = Ac, or Bz, or Bn.

The octa-O-acetyl-α,β-lactose 11 (α∶β = 7∶1; which is a precursor of the phosphodisaccharide 6; Scheme 2) was prepared by conventional acetylation of α-lactose and then converted to the hemiacetal 12 (83%; α∶β = 4∶1) by anomeric de-O-acylation ^3–6,8–10 with dimethylamine in CH₃CN–THF. H-Phosphonylation ^3–6,8–10 of compound 12 with triimidazolylphosphine (prepared in situ from PCl₃, imidazole and Et₃N) followed by mild hydrolysis produced a mixture of α- and β-linked H-phosphonates 13 (α∶β = 4∶1, as evinced from ¹H and ³¹P NMR spectra, see Experimental section), which were not separable by flash-column chromatography. This mixture was converted to the pure α-(H-phosphonate) 14 (48% based on the hemiacetal 12) by treatment with H₃PO₃ in acetonitrile. This procedure was developed first for the preparation ¹⁰ of pure 2,3,4,6-tetra-O-benzoyl-α-D-galactopyranosyl H-phosphonate and utilizes the higher reactivity of the β-linked glycosyl H-phosphonate, converting it to either the α-linked isomer (as a result of S_N2-attack), or easily separable hemiacetal derivative (product of acid-catalyzed cleavage of the H-phosphonate group).

Scheme 2 Reagents: i, Me₂NH, MeCN–THF; ii, (a) triimidazolylphosphine, MeCN; (b) Et₃NHHCO₃, water (pH 7); iii, H₃PO₃, MeCN; iv, (a) dec-9-en-1-ol, trimethylacetyl chloride, pyridine; (b) I₂, pyridine–water; v, NaOMe, MeOH.

Synthesis of the protected benzyl β-D-galactopyranosyl-(1→4)-α-D-altropyranoside 22 (which is a precursor of the phosphodisaccharide 7; Scheme 3) was performed from benzyl 2,3,6-tri-O-benzoyl-α-D-altropyranoside 20 and acetobromogalactose 21. The altropyranoside 20 in turn was prepared starting from benzyl α-D-mannopyranoside, which was converted first to the 2-O-benzoate 17 (65%) by 4,6-O-isopropylidenation ¹¹ with 2-methoxypropene ( → 16) followed by selective benzoylation ¹² with benzoyl cyanide in the presence of Et₃N. Successive reaction with triflic anhydride in CH₂Cl₂ in the presence of pyridine led to the triflate 18, which reacted with tetrabutylammonium benzoate (Bu₄NOBz) in toluene (60 °C) to give the altroside 19 (73%). The D-altro-configuration of the derivative 19 was confirmed by the characteristic values of J_2,3 = J_3,4 = 3.0 Hz in ¹H NMR spectrum. Further, compound 19 was converted to the glycosyl acceptor 20 (67%) by acid hydrolysis followed by selective 6-O-benzoylation with benzoyl cyanide.

Scheme 3 Reagents: i, BzCN, Et₃N, MeCN; ii, Tf₂O, CH₂Cl₂–pyridine; iii, Bu₄NOBz, toluene; iv, 80% AcOH; v, AgOTf, Ag₂CO₃, MS 4 Å, CH₂Cl₂; vi, H₂, Pd(OH)₂/C, THF; vii, (a) triimidazolylphosphine, MeCN; (b) Et₃NHHCO₃, water (pH 7); viii, H₃PO₃, MeCN; ix, (a) dec-9-en-1-ol, trimethylacetyl chloride, pyridine; (b) I₂, pyridine–water; x, NaOMe, MeOH.

Glycosylation of the acceptor 20 with the bromide 21 in the presence of silver triflate (AgOTf ), silver carbonate and molecular sieves 4 Å in dichloromethane provided the disaccharide 22 in 52% yield. Hydrogenolysis of compound 22 over Pd(OH)₂/C afforded a mixture of α- and β-hemiacetals 24 in the ratio α∶β = 0.8∶1 (confirmed by ¹H NMR data, see Experimental section). Probably, the mutarotation was facilitated because of unfavourable 1,3-synaxial interaction between 1-OH and 3-benzoate in the α-hemiacetal. The anomeric mixture 24 was converted to the pure α-(H-phosphonate) 23 using the same procedure as described for the H-phosphonate 14: i.e., the reaction with triimidazolylphosphine and mild hydrolysis ( → 25) followed by treatment with H₃PO₃ in CH₃CN. This produced the H-phosphonate 23 (35% based on the disaccharide 22) along with the recovered hemiacetal 24 (49%).

The hepta-O-acetyl-β-D-galactopyranosyl-(1→4)-α-D-rhamnopyranose 29 (which is a precursor of the phosphodisaccharide 8; Scheme 4) was synthesized using acetobromogalactose 21 and methyl 2,3-O-isopropylidene-α-D-rhamnopyranoside ¹³27 as starting materials. Their coupling in the presence of Hg(CN)₂–HgBr₂ in acetonitrile–toluene gave the disaccharide 28 (74%), which was converted to the crystalline heptaacetate 29 in 69% yield by acid hydrolysis followed by acetolysis/acetylation ¹⁴ with 1.32% (v/v) H₂SO₄ in acetic anhydride.

Scheme 4 Reagents: i, Hg(CN)₂, HgBr₂, MeCN–PhMe; ii, (a) aq. TFA, CHCl₃; (b) H₂SO₄, Ac₂O; iii, Me₂NH, MeCN–THF; iv, (a) triimidazolylphosphine, MeCN; (b) Et₃NHHCO₃, water (pH 7); v, (a) dec-9-en-1-ol, trimethylacetyl chloride, pyridine; (b) I₂, pyridine–water; vi, NaOMe, MeOH.

The hepta-O-benzoyl-β-D-fucopyranosyl-(1→4)-α-D-mannopyranose 37 (which is a precursor of the phosphodisaccharide 9; Scheme 5) was prepared in 62% yield by the glycosylation of the D-mannose tetrabenzoate ³36 with the α-D-fucosyl trichloroacetimidate 35 in the presence of trimethylsilyl (TMS) triflate. A small proportion of the isomeric α-linked disaccharide 40 (13%; a precursor of the phosphodisaccharide 10; Scheme 6) was also isolated from the reaction mixture. The trichloroacetimidate 35 in turn was synthesized from D-fucose by consecutive standard benzoylation ( → 33), anomeric deprotection with Me₂NH ( → 34; 61%) and the reaction (93% yield) with trichloroacetonitrile in the presence of 1,8–diazabicyclo[5.4.0]undec-7-ene (DBU).¹⁵

Scheme 5 Reagents: i, Me₂NH, MeCN–THF; ii, CCl₃CN, DBU, CH₂Cl₂; iii, TMS triflate, MS 4 Å, CH₂Cl₂; iv, (a) triimidazolylphosphine, MeCN; (b) Et₃NHHCO₃, water (pH 7); v, (a) dec-9-en-1-ol, trimethylacetyl chloride, pyridine; (b) I₂, pyridine–water; vi, NaOMe, MeOH.

Scheme 6 Reagents: i, Me₂NH, MeCN–THF; ii, (a) triimidazolylphosphine, MeCN; (b) Et₃NHHCO₃, water (pH 7); iii, (a) dec-9-en-1-ol, trimethylacetyl chloride, pyridine; (b) I₂, pyridine–water; iv, NaOMe, MeOH.

The β-configuration of newly formed glycosidic linkages in the disaccharides 22, 28 and 37 followed from the characteristic values of J_1′,2′ (7.5–8.0 Hz) in ¹H NMR spectra. For the α-D-fucoside 40 the corresponding value is J_1′,2′ = 3.0 Hz.

In contrast to the anomeric de-O-acylation of the peracetylated lactose 11 (see above), similar reaction of the disaccharide heptaacetate 29 and heptabenzoates 37 and 40 with dimethylamine in CH₃CN–THF afforded the pure α-hemiacetal derivatives 30, 38 and 42 (66–90%), respectively. Compounds 30, 38 and 42 were then treated with triimidazolylphosphine followed by mild hydrolysis to produce the α-linked glycosyl H-phosphonates 31, 39 and 43, respectively, in 70–97% yield.

The structures of all the prepared disaccharide H-phosphonates were confirmed by NMR and mass spectrometric data (see Experimental section). For example, signals characteristic of the H-phosphonate group [δ_P 0.77; δ_H 5.67 (dd, J_1,2 3.4, J_1,P 8.8, 1-H), 6.89 (d, ¹J_H,P 637.8, HP)] were present in the ³¹P and ¹H NMR spectra of the derivative 14. The α-configuration of the D-glucopyranosyl residue followed from the characteristic value of J_1,2. The main signal in the (electrospray) ES(−) mass spectrum corresponded to the pseudomolecular ion (m/z 698.9, [M − Et₃N − H]⁻) for the compound. The structures 23, 31, 39 and 43 were established in similar manner apart from that the α-configuration of the D-Altp (in compound 23), D-Rhap (in compound 31) and D-Manp (in compounds 39 and 43) residues followed from the characteristic positions of the 3- and 5-H resonances in ¹H NMR spectra. The chemical shifts of these signals were close to those of 3- and 5-H of the disaccharide derivatives 22 (containing a benzyl 2,3,6-tri-O-benzoyl-α-D-altropyranoside moiety), 29 (containing a 1,2,3-tri-O-acetyl-α-D-rhamnopyranose moiety) and 37 and 40 (both containing 1,2,3,6-tetra-O-benzoyl-α-D-mannopyranose moieties), respectively.

The glycosyl H-phosphonates 14, 23, 31, 39 and 43 were converted to the protected phosphodiesters 15, 26, 32, 41 and 44 (75-96% yield), respectively, by their condensation with dec-9-en-1-ol in pyridine in the presence of trimethylacetyl chloride followed by oxidation of the resulting H-phosphonic diesters with iodine in aq. pyridine. The deprotected phosphodisaccharides 6–10 were prepared from the derivatives 15, 26, 32, 41 and 44, respectively, by de-O-acylation with 0.05 mol dm⁻³ methanolic sodium methoxide in 88–100% yield.

