Faculty of Science, Department of Chemistry, University of Hamburg, Martin-Luther-King-Platz 6, D-20146 Hamburg, Germany <chunk>Chemical synthesis using enzymatically generated building units for construction of the human milk pentasaccharides sialyllacto-</chunk> <chunk italic="yes">N</chunk> <chunk>-tetraose and sialyllacto-</chunk> <chunk italic="yes">N</chunk> <chunk>-neotetraose epimer</chunk> block synthesis human milk oligosaccharides sialyllacto- N -neotetraose epimer sialyllacto- N -tetraose trisaccharide thioglycoside donors α,2-3- and α,2-6-sialylated lactosaminide precursor structures obtained by various enzymatic procedures could be used for glycosylations employing triflic acid/ N -iodosuccinimide. Easily accessible selectively protected lactoside derivatives served as acceptor disaccharides to give the corresponding human milk pentasaccharides in good yields. These were characterized by spectroscopic means in the form of their peracetylated derivatives.
<chunk>Introduction</chunk> From an inspection of contemporary syntheses of biologically and medicinally relevant oligosaccharides, it is evident that the majority is performed either by classical chemical methods or exclusively by enzymatic procedures. Even although considerable progress has been reported during the recent decades, every synthesis of a complex heterooligosaccharide still represents a challenge. To arrive at an oligosaccharide structure with specific patterns of substitution and defined regio- and stereochemical layout, all the presently available procedures need to be checked for efficiency with respect to not only all the above points, but also the efforts required at the purification steps as well as the yields. Whilst both approaches can be employed advantageously in certain cases, in others this is certainly not so. For instance, for structures that contain glycosamino units, it has been demonstrated that these units can be introduced by classical methods in high yields and with good to excellent stereochemical control. In case of glucosylations and galactosylations, and in particular for β-galactosylations, classical and enzymatic methods are almost equal in terms of stereoselectivity and transfer efficiency. By contrast, for syntheses of sialylated structures, enzymatic procedures are still considerably superior to classical chemical sialylations with respect to both stereochemical outcome and preparative input. The use of both procedures in a synergistic mode should also be considered. One of the general approaches ideally suited in such cases is the block synthesis method. Moreover, in recent years a number of combined chemical and chemoenzymatic syntheses have been reported . As a proof of principle, we were interested to employ some trisaccharide building units previously obtained by enzymatic routes in block syntheses en route to interesting structures. To this end, two human milk pentasaccharides of prominent importance, sialyllacto- N -tetraose ( 1 ) and an epimer of sialyllacto- N -neotetraose ( 2 ) ( ) were selected as target molecules. Both these pentasaccharides, Neu5Acα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc ( 1 ) and Neu5Acα2-6Galβ1-4GlcNAcβ1-4Galβ1-4Glc ( 2 ), are dominant constituents of complex human milk oligosaccharides ( ). They are considered to play a major role in immuno defense against bacterial and viral infections in the gastrointestinal tract of infants . It is thought that they effectively inhibit bacterial adhesion to epithelial surfaces and so block the first stages of infection processes. Thus, these human milk oligosaccharides are considered as soluble receptor analogues of epithelial cell surfaces .
<chunk>Results and Discussion</chunk> Previously, we reported the chemoenzymatic synthesis of the 3-sialylated lactosamine derivative 3 obtained by the enzymatic β-galactosylation of the 2-azidothioglucoside with p -nitrophenyl β-galactopyranoside and β-galactosidase ( Bovin testes ). The subsequent transsialylation was carried out with p -nitrophenyl sialoside (pNp-αNeu5Ac) and either sialidase from Salmonella typhimurium or from Newcastle disease virus . Recently, a more effective higher yielding transfer has been reported in which sialylation with recombinant transsialidase ( Trypanosoma cruzi ) gave the trisaccharide 3 in 32% yield . Treatment of 3 with methanol and acidic ion exchange resin led to the methyl ester (for the method cf. lit. ) which was then peracetylated to give trisaccharide 4 as the donor building block. For formation of the disaccharide acceptor 6 , a straight-forward three-step standard reaction sequence was used . Methyl β-lactoside was isopropylidenated at 3′,4′-position with dimethoxypropane and p -toluene sulfonic acid in DMF/acetone. Peracetylation (Ac 2 O/Py) and subsequent cleavage of the isopropylidene group with 80% acetic acid at 80 °C gave the diol acceptor 6 . Since it is known that in galactopyranosyl structures the nucleophilicity of 3-OH considerably exceeds that of the 4-OH-group, further protecting group manipulations were not required. Glycosylation of 4 by 6 catalyzed by N -iodosuccinimide and trifluoromethane sulfonic acid (as introduced by van Boom et al. ) gave the β,1-3-linked pentasaccharide 7 in 61% yield. About 5% of the corresponding α,1-3-linked compound and ca. 7% of the bis (β,1-3- and β,1-4-) linked octasaccharide were observed as side products and separated by chromatography but these were not further characterized. Reduction of the 2′′′-azido to the 2′′′-amino group with nickel boride followed by peracetylation gave 8 in 81% yield ( ). 1 H NMR spectrum contained a doublet for H-1″ at δ 4.96 ( J 1″2″ = 8.0 Hz) and a down field shifted doublet for H-4′ at δ 5.37 ( J 3′4′ = 2.9 Hz). A similar approach was employed for the synthesis of the protected epimer of sialyllacto- N -neotetraose 14 . β-Galactosylation of 2-azidothioglucoside with p -nitrophenyl β-galactopyranoside and β-galactosidase ( Bacillus circulans ) gave the β,1-3-linked isolactosamine derivative. Further sialylation at position 3′-OH with pNp-αNeu5Ac and either sialidase from Vibrio cholerae or Clostridium perfringens afforded the α,2-6-sialylated trisaccharide 9 exclusively . Later studies showed that 9 could be obtained in an enhanced yield of 32% by transsialylation with recombinant transsialidase ( Trypanosama cruzi ) . Formation of the methyl ester and peracetylation led to the trisaccharide donor building block 10 . Synthesis of the disaccharide acceptor in this case started from methyl β-lactoside, which was transformed into its 4′,6′-benzylidene-protected derivative 11 in almost quantitative yield by transacetalization with benzaldehyde dimethylacetal in acetonitrile under p -toluenesulfonic acid catalysis. Subsequent peracetylation with acetic anhydride/pyridine, selective cleavage of the benzylidene group with 80% acetic acid at 90 °C and finally treatment with tert -butyldiphenylsilyl chloride and imidazole in DMF afforded the disaccharide 12 (cf. references ). After activation of the trisaccharide donor 10 with N -iodosuccinimide and trifluoromethanesulfonic acid, the disaccharide acceptor unit 12 could be glycosylated to give the β,1-4-linked pentasaccharide derivative 13 in 53% yield. In addition, the corresponding α,1-4-linked pentasaccharide was obtained in 8% yield. Finally, the azido group was reduced by the nickel boride method with sodium borohydride, nickel chloride and boric acid . During this step partial cleavage of the tert -butyldiphenylsilyl groups was also observed. Complete removal was achieved with trifluoroacetic acid in dichloromethane. For characterization purposes, peracetylation was carried out to give the completely protected pentasaccharide 14 in 67% yield ( ). As evident from a comparison of the 1 H NMR data of 14 with the precursor tri- and disaccharide units 10 and 12 , the novel characteristic doublet for the anomeric H-1″ of the β-GlcNAc unit at δ 5.12 ( J 1″2″ = 8.2 Hz) as well as the downfield shift Δδ 0.15 of H-4′ to δ 4.14 compared to 12 were in accord with structure of the target pentasaccharide.