The structures of the compounds 6–10 and the protected phosphodiesters 15, 26, 32, 41 and 44 were confirmed by NMR and mass spectrometric data. The ³¹P NMR spectra exhibited single signals [δ_P between −1.35 and −1.96 for the deprotected compounds 6–10 (in D₂O) and between −1.67 and −3.12 for the protected phosphodiesters (in CDCl₃)], which are characteristic for glycoside-linked phosphodiesters.^3–6,8–10 The presence of a (1→1)-phosphodiester linkage at the reducing terminus of each of the disaccharides 6–10 was confirmed by the C-1 and C-2 signals of the corresponding monosaccharide residue and the dec-9-enyl unit in the ¹³C NMR spectra (Table 1). These signals were shifted as a result of the α- and β-effects of phosphorylation and were coupled with phosphorus (or broadened).

Table 1 ¹³C and ³¹P NMR data [δ_C and δ_P in ppm; J_C,P and J_C,H in Hz; spectra recorded in D₂O] and ESMS(−) data (m/z) for the phosphodisaccharides 6–10

Residue	Atom	6 ^a	7 ^a	8 ^b	9 ^a	10 ^a
a Additional signals of Et3NH^+ [δC 9.20–9.37 (CH3) and δC 47.41–47.63 (CH2)] were present. b Additional signals of CC H2C [δC 25.95–26.26, 29.19–30.09 and 34.16–34.44] were present. c Corresponds to the pseudomolecular ions [M − Et3N − H]^−. For compounds 6 and 7 (triethylammonium salt), C28H56NO14P requires M, 661.34 (expected m/z, 559.14); for compounds 8–10 (triethylammonium salt), C28H56NO13P requires M, 645.35 (expected m/z, 534.15).
Dec-9-enyl	OC H2CH2	67.20d	67.82d	67.76d	67.45br	67.01br
		J C,P 4.0	J C,P ≈6	J C,P ≈6
	OCH2C H2	31.15d	30.96d	30.87d	30.95br	31.03d
		J C,P 9.0	J C,P 8.8	J C,P 5.9		J C,P 8.3
	–CH[double bond, length half m-dash]	140.32	141.64	141.54	140.87	141.52
	[double bond, length half m-dash]CH2	115.10	115.09	115.00	115.00	114.98
Aldose	C-1	95.71d	96.76br	96.70br	96.69br	96.78d
		J C,P 6.9				J C,P 5.8
			J C,H 171.3	J C,H 169.7	J C,H 171.0	J C,H 170.5
	C-2	72.10d	71.54d	71.18d	70.89d	71.29d
		J C,P 7.7	J C,P 10.0	J C,P 6.9	J C,P 7.2	J C,P 7.5
	C-3	72.23	70.95	69.61	69.76	70.54
	C-4	78.77	74.38	82.62	77.20	76.98
	C-5	72.39	69.78	69.44	73.12	73.30
	C-6	60.65	61.53	17.82	61.10	61.30
Aldose′	C-1′	103.85	105.04	104.27	103.87	102.08
			J C,H 162.5	J C,H 161.0	J C,H 160.5	J C,H 171.0
	C-2′	71.83	71.96	72.09	71.45	69.64
	C-3′	73.58	73.67	73.61	73.71	71.23
	C-4′	69.51	69.96	69.72	72.12	72.57
	C-5′	76.25	76.21	76.37	72.01	68.22
	C-6′	61.93	62.20	62.18	16.34	16.42
Phosphate	P	−1.96	−1.35	−1.50	−1.41	−1.66
	m/z ^c	559.34	558.90	543.25	543.10	543.10

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The α-configuration of the D-glucopyranosyl phosphate fragments in compounds 6 and 15 was evident from the characteristic values of J_1,2 = 3.4–3.5 Hz in the ¹H NMR spectra (see Experimental section). The α-configuration of the D-altropyranosyl residue in the phosphodisaccharide 7 followed from the characteristic value† of ¹J_C,H = 171.3 Hz for the signal of C-1 and the characteristic position of the C-5 resonance of D-Altp in the ¹³C NMR spectrum (Table 1). The chemical shift of the C-5 signal (δ_C 69.78) is fairly close to that of C-5 (δ_C 70.00)‡ of methyl α-D-altropyranoside.¹⁷

The α-configuration of the D-mannopyranosyl phosphate fragments in compounds 9 and 10 and of the D-rhamnopyranosyl phosphate in compound 8 was confirmed by 1) the characteristic values † of ¹J_C,H for the signals of C-1 and 2) the characteristic positions of the C-3 and C-5 resonances of D-Manp and D-Rhap residues, respectively, in the ¹³C NMR spectra (see Table 1). The chemical shifts of the signals of C-3 and -5 of D-Manp and C-3 of D-Rhap (i.e., 6-deoxy-D-mannose) are close to those of C-3 and C-5 of α-D-mannopyranosyl phosphate ¹⁸ taking into account the influence of the glycosyl substituents at position 4. The chemical shift of C-5 resonance (δ_C 69.44) of D-Rhap in compound 8 is very close to that of C-5 (δ_C 69.40)§ of methyl α-D-rhamnopyranoside.¹⁷

The α-configuration of the glycosyl phosphate linkages in the protected derivatives 26, 32, 41 and 44 followed from the characteristic positions of 1-, 3- and 5-H resonances in their ¹H NMR spectra (see Experimental section).

The molecular masses of the phosphodiesters 6–10, 15, 26, 32, 41 and 44 were confirmed by electrospray mass spectrometry. The signals in the ES(−) mass spectra corresponded to the pseudomolecular ions for the disaccharide phosphates (see Table 1 and Experimental section). A biochemical evaluation of compounds 6–10 will be published elsewhere ¹⁹ in due course.

Experimental

General procedures

Optical rotations were measured with a Perkin-Elmer 141 polarimeter; [α]_D-values are given in units of 10⁻¹ deg cm² g⁻¹. NMR spectra (¹H at 200 and 500 MHz, ¹³C{¹H} at 50.3 and 125 MHz, and ³¹P{¹H} at 81 and 202.5 MHz) were recorded with Bruker AM-200 and AM-500 spectrometers for solutions in CDCl₃, unless otherwise indicated. Chemical shifts (δ in ppm) are given relative to those for Me₄Si (for ¹H and ¹³C) and external aq. 85% H₃PO₄ (for ³¹P); J-values are given in Hz. ES mass spectra were recorded with a Micromass Quattro system (Micromass Biotech, UK). TLC was performed on Kieselgel 60 F₂₅₄ (Merck) with A, toluene–ethyl acetate (95∶5); B, toluene–ethyl acetate (9∶1); C, toluene–ethyl acetate (7∶3); D, toluene–ethyl acetate (3∶7); E, dichloromethane–methanol (95∶5); F, chloroform–methanol (8∶2); and G, chloroform–methanol–water (10∶10∶3) as developers and detection under UV light or by charring with sulfuric acid–water–ethanol (15∶85∶5). Flash-column chromatography (FCC) was performed on Kieselgel 60 (0.040–0.063 mm) (Merck). Dichloromethane, acetonitrile and toluene were freshly distilled from CaH₂. Solutions worked up were concentrated under reduced pressure at < 40 °C.

2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl-(1→4)-2,3,6-tri-O-acetyl-α,β-D-glucopyranose 12

To a solution of 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-(1→4)-1,2,3,6-tetra-O-acetyl-α,β-D-glucopyranose 11 (1 g, 1.47 mmol) [prepared by standard acetylation of α-lactose with Ac₂O in pyridine at 0 °C; δ_H (200 MHz) (inter alia) 1.94, 1.98, 2.10, 2.13 and 2.15 (15 H, 5 × s, 5 × Ac), 2.03 (9 H, s, 3 × Ac), 3.79 (t, J_3,4 = J_4,5 = 9.6, 4-H^α), 3.86 (1 H, dt, J_5′,6′ 6.6, 5′-H), 3.93–4.17 (4 H, m, 5-H, 6-H^a and 6′-H₂), 4.42 (1 H, dd, J_5,6b 1.6, J_6a,6b 12.4, 6-H^b), 4.45 (d, J_1′,2′ 7.9, 1′-H^α), 4.54 (d, J_1′,2′ 8.2, 1′-H^β), 4.94 (1 H, dd, J_3′,4′ 3.4, 3′-H), 4.98 (dd, J_2,3 9.9, 2-H^α), 5.10 (dd, J_2′,3′ 10.6, 2′-H^α), 5.33 (1 H, dd, J_4′,5′ 0.5, 4′-H), 5.44 (dd, 3-H^α), 5.65 (d, J_1,2 7.1, 1-H^β) and 6.22 (d, J_1,2 3.6, 1-H^α); α∶β ≈ 7∶1] in acetonitrile (6 cm³) was added 2 mol dm⁻³ Me₂NH in THF (4 cm³; 7.96 mmol) and the mixture was kept at rt with monitoring by TLC (solvent D). After 4–9 h the mixture was concentrated to dryness and acetonitrile was evaporated off from the residue. FCC [ethyl acetate–toluene, (2∶8) → (8∶2)] of the residue gave the disaccharide α,β-hemiacetal12 (0.779 g, 83%) as an amorphous solid, [α]²⁵_D +35.2 (c 1.06, CHCl₃) (Found: C, 48.8; H, 5.6. C₂₆H₃₆O₁₈ requires C, 49.1; H, 5.7%); δ_H (200 MHz) (inter alia) 1.96, 2.03, 2.04, 2.05, 2.07, 2.12 and 2.15 (21 H, 7 × s, 7 × Ac), 3.75 (dd, J_4,5 9.3, 4-H^α), 3.86 (1 H, dt, J_5′,6′ 6.3, 5′-H), 4.00–4.22 (4 H, m, 5-H, 6-H^a and 6′-H₂), 4.47 (d, J_1′,2′ 7.7, 1′-H^β), 4.48 (1 H, dd, J_5,6b 3.4, J_6a,6b 11.2, 6-H^b), 4.49 (d, J_1′,2′ 7.9, 1′-H^α), 4.76 (m, 1- and 2-H^β), 4.81 (dd, 2-H^α), 4.94 (1 H, dd, J_3′,4′ 3.2, 3′-H), 5.09 (dd, J_2′,3′ 10.6, 2′-H^β), 5.11 (dd, J_2′,3′ 10.5, 2′-H^α), 5.22 (t, J_2,3 = J_3,4 = 9.3, 3-H^β), 5.34 (1 H, dd, J_4′,5′ 0.5, 4′-H), 5.36 (d, J_1,2 3.4, 1-H^α) and 5.51 (t, J_2,3 = J_3,4 = 9.7, 3-H^α); α:β = 4∶1.

2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl-(1→4)-2,3,6-tri-O-acetyl-α-D-glucopyranosyl hydrogenphosphonate, triethylammonium salt 14

To a stirred solution of imidazole (0.65 g, 9.56 mmol) in acetonitrile (10 cm³) at 0 °C was added phosphorus trichloride (0.25 cm³, 2.87 mmol) followed by Et₃N (1.4 cm³, 10.04 mmol). The mixture was stirred for 20 min, after which a solution of compound 12 (0.304 g, 0.478 mmol) in MeCN (10 cm³) was added dropwise over a period of 10–15 min at 0 °C. The mixture was stirred at rt for 30–40 min and quenched with 1 mol dm⁻³ triethylammonium (TEA) hydrogen carbonate (pH 7; 4 cm³). The clear solution was stirred for 15 min, CH₂Cl₂ (100 cm³) was added and the organic layer was washed in turn with ice-cold water (2 × 40 cm³) and cold 0.5 mol dm⁻³ TEA hydrogen carbonate (2 × 40 cm³), dried by filtration through cotton wool, and concentrated to give the α,β-(H-phosphonate) 13, δ_P 0.43 (P^α) and 1.20 (P^β); δ_H (200 MHz) (inter alia) 5.20 (dd, J_1,2 7.7, J_1,P 9.1, 1-H^β), 5.67 (dd, J_1,2 3.4, J_1,P 8.8, 1-H^α), 6.88 (d, ¹J_H,P 644.0, H-P^β) and 6.91 (d, ¹J_H,P 637.8, H-P^α); α∶β = 4∶1.

The residue was dissolved in CH₃CN (15 cm³) and anhydrous H₃PO₃ (0.67 g, 8.17 mmol) was added. The mixture was stirred at rt for 19 h, then diluted with CH₂Cl₂ (100 cm³) and washed successively with cold saturated aq. NaHCO₃ (2 × 40 cm³) and cold 0.5 mol dm⁻³ aq. TEA hydrogen carbonate (2 × 40 cm³). The organic phase (containing the hemiacetal 12) was discarded. The aqueous washings were then combined, and extracted with CH₂Cl₂ (4 × 40 cm³). The combined organic washings were dried by filtration through cotton wool, and concentrated to produce the α-hydrogenphosphonate 14 (0.184 g, 48%) as a chromatographically homogeneous amorphous solid, [α]²⁶_D +41.8 (c 0.97, CHCl₃); δ_H (200 MHz) 1.32 (9 H, t, 3 × MeCH₂), 1.92, 2.00, 2.02, 2.07 and 2.10 (15 H, 5 × s, 5 × Ac), 1.99 (6 H, s, 2 × Ac), 3.04 (6 H, q, 3 × MeCH₂), 3.74 (1 H, t, J_3,4 = J_4,5 = 9.6, 4-H), 3.84 (1 H, t, J_5′,6′ 6.7, 5′-H), 3.97–4.20 (4 H, m, 5-H, 6-H^a and 6′-H₂), 4.41 (1 H, d, J_1′,2′ 7.7, 1′-H), 4.42 (1 H, br d, J_6a,6b 10.9, 6-H^b), 4.84 (1 H, dd, J_1,2 3.4, 2-H), 4.90 (1 H, dd, J_3′,4′ 3.3, 3′-H), 5.06 (1 H, dd, J_2′,3′ 10.3, 2′-H), 5.30 (1 H, d, 4′-H), 5.44 (1 H, t, J_2,3 9.6, 3-H), 5.67 (1 H, dd, J_1,P 8.8, 1-H) and 6.89 (1 H, d, J_H,P 637.8, HP); δ_P 0.77; ESMS(−) data: m/z 698.9 (100%, [M − Et₃N − H]⁻) (expected m/z, 699.08. C₃₂H₅₂NO₂₀P requires M, 801.28).

Dec-9-enyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-(1→4)-2,3,6-tri-O-acetyl-α-D-glucopyranosyl phosphate, triethylammonium salt 15

A mixture of the H-phosphonate 14 (126 mg, 0.16 mmol) and dec-9-en-1-ol (0.056 cm³, 0.31 mmol) was dried by evaporation of pyridine (3 × 2 cm³) therefrom. The residue was dissolved in pyridine (1 cm³), trimethylacetyl chloride (0.048 cm³, 0.39 mmol) was added, and the mixture was stirred at rt for 10–15 min, whereafter a freshly prepared solution of iodine (80 mg, 0.314 mmol) in pyridine–water (95∶5; 2 cm³) was added. After 30 min, CH₂Cl₂ was added and the solution was washed successively with ice-cold 1 mol dm⁻³ aq. Na₂S₂O₃ and cold 0.5 mol dm⁻³ aq. TEA hydrogen carbonate, dried by filtration through cotton wool, and concentrated. FCC [CH₂Cl₂–MeOH–Et₃N, (99∶0∶1) → (89∶10∶1)] of the residue gave the phosphodiester 15 (144 mg, 96%) as an amorphous solid, [α]²⁵_D +37 (c 0.96, CHCl₃); δ_H (200 MHz) 1.20–1.32 (10 H, m, 5 × CH₂), 1.29 (9 H, t, 3 × MeCH₂), 1.57 (2 H, tt, J 6.9, OCH₂CH₂CH₂), 1.93, 2.01, 2.02, 2.09 and 2.12 (15 H, 5 × s, 5 × Ac), 2.00 (8 H, s, 2 × Ac and m, CH₂CH₂CH=), 3.03 (6 H, q, 3 × MeCH₂), 3.76 (1 H, t, J_3,4 = J_4,5 = 9.6, 4-H), 3.83 (1 H, t, J_5′,6′ 6.7, 5′-H), 3.85 (2 H, m, OCH₂CH₂), 3.98–4.12 (3 H, m, 6-H^a and 6′-H₂), 4.17 (1 H, ddd, J_5,6a 2.4, 5-H), 4.41 (1 H, d, J_1′,2′ 7.7, 1′-H), 4.45 (1 H, dd, J_5,6b 1.3, J_6a,6b 12.0, 6-H^b), 4.83 (1 H, ddd, J_1,2 3.4, J_2,P 1.9, 2-H), 4.90 (1 H, dd, 3′-H), 4.86 (1 H, dd, ²J_H,H 1.4, ³J_H,H-Z 10.3, H CH[double bond, length half m-dash]CH), 4.95 (1 H, dd, ³J_H,H-E 17.0, HCH[double bond, length half m-dash]CH), 5.07 (1 H, dd, J_2′,3′ 10.4, 2′-H), 5.31 (1 H, d, J_3′,4′ 3.2, 4′-H), 5.45 (1 H, t, J_2,3 9.6, 3-H), 5.63 (1 H, dd, J_1,P 8.0, 1-H) and 5.77 (1 H, ddt, J_H,CH2 6.7, CH₂CH[double bond, length half m-dash]CH₂); δ_P −1.67; ESMS(−): m/z 853.0 (100%, [M − Et₃N − H]^–) (expected m/z, 853.22. C₄₂H₇₀NO₂₁P requires M, 955.42).

Dec-9-enyl β-D-galactopyranosyl-(1→4)-α-D-glucopyranosyl phosphate, triethylammonium salt 6

To a solution of compound 15 (138 mg) in MeOH (15 cm³) was added 0.5 mol dm⁻³ methanolic NaOMe (1.7 cm³). The mixture (now 0.05 mol dm⁻³ in NaOMe) was kept at room temperature for 1 h, whereafter it was deionized with Dowex 50W-X4 (H⁺) resin, filtered, and immediately neutralized with Et₃N. The solution was concentrated and methanol was evaporated off from the residue. The phosphodiester 6 (96 mg, 100%) was thereby obtained as an amorphous solid, [α]²⁵_D +54.5 (c 1, MeOH); δ_H (200 MHz; D₂O) (inter alia) 1.16 (9 H, t, 3 × MeCH₂), 1.19 (10 H, m, 5 × CH₂), 1.48 (2 H, tt, J 6.9, OCH₂CH₂CH₂), 1.91 (2 H, dt, J 6.6, CH₂CH₂CH[double bond, length half m-dash]), 3.06 (6 H, q, 3 × MeCH₂), 4.34 (1 H, d, J_1′,2′ 7.6, 1′-H), 5.33 (1 H, dd, J_1,2 3.5, J_1,P 7.1, 1-H) and 5.71 (1 H, ddt, J_H,CH2 6.6, J_H,H-Z 10.2, J_H,H-E 17.0, CH₂CH[double bond, length half m-dash]CH₂); δ_C, δ_P and ESMS(−) data: see Table 1.

Benzyl 2-O-benzoyl-4,6-O-isopropylidene-α-D-mannopyranoside 17

To a stirred solution of benzyl 4,6-O-isopropylidene-α-D-mannopyranoside 16 (3.1 g, 10 mmol) [prepared from benzyl α-D-mannopyranoside and 2-methoxypropene in 85% yield, [α]²⁵_D +85 (c 1, CHCl₃), R_f 0.3 (solvent E ) (Found: C, 61.8; H, 7.2. C₁₆H₂₂O₆ requires C, 61.9; H, 7.1%) as described for the preparation of methyl 4,6-O-isopropylidene-α-D-mannopyranoside ¹¹] and BzCN (1.57 g, 12 mmol) in acetonitrile (20 cm³) was added Et₃N (0.025 cm³). After 30 min, methanol was added, the reaction mixture was concentrated, and toluene was evaporated off from the residue. FCC (solvent A) gave the monobenzoate17 (3.15 g, 76%) as an amorphous solid, [α]²⁵_D +48 (c 1, CHCl₃); R_f 0.2 (solvent B) (Found: C, 66.25; H, 6.3. C₂₃H₂₆O₇ requires C, 66.65; H, 6.3%); δ_H (200 MHz) 1.49 and 1.61 (6 H, 2 × s, 2 × Me), 3.73-3.93 (3 H, m, 5-H and 6-H₂), 4.07 (1 H, t, J_3,4 = J_4,5 = 9.0, 4-H), 4.26 (1 H, dd, J_2,3 3.4, 3-H), 4.53 and 4.73 (2 H, AB q, J 11.7, CH₂Ph), 5.00 (1 H, d, J_1,2 1.3, 1-H), 5.50 (1 H, dd, 2-H) and 7.15–8.15 (10 H, m, 2 × Ph).