<chunk>Conclusion</chunk> In this contribution chemoenzymatically generated sialyl α,2-3- and sialyl α,2-6-glycosylated thiophenol 2-azido-lactose derivatives were employed as precursors for sialylated lactosaminide donor substituents in triflic acid/ N -iodosuccinimide glycosylations. With easily accessible selectively unprotected lactose acceptor glycosides the pentasaccharide structures sialyllacto- N -tetraose and the epimer of sialyllacto- N -neotetraose could be obtained in good yields, and subsequently transformed into their peracetylated derivatives for structure elucidation. Thus, a combination of enzymatic and purely chemical procedures was shown to be advantageous in the preparation of complex oligosaccharides.
<chunk>Experimental</chunk> For general methods cf. reference . The NMR data for the saccharide rings in the pentasaccharides 7 , 8 , 13 and 14 are denoted according to the Roman numberals I-V from the reducing end, as depicted for compounds 8 and 14 ( ): Methyl O -(methyl-5-acetamido-4,7,8,9-tetra- O -acetyl-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosylonate)-(2-3)- O -(2,4,6-tri- O -acetyl-β-D-galactopyranosyl)-(1-3)- O -(4,6-di- O -acetyl-2-azido-2-deoxy-β-D-glucopyranosyl)-(1-3)- O -(2,4,6-tri- O -acetyl-β-D-galactopyranosyl)-(1-4)-2,3,6-tri-O-acetyl-β-D-glucopyranoside (7): Glycosylation was carried out as described for the synthesis of compound 13 from compound 4 (95 mg, 83 μmol) as donor and compound 6 (50 mg, 86 μmol) as acceptor. The pentasccharide derivative 7 was obtained as a colorless amorphous solid; 83 mg (61%); [α] D 20 = −3.5 ( c 0.1, CHCl 3 ); 1 H NMR (500 MHz, CDCl3) δ 5.75 (dt, 1 H, H- 8 V ), 5.72 (d, 1 H, NH), 5.39 (d, J 3,4 = 2.8 Hz, 1 H, H -4 II ), 5.36 (dd, J 7,8 = 9.3 Hz, 1H, H -7 V ), 5.15 (dd, J 2,3 = J 3,4 = 9.4 Hz, 1 H, H -3 I ), 5.06 (dd, J 1,2 = 8.0 Hz, J 2,3 = 10.2 Hz, H -2 IV ), 4.99–4.97 (m, 2 H, H -2 II , H -4 III ), 4.96 (dd, J 1,2 = 7.8 Hz, J 2,3 = 9.6 Hz, 1 H, H -2 I ), 4.91 (dd, J 1,2 = 8.0 Hz, J 2,3 = 10.2 Hz, 1 H, H -1 IV ), 4.87 (d, J 3,4 = 2.6 Hz, H -4 IV ), 4.83 (dt, J 3eq,4 = 4.4 Hz, J 3ax,4 = J 4,5 = 12.1 Hz, 1 H, H -4 V ), 4.75 (dd, J 5,6 = 10.1 Hz, J 6,7 = 2.2 Hz, H -6 V ), 4.62 (dd, 1 H, H -3 III ), 4.57 (dd, J 2,3 = 10.0 Hz, J 3,4 = 2.8 Hz, 1 H, H -3 IV ), 4.51 (d, J 1,2 = 7.8 Hz, 1 H, H -1 I ), 4.49 (dd, 1 H, H -6 V ), 4.39 (d, J 1,2 = 8.0 Hz, 1 H, H -1 II ), 4.37 (d, J 1,2 = 8.1 Hz, 1 H, H -1 III ), 4.29 (dd, J 9a,9b = 12.4 Hz, J 8,9b = 2.4 Hz, H -9b V ), 4.24 (dd, J 6a,6b = 12.6 Hz, J 5,6a = 6.6 Hz, 1 H, H -6a III ), 4.17 (dd, J 5,6 = 10.2 Hz, H -5 V ), 4.13 (d, 1 H, NH), 3.75 (s, 3 H, COOCH 3 V ), 4.00 (dd, J 8,9a = 5.8 Hz, 1 H, H -9a V ), 3.72 (bt, 1 H, H -5 IV ), 3.59 (m, 1 H, H -5 I ), 3.47–3.44 (m, 2 H, H -3 II , H -6a IV ), 3.39 (dd, J 6a,6b = 11.0 Hz, J 5,6b = 7.8 Hz, H -6b IV ), 2.61 (dd, J 3ax,3eq = 12.4 Hz, J 3eq,4 = 4.8 Hz, 1 H, H -3eq V ), 2.17–1.33 (15s, 45 H, 14 OAc, 1 NAc), 1.91 (dd, J 3ax,4 = 11.8 Hz, 1 H, H -3ax V ). C 67 H 92 N 4 O 43 (1641.45): Found C, 49.33; H, 5.59; N, 3.62. Calculated C; 49.02; H, 5.65; N, 3.41. MALDI-TOF: 1664.44 (M+Na) + ; 1680.59 (M+K) + . Methyl O -(methyl-5-acetamido-4,7,8,9-tetra- O -acetyl-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosylonate)-(2-3)- O -(2,4,6-tri- O -acetyl-β-D-galactopyranosyl)-(1-3)- O -(4,6-di- O -acetyl-2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1-3)- O -(2,4,6-tri- O -acetyl-β-D-galactopyranosyl)-(1-4)-2,3,6-tri-O-acetyl-β-D-glucopyranoside (8): Reduction and peracetylation of compound 7 was carried out as described for 14 . Thus, from 80 mg (49 μmol) of 7 , 65 mg (81%) of 8 was obtained as a colorless amorphous solid; [α] D 20 = −12.7 ( c 0.5, CHCl 3 ); 1 H NMR (500 MHz, CDCl 3 ) δ 5.74 (dt, 1 H, H- 8 V ), 5.72 (d, 1 H, NH), 5.38 (d, J 3,4 = 2.8 Hz, 1 H, H- 4 II ), 5.33 (dd, J 7,8 = 9.3 Hz, 1H, H- 7 V ), 5.12 (dd, J 2,3 = J 3,4 = 9.6 Hz, 1 H, H -3 I ), 5.05 (dd, J 1,2 = 8.0 Hz, J 2,3 = 10.2 Hz, H -2 IV ), 4.99 (dd, J 1,2 = 8.0 Hz, J 2,3 = 9.7 Hz, 1 H, H -2 II ), 4.97 (m, 2 H, H -4 III ), 4.95 (dd, J 1,2 = 8.0 Hz, J 2,3 = 9.8 Hz, 1 H, H -2 I ), 4.91 (dd, J 1,2 = 7.9 Hz,1 H, H -1 IV ), 4.86 (dt, J 3eq,4 = 4.6 Hz, J 3ax,4 = J 4,5 = 12.0 Hz, 1 H, H -4 V ), 4.83 (d, J 3,4 = 2.6 Hz, H -4 IV ), 4.75 (dd, J 5,6 = 9.9 Hz, J 6,7 = 1.8 Hz, H -6 V ), 4.61 (dd, 1 H, H- 3 III ), 4.57 (dd, J 2,3 = 9.8 Hz, J 3,4 = 2.6 Hz, 1 H, H -3 IV ), 4.53 (dd, 1 H, H -6 I ), 4.51 (d, J 1,2 = 8.0 Hz, 1 H, H -1 I ), 4.39–4.37 (m, 2 H, H -1 II , H -1 III ), 4.32 (dd, J 9a,9b = 12.3 Hz, J 8,9b = 2.1 Hz, H -9b V ), 4.26 (dd, J 6a,6b = 12.2 Hz, J 5,6a = 6.1 Hz, 1 H, H- 6a III ), 4.13 (dd, J 5,6 = 9.8 Hz, H -5 V ), 4.13 (d, 1 H, NH), 4.00 (dd, J 8,9a = 5.8 Hz, 1 H, H -9a V ), 3.75 (s, 3 H, COOCH 3 V ), 3.71 (bt, 1 H, H -5 IV ), 3.55 (m, 1 H, H -5 I ), 3.46–3.44 (m, 2 H, H -6a IV , H -3 II ), 3.43 (dd, J 6a,6b = 11.0 Hz, J 5,6b = 7.8 Hz, H -6b IV ), 2.59 (dd, J 3ax,3eq = 12.2 Hz, J 3eq,4 = 4.6 Hz, 1 H, H -3eq V ), 2.21–1.35 (17s, 51 H, 15 OAc, 2 NAc), 1.93 (dd, J 3ax,4 = 12.0 Hz, 1 H, H -3ax V ). C 69 H 96 N 2 O 44 (1657.49): Found C, 49.90; H, 5.69; N, 1.57. Calculated C, 50.00; H, 5.84; N, 1.69. MALDI-TOF: 1680.48 (M+Na) + ; 1696.59 (M+K) + . Methyl O -(methyl-5-acetamido-4,7,8,9-tetra- O -acetyl-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosylonate)-(2-6)- O -(2,3,4-tri- O -acetyl-β-D-galactopyranosyl)-(1-4)- O -(3,6-di- O -acetyl-2-azido-2-deoxy-β-D-glucopyranosyl)-(1-4)- O -(2,3-di- O -acetyl-6- O-tert -butyldiphenylsilyl-β-D-galactopyranosyl)-(1-4)- O -2,3,6-tri- O -acetyl-β-D-glucopyranoside (13): A solution of trisaccharide 10 (68 mg, 60 μmol) and disaccharide 12 (56 mg, 70 μmol) in anhydrous toluene (2 mL) was cooled to −40 °C. N -Iodosuccinimide (20 mg, 94 μmol), molecular sieves (4 Å, 200 mg) were added, and after cooling a saturated solution of trifluoromethane sulfonic acid in CCl 4 (ca. 2 M, 50 μL) was added with vigorous stirring. The mixture was gradually warmed over 2.5 h to −10 °C. Ethyl acetate (20 mL) was added and the reaction quenched by addition of a saturated aqueous NaHCO 3 solution (10 mL). After filtration through Celite, the phases were separated. The organic phase was washed with aqueous Na 2 S 2 O 3 solution (10 mL), dried over MgSO 4 , evaporated and the residue purified by flash chromatography with petroleum ether/ethyl acetate 2:1. Compound 7 was obtained as a colorless amorphous solid; 58 mg (53%). [α] D 20 = −21.6 ( c 0.3, CHCl 3 ); 1 H NMR (500 MHz, CDCl 3 ) δ 7.70–7.24 (m, 10, Ph), 5.74 (dd, 1 H, H -4 IV ), 5.70 (ddd, 1 H, H -8 V ), 5.61 (dd, J 2,3 = 10.2 Hz, 1 H, H -2 IV ), 5.44 (dd, J 7,8 = 9.0 Hz, 1 H, H -7 V ), 5.26 (dd, 1 H, H -3 IV ), 5.22 (t, J 2,3 = J 3,4 = 9.8 Hz, 1 H, H -3 I ), 5.10 (dd, J 1,2 = 8.0 Hz, J 2,3 = 9.9 Hz, 1 H, H -2 II ),4.93 (t, J 2,3 = 9.8 Hz, 1 H, H -2 I ), 4.87 (2d, 2 H, H -1 IV , H -3 III ), 4.85 (ddd, J 4,5 = 10.2 Hz, 1 H, H -4 V ), 4.65 (dd, 1 H, H -3 II ), 4.61 (d, J 3,4 = 4.0 Hz, H -4 II ), 4.59 (dd, J 9a,9b = 12.2 Hz, 1 H, H -9a V ), 4.51 (d, 1 H, H -1 III ), 4.49 (d, J 1,2 = 8.0 Hz, 1 H, H -1 II ), 4.48 (dd, 1 H, H -6b III ), 4.46 (d, J 1,2 = 10.0 Hz, 1 H, H -1 I ), 4.42 (dd, 1 H, H -6b I ), 4.34 (dd, 1 H, H -9b V ), 4.32 (d, 1 H, N H V ), 4.29 (ddd, J 5,6 = 10.6 Hz, 1 H, H -5 V ), 4.20 (dd, 1 H, H -6b III ), 4.13 (m, 2 H, H -6 V , H -6b II ), 4.10 (dd, J 5,6a = 5.6 Hz, J 6a,6b = 12.4 Hz, 1 H, H -6a I ), 4.08 (dd, J 5,6a = J 6a,6b = 6.0 Hz, 1 H, H -6a II ), 4.06 (dd, 1 H, H -6b IV ), 3.88 (ddd, 1 H, H -5 IV ), 3.83 (m, 2 H, H -5I, H -4 III ), 3.78 (s, 3 H, OCH 3 ), 3.77 (t, J 3,4 = 9.9 Hz, 1 H, H -4 I ), 3.62 (ddd, J 4,5 = 10.0 Hz, J 5,6a = 5.5 Hz, J 5,6b = 2.0 Hz, 1 H, H -5 I ), 3.61 (dd, 1 H, H -6b IV ), 3.59 (ddd, 1 H, H -5 III ), 3.56 (dd, J 2,3 = 10.2 Hz, 1 H, H -2 III ), 3.33 (s, 3 H, C H 3 V ), 2.69 (dd, J 3eq ,3 ax = 12.7 Hz, J 3eq,4 = 4.6 Hz, 1 H, H -3 eq V ), 2.15–1.36 (14s, 42 H, 13 OAc, 1 NAc), 2.03 (dd, J 3ax,4 = 12.0 Hz, 1 H, H -3 ax V ), 1.01 (s, 9 H, SiCCH 3 ). C 81 H 108 N 4 O 42 Si (1837.81): Found C, 53.89; H, 6.34; N, 2.66. Calculated C, 52.94; H, 5.92; 3.04. MALDI-TOF: 1860.80 (M+Na) + ; 1876.91 (M+K) + . The α,1 III -4 II -anomer of 7 was obtained as colorless syrup (9 mg, 8%) and not further characterized. Methyl O -(methyl-5-acetamido-4,7,8,9-tetra- O -acetyl-3,5-dideoxy-α-D- glycero -D- galacto -2-nonulopyranosylonate)-(2-6)- O -(2,3,4-tri- O -acetyl-β-D-galactopyranosyl)-(1-4)- O -(3,6-di- O -acetyl-2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1-4)- O -(2,3,6-tri- O- acetyl-β-D-galactopyranosyl)-(1-4)- O -2,3,6-tri-O-acetyl-β-D-glucopyranoside (14): Compound 13 (53 mg, 29 μmol), NiCl 2 ·6H 2 O (105 mg, 450 μmol) and boric acid (55 mg, 900 μmol) were dissolved in ethanol (3 mL). Under vigorous stirring a suspension of sodium borohydride (28 mg, 750 μmol) in ethanol (1 mL) was added with the temperature maintained at 20 °C. After 30 min ethanol (6 mL) and acetic acid (3 mL) were added. Then the mixture was co-distilled three times with toluene (5 mL each), and then the residue dissolved in dichloromethane (10 mL). After washing with diluted aqueous KHSO 4 solution (5 mL), saturated aqueous NaHCO 3 solution (5 mL), and water (5 mL), the organic phase was dried (MgSO 4 ) and evaporated to dryness. The resulting material was treated with dichloromethane/trifluoroacetic acid (9:1, 2 mL) for 1 h at room temperature, then co-distilled three times with toluene (5 mL each) and dried under high vacuum. The residue was treated with acetic anhydride (1 mL) and pyridine (5 mL) for 10 h, then co-distilled three times with toluene (5 mL each). Purification by flash chromatography (toluene/acetone 3:1) gave 14 (35 mg, 67%) as a colorless amorphous solid; [α] D 20 = −31.2 ( c 0.4, CHCl 3 ); 1 H NMR (500 MHz, CDCl 3 ) δ 7.72–7.25 (m, 10, Ph), 5.72 (ddd, 1 H, H -8 V ), 5.70 (dd, 1 H, H -4 IV ), 5.59 (dd, J 2,3 = 9.9 Hz, 1 H, H -2 IV ), 5.43 (dd, J 7,8 = 8.8 Hz, 1 H, H -7 V ), 5.32 (d, J 3,4 = 4.0 Hz, H -4 II ), 5.23 (dd, 1 H, H -3 IV ), 5.21 (t, J 2,3 = J 3,4 = 10.2 Hz, 1 H, H -3 I ), 5.11 (dd, J 1,2 = 7.8 Hz, J 2,3 = 9.8 Hz, 1 H, H -2 II ), 4.90 (t, J 2,3 = 10.0 Hz, 1 H, H -2 I ), 4.88 (d, 1 H, H -1 IV ), 4.86 (m, 2 H, H -3 III , H -4 V ), 4.63 (dd, 1 H, H -3 II ), 4.59 (dd, J 9a,9b = 11.9 Hz, 1 H, H -9a V ), 4.53 (d, J 1,2 = 8.0 Hz, 1 H, H -1 II ), 4.50 (d, 1 H, H -1 III ), 4.47 (m, 2 H, H -6b III , H -1 I ), 4.39 (dd, 1 H, H -6b I ), 4.32 (d, 1 H, N H ), 4.31 (dd, 1 H, H -9b V ), 4.27 (ddd, J 5,6 = 10.3 Hz, 1 H, H -5 V ), 4.18 (dd, 1 H, H -6b III ), 4.11 (dd, 1 H, H -6 V ), 4.09 (dd, J 5,6a = 5.8 Hz, J 6a,6b = 12.2 Hz, 1 H, H -6a I ), 4.01 (dd, 1 H, H -6b IV ), 3.92 (ddd, 1 H, H -5 IV ), 3.87 (dd, J 5,6a = J 6a,6b = 6.0 Hz, 1 H, H -6a II ), 3.85 (dd, 1 H, H -6b II ), 3.82 (m, 2 H, H -5 II , H -4 III ), 3.77 (s, 3 H, OCH 3 ), 3.75 (t, J 3,4 = 10.1 Hz, 1 H, H -4 I ), 3.65 (ddd, J 4,5 = 9.8 Hz, J 5,6a = 5.6 Hz, J 5,6b = 1.8 Hz, 1 H, H -5 I ), 3.59 (m, 2 H, H -6b IV , H -5 III ), 3.57(dd, J 2,3 = 10.0 Hz, 1 H, H -2 III ), 3.31 (s, 3 H, C H 3 V ), 2.70 (dd, J 3eq,3ax = 12.5 Hz, J 3e,4 = 4.4, 1 H, H -3eq V ), 2.17–1.33 (16s, 48 H, 14 OAc, 2 NAc), 2.01 (dd, J 3ax,4 = 12.0 Hz, 1 H, H -3ax V ), 1.00 (s, 9 H, SiCCH 3 ). C 69 H 96 N 2 O 44 (1657.49): Found C, 49.86; H, 5.77; N, 1.65. Calculated C, 50.00; H, 5.84; N, 1.69. MALDI-TOF: 1680.39 (M+Na) + ; 1696.59(M+K) + .
Structures of pentasaccharides 1 and 2 .
Roman numbering of saccharide units in all pentasaccharides for NMR assignment.
Preparation of pentasaccharide 8 . 1) MeOH, acidic ion exchange resin; 2) Ac 2 O, pyridine; 3) 80% HOAc, 90 °C; 4) NIS, CF 3 SO 3 H, 61%; 5) NiCl 2 •6H 2 O, H 3 BO 3 , EtOH, then NaBH 4 , EtOH and acidic workup. Preparation of pentasaccharide 14 . 1) MeOH, acidic ion exchange resin; 2) Ac 2 O, pyridine; 3) 80% HOAc, 90 °C; 4) TBDPSCl, imidazole, DMF; 5) NIS, CF 3 SO 3 H, 53%; 6) NiCl 2 •6H 2 O, H 3 BO 3 , EtOH, then NaBH 4 , EtOH, then acidic workup; 7) CF 3 CO 2 H, CH 2 Cl 2 . VSXGAIHLUJPKPA-MLKJWYSASA-N InChI=1S/C23H34O16/c1-9(24)32-7-14-16(29)17(30)19(34-11(3)26)23(37-14)39-18-15(8-33-10(2)25)38-22(31-6)21(36-13(5)28)20(18)35-12(4)27/h14-23,29-30H,7-8H2,1-6H3/t14-,15-,16+,17+,18-,19-,20+,21-,22-,23+/m1/s1 CC(=O)OC[C@@H]1[C@@H]([C@@H]([C@H]([C@@H](O1)O[C@@H]2[C@@H](COC(=O)C)O[C@H]([C@@H]([C@H]2OC(=O)C)OC(=O)C)OC)OC(=O)C)O)O [C@H]1([C@@H]([C@H]([C@@H](O[C@@H]1COC(=O)C)O[C@H]2[C@@H]([C@H]([C@@H](O[C@@H]2COC(=O)C)OC)OC(=O)C)OC(=O)C)OC(=O)C)O)O |(340.78,-144.83,;347.98,-157.3,;361.91,-153.65,;375.76,-157.3,;368.56,-144.83,;354.63,-148.48,;347.43,-136.01,;361.33,-132.28,;371.48,-142.43,;385.34,-138.71,;367.77,-156.29,;388.23,-150.1,;413.07,-156.8,;420.27,-144.33,;434.2,-148.29,;448.05,-144.33,;440.85,-156.8,;426.92,-152.84,;419.72,-165.31,;432.19,-172.51,;432.19,-186.86,;444.62,-194.04,;419.76,-194.04,;460.52,-151.53,;460.52,-165.88,;441.4,-135.81,;455.75,-135.81,;462.93,-123.39,;462.93,-148.24,;408.36,-147.1,;404.19,-160.83,;414,-171.31,;390.22,-164.09,;369.11,-166.12,;383.46,-166.12,;390.64,-153.69,;390.64,-178.55,;334.07,-153.57,;340.78,-128.43,)| AuxInfo=1/0/N:11,35,39,24,28,31,7,29,10,34,38,23,27,6,17,1,2,16,3,15,20,19,4,9,33,37,22,26,12,13,30,8,32,36,21,25,5,18,14/it:im/rA:39nC.eC.oC.eC.oOC.eCOOCCOOOC.oC.oC.eOC.eC.eOOCCOOCCCOCOOCCOOCC/rB:s1;s2;s3;s4;s1s5;s6;s7;;s8d9;s10;s1;s2;s4;;s14s15;s16;s17;s18;s15s19;s15;;s21d22;s23;s20;;s25d26;s27;s17;s19;s30;s29;;s32d33;s34;s3;;s36d37;s38;/rC:340,7799,-144,8292,0;347,9799,-157,2999,0;361,9102,-153,6503,0;375,7557,-157,2999,0;368,5568,-144,8292,0;354,6255,-148,4774,0;347,4255,-136,