Benzyl 2,3-di-O-benzoyl-4,6-O-isopropylidene-α-D-altropyranoside 19

Triflic anhydride (2.05 cm³, 12.2 mmol) was added dropwise to a cooled (0 °C) stirred solution of compound 17 (2.53 g, 6.11 mmol) in CH₂Cl₂ (50 cm³) containing pyridine (3.85 cm³, 48.9 mmol), and then the reaction mixture was allowed to warm to rt. After 1 h, the mixture was diluted with CH₂Cl₂, washed successively with ice-cold 0.1 mol dm⁻³ HCl, ice-cold saturated aq. NaHCO₃ and water, and dried by filtration through cotton wool. The filtrate was concentrated to dryness and toluene was evaporated off from the residue to produce the triflate 18 [R_f 0.5 (solvent B), δ_H (200 MHz) 1.45 and 1.58 (6 H, 2 × s, 2 × Me), 3.83–3.93 (3 H, m, 5-H and 6-H₂), 4.28 (1 H, t, J_3,4 = J_4,5 = 9.1, 4-H), 4.57 and 4.72 (2 H, AB q, J 11.7, CH₂Ph), 5.02 (1 H, d, J_1,2 1.1, 1-H), 5.25 (1 H, dd, J_2,3 3.6, 3-H), 5.65 (1 H, dd, 2-H) and 7.10–8.10 (10 H, m, 2 × Ph)].

A solution of tetrabutylammonium benzoate (3.63 g, 10 mmol; dried beforehand by evaporation of anhydrous toluene therefrom) in toluene (20 cm³) was added to a solution of the triflate 18 in the same solvent (30 cm³). The reaction mixture was stirred at 60 °C for 7 h, then diluted with CH₂Cl₂, washed successively with saturated aq. NaHCO₃ and water, dried by filtration through cotton wool and concentrated. FCC (solvent A) gave the altroside19 (2.3 g, 73%), mp 142–144 °C (from diethyl ether–hexane); [α]²⁵_D +16 (c 1, CHCl₃); R_f 0.5 (solvent B) (Found: C, 69.8; H, 5.9. C₃₀H₃₀O₈ requires C, 69.5; H, 5.8%); δ_H (200 MHz) 1.35 and 1.61 (6 H, 2 × s, 2 × Me), 3.90 (1 H, t, J_5,6a = J_6a,6b = 9.6, 6-H^a), 3.98 (1 H, dd, J_5,6b 5.7, 6-H^b), 4.26 (1 H, dd, J_4,5 9.6, 4-H), 4.43 (1 H, dt, 5-H), 4.53 and 4.82 (2 H, AB q, J 11.1, CH₂Ph), 5.03 (1 H, d, J_1,2 1.1, 1-H), 5.39 (1 H, dd, 2-H), 5.55 (1 H, t, J_2,3 = J_3,4 = 3.0, 3-H) and 7.15–8.15 (15 H, m, 3 × Ph).

Benzyl 2,3,6-tri-O-benzoyl-α-D-altropyranoside 20

A solution of the altroside 19 (2.49 g, 4.8 mmol) in 80% aq. acetic acid (50 cm³) was heated at 60 °C for 1 h, whereafter the mixture was concentrated and toluene was twice evaporated off from the residue. The residue was dissolved in acetonitrile (50 cm³) and BzCN (0.63 g, 4.82 mmol) and Et₃N (0.025 cm³) were added to the solution. After 30 min, methanol was added, the reaction mixture was concentrated, and toluene was evaporated off from the residue. FCC (solvent A) gave the tribenzoate20 (1.87 g, 67%), mp 140–142 °C (from diethyl ether–hexane); [α]²⁵_D −6.5 (c 1, CHCl₃); R_f 0.25 (solvent B) (Found: C, 70.4; H, 5.1. C₃₄H₃₀O₉ requires C, 70.1; H, 5.2%); δ_H (200 MHz; CDCl₃ + D₂O) 4.28 (1 H, dd, J_4,5 9.7, 4-H), 4.48 (1 H, ddd, J_5,6a 2.3, 5-H), 4.58 and 4.83 (2 H, AB q, J 10.8, CH₂Ph), 4.66 (1 H, dd, J_6a,6b 12.0, 6-H^a), 4.78 (1 H, dd, J_5,6b 4.0, 6-H^b), 5.10 (1 H, d, J_1,2 1.0, 1-H), 5.42 (1 H, dd, 2-H), 5.62 (1 H, t, J_2,3 = J_3,4 = 3.2, 3-H) and 7.15–8.15 (20 H, m, 4 × Ph).

Benzyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-(1→4)-2,3,6-tri-O-benzoyl-α-Dl-altropyranoside 22

A solution of acetobromogalactose 21 (1.03 g, 2.5 mmol) in CH₂Cl₂ (15 cm³) was added dropwise to a stirred mixture of the altroside 20 (0.73 g, 1.25 mmol), Ag₂CO₃ (1.37 g, 5.0 mmol), AgOTf (0.64 g, 2.5 mmol) and freshly activated molecular sieves 4 Å (powder, 5 g) in boiling dichloromethane (30 cm³). The reaction mixture was stirred under reflux for 2.5 h and then at rt for 20 h. The solids were filtered off and the filtrate was concentrated. FCC [diethyl ether–hexane, (1∶1) → (2∶1)] gave the disaccharide22 (0.59 g, 52%) as an amorphous solid, [α]²⁵_D +12 (c 1, CHCl₃); R_f 0.45 (solvent C ) (Found: C, 63.1; H, 5.4. C₄₈H₄₈O₁₈ requires C, 63.15; H, 5.3%); δ_H (200 MHz) 1.91 (6 H, s, 2 × Ac), 1.93 and 2.01 (6 H, 2 × s, 2 × Ac), 3.82–3.99 (3 H, m, 5′-H and 6′-H₂), 4.32 (1 H, dd, J_4,5 9.5, 4-H), 4.44 (1 H, dd, J_5,6a 4.5, J_6a,6b 12.5, 6-H^a), 4.52 and 4.81 (2 H, AB q, J 11.6, CH₂Ph), 4.58–4.70 (3 H, m, 1′-, 5-H and 6-H^b), 4.91 (1 H, dd, J_2′,3′ 10.5, 3′-H), 5.02 (1 H, br s, 1-H), 5.15 (1 H, dd, J_1′,2′ 7.5, 2′-H), 5.25 (1 H, d, J_3′,4′ 3.5, 4′-H), 5.46 (1 H, d, 2-H), 5.66 (1 H, t, J_2,3 = J_3,4 = 3.3, 3-H) and 7.15–8.15 (20 H, m, 4 × Ph).

2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl-(1→4)-2,3,6-tri-O-benzoyl-α-D-altropyranosyl hydrogenphosphonate, triethylammonium salt 23

A solution of the disaccharide 22 (0.455 g, 0.499 mmol) in THF (5 cm³) containing 20% Pd(OH)₂/C (200 mg) was shaken under a slight overpressure of H₂ at rt for 2 h. The catalyst was filtered off through a Celite pad and the filtrate was concentrated to give the α,β-hemiacetal 24 (0.39 g, 95%) as an amorphous solid [R_f 0.3 (solvent C ); δ_H (500 MHz) 1.89–2.03 (12 H, m, 4 × Ac), 3.78–3.92 (3 H, m, 5′-H and 6′-H₂), 4.30 (dd, J_4,5 8.5, 4-H^β), 4.39 (dd, J_4,5 8.5, 4-H^α), 4.42 (dt, J_5,6a = J_5,6b = 3.0, 5-H^β), 4.52 (dd, J_5,6a 3.0, J_6a,6b 11.7, 6-H^a), 4.61 (d, J_1′,2′ 7.0, 1′-H^α), 4.67 (d, J_1′,2′ 7.0, 1′-H^β), 4.70–4.78 (m, 5-H^α and 6-H^b), 4.92 (dd, J_3′,4′ 3.0, 3′-H^α), 4.95 (dd, J_3′,4′ 3.0, 3′-H^β), 5.13 (dd, J_2′,3′ 9.0, 2′-H^α), 5.17 (dd, J_2′,3′ 9.0, 2′-H^β), 5.22 (d, 4′-H^α), 5.26 (d, 4′-H^β), 5.30 (br s, 1-H^β), 5.39 (br s, 1-H^α), 5.46 (d, 2-H^β), 5.51 (d, 2-H^α), 5.69 (t, J_2,3 = J_3,4 = 2.8, 3-H^β), 5.79 (t, J_2,3 = J_3,4 = 2.8, 3-H^α) and 7.20–8.20 (15 H, m, 3 × Ph); α∶β = 0.8∶1].