0067,0;361,3348,-132,2797,0;385,3428,-138,7126,0;371,4818,-142,4266,0;367,7677,-156,2877,0;340,7799,-128,4292,0;334,0706,-153,5730,0;388,2264,-150,0999,0;420,2722,-144,3292,0;413,0722,-156,7999,0;426,9181,-152,8427,0;440,8477,-156,7999,0;448,0494,-144,3292,0;434,2025,-148,2850,0;408,3629,-147,1022,0;413,9993,-171,3096,0;404,1930,-160,8330,0;390,2169,-164,0872,0;441,4025,-135,8143,0;462,9275,-123,3868,0;455,7524,-135,8142,0;462,9275,-148,2417,0;419,7181,-165,3135,0;460,5202,-151,5292,0;460,5202,-165,8792,0;432,1888,-172,5135,0;444,6163,-194,0385,0;432,1888,-186,8635,0;419,7614,-194,0385,0;369,1102,-166,1211,0;390,6352,-153,6936,0;383,4602,-166,1211,0;390,6352,-178,5485,0; C23H34O16 CC(=O)O* OAc,CO* OMe CDK 04212621462D 0 0 0 0 0 999 V3000 M V30 BEGIN CTAB M V30 COUNTS 39 40 0 0 1 M V30 BEGIN ATOM M V30 1 C 340.77991 -144.82919 0 0 CFG=1 M V30 2 C 347.97992 -157.29994 0 0 CFG=2 M V30 3 C 361.91022 -153.65033 0 0 CFG=1 M V30 4 C 375.75568 -157.29994 0 0 CFG=2 M V30 5 O 368.55676 -144.82919 0 0 M V30 6 C 354.62549 -148.47742 0 0 CFG=1 M V30 7 C 347.42548 -136.00667 0 0 M V30 8 O 361.33478 -132.27966 0 0 M V30 9 O 385.3428 -138.71255 0 0 M V30 10 C 371.48178 -142.42662 0 0 M V30 11 C 367.76773 -156.28767 0 0 M V30 12 O 340.77991 -128.4292 0 0 M V30 13 O 334.07059 -153.57295 0 0 M V30 14 O 388.22644 -150.09995 0 0 M V30 15 C 420.27219 -144.32919 0 0 CFG=2 M V30 16 C 413.0722 -156.79994 0 0 CFG=2 M V30 17 C 426.91809 -152.84274 0 0 CFG=1 M V30 18 O 440.84772 -156.79994 0 0 M V30 19 C 448.04944 -144.32919 0 0 CFG=1 M V30 20 C 434.20245 -148.28502 0 0 CFG=1 M V30 21 O 408.36285 -147.10219 0 0 M V30 22 O 413.99933 -171.30957 0 0 M V30 23 C 404.19302 -160.83299 0 0 M V30 24 C 390.21689 -164.08722 0 0 M V30 25 O 441.40247 -135.81425 0 0 M V30 26 O 462.92746 -123.38678 0 0 M V30 27 C 455.75244 -135.81424 0 0 M V30 28 C 462.92746 -148.24171 0 0 M V30 29 C 419.71811 -165.31351 0 0 M V30 30 O 460.5202 -151.52919 0 0 M V30 31 C 460.5202 -165.87918 0 0 M V30 32 O 432.18884 -172.5135 0 0 M V30 33 O 444.6163 -194.0385 0 0 M V30 34 C 432.18884 -186.86349 0 0 M V30 35 C 419.76138 -194.0385 0 0 M V30 36 O 369.1102 -166.12108 0 0 M V30 37 O 390.63519 -153.6936 0 0 M V30 38 C 383.46021 -166.12106 0 0 M V30 39 C 390.63519 -178.54852 0 0 M V30 END ATOM M V30 BEGIN BOND M V30 1 1 1 2 M V30 2 1 2 3 M V30 3 1 3 4 M V30 4 1 4 5 M V30 5 1 5 6 M V30 6 1 6 1 M V30 7 1 6 7 M V30 8 1 8 10 M V30 9 2 9 10 M V30 10 1 10 11 M V30 11 1 7 8 M V30 12 1 1 12 M V30 13 1 2 13 M V30 14 1 4 14 M V30 15 1 15 16 M V30 16 1 16 17 M V30 17 1 17 18 M V30 18 1 18 19 M V30 19 1 19 20 M V30 20 1 20 15 M V30 21 1 14 16 M V30 22 1 21 23 M V30 23 2 22 23 M V30 24 1 23 24 M V30 25 1 15 21 M V30 26 1 25 27 M V30 27 2 26 27 M V30 28 1 27 28 M V30 29 1 20 25 M V30 30 1 17 29 M V30 31 1 30 31 M V30 32 1 19 30 M V30 33 1 32 34 M V30 34 2 33 34 M V30 35 1 34 35 M V30 36 1 29 32 M V30 37 1 36 38 M V30 38 2 37 38 M V30 39 1 38 39 M V30 40 1 3 36 M V30 END BOND M V30 END CTAB M END BVKILKCBYKMDBW-ABBDDOLJSA-N InChI=1S/C39H52O16Si/c1-22(40)47-20-30-32(34(50-24(3)42)36(52-26(5)44)37(46-9)54-30)55-38-35(51-25(4)43)33(49-23(2)41)31(45)29(53-38)21-48-56(39(6,7)8,27-16-12-10-13-17-27)28-18-14-11-15-19-28/h10-19,29-38,45H,20-21H2,1-9H3/t29-,30-,31+,32-,33+,34+,35-,36-,37-,38+/m1/s1 CC(=O)OC[C@@H]1[C@H]([C@@H]([C@H]([C@H](OC)O1)OC(=O)C)OC(=O)C)O[C@H]2[C@@H]([C@H]([C@H]([C@@H](CO[Si](C3=CC=CC=C3)(C4=CC=CC=C4)C(C)(C)C)O2)O)OC(=O)C)OC(=O)C [C@H]1([C@@H]([C@H]([C@@H](O[C@@H]1CO[Si](C2=CC=CC=C2)(C3=CC=CC=C3)C(C)(C)C)O[C@H]4[C@@H]([C@H]([C@@H](O[C@@H]4COC(=O)C)OC)OC(=O)C)OC(=O)C)OC(=O)C)OC(=O)C)O |(392.71,-163.56,;399.91,-176.03,;413.84,-172.38,;427.68,-176.03,;420.48,-163.56,;406.55,-167.21,;399.35,-154.74,;404.58,-141.33,;418.76,-139.16,;423.98,-125.79,;443.57,-122.84,;450.84,-104.14,;438.29,-88.57,;418.31,-91.5,;411.43,-110.21,;418.44,-153.5,;400.85,-163.2,;400.42,-183.31,;417.62,-193.77,;435.28,-184.15,;435.77,-164.11,;432.84,-141.97,;437.44,-155.56,;442.31,-131.19,;446.91,-144.78,;441.15,-168.83,;465.12,-172.53,;472.32,-160.06,;486.25,-164.02,;500.1,-160.06,;492.9,-172.53,;478.97,-168.57,;471.77,-181.05,;484.24,-188.25,;484.24,-202.6,;496.67,-209.77,;471.81,-209.77,;512.57,-167.26,;512.57,-181.61,;493.45,-151.55,;507.8,-151.55,;514.98,-139.12,;514.98,-163.97,;460.42,-162.83,;456.25,-176.56,;466.05,-187.04,;442.27,-179.82,;421.04,-184.85,;435.39,-184.85,;442.56,-172.43,;442.56,-197.28,;386,-172.3,;375.85,-182.45,;379.57,-196.31,;361.99,-178.74,;392.71,-147.16,)| AuxInfo=1/0/N:56,30,45,34,49,23,24,25,52,14,19,13,15,18,20,12,16,17,21,50,7,55,29,44,33,48,9,11,6,38,1,37,2,36,3,41,40,4,22,54,28,43,32,47,26,51,53,8,27,42,31,46,5,39,35,10/E:(6,7,8)(10,11)(12,13,14,15)(16,17,18,19)(27,28)/it:im/rA:56nC.eC.oC.eC.oOC.eCOCSiCCCCCCCCCCCCCCCOOOCCOOCCOC.oC.oC.eOC.eC.eOOCCOOCCCOCOOCC/rB:s1;s2;s3;s4;s1s5;s6;s7;;s8s9;s10;d9;s12;d13;s14;s9d15;d11;s17;d18;s19;s11d20;s10;s22;s22;s22;s1;s2;;s27d28;s29;s3;;s31d32;s33;s4;;s35s36;s37;s38;s39;s36s40;s36;;s42d43;s44;s41;;s46d47;s48;s38;s40;s51;s50;;s53d54;s55;/rC:392,7074,-163,5610,0;399,9074,-176,0318,0;413,8377,-172,3822,0;427,6832,-176,0318,0;420,4843,-163,5610,0;406,5530,-167,2093,0;399,3530,-154,7385,0;404,5800,-141,3292,0;423,9764,-125,7875,0;418,7647,-139,1576,0;418,4401,-153,5039,0;443,5659,-122,8442,0;450,8396,-104,1399,0;438,2903,-88,5670,0;418,3103,-91,5038,0;411,4271,-110,2146,0;400,8522,-163,1977,0;400,4247,-183,3111,0;417,6193,-193,7672,0;435,2757,-184,1462,0;435,7718,-164,1056,0;432,8367,-141,9684,0;437,4384,-155,5606,0;442,3069,-131,1871,0;446,9087,-144,7793,0;392,7074,-147,1610,0;385,9981,-172,3048,0;379,5652,-196,3128,0;375,8511,-182,4518,0;361,9901,-178,7377,0;421,0377,-184,8529,0;442,5627,-172,4255,0;435,3877,-184,8529,0;442,5627,-197,2804,0;441,1539,-168,8318,0;472,3247,-160,0610,0;465,1247,-172,5318,0;478,9706,-168,5746,0;492,9002,-172,5318,0;500,1019,-160,0610,0;486,2549,-164,0169,0;460,4153,-162,8340,0;466,0518,-187,0414,0;456,2455,-176,5648,0;442,2694,-179,8191,0;493,4550,-151,5461,0;514,9800,-139,1186,0;507,8049,-151,5461,0;514,9800,-163,9736,0;471,7706,-181,0453,0;512,5727,-167,2610,0;512,5727,-181,6110,0;484,2414,-188,2453,0;496,6688,-209,7703,0;484,2414,-202,5953,0;471,8139,-209,7703,0; C39H52O16Si CC(C)(C)[Si](C1=CC=CC=C1)(C2=CC=CC=C2)O* OTBDPS,CC(=O)O* OAc,CO* OMe CDK 04212621462D 0 0 0 0 0 999 V3000 M V30 BEGIN CTAB M V30 COUNTS 56 59 0 0 1 M V30 BEGIN ATOM M V30 1 C 392.7074 -163.56104 0 0 CFG=1 M V30 2 C 399.90741 -176.03178 0 0 CFG=2 M V30 3 C 413.83771 -172.38217 0 0 CFG=1 M V30 4 C 427.68317 -176.03178 0 0 CFG=2 M V30 5 O 420.48425 -163.56104 0 0 M V30 6 C 406.55298 -167.20926 0 0 CFG=1 M V30 7 C 399.35297 -154.73851 0 0 M V30 8 O 404.57996 -141.32919 0 0 M V30 9 C 423.97638 -125.78746 0 0 M V30 10 Si 418.76468 -139.15759 0 0 M V30 11 C 418.44012 -153.50392 0 0 M V30 12 C 443.56586 -122.84421 0 0 M V30 13 C 450.8396 -104.13986 0 0 M V30 14 C 438.29028 -88.56699 0 0 M V30 15 C 418.31033 -91.50381 0 0 M V30 16 C 411.42706 -110.2146 0 0 M V30 17 C 400.85223 -163.19769 0 0 M V30 18 C 400.42468 -183.31111 0 0 M V30 19 C 417.61926 -193.76717 0 0 M V30 20 C 435.27567 -184.14621 0 0 M V30 21 C 435.77179 -164.10559 0 0 M V30 22 C 432.8367 -141.96843 0 0 M V30 23 C 437.43845 -155.56058 0 0 M V30 24 C 442.30695 -131.18713 0 0 M V30 25 C 446.90872 -144.77928 0 0 M V30 26 O 392.7074 -147.16104 0 0 M V30 27 O 385.99808 -172.30479 0 0 M V30 28 O 379.56516 -196.31281 0 0 M V30 29 C 375.85107 -182.45177 0 0 M V30 30 C 361.99005 -178.73772 0 0 M V30 31 O 421.03769 -184.85294 0 0 M V30 32 O 442.56268 -172.42546 0 0 M V30 33 C 435.3877 -184.85294 0 0 M V30 34 C 442.56268 -197.2804 0 0 M V30 35 O 441.15393 -168.83179 0 0 M V30 36 C 472.32468 -160.06104 0 0 CFG=2 M V30 37 C 465.12469 -172.53178 0 0 CFG=2 M V30 38 C 478.97058 -168.57458 0 0 CFG=1 M V30 39 O 492.90021 -172.53178 0 0 M V30 40 C 500.10193 -160.06104 0 0 CFG=1 M V30 41 C 486.25494 -164.01686 0 0 CFG=1 M V30 42 O 460.41534 -162.83403 0 0 M V30 43 O 466.05182 -187.04141 0 0 M V30 44 C 456.24551 -176.56483 0 0 M V30 45 C 442.26938 -179.81906 0 0 M V30 46 O 493.45496 -151.5461 0 0 M V30 47 O 514.97998 -139.11862 0 0 M V30 48 C 507.80493 -151.54608 0 0 M V30 49 C 514.97998 -163.97356 0 0 M V30 50 C 471.7706 -181.04535 0 0 M V30 51 O 512.57269 -167.26103 0 0 M V30 52 C 512.57269 -181.61104 0 0 M V30 53 O 484.24136 -188.24535 0 0 M V30 54 O 496.66882 -209.77034 0 0 M V30 55 C 484.24136 -202.59534 0 0 M V30 56 C 471.8139 -209.77034 0 0 M V30 END ATOM M V30 BEGIN BOND M V30 1 1 1 2 M V30 2 1 2 3 M V30 3 1 3 4 M V30 4 1 4 5 M V30 5 1 5 6 M V30 6 1 6 1 M V30 7 1 6 7 M V30 8 1 8 10 M V30 9 1 9 10 M V30 10 1 10 11 M V30 11 2 9 12 M V30 12 1 12 13 M V30 13 2 13 14 M V30 14 1 14 15 M V30 15 2 15 16 M V30 16 1 9 16 M V30 17 2 11 17 M V30 18 1 17 18 M V30 19 2 18 19 M V30 20 1 19 20 M V30 21 2 20 21 M V30 22 1 11 21 M V30 23 1 10 22 M V30 24 1 22 23 M V30 25 1 22 24 M V30 26 1 22 25 M V30 27 1 7 8 M V30 28 1 1 26 M V30 29 1 27 29 M V30 30 2 28 29 M V30 31 1 29 30 M V30 32 1 2 27 M V30 33 1 31 33 M V30 34 2 32 33 M V30 35 1 33 34 M V30 36 1 3 31 M V30 37 1 4 35 M V30 38 1 36 37 M V30 39 1 37 38 M V30 40 1 38 39 M V30 41 1 39 40 M V30 42 1 40 41 M V30 43 1 41 36 M V30 44 1 35 37 M V30 45 1 42 44 M V30 46 2 43 44 M V30 47 1 44 45 M V30 48 1 36 42 M V30 49 1 46 48 M V30 50 2 47 48 M V30 51 1 48 49 M V30 52 1 41 46 M V30 53 1 38 50 M V30 54 1 51 52 M V30 55 1 40 51 M V30 56 1 53 55 M V30 57 2 54 55 M V30 58 1 55 56 M V30 59 1 50 53 M V30 END BOND M V30 END CTAB M END <chunk>Acknowledgements</chunk> Support of these studies by the Deutsche Forschungsgemeinschaft (SFB 470, A5) and the Fonds der Chemischen Industrie is gratefully acknowledged. J. Org. Chem. Carbohydr. Res. Angew. Chem., Int. Ed. Engl. Carbohydr. Res. Infect. Immun. Z. Ernährungswiss. J. Org. Chem. Org. Biomol. Chem. Chem. Ber. Carbohydr. Res. Tetrahedron Lett. Chem. Ber. Chem. Ber. J. Org. Chem. Bioorg. Med. Chem. Lett. Eur. J. Org. Chem. This is an Open Access article under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The license is subject to the Beilstein Journal of Organic Chemistry terms and conditions: (http://www.beilstein-journals.org/bjoc)