The reaction of the compound 24 (0.39 g, 0.474 mmol) with PCl₃ (0.165 cm³, 1.89 mmol), imidazole (0.45 g, 6.62 mmol) and Et₃N (0.99 cm³, 7.09 mmol) in CH₃CN (10 cm³), followed by hydrolysis with 1 mol dm⁻³ aq. TEA hydrogen carbonate (2.5 cm³), was accomplished as described for the preparation of the disaccharide H-phosphonate 13. After work-up, the solution was concentrated and acetonitrile was evaporated off from the residue. The residue was dissolved in the same solvent (5 cm³) and anhydrous H₃PO₃ (0.39 g, 4.73 mmol) was added to the solution. The reaction mixture was kept at rt for 20 h, then diluted with CH₂Cl₂ (50 cm³) and washed successively with saturated aq. NaHCO₃ and 0.5 mol dm⁻³ aq. TEA hydrogen carbonate, dried by filtration through cotton wool, and concentrated. FCC [CH₂Cl₂–MeOH, (99∶1) → (80∶20)] gave the H-phosphonate 23 (0.175 g, 35% from the disaccharide 22) as an amorphous solid, [α]²⁵_D +1 (c 1, CHCl₃); R_f 0.35 (solvent F ); δ_H (200 MHz) 1.20 (9 H, t, 3 × MeCH₂), 1.91, 1.92, 1.93 and 2.02 (12 H, 4 × s, 4 × Ac), 2.91 (6 H, q, 3 × MeCH₂), 3.80–3.88 (2 H, m, 5′-H and 6′-H^a), 3.93 (1 H, dd, J_5′,6b′ 7.8, J_6a′,6b′ 13.5, 6′-H^b), 4.35 (1 H, dd, J_4,5 9.6, 4-H), 4.46 (1 H, dd, J_6a,6b 11.6, 6-H^a), 4.64 (1 H, d, J_1′,2′ 7.7, 1′-H), 4.71 (1 H, dd, J_5,6b 1.0, 6-H^b), 4.88 (1 H, ddd, J_5,6a 3.7, 5-H), 4.91 (1 H, dd, J_3′,4′ 3.2, 3′-H), 5.13 (1 H, dd, J_2′,3′ 10.6, 2′-H), 5.23 (1 H, d, 4′-H), 5.44 (1 H, d, 2-H), 5.67 (1 H, t, J_2,3 = J_3,4 = 2.8, 3-H), 5.72 (1 H, d, J_1,P 8.5, 1-H), 7.02 (1 H, d, J_H,P 640.0, HP) and 7.40–8.20 (15 H, m, 3 × Ph); δ_P 0.58; ESMS(−): m/z 884.9 (100%, [M − Et₃N − H]⁻) (expected m/z, 885.008. C₄₇H₅₈NO₂₀P requires M, 987.208). Also isolated was the disaccharide hemiacetal 24 (0.2 g, 49% recovery).

Dec-9-enyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-(1→4)-2,3,6-tri-O-benzoyl-α-D-altropyranosyl phosphate, triethylammonium salt 26

This compound was prepared by condensation of the glycobiosyl H-phosphonate 23 (98 mg, 0.10 mmol) and dec-9-en-1-ol (0.053 cm³, 0.30 mmol) in pyridine (1 cm³) in the presence of trimethylacetyl chloride (0.037 cm³, 0.30 mmol), followed by oxidation with iodine (51 mg, 0.20 mmol) in pyridine–water (95∶5; 2 cm³) as described for the synthesis of the phosphodiester 15. FCC [CH₂Cl₂–MeOH, (99∶1) → (80∶20)] gave the phosphodiester 26 (85 mg, 75%) as an amorphous solid, [α]²⁵_D +2 (c 1, CHCl₃); R_f 0.5 (solvent F ); δ_H (200 MHz) 1.25 (19 H, m, 3 × MeCH₂ and 5 × CH₂), 1.48 (2 H, tt, J 6.9, OCH₂CH₂CH₂), 1.95 (6 H, s, 2 × Ac), 1.97 and 2.00 (6 H, 2 × s, 2 × Ac), 2.03 (2 H, m, CH₂CH₂CH[double bond, length half m-dash]), 2.95 (6 H, q, 3 × MeCH₂), 3.74–3.96 (5 H, m, 5′-H, 6′-H₂ and OCH₂CH₂), 4.38 (1 H, dd, J_4,5 9.6, 4-H), 4.46 (1 H, dd, J_5,6a 3.0, J_6a,6b 11.8, 6-H^a), 4.64 (1 H, d, J_1′,2′ 7.8, 1′-H), 4.72 (1 H, dd, J_5,6b 1.0, 6-H^b), 4.87–4.95 (3 H, m, 3′-, 5-H and H CH[double bond, length half m-dash]CH), 4.98 (1 H, dd, ²J_H,H 1.6, ³J_H,H-E 16.8, HCH[double bond, length half m-dash]CH), 5.12 (1 H, dd, J_2′,3′ 10.1, 2′-H), 5.23 (1 H, d, J_3′,4′ 3.1, 4′-H), 5.50 (1 H, d, J_2,3 3.0, 2-H), 5.64 (1 H, d, J_1,P 7.7, 1-H), 5.68 (1 H, dd, J_3,4 3.5, 3-H), 5.80 (1 H, ddt, J_H,CH2 6.6, ³J_H,H-Z 10.9, CH₂CH[double bond, length half m-dash]CH₂) and 7.40–8.25 (15 H, m, 3 × Ph); δ_P −2.79; ESMS(−): m/z 1039.0 (100%, [M − Et₃N − H]⁻) (expected m/z, 1039.14. C₅₇H₇₆NO₂₁P requires M, 1141.344).

Dec-9-enyl β-D-galactopyranosyl-(1→4)-α-D-altropyranosyl phosphate, triethylammonium salt 7

To a solution of compound 26 (50 mg) in MeOH (1.8 cm³) was added 0.5 mol dm⁻³ methanolic NaOMe (0.2 cm³). The mixture (now 0.05 mol dm⁻³ in NaOMe) was kept at 0 °C for 16 h and then at room temperature for 8 h, whereafter it was deionized with Dowex 50W-X4(H⁺) resin, filtered and immediately neutralized with Et₃N. After concentration, water (3 × 5 cm³) was evaporated off from the residue to remove methyl benzoate. The phosphodiester 7 (28 mg, 96%) was thereby obtained as an amorphous solid, [α]²⁵_D +36 (c 1, MeOH); R_f 0.65 (solvent G ); δ_H (200 MHz; D₂O) (inter alia) 1.15 (9 H, t, 3 × MeCH₂), 1.23 (10 H, m, 5 × CH₂), 1.52 (2 H, tt, J 6.9, OCH₂CH₂CH₂), 1.94 (2 H, dt, J 6.7, CH₂CH₂CH[double bond, length half m-dash]), 3.10 (6 H, q, 3 × MeCH₂), 4.41 (1 H, d, J_1′,2′ 7.0, 1′-H), 5.21 (1 H, br d, J_1,P 6.6, 1-H) and 5.83 (1 H, ddt, J_H,CH2 6.7, J_H,H-Z 10.1, J_H,H-E 18.0, CH₂CH[double bond, length half m-dash]CH₂); δ_C, δ_P and ESMS(−) data: see Table 1.

Methyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-(1→4)-2,3-O-isopropylidene-α-D-rhamnopyranoside 28

A solution of acetobromogalactose 21 (1.49 g, 3.58 mmol) in acetonitrile–toluene (10∶3; 13 cm³) was added to a stirred mixture of methyl 2,3-O-isopropylidene-α-D-rhamnopyranoside ¹³27 (0.318 g, 1.45 mmol), Hg(CN)₂ (0.9 g, 3.58 mmol) and HgBr₂ (0.64 g, 1.79 mmol) in the same mixed solvent (5 cm³). After being stirred at rt for 16 h, the reaction mixture was diluted with CH₂Cl₂ (50 cm³), washed successively with 1 mol dm⁻³ aq. KBr, saturated aq. NaHCO₃, and water, dried by filtration through cotton wool, and concentrated. FCC [toluene–ethyl acetate, (8∶2)] of the residue gave the disaccharide derivative28 (0.59 g, 74%), mp 126–129 °C (from ethanol); [α]²²_D +25.7 (c 1, CHCl₃) (Found: C, 52.5; H, 6.6. C₂₄H₃₆O₁₄ requires C, 52.6; H, 6.6%); δ_H (200 MHz) 1.23 (3 H, d, J_5,6 6.3, 6-H₃), 1.32 and 1.50 (6 H, 2 × s, CMe₂), 2.00, 2.05, 2.07 and 2.15 (12 H, 4 × s, 4 × Ac), 3.34 (3 H, s, OMe), 3.36 (1 H, dd, J_3,4 7.2, 4-H), 3.63 (1 H, dq, J_4,5 9.8, 5-H), 3.88 (1 H, t, J_5′,6′ 6.6, 5′-H), 4.06 (1 H, d, J_2,3 5.7, 2-H), 4.14 (2 H, d, 6′-H₂), 4.24 (1 H, dd, 3-H), 4.65 (1 H, d, J_1′,2′ 8.0, 1′-H), 4.81 (1 H, s, 1-H), 5.00 (1 H, dd, J_3′,4′ 3.2, 3′-H), 5.21 (1 H, dd, J_2′,3′ 10.3, 2′-H) and 5.36 (1 H, d, 4′-H).

2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl-(1→4)-1,2,3-tri-O-acetyl-α-D-rhamnopyranose 29

To a stirred solution of the disaccharide 28 (0.728 g) in chloroform (36 cm³) was added 90% aq. trifluoroacetic acid (4 cm³). Stirring was continued for 2 h, whereafter the solution was concentrated and toluene was twice evaporated off from the residue. The residue was then dissolved in Ac₂O (5 cm³) and a sulfuric acid–acetic anhydride mixture [1∶50 (v/v); 10.2 cm³] was added. The solution was stirred at rt for 2 h, whereafter it was diluted with CH₂Cl₂ (100 cm³), washed successively with water, saturated aq. NaHCO₃, and water, dried by filtration through cotton wool, and concentrated. Toluene was twice evaporated off from the residue. FCC (solvent B → solvent C ) of the residue gave the heptaacetate29 (0.57 g, 69%), mp 145–148 °C (from ethanol); [α]²¹_D +36.8 (c 0.99, CHCl₃) (Found: C, 50.6; H, 5.9. C₂₆H₃₆O₁₇ requires C, 50.3; H, 5.9%); δ_H (200 MHz) 1.30 (3 H, d, J_5,6 6.1, 6-H₃), 1.96, 2.02, 2.03, 2.04 and 2.14 (15 H, 5 × s, 5 × Ac), 2.13 (6 H, s, 2 × Ac), 3.63 (1 H, t, J_3,4 = J_4,5 = 9.4, 4-H), 3.82 (1 H, dq, 5-H), 3.87 (1 H, ddd, J_5′,6a′ 7.1, 5′-H), 4.02 (1 H, dd, J_6a′,6b′ 11.0, 6′-H^a), 4.16 (1 H, dd, J_5′,6b′ 6.4, 6′-H^b), 4.58 (1 H, d, J_1′,2′ 7.8, 1′-H), 4.97 (1 H, dd, J_3′,4′ 3.4, 3′-H), 5.14 (1 H, dd, J_2′,3′ 10.3, 2′-H), 5.19 (1 H, dd, J_2,3 3.3, 2-H), 5.28 (1 H, dd, 3-H), 5.33 (1 H, dd, J_4′,5′ 0.5, 4′-H) and 5.94 (1 H, d, J_1,2 1.9, 1-H).

2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl-(1→4)-2,3-di-O-acetyl-α-D-rhamnopyranose 30

This compound was prepared from compound 29 (0.307 g) as described for the hemiacetal derivative 12. Two consecutive FCC separations [toluene–ethyl acetate, (55∶45) → (8∶2) and solvent B → solvent D] gave the disaccharide α-hemiacetal30 (0.19 g, 66%) as an amorphous solid, [α]²¹_D +11 (c 0.97, CHCl₃) (Found: C, 49.4; H, 6.0. C₂₄H₃₄O₁₆ requires C, 49.8; H, 5.9%); δ_H (200 MHz) 1.28 (3 H, d, J_5,6 6.2, 6-H₃), 1.96, 2.01, 2.03, 2.04, 2.12 and 2.14 (18 H, 6 × s, 6 × Ac), 3.59 (1 H, t, J_3,4 = J_4,5 = 9.4, 4-H), 3.87 (1 H, t, J_5′,6a′ = J_5′,6b′ = 6.5, 5′-H), 4.00 (1 H, dq, 5-H), 4.01 (1 H, dd, 6′-H^a), 4.16 (1 H, dd, J_6a′,6b′ 11.0, 6′-H^b), 4.57 (1 H, d, J_1′,2′ 7.8, 1′-H), 4.97 (1 H, dd, J_3′,4′ 3.3, 3′-H), 5.08 (1 H, d, J_1,2 1.9, 1-H), 5.13 (1 H, dd, J_2′,3′ 10.5, 2′-H), 5.21 (1 H, dd, J_2,3 3.5, 2-H), 5.32 (1 H, d, 4′-H) and 5.33 (1 H, dd, 3-H).

2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl-(1→4)-2,3-di-O-acetyl-α-D-rhamnopyranosyl hydrogenphosphonate, triethylammonium salt 31

This compound was prepared by the reaction of the hemiacetal 30 (0.184 g, 0.32 mmol) with PCl₃ (0.166 cm³, 1.91 mmol), imidazole (0.433 g, 6.36 mmol) and Et₃N (0.93 cm³, 6.7 mmol) in acetonitrile (15 cm³) followed by hydrolysis as described for the H-phosphonate 13. For work-up, the reaction mixture was diluted with CH₂Cl₂ (100 cm³) and washed successively with cold saturated aq. NaHCO₃ (2 × 40 cm³) and cold 0.5 mol dm⁻³ aq. TEA hydrogen carbonate (2 × 40 cm³). The organic phase was discarded. The aqueous washings were combined, and extracted with CH₂Cl₂ (4 × 20 cm³). The combined organic washings were dried by filtration through cotton wool, and concentrated to produce the H-phosphonate 31 (0.165 g, 70%) as a chromatographically homogeneous amorphous solid, [α]²⁵_D +23.1 (c 1.06, CHCl₃); δ_H (200 MHz) 1.27 (3 H, d, J_5,6 6.3, 6-H₃), 1.30 (9 H, t, 3 × MeCH₂), 1.94, 1.95, 2.00, 2.02, 2.08 and 2.11 (18 H, 6 × s, 6 × Ac), 3.03 (6 H, q, 3 × MeCH₂), 3.56 (1 H, t, J_3,4 = J_4,5 = 9.4, 4-H), 3.83 (1 H, t, J_5′,6a′ = J_5′,6b′ = 6.6, 5′-H), 3.98 (1 H, dq, 5-H), 3.99 (1 H, dd, 6′-H^a), 4.12 (1 H, dd, J_6a′,6b′ 11.0, 6′-H^b), 4.54 (1 H, d, J_1′,2′ 7.7, 1′-H), 4.94 (1 H, dd, J_3′,4′ 3.3, 3′-H), 5.10 (1 H, dd, J_2′,3′ 10.5, 2′-H), 5.21 (1 H, dd, J_1,2 1.8, 2-H), 5.30 (1 H, d, 4′-H), 5.31 (1 H, dd, J_2,3 3.6, 3-H), 5.44 (1 H, dd, J_1,P 8.8, 1-H) and 5.44 (1 H, d, J_H,P 642.0, HP); δ_P −0.10; ESMS(−): m/z 641.0 (100%, [M − Et₃N − H]⁻) (expected m/z, 641.07. C₃₀H₅₀NO₁₈P requires M, 743.27).

Dec-9-enyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-(1→4)-2,3-di-O-acetyl-α-D-rhamnopyranosyl phosphate, triethylammonium salt 32

This compound was prepared by condensation of the H-phosphonate 31 (0.14 g, 0.19 mmol) and dec-9-en-1-ol (0.067 cm³, 0.38 mmol) in pyridine (1 cm³) in the presence of trimethylacetyl chloride (0.058 cm³, 0.47 mmol) followed by oxidation with iodine (0.096 g, 0.376 mmol) in pyridine–water (95∶5; 2 cm³) as described for the synthesis of the phosphodiester 15. FCC [CH₂Cl₂–MeOH–Et₃N, (99∶0∶1) → (89∶10∶1)] gave the phosphodiester 32 (0.148 g, 88%) as an amorphous solid, [α]²⁷_D +12.4 (c 1.09, CHCl₃); δ_H (200 MHz) 1.18 (3 H, d, J_5,6 6.0, 6-H₃), 1.24 (9 H, t, 3 × MeCH₂), 1.25 (10 H, m, 5 × CH₂), 1.53 (2 H, tt, J 6.9, OCH₂CH₂CH₂), 1.89, 1.90, 1.97, 1.98, 2.03 and 2.07 (18 H, 6 × s, 6 × Ac), 1.96 (2 H, dt, J 6.8, CH₂CH₂CH[double bond, length half m-dash]), 3.00 (6 H, q, 3 × MeCH₂), 3.51 (1 H, dd, J_4,5 9.7, 4-H), 3.71–3.86 (4 H, m, 5′-H, 6′-H^a and OCH₂CH₂), 3.97 (1 H, dq, 5-H), 4.08 (1 H, dd, J_5′,6b′ 6.6, J_6a′,6b′ 11.2, 6′-H^b), 4.52 (1 H, d, J_1′,2′ 7.7, 1′-H), 4.84 (1 H, dd, ²J_H,H 1.6, ³J_H,H-Z 9.3, H CH[double bond, length half m-dash]CH), 4.90 (1 H, dd, ³J_H,H-E 17.0, HCH[double bond, length half m-dash]CH), 4.92 (1 H, dd, 3′-H), 5.08 (1 H, dd, J_2′,3′ 10.5, 2′-H), 5.19 (1 H, dd, J_2,3 3.3, 2-H), 5.25 (1 H, d, J_3′,4′ 4.0, 4′-H), 5.26 (1 H, dd, J_3,4 9.4, 3-H), 5.34 (1 H, dd, J_1,2 1.5, J_1,P 8.8, 1-H) and 5.73 (1 H, ddt, J_H,CH2 6.8, CH₂CH[double bond, length half m-dash]CH₂); δ_P −3.12; ESMS(−): m/z 795.3 (100%, [M − Et₃N − H]⁻) (expected m/z, 795.21. C₄₀H₆₈NO₁₉P requires M, 897.41).

Dec-9-enyl β-D-galactopyranosyl-(1→4)-α-D-rhamnopyranosyl phosphate, ammonium salt 8

De-O-acetylation of compound 32 (74 mg) with 0.05 mol dm⁻³ NaOMe in methanol (3 h at rt) followed by work-up, as described in the preparation of the phosphodiester 6, produced a crude product, which then was applied to a column (18 × 1.5 cm) of Fractogel TSK DEAE-650 (S) (HCO₃⁻-form) (Merck) eluted with a linear gradient of NH₄HCO₃ (0 → 0.1 mol dm⁻³) in 3∶2 water–propan-2-ol at 1 cm³ min⁻¹ to afford the phosphodiester 8 (41 mg, 88%) as an amorphous solid, [α]²⁶_D +22.4 (c 0.99, MeOH); δ_H (200 MHz; D₂O) (inter alia) 1.22–1.45 (13 H, m, 6-H₃ and 5 × CH₂), 1.63 (2 H, tt, J 6.9, OCH₂CH₂CH₂), 2.05 (2 H, dt, J 6.9, CH₂CH₂CH[double bond, length half m-dash]), 4.48 (1 H, d, J_1′,2′ 7.0, 1′-H), 5.32 (1 H, br d, J_1,P 6.3, 1-H) and 5.91 (1 H, m, CH₂CH[double bond, length half m-dash]CH₂); δ_C, δ_P and ESMS(−) data: see Table 1.

2,3,4-Tri-O-benzoyl-α,β-D-fucopyranose 34

To a solution of 1,2,3,4-tetra-O-benzoyl-α-D-fucopyranose 33 (0.5 g, 0.861 mmol) [prepared by standard benzoylation of α-D-fucose with benzoyl chloride in pyridine–chloroform; δ_H (200 MHz) 1.34 (3 H, d, J_5,6 6.4, 6-H₃), 4.67 (1 H, q, 5-H), 5.92 (1 H, d, 4-H), 6.01 (1 H, dd, J_2,3 10.8, 2-H), 6.14 (1 H, dd, J_3,4 3.0, 3-H), 6.91 (1 H, d, J_1,2 3.3, 1-H) and 7.17–8.28 (20 H, m, 4 × Ph)] in acetonitrile (3 cm³) was added 2 mol dm⁻³ Me₂NH in THF (4.3 cm³; 8.61 mmol) and the mixture was kept at rt with monitoring by TLC (solvents B and C ). After 16–24 h, the mixture was concentrated to dryness and acetonitrile was evaporated off from the residue. FCC [toluene–ethyl acetate, (99∶1) → (85∶15)] gave the unchanged starting material 33 (0.079 g, 16% recovery) and the hemiacetal34 (0.25 g, 61%; amorphous solid), [α]²²_D +247.4 (c 1, CHCl₃) (Found: C, 68.3; H, 5.1. C₂₇H₂₄O₈ requires C, 68.1; H, 5.1%); δ_H (200 MHz; CDCl₃ + D₂O) 1.26 (d, J_5,6 6.4, 6-H^α), 1.35 (d, J_5,6 6.3, 6-H^β), 4.12 (dq, J_4,5 0.8, 5-H^β), 4.67 (q, H-5^α), 5.06 (d, J_1,2 6.8, 1-H^β), 5.66–5.85 (m, 1-H^α, 2-H, 3-H^β and 4-H), 6.09 (dd, J_2,3 10.6, J_3,4 3.2, 3-H^α) and 7.01–8.40 (15 H, m, 3 × Ph); α∶β = 5∶1.

2,3,4-Tri-O-benzoyl-α-D-fucopyranosyl trichloroacetimidate 35

To a stirred solution of the hemiacetal 34 (0.228 g, 0.48 mmol) and CCl₃CN (2 cm³, 20 mmol) in dichloromethane (4 cm³) cooled to 0 °C was added DBU (0.072 cm³, 0.48 mmol) under argon. The mixture was stirred for 2 h at 0 °C and then concentrated. FCC (solvent A) of the residue gave the α-fucopyranosyl trichloroacetimidate 35 (0.277 g, 93%) as an amorphous solid, [α]²²_D +209.4 (c 1, CHCl₃); δ_H (200 MHz) 1.34 (3 H, d, J_5,6 6.5, 6-H₃), 4.66 (1 H, dq, 5-H), 5.89 (1 H, dd, J_4,5 0.5, 4-H), 5.92 (1 H, dd, J_2,3 10.5, 2-H), 6.06 (1 H, dd, J_3,4 3.2, 3-H), 6.86 (1 H, d, J_1,2 3.3, 1-H), 7.10–8.23 (15 H, m, 3 × Ph) and 8.60 (1 H, s, HN); δ_C 16.19 (C-6), 67.95 (C-5), 68.05 (C-2), 68.66 (C-3), 71.29 (C-4), 90.91 (CCl₃), 94.11 (C-1), 128.30–133.48 (Ph), 160.78 (C=NH) and 165.62–165.83 (C=O); ESMS(+): m/z 459.2 (100%, [M − CCl₃CONH]⁺) (expected m/z, 459.47. C₂₉H₂₄Cl₃NO₈ requires M, 620.86).

2,3,4-Tri-O-benzoyl-β-D-fucopyranosyl-(1→4)-1,2,3,6-tetra-O-benzoyl-α-D-mannopyranose 37 and 2,3,4-tri-O-benzoyl-α-D-fucopyranosyl-(1→4)-1,2,3,6-tetra-O-benzoyl-α-D-mannopyranose 40

A mixture of the trichloroacetimidate 35 (0.554 g, 0.89 mmol), 1,2,3,6-tetra-O-benzoyl-α-D-mannopyranose ³36 (0.638 g, 1.07 mmol) and freshly activated molecular sieves 4 Å (powder, 1 g) in dry dichloromethane (5 cm³) was stirred under argon for 30 min. TMS triflate (0.046 cm³, 0.22 mmol) was then added and the mixture was cooled to −70 °C. Stirring was continued for a further 1.5 h, while the mixture slowly warmed to −10 °C. The reaction was quenched with a few drops of N,N-diisopropylethylamine, the solids were filtered off, and the solvent was removed under reduced pressure. FCC (toluene → solvent B) of the residue gave a mixture of the disaccharides 37 and 40, which were then separated by further FCC [dichloromethane–ethyl acetate, (100∶0) → (98∶2)]. That provided, first, the β-linked disaccharide37 (0.582 g, 62%) as an amorphous solid, [α]²³_D +118 (c 1.03, CHCl₃) (Found: C, 69.6; H, 4.7. C₆₁H₅₀O₁₇ requires C, 69.4; H, 4.8%); δ_H (200 MHz) 0.82 (3 H, d, J_5′,6′ 6.3, 6′-H₃), 3.56 (1 H, q, 5′-H), 4.27 (1 H, ddd, J_5,6a 2.6, 5-H), 4.45 (1 H, dd, J_6a,6b 12.7, 6-H^a), 4.68 (1 H, dd, J_5,6b 1.8, 6-H^b), 4.69 (1 H, J_3,4 = J_4,5 = 9.7, 4-H), 4.96 (1 H, d, J_1′,2′ 7.9, 1′-H), 5.39 (1 H, dd, J_3′,4′ 3.3, 3′-H), 5.48 (1 H, d, 4′-H), 5.70 (1 H, dd, J_2′,3′ 10.3, 2′-H), 5.85 (1 H, dd, J_2,3 3.3, 2-H), 5.99 (1 H, dd, 3-H), 6.48 (1 H, d, J_1,2 1.7, 1-H) and 7.08–8.22 (35 H, m, 7 × Ph). Continued elution gave the α-linked disaccharide40 (0.126 g, 13%) as an amorphous solid, [α]²³_D +124 (c 0.93, CHCl₃) (Found: C, 69.9; H, 5.1%); δ_H (200 MHz) 1.13 (3 H, d, J_5′,6′ 6.6, 6′-H₃), 4.42–4.56 (2 H, m, 5- and 5′-H), 4.66 (1 H, dd, J_5,6a 2.7, J_6a,6b 12.3, 6-H^a), 4.91 (1 H, J_3,4 = J_4,5 = 9.3, 4-H), 4.93 (1 H, dd, J_5,6b 0.5, 6-H^b), 5.67–5.94 (6 H, 1′-, 2-, 2′-, 3-, 3′- and 4′-H), 6.55 (1 H, d, J_1,2 2.0, 1-H) and 7.05–8.28 (35 H, m, 7 × Ph).

2,3,4-Tri-O-benzoyl-β-D-fucopyranosyl-(1→4)-2,3,6-tri-O-benzoyl-α-D-mannopyranose 38

This compound was prepared from compound 37 (0.307 g) as described for the hemiacetal derivative 34. FCC [toluene → solvent C ] gave the disaccharide α-hemiacetal38 (0.25 g, 90%) as an amorphous solid, [α]²⁵_D +123.3 (c 1.06, CHCl₃) (Found: C, 68.1; H, 5.0. C₅₄H₄₆O₁₆ requires C, 68.2; H, 4.9%); δ_H (200 MHz) 0.83 (3 H, d, J_5′,6′ 6.2, 6′-H₃), 3.53 (1 H, q, 5′-H), 4.07 (1 H, d, J_1,OH 4.1, 1-OH), 4.33–4.46 (2 H, m, 5-H and 6-H^a), 4.58 (1 H, J_3,4 = J_4,5 = 9.6, 4-H), 4.77 (1 H, dd, J_5,6b 0.6, J_6a,6b 12.5, 6-H^b), 4.95 (1 H, d, J_1′,2′ 7.9, 1′-H), 5.35 (1 H, d, J_1,2 1.8, 1-H), 5.42 (1 H, dd, J_2′,3′ 10.2, 3′-H), 5.46 (1 H, d, J_3′,4′ 3.2, 4′-H), 5.62–5.75 (2 H, m, 2- and 2′-H), 5.95 (1 H, dd, J_2,3 3.0, 3-H) and 6.98–8.22 (30 H, m, 6 × Ph).

2,3,4-Tri-O-benzoyl-β-D-fucopyranosyl-(1→4)-2,3,6-tri-O-benzoyl-α-D-mannopyranosyl hydrogenphosphonate, triethylammonium salt 39

This compound was prepared from the hemiacetal 38 (0.208 g, 0.219 mmol) as described for the H-phosphonate derivative 13. This produced the disaccharide hydrogenphosphonate 39 (0.232 g, 95%) as a chromatographically homogeneous amorphous solid, [α]²⁵_D +102.4 (c 0.96, CHCl₃); δ_H (200 MHz) 0.81 (3 H, d, J_5′,6′ 6.3, 6′-H₃), 1.27 (9 H, t, 3 × MeCH₂), 3.01 (6 H, q, 3 × MeCH₂), 3.49 (1 H, q, 5′-H), 4.37 (1 H, ddd, J_5,6a 2.7, 5-H), 4.43 (1 H, dd, J_6a,6b 12.9, 6-H^a), 4.53 (1 H, J_3,4 = J_4,5 = 9.5, 4-H), 4.65 (1 H, dd, J_5,6b 1.4, 6-H^b), 4.87 (1 H, d, J_1′,2′ 7.9, 1′-H), 5.34 (1 H, dd, J_3′,4′ 3.4, 3′-H), 5.42 (1 H, d, 4′-H), 5.64 (1 H, dd, J_2′,3′ 10.4, 2′-H), 5.67 (1 H, dd, J_1,2 2.0, 2-H), 5.70 (1 H, dd, J_1,P 7.7, 1-H), 5.88 (1 H, dd, J_2,3 3.3, 3-H), 7.01 (1 H, d, J_H,P 636.9, HP) and 7.05–8.15 (30 H, m, 6 × Ph); δ_P 0.11; ESMS(−): m/z 1012.8 (100%, [M − Et₃N − H]⁻) (expected m/z, 1013.17. C₆₀H₆₂NO₁₈P requires M, 1115.37).

Dec-9-enyl 2,3,4-tri-O-benzoyl-β-D-fucopyranosyl-(1→4)-2,3,6-tri-O-benzoyl-α-D-mannopyranosyl phosphate, triethylammonium salt 41

This compound was prepared by condensation of the H-phosphonate 39 (0.12 g, 0.107 mmol) and dec-9-en-1-ol (0.038 cm³, 0.215 mmol) in pyridine (1 cm³) in the presence of trimethylacetyl chloride (0.033 cm³, 0.268 mmol) followed by oxidation with iodine (0.055 g, 0.215 mmol) in pyridine–water (95∶5; 2 cm³) as described for the synthesis of the phosphodiester 15. FCC [CH₂Cl₂–MeOH–Et₃N, (99∶0∶1) → (89∶10∶1)] gave the phosphodiester 41 (0.108 g, 80%) as an amorphous solid, [α]²⁵_D +82.2 (c 1, CHCl₃); δ_H (200 MHz) 0.84 (3 H, d, J_5′,6′ 6.3, 6′-H₃), 1.24 (10 H, m, 5 × CH₂), 1.30 (9 H, t, 3 × MeCH₂), 1.58 (2 H, tt, J 6.9, OCH₂CH₂), 1.97 (2 H, dt, J 6.9, CH₂CH₂CH[double bond, length half m-dash]), 3.05 (6 H, q, 3 × MeCH₂), 3.47 (1 H, q, 5′-H), 3.90 (2 H, m, OCH₂CH₂), 4.37–4.49 (2 H, m, 5-H and 6-H^a), 4.55 (1 H, J_3,4 = J_4,5 = 9.4, 4-H), 4.64 (1 H, dd, J_5,6b 1.1, J_6a,6b 11.7, 6-H^b), 4.87 (1 H, d, J_1′,2′ 7.8, 1′-H), 4.89 (1 H, dd, ²J_H,H 1.3, ³J_H,H-Z 10.4, H CH[double bond, length half m-dash]CH), 4.95 (1 H, dd, ³J_H,H-E 17.2, HCH[double bond, length half m-dash]CH), 5.33 (1 H, dd, J_2′,3′ 10.4, 3′-H), 5.43 (1 H, d, J_3′,4′ 3.3, 4′-H), 5.65 (1 H, dd, J_1,P 8.3, 1-H), 5.66 (1 H, dd, 2′-H), 5.74 (1 H, dd, J_1,2 2.5, 2-H), 5.78 (1 H, ddt, J_H,CH2 6.9, CH₂CH[double bond, length half m-dash]CH₂), 5.91 (1 H, dd, J_2,3 3.4, 3-H) and 7.05–8.10 (30 H, m, 6 × Ph); δ_P −2.83; ESMS(−): m/z 1166.9 (100%, [M − Et₃N − H]⁻) (expected m/z, 1167.31. C₇₀H₈₀NO₁₉P requires M, 1269.51).

Dec-9-enyl β-D-fucopyranosyl-(1→4)-α-D-mannopyranosyl phosphate, triethylammonium salt 9

De-O-benzoylation of compound 41 (101 mg) with 0.05 mol dm⁻³ NaOMe in methanol (16 h at rt) followed by work-up, as described in the preparation of the phosphodiester 7, gave the phosphodiester 9 (51 mg, 99%) as an amorphous solid, [α]²⁸_D +22.5 (c 0.99, MeOH); δ_H (D₂O) (inter alia) 1.22–1.39 (22 H, m, 6′-H₃, 3 × MeCH₂ and 5 × CH₂), 1.58 (2 H, tt, J 6.9, OCH₂CH₂CH₂), 2.01 (2 H, dt, J 6.9, CH₂CH₂CH[double bond, length half m-dash]), 3.15 (6 H, q, 3 × MeCH₂), 4.38 (1 H, d, J_1′,2′ 7.6, 1′-H), 5.37 (1 H, br d, J_1,P 7.1, 1-H) and 5.83 (1 H, m, CH₂CH[double bond, length half m-dash]CH₂); δ_C, δ_P and ESMS(−) data: see Table 1.

2,3,4-Tri-O-benzoyl-α-D-fucopyranosyl-(1→4)-2,3,6-tri-O-benzoyl-α-D-mannopyranose 42

This compound was prepared from compound 40 (160 mg) as described for the hemiacetal derivative 34. FCC [toluene → solvent C ] gave the disaccharide α-hemiacetal40 (99 mg, 69%) as an amorphous solid, [α]²⁶_D +54.7 (c 1.02, CHCl₃) (Found: C, 68.0; H, 5.0. C₅₄H₄₆O₁₆ requires C, 68.2; H, 4.9%); δ_H (200 MHz) 1.13 (3 H, d, J_5′,6′ 6.4, 6′-H₃), 3.83 (1 H, d, J_1,OH 4.3, 1-OH), 4.52 (1 H, q, 5′-H), 4.60 (1 H, ddd, J_5,6b 0.7, 5-H), 4.65 (1 H, dd, J_5,6a 2.6, 6-H^a), 4.81 (1 H, J_3,4 = J_4,5 = 9.6, 4-H), 5.01 (1 H, dd, J_6a,6b 11.2, 6-H^b), 5.40 (1 H, d, J_1,2 1.8, 1-H), 5.68 (1 H, dd, 2-H), 5.75 (1 H, dd, J_2,3 3.4, 3-H), 5.76–5.85 (4 H, m, 1′-, 2′-, 3′- and 4′-H) and 7.10–8.25 (30 H, m, 6 × Ph).

2,3,4-Tri-O-benzoyl-α-D-fucopyranosyl-(1→4)-2,3,6-tri-O-benzoyl-α-D-mannopyranosyl hydrogenphosphonate, triethylammonium salt 43

This compound was prepared from the hemiacetal 42 (88 mg, 0.092 mmol) as described for the H-phosphonate derivative 13. This produced the disaccharide hydrogenphosphonate 43 (100 mg, 97%) as a chromatographically homogeneous amorphous solid, [α]²¹_D +61 (c 1.06, CHCl₃); δ_H (200 MHz) 1.08 (3 H, d, J_5′,6′ 6.3, 6′-H₃), 1.29 (9 H, t, 3 × MeCH₂), 3.01 (6 H, q, 3 × MeCH₂), 4.47 (1 H, q, 5′-H), 4.57–4.68 (2 H, m, 5-H and 6-H^a), 4.79 (1 H, J_3,4 = J_4,5 = 9.4, 4-H), 4.93 (1 H, dd, J_5,6b 2.8, J_6a,6b 12.9, 6-H^b), 5.67 (1 H, dd, J_1,2 2.0, J_2,3 3.0, 2-H), 5.70–5.82 (6 H, m, 1-, 1′-, 2′-, 3-, 3′- and 4′-H), 7.10 (1 H, d, J_H,P 640.4, HP) and 6.92–8.23 (30 H, m, 6 × Ph); δ_P 0.57; ESMS(−): m/z 1013.0 (100%, [M − Et₃N − H]⁻) (expected m/z, 1013.17. C₆₀H₆₂NO₁₈P requires M, 1115.37).

Dec-9-enyl 2,3,4-tri-O-benzoyl-α-D-fucopyranosyl-(1→4)-2,3,6-tri-O-benzoyl-α-D-mannopyranosyl phosphate, triethylammonium salt 44

This compound was prepared by condensation of the H-phosphonate 43 (100 mg, 0.09 mmol) and dec-9-en-1-ol (0.035 cm³, 0.197 mmol) in pyridine (1 cm³) in the presence of trimethylacetyl chloride (0.03 cm³, 0.246 mmol) followed by oxidation with iodine (50 mg, 0.197 mmol) in pyridine–water (95∶5; 2 cm³) as described for the synthesis of the phosphodiester 15. FCC [CH₂Cl₂–MeOH–Et₃N, (99∶0∶1) → (87∶12∶1)] gave the phosphodiester 44 (95 mg, 78%) as an amorphous solid, [α]²⁶_D +46 (c 0.99, CHCl₃); δ_H (200 MHz) 1.04 (3 H, d, J_5′,6′ 6.3, 6′-H₃), 1.23 (10 H, m, 5 × CH₂), 1.31 (9 H, t, 3 × MeCH₂), 1.64 (2 H, tt, J 6.9, OCH₂CH₂), 1.99 (2 H, dt, J 6.9, CH₂CH₂CH[double bond, length half m-dash]), 3.10 (6 H, q, 3 × MeCH₂), 4.00 (2 H, m, OCH₂CH₂), 4.44 (1 H, q, 5′-H), 4.57–4.70 (2 H, m, 5-H and 6-H^a), 4.79 (1 H, J_3,4 = J_4,5 = 9.8, 4-H), 4.89 (2 H, br d, 6-H^b and H CH[double bond, length half m-dash]CH), 4.96 (1 H, dd, ²J_H,H 1.9, ³J_H,H-E 17.0, HCH[double bond, length half m-dash]CH), 5.65–5.83 (8 H, m, 1-, 1′-, 2-, 2′-, 3-, 3′-, 4′-H and CH₂CH[double bond, length half m-dash]CH₂) and 7.00–8.25 (30 H, m, 6 × Ph); δ_P −2.60; ESMS(−): m/z 1166.9 (100%, [M − Et₃N − H]⁻) (expected m/z, 1167.31. C₇₀H₈₀NO₁₉P requires M, 1269.51).

Dec-9-enyl α-D-fucopyranosyl-(1→4)-α-D-mannopyranosyl phosphate, triethylammonium salt 10

De-O-benzoylation of compound 44 (95 mg) with 0.05 mol dm⁻³ NaOMe in methanol (16 h at rt) followed by work-up, as described in the preparation of the phosphodiester 7, gave the phosphodiester 10 (48 mg, 100%) as an amorphous solid, [α]²⁸_D +69.8 (c 0.98, MeOH); δ_H (200 MHz; D₂O) (inter alia) 1.23 (3 H, d, J_5′,6′ 6.6, 6′-H₃), 1.26-1.40 (19 H, m, 3 × MeCH₂ and 5 × CH₂), 1.61 (2 H, tt, J 6.5, OCH₂CH₂CH₂), 2.04 (2 H, dt, J 7.0, CH₂CH₂CH[double bond, length half m-dash]), 3.20 (6 H, q, 3 × MeCH₂), 5.20 (1 H, br s, 1′-H), 5.42 (1 H, br d, J_1,P 7.6, 1-H) and 5.83 (1 H, m, CH₂CH[double bond, length half m-dash]CH₂); δ_C, δ_P and ESMS(−) data: see Table 1.

Acknowledgements

This work and I. A. I. were supported by a Wellcome Trust International Grant. The research of A. V. N. was supported by an International Research Scholar’s award from the Howard Hughes Medical Institute. One of us (A. J. R.) thanks the BBSRC for the award of a studentship.

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Footnotes

† The value of ¹J_C1,H1 ≈ 170 Hz is typical for α-D-derivatives. For the β-D-glycosyl residues the value is about 160 Hz: for β-D-Galp in compound 7, ¹J_C1′,H1′ = 162.5 Hz (Table 1) (see also refs. 3, 4, 9 and 16).

‡ For methyl β-D-altropyranoside, δ_C-5 = 75.60.¹⁷

§ For methyl β-D-rhamnopyranoside, δ_C-5 = 73.60.¹⁷